Search

Background and Purpose: Nasal sensory nerves play an important role in symptoms associated with rhinitis triggered by environmental stimuli. Here, we propose that TRPV1 is pivotal in nasal sensory nerve activation and assess the potential of SB-705498 as an intranasal therapy for rhinitis., Experimental Approach: The inhibitory effect of SB-705498 on capsaicin-induced currents in guinea pig trigeminal ganglion cells innervating nasal mucosa was investigated using patch clamp electrophysiology. A guinea pig model of rhinitis was developed using intranasal challenge of capsaicin and hypertonic saline to elicit nasal secretory parasympathetic reflex responses, quantified using MRI. The inhibitory effect of SB-705498, duration of action and potency comparing oral versus intranasal route of administration were examined., Key Results: SB-705498 concentration-dependently inhibited capsaicin-induced currents in isolated trigeminal ganglion cells (pIC50 7.2). In vivo, capsaicin ipsilateral nasal challenge (0.03-1 mM) elicited concentration-dependent increases in contralateral intranasal fluid secretion. Ten per cent hypertonic saline initiated a similar response. Atropine inhibited responses to either challenge. SB-705498 inhibited capsaicin-induced responses by ∼50% at 10 mg·kg⁻¹ (oral), non-micronized 10 mg·mL⁻¹ or 1 mg·mL⁻¹ micronized SB-705498 (intranasal) suspension. Ten milligram per millilitre intranasal SB-705498, dosed 24 h prior to capsaicin challenge produced a 52% reduction in secretory response. SB-705498 (10 mg·mL⁻¹, intranasal) inhibited 10% hypertonic saline responses by 70%., Conclusions and Implications: The paper reports the development of a guinea pig model of rhinitis. SB-705498 inhibits capsaicin-induced trigeminal currents and capsaicin-induced contralateral nasal secretions via oral and intranasal routes; efficacy was optimized using particle-reduced SB-705498. We propose that TRPV1 is pivotal in initiating symptoms of rhinitis., (© 2013 GlaxoSmithKline. British Journal of Pharmacology © 2013 The British Pharmacological Society.)

Background and Purpose: Intraglandular injection of botulinum toxin (BoNT) leads to a transient denervation of the submandibular gland and this is associated with reduced salivary secretion. The purpose of the present study was to verify whether temporary acinar atrophy occurs simultaneously with chemical denervation of the glands., Experimental Approach: Tissue specimens of the right submandibular gland taken from 18 Wistar rats after intraglandular injection of BoNT A, BoNT B, or a combination of both were examined. As a sham control, an equivalent volume of saline was injected into the left submandibular gland. Morphometric measurements, immunohistochemistry, electron microscopy and western blot analysis were used to analyse the morphological and functional changes of the denervated glands., Key Results: Morphological and ultrastructural analyses of the cell organelles and secretory granula showed a clear atrophy of the acini, which was more prominent in glands injected with the combination of BoNT/A and B. Morphometric measurements of the glandular acini revealed a significant reduction of the area of the acinar cells after injection of BoNT (P=0.031). The expression of amylase was significantly reduced in BoNT treated glands., Conclusions and Implications: Intraglandular application of BoNT induces structural and functional changes of the salivary glands indicated by glandular atrophy. These effects may be due to glandular denervation induced by the inhibition of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) involved in acetylcholine release at the neuroglandular junction and also specially inhibition of those involved in exocytosis of the granula of the acinar cells.

Theta-frequency (4-12 Hz) electroencephalographic activity is thought to play a role in mechanisms mediating sensory and mnemonic processing in the entorhinal cortex and hippocampus, but the effects of acetylcholine on excitatory synaptic inputs to the entorhinal cortex are not well understood. Field excitatory postsynaptic potentials (fEPSPs) evoked by stimulation of the piriform (olfactory) cortex were recorded in the medial entorhinal cortex during behaviors associated with theta activity (active mobility) and were compared with those recorded during nontheta behaviors (awake immobility and slow wave sleep). Synaptic responses were smaller during behavioral activity than during awake immobility and sleep, and responses recorded during movement were largest during the negative phase of the theta rhythm. Systemic administration of cholinergic agonists reduced the amplitude of fEPSPs, and the muscarinic receptor blocker scopolamine strongly enhanced fEPSPs, suggesting that the theta-related suppression of fEPSPs is mediated in part by cholinergic inputs. The reduction in fEPSPs was investigated using in vitro intracellular recordings of EPSPs in Layer II neurons evoked by stimulation of Layer I afferents. Constant bath application of the muscarinic agonist carbachol depolarized membrane potential and suppressed EPSP amplitude in Layer II neurons. The suppression of EPSPs was not associated with a substantial change in input resistance, and could not be accounted for by a depolarization-induced reduction in driving force on the EPSP. The GABA(A) receptor-blocker bicuculline (50 microM) did not prevent the cholinergic suppression of EPSPs, suggesting that the suppression is not dependent on inhibitory mechanisms. Paired-pulse facilitation of field and intracellular EPSPs were enhanced by carbachol, indicating that the suppression is likely due to inhibition of presynaptic glutamate release. These results indicate that, in addition to well known effects on postsynaptic conductances that increase cellular excitability, cholinergic activation in the entorhinal cortex results in a strong reduction in strength of excitatory synaptic inputs from the piriform cortex.

Background and Purpose: The functional roles of M(2) and M(3) muscarinic receptors in neurogenic cholinergic contractions in gastrointestinal tracts remain to be elucidated. To address this issue, we studied cholinergic nerve-induced contractions in the ileum using mutant mice lacking M(2) or M(3) receptor subtypes., Experimental Approach: Contractile responses to transmural electrical (TE) stimulation were isometrically recorded in ileal segments from M(2)-knockout (KO), M(3)-KO, M(2)/M(3)-double KO, and wild-type mice., Key Results: TE stimulation at 2-50 Hz frequency-dependently evoked a fast, brief contraction followed by a slower, longer one in wild-type, M(2)-KO or M(3)-KO mouse preparations. Tetrodotoxin blocked both the initial and later contractions, while atropine only inhibited the initial contractions. The initial cholinergic contractions were significantly greater in wild-type than M(2)-KO or M(3)-KO mice; the respective mean amplitudes at 50 Hz were 91, 74 and 68 % of 70mM K(+)-induced contraction. Pretreatment with pertussis toxin blocked the cholinergic contractions in M(3)-KO but not in M(2)-KO mice. Cholinergic contractions also remained in wild-type preparations, but their sizes were reduced by 20-30 % at 10-50 Hz. In M(2)/M(3)-double KO mice, TE stimulation evoked only slow, noncholinergic contractions, which were significantly greater in sizes than in any of the other three mouse strains., Conclusion and Implications: These results demonstrate that M(2) and M(3) receptors participate in mediating cholinergic contractions in mouse ileum with the latter receptors assuming a greater role. Our data also suggest that the lack of both M(2) and M(3) receptors causes upregulation of noncholinergic excitatory innervation of the gut smooth muscle.

Background: Secretion of calcitonin gene-related peptide (CGRP) from trigeminal nerves and vasoactive intestinal peptide (VIP) from parasympathetic nerves is involved in the pathophysiology of migraine and rhinosinusitis. Analysis of these neuropeptides in human saliva samples can be used as markers of trigeminal and parasympathetic nerve activity in patients between and during attacks as well as in response to specific treatments., Objective: To compare the amount of trigeminal sensory and parasympathetic nerve activation by measuring CGRP and VIP levels in the saliva of subjects experiencing noninfectious allergic rhinosinusitis, migraine with sinus symptoms, and no symptoms., Methods: Subjects were enrolled in three groups. Group A: subjects without a history of migraine, "sinus" headache, or allergic rhinosinusitis within the previous 6 months. Group B: subjects with chronic recurrent noninfectious rhinosinusitis and no history of migraine or "sinus" headache. Group C: subjects with self-described "sinus" headaches whose symptoms met International Headache Society diagnostic criteria (1.1 or 1.2) for migraine. The total amount of CGRP and VIP present in saliva collected under normal, pathological, and therapeutic conditions was determined by radioimmunoassay. Neuropeptide levels were normalized to total volume and amount of protein, and levels were correlated to onset and change in clinical symptoms., Results: Total volume, total protein, and CGRP and VIP levels did not significantly change in saliva collected on consecutive days in the clinic and at the subject's home, respectively. No appreciable change in baseline salivary levels of CGRP and VIP was detected in control subjects. However, baseline salivary levels of CGRP and VIP were significantly elevated between attacks in allergic rhinosinusitis and migraine subjects compared to control values. For rhinosinusitis subjects, the amount of CGRP and VIP during attacks returned to baseline values following treatment with pseudoephedrine and relief of symptoms. Similarly, CGRP and VIP levels during a migraine headache were significantly reduced within 2 hours after sumatriptan treatment and reported symptom relief., Conclusion: Correlation of CGRP and VIP saliva levels observed in our study supports physiologically coordinated regulation of trigeminal and parasympathetic nerve activation in allergic rhinosinusitis and migraine patients between and during attacks as well as following treatment. Furthermore, our findings demonstrate that analysis of human saliva neuropeptides may provide a semiquantitative index of pathological and therapeutic states and, therefore, function as a clinical model for studying neuronal mechanisms involved in migraine and rhinosinusitis.

1. Various types of anticholinesterasic agents have been used to improve the daily activities of Alzheimer's disease patients. It was recently demonstrated that Galantamine, described as a molecule with anticholinesterasic properties, is also an allosteric enhancer of human alpha4beta2 neuronal nicotinic receptor activity. We explored its effect on the human alpha7 neuronal nicotinic acetylcholine receptor (nAChR) expressed in Xenopus oocytes. 2. Galantamine, at a concentration of 0.1 microM, increased the amplitude of acetylcholine (ACh)-induced ion currents in the human alpha7 nAChR expressed in Xenopus oocytes, but caused inhibition at higher concentrations. The maximum effect of galantamine, an increase of 22% in the amplitude of ACh-induced currents, was observed at a concentration of 250 microM Ach. 3. The same enhancing effect was obtained in oocytes transplanted with Torpedo nicotinic acetylcholine receptor (AChR) isolated from the electric organ, but in this case the optimal concentration of galantamine was 1 microM. In this case, the maximum effect of galantamine, an increase of 35% in the amplitude of ACh-induced currents, occurred at a concentration of 50 microM ACh. 4. Galantamine affects not only the activity of post-synaptic receptors but also the activity of nerve terminals. At a concentration of 1 microM, quantal spontaneous events, recorded in a cholinergic synapse, increased their amplitude, an effect which was independent of the anticholinesterasic activity associated with this compound. The anticholinesterasic effect was recorded in preparations treated with a galantamine concentration of 10 microM. 5. In conclusion, our results show that galantamine enhances human alpha7 neuronal nicotinic ACh receptor activity. It also enhances muscular AChRs and the size of spontaneous cholinergic synaptic events. However, only a very narrow range of galantamine concentrations can be used for enhancing effects.

1 This double-blind, cross-over, placebo-controlled study on six healthy male volunteers was designed to evaluate the effects of alpha2-adrenoceptor antagonism on cardiac parasympathetic regulation. 2 The subjects received atipamezole intravenously as a three-step infusion, which aimed at steady-state serum concentrations of 10, 30 and 90 ng ml(-1) at 50-min intervals. 3 Drug effects were assessed with repeated recordings of blood pressure and electrocardiogram, in which the high-frequency (0.15-0.40 Hz) R-R interval variation is supposed to reflect cardiac parasympathetic efferent neuronal activity. 4 At the end of the three steps of the infusion, the mean (+/-SD) concentrations of atipamezole were 10.5 (3.9), 26.8 (5.6) and 81.3 (21.1) ng ml(-1). 5 Within this concentration range, atipamezole appeared to reduce slightly the high-frequency R-R interval fluctuations, indicating a minor vagolytic effect in the heart. 6 Atipamezole increased systolic and diastolic arterial pressure, on average by 20 and 14 mmHg (maxima at the second step of the infusion), which evidently reflects an overall sympathetic augmentation.

1. In the present study, we examined the effect of E-ring 8-isoprostanes on cholinergic neurotransmission in guinea-pig trachea and identified the receptor(s) involved. As isoprostanes are isomeric with prostaglandins, PGE(2) and sulprostone (a selective EP(3)-receptor agonist) were examined in parallel. 2. 8-Iso-PGE(1), 8-iso-PGE(2) (0.1 nm-1 microM), sulprostone (1 nm-1 microM) and PGE(2) (1 microM) suppressed EFS-evoked [(3)H]ACh release from guinea-pig trachea in a concentration-dependent manner, producing 39.5, 53.9, 61.2 and 59.9% inhibition, respectively, at 1 microM. It should be noted that an established maximum effective concentration was not determined. 3. Neither SQ 29,548 (1 microm; a TP-receptor antagonist) nor AH 6809 (10 microM; an EP(1)-/EP(2)-/DP-receptor antagonist) reversed the inhibitory effect of these compounds. 4. L-798,106, a novel and highly selective EP(3)-receptor antagonist, produced a parallel shift to the right of the concentration-response curves that described the inhibitory action of sulprostone on EFS-evoked contractile responses in guinea-pig vas deferens (an established EP(3)-receptor-expressing tissue), from which a mean pA(2) of 7.48 was derived. On guinea-pig trachea, L-798,106 also antagonised sulprostone-induced inhibition of EFS-induced twitch responses, with similar potency (mean pA(2)=7.82). 5. The inhibitory effects of 8-iso-PGE(1), 8-iso-PGE(2), sulprostone and PGE(2) on EFS-induced [(3)H]ACh release was blocked by L-798,106 at a concentration (10 microM) that binds only weakly to human recombinant EP(1)-, EP(2)- and EP(4)-receptor subtypes expressed in HEK 293 cells. 6. These data suggest that E-ring 8-isoprostanes, PGE(2) and sulprostone inhibit EFS-evoked [(3)H]ACh release from cholinergic nerves innervating guinea-pig trachea, by interacting with prejunctional prostanoid receptors of the EP(3)-subtype.

To lesion the cholinergic input to the hippocampus, rats received injections of 192 IgG-saporin into the medial septum/vertical limb of the diagonal band (MS/VDB). The lesions produced near-total loss of choline acetyltransferase (ChAT)-positive neurons in the MS/VDB. The loss was accompanied, however, by only partial decreases (to 40% of control levels) in acetylcholine (ACh) release in the hippocampus. Moreover, ACh release in the hippocampus increased when lesioned and control rats were tested on a spontaneous alternation task, indicating that there was significant residual cholinergic function in the hippocampus. The lesions were sufficient to impair spontaneous alternation scores. However, this impairment could be reversed by either systemic or intra-hippocampal injections of the indirect cholinergic agonist, physostigmine, providing additional evidence of residual and effective cholinergic functions in the hippocampus of lesioned rats. Moreover, systemic injections of physostigmine at doses that produced mild tremors in control rats led to more severe tremors in the lesioned rats, suggesting upregulation of cholinergic mechanisms after saporin lesions, likely in brain areas other than the hippocampus. Thus, these findings provide evidence for decreases in cholinergic input to the hippocampus accompanied by deficits on a spontaneous alternation tasks. The findings also provide evidence for considerable residual cholinergic input to the hippocampus after saporin lesions of the MS/VDB. Together, the results suggest that 192 IgG-saporin lesions of the MS/VDB, using methods often employed, do not fully remove septohippocampal cholinergic input to the hippocampus but are nonetheless sufficient to produce impairments on a task impaired by hippocampal lesions.

Administration of muscarinic cholinergic antagonists such as scopolamine impairs the acquisition of contextual fear conditioning, but the role of the basal forebrain (BF) cholinergic system in consolidation is unclear. To test the hypothesis that BF cholinergic neurons are critical for acquisition and consolidation of fear conditioning, male Sprague-Dawley rats with 192 IgG-saporin lesions of the entire cholinergic BF made either before or after fear conditioning were tested for conditioned fear to context and tone by assessing freezing and 22 kHz ultrasonic vocalization (USV) responses. Spatial learning in a 1-day water maze task provided a comparison for effects of the BF lesions on fear conditioning. In the test phase, neither pre-training nor posttraining BF lesions affected freezing to the context or tone. During both training and testing, pre-lesioned rats were impaired in production of USVs associated with fear. Postlesioned rats emitted fewer USVs only during testing. Acquisition of a spatial water maze task was mildly impaired in lesioned rats, although probe trial and cued performance was unimpaired. Nevertheless, these data suggest that conditioned fear-induced USVs are more sensitive to the loss of BF cholinergic neurons than is conditioned fear-induced freezing. The failure of BF cholinergic lesions to impair contextual fear conditioning indicates that scopolamine-induced impairments in fear conditioning may not be mediated by affecting cholinergic input to the hippocampus and neocortex.

1. Cardiac vagal outflow is the major factor determining the magnitude of heart rate (HR) variability analysed by traditional time and frequency domain methods. New analysis techniques, such as fractal and complexity methods, have been developed to probe non-linear features in HR behaviour that may not be detectable by traditional methods. 2. We investigated the effects of vagal blockade (glycopyrrolate i.v. 5 microg kg-1 h-1 for 2 h, n = 8 vs. unmedicated control group, n = 8) and various breathing patterns (n = 12) on two non-linear measures of HR variability--detrended fluctuation analysis (DFA) and approximate entropy (ApEn)--in healthy male volunteers. 3. Glycopyrrolate decreased the mean (+/-SD) ApEn from 1.46 +/- 0.18 to 0.85 +/- 0.24 (P = 0.001 in comparison with the control group), and increased the short-term (alpha 1) and intermediate-term (alpha 2) fractal scaling exponents of DFA, alpha 1 from 0.96 +/- 0.19 to 1.43 +/- 0.29 (P = 0.003) and alpha 2 from 1.13 +/- 0.10 to 1.34 +/- 0.14 (P < 0.001). 4. Decrease in fixed respiration rate from 15 to 6 breaths min-1 increased alpha 1 from 0.83 +/- 0.25 to 1.18 +/- 0.27 (P < 0.001), but decreased alpha 2 from 0.88 +/- 0.09 to 0.45 +/- 0.17 (P < 0.001) and ApEn from 1.26 +/- 0.12 to 1.10 +/- 0.14 (P = 0.028). Rapid breathing (24 min-1) had no influence on these non-linear measures of HR variability. Hyperventilation (15 min-1, tidal volume increased voluntarily by 0.5 l) decreased alpha 1 from 0.83 +/- 0.25 to 0.66 +/- 0.28 (P = 0.002) but did not affect alpha 2 or ApEn. 5. To conclude, vagal blockade alters the fractal scaling properties of R-R intervals (alpha 1, alpha 2) and reduces the complexity (ApEn) of HR behaviour. Both the fractal and complexity measures of HR variability can also be influenced by changes in the breathing pattern.

1. This study investigates the mechanisms accounting for the adverse cholinergic effects of the antitumour drug irinotecan. The activity of irinotecan and its active metabolite, 7-ethyl-10-hydroxy-camptothecin (SN-38), was assayed in models suitable for pharmacological studies on cholinergic system. 2. Irinotecan moderately inhibited human or electric eel acetylcholinesterase activity, SN-38 had no effect, whereas physostigmine blocked both the enzymes with high potency and efficacy. 3. Irinotecan and SN-38 did not affect spontaneous or electrically-induced contractile activity of human colonic muscle. Acetylcholine and dimethylphenylpiperazinium (DMPP) caused phasic contractions or relaxations, respectively. Physostigmine enhanced the motor responses elicited by electrical stimulation. 4. Although irinotecan and SN-38 did not modify the basal contractile activity of guinea-pig ileum longitudinal muscle strips, irinotecan 100 microM moderately enhanced cholinergic twitch contractions. Acetylcholine or DMPP caused phasic contractions, whereas physostigmine enhanced the twitch responses. Electrically-induced [(3)H]-acetylcholine release was reduced by irinotecan (100 microM) or physostigmine (0.1 microM). 5. Intravenous irinotecan stimulated gastric acid secretion in rats, but no effects were obtained with SN-38, physostigmine or i.c.v. irinotecan. Hypersecretion induced by irinotecan was partly prevented by ondansetron, and unaffected by capsazepine. In the presence of atropine, vagotomy and systemic or vagal ablation of capsaicin-sensitive afferent fibres, irinotecan did not stimulate gastric secretion. 6. The present results indicate that irinotecan and SN-38 do not act as specific acetylcholinesterase blockers or acetylcholine receptor agonists. It is rather suggested that irinotecan promotes a parasympathetic discharge to peripheral organs, mediated by capsaicin-sensitive vagal afferent fibres, and that serotonin 5-HT(3) receptors are implicated in the genesis of vago-vagal reflex triggered by irinotecan.

1. The effect of ophiopogonin-D (OP-D), a steroidal glycoside and an active component of Bakumondo-to, a Chinese herbal antitussive, on neurones acutely dissociated from paratracheal ganglia of 2-week-old Wistar rats was investigated using the nystatin-perforated patch recording configuration. 2. Under current-clamp conditions, OP-D (10 microM) hyperpolarized the paratracheal neurones from a resting membrane potential of -65.7 to -73.5 mV. 3. At the concentration of 1 microM and above, OP-D concentration-dependently activated an outward current accompanied by an increase in the membrane conductance under voltage-clamp conditions at a holding potential of -40 mV. 4. The reversal potential of the OP-D-induced current (I(OP-D)) was -79.4 mV, which is close to the K(+) equilibrium potential of -86.4 mV. The changes in the reversal potential for a 10 fold change in extracellular K(+) concentration was 53.1 mV, indicating that the current was carried by K(+). 5. The I(OP-D) was blocked by an extracellular application of 1 mM Ba2+ by 59.0%, but other K(+) channel blockers, including 4-aminopyridine (3 mM), apamin (1 microM), charybdotoxin (0.3 microM), glibenclamide (1 microM), tolbutamide (0.3 mM) and tetraethylammonium (10 mM), did not inhibit the I(OP-D). 6. OP-D also inhibited the ACh- and bradykinin-induced depolarizing responses which were accompanied with firing of action potentials. 7. The results suggest that OP-D may be of benefit in reducing the excitability of airway parasympathetic ganglion neurones and consequently cholinergic control of airway function and further, that the hyperpolarizing effect of OP-D on paratracheal neurones via an activation of K(+) channels might explain a part of mechanisms of the antitussive action of the agent.

We investigated the ability of the cannabinoid agonists CP55,940 (CB(1)/CB(2)) and anandamide (endogenous cannabinoid) to modulate electrical field stimulation (EFS)-induced acetylcholine (ACh) release from parasympathetic nerve terminals innervating guinea-pig trachea. We assessed whether modulation of transmitter release translated to an impact on functional responses by investigating the effect of these agents on contractile responses evoked by EFS and ACh. Furthermore, we evaluated the ability of these compounds to elicit bronchodilation in pre-contracted guinea-pig tracheal strips. CP55,940 and anandamide significantly inhibited EFS-evoked ACh release (maximal inhibition of 35.1+/-2.9% and 33.4+/-6.4% at 1 microM, P<0.05, respectively). The CB(1) receptor antagonist SR 141716A (1 microM), had no effect on ACh release and failed to reverse the inhibitory effect of CP55,940 (1 microM). Paradoxically, CP55,940 had no significant effect on EFS-evoked cholinergic contractile responses. Furthermore, CP55,940 did not relax pre-contracted tracheal strips or affect contractile responses to exogenous ACh. This lack of activity on smooth muscle tone is consistent with the fact that no detectable specific binding of [(3)H] CP55,940 was found in tracheal homogenates. These data suggest that cannabinoid agonists inhibit ACh release from cholinergic nerve terminals via activation of CB(2) receptors but that this inhibitory action does not impact on functional responses such as cholinergic contraction.

Neurally evoked contractions and release of (3)H- acetylcholine (ACh) during electrical field stimulation were measured in rat urinary bladder strips. The alpha(1) agonist phenylephrine (PE, 2-8 microM) increased the amplitude of neurally evoked contractions, facilitated the release of ACh and increased the baseline tone of the bladder strips. The PE-induced facilitation of the contractions did not significantly change during a prolonged exposure to PE (120 min), whereas the PE-induced rise in baseline tone gradually decreased to 65% of the initial value. Low concentrations of specific alpha(1A) antagonists, 5-methyl urapidil (5-MU), REC15/2739 and WB-4101 competitively inhibited the facilitation of the neurally-evoked contractions (pA(2:) 8.77; 9.59 and 9.62, respectively), whereas higher concentrations of 5-MU (IC(50): 48 nM) were required to suppress the PE-rise in baseline. WB-4101 (100 microM) inhibited the PE-induced facilitation of ACh release. The irreversible alpha(1B) antagonist chloroethyl-clonidine (CEC, 10-50 microM) inhibited the PE-evoked rise in base line tone, but did not affect the PE-induced facilitation of the neurally evoked contractions nor the facilitation of ACh release. However, CEC increased the area and amplitude of the neurally-evoked contractions by 261+/-33 and 47.2+/-8.4%, respectively. Atropine significantly inhibited the CEC evoked increase in area and amplitude of the electrically evoked contractions (76.5+/-4.8 and 40.8+/-3%, respectively) indicating that CEC facilitated the cholinergic responses of the electrically stimulated bladder strips. It is concluded that alpha(1A) and CEC sensitive alpha(1B) and/or alpha(1D) adrenoceptors are expressed in the rat bladder in different locations. On the cholinergic nerve terminals alpha(1A) adrenoceptors mediate prejunctional facilitation, whereas postjunctional alpha(1B)/alpha(1D) adrenoceptors mediate smooth muscle contraction.

The actions of omega-conotoxin (omega-CTX) and diltiazem on adrenergic and purinergic components of double peaked vasoconstrictor responses to periarterial nerve stimulation have been investigated in the isolated, perfused canine splenic arterial preparation. Double peaked vasoconstrictions (biphases of vasoconstrictors) were consistently observed in the conditions of 30 s trains of pulses at 1 - 10 Hz frequencies. omega-CTX (1 - 30 nM) produced similar inhibitory effects on the first phase and second phase responses in a dose-related manner. Thirty nM omega-CTX almost completely inhibited the biphasic vasoconstrictions at any used frequencies but did not affect the vasoconstrictor responses to exogenous applied ATP (0.01 - 1 micromol) and noradrenaline (0.03 - 3 nmol). Intraluminal application of a large dose of diltiazem (3 - 10 microM) also produced a dose-dependent inhibitory effect on biphasic vasoconstrictions at any used frequencies. Three microM diltiazem exerted rather a larger inhibitory effect on the second phase than the first phase response at low frequencies (1 - 3 Hz), but a similar inhibition on first and second phasic responses at high frequencies (6 - 10 Hz). An extremely high dose of diltiazem (10 microM) almost completely inhibited the biphasic vasoconstrictor responses to nerve stimulation, and slightly inhibited the contractile responses to exogenous applied ATP (0.01 - 1 micromol) and noradrenaline (0.03 - 3 nmol). The present results indicate that omega-CTX selectively acts prejunctionally to inhibit the release of transmitters from sympathetic nerve terminals, and omega-CTX-sensitive calcium channels may produce a parallel controlling of purinergic and adrenergic components of sympathetic cotransmission. A large dose of diltiazem has inhibitory effects on both prejunctional and postjunctional sympathetic co-transmission. British Journal of Pharmacology (2000) 129, 47 - 52

1. The mechanisms and the subtypes of muscarinic receptors implicated in the cardiovascular effects of physostigmine were investigated in conscious normotensive and spontaneously hypertensive rats. 2. Intravenous (i.v.) physostigmine (50 microg kg-1) induced in both strains a long pressor response, accompanied by a bradycardia. This pressor response was larger in spontaneously hypertensive (+41+/-6 mmHg) than in Wistar-Kyoto (+25+/-2 mmHg) rats (P<0.05). 3. Pretreatment with atropine sulphate (0.4 mg kg-1 i.v.), completely abolished the physostigmine-induced pressor response in both normotensive and hypertensive rats. In both strains, the physostigmine pressor response was significantly reduced by the systemic administration of either an alpha1-adrenoceptor antagonist (prazosin, 1 mg kg-1) or a V1A-vasopressin receptor antagonist (AVPX, 20 microg kg-1). This physostigmine pressor effect was completely abolished in both strains when both antagonists were administered concomitantly. 4. In WKY rats, the pressor response to physostigmine (50 microg kg-1 i.v.) was inhibited in a dose-dependent manner by i. c.v. administration of atropine (ID50=3.70 nmoles), the M1 receptor antagonist pirenzepine (ID50=10.71 nmoles), the M2 receptor antagonist methoctramine (ID50=4.31 nmoles), the M3 receptor antagonist p-F-HHSiD (ID50=60.52 nmoles) and the M4 receptor antagonist tropicamide (ID50=214.20 nmoles). In the hypertensive strain, the ID50 were found to be significantly higher for atropine (7.34 nmoles), pirenzepine (21.60 nmoles) and p-F-HHSiD (139.50 nmoles) (P<0.05). 5. The present results indicate that physostigmine acts in normotensive and spontaneously hypertensive rats, through stimulation of both central M2 and M1 cholinoceptors to induce a rise in blood pressure mediated by an increase in plasma vasopressin and sympathetic outflow. Moreover, our results suggest that some modifications of the M1 receptor subtypes in terms of expression or affinity could be responsible for the hyper-responsiveness of the hypertensive strain to cholinomimetic agents.

1. In the rat corpus cavernosum (CC), the distribution of immunoreactivity for neuronal and endothelial NO synthase (nNOS and eNOS), and the pattern of NOS-immunoreactive (-IR) nerves in relation to some other nerve populations, were investigated. Cholinergic nerves were specifically immunolabelled with antibodies to the vesicular acetylcholine transporter protein (VAChT). 2. In the smooth muscle septa surrounding the cavernous spaces, and around the central and helicine arteries, the numbers of PGP- and tyrosine hydroxylase (TH)-IR terminals were large, whereas neuropeptide Y (NPY)-, VAChT-, nNOS-, and vasoactive intestinal polypeptide (VIP)-IR terminals were found in few to moderate numbers. 3. Double immunolabelling revealed that VAChT- and nNOS-IR terminals, VAChT- and VIP-IR terminals, nNOS-IR and VIP-IR terminals, and TH- and NPY-IR terminals showed coinciding profiles, and co-existence was verified by confocal laser scanning microscopy. TH immunoreactivity was not found in VAChT-, nNOS-, or VIP-IR nerve fibres or terminals. 4. An isolated strip preparation of the rat CC was developed, and characterized. In this preparation, cumulative addition of NO to noradrenaline (NA)-contracted strips, produced concentration-dependent, rapid, and almost complete relaxations. Electrical field stimulation of endothelin-1-contracted preparations produced frequency-dependent responses: a contractile twitch followed by a fast relaxant response. After cessation of stimulation, there was a slow relaxant phase. Inhibition of NO synthesis, or blockade of guanylate cyclase, abolished the first relaxant phase, whereas the second relaxation was unaffected. 5. The results suggest that in the rat CC, nNOS, VAChT- and VIP-immunoreactivities can be found in the same parasympathetic cholinergic neurons. Inhibitory neurotransmission involves activation of the NO-system, and the release of other, as yet unknown, transmitters.

The leaf and seed of Piper guineense were separately extracted and the effects of repeated treatments on rat uterine contractions and their chemical composition were investigated. Repeated treatments with the extracts enhanced spontaneous uterine muscle contractions in a dose related pattern for up to 14 days which was followed by a marked decrease in uterine response in those treated for 30 days. Similarly, the pretreatment with the extracts maximally enhanced oxytocin induced uterine contraction in those rats treated for 3 days, this was followed by a gradual reversal of the effect when treatment was repeated for up to 14 days. This enhanced uterine contraction was inhibited following treatment for 30 days. In a further study, treatment with the extracts induced a significant increase in the uterine weight similar to that seen with oestradiol. It is therefore concluded that the leaf and seed extracts of Piper guineense possess oestrogenic and oxytocic properties which might justify their usage in Nigerian traditional medicine.

1. The modulation of high-voltage-activated (HVA) Ca2+ channels by acetylcholine (ACh) was studied in the paratracheal ganglion cells acutely dissociated from 2-week-old Wistar rats by use of the nystatin perforated patch recording configuration under voltage-clamp conditions. 2. ACh inhibited the HVA Ca2+ currents in a concentration- and voltage-dependent manner. 3. The inhibition was mimicked by a muscarinic agonist, oxotremorine. Pirenzepine and methoctramine produced parallel shifts to the right in the ACh concentration-response curves. Schild analysis of the ACh concentration-ratios yield pA2 values for pirenzepine and methoctramine of 6.85 and 8.57, respectively, suggesting the involvement of an M2 receptor. 4. Nifedipine, omega-conotoxin-GVIA and omega-conotoxin-MVIIC reduced the HVA I(Ca) by 16.8, 59.2 and 6.3%, respectively. A current insensitive to all of these Ca2+ antagonists, namely 'R-type', was also observed. The results indicated the existence of L-, N-, P/Q-, and R-type Ca2+ channels. 5. The ACh-sensitive current component was markedly reduced in the presence of omega-conotoxin-GVIA, but not with both nifedipine and omega-conotoxin-MVIIC. ACh also inhibited the R-type HVA I(Ca) remaining in saturating concentrations of nifedipine, omega-conotoxin-GVIA and omega-conotoxin-MVIIC. 6. The inhibitory effect of ACh was prevented by pretreatment with pertussis toxin. 7. It was concluded that ACh selectively reduces both the N- and R-type Ca2+ channels, by activating pertussis toxin sensitive G-protein through the M2 muscarinic receptor in paratracheal ganglion cells.

1. In the Fisher 344 rat, tachykinins have been shown to cause the release of 5-hydroxytryptamine (5-HT) from airway mast cells, which then causes direct smooth muscle activation as well as the release of acetylcholine from cholinergic nerves. The aim of the present study was to examine the modulatory effects of 5-HT receptors on the neurokinin A (NKA)-induced release of endogenous 5-HT and airway smooth muscle contraction in the isolated Fisher 344 rat trachea. 2. The selective 5-HT2 receptor antagonist ketanserin (0.1 microM) produced an almost complete inhibition of the contractions caused by NKA (n=4, P<0.0001, two-way ANOVA), and a significant rightward shift of the concentration-response curve to 5-HT (n=8, P<0.001, two-way ANOVA). 3. The partial agonist for 5-HT1A receptors, 8-OH-DPAT (1 microM), and the full agonist for 5-HT1 receptors, 5-CT (0.3 microM), potentiated the submaximal contractions induced by the 5-HT2 receptor agonist alpha-methyl-5-HT (0.1 microM) (n=4; P<0.005 and P<0.05, respectively). 8-OH-DPAT (1 microM), as well as the 5-HT1A receptor antagonists pMPPI, SDZ 216525 and NAN-190 (0.1 microM each), caused significant inhibition of the tracheal contractions induced both by NKA (10 nM-3 microM) and 5-HT (10 nM-10 microM) (n=4-10). This suggests that activation of 5-HT1A receptors potentiates the 5-HT2 receptor-mediated contractions. 4. SDZ 216525 (0.1 microM) significantly reduced the maximal contraction produced by 1 microM NKA (n=10, P< 0.001), without affecting the release of endogenous 5-HT. These data rule out the involvement of a 5-HT1A receptor-mediated positive feedback mechanism of the 5-HT release from mast cells. 5. Even in the presence of atropine (1 microM), 8-OH-DPAT (1 microM) further reduced the maximal NKA-induced contraction (n=4, P<0.0001), while the contractions of the rat isolated trachea induced by electrical field stimulation and the concentration-response curve to carbachol were unaffected by pMPPI (0.1 microM), SDZ 216525 (0.1 microM), NAN-190 (0.1 microM) and 8-OH-DPAT (1 microM) (n=4-6). These data demonstrate that the 5-HT1A receptor-mediated potentiation of contractile responses is not due to nonspecific inhibition of airway smooth muscle contraction or to modulation of postganglionic nerve activation. 6. The selective 5-HT1B/1D receptor antagonist GR 127935, the selective 5-HT3 receptor antagonist tropisetron and the selective 5-HT4 receptor antagonists SB 204070 and GR 113808 (0.1 microM each) had no effect on the concentration-response curve for NKA (n=6-10), ruling out the involvement of 5-HT1B/1D, 5-HT3 and 5-HT4 receptors. 7. The alpha-adrenoreceptor antagonist phentolamine (1 microM) had no effect on the 5-HT-induced contractions (n=4), ruling out the involvement of alpha-adrenoreceptors. 8. In conclusion, the tachykinin-induced contraction of the F334 rat isolated trachea is mediated by the stimulation of 5-HT2 receptors. Activation of 5-HT1A receptors located on airway smooth muscle potentiates the direct contractile effects of 5-HT2 receptor activation. The 5-HT1B/1D, 5-HT3 and 5-HT4 receptors are not involved in the NKA-induced contraction of rat airways.

1. We have demonstrated recently that exogenous prostaglandin E2 (PGE2) inhibits electrical field stimulation (EFS)-induced acetylcholine (ACh) release from parasympathetic nerve terminals innervating guinea-pig trachea. In the present study, we have attempted to characterize the pre-junctional prostanoid receptor(s) responsible for the inhibitory action of PGE2 and to assess whether other prostanoids modulate, at a prejunctional level, cholinergic neurotransmission in guinea-pig trachea. To this end, we have investigated the effect of a range of both natural and synthetic prostanoid agonists and antagonists on EFS-evoked [3H]-ACh release. 2. In epithelium-denuded tracheal strips pretreated with indomethacin (10 microM), PGE2 (0.1 nM-1 microM) inhibited EFS-evoked [3H]-ACh release in a concentration-dependent manner with an EC50 and maximal effect of 7.62 nM and 74% inhibition, respectively. Cicaprost, an IP-receptor agonist, PGF2alpha and the stable thromboxane mimetic, U46619 (each at 1 microM), also inhibited [3H]-ACh release by 48%, 41% and 35%, respectively. PGD2 (1 microM) had no significant effect on [3H]-ACh release. 3. The selective TP-receptor antagonist, ICI 192,605 (0.1 microM), completely reversed the inhibition of cholinergic neurotransmission induced by U-46619, but had no significant effect on similar responses effected by PGE2 and PGF2alpha. 4. A number of EP-receptor agonists mimicked the ability of PGE2 to inhibit [3H]-ACh release with a rank order of potency: GR63799X (EP3-selective) > PGE2 > M&B 28,767 (EP3 selective) > 17-phenyl-omega-trinor PGE2 (EP1-selective). The EP2-selective agonist, AH 13205 (1 microM), did not affect EFS-induced [3H]-ACh release. 5. AH6809 (10 microM), at a concentration 10 to 100 times greater than its pA2 at DP-, EP1- and EP2-receptors, failed to reverse the inhibitory effect of PGE2 or 17-phenyl-omega-trinor PGE2 on [3H]-ACh release. 6. These results suggest that PGE2 inhibits [3H]-ACh release from parasympathetic nerves supplying guinea-pig trachea via an interaction with prejunctional prostanoid receptors of the EP3-receptor subtype. Evidence for inhibitory prejunctional TP- and, possibly, IP-receptors was also obtained although these receptors may play only a minor role in suppressing [3H]-ACh release when compared to receptors of the EP3-subtype. However, the relative importance of the different receptors will depend not only on the sensitivity of guinea-pig trachea to prostanoids but on the nature of the endogenous ligands released locally that have activity on parasympathetic nerves.

The novel peptide, nociceptin and the mu-opioid agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) produced a concentration-dependent inhibition of electrical field stimulation (EFS)-evoked release of acetylcholine (ACh) from cholinergic nerves innervating guinea-pig trachea. The non-selective opioid receptor antagonist, naloxone, did not antagonize the inhibitory action of nociceptin under conditions where the inhibition of ACh release evoked by DAMGO was completely reversed. It is suggested that DAMGO and nociceptin can inhibit cholinergic, parasympathetic neurotransmission to the airways via the activation of classical (naloxone-sensitive) and novel (naloxone-insensitive) opioid receptors, respectively.

1. The airway and pulmonary vascular effects of adrenomedullin were studied in the guinea-pig isolated trachea, main bronchi and pulmonary artery in vitro and compared to the effects of calcitonin gene-related peptide (CGRP). 2. In tracheal rings, CGRP (1 nM to 1 microM) potentiated the cholinergic contractions induced by electrical field stimulation (EFS) at 5 Hz in a concentration-dependent manner. At a concentration of 1 microM, CGRP slightly decreased the responses to log EFS frequency, producing 50% of the maximum contraction from a control value of 0.77 +/- 0.10 Hz to 0.54 +/- 0.05 Hz without a significant effect on the concentration-response curves to acetylcholine (ACh). In contrast, adrenomedullin (1 nM to 1 microM) did not alter either EFS-induced cholinergic or ACh-induced contractions. 3. In bronchial strips, CGRP (1 nM to 1 microM) slightly reduced both the non-adrenergic non-cholinergic (NANC) contraction induced by EFS at 10 Hz and the substance P (1 microM)-induced contraction in a concentration-dependent manner, whereas adrenomedullin (1 nM to 1 microM) was without effect. 4. Neither CGRP (1 microM) nor adrenomedullin (1 microM) altered NANC relaxation induced by EFS at 5 Hz in tracheal rings precontracted with histamine (10 microM). 5. Adrenomedullin (1 nM to 1 microM) and CGRP (1 nM to 1 microM) induced a concentration-dependent relaxation of the histamine (10 microM)- and prostaglandin F2 alpha (10 microM)-precontracted pulmonary arterial rings with intact endothelium with a similar potency. 6. Neither removal of the endothelium nor NG-nitro-L-arginine methyl ester (100 microM) altered the vasorelaxant effects of adrenomedullin (1 nM to 1 microM) and CGRP (1 nM to 1 microM). 7. The putative CGRP receptor antagonist, CGRP8-37 (1 microM to 10 microM) concentration-dependently attenuated the CGRP (3 nM to 30 nM)-induced vasorelaxant actions, whereas it had no effect on the relaxation of vessel rings induced by adrenomedullin (3 nM to 30 nM). 8. These results suggest that adrenomedullin is a potent vasodilator of the pulmonary artery without any bronchomotor effect in the guinea-pig lung, and that the vasorelaxant actions of adrenomedullin are not mediated via the activation of CGRP1 receptors.

1. Previous studies have provided evidence that activation of beta-adrenoceptors on cholinergic nerve terminals can inhibit neurotransmission in the airways. However, in most cases, this conclusion has been based on indirect evidence obtained from mechanical experiments where changes in airways smooth muscle tone were measured. 2. We have assessed whether modulation of cholinergic neurotransmission by beta-adrenoceptor agonists is due to a pre- or post-junctional action by investigating the effect of isoprenaline on contractile responses evoked by exogenous acetylcholine (ACh) and electrical field stimulation (EFS; 4 Hz, 40 V, 0.5 ms pulse width every 15 s), and on EFS-induced ACh release from cholinergic nerves innervating guinea-pig and human trachea. Furthermore, the subtype of beta-adrenoceptor which modulates neurotransmission and the potential role of cyclic AMP in this response were evaluated. 3. In guinea-pig trachea, isoprenaline (1 nM-1 microM) inhibited the contractile response evoked by exogenous ACh (1 microM) to a similar extent to that evoked by EFS (EC50 = 19.9 and 23 nM, respectively). 4. In epithelium-denuded guinea-pig strips treated with indomethacin (10 microM), isoprenaline significantly enhanced EFS-induced ACh release from cholinergic nerve terminals (by 36% at 0.3 microM). This effect was blocked by propranolol and ICI 118, 551 (each 0.1 microM). In contrast, isoprenaline failed to affect EFS-induced ACh release from parasympathetic nerves innervating human trachea. 5. To evaluate the role of cyclic AMP in the beta-adrenoceptor-induced facilitation of cholinergic neurotransmission, the effects of various cyclic AMP elevating drugs on ACh release were studied. Forskolin (10 microM) significantly augmented (by 17%) EFS-induced ACh release, an effect which was not reproduced by 1,9-dideoxyforskolin (10 microM) which does not activate adenylyl cyclase. Similarly, the cyclic AMP analogue, 8-bromo-cyclic AMP (1 mM) and cholera toxin (1 microgram ml-1) facilitated ACh output by 22 and 47% respectively, whereas prostaglandin E2 (PGE2, 0.1 nM-1 microM) inhibited this response (by 67% at 1 microM). 6. Zardaverine (10 microM), a dual inhibitor of the phosphodiesterase (PDE)3 and PDE4 isoenzyme families, did not affect EFS-induced ACh release and failed to facilitate the actions of either isoprenaline or PGE2. Similarly, neither SK&F 94120 (10 microM) nor rolipram (10 microM), selective inhibitors of PDE3 and PDE4 respectively, significantly affected the release of ACh in response to EFS. 7. The result of this study suggests that isoprenaline facilitates cholinergic neurotransmission in guinea-pig, but not human, trachea by activation of pre-junctional beta 2-adrenoceptors, an effect that may be mediated via activation of the cyclic AMP/cyclic AMP-dependent protein kinase cascade. Furthermore, the data presented herein illustrate the need to undertake direct measurements of neurotransmitter release when examining the effect of agents purported to act pre-junctionally.

1. Orbital parasympathetic innervation normally provides prejunctional muscarinic inhibition of sympathetic neurotransmission without activation of excitatory muscarinic receptors located on the innervated smooth muscle. The present study examines the role of acetylcholinesterase (AChE) in limiting the effects of parasympathetically released acetycholine to prejunctional receptors. 2. Urethane anaesthetized rats were placed in a stereotaxic frame, and parasympathetic activation was achieved by electrical stimulation (20 Hz, < 2.0 V) of the ipsilateral superior salivatory nucleus. Drugs were administered through a femoral venous cannula. Superior tarsal smooth muscle responses were measured by recording eyelid tension. 3. Parasympathetic stimulation alone caused a small decrease in resting tension; previous studies have shown this to be attributable to attention of resting sympathetic tone. Parasympathetic activation following physostigmine administration, however, evoked a large contractile response. Contractions were resistant to atropine but were blocked by gallamine, guanethidine, and phentolamine. 4. We conclude that AChE inhibition results in conversion of orbital parasympathetic nerve function from inhibition of sympathetic neurotransmission to smooth muscle excitation. This occurs as a result of cholinergic activation of excitatory nicotinic receptors on sympathetic varicosities, which elicit the release of noradrenaline.

1. The Ca2+ channel subtypes of the autonomic nerves of guinea-pig atria were elucidated by monitoring the effects of specific Ca2+ channel blockers on the negative and positive inotropic responses associated respectively, with stimulation of the parasympathetic and sympathetic nerves. 2. In left atria paced at 2-4 Hz, the negative inotropic effect induced by field stimulation of parasympathetic nerves (in the presence of propranolol) was abolished by omega-conotoxin MVIIC, a blocker of N-type and OPQ subfamily Ca2+ channels. omega-Conotoxin GVIA (an N-type blocker), omega-agatoxin IVA (a P-type blocker), nifedipine (an L-type blocker) and Ni2+ (a T- and R-type blocker) were much less effective. 3. The positive inotropic response resulting from field stimulation of the sympathetic nerves (in the presence of atropine) was abolished by both omega-conotoxins, while omega-agatoxin IVA, nifedipine and Ni2+ were ineffective. 4. In the spontaneously beating right atria, the early negative inotropic effect produced by 1,1-dimethyl-4-phenylpiperazinium was abolished by omega-conotoxin MVIIC, whereas the late positive inotropic effect was partially reduced, but not abolished, by a high concentration of omega-conotoxin GVIA. 5. None of the peptide toxins affected the chronotropic and the inotropic responses evoked by carbachol and isoprenaline. 6. These results suggested that, under physiological conditions, the release of acetylcholine from parasympathetic nerves is dominated by an OPQ subfamily Ca2+ channel while that of noradrenaline from sympathetic nerves is controlled by an N-type Ca2+ channel. Ligand-induced noradrenaline release appeared to recruit additional type(s) of Ca2+ channel.

1. The effect of activation of alpha 1-adrenoceptors on acetylcholine (ACh) release and neurally evoked contractile responses induced by electrical field stimulation was investigated in smooth muscle strips from the rat urinary bladder. 2. Neurogenic contractions were facilitated by the alpha 1-adrenoceptor agonists, phenylephrine (PE) (2-128 microM) and methoxamine (2-128 microM) in a dose-dependent manner. These agents also increased small amplitude spontaneous contractions of bladder strips and in 10% of strips increased basal tone. However, contractions elicited by exogenous ACh (1-10 microM) were not affected by alpha 1-agonists. 3. The magnitude of the PE facilitation was higher at lower frequencies (1-5 Hz) or at submaximal intensities of stimulation and at lower Ca2+ concentrations (0.5-1 mM). The selective alpha 1-adrenoceptor antagonist, terazosin (TRZ) (0.05-1 microM), competitively inhibited (pA2 value: 8.6) the PE facilitation of the neurally evoked contractions but not the PE induced increase of spontaneous contractions. 4. [3H]-noradrenaline (NA) and [14C]-ACh release evoked by electrical field stimulation were increased (140% and 173%, respectively) by 2 microM PE. TRZ (0.05-0.1 microM) blocked the PE facilitation of ACh release but not the facilitation of NA release. TRZ alone did not alter the release of ACh or NA nor the amplitude of the neurogenic contractions. 5. PE (2 microM) did not alter the basal release of ACh but did increase (by 180%) the basal release of NA. Desipramine (2 microM) blocked this effect of PE and also the PE-facilitation of evoked ACh and NA release. 6. It is concluded that cholinergic terminals in the rat urinary bladder exhibit alpha 1-adrenoceptors which can facilitate the release of transmitter. However, under the conditions of our experiments it appears that cholinergic transmission is not modulated by alpha 1 adrenergic mechanisms. Further studies are necessary to determine whether these receptors can be activated by endogenous noradrenaline released within the bladder.

The effects of low and high doses of atropine on respiratory sinus arrhythmia were assessed to ascertain whether any differences exist in the degree of parasympathetic control on cardiac rhythm between two ethnic groups. The standard deviation of the R-R interval (the R peak-to-peak interval of two QRS complexes of an electrocardiogram) has been used as a noninvasive parameter to measure the degree of parasympathetic cardiac control. Nine black and nine white healthy male volunteers took part in study after approval by the Research and Ethics Committee of the Medical University of South Africa. Thirty consecutive electrocardiographic complex were recorded during each of the following: 3 deep inspirations and expirations, incremental cumulative atropine injections of 0.001 mg/kg until 0.005 mg/kg, followed by two injections of 0.01 mg/kg and 0.015 mg/kg of atropine each. After each of the last two injections the deep inspiration and expiration procedure was repeated. No significant differences could be found at any stage for any parameter between the groups. In both groups the R-R interval variation increased threefold during voluntary induced respiratory sinus arrhythmia. This effect was blocked after 0.01 mg/kg of atropine. The degree of parasympathetic control was not affected by any respiratory maneuver, but was affected by atropine. A significant inverse quadratic relationship was found between parasympathetic control and heart rate change (R = .984 for whites, and R = .905 for blacks). A poor correlation was found between the R-R interval variation and heart rate changes. In conclusion, ethnicity does not affect parasympathetic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

1. We have previously shown that endothelin-1-induced contraction of mouse isolated tracheal smooth muscle was mediated via both ETA and ETB receptors. In the current study, we have investigated endothelin-1-induced potentiation of cholinergic nerve-mediated contractions in mouse isolated trachea and have characterized pharmacologically the endothelin receptors mediating this response. 2. Electrical field stimulation (EFS; 70 V, 0.5 ms duration, 10s train, 0.1-60 Hz) of mouse isolated trachea caused frequency-dependent, monophasic contractions (magnitude of contraction of 60 Hz was 56 +/- 4% Cmax (n = 6), where Cmax is the contractile response to 10 microM carbachol). EFS-induced contractions were abolished by either 0.1 microM atropine or 3 microM tetrodotoxin, but were not affected by 1 microM hexamethonium, indicating that they were induced by stimulation of postganglionic cholinergic nerves. In contrast, contractions induced by exogenously applied acetylcholine were inhibited by atropine, but not by either tetrodotoxin or hexamethonium. 3. The ETB receptor-selective agonist, sarafotoxin S6c, caused marked concentration-dependent potentiation of EFS-induced contractions in mouse isolated tracheal segments. At 0.1 nM, sarafotoxin S6c exerted no direct contractile effect, but significantly increased a standard EFS-induced contraction of 20% Cmax by 8 +/- 2% Cmax (i.e. 1.4 fold, n = 5, P < 0.05). At higher concentrations, 10 nM sarafotoxin S6c induced a large, transient contraction (peak response of 74 +/- 2% Cmax at 10 min; 3 +/- 2% Cmax at 45 min) and enhanced the standard EFS-induced contraction by 30 +/- 4% Cmax (i.e. 2.5 fold, n = 5, P < 0.01). In contrast, 10 nM sarafotoxin S6c did not enhance contractile responses to exogenously applied acetylcholine(n = 6).4. Endothelin-1 also modulated EFS-induced contractions. At 0.1 nM, endothelin-1 exerted no direct contractile effect, but significantly increased the standard EFS-induced contraction of 20%Cmax, by 7 +/- 2%Cma, (i.e. 1.35 fold, n = 5, P<0.05). At 1 nM, endothelin-l induced a small, sustained contraction(16 +/- 3%Cmo) and increased the standard EFS-induced contraction by 19 +/- 2%Cmax (i.e. 1.95 fold,n = 5, P <0.01). Finally, 10 nM endothelin-1 induced a large, sustained contraction (98 +/- 8%Cma), but the EFS-induced contraction was significantly reduced from 20%Cmax to 6 +/- 4%Cmax (n = 6, P <0.05).In contrast, in the presence of 3 microM BQ-123 (ETA receptor-selective antagonist), 1O nM endothelin-1 induced a transient contraction mediated via ETB receptors (peak response of 59 +/- 10%Cmax at 10 min;8 +/- 2%Cmax at 45 min). Under these conditions, the standard EFS-induced contraction was increased by 26+/- l%Cmax (i.e. 2.3 fold, n = 6, P<0.01).5. The potentiation of EFS-induced contractions produced by 1 nM endothelin-1 was not mediated by ETA receptors, since 3 microM BQ-123 did not diminish this effect (n = 6). Furthermore, 1 nM endothelin-1 did not potentiate EFS-induced contractions in preparations in which the function of the ETB receptor effector system had been attenuated by desensitization (n = 6).6. In summary, endothelin-1 potentiates cholinergic nerve-mediated contractions in mouse isolated trachea, apparently by activating prejunctional ETB receptors. This neuronal pathway offers an additional mechanism through which endothelin-1 may elevate bronchomotor tone.

1. The local cardiac actions of substance P were examined in isolated perfused hearts and atria of the guinea-pig. 2. In both hearts and right atria, substance P caused negative inotropic and chronotropic effects. 3. Atropine (10(-6) M) or depletion of acetylcholine, by electrical stimulation and hemicholinium-3 perfusion, significantly attenuated the negative inotropic and chronotropic effects of substance P. alpha- and beta-adrenoceptor blockade by nadolol and phentolamine (10(-6) M each) did not prevent the negative inotropic and chronotropic effects of substance P. This indicates that cholinergic neurones, but not adrenergic neurones, partially mediate the effects of substance P. 4. There was no significant difference in the effects of substance P observed between groups with acetylcholine depletion and with cholinoceptor blockade. This suggests that substance P elicits its effects mainly through release of acetylcholine. 5. These results indicate that substance P has negative inotropic and chronotropic effects in guinea-pig hearts and right atria mediated partly by release of acetylcholine. Substance P also appears to have direct effects on cardiac tissue.

1. In pentobarbitone anaesthetized dogs, preganglionic stimulation of the superior cervical sympathetic nerve (15V, 1 ms, 10 Hz) induced marked reduction of nasal arterial blood flow, whereas parasympathetic nerve stimulation (5 V, 1 ms, 10-30 Hz) evoked frequency-dependent vasodilatation. 2. Sympathetic nerve stimulation for 3 min at 10 Hz evoked significant (P < 0.05) and prolonged attenuation of the vasodilator response to subsequent parasympathetic stimulation. Pretreatment with phentolamine (0.5 mg kg-1 h-1), propranolol (1 mg kg-1) and atropine (0.5 mg kg-1) reduced the vasoconstrictor effect of sympathetic stimulation by 35 +/- 4% whereas the parasympathetic nerve-evoked vasodilatation was not significantly modified. Atropine-resistant parasympathetic vasodilatation remained significantly attenuated for more than 30 min after non-adrenergic sympathetic nerve-evoked vasoconstriction. 3. Vasodilator effects of exogenous vasoactive intestinal polypeptide and peptide histidine isoleucine and vasoconstrictor effects of exogenous neuropeptide Y (NPY) and the NPY analogue [Leu31, Pro34] NPY (Y1-receptor agonist, 8 nmol kg-1), were not altered by adrenoceptor antagonists and atropine f1p4eas the effects of exogenous noradrenaline and acetylcholine were virtually abolished. Attenuation of parasympathetic-evoked vasodilatation could be mimicked by exogenous NPY (8 nmol kg-1) and the NPY analogue, N-acetyl [Leu28, Leu31] NPY 24-36 (Y2-receptor agonist, 20 nmol kg-1) but not by exogenous Y1-receptor agonist. The Y2-receptor agonist did not show significant vasoconstrictor action. 4. It is concluded that sympathetic nerve stimulation attenuates parasympathetic vasodilatation via NPY release acting on prejunctional Y2 receptors.

In human isolated detrusor strips, submaximal contractile responses evoked by electrical stimulation were resistant to hexamethonium (30 microM) and abolished by tetrodotoxin (0.6 microM) and hyoscine (1 microM), indicating the activation of postganglionic cholinergic nerves. In methysergide (1 microM) and ondansetron (3 microM) pretreated tissues, 5-hydroxytryptamine (5-HT) (0.3 nM-1 microM) caused a concentration-dependent increase in the amplitude of contractions (pEC50 = 8.1), which was antagonized by the selective 5-HT4 receptor antagonist GR 113808 (3, 10 and 30 nM) in a competitive manner. Schild analysis yielded a pA2 estimate of 8.9, a value comparable to that reported for GR 113808 in other animal and human peripheral tissues (8.8-9.7). Our findings indicate that neuromuscular cholinergic transmission in human isolated detrusor muscle is facilitated by neural 5-HT receptors belonging to the 5-HT4 subtype. The human urinary bladder can thus be regarded as an additional site in which 5-HT4 receptors are distributed.

1. The synthetic cationic polypeptide, poly-L-arginine (0.03-1 mg ml-1) induced concentration-dependent contraction of guinea-pig and rat isolated trachea. In guinea-pig isolated trachea, this response was attenuated in the presence of the muscarinic cholinoceptor antagonist, atropine (0.1 microM) and augmented by the acetylcholinesterase inhibitor, ecothiophate (0.1 microM). The neuronal sodium channel blocker, tetrodotoxin (3 microM) failed to alter the contractile response to poly-L-arginine and acetylcholine. 2. The contractile response to poly-L-arginine in rat isolated trachea was inhibited in the presence of atropine (0.1 microM) and the 5-hydroxytryptamine (5-HT) receptor antagonist, methysergide (1 microM). Treatment of rat tracheal preparations with capsaicin (100 microM) or tetrodotoxin (3 microM) failed to alter the contractile response to poly-L-arginine. In contrast, ecothiophate (0.1 microM) augmented the contractile response to poly-L-arginine in rat isolated trachea. 3. Electrical field stimulation (5 Hz, 2 min) of epithelium-denuded guinea-pig tracheal preparations preloaded with [3H]-choline resulted in a contractile response and the simultaneous efflux of radioactivity into the superfusate. Both these responses were abolished in the presence of tetrodotoxin (1.5 microM). Poly-L-arginine (1 mg ml-1) also increased the efflux of total radioactivity from epithelium-denuded guinea-pig isolated tracheal preparations preloaded with [3H]-choline, but this response was tetrodotoxin-insensitive. The negatively charged polyanion, heparin (1 mg ml-1) failed to increase significantly the efflux of radioactivity from epithelium-denuded preparations. 4.In conclusion, the synthetic cationic polypeptide, poly-L-arginine, caused contraction of guinea-pig isolated tracheal preparations via the release of acetylcholine from parasympathetic nerves. Similarly,poly-L-arginine-induced contraction of rat isolated trachea is secondary to the release of acetylcholine from parasympathetic nerves and/or the release of mast cell-derived 5-HT.

1. The effects of superimposed and continuous sympathetic nerve stimulation on submandibular parasympathetic salivation were investigated in anaesthetized dogs. 2. Superimposed sympathetic nerve stimulation (1-2 min) initially enhanced and later inhibited salivary secretion induced by parasympathetic nerve stimulation (2-8 Hz) in glands with uncontrolled blood supply or constant-flow vascular perfusion. Propranolol (0.05 mg kg-1, i.a.) did not affect the diphasic sympathetic action whereas phentolamine (0.1 mg kg-1, i.a.) abolished it. Prazosin (0.025 mg kg-1, i.a.) greatly lessened the initial enhancement while yohimbine (0.025 mg kg-1, i.a.) alleviated the late inhibition. 3. Salivary secretion, induced by parasympathetic nerve stimulation (4 Hz) or acetylcholine infusion (10 micrograms kg-1 min-1, i.a.), was abolished by atropine (0.05 mg kg-1, i.a.), increased by phenylephrine infusion (0.25 microgram kg-1 min-1, i.a.) and depressed by clonidine infusion (0.75 microgram kg-1 min-1, i.a.). Hexamethionium (12.5 mg kg-1, i.a.) abolished the nerve-induced secretion but had no effect on the acetylcholine-induced secretion. 4. Continuous background sympathetic nerve stimulation decreased parasympathetic nerve-induced salivary secretion in glands with uncontrolled blood supply or constant-flow vascular perfusion. 5. These results show that parasympathetic salivation can be modified by the sympathetic system at the postsynaptic level; enhancement is via alpha 1-adrenoceptors whereas inhibition is via alpha 2-adrenoceptors.

1. Exogenous tachykinins modulate cholinergic neurotransmission in rabbit and guinea-pig airways. We have investigated the effect of selective tachykinin receptor agonists and antagonists on cholinergic neurotransmission evoked by electrical field stimulation (EFS) of bronchial rings in rabbit, guinea-pig and human airways in vitro to assess which type of tachykinin receptor is mediating this facilitatory effect. 2. Bronchial rings were set up for isometric tension recording. Contractile responses to EFS (60 V, 0.4 ms, 2 Hz for 10 s every min) and exogenous acetylcholine (ACh) were obtained and the effects of selective tachykinin agonists and antagonists were investigated. 3. In rabbit bronchi the endogenous tachykinins, substance P (SP) and neurokinin A (NKA) (10 nM) potentiated cholinergic responses to EFS (by 287.6 +/- 121%, P < 0.01 and 181.4 +/- 56.5%, P < 0.001 respectively). 4. The NK1 receptor selective agonist, [Sar9]SP sulphone (10 nM) evoked a maximal facilitatory action on cholinergic responses of 334.9 +/- 63% (P < 0.01) (pD2 = 8.5 +/- 0.06) an effect which was blocked by the selective NK1-receptor antagonist, CP 96,345 (100 nM) (P < 0.05) but not by the NK2 receptor antagonist, MEN 10,376 (100 nM). The NK2 receptor selective agonist, [beta Ala8]NKA(4-10) (10 nM), produced a maximum enhancement of 278 +/- 83.5% (P < 0.01) (pD2 = 8.7 +/- 0.1) an effect which was blocked by MEN 10,376 (100 nM) (P < 0.05) and not by CP 96,345. [MePhe7]NKB, an NK3 receptor selective agonist was without effect. 5. The rank order of potency of NK2 receptor antagonists against enhancement of cholinergic responses by [Beta Ala8]NKA(4-10) was MEN 10,376> L 659,877> R 396. This pattern together with the observation of the full agonist activity of MDL 28,564 indicates that the NK2 receptors in the rabbit bronchus are similar to those which are present in the rabbit pulmonary artery.6. Neither [Sar9]SP sulphone (5 nM) nor [Beta Ala8]NKA(4- 10) (1 nM) had any effect on contractile responses to ACh (10 MicroM) suggesting a pre-junctional mechanism of action.7. By contrast, in guinea-pig bronchi only the NK1-receptor agonist [Sar9]SP sulphone (3 nM) was effective in enhancing cholinergic neurotransmission but the effect was relatively small (maximal enhancement 25.7 +/- 5.5%, P<0.01). In human bronchial rings all the selective neurokinin agonists were without effect on cholinergic neurotransmission.8. These results suggest that tachykinins may play an important role in modulating cholinergic neurotransmission in rabbit (via NK1 and NK2 receptors) and guinea-pig airways (via NK1 receptor) but have no demonstrable effect on human airways

1. The mechanisms behind bradykinin-induced effects in the airways are considered to be largely indirect. The role of cholinergic nerves and eicosanoids, and their relationship in these mechanisms were investigated in guinea-pigs. 2. The role of cholinergic nerves was studied in animals given atropine (1 mg kg-1, i.v.), hexamethonium (2 mg kg-1, i.v.), or vagotomized. To study the role of eicosanoids, animals were pretreated with a thromboxane A2 (TxA2) receptor antagonist (ICI 192,605; 10(-6) mol kg-1, i.v.) or with a leukotriene (LT) receptor C4/D4/E4 antagonist (ICI 198,615; 10(-6) mol kg-1, i.v.). 3. After pretreatment with a drug, bradykinin (150 nmol) was instilled into the tracheal lumen. We measured both airway insufflation pressure (Pi), to assess airway narrowing, and the content of Evans blue dye in airway tissue, to assess plasma exudation. 4. Bradykinin instillation into the trachea caused an increase in Pi and extravasation of Evans blue dye. The increase in Pi was significantly attenuated by atropine or the TxA2 receptor antagonist, but not by hexamethonium, vagotomy or the LT receptor antagonist. 5. The bradykinin-induced exudation of Evans blue dye was significantly attenuated in the intrapulmonary airways by the TxA2 receptor antagonist, but not by atropine, hexamethonium, cervical vagotomy or the LT receptor antagonist. 6. A thromboxane-mimetic U-46619 (20 nmol kg-1, i.v. or 10 nmol intratracheally), caused both an increase in Pi and extravasation of Evans blue dye at all airway levels. Atropine pretreatment slightly attenuated the peak Pi after the intratracheal administration of U-46619, but not after i.v. administration. 7. We conclude that peripheral cholinergic nerves are involved in bradykinin-induced airflow obstruction but not plasma exudation, and that TxA2 is involved in both airflow obstruction and airway plasma exudation induced by bradykinin given via the airway route. TxA2-induced airflow obstruction is mediated only to a minor degree, via the release of acetylcholine in the airways.

1. The role of endogenous nitric oxide (NO) as a modulator of enteric neurotransmission was investigated in longitudinal muscle myenteric plexus (LMMP) preparations of guinea-pig isolated ileum. 2. In tissues previously incubated with [3H]-choline, exogenous NO inhibited electrically-evoked [3H]-choline overflow as well as responses to exogenous agonists, indicating that NO has the potential of neuromodulation both pre- and postjunctionally. 3. A series of NO synthase inhibitors enhanced contractile responses to nerve stimulation indicating inhibitory neuromodulation by endogenous NO. 4. The potency order of the NO synthase inhibitors and their consistent effects after dexamethasone, on responses to nerve stimulation, indicate action on a constitutive NO synthase. 5. Responses enhanced by NO synthase inhibitors were inhibited by the substance P receptor antagonist, spantide, suggesting a neuromodulatory influence on substance P-like neurotransmission by the endogenous NO. 6. NO synthase inhibition did not modify contractile responses to application of acetylcholine or substance P, or [3H]-choline overflow, indicating that endogenous NO mainly has a prejunctional inhibitory action on substance P-like neurotransmission. Nor did it modify responses to direct electrical muscle stimulation in the presence of tetrodotoxin. This suggests a prejunctional enhancing effect by NO synthesis inhibition. 7. Evidence for endogenous NO modulation of acetylcholine release was obtained when NO synthase inhibition modified atropine-sensitive, nerve-mediated contractile responses. However, [3H]-choline overflow was unaltered by NO synthase inhibition. 8. NO synthase inhibition did not modify responses to inhibitory neurotransmission. 9. The findings suggest that endogenous NO inhibits substance P-like motor neurotransmission, probably via prejunctional mechanisms. Cholinergic transmission may also be reduced by endogenous NO, acting prejunctionally.

The power spectrum of instantaneous heart rate fluctuations was used to determine the optimal doses of atropine that induce a maximal vagolytic or vagomimetic effect. In a crossover placebo controlled study, eight volunteers received increasing bolus doses of intravenous atropine (0.1 to 2.3 mg per subject) or placebo, and frequency bands of the power spectrum were integrated. During atropine administration a significant bimodal dose dependence was observed for the respiratory peak (0.2 to 0.4 Hz, p = 0.0006), the midfrequency band (0.09 to 0.15 Hz, p = 0.0035), and mean heart rate (p < 0.0001). Low doses (< 0.4 mg per subject) increased the respiratory and midfrequency band power, with maximal response at 0.2 mg per subject. Larger doses of atropine, 0.5 to 2.3 mg per subject, markedly reduced the power in all frequency bands in a dose-dependent way. The corresponding changes in mean heart rate were simultaneous, but in the opposite direction. We suggest that the respiratory peak of the power spectrum can be used to optimize drug effects on cardiac parasympathetic control.

To investigate the role of muscarinic cholinergic mechanisms in mediating the pancreatic and pituitary hormonal responses to hypoglycaemia, six normal subjects were studied during acute insulin-induced hypoglycaemia under control conditions, and during blockade with intravenous atropine. During atropine blockade the response of pancreatic polypeptide was suppressed while the maximum response of plasma glucagon was significantly higher. The increment in plasma vasopressin was also increased significantly during cholinergic blockade. During blockade with atropine the responses of plasma prolactin was reduced, with a slight but significant reduction in the growth hormone response, and although a similar maximum response of plasma ACTH was achieved, this rise was delayed. These results implicate involvement of a cholinergic muscarinic inhibitory and stimulatory mechanisms in regulating the responses of pancreatic and pituitary hormones to hypoglycaemia.

1. Intravenous administration of leukotriene C4 (LTC4) and LTD4 (1-10 nmol kg-1) caused a dose-dependent increase in secretion of glandular-kallikrein in the bronchial washings of guinea-pigs, as measured by cleavage of a synthetic substrate and the formation of kinin. LTC4 was more potent than LTD4 and pilocarpine was much less potent than peptide leukotrienes on a molecular basis. 2. The increases in levels of glandular-kallikrein in the bronchial washings that were induced by LTC4 (3 nmol kg-1, i.v.) were almost completely inhibited by pretreatment with an antagonist of leukotrienes (ONO-1078), with an antagonist of thromboxane (S-1452), with an inhibitor of thromboxane synthetase (OKY-046), with indomethacin, with atropine or with scopolamine. These results indicate that the LTC4-induced increase in levels of glandular-kallikrein may have been mediated by the formation of thromboxane and the release of acetylcholine. 3. The increases in levels of glandular-kallikrein in the bronchial washings induced by STA2 (20 pmol kg-1, i.v.), a stable analogue of thromboxane A2, were completely blocked by pretreatment with atropine, whereas increases induced by pilocarpine (41 mumol kg-1, i.v.) were not blocked by pretreatment with indomethacin, although such increases were inhibited by atropine. This result indicates that secretion of kallikrein stimulated by LTC4 may have been mediated by the successive formation of thromboxane A2 and release of acetylcholine. 4. Intravenous administration of bradykinin (3-30 nmol kg-1) caused a dose-dependent increase in levels of glandular-kallikrein in the bronchial washings. This increase was completely inhibited by pretreatment with atropine, with indomethacin or with an antagonist of thromboxane.5. The increases in levels of glandular-kallikrein in the bronchial washings induced by LTC4 (3 nmol kg'- , i.v.) and pilocarpine (41 flmol kg- 1, i.v.) were significantly inhibited by pretreatment with an antagonist of bradykinin. These results suggest that intravenous LTC4 may increase secretion of glandular-kallikrein via formation of thromboxane A2 and release of acetylcholine in that order, and kinin released by kallikrein may enhance the rate of secretion of glandular-kallikrein.

1 The objective of the study was to investigate the central vagal and local axon reflex components of bronchoconstrictor responses evoked by inhalation of capsaicin aerosol in anaesthetized guinea-pigs. This was accomplished by comparing the effects of bilateral vagotomy, atropine and the peripherally-acting polar enkephalin analogue, BW443C81, on bronchoconstrictor responses evoked by capsaicin. The effects of codeine were also determined. 2 Aerosols of capsaicin were generated from a 0.9 microgram ml-1 solution. Inhalation of capsaicin aerosol in 5, 10 and 15 breaths evoked dose-related bronchoconstrictor responses. The responses were immediate in onset and of extended duration. 3 Capsaicin-induced bronchoconstrictor responses were significantly inhibited following bilateral vagotomy or atropine (0.3 mg kg-1, i.v.) pretreatment by 46% +/- 14% (P less than 0.05) and 59% +/- 13% (P less than 0.01), respectively. 4 Administration of BW443C81 by intravenous infusion (3, 30 and 100 micrograms kg-1 min-1) caused a significant inhibition of capsaicin-induced bronchoconstrictor responses which achieved a greater maximum than either bilateral vagotomy or atropine. Codeine (100 micrograms kg-1 min-1, i.v.) did not significantly inhibit the bronchoconstrictor responses. 5 Inhibition of capsaicin-induced bronchoconstrictor responses by BW443C81 (30 micrograms kg-1 min-1, i.v.) was significantly (P less than 0.05) reduced by the peripherally-acting opioid antagonist N-methyl nalorphine (100 micrograms kg-1 min-1, i.v.). 6 These results show that capsaicin-induced bronchoconstrictor responses are mediated by at least two mechanisms, a vagal and/or cholinergic reflex pathway and a non-cholinergic pathway. BW443C81, but not codeine, significantly inhibited (P < 0.005) both mechanisms of capsaicin-induced bronchoconstriction probably by an action on peripheral opioid receptors located on vagal sensory nerves.

To determine whether hyperinsulinaemia of human obesity is dependent on the activity of the parasympathetic nervous system, and whether activation of the parasympathetic nervous system plays a role in glucose-induced thermogenesis, the metabolic effect of a continuous intravenous glucose infusion [44.4 mumol kg-1 body weight (bw) min-1] with or without atropine infusion was assessed in 11 obese patients and 10 lean controls. Compared with lean controls, obese patients had increased basal and glucose-stimulated plasma insulin and C-peptide concentrations and increased plasma glucose concentrations during glucose infusion. Glucose oxidation during i.v. glucose was lower in obese patients than in lean controls. Glucose-induced thermogenesis was similar in obese patients and in lean controls. Atropine infusion did not affect basal plasma glucose, insulin or free fatty acid concentrations nor glucose-stimulated plasma glucose, insulin, C-peptide, glucagon or free fatty acid concentrations in both groups of subjects. Glucose and lipid oxidation rates and glucose-induced thermogenesis were also unaffected by atropine administration. It is concluded that (1) glucose-stimulated hyperinsulinaemia in human obesity is not dependent on a hyperactivity of the parasympathetic nervous system, which indicates that human obesity is different from most animal models of obesity; (2) glucose-induced thermogenesis is similar in obese and lean subjects when a similar load of glucose is administered; (3) inhibition of the parasympathetic nervous system does not affect the thermic effect of i.v. glucose.

1. The effects of vasoactive intestinal polypeptide (VIP) antagonists [AC-Tyr1, D-Phe2]-GRF(1-29)-NH2 and [4-Cl-D-Phe6, Leu17]-VIP on excitatory neuroeffector transmission in the dog and cat trachea were investigated by use of microelectrode, double sucrose-gap and tension recording methods. 2. In the dog trachea, repetitive stimuli at high frequency (20 Hz) markedly enhanced the amplitude of contraction, the amplitude of contractions evoked by 50 stimuli at 20 Hz relative to that evoked by 5 stimuli being 14.2 +/- 3.8 times (n = 7, +/- s.d.). In the cat, the summation was much less marked, the amplitude of contractions evoked by 50 stimuli relative to that evoked by 5 stimuli being only 2.1 +/- 0.6 times (n = 5, +/- s.d.). Neither VIP antagonist had any effect on the relationship between the number of stimuli at 20 Hz and the relative amplitude of contraction in the dog trachea, but did enhance the amplitude of contractions to 1.1-1.5 times control in the cat trachea. 3. VIP antagonists dose-dependently enhanced the amplitude of excitatory junction potentials (e.j.ps) evoked by a single stimulus in the cat trachea, without changing the resting membrane potential or input membrane resistance of the smooth muscle cells. However, neither antagonist had any effect on the amplitude of the e.j.p. in the dog trachea. 4. Neither VIP antagonist had any effect on the post-junctional response of smooth muscle cells to exogenously applied acetylcholine (ACh; 10(-9)-10(-5) M) in the dog or cat trachea.5. In the cat trachea, VIP (10-11 M) suppressed the ej.p. amplitude to 0.74 +/- 0.09 times the control value (n = 6). However, after pretreatment of the tissue with the VIP antagonists [Ac-Tyr', D-Phe2]-GRF(1-29)- H2 (10-8M) and [4-Cl-D-Phe6, Leu17]-VIP (10-8M), VIP (10-11 M) did not suppress the ej.p. amplitude, indicating that VIP antagonists block the presynaptic inhibitory action of exogenous VIP.6. In parallel with the enhancement of contraction, ej.ps showed marked summation when repetitive field stimulations were applied at high frequency (20 Hz) in the dog trachea. The relationship between the relative amplitude of the ej.p. and number of stimuli at 20 Hz was linear and the slope was 2.2 +/- 0.3 mV/stimulation. VIP antagonists did not affect this relationship. However, in the cat trachea, summation of ej.ps was not at all marked and a linear relationship was not observed with the double sucrose-gap method. Incubation of the cat tracheal tissue with either of the VIP antagonists (10-8 or 10-7M) markedly enhanced the summation of ej.ps evoked by repetitive field stimulation at 20 Hz, and after the treatment a linear relationship between the number of stimuli and the amplitude of ej.ps was observed, the slopes being 0.6 +/- 0.1 (n = 8) and 0.55 +/- 0.1 mV/stimulation (n = 5), respectively.7. These results indicate that both VIP antagonists, [Ac-Tyr', D-Phe2]-GRF(1-29)-NH2 and [4-Cl-DPhe6, Leu17]-VIP, have a prejunctional action accelerating the excitatory neuroeffector transmission, presumably by enhancing transmitter release from the vagus nerves in the cat, but not in the dog trachea.

1. This study was designed to assess the effects of exogenous neuropeptide Y (NPY) on cardiac contractility and coronary blood flow (CBF) in anaesthetized dogs and to evaluate the effect of NPY on the responses to sympathetic and parasympathetic stimulation and to inotropic agents. 2. Bolus doses of NPY (500 micrograms), administered via the femoral vein, increased mean arterial pressure. The pressor effect was associated with reductions in heart rate, CBF and cardiac contractility. 3. The effects of NPY (500 micrograms) on contractility and CBF were compared with that of vasopressin (VP) (1 unit). For similar reductions in CBF, NPY and VP had similar negative inotropic effects. Thus, it is likely that the negative inotropic response to NPY is not due to a direct effect of NPY on the heart muscle but is largely due to coronary vasoconstriction. 4. In the presence of NPY (500 micrograms, i.v.), there was an inhibition of the positive inotropic response to stimulation of the left cardiac sympathetic nerve (2 or 5 Hz, 0.05 ms). NPY also inhibited the negative inotropic response and chronotropic response to vagal stimulation (2, 3 or 5 Hz, 0.05 ms). 5. Dose-response curves were obtained for the inotropic, chronotropic and pressor responses to cumulative infusions of noradrenaline (n = 6) and dobutamine (n = 6). NPY had no effect on these dose-response curves. 6. The effect of NPY on the responses to salbutamol and impromidine was assessed. NPY did not alter the positive inotropic, chronotropic or depressor responses to these agonists. 7. Our results indicate that NPY has direct, postsynaptic vasoconstrictor activity and the reduction in myocardial contractility and heart rate are due to a combination of coronary vasoconstriction, baroreceptor reflex activation and presynaptic inhibition of transmitter release from sympathetic nerves. The welldocumented inhibitory effect of NPY on the chronotropic response to parasympathetic stimulation was confirmed and similar inhibition of the inotropic response to both sympathetic and parasympathetic stimulation was demonstrated. Since there was no modulation of the inotropic effects of exogenous alpha- and beta-adrenoceptor or H2-receptor agonists, it is concluded that the effects of NPY on myocardial contractility are exerted presynaptically.

1. We studied the effect of hyperosmolarity on human isolated airways because a better understanding of the effect of hyperosmolarity on the human airway wall may improve insight into the pathophysiology of hyperosmolarity-induced bronchoconstriction in asthma. 2. In cartilaginous bronchial rings dissected from fresh human lung tissue, hyperosmolar krebs-Henseleit buffer (450 mosM, extra sodium chloride added) evoked a biphasic response: a rapid relaxation phase (peak after 5.0 +/- 0.3 min) followed by a slow contraction phase (peak after 25.4 +/- 0.8 min). 3. With the histamine (H1) receptor antagonist mepyramine, the contraction phase was reduced to 41.2% of the control value (P less than 0.001), with atropine to 50.0% (P less than 0.01), with the local anaesthetic lignocaine to 48.7% (P less than 0.05) and with mepyramine together with atropine to 19.2% (P less than 0.001). 4. With the inhibitor of neutral metalloendopeptidase, phosphoramidon, the contraction phase increased to 128.0% of the control value (P less than 0.05) and after removal of the epithelium to 131.8% (P less than 0.05). 5. Indomethacin, the leukotriene C4/D4 (LTC4/D4) antagonist FPL 55712 or the blocker of nerve conduction, tetrodotoxin, had no effect on the contractile phase. 6. The relaxation phase was not altered by any of these drugs nor by epithelial denudation. The relaxation phase was also unchanged in the presence of alpha-chymotrypsin, which degrades muscle relaxing peptides such as vasoactive intestinal peptide. 7. Hyperosmolar buffer slightly increased the sensitivity and maximal response to methacholine as well as the cholinergic twitch to electric field stimulation. 8. We conclude that hyperosmolarity releases acetylcholine, histamine and neuropeptides in the human airway wall in sufficient quantities to contract airway smooth muscle. This release itself or its effect on airway muscle is modulated by the airway epithelium. The mechanism of the relaxation phase may be an unknown smooth muscle relaxing substance or a direct effect on the airway muscle, related to ion fluxes.

1. The effects of muscarinic antagonists considered to be selective for M1 receptors (pirenzepine) and for M2 receptors (gallamine and methoctramine) were used to investigate the existence of prejunctional muscarinic receptors on cholinergic nerves in the rat lung. The tracheal tube preparation was used in vitro, and contraction of the trachealis muscle was induced by electrical field stimulation (EFS) and by application of an exogenous muscarinic agonist (pilocarpine), and measured as an increase in intraluminal pressure in the tube. 2. The muscarinic antagonists, gallamine and methoctramine, enhanced the contractions induced by nerve stimulation, while contractions elicited by exogenous application of pilocarpine were inhibited by the antagonists. 3. In contrast, pirenzepine blocked contractions induced by both EFS and pilocarpine in a dose-dependent manner (EC50 0.1 microM) due to blockade of the postjunctional muscarinic receptors on airway smooth muscle. Potentiation of the response to EFS was never seen with this antagonist. 4. The muscarinic agonist, pilocarpine, caused a slow maintained increase in tone of the tracheal tube and at the same time reduced the contractions induced by EFS. This inhibitory effect was blocked by gallamine and methoctramine. 5. The results suggest that prejunctional inhibitory muscarinic receptors may be localised on the parasympathetic cholinergic nerve terminals innervating tracheal smooth muscle in the rat. This confirms previous findings obtained by measuring transmitter release in this species. The present results suggest that these receptors are of the M2 subtype. Blockade of these autoreceptors with gallamine or methoctramine would increase the output of acetylcholine (ACh) and thereby enhance the nerve-induced contraction of tracheal smooth muscle.

1. A quaternary derivative of (-)-hyoscyamine, phenthonium (Phen) induced a concentration-dependent increase in the rate of spontaneous quantal release of acetylcholine (ACh) at the mammalian neuromuscular junction, as shown by intracellular recordings of the miniature endplate potentials (m.e.p.ps) in rat diaphragm muscles. 2. The prejunctional effect of Phen (10-50 microM) was reversible, unrelated to temperature (22 degrees-35 degrees C), unaltered by either changes in [Ca2+]o or by high [Mg2+]o, and was not induced by membrane depolarization. 3. Simultaneously, Phen reduced the amplitude of m.e.p.ps by a postjunctional action. 4. The muscarinic agonist oxotremorine did not prevent the increase in m.e.p.p. frequency induced by Phen. Cholinesterase inhibition with neostigmine potentiated the prejunctional effect induced by a low (20 microM) but not a high (50 microM) concentration of Phen. 5. The increase in m.e.p.p. frequency induced by Phen was not influenced by previous incubation with either atropine (0.01-10 microM) or (+)-tubocurarine (0.05-0.1 microM). Each antagonist however, intensified the postjunctional effect of Phen. 6. Phen (20 microM) did not influence the quantal contents of e.p.ps in cut-muscle preparations or in the presence of high [Mg2+]o. A high concentration of Phen (50 microM) increased the rundown of e.p.p. trains evoked at 10-50 Hz. 7. The results indicate that the facilitatory prejunctional action of Phen cannot be explained by an antimuscarinic activity. A possible interaction of the antagonist with putative prejunctional nicotinic cholinoceptors however, was not excluded.

1. Opioid receptors have been localised on sensory fibres in the vagus nerve and opioids have previously been shown to inhibit non-adrenergic, non-cholinergic (NANC) neurotransmission in guinea-pig bronchi in vitro and in vivo. We have now investigated whether an inhibitory effect could be demonstrated on cholinergic neurotransmission. 2. Electrical field stimulation (EFS) (8 Hz, 0.5 ms, 40 V for 20 s) produced only a rapid, cholinergic response in the upper trachea but in the lower trachea and main bronchi a cholinergic response which was atropine-sensitive and a longer lasting NANC contraction that was atropine-insensitive was demonstrated. This slow contraction could be blocked by tetrodotoxin and capsaicin pretreatment. 3. [D-Ala2, NMePhe4, Gly-ol5]enkephalin (DAMGO), a selective mu-opioid receptor agonist, inhibited the cholinergic response to EFS at 8 Hz in a dose-dependent manner in main bronchi (IC50 = 113 nM with a maximal inhibition of 35.7 +/- 5.6% 10 microM, n = 5). In the lower trachea, DAMGO inhibited the cholinergic response to a similar extent (inhibition of 35.8 +/- 3.5% at 10 microM, n = 5). However, DAMGO had no effect on the contractile response to exogenously applied acetylcholine in the main bronchi. By contrast, opioids had no inhibitory effect on cholinergic neurotransmission in the upper trachea. DAMGO (1 microM) inhibited the cholinergic response to EFS in a frequency-dependent manner in the main bronchi with greater inhibition at lower frequencies of stimulation. 4. The delta-opioid receptor agonist [D-Pen2, D-Pen5]enkephalin (DPDPE) significantly inhibited the cholinergic component of the constrictor response to EFS at 8 Hz in the bronchi but at the highest dose used (10 microM). U-50,488H, a Kappa-receptor agonist, had no inhibitory effect on the cholinergic constrictor component in the main bronchi (10microM). 5. DAMGO also inhibited the NANC responses to EFS in the main bronchi in a dose-dependent manner (with an IC50 = 36 nm and a maximal inhibition of 63.4 + 8.3%, at 1 microM, n = 5). DAMGO had no effect on contractile responses to exogenously applied substance P (SP). DPDPE (10 microM) was less effective in inhibition of the NANC bronchoconstriction with a maximal inhibition of 29.2 + 4.2% (n = 7), and U-50,488H (1O microM) had no inhibitory effect. 6. After capsaicin pretreatment, which depleted sensory nerves of neuropeptides, the inhibitory effect of DAMGO (1 microM) on cholinergic constriction in main bronchi at 8 Hz was only 13.4 + 1.9% (n = 13) compared with 32.9 + 4.0% (n = 9) inhibition in vehicle-treated controls (P < 0.001). 7. Opioids may reduce the cholinergic neural responses in airways partly via an inhibitory action on excitatory NANC nerves and partly by a direct effect on cholinergic neurotransmission. The opioid receptor involved is of the mu-opioid receptor subtype.