Search

Background and Purpose: While arachidonyl ethanolamine (anandamide) produces pharmacological effects mediated by cannabinoid CB1 receptors, it is also an agonist at the transient receptor potential vanilloid type 1 (TRPV1) ion channel. This study examined the cellular actions of anandamide in the midbrain periaqueductal grey (PAG), a region implicated in the analgesic actions of cannabinoids, and which expresses both CB1 receptors and TRPV1.Experimental Approach: In vitro whole cell patch clamp recordings of glutamatergic excitatory postsynaptic currents (EPSCs) were made from rat and mouse PAG slices.Key Results: Capsaicin (1 µM) increased the rate, but not the amplitude of miniature EPSCs in subpopulations of neurons throughout the rat and mouse PAG. Capsaicin had no effect on miniature EPSCs in PAG neurons from TRPV1 knock-out mice. In mouse PAG neurons, anandamide (30 µM) had no effect on the rate of miniature EPSCs alone, or in the presence of either the CB1 antagonist AM251 (3 µM) or the TRPV1 antagonist iodoresiniferatoxin (300 nM). Anandamide produced a decrease in miniature EPSC rate in the presence of the fatty acid amide hydrolase (FAAH) inhibitor URB597 (1 µM). By contrast, anandamide produced an increase in miniature EPSC rate in the presence of both URB597 and AM251, which was absent in TRPV1 knock-out mice.Conclusions and Implications: These results suggest that the actions of anandamide within PAG are limited by enzymatic degradation by FAAH. FAAH blockade unmasks both presynaptic inhibition and excitation of glutamatergic synaptic transmission which are mediated via CB1 receptors and TRPV1 respectively. [ABSTRACT FROM AUTHOR]

Key Points: The midbrain periaqueductal grey (PAG) forms part of an endogenous analgesic system which is tightly regulated by the neurotransmitter GABA. The role of endocannabinoids in regulating GABAergic control of this system was examined in rat PAG slices. Under basal conditions GABAergic neurotransmission onto PAG output neurons was multivesicular. Activation of the endocannabinoid system reduced GABAergic inhibition by reducing the probability of release and by shifting release to a univesicular mode. Blockade of endocannabinoid system unmasked a tonic control over the probability and mode of GABA release. These findings provides a mechanistic foundation for the control of the PAG analgesic system by disinhibition., Abstract: The midbrain periaqueductal grey (PAG) has a crucial role in coordinating endogenous analgesic responses to physiological and psychological stressors. Endocannabinoids are thought to mediate a form of stress-induced analgesia within the PAG by relieving GABAergic inhibition of output neurons, a process known as disinhibition. This disinhibition is thought to be achieved by a presynaptic reduction in GABA release probability. We examined whether other mechanisms have a role in endocannabinoid modulation of GABAergic synaptic transmission within the rat PAG. The group I mGluR agonist DHPG ((R,S)-3,5-dihydroxyphenylglycine) inhibited evoked IPSCs and increased their paired pulse ratio in normal external Ca 2+ , and when release probability was reduced by lowering Ca 2+ . However, the effect of DHPG on the coefficient of variation and kinetics of evoked IPSCs differed between normal and low Ca 2+ . Lowering external Ca 2+ had a similar effect on evoked IPSCs to that observed for DHPG in normal external Ca 2+ . The low affinity GABA A receptor antagonist TPMPA ((1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid) inhibited evoked IPSCs to a greater extent in low than in normal Ca 2+ . Together these findings indicate that the normal mode of GABA release is multivesicular within the PAG, and that DHPG and lowering external Ca 2+ switch this to a univesicular mode. The effects of DHPG were mediated by mGlu5 receptor engagement of the retrograde endocannabinoid system. Blockade of endocannabinoid breakdown produced a similar shift in the mode of release. We conclude that endocannabinoids control both the mode and the probability of GABA release within the PAG., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Background and Purpose: Endogenous cannabinoids (endocannabinoids) in the periaqueductal grey (PAG) play a vital role in mediating stress-induced analgesia. This analgesic effect of endocannabinoids is enhanced by pharmacological inhibition of their degradative enzymes. However, the specific effects of endocannabinoids and the inhibitors of their degradation are largely unknown within this pain-modulating region., Experimental Approach: In vitro electrophysiological recordings were conducted from PAG neurons in rat midbrain slices. The effects of the major endocannabinoids and their degradation inhibitors on inhibitory GABAergic synaptic transmission were examined., Key Results: Exogenous application of the endocannabinoid, anandamide (AEA), but not 2-arachidonoylglycerol (2-AG), produced a reduction in inhibitory GABAergic transmission in PAG neurons. This AEA-induced suppression of inhibition was enhanced by the fatty acid amide hydrolase (FAAH) inhibitor, URB597, whereas a 2-AG-induced suppression of inhibition was unmasked by the monoacylglycerol lipase (MGL) inhibitor, JZL184. In addition, application of the CB1 receptor antagonist, AM251, facilitated the basal GABAergic transmission in the presence of URB597 and JZL184, which was further enhanced by the dual FAAH/MGL inhibitor, JZL195., Conclusions and Implications: Our results indicate that AEA and 2-AG act via disinhibition within the PAG, a cellular action consistent with analgesia. These actions of AEA and 2-AG are tightly regulated by their respective degradative enzymes, FAAH and MGL. Furthermore, individual or combined inhibition of FAAH and/or MGL enhanced tonic disinhibition within the PAG. Therefore, the current findings support the therapeutic potential of FAAH and MGL inhibitors as a novel pharmacotherapy for pain., (© 2014 The British Pharmacological Society.)

Background and Purpose: Menthol, a naturally occurring compound in the essential oil of mint leaves, is used for its medicinal, sensory and fragrant properties. Menthol acts via transient receptor potential (TRPM8 and TRPA1) channels and as a positive allosteric modulator of recombinant GABAA receptors. Here, we examined the actions of menthol on GABAA receptor-mediated currents in intact midbrain slices., Experimental Approach: Whole-cell voltage-clamp recordings were made from periaqueductal grey (PAG) neurons in midbrain slices from rats to determine the effects of menthol on GABAA receptor-mediated phasic IPSCs and tonic currents., Key Results: Menthol (150-750 μM) produced a concentration-dependent prolongation of spontaneous GABAA receptor-mediated IPSCs, but not non-NMDA receptor-mediated EPSCs throughout the PAG. Menthol actions were unaffected by TRPM8 and TRPA1 antagonists, tetrodotoxin and the benzodiazepine antagonist, flumazenil. Menthol also enhanced a tonic current, which was sensitive to the GABAA receptor antagonists, picrotoxin (100 μM), bicuculline (30 μM) and Zn(2+) (100 μM), but unaffected by gabazine (10 μM) and a GABAC receptor antagonist, 1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid hydrate (TPMPA; 50 μM). In addition, menthol potentiated currents induced by the extrasynaptic GABAA receptor agonist THIP/gaboxadol (10 μM)., Conclusions and Implications: These results suggest that menthol positively modulates both synaptic and extrasynaptic populations of GABAA receptors in native PAG neurons. The development of agents that potentiate GABAA -mediated tonic currents and phasic IPSCs in a manner similar to menthol could provide a basis for novel GABAA -related pharmacotherapies., (© 2014 The British Pharmacological Society.)

Cholecystokinin modulates pain and anxiety via its functions within brain regions such as the midbrain periaqueductal gray (PAG). The aim of this study was to examine the cellular actions of cholecystokinin on PAG neurons. Whole-cell patch clamp recordings were made from rat midbrain PAG slices in vitro to examine the postsynaptic effects of cholecystokinin and its effects on synaptic transmission. Sulfated cholecystokinin-(26-33) (CCK-S, 100-300 nM), but not non-sulfated cholecystokinin-(26-33) (CCK-NS, 100-300 nM) produced an inward current in a sub-population of opioid sensitive and insensitive PAG neurons, which did not reverse over a range of membrane potentials. The CCK-S-induced current was abolished by the CCK1 selective antagonist devazepide (100 nM), but not by the CCK2 selective antagonists CI988 (100 nM, 1 μM) and LY225910 (1 μM). CCK-S, but not CCK-NS produced a reduction in the amplitude of evoked GABA(A)-mediated inhibitory postsynaptic currents (IPSCs) and an increase in the evoked IPSC paired-pulse ratio. By contrast, CCK-S had little effect on the rate and amplitude of TTX-resistant miniature IPSCs under basal conditions and when external K(+) was elevated. The CCK-S-induced inhibition of evoked IPSCs was abolished by the cannabinoid CB1 receptor antagonist AM251 (3 μM), the mGluR5 antagonist MPEP (10 μM) and the 1, 2-diacylglycerol lipase (DAGLα) inhibitor tetrahydrolipstatin (10 μM). In addition, CCK-S produced an increase in the rate of spontaneous non-NMDA-mediated, TTX-dependent excitatory postsynaptic currents (EPSCs). These results suggest that cholecystokinin produces direct neuronal depolarisation via CCK1 receptors and inhibits GABAergic synaptic transmission via action potential-dependent release of glutamate and mGluR5-induced endocannabinoid signaling. Thus, cholecystokinin has cellular actions within the PAG that can both oppose and reinforce opioid and cannabinoid modulation of pain and anxiety within this brain structure.

Substance P is thought to play an essential role in several forms of supraspinally mediated analgesia. The actions of substance P on synaptic transmission within descending analgesic pathways, however, are largely unknown. Here, we used whole-cell recordings from rat midbrain slices to examine the effects of substance P on GABAergic and glutamatergic transmission within the periaqueductal gray (PAG), a key component of a descending analgesic pathway that projects via the rostral ventromedial medulla (RVM) to the spinal cord dorsal horn. We found that substance P reversibly decreased the amplitude and increased the paired-pulse ratio of evoked IPSCs recorded from identified PAG-RVM projection neurons and from unidentified PAG neurons. Substance P had no effect on miniature IPSCs, implying an indirect mode of action. The effects of substance P were abolished by metabotropic glutamate type 5 and cannabinoid CB1 receptor antagonists, but unaltered by NMDA, GABA(B), mu,delta-opioid, adenosine A(1), and 5HT(1A) receptor antagonists. Consistent with a role for endogenous glutamate in this process, substance P increased the frequency of action potential-dependent spontaneous EPSCs. Moreover, the effect of substance P on evoked IPSCs was mimicked and occluded by a glutamate transport inhibitor. Finally, these effects were dependent on postsynaptic G-protein activation and diacylglycerol lipase activity, suggesting the requirement for retrograde signaling by the endocannabinoid 2-arachidonoylglycerol. Thus, substance P may facilitate descending analgesia in part by enhancing glutamate-mediated excitation and endocannabinoid-mediated disinhibition of PAG-RVM projection neurons.

Glutamate spillover regulates GABAergic synaptic transmission at several CNS synapses via presynaptic ionotropic and metabotropic glutamate receptors (mGluRs). We have previously demonstrated that activation of group I-III mGluRs inhibits GABAergic transmission in the midbrain periaqueductal gray (PAG), a region involved in organizing behavioral responses to threat, stress, and pain. Here, we examined the role of glutamate spillover in the modulation of GABAergic transmission in the PAG. Using whole-cell recordings from rat PAG slices, we found that evoked IPSCs were reduced by the nonspecific glutamate transport blockers DL-threo-beta-benzyloxyaspartic acid (TBOA) and L-trans-pyrrolidine-2,4-dicarboxylic acid, but not by the glial GLT1-specific blocker dihydrokainate. In contrast, TBOA had no effect on evoked IPSCs when glutamate uptake into the postsynaptic neuron was selectively impaired. TBOA increased the paired-pulse ratio of evoked IPSCs and reduced the rate but not the amplitude of spontaneous miniature IPSCs. The effect of TBOA on evoked IPSCs was abolished by the broad-spectrum mGluR antagonist (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (100 microM), reduced by the mGluR5-specific antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) and mimicked by the mGluR1/5 agonist (RS)-3,5-dihydroxyphenylglycine (DHPG). Furthermore, the effects of both TBOA and DHPG were reduced by the cannabinoid CB1 receptor antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (AM251). Finally, although MPEP and AM251 had no effect on single evoked IPSCs, they increased evoked IPSCs during repetitive stimulation. These results indicate that neuronal glutamate transporters limit mGluR5 activation and endocannabinoid signaling, but may be overwhelmed during conditions of elevated glutamate release. Thus, neuronal glutamate transporters play a key role in regulating endocannabinoid-mediated cross talk between glutamatergic and GABAergic synapses within the PAG.

The postsynaptic actions of substance P on rat midbrain periaqueductal grey (PAG) neurons were examined using whole-cell patch-clamp recordings in brain slices. Substance P produced an inward current in a subpopulation (60%) of PAG neurons. The substance P induced current was concentration dependent (EC50=27 nM) and was reduced by the NK1, NK2 and NK3 antagonists L-732,138 (20 microM), GR 159897 (3 microM) and SB 218795 (3 microM). The selective NK1, NK2 and NK3 agonists [Sar9,Met(O2)11]-Substance P (100 nM), GR 64349 (300-500 nM) and senktide (300 nM) also produced inward currents in subpopulations of neurons. A greater proportion of substance P-sensitive neurons (70%) than substance P-insensitive neurons (31%) responded to the mu/delta opioid agonist met-enkephalin (10 microM). Substance P reduced the outward current produced by met-enkephalin. The reversal potential of the substance P induced current varied from -5 mV to below -140 mV in the absence of met-enkephalin, and was -105 mV in the presence of met-enkephalin. These results indicate that substance P acts via NK1, NK2 and NK3 receptors to excite subpopulations of opioid-sensitive and insensitive PAG neurons by increasing a non-selective cation conductance and by reducing a K+ current. In addition, substance P has anti-opioid actions that are largely mediated by a reduction in the opioid induced K+ current.

The effect of metabotropic glutamate receptor (mGluR) activation on GABAergic synaptic transmission in rat periaqueductal grey (PAG) neurons was examined using whole-cell patch-clamp recordings in brain slices. The selective groups I, II and III mGluR agonists DHPG (10-30 microM), DCG-IV (1-3 microM) and L-AP4 (10-30 microM) inhibited electrically evoked GABA(A) mediated inhibitory postsynaptic currents (IPSCs) in all PAG neurons tested. DCG-IV and L-AP4 also reduced the frequency of spontaneous IPSCs, while DHPG produced both increases and decreases in spontaneous IPSC frequency in a dose dependent manner. In the presence of TTX, DHPG, DCG-IV and L-AP4 all reduced the frequency of spontaneous miniature IPSCs, but had no effect on their amplitudes. The DHPG, DCG-IV and L-AP4 effects on miniature IPSCs were dose dependent (EC(50)s=1.4, 0.055 and 0.52 microM, respectively) and were reduced by the selective mGluR antagonists MCPG, EGLU and MSOP, respectively. These results indicate that GABAergic synaptic transmission within the PAG is reduced by groups I, II and III mGluR activation via a presynaptic mechanism and is increased by group I mGluR activation via an action potential dependent mechanism. The finding of convergent groups I, II and III mGluR-mediated inhibition of synaptic transmission is novel and indicates that all groups of mGluRs have the potential to modulate the constellation of analgesic, behavioural and autonomic functions within the PAG.

The present study investigated whether mechanical allodynia following contusive spinal cord injury (SCI) of the thoracic segments 12 and 13 of the rat was associated with a reduction in gamma-aminobutyric acid (GABA)ergic inhibition adjacent to the site of injury. Five to 7 days following SCI, extracellular recordings were obtained from dorsal horn neurones located 1-2 segments caudal to the injury, in non-allodynic and allodynic halothane anaesthetised rats and from comparable neurones in normal rats. To assess spinal GABAergic inhibition in the three groups of animals, spontaneous and evoked cell firing rates were recorded before, during and after microiontophoretic application of the GABA(A) receptor antagonist bicuculline. Administration of bicuculline to normal animals resulted in significant and reversible increases in the receptive field size, spontaneous firing rate, response to brushing and pinching the skin and afterdischarge activity of dorsal horn neurones, as well as decreasing paired-pulse depression of responses evoked by transcutaneous electrical stimulation. In non-allodynic SCI animals, bicuculline ejection led to significant changes in receptive field size, paired-pulse depression and responses to brush and pinch stimulation that were comparable to those observed in normal animals. By contrast, in allodynic SCI animals, bicuculline ejection had little or no effect on dorsal horn neurone responses to mechanical skin stimuli and paired-pulse depression despite reliably blocking the inhibition of cell firing produced by similarly applied GABA. The demonstration of reduced GABAergic inhibition predominantly in the allodynic SCI rats suggests that such a deficiency contributed to this pain-related behaviour acutely following SCI.

1 Patch clamp recordings were made from periaqueductal grey (PAG) neurons in vitro to investigate the cellular actions of opioids in wild-type C57B16/J mice and mutant mice lacking the first exon of the micro -opioid (MOP) receptor. 2 In wild-type mice, the kappa-(KOP) agonist U-69593 (300 nM) and the mixed micro /delta-opioid agonist met-enkephalin (10 micro M), but not the delta-(DOP) agonist deltorphin (300 nM), reduced the amplitude of evoked GABA(A)-mediated inhibitory postsynaptic currents (IPSCs). Met-enkephalin and U-69593 also reduced the rate of spontaneous miniature IPSCs, but had no effect on their amplitude and kinetics. In micro -receptor-deleted mice, only U-69593 (300 nM) reduced the amplitude of evoked IPSCs. 3 In wild-type mice, the MOP agonist DAMGO (3 micro M) produced an outward current in 76% of the neurons. Deltorphin and U-69593 produced outward currents in 24 and 32% of the neurons, respectively. In micro -receptor-deleted mice, deltorphin and U-69593 produced similar outward currents in 32 and 27% of the neurons, respectively, while DAMGO was without effect. All neurons in both the wild-type and micro -receptor-deleted mice responded with similar outward currents to either the GABA(B) receptor agonist baclofen (10 micro M), or the opioid-like receptor ORL1 (NOP) agonist nociceptin (300 nM). 4 The DAMGO-, deltorphin-, U-69593-, baclofen- and nociceptin-induced currents displayed inward rectification and reversed polarity at -109 to -116 mV. 5 These findings indicate that micro -, delta- and kappa-opioid receptor activation has complex pre- and postsynaptic actions within the mouse PAG. This differs to the rat PAG where only micro -opioid receptor actions have been observed.

The firing of neurones in spinal segments adjacent to a contusive T13 spinal cord injury was characterised in anaesthetised rats. Three groups of rats were examined: (1) allodynic spinally injured, (2) non-allodynic spinally injured and (3) normal, uninjured. Spinal cord field potentials evoked by electrical dorsal root stimulation and the responses of 207 dorsal horn neurones to mechanical stimuli applied to the skin were studied. Within the lesioned spinal segment few active neurones were encountered and field potentials were absent. Depolarising field potentials recorded rostral to the lesion were reduced in both allodynic and non-allodynic animals compared to uninjured controls, while those recorded in caudal segments were enhanced in allodynic animals. Neuronal recordings revealed that allodynia was associated with exaggerated responses, including afterdischarges, to innocuous and noxious mechanical stimuli in a proportion of wide dynamic range, but not low threshold, neurones. These changes were observed both rostral and caudal to the site of injury. The results suggest that an increased responsiveness of some dorsal horn neurones in segments neighbouring a contusive spinal cord injury may contribute to the expression of mechanical allodynia. It is proposed that a relative lack of inhibition underlies altered cell responses.

The healing of tissue involves a wide range of molecular, cellular, and physiological events that are coordinated in a temporally specific manner. The cellular transcription factor early growth response factor 1 (Egr-1) is expressed minutes after acute injury and serves to stimulate the production of a class of growth factors whose role is to promote tissue repair. We have studied the effects of Egr-1 expression at the site of dermal wounding in rodents. We find that Egr-1 promotes angiogenesis in vitro and in vivo, increases collagen production, and accelerates wound closure. These results show that Egr-1 gene therapy accelerates the normal healing process and raises the potential use of this therapeutic transcription factor for any aspect of tissue repair.

1 The cardioprotective effect of N-[(1S, trans)-2-hydroxycyclopentyl]adenosine (GR79236), an adenosine A1 receptor agonist, was compared with that produced by ischaemic preconditioning in an anaesthetized rabbit model of myocardial ischaemia and reperfusion. In addition, we examined the effect of different body core temperatures on GR79236- or ischaemic preconditioning-induced cardioprotection when administered prior to ischaemia, and on cardioprotection induced by GR79236 administered 10 min prior to the onset of reperfusion. 2 When rabbits were subjected to 30 min occlusion of the left coronary artery, followed by 2 h reperfusion, GR79236 (3 x 10(-8) mol kg-1 i.v. (10.5 microg kg-1 i.v.)) or ischaemic preconditioning (5 min ischaemia followed by 5 min reperfusion), administered or applied 10 min prior to the occlusion, significantly limited the development of infarction. The cardioprotective effect of ischaemic preconditioning was significantly greater than that seen after administration of GR79236. Pre-treatment with the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 3.3 x 10(-6) mol kg-1 (1 mg kg-1 i.v.)), prevented the cardioprotective effect of GR79236, but not that of ischaemic preconditioning. 3 Maintaining body core temperature at 38.5 degrees C rather than at 37.0 degrees C did not influence infarct size in control groups of rabbits, but reduced the cardioprotective effect of GR79236 when administered 10 min prior to occlusion or 10 min prior to the onset of reperfusion. The cardioprotective effect of ischaemic preconditioning was not temperature-dependent. 4 In conclusion, myocardial protection conferred by GR79236 in anaesthetized rabbits is mediated via adenosine A1 receptors. Myocardial protection can be conferred when GR79236 is administered before the onset of ischaemia or reperfusion, and is reduced when body core temperature is maintained at 38.5 degrees C rather than at 37.0 degrees C. In contrast, myocardial protection conferred by ischaemic preconditioning is not reduced by adenosine A1 receptor blockade, or by maintaining body core temperature at 38.5 degrees C rather than at 37.0 degrees C. These findings point to distinct differences in the mechanisms of induction of myocardial protection by adenosine A1 receptor agonist and ischaemic preconditioning. They also highlight the need for careful control of body core temperature when investigating the phenomenon of cardioprotection.

The cardioprotective effects of the selective adenosine A1-receptor agonist, GR79236 (N-[(1S, trans)-2-hydroxycyclopentyl]adenosine), were examined in a porcine model of myocardial ischaemia-reperfusion injury. When pigs were subjected to a 50-min coronary artery occlusion followed by 3-h reperfusion, GR79236 (10 nmol/kg, i.v.) significantly reduced infarct size whether given 10 min before the onset of ischaemia or reperfusion. This effect was independent of the bradycardia induced by GR79236, as it was also observed in animals in which heart rate was maintained by electrical pacing. However, GR79236 administered 10 min after reperfusion did not reduce infarct size. GR79236 had no effect on the incidence or outcome of ventricular dysrhythmias in this pig model of infarction. Similarly, ischaemic preconditioning (IPC, 2 x 10-min ischaemia and 10-min reperfusion) significantly reduced infarct size. The selective adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 3.3 micromol/kg, i.v.), abolished the haemodynamic and cardioprotective effects of GR79236 and the cardioprotective effects of IPC in anaesthetised pigs. In conclusion, GR79236 exerted a marked cardioprotective effect in a porcine model of myocardial ischaemia-reperfusion injury, provided that it was administered before reperfusion. This suggests that GR79236 may have clinical utility in the treatment of various aspects of ischaemic heart disease.

The cardiovascular profile of the angiotensin AT1 receptor antagonist, GR138950, and the influence of potential compensatory homeostatic mechanisms on this profile, were investigated in renal artery ligated hypertensive (RALH) rats. GR138950 caused a marked reduction in blood pressure associated with immediate tachycardia in conscious RALH rats. The antihypertensive action of GR138950 appeared biphasic; an immediate fall in blood pressure, which plateaued within 1 h, and which was followed by a further slow decline that reached maximum between 5-7 h after administration. The tachycardia caused by GR138950 was attenuated by atenolol and was abolished by combined pretreatment with atenolol and atropine methyl nitrate. However, the antihypertensive profile of GR138950 was unchanged by these pretreatments. The resting blood pressure and the antihypertensive effect of GR138950, in RALH rats, were unaffected by the vasopressin V1 receptor antagonist, [beta-mercapto-beta,beta-cyclopentamethylene propionyl(1)-O-Me-Tyr2,Arg8]-vasopressin. Thus, vasopressinergic mechanisms are not involved in either maintaining blood pressure in RALH rats, or in compensating for the fall in blood pressure caused by GR138950. In anaesthetized RALH rats, GR138950 caused a marked fall in blood pressure that was accompanied by an increase in heart rate along with sustained increases in renal and splanchnic sympathetic nerve activity. In summary, the biphasic fall in blood pressure evoked by GR138950 in RALH rats can not be explained on the basis of changes in autonomic control of the heart, alteration of vasopressin-mediated vasoconstrictor mechanisms or overall suppression of central sympathetic outflow. Rather, increased vasoconstrictor tone might serve to oppose the initial fall in blood pressure.

Brief renal ischemia-reperfusion is reported to precondition the myocardium; however, the underlying mechanisms are unknown. This phenomenon was, therefore, investigated using an in vivo rabbit model of acute myocardial infarction. Characterization of the mechanisms involved was performed using the nonselective adenosine receptor antagonist 8-(p-sulfophenyl)theophylline (8-SPT) and the ATP-sensitive potassium (KATP) channel blocker sodium 5-hydroxydecanoate (5-HD). Pentobarbital-anesthetized rabbits underwent a left thoracotomy and pericardiotomy. A laparotomy was then performed to expose the left renal artery. Animals were either preconditioned with a 10-min occlusion of the renal artery followed by 10 min of reperfusion or underwent a 20-min sham period of anesthesia. Subsequently, the left coronary artery was then occluded for 30 min and reperfused for 2 h. Infarct-to-risk ratio was limited from 32.7 +/- 4.0% (n = 12) in controls to 17.8 +/- 3.0% (n = 9; P = 0.002) in preconditioned hearts. Protection was abolished by 7.5 mg/kg iv 8-SPT (36.7 +/- 3.7%; n = 6) or 5 mg/kg iv 5-HD (33.1 +/- 4. 4%; n = 6) administered before preconditioning. 8-SPT (40.0 +/- 4. 4%; n = 6) or 5-HD (40.5 +/- 4.2%; n = 6) did not affect infarct-to-risk ratio in sham controls. Thus activation of both adenosine receptors and KATP channels appears to be involved in acute renal preconditioning of the myocardium.

1. The potential influences of nitric oxide (NO) and prostaglandins on the renal effects of angiotensin II (Ang II) have been investigated in the captopril-treated anaesthetized rat by examining the effect of indomethacin or the NO synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), on the renal responses obtained during infusion of Ang II directly into the renal circulation. 2. Intrarenal artery (i.r.a.) infusion of Ang II (1-30 ng kg(-1) min(-1)) elicited a dose-dependent decrease in renal vascular conductance (RVC; -38+/-3% at 30 ng kg(-1) min(-1); P < 0.01) and increase in filtration fraction (FF; +49+/-8%; P < 0.05) in the absence of any change in carotid mean arterial blood pressure (MBP). Urine output (Uv), absolute (UNaV) and fractional sodium excretion (FENa), and glomerular filtration rate (GFR) were unchanged during infusion of Ang II 1-30 ng kg(-1) min(-1) (+6+/-17%, +11+/-17%, +22+/-23%, and -5+/-9%, respectively, at 30 ng kg(-1) min(-1)). At higher doses, Ang II (100 and 300 ng kg(-1) min(-1)) induced further decreases in RVC, but with associated increases in MBP, Uv and UNaV. 3. Pretreatment with indomethacin (10 mg kg(-1) i.v.) had no significant effect on basal renal function, or on the Ang II-induced reduction in RVC (-25+/-7% vs -38+/-3% at Ang II 30 ng kg(-1) min(-1)). In the presence of indomethacin, Ang II tended to cause a dose-dependent decrease in GFR (-38+/-10% at 30 ng kg(-1) min(-1)); however, this effect was not statistically significant (P=0.078) when evaluated over the dose range of 1-30 ng kg(-1) min(-1), and was not accompanied by any significant changes in Uv, UNaV or FENa (-21+/-12%, -18+/-16% and +36+/-38%, respectively). 4. Pretreatment with L-NAME (10 microg kg(-1) min(-1) i.v.) tended to reduce basal RVC (control -11.8+/-1.4, +L-NAME -7.9+/-1.8 ml min(-1) mmHg(-1) x 10(-2)), and significantly increased basal FF (control +15.9+/-0.8, +L-NAME +31.0+/-3.7%). In the presence of L-NAME, renal vasoconstrictor responses to Ang II were not significantly modified (-38+/-3% vs -35+/-13% at 30 ng kg(-1) min(-1)), but Ang II now induced dose-dependent decreases in GFR, Uv and UNaV (-51+/-11%, -41+/-14% and -31+/-17%, respectively, at an infusion rate of Ang II, 30 ng kg(-1) min(-1)). When evaluated over the range of 1-30 ng kg(-1) min(-1), the effect of Ang II on GFR and Uv were statistically significant (P < 0.05), but on UNaV did not quite achieve statistical significance (P=0.066). However, there was no associated change in FENa observed, suggesting a non-tubular site of interaction between Ang II and NO. 5. In contrast to its effects after pretreatment with L-NAME alone, Ang II (1-30 ng kg(-1) min(-1)) failed to reduce renal vascular conductance in rats pretreated with the combination of L-NAME and the selective angiotensin AT1 receptor antagonist, GR117289 (1 mg kg(-1) i.v.). This suggests that the renal vascular effects of Ang II are mediated through AT1 receptors. Over the same dose range, Ang II also failed to significantly reduce GFR or Uv. 6. In conclusion, the renal haemodynamic effects of Ang II in the rat kidney appear to be modulated by cyclooxygenase-derived prostaglandins and NO. The precise site(s) of such an interaction cannot be determined from the present data, but the data suggest complex interactions at the level of the glomerulus.

We examined the effects of puff application of endothelin (ET)-1 on the induction of long-term potentiation (LTP) and heterosynaptic long-term depression (LTD) in hippocampal CA1 slices. ET-1 applied 2 min prior to tetanus blocked the induction of LTP, but facilitated the induction of heterosynaptic LTD. These ET-1 effects on synaptic plasticity were dose-dependent, and not due to a generalized depression of baseline responses. ET-1 did not alter NMDA receptor-mediated responses. These data provide the first evidence that endothelin modulates activity-dependent synaptic plasticity, and the potency of these effects suggests that endogenous ET-1 may play an important role in regulating memory storage processes.

1. The effect of systemic administration of the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME) on the antihypertensive effects of the angiotensin AT1 receptor antagonist, GR138950, the angiotensin-converting enzyme (ACE) inhibitor, enalapril, or hydralazine has been evaluated in unrestrained, conscious renal artery ligated hypertensive (RALH) rats. The effect of the phosphodiesterase type V inhibitor, zaprinast on the antihypertensive effect of GR138950 in RALH rats was also examined. The effect of GR138950 on blood pressure, and plasma and urine cyclic GMP levels was compared to that of zaprinast in conscious RALH rats. 2. GR138950, enalapril or hydralazine caused marked reductions in blood pressure associated with immediate tachycardia in conscious RALH rats. L-NAME pretreatment attenuated the antihypertensive effects of GR138950 or enalapril but not that of hydralazine in conscious RALH rats. The initial tachycardia caused by GR138950 or enalapril but not hydralazine was attenuated by L-NAME pretreatment. L-NAME alone caused a transient (20 min) pressor response and a prolonged (6 h) bradycardia in conscious RALH rats. 3. Pretreatment with indomethacin did not affect the cardiovascular effect of GR138950 in conscious RALH rats. Indomethacin alone did not significantly change basal blood pressure or heart rate in RALH rats. 4. Zaprinast pretreatment did not affect the antihypertensive effect of GR138950 in conscious RALH rats but potentiated the depressor response to sodium nitroprusside. Zaprinast alone caused a small reduction in basal blood pressure but did not change basal heart rate in RALH rats. 5. The antihypertensive effect of GR138950 was not associated with an increase in plasma or urine cyclic GMP levels in conscious RALH rats, whereas zaprinast caused a small fall in blood pressure associated with increases in plasma and urine cyclic GMP. 6. The ability of L-NAME to inhibit the antihypertensive action of GR138950 or enalapril suggests that these agents release nitric oxide (NO) and/or enhance the cardiovascular effects of NO as part of their mechanism of action. However, the inability of zaprinast to potentiate the antihypertensive effects of GR138950 and the finding that GR138950 did not increase urine and plasma cyclic GMP levels are not consistent with this view. Attenuation of the response to GR138950 or enalapril, but not hydralazine, suggests a selective interaction between L-NAME and inhibitors of the renin-angiotensin system, although the nature of this interaction is unknown.

1. The antagonist activity of the angiotensin AT1 receptor antagonist, GR138950, and its haemodynamic effects have been investigated in beagle dogs. 2. In four anaesthetized dogs, GR138950 (0.1, 1 and 10 mg kg-1 i.v.), displaced dose-response curves to angiotensin II (AngII) rightwards, in a parallel manner; GR138950, at 1 mg kg-1 i.v., produced a 15-fold displacement of the AngII dose-response curve. In four conscious dogs, GR138950 (1 mg kg-1 i.v. or p.o.) antagonized AngII, and produced rightward and parallel displacements of the AngII dose-pressor response curves. Maximum displacements of approximately 33- and 16-fold occurred at 1 h after intravenous and 5 h after oral administration, respectively. The inhibitory effect of GR138950 was long lasting; AngII dose-pressor response curves were still displaced by 10-fold from control values, 24 h after intravenous or oral administration of GR138950. 3. In comparison with its vehicle, GR138950 (0.1-10 mg kg-1) caused a significant decrease in resting blood pressure as a consequence of a significant decrease in total peripheral resistance in anaesthetized dogs. Effects on cardiac output, stroke volume and heart rate were not significantly different between GR138950- or vehicle-treated dogs. 4. Compared with vehicle, GR138950 administration did not significantly affect blood flow to the mesenteric and femoral vascular beds but did significantly increase blood flow to the renal vascular bed. Vascular resistance of the femoral bed was unaffected but that of the mesenteric and renal vascular beds was significantly reduced by GR138950, compared with the changes produced by vehicle treatment. 5. Compared with vehicle, left ventricular end diastolic pressure was significantly reduced by GR138950, but GR138950 had no significant effect on indices of cardiac contractility (+dP/dtmax and +dP/dt@40) or cardiac relaxation during early diastole (-dP/dt). 6. In conclusion, GR138950 acts as a competitive, surmountable antagonist at vascular angiotensin II receptors in beagle dogs. GR138950 reduced arterial blood pressure by reducing total peripheral resistance, attributable partly to reduction in resistance in the mesenteric and renal vascular beds. GR138950 reduced left ventricular end diastolic pressure whilst having no direct effect on cardiac contractility or relaxation.

1. The angiotensin AT1 receptor antagonist, GR138950, produces a long-lasting antihypertensive effect in conscious renal artery ligated hypertensive (RALH) rats but this effect does not correlate temporally with its antagonist profile against angiotensin II (AII). In the present experiments we have compared the inhibitory profiles of GR138950 and enalapril, against angiotensin I (AI), with their respective antihypertensive activities. 2. GR138950 (1 mg kg-1, i.a.) and enalapril (3 mg kg-1, i.a.) reduced blood pressure in RALH rats to a similar degree. Maximum reductions in blood pressure occurred approximately 5-24 h and 3-5 h after administration, respectively. The antihypertensive effect of GR138950 lasted for 24-48 h. However, the effect of enalapril lasted for only 5-24 h. 3. In conscious normotensive rats, inhibition of AI-induced pressor responses was maximal 1 h after systemic administration of GR138950 and enalapril. Dose-response curves to AI were displaced to the right, in a parallel manner, 1406 and 102 fold by GR138950 (1 mg kg-1, i.a.) and enalapril (3 mg kg-1 i.a.), respectively. The inhibitory effect of enalapril lasted for < 24 h whereas that of GR138950 lasted for up to 48 h. 4. Contractile responses to AI were extensively inhibited in aortae removed from either RALH rats or normotensive rats, 1 and 5 h after administration of GR138950 (1 mg kg-1, i.a.). Responses were still significantly reduced 24 h after administration but had returned to control levels after 48 h. Enalapril pretreatment (3 mg kg-1, i.a.) did not inhibit contractile responses to AI in aortae isolated from normotensive rats at any time point. 5. These experiments confirm that GR138950 is an effective and long-lasting antihypertensive agent. GR138950 was a more potent and longer lasting antagonist against AI than has previously been found against AII, and the duration of its antihypertensive activity coincides better with its blockade of responses to AI. Blockade of the effects of AII generated locally within the vascular wall might play an important role in the antihypertensive profile of GR138950.

This paper describes the renal pharmacology of the novel, specific, non-peptide angiotensin AT1 receptor antagonist, GR138950 (1-[[3-bromo-2-[2-[[(trifluoromethyl) sulphonyl] amino] phenyl]-5-benzofuranyl] methyl]-4-cyclopropyl-2-ethyl-1H-imidazole-5- carboxamide). When administered to anaesthetised salt-replete dogs, GR138950 caused renal vasodilatation and significant increases in sodium and urine excretion. No change in glomerular filtration rate was observed indicating that the natriuresis was a consequence of inhibition of tubular sodium reabsorption. Qualitatively similar but less marked changes in renal function were observed in response to the angiotensin converting enzyme inhibitor, captopril, although in contrast to GR138950, captopril also caused a small but significant fall in mean blood pressure. Intra-renal artery infusion of exogenous angiotensin II resulted in dose-related renal vasoconstriction and decreases in urine excretion, sodium excretion, fractional excretion of sodium and glomerular filtration rate. These renal effects of angiotensin II were all markedly antagonised by GR138950. We conclude that GR138950 is an effective antagonist of the renal haemodynamic and excretory actions of endogenous and exogenous angiotensin II.

The antagonist activity of GR138950 (1-[[3-bromo-2-[2-[[(trifluoromethyl)sulphonyl]amino]phenyl]-5- benzofuranyl]methyl]-4-cyclopropyl-2-ethyl-1H-imidazole-5-carboxamide) was investigated at angiotensin AT1 receptors and AT2 receptors in vitro and on blood pressure in conscious rats. GR138950 suppressed and displaced angiotensin II (AII) concentration-effect curves in the rabbit isolated aorta (pKb approximately 9.0-9.7) but had no effect against phenylephrine or serotonin induced-contractions. GR138950 competed with [3H]-AII for angiotensin AT1 receptors in rat liver membranes (pKi = 9.09). GR138950 had no apparent affinity for angiotensin AT2 receptors (bovine cerebellum; pKi < 6.0). GR138950 (1 mg/kg i.a. and p.o.) inhibited pressor responses to AII, but not phenylephrine, in conscious normotensive rats. Parallel displacements in AII dose-response curves occurred without any reduction in the maximum response to AII. The antagonist activity of GR138950 lasted for up to 24 h. GR138950 (> 0.03 mg/kg i.a., > 0.3 mg/kg p.o.) significantly reduced diastolic blood pressure (DBP) in renal artery ligated hypertensive rats. DBP was reduced maximally, 5 to 7 h after administration and the antihypertensive effect of GR138950 lasted for up to 48 h. Daily administration (5 days) of GR138950 to renal artery ligated hypertensive rats produced a sustained reduction in DBP. Acute administration of GR138950 (1 mg/kg i.a.) also significantly reduced DBP in spontaneously hypertensive rats but not in normotensive rats. Heart rate was little changed in renal artery ligated hypertensive rats, normotensive rats and spontaneously hypertensive rats. These experiments demonstrate that GR138950 is a potent, selective and specific angiotensin AT1 receptor antagonist that is orally active and reduces DBP in conscious hypertensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Currently available antagonists and agonists cannot distinguish between angiotensin AT1 receptor subtypes. 2. We synthesized a series of compounds selected on the basis of having the most diverse structural features with respect to losartan (DuP753), the prototype non-peptide AT1 receptor antagonist. Using a radioligand-receptor binding assay and membranes prepared from COS-M6 cells transfected with individual AT1 receptor subtypes, we determined whether any of these compounds could distinguish between the receptor subtypes. 3. The diversity of the structural features of this series of compounds was reflected by the wide range of affinities (pIC50 values) displayed towards competing with [125I]-Sar1Ile8 angiotensin II for binding to the AT1 receptors. 4. Direct comparisons of the pIC50 values of individual compounds for rat AT1A, AT1B and human AT1 receptors revealed only minor differences. 5. It is concluded that compounds based structurally on losartan are unlikely to distinguish between these receptors.

1. The effect of GR117289, an angiotensin AT1 receptor antagonist, on diastolic blood pressure (DBP) was determined in angiotensin-dependent and angiotensin-independent models of hypertension in rats. In addition, the antagonist profile of GR117289 at angiotensin AT1 receptors was determined in conscious renal hypertensive rats and conscious normotensive rats, dogs and marmosets. 2. Intra-arterial and oral administration of GR117289 (0.3-3 mg kg-1, i.a.; 1-10 mg kg-1, p.o.) to 6-day left renal artery ligated hypertensive (RALH) rats (DBP > 140 mmHg) produced significant, dose-related reductions in DBP with little apparent effect on heart rate (< 15%). The antihypertensive effect of GR117289 developed progressively over several hours and with some doses persisted for 24-48 h after administration. 3. Administration of GR117289 (1 mg kg-1, i.a.) on 5 consecutive days to RALH rats reduced DBP on each day. The antihypertensive effect of GR117289 was not cumulative as DBP had almost returned to base-line values, 24 h after administration of each dose. 4. A dose of GR117289 (3 mg kg-1, i.a.), which produced a substantial reduction in DBP (about 70 mmHg) in RALH rats, was administered to rats in which blood pressure was elevated either by unilateral renal artery clipping, sustained infusion of angiotensin II (AII), DOCA-salt administration or genetic inbreeding. GR117289 reduced DBP in rats in which the renin-angiotensin system was activated by renal artery clipping or AII infusion but had little effect in normotensive rats, DOCA-salt rats and SHR. 5. Systemic administration of All to RALH rats and to normotensive rats, dogs and marmosets elicited reproducible pressor responses in all species. Systemic or oral administration of GR1 17289 (3 mg kg-1)inhibited the pressor responses produced by All, resulting in parallel, rightward displacements of All dose-response curves.6. Maximal displacements of All dose-response curves occurred 1 h and 1-7 h after systemic and oral administration, respectively. GR1 17289 produced a 32-246 fold displacement after systemic administration and a 4-12 fold displacement after oral administration. The effect in dogs was short lasting after systemic administration but the effect of GRI17289 lasted for up to 24 h in rats and marmosets and for up to 24 h after oral administration in all species. The antagonist activity appeared specific for angiotensin receptors as GRi17289 did not inhibit pressor responses to phenylephrine or vasopressin.7. These experiments demonstrate that GRI 17289 reduces blood pressure in conscious hypertensive rats after both systemic and oral administration, and is an effective antagonist at angiotensin AT1 receptors in conscious rats, dogs and marmosets.

The effects of the potassium (K+) channel opener, cromakalim, on skeletal muscle performance were studied in a model of acute hindlimb ischaemia in the anaesthetized rat. Twitch contractions to direct electrical stimulation of the extensor digitorum and tibialis anterior skeletal muscles were recorded following administration of cromakalim (10-100 micrograms kg-1 i.v.) under normal and reduced whole limb blood flow. With normal blood flow, twitch responses (0.5 and 1 Hz) of the hindlimb skeletal muscles were sustained for > 30 min. Controlled adjustment of the perfusion pressure in the contralateral hindlimb to 45, 30 or 0 mm Hg by partial or total occlusion of the abdominal aorta produced a pressure-related fall in flow to the working hindlimb, and a corresponding increase in the rate of muscle fatigue. Cromakalim (10-100 micrograms kg-1 i.v.) produced a dose-dependent reduction in mean carotid arterial blood pressure, femoral arterial pressure and hindlimb vascular resistance together with an increase in iliac artery blood flow and heart rate, but did not attenuate skeletal muscle fatigue under the different conditions of muscle work and ischaemia employed. A similar profile was observed with levcromakalim (15 micrograms kg-1 i.v.), the active enantiomer of cromakalim. These results demonstrate that in the direct muscle-stimulated hindlimb of the anaesthetized rat, the K+ channel opener cromakalim does not prevent acute ischaemia-induced skeletal muscle fatigue. The previous observation that K+ channel openers improve nutritive blood flow in a chronic model of rat hindlimb ischaemia is not reflected by an improvement in muscle function in the present study.

1. Experiments were performed using the selective AT1 receptor antagonist, GR117289, and the selective AT2 receptor antagonist, PD123177, to assess the relative importance of AT1 versus AT2 receptors in mediating the renal effects of angiotensin II (AII) in vivo, in salt-replete pentobarbitone-anaesthetized dogs. 2. The AT1 receptor antagonist, GR117289 (0.5 mg kg-1 + 1 microgram kg-1 min-1, i.v.), caused renal vasodilatation, characterized by a mean increase of 21 +/- 5% in renal blood flow, 45 min post-dose. GR117289 also caused a fall in mean blood pressure (12 +/- 4%), but despite this, sodium and urine excretion were not reduced. Indeed, there was a tendency for urine output and sodium excretion to increase, although the changes were not statistically significant. GR117289 caused a reduction in plasma aldosterone levels (-35 +/- 16%) 45 min post-dose, despite increasing plasma renin activity (+ 173 +/- 42%). In contrast to GR117289, the AT2 receptor antagonist, PD123177 (20 micrograms kg-1 min-1 intra-renal artery; i.r.a.) caused no significant change in blood pressure, renal blood flow, or sodium and urine excretion, indicating that the renal effects of endogenous AII in these salt-replete animals are mediated predominantly by AT1 receptors. 3. Intra-renal artery infusion of AII (1-300 ng kg-1 min-1) caused dose-related renal vasoconstriction, and decreases in urine output, sodium excretion, fractional excretion of sodium, and glomerular filtration rate (GFR). The AT1 receptor antagonist, GRI 17289 (0.5 mg kg-1 + 1 microg kg-1 min-1, i.v.)antagonized these renal effects of AII, causing 15-38 fold rightward displacements of mean dose response curves for these parameters. In contrast, PD123177 (20 microg kg-1 min-1, i.r.a.) failed to antagonize the renal haemodynamic and excretory effects of lower doses of All (1-10 ng kg-1 min-1,i.r.a.). However, at higher doses of AII (30-300 ng kg-l min-1, i.r.a.), while PD123177 still failed to antagonize the effects of the peptide on urine output, sodium excretion and GFR, it did cause a small,but significant, degree of inhibition of All-induced renal vasoconstriction. In addition, at a higher dose(50 microg kg-1 min-1, i.r.a.), PD123177 caused a greater degree of antagonism of AII-induced renal vasoconstriction, while renal excretory responses to AII remained unaffected.4. This study shows that the renal haemodynamic and excretory effects of AII in salt-replete anaesthetized dogs are mainly mediated by angiotensin AT1 receptors. However, the inhibitory effect of PD123177 on renal vasoconstrictor responses to high doses of AII, raises the possibility that functionally important AT2 receptors are present in the canine renal vasculature.

1. The role played by the endothelium-derived relaxing factor (EDRF), nitric oxide (NO) in the regulation of blood flow to the skeletal muscle vasculature of the dog skinned hindlimb has been determined by examining the effects of the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) upon (i) basal iliac artery blood flow, (ii) vasodilator responses to endothelium-dependent and -independent agonists and (iii) reactive hyperaemic responses to arterial occlusion. 2. L-NAME (0.1-3 mg min-1) infused directly into the iliac artery dose-dependently reduced basal iliac artery blood flow by a maximum of 48.6 +/- 6.9% (n = 4) and also increased mean systemic arterial blood pressure by 25.6 +/- 5.0 mmHg (n = 4) (at 3 mg min-1 L-NAME). 3. Over the same dose range, L-NAME also inhibited the peak vasodilator responses to intra-arterially administered, submaximal bolus doses of the endothelium-dependent agonists, bradykinin (3-300 ng) and acetylcholine (30-300 ng) by approximately 40%. In contrast, peak vasodilator responses to the endothelium-independent agonists, sodium azide (3-30 micrograms) and adenosine (0.3-1 mg), and peak reactive hyperaemic responses to arterial occlusion (60 s) were largely unaffected by L-NAME. 4. The dose-related effects of L-NAME on basal iliac artery blood flow, mean systemic arterial blood pressure and endothelium-dependent vasodilator responses were significantly attenuated by pretreatment with L-arginine (100 mg min-1) followed by co-infusion of L-arginine (100 mg min-1) with L-NAME. 5. In conclusion, these data suggest that NO plays some role in regulating basal blood flow and in mediating the vasodilator responses to the endothelium-dependent agonists bradykinin and acetylcholine in the skeletal muscle vasculature of the dog hindlimb. The substantial component (~60%) of the peak vasodilator responses to bradykinin and acetylcholine, unaffected by L-NAME, may be independent of NO, or be mediated by an alternative EDRF-dependent but L-NAME-insensitive mechanism.

1. This paper describes the effects of GR117289 (1-[[3-bromo-2-[2-(1H-tetrazol-5-yl)phenyl]-5-benzo-furanyl]methyl ]-2-butyl-4-chloro-1H-imidazole-5-carboxylic acid) at angiotensin receptors and binding sites in rabbit aorta, rat liver and bovine cerebellum preparations in vitro. 2. In rabbit isolated aortic strips, GR117289 (0.3, 1 and 3 nM) caused a concentration-related, insurmountable suppression of the concentration-response curve to angiotensin II (AII). When the contact time was increased, a greater degree of antagonism of AII was observed, suggesting that GR117289 is slow to reach equilibrium. A pKB of 9.8 +/- 0.1 was calculated for GR117289 after 3 h incubation. GR117289 (1 microM) did not affect contractile responses to phenylephrine or 5-hydroxytryptamine (5-HT) in the rabbit aorta. 3. GR117289 (1 nM) alone caused a marked suppression and a slight rightward displacement of the AII concentration-response curve. Co-incubation with the competitive, surmountable AT1 receptor antagonist, losartan (10 nM, 100 nM and 1 microM), resulted in a concentration-related upward and rightward displacement of the concentration-response curve to subsequently administered AII. In separate experiments in which preparations were pre-incubated with GR117289 (1 nM), subsequent addition of losartan (1 microM) for 2, 15 or 45 min caused a further, but similar, rightward displacement of the concentration-response curve to subsequently administered AII with a time-dependent increase in the maximum response.4. Suppression of All-induced contractile responses, caused by superfusion with GRI17289 (0.3, 1 or 3 nM) was not reversed by continuously washing the tissues for 3 h; in fact, the potency of GRI 17289 was slightly enhanced after this period.5. In rat liver membranes, GRI17289 was a potent competitor with [3H]-AII for AT, binding sites(pKi = 8.7 +/- 0.1) but in bovine cerebellum membranes, it was a very weak competitor for AT2 binding sites (pKi<6). Pre-incubation of rat liver membranes with GRI17289 had little effect on its affinity(pKi = 9.1 +/- 0.21), but increasing the concentration of bovine serum albumen in the assay buffer from 0.001% to 0.1% w/v decreased affinity (pKi= 7.5 +/- 0.1).6. In saturation binding experiments in rat liver membranes, GRI 17289 (12 nM) increased the Kd of[3H]-AII from 0.28 +/- 0.06 nM to 0.37 +/- 0.02 nM, and decreased Bm. from 10.0 +/- 0.1 to 5.6 +/-0.3 fmol mg' tissue. In other experiments, GR1 17289 (1 jIM) did not alter the rate of dissociation of[3H]-AII from AT1 binding sites, following addition of excess unlabelled All.7. In rabbit aorta vascular smooth muscle membranes, GR1 17289 competed with ['25I]-Sar'1le8 All for binding to AT, binding sites. In the presence of 0.1% w/v bovine serum albumen, a pIC50 of 7.6 +/- 0.1 was calculated. Under the same conditions, but with rat liver membranes, a pIC50 of 7.8 +/- 0.1 was determined.8. Taken together, these results show that GRI17289 is a potent, specific, selective and insurmountable antagonist at angiotensin AT, receptors. Its profile in the rabbit aorta is consistent with the proposalthat GRI17289 is a slowly reversible (pseudo-irreversible) antagonist at these receptors.

1. The effects of endothelium removal and of a number of pharmacological agents known to modify endothelial cell function on the contractile response of rabbit isolated basilar arteries to 5-hydroxytryptamine (5-HT) and other vasoconstrictors were studied. 2. Endothelium removal slightly reduced the contractile response to potassium chloride (40 mM) but markedly augmented and potentiated contractions to 5-HT (1 nM-10 microM). 3. L-NG-nitro-arginine (L-NOARG, 1-30 microM), an inhibitor of nitric oxide formation in vascular endothelial cells, evoked endothelium-dependent contraction, and augmented and potentiated contractions to 5-HT in endothelium-intact but not endothelium-denuded tissues. Prior incubation with L-arginine (1 mM), but not D-arginine (1 mM), abolished these effects of L-NOARG (1 microM). L-NOARG (30 microM) also augmented contractions of endothelium-intact tissues to noradrenaline, prostaglandin F2 alpha, and to a lesser degree endothelin-1. 4. Neither glibenclamide (3 microM) nor N-ethylmaleimide (1 microM), putative inhibitors of the effects of endothelium-derived hyperpolarizing factor (EDHF) and of agonist-stimulated endothelium-derived relaxing factor (EDRF) release respectively, had any effect on either resting tension or the contractile response to 5-HT. In some tissues indomethacin (3 microM), a cyclo-oxygenase inhibitor, produced a small contraction and augmented the contractile response to 5-HT, but in most cases indomethacin was without effect. 5. In endothelium-intact tissues precontracted with uridine 5'-triphosphate (UTP; 100 microM), 5-HT did not evoke relaxation but rather caused further contraction. Under the same conditions acetylcholine (0.01-10 microM) evoked endothelium-dependent relaxation.6. These data demonstrate that the endothelium profoundly depresses contractions of rabbit isolated basilar artery to 5-HT, and that this phenomenon can be fully accounted for by the release of an L-NOARG-sensitive relaxing factor. Neither glibenclamide-sensitive EDHF nor cyclo-oxygenase products plays a major role. As we could find no evidence that 5-HT stimulates the production of EDRF per se, and L-NOARG caused endothelium-dependent contraction and augmented contractions to other vasoconstrictor agents, it seems likely that a basal release of EDRF underlies this phenomenon.

Experiments were performed in anesthetized cats to compare the characteristics of angiotensin receptors in the renal and mesenteric vascular beds. Injection of either angiotensin II (Ang II; 0.3-30 ng) or angiotensin III (Ang III; 0.3-30 ng) directly into the superior mesenteric or renal artery caused dose-related, reproducible reductions in mesenteric and renal blood flow, respectively. Ang II and Ang III were equipotent as vasoconstrictors in both vascular beds. The peptide angiotensin receptor antagonists saralasin and Ile7-Ang III (1 microgram/kg/min intravenously, i.v.) and the nonpeptide angiotensin receptor antagonist DuP 753 (3 mg/kg plus 20 micrograms/kg/min i.v.) caused a rightward displacement of dose-response curves to Ang II or Ang III in both the mesenteric and renal vasculature. Vasoconstrictor responses to Ang II or Ang III in either vascular bed were blocked to similar extents by each antagonist. In separate cats, the dose of the antagonists required to cause a 10-fold rightward displacement of the Ang II dose-response curve (DR10) in both vascular beds was determined. The DR10 values indicated that the potency of each antagonist was similar in the renal and mesenteric vascular beds. These results provide no evidence to suggest that angiotensin receptors mediating vasoconstriction in the renal and mesenteric vasculature have different characteristics.

In vitro differentiation of the mouse neuroblastoma-rat glioma hybrid cell line, NG-108-15, with dimethyl sulphoxide (1.5%) and low serum (0.5%), produced a marked increase in the number of angiotensin II receptors, from a level at the limit of sensitivity using labelled angiotensin II with a high specific activity ([125I]angiotensin II), in undifferentiated cells, to a Bmax of 1077 (1070-1268) fmol/mg in 5-day-differentiated cells. The affinity (Kd) of radiolabelled angiotensin II for the receptors in differentiated cells was 8.1 (7.5-10) nM. The recently available selective non-peptide antagonists, DuP 753 and PD 123177 and the peptide analogues of angiotensin II, CGP 42112A and p-aminophenylalanine6 angiotensin II, were used to characterize the angiotensin II receptors by competing for 125I-[Sar1-Ile8]angiotensin II binding to membranes prepared from undifferentiated and differentiated cells. The predominant angiotensin II receptor subtype expressed by undifferentiated cells was AT1 and after differentiation AT2. This change in receptor expression was evident 2 days after initiation of differentiation, was maximal at 4-5 days and was stable for at least 8 days. Administration of angiotensin II induced intracellular Ca2+ mobilization in both undifferentiated and differentiated cells. This was antagonised by the selective AT1 antagonist, DuP 753, indicating an action at the AT1 receptor subtype in both undifferentiated and differentiated cells. The selective AT2 antagonist, PD 123177 was without effect on the angiotensin II induced increase in intracellular Ca2+. This effect of DuP 753 on Ca2+ was specific for angiotensin II since the drug had no effect on bradykinin induced increases in intracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Incubation of cultured rat glomerular mesangial cells with dopamine caused an increase in cyclic AMP formation in a concentration-dependent manner (Ka apparent 2.2 microM). The selective dopamine D1 receptor agonists, fenoldopam, SKF 38393 and (+/-)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (6,7-ADTN) also produced concentration-dependent increases in cyclic AMP with mean Ka apparent values of 0.04 microM, 0.02 microM and 1.02 microM, respectively. Although fenoldopam and SKF 38393 were more potent than dopamine, they were partial agonists with efficacies, relative to dopamine, of approximately 60 and 35%, respectively. The dopamine analogue, 6,7-ADTN, in contrast, behaved as a full agonist. Dopamine-stimulated cAMP formation was inhibited in a concentration-dependent manner by the D1-selective antagonist, SCH 23390, with a Ki of 0.06 nM. In contrast, the D2-selective antagonist, domperidone, was four orders of magnitude less potent than SCH 23390, having a Ki of 2072 nM. In addition, SCH 23388, the stereoisomer of SCH 23390, was observed to be two orders of magnitude less potent than SCH 23390, indicating the stereoselective nature of the receptor. The potency series for the selective agonists and antagonists is the same as that described, using identical experimental conditions, for the D1 receptor expressed by a cell line of central origin confirming that the peripheral DA1 and the central D1 dopamine receptor are pharmacologically similar.

Experiments were performed in anaesthetized dogs to characterize the renal effects of the selective dopamine DA1-receptor agonist, fenoldopam. Intrarenal artery infusion of fenoldopam (0.01-10 micrograms/kg per min) caused dose-related renal vasodilation. At low doses (0.01-0.3 micrograms/kg per min), renal vasodilation occurred without concomitant falls in blood pressure but was accompanied by increased urine output. This diuresis was most probably a result of reduced tubular reabsorption since glomerular filtration rate was not increased. Both fenoldopam-induced renal vasodilation and diuresis were blocked to a similar extent by the selective dopamine DA1-receptor antagonist, SCH 23390 (30 micrograms/kg, intravenously), suggesting that both effects were mediated by dopamine DA1-receptors. In the presence of the angiotensin converting enzyme inhibitor, captopril (1 mg/kg, intravenously, + 20 micrograms/kg per min, intrarenal artery), fenoldopam (0.01-0.3 micrograms/kg per min) significantly increased fractional excretion of sodium, despite reducing blood pressure; neither of these effects were observed in captopril-free dogs. These observations support the view that the inhibitory effect of fenoldopam on tubular function, and its vasodepressor activity, may be opposed by angiotensin II resulting from fenoldopam-induced renin release.

We compared the abilities of verapamil and nicardipine to protect the porcine myocardium from the consequences of ischaemia-reperfusion in vivo. Infusion of verapamil (50 micrograms/kg) into the left anterior descending coronary artery LAD (i.c.a.), in 15 min immediately before ligation depressed regional contractile function, reduced infarct size by 80%, and enabled contractile function to recover partially during reperfusion. Verapamil (10 micrograms/kg i.c.a.) did not depress contractile function before ligation or permit its recovery during reperfusion, despite reducing infarct size by 80%. Lower doses of verapamil were not cardioprotective. Nicardipine (10 and 30 micrograms/kg i.c.a.) depressed contractile function before ligation but did not permit its recovery during reperfusion. Nicardipine did not reduce infarct size development. Thus, drug-induced negative inotropic activity (which presumably reflects myocardial calcium channel blockade) and cardioprotection are not linked. Verapamil can markedly reduce infarct size development at a dose that exerts no detectable negative inotropic activity. This cardioprotective effect of verapamil was greatly reduced by intravenous (i.v) pretreatment with aspirin (30 mg/kg), which alone did not alter infarct size development. Thus, the cardioprotective effect of verapamil (10 micrograms/kg i.c.a.) appears to be mediated by a cyclooxygenase product, possibly prostacyclin.

1. The ability of the putative, selective post-junctional alpha 2-adrenoceptor antagonist, SK&F 104078 to antagonize the effects of structurally-diverse agonists at pre-junctional alpha 2-adrenoceptors in the guinea-pig ileum and rat vas deferens in vitro and in the rat heart in vivo, and at post-junctional alpha 2-adrenoceptors in the rabbit ear vein in vitro, was examined. Results obtained with SK&F 104078 were compared with those obtained with yohimbine. 2. Xylazine and B-HT933 each caused a concentration-dependent inhibition of the field-stimulation-evoked twitch responses of the guinea-pig ileum and rat vas deferens. SK&F 104078 did not antagonize either agonist in the guinea-pig ileum and exerted only minimal blocking activity against xylazine in the rat vas deferens. In contrast, SK&F 104078 competitively antagonized B-HT933 in the rat vas deferens (pA2 = 6.45). Yohimbine competitively antagonized both agonists in each tissue (pA2 values ranged between 7.46 and 7.88). 3. In the pithed rat xylazine and B-HT933, injected intravenously, caused a dose-dependent reduction in the tachycardia elicited by stimulation of the cardiac preganglionic sympathetic nerves. SK&F 104078 (10 mg kg-1, i.v.) caused a 20-30 fold rightward displacement of the dose-response curve to xylazine, but did not affect responses to B-HT933. Yohimbine (1 mg kg-1, i.v.) antagonized both agonists to a similar degree. 4. In the rabbit ear vein xylazine, B-HT933, noradrenaline and UK 14304 elicted vasoconstrictor responses. Prazosin was without effect, but in contrast, SK&F 104078 was a competitive antagonist of each of the agonists (pA2 values ranged between 6.63 and 6.72). Yohimbine also competitively antagonized each of the agonists in this preparation (pA2 values ranged between 7.81 and 8.07). 5. SK&F 104078 was also a competitive antagonist (pA2 = 6.20) against noradrenaline at post-junctional alpha 1-adrenoceptors in the rabbit aorta. 6. These data show that SK&F 104078 is a competitive antagonist at post-junctional alpha l- and alpha 2-adrenoceptors. Its antagonist potency at pre-junctional alpha 2-adrenoceptors is agonist- and tissuedependent. Yohimbine does not discriminate between pre- and post-junctional alpha 2-adrenoceptors. The findings are discussed in terms of the possible existence of subclasses of OC2-adrenoceptors.

The aim of this study was to investigate whether the pentobarbitone anesthetized cat is a suitable preparation in which to characterize renal tubular dopamine receptors. Intravenous infusion of dopamine (10-100 micrograms/kg/min) resulted in a dose-related increase in mean blood pressure (MBP), urine output, sodium excretion (UNaV), and fractional sodium excretion (FENa). This diuretic effect occurred despite little change in glomerular filtration rate, suggesting that it is a consequence of decreased tubular reabsorption. Dopamine (10-100 micrograms/kg/min, i.v.) also induced marked dose-related falls in renal vascular conductance; however, renal blood flow was not significantly reduced. The increases in MBP, urine output, UNaV, and FENa induced by dopamine (10-100 micrograms/kg/min, i.v.), were unaffected by pretreatment of cats with either the selective dopamine DA1 or DA2 receptor antagonists, SCH 23390 (30 micrograms/kg, i.v.), or domperidone (100 micrograms/kg, i.v.) respectively. In contrast, pretreatment of cats with the nonselective alpha-adrenoceptor antagonist, phentolamine (1 mg/kg, i.v.) prevented dopamine-induced increases in urine output and MBP. Infusion of the selective dopamine DA1 receptor agonist fenoldopam (0.01-10 micrograms/kg/min) into the left renal artery failed to increase left renal vascular conductance, or left kidney urine output, UNaV, or FENa. In conclusion, this study provides no evidence for the involvement of renal tubular dopamine receptors in dopamine-induced diuresis in anesthetized cats. Rather, the diuretic effect appears to be linked to stimulation of alpha-adrenoceptors.

1 Spinal alpha-adrenoreceptors involved in cardiovascular control have been investigated using selective alpha-adrenoreceptor agonists and antagonists in urethane-anaesthetized rats. 2 Intrathecal injections of clonidine, alpha-methylnoradrenaline, guanfacine and M7 at the C7-T1 level reduced blood pressure and heart rate. In contrast, phenylephrine, 5-hydroxytryptamine and procaine had little or no effect. These results suggest the involvement of spinal alpha 2-adrenoreceptors. 3 The fall in blood pressure produced by clonidine appeared to be attributable to a reduction in heart rate and stroke volume. Lower body vascular resistance was unchanged. 4 The clonidine-induced bradycardia was antagonised by prazosin, WB4101, piperoxan or yohimbine. Their relative potencies suggest that alpha 1-rather then alpha 2-adrenoreceptors mediate this response. 5 piperoxan and yohimbine clearly prevented the clonidine-induced fall in blood pressure; prazosin and WB4101 also appeared to antagonise clonidine but these results were complicated by the fact that these antagonists themselves reduced blood pressure. 6 It was difficult to interpret these results simply in terms of alpha 1- or alpha 2-adrenoreceptors. Thus spinal alpha-adrenoreceptors may be different from peripheral alpha 1- or alpha 2-adrenoreceptors.

The alpha-adrenoceptor blocking potency of WB 4101 at alpha 1- and alpha 2-adrenoceptors has been investigated in pithed rats. WB 4101 was approximately 97 times more potent at antagonizing the vasopressor responses produced by the selective alpha 1-adrenoceptor agonist phenylephrine, than those produced by the selective alpha 2-adrenoceptor agonist M-7. A dose of WB 4101 (3 mg/kg) that caused extensive blockade of vascular alpha 1-adrenoceptors, but little or no blockade of vascular alpha 2-adrenoceptors, exerted no significant blockade of the presynaptic alpha 2-adrenoceptors in the rat heart. The results support the view that WB 4101 is a highly selective antagonist at alpha 1-adrenoceptors in vivo.

Prenalterol exerted agonist activity in cat, but not guinea-pig, isolated atria, which contain predominantly beta 1-adrenoceptors. Prenalterol relaxed K+ -contracted rat uterus, but not histamine-contracted cat lung strips; both contain predominantly beta 2-adrenoceptors. The effect of prenalterol in rat uterus was antagonised by the selective beta 2-adrenoceptor antagonist ICI 118551 but not by the selective beta 1-adrenoceptor antagonist atenolol. Thus the ability of prenalterol to exert beta-adrenoceptor activity is tissue-dependent, rather than beta-adrenoceptor subtype-dependent.

1 The central alpha-adrenoceptors responsible for mediating clonidine-induced sedation in rats have been characterized according to their sensitivity to alpha-adrenoceptor agonists and antagonists.2 Clonidine, injected intraperitoneally or intracerebroventricularly, caused dose-dependent sedation, both in terms of a reduction in the time that rats could remain on an accelerating rotarod and in terms of overt sedation assessed visually. Following intracerebroventricular injection, xylazine, naphazoline and methoxamine, but not phenylephrine, produced similar effects.3 The sedation caused by intraperitoneal injection of clonidine was antagonized by intracerebroventricularly injected phentolamine, yohimbine, piperoxan and tolazoline but not by labetalol, thymoxamine or prazosin.4 The relative potencies of the agonists in causing sedation and of the antagonists in inhibiting the sedative effect of clonidine clearly demonstrated that the central alpha-adrenoceptors mediating clonidine-induced sedation are the same as the peripheral presynaptic alpha(2)-adrenoceptors.5 All the alpha-adrenoceptor agonists caused hypothermia after intracerebroventricular injection, but their order of potency was different from that in producing sedation. The hypothermic effect of intraperitoneally injected clonidine was little affected by any of the antagonists administered intracerebroventricularly. No conclusions could be drawn concerning the type of receptor responsible for mediating hypothermia.

The effects of dopamine receptor and alpha-adrenoceptor agonists and antagonists on the stimulation-evoked overflow of radioactivity from strips of dog saphenous vein previously loaded with [3H]-noradrenaline have been examined alone and in combination. In the presence of neuronal and extraneuronal catecholamine uptake inhibitors, noradrenaline (0.1-1 X 10(-6)M) and dopamine (0.01-1 X 10(-6)M) both inhibited the stimulation-evoked overflow of radioactivity. Sulpiride (1 X 10(-6)M) was without effect and prazosin (1 X 10(-7)M) had little effect on stimulation-evoked overflow but yohimbine enhanced it approximately 2 fold; the effect of yohimbine was similar at concentrations of 1 X 10(-7) and 1 X 10(-6)M. Sulpiride abolished the inhibitory effect of dopamine on stimulation-evoked overflow, but was without effect against noradrenaline. When allowance was made for the effects of yohimbine, alone, on overflow, yohimbine (1 X 10(-7)M) had no effect against dopamine and minimal effects against noradrenaline. A similar result was obtained when the concentration of yohimbine was increased to 1 X 10(-6)M. Prazosin did not antagonize the effect of noradrenaline. In the absence of the uptake inhibitors, clonidine (0.01-1 X 10(-5)M) inhibited stimulation-evoked overflow of radioactivity. Yohimbine (1 X 10(-6)M) was without effect on its own and antagonized the effects of clonidine at a concentration of 0.1 X 10(-5)M, but not at 0.01 or 1.0 X 10(-5)M. These findings suggest that dopamine inhibits overflow by stimulating presynaptic dopamine receptors on the terminals of the noradrenergic nerves supplying the dog saphenous vein. The interaction between yohimbine and noradrenaline is discussed in terms of the current concepts of control of transmitter release mediated via presynaptic alpha 2-adrenoceptors.