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The effect of adrenaline, noradrenaline, and isoprenaline was studied in vitro on the spirally-cut trachea and in anaesthetized animals in vivo. In the in vitro studies, all agents caused a relaxation of the tracheal muscles, and the relative potencies were in descending order, isoprenaline (142.2), adrenaline (21.9), and noradrenaline (1.0). After pretreatment for 20 minutes with propranolol (> 0.5 μg/ml) or INPEA (> 3 μg/ml) adrenaline and noradrenaline caused a contraction of the tracheal muscles. The effect of noradrenaline was 0.44 times that of adrenaline. The constrictor effect of adrenaline and noradrenaline was antagonized or completely inhibited by the α-receptor blocking agents phenoxy-benzamine and dibenamine in a concentration of 1–5 μg/ml. Isoprenaline did not produce any contraction of the tracheal muscles after pretreatment with propranolol or INPEA. In the in vivo studies, the animals were anaesthetized with pentobarbital sodium (50 mg/kg intraperitoneally). Intravenous injections were made into the right jugular vein. Changes in tidal volume were recorded according to the method of KONZETT & ROSSLER. Adrenaline, in the doses 2–4 μg/kg, produced a weak bronchoconstriction that was enhanced after pretreatment of the animals with propranolol (2 mg/kg). The bronchoconstrictor effect of adrenaline and noradrenaline was not affected by atropine but was inhibited by phenoxybenzamine or dibenamine (1–2 mg/kg). Isoprenaline in doses up to 0.1 mg/kg did not produce any bronchoconstriction after pretreatment with propranolol. The results obtained suggest that the smooth muscles in the guinea-pig trachea and lung contain both α- and β-receptors, the β-receptor being dominant.

The study was undertaken to characterize the effects of porcine galanin (pGal) and some of its analogues on rat gastric fundus muscle strips. pGal, galantide (M15) and pGal(1-14)-[Abu8]SCY-I evoked reproducible concentration-dependent contractions in concentrations of 1-300, 3-1,000 and 100-3,000 nM, respectively, with EC50 values of 13, 70 and 187 nM. Hill's coefficient for pGal is 1.03, indicating an interaction of one pGal molecule with one receptor, fulfilling criteria of classical receptor theory. For M15 and pGal(1-14)-[Abu8]SCY-I, Hill's coefficients are significantly different from 1, namely 0.73 and 1.56, so that one drug molecule may not interact with one receptor. The stimulatory effects of pGal were not modified by dibenamine 10 microM or glybenclamide 1 or 10 microM. Diltiazem 0.1, 1 and 10 microM, papaverine 0.1, 10 microM or dibutyryl cAMP (dib cAMP) 100 and 300 microM, blocked the contraction to pGal in a concentration-dependent manner, indicating an important role for the influx of extracellular calcium ions and regulation by cAMP the pGal-evoked contraction. Diltiazem, dibutyryl cAMP and papaverine were not competitive antagonists of pGal in the stomach smooth muscle.

Adrenaline and isoproterenol elicited nearly maximal relaxation of the colon even in small doses, whereas increase in the doses caused greater relaxation in the duodenum. In the colon, these drugs prevented, to a great extent the contraction induced by acetylcholine (ACh) and serotonin but in the duodenum were totally ineffective. Dibenamine and propranolol reduced adrenaline- and isoproterenol-induced relaxation in the duodenum, though propranolol decreased the relaxation caused by isoproterenol. Atropine prevented ACh-induced contraction in both the colon and duodenum in the same way. After 2-bromolysergic acid diethylamide, duodenal contraction caused by ACh or serotonin decreased by over 70%; however, the contraction of the colon was not significantly inhibited. Methysergide had similar effects, but to a lesser degree. In calcium-free bathing fluid without addition of Na2EDTA, ACh and prostaglandin E1 elicited contraction in the colon, but not in the duodenum.

INTRODUCTION In previous papers (Markee, Sawyer and Hollinshead, 1946, 1947 a, b) we have reported the results of investigations of the mechanism by which the central nervous system exerts its control over the release of luteinizing hormone in the rabbit. Using unipolar and bipolar electrical stimulation of known voltage and current we found that direct stimulation of the hypophysis by a unipolar electrode was effective in producing ovulation in the rabbit only with relatively strong currents, which gave signs of considerable spread to the central nervous system. On the other hand, localized bipolar stimulation of the hypothalamic region with a current which was ineffective when applied by the same electrode directly to the hypophysis, produced ovulation in three out of four animals. From these results we concluded that the pathway of excitation from the hypothalamus to the anterior lobe of the hypophysis was probably not through thehypothalamico-hypophyseal nerve tract

The neuropharmacological mechanisms underlying the action of milbemycin oxime on the motility of Angiostrongylus cantonensis and Dirofilaria immitis were examined in vitro. In A. cantonensis, milbemycin oxime caused inhibitory effects at low concentrations ofor = 10(-9) g/ml, and paralysis was elicited at 10(-8) - 10(-6) g/ml. The paralysis was antagonized by picrotoxin and bicuculline but not by dibenamine. In addition, stimulatory effects were observed when the antibiotic was used at higher concentrations of 3-5 x 10(-6) g/ml, and the action was antagonized by strychnine. Both effects were also observed in the preparation contracted by eserine or pyrantel. When D. immitis was treated with milbemycin oxime at concentrations of 10(-7) and 3-5 x 10(-6) g/ml, only slight inhibitory and stimulatory effects, respectively, were observed. These effects were partially antagonized by picrotoxin and strychnine, respectively. These results suggest that the inhibitory and stimulatory actions of milbemycin oxime are caused through gabergic and cholinergic mechanisms in A. cantonensis and D. immitis.

The age changes of neural control over the function of the thyroid, adrenal cortex and testicles were examined in adult (6 months) and old (28 months) male rats. In old age, there was a weakening of adrenergic control of thyroidogenesis, alpha-adrenergic and M-cholinergic control of glucocorticoid function of the adrenal cortex and reduction of adrenergic and M-cholinergic influences in the regulation of steroidogenic function of the testicles.

The effects of irreversible alpha1-adrenoceptor antagonists, SZL-49 (an alkylating analogue of prazosin), dibenamine and benextramine on contractions to noradrenaline (NA) in longitudinal and circular muscle of human epididymal vas deferens were investigated. Competitive alpha1-adrenoceptor antagonists were also used to further characterize the alpha1-adrenoceptor subtype stimulated by NA in longitudinal and circular muscle. NA evoked concentration-dependent contractions of both muscle types (pD2; 5.4 and 5.2 respectively). The contraction of circular muscle was comparatively more sensitive than that of longitudinal muscle to pretreatment with SZL-49. In contrast, dibenamine or benextramine produced comparable effects in both muscle types. The relationship between receptor occupancy and contraction in either longitudinal or circular muscle was nonlinear, with half-maximal response requiring similar receptor occupancy (longitudinal muscle 14%, circular muscle 16%). Maximal response in both muscle types occurred with little or no receptor reserve (10%). The competitive alpha1-adrenoceptor antagonists produced dextral shifts of the dose-response curves to NA in longitudinal and circular muscle. The inhibitory potencies, estimated from the apparent pKB values were significantly different in longitudinal and circular muscle respectively for either WB 4101 (pKB, 8.6 and 9.5) or RS-17053 (pKB, 7.1 and 9.0) but not for Rec 15/2739 (pKB, 9.2 and 9.8) or HV 723 (pKB, 8.3 and 8.4). In conclusion, the potency profile of the competitive alpha1-adrenoceptor antagonists and the lack of different receptor reserves for NA in the muscle types suggest that the discriminatory effects of SZL-49 is primarily due to a predominance of the alpha1L-adrenoceptor subtype in longitudinal muscle and alpha1A-subtype in circular muscle.

In addition to the existence of complex memory (similar to the implicit nondeclarative memory of Squire), the existence of a phylogenetically old apparatus of a memory of situations (SMA) is supposed, which is to some extent comparable with the declarative memory of Squire. During actual sensory information the SMA generates a general frame and forms a general ‘mark’, indicating whether a given information has its origin inside or outside the body, and whether it is new or known. The procedure of this marking process can be explained as the time-depending arrest of a copy of the actual original information-transporting signal ‘shower’; this copy must last until the feedback from thalamocortical centers indicates the termination of the processing of the original signal showers. The arrest of the shower copies is the performance of neuronal networks of the entorhinal cortex (EC) and the gyrus dentatus (GD). The psychopathological and biochemical analyses of experimental dibenamine psychosis show a different effect of dibenamine on the noradrenaline (NA) receptors of the EC and GD, respectively: these effects are responsible for the repeated perception cycles of a single situation. N,N-Dibencylamine blocks the postsynaptic α1-receptors of the EC without influencing the β-receptors of the GD. Thus the interaction between EC and GD is changed: instead of new scenes, perceptions that have just been experienced get repeated presence and the quality of familiarity. The prolonged arrest of shower copies simultaneously blocks the entrance of new signal showers from the EC to the GD. No information-transporting signal showers can come in as long as the arrest lasts. In case of a disturbance in NA-dependent actions within the EC and the GD, the duration of arrest of information-transporting signal showers is shortened. Thus the formal frame of experience receives the quality of novelty instead of familiarity, and in addition the qualities of uncertainty, vagueness, and alienity. These very changes in perception and experience represent the basic disturbance of schizophrenia. All the symptoms of schizophrenia may be explained by this basic disturbance. The analysis of biochemical aspects turns attention to the energetic situation of NA and N-methly-d-aspartate systems. These considerations suggest a genetic background of the basic disturbance of schizophrenia: transmitter effects on membranes of neurons and possibly also on glial cells, and energy supply of these effects may be predetermined genetically. It may be assumed that the compensation of such membrane-dependent disturbances will be possible within wide areas of the neural network, except for the ‘bottleneck’ of the overlapping region of the iso- and allocortex.

The dentate gyrus neurons in the hippocampus contain a high density of both mineralocorticoid and adrenergic receptors. By in vivo extracellular recording from adrenalectomized rats we investigated the possible relationships between the two systems with regard to neuronal excitability and activity-dependent synaptic plasticity. Pretreatment with aldosterone significantly enhanced both basal neuronal excitability and tetanically evoked synaptic plasticity in adrenalectomized, but not sham-operated rats. The enhancement was blocked by spironolactone, indicating a mineralocorticoid receptor-dependent effect. The adrenomedullary hormone epinephrine also significantly enhanced synaptic plasticity via activation of beta-adrenergic receptors. Beta-adrenergic antagonist propranolol, infused directly into the dentate gyrus granule cell layer, significantly reduced the effect of aldosterone on neuronal excitability and partly canceled the aldosterone-enhanced synaptic plasticity. No effect of propranolol was found after its amygdaloid infusion. The mineralocorticoid receptor antagonist spironolactone did not affect the epinephrine-induced effects. These results indicate that the pretreated adrenal steroids interact with the catecholaminergic system in the dentate gyrus of adrenalectomized rats and that the functional beta-adrenergic pathway is involved in the mechanism of mineralocorticoid-induced cellular effects in vivo.

The determination of the affinity and efficacy of an agonist in functional (as opposed to radioligand binding) experiments is necessary to explain its potency. Using modeled dose-response data both in their ideal form and with an added average deviation as well as previously published experimental data, a variety of analytical approaches were compared which differed in goodness-of-fit, ease of handling, and range of successful application. A nonlinear curve-fitting algorithm that analyzed several dose-response curves simultaneously and fitted them to an extended version of the operational model of Black and Leff (1983) (Proc R Soc Lond B 220: 141–162) was demonstrated to be superior to the other approaches using the above criteria. However, judging from the limitations of the analytical approaches, claims of efficacy or affinity differences between agonists that are based on less than 10-fold numerical differences in the same behavioral paradigm should be viewed with skepticism. It was also found that simple inspection of dose-response curves obtained before and after administration of an insurmountable antagonist give estimates of fair accuracy under most circumstances.

1. This paper describes aspects of the functional antagonism between the responses mediated by activated alpha 1-adrenoceptors and adenosine A2 receptors in the adventitia- and endothelium-denuded aorta of the rabbit. 2. Adenosine A2 receptor agonists relaxed aortic rings pre-contracted with phenylephrine. The relaxation response was agonist concentration-dependent and saturable. The respective contractile and relaxation responses were stable, reproducible, and reversible. 3. Increasing the phenylephrine concentration caused a progressive attenuation of the action of adenosine A2 receptor agonists, consisting of a decreased maximal response and a dextral shift of the adenosine agonist concentration-response curve. This functional antagonism could be completely reversed upon removal of adenosine by either the addition of adenosine deaminase or by wash-out of the adenosine agonist from the tissue. The relaxation response to the adenosine A2 receptor partial agonists, N6-cyclohexyladenosine and R-(-)-N6-(2-phenylisopropyl)adenosine, was abolished at higher phenylephrine concentrations (e.g. 30 EC50). 4. A 1000 fold increase in the adenosine concentration was required to shift the value of the EC50 of phenylephrine six fold, while a similar increase in the value of the EC50 of adenosine could be elicited by only a 32 fold increase in the phenylephrine concentration. A 30 fold increase in the phenylephrine concentration shifted the value of the EC50 of 5'-N-ethylcarboxamidoadenosine four fold. 5. Analysis of the functional antagonism between the responses mediated by these receptors using the Black & Leff (1983) operational model of agonism allowed for the estimation of the agonist dissociation constant, KA, and the apparent efficacy, tau, for both phenylephrine and adenosine A2 receptor agonists. Increasing the concentration of phenylephrine reduced the value of tau for adenosine agonists in a concentration-dependent and saturable manner. Similarly, increasing the concentration of adenosine reduced the value of tau for phenylephrine in a concentration-dependent and saturable manner. The phenylephrine KA value obtained by the method of functional antagonism (1.9 microM) was similar to that obtained by the receptor inactivation method (2.1 microM). 6. Partial occlusion of the alpha 1-adrenoceptor by the alkylating agent, dibenamine, demonstrated that the magnitude of the adenosine A2 receptor-mediated relaxation was inversely proportional to the number of functional alpha 1-adrenoceptors. 7. It is concluded that the magnitude of functional antagonism is proportional to the stimulus elicited through either receptor. We propose that this tissue preparation and pair of receptors is a good model to study quantitative aspects of functional antagonism between activated receptors.

Scatchard analyses of 3H-prazosin binding in rat brain membranes showed biphasic curves, which identified the presence of alpha 1High- and alpha 1Low-affinity sites. The alpha 1High-affinity site was completely inhibited by 0.1 microM phenoxybenzamine. On the other hand, 3H-prazosin binding in rat spleen membranes resulted in linear curves that were identical to the binding curve for the alpha 1High-affinity site in the brain. The displacement potencies of alpha 1-adrenergic antagonists were characterized by 3H-prazosin binding to alpha 1High-affinity sites in the rat spleen and brain and alpha 1Low-affinity sites in the brain in the presence of 0.1 microM of phenoxybenzamine. The affinities of WB-4101, phenoxybenzamine, phentolamine, chlorpromazine, labetalol and nifedipine for brain alpha 1High-affinity sites were significantly higher than those in the spleen. The affinities of most ligands for alpha 1Low-affinity sites were significantly lower than those for both alpha 1High-affinity sites in the brain and spleen, but chlorethylclonidine was significantly selective for alpha 1Low-affinity sites, and bunazosin, dibenamine and 5HT had the same affinities for the alpha 1Low- and both alpha 1High-affinity sites. These results show that two alpha 1-adrenoceptor subtypes, alpha 1High- and alpha 1Low-affinity, are present in the rat brain and that a different alpha 1High-subtype, exists in the rat spleen.

5-Hydroxytryptamine (5-HT), and the selective alpha-1 agonists phenylephrine (PE) and oxymetazoline (OXY), were used to study the effects of simultaneous coactivation of the 5-HT2 and alpha-1 adrenergic receptors, respectively, on the contractile responses of isolated rat aortic rings. Dissociation constants (KA) were determined for each of the agonists at their respective receptor subtypes. The KA values for PE and OXY at the alpha-1 receptor were 316 nM and 1.82 microM, respectively, while the KA for 5-HT at the 5-HT2 receptor was 478 nM. Concentration-response curves for each agonist were analyzed by the Black and Leff operational model of pharmacological agonism to determine efficacy (tau) and slope factor values. The estimated tau for PE (16.02) was much greater than the tau for either OXY (4.15) or 5-HT (2.95), which had similar efficacies. Using a previously described drug concentration paradigm, a mutual-effect amplification of the 5-HT-induced contractile response was observed with mixtures of 5-HT and PE, whereas mixtures of OXY and 5-HT elicited a mutual-effect amplification of the observed response to OXY alone. In both cases, the theoretical concentration-response curve constructed using the Poch and Holzmann method of equiactive substitution demonstrated that mutual-effect amplification was largely the result of simple additivity of agonist effects. In addition, estimates of tau and slope factor determined from the Black and Leff equation were substituted into the Leff model of mutual-effect amplification and used to accurately predict the location of the concentration-response curves elicited by mixtures of 5-HT and PE, as well as mixtures of OXY and 5-HT. This represents the first time a mathematical model has been used to accurately predict the outcome of coactivation of the alpha-1 and 5-HT2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

5-Hydroxytryptamine (5-HT) and 2,5-dimethoxy-4-methyl-amphetamine (DOM) are potent agonists on isolated ovine uterine arteries in late pregnancy. Similar pA2 values (8.56 and 8.33, respectively) of ketanserin, tested against 5-HT and DOM, indicate that responses produced by both agonists are mediated by the 5-HT2 receptor. The contractions produced by 8-OH-DPAT and 2-methyl-5-HT were also blocked by ketanserin (10(-8) M) with the dissociation constants (KB) being 2.49 and 2.88 nM, respectively. This provides evidence that these agonists are activating 5-HT2 receptors in the ovine uterine artery. DOM was more potent than 5-HT, but had a similar efficacy to that of 5-HT. The greater affinity of DOM may explain its greater potency. The dissociation constants (KA) of 5-HT and DOM acting on 5-HT receptors were determined by analysis of concentration-response data before and after fractional inactivation of receptors with dibenamine. The mean KA values for 5-HT and DOM were 3.7 +/- 0.7 x 10(-7) and 1.8 +/- 0.3 x 10(-7) M, respectively. Assessment of receptor occupancy vs. functional response demonstrated little or no receptor reserve in this tissue. Several other 5-HT receptor agonists caused contractions but were much less potent than 5-HT. The order of potency of these agonists was determined to be DOM greater than 5-HT greater than or equal to alpha-methyl-5-HT much greater than 8-hydroxy-dipropylaminotetralin (8-OH-DPAT) greater than 2-methyl-5-HT greater than 1-(3-chlorophenyl) piperazine (mCPP) greater than m-trifluoromethyl-phenylpiperazine (TFMPP).(ABSTRACT TRUNCATED AT 250 WORDS)

Clonidine (150 Μg/kg s. c.) depressed avoidance conditioned reflexes in Long-Evans rats. Some α-adrenoceptor blocking agents piperoxan, tolazoline, phentolamine, dibenamine and phenoxybenzamine also depressed these reflexes. Yohimbine (1–2 mg/kg) was found to have no significant effect. Atropine (10 to 15 mg/kg i. p.) antagonized the depression of conditioned avoidance reflexes induced by α-adrenoceptor blocking agents as well as the depression produced by clonidine. Yohimbine (1–2 mg/kg) reduced the depressing effects of clonidine on avoidance conditioned reflexes. In rats treated with piperoxan (10 mg/kg) the effects of clonidine were reduced. In these rats the depression of conditioned reflexes was less than in rats treated with clonidine or piperoxan alone. These experiments suggest that clonidine depressed avoidance conditioned reflexes in rats by activating central α-adrenoceptors. It is proposed that α-adrenoceptor blocking agents might act as partial agonists.

Protracted anaphylactic shock of guinea pigs led to death in over 90% of the animals, and good protection was obtained with an infusion of adrenaline after dibenamine pretreatment. Adrenaline alone, in doses which prevented the anaphylactic fall of arterial blood pressure, had no beneficial effect. Practolol abolished the therapeutic action of the combination of dibenamine/adrenaline. Stimulation of β-receptors by isoproterenol did not increase the survival rate. With dopamine, however, a significant prolongation of the survival times was obtained.

Among different carrageenins, kappa-carrageenans were found to be thrombogenic, whereas lambda-carrageenins were inactive in this respect, although the latter substances exerted a stronger edemogenic activity. Kappa-carrageenin (Sigma) was the most potent thrombogen. As the consequence of thrombosis tail infarction became visible some minutes after i.v. administration, but it was delayed for about 3 hours after the i.p. route and for about 6 hours after subplantar injection. Infarction frequency as well as extent of infarction was inhibited by heparin, cyproheptadine, phentolamine and dibenamine. Other substances like aspirin and dipyridamole showed no or only weak effects. Advantages of the carrageenin-induced thrombosis model in rats and mice are: (i) simple induction in small laboratory animals, (ii) easy observation and quantification all the time without killing the animals, and (iii) possible external testing of antithrombotic agents by applying substances on the tail.

The fluid permeability of the isolated ciliary epithelium of the rabbit eye to adrenergic agonist's has been measured in paired normal and ganglionectomized tissues. The agonists norepinephrine (NE), epinephrine (E), phenylephrine (P) and isoproterenol (ISO) were used at concentrations from 10(-12) to 10(6)M in the presence and absence of cocaine 2.6 or 26x10(-6)M (C, to prevent uptake of agonist in the synapse); iproniazid (I) 3.6 x 10(-5)M, or clorgyline (CL) 5x10(-5)M (to inhibit 10(-5)M, or clorgyline (CL) 5x10(-5)M (to inhibit monoamine oxidase); dibenamine, (D) 1x10(-5) (to block extraneuronal uptake) and/or 1x10(-8)M phenoxybenzamine (PH) (an alpha-adrenergic antagonist). No supersensitivity to adrenergic agonists was found in ganglionectomized tissues. Normal tissues showed an increased fluid permeability after agonist alone with the other of efficacy at 10(-8)M being ISO greater than NE greater than E greater than P, while in ganglionectomized tissues the order at 10(-8)M was E = NE greater than ISO = P. Normal tissues showed a sensitivity to agonists at 10(-8)M in order E greater than NE greater than P greater than ISO after treatment with C, I and D, while ganglionectomized tissues revealed a sensitivity to NE greater than P greater than ISO greater than E, even though the quantitative response after C, I and D was reduced. It appears, therefore, that both alpha- and beta-receptors exist in the ciliary epithelium, and although ganglionectomy alters the tissues responsiveness to different adrenergic agonists it is not the site of the supersensitivity seen in the whole eye.

Dopamine and apomorphine have been compared with regard to contraction and relaxation of aortic strips prepared from rats of different ages. Both dopamine and apomorphine contracted aortic strips from older (9–12 weeks) rats to greater maximal tension than preparations from younger (3–5 weeks) animals. Contraction in response to both agonists could be blocked by the α-blocker, Dibenamine. In contrast to dopamine, contraction by apomorphine was associated with the development of tachyphylaxis. Both apomorphine and dopamine relaxed the aorta after alpha blockade, however, the relaxation produced by these drugs differed in two major aspects. First, dopamine-induced relaxation was greatest in aortic strips from young rats compared to older rats whereas relaxation with apomorphine was not age dependent. Second, dopamine-induced relaxation was abolished by propanolol but not haloperidol whereas apomorphine relaxation was only blocked by haloperidol. These data establish that the aortic relaxation caused by dopamine is most likely a β-adrenergically mediated response whereas that produced by apomorphine is not. Moreover, only dopamine was able to increase the concentration of rat aortic cyclic AMP. Should these data be applicable to other vascular beds or species, it is possible that the vascular effects of dopamine will be influenced by age and drugs which impinge on cyclic nucleotide disposition.

Further studies have been made on the differential blockade produced by 2-halogenoalkylamines against the pressor responses produced by monoamines in the rat. Results of the present study are discussed with reference to previous ideas on the existance of a pool of noradrenaline - sensitive, phenoxybenzamine - resistant alpha receptors in the cardiovascular system of the rat.

The relation of the amount of α 1 -adrenoceptors ( α 1 -R) with contraction to norepinephrine (NE) through α 1 -R in rat vas deferens was examined by means of radiobinding assays. Treatment with dibenamine decreased the maximal contraction to NE with a decrease in the amount of α 1 R but the relation was not linear. The contractile response disappeared completely when 20% of the α 1 R still remained. Moreover, culture of dibenamine-pretreated muscle restored the contraction to NE without a significant increase in the amount of α 1 -R in the muscle. These findings suggest that some α 1 -R are ‘silent’ in the contraction of rat vas deferens in response to NE under physiological conditions.

The motility of smooth muscles excised from the wall of bovine teats was studied "in vitro". These muscle preparations often show spontaneous rhythmic contractions. Administration of isoprenaline results in relaxation and decreased spontaneous motility, these effects being blocked by propranolol. Noradrenaline elicits contraction and stimulates rhythmical activity, these effects being inhibited by dibenamine. The effect of adrenaline is variable as it induces contraction or inhibition or biphasic responses. Dibenamine blocked contractions, whereas propranolol inhibited relaxations. It appears that alpha and beta adrenoceptors are present in teat muscles. Stimulation of the former elicits activation, whereas stimulation of the latter results in inhibition. These "in vitro" results are largely in agreement with "in vivo" responses described previously.

1. Either electrical nervous stimulation or adrenergic monoamines having alpha effects caused melanosome aggregation in guppy melanophores. 2. Some beta agonistic drugs were also found to be pigment-aggregating. 3. Various alpha adrenergic antagonists eliminated these melanin-aggregating effects. 4. On the other hand, beta adrenergic blocking agents were not effective in blocking these effects. 5. Postsynaptic adrenoceptors in melanophores which mediate pigment aggregation appear to be alpha adrenergic.

Using a food-reinforced two-lever operant method, rats (n = 5) could be trained to discriminate 10 mg/kg cocaine from saline. Stimulus generalization experiments with lower doses (0.31–5.0 mg/kg) revealed that the cocaine cue is a dose-related phenomenon. Neuroleptic drugs were found relatively ineffective as possible antagonists of the cocaine cue, and no antagonistic effect whatsoever was obtained with dibenamine, propranolol, cyproheptadine and methysergide. Amphetamine (1.25 mg/kg) and apomorphine (0.31 mg/kg) were generalized with cocaine, and a dopaminergic involvement is discussed.

A number of drugs classed as calcium antagonists, spasmolytics, non-specific receptor antagonists or receptor antagonists with multiple sites of action were tested to determine whether they prevent the stimulation of phosphatidylinositol turnover caused in various tissues by the activation of receptors which increase cell-surface Ca2+ permeability. The experiments were done with fragments of longitudinal smooth muscle from guinea-pig ileum; these were incubated in vitro with 32Pi and either 100 muM-carbamoylcholine or 100 muM-histamine, in the presence of antagonistic drugs at concentrations at least sufficient to cause complete blockade of smooth-muscle contraction. The phosphatidylinositol response to carbamoylcholine was not changed by cinchocaine, papaverine, nifedipine, dibenamine, amethocaine, cinnarizine, lidoflazine, methoxyverapamil, prenylamine or two antimuscarinic alkane-bis-ammonium compounds, and the response to histamine was unaffected by the first four drugs. In contrast, phenoxybenzamine prevented the increase in phosphatidylinositol labelling caused by either carbamoylcholine or histamine. The insensitivity of the phosphatidylinositol response to most of the drugs provides further experimental support for the conclusion that the receptor-stimulated phosphatidylinositol breakdown which initiates the increase in phosphatidylinositol turnover is not caused by an increase in intracellular Ca2+. The simplest interpretation of the available information appears to be that phosphatidylinositol breakdown plays a role in the coupling between the receptor-agonist interaction and the opening of cell-surface Ca2+ gates [Michell, R. H. (1975) Biochim. Biophys. Acta 415, 81-147]. If this is correct, then phenoxybenzamine must exert its inhibitory effects on phosphatidylinositol breakdown early in this sequence of events, but the drugs must act at a stage later than phosphatidylinositol breakdown. The unexpected difference in the effects of dibenamine and phenoxybenzamine, which are chemically very similar, may provide a useful experimental tool with which to explore the way in which activated receptors provoke the opening of cell-surface Ca2+ gates.

Data obtained in radioligand - binding studies on guinea pig and mouse ileum were compared with those on the contractile responses to the muscarinic agonists methacholine (MCh), oxotremorine, bethanechol (BCh) and pilocarpine. Guinea pig ileum has a large amount of binding sites for 3 H-quinuclidinyl benzilate (QNB), a muscarinic antagonist, (102 fmoles/mg tissue), whereas mouse ileum has a small amount of these binding sites (19 fmoles/mg tissue). MCh, oxotremorine and BCh were full agonists in the contractile responses ofboth ilea, but pilocarpine was full agonist only in guinea pig ileum. After treatment with dibenamine (0.15 mM) (for 15 min at 30°C) guinea pig ileum exhibited maximal responses to MCh, oxotremorine and BCh, but not to pilocarpine, although the ED 50 values for the agonists in the muscle were increased by this treatment. The amount of binding sites of 3 H-QNB in dibenamine-treated guinea pig ileum was 55% of that in untreated ileum. When the amount of 3 H-QNB-binding sites in mouse ileum was decreased to 54% by dibenamine-treatment for 15 min, the maximal contractions induced by MCh, oxotremorine and BCh were also decreased to some degree. In low Ca 2+ medium, pilocarpine became a partial agonist in the contractile responses of guinea pig ileum. The following conclusions were drawn from these findings: 1. 1. The spare receptors proposed by Stephenson actually exist, and change in the amount of spare receptors, which can be brought about by dibenamine-treatment, or in low Ca 2+ medium, can change the ED 50 value of agonists. 2. 2. In a single tissue, the amount of spare receptors for different agonists differ, probably depending on the efficacy of the agonists. 3. 3. For a single agonist, the amount of spare receptors in a tissue differs from species to species, presumably depending on the total amounts of receptors.

The binding of lanthanum to murine neuroblas-toma cells (clone N1E-115) was studied by means of electron microscopy and x-ray microanalysis. Lanthanum bound only to cellular membranes, in a nonuniform manner. This lanthanum binding was reduced by pharmacological agents that affect muscarinic receptors of these cells or the function of the receptors. These results suggest that this binding of lanthanum is to sites closely related to muscarinic receptors in the cells.

The adrenergic receptors in the isolated feline middle cerebral artery were characterized pharmacologically using a sensitive system for recording circular contractions in vitro. Epinephrine. norepinephrine, isoproterenol. and phenylephrine contracted the vessel in the mentioned order of potency. Together with the inhibitory patterns obtained with graded doses of piperoxan (reversible competitive inhibition) and dibenamine or phenoxybenzamine (irreversible competitive inhibition), these results showed that the contraction was mediated by alpha receptors. With piperoxan and norepinephrine, the mean value for pA 2 was 7.06 and for K H 1.24 x 10 -7 M. The mean value for K A calculated for norepinephrine before and after partial irreversible blockade of the alpha receptors with phenoxybenzamine was 1.73 x 10 -6 M. The norepinephrine response was not directly proportional to the amount of receptors occupied; ED 50 was reached when only about 11% of the receptors were occupied and the E Am response was obtained when 75% of the receptors were occupied. Dilation was achieved only after an active tonic contraction had been induced (with 5-hydroxytryptamine) in the vessels, and the order of potency was isoproterenol > norepinephrine - epinephrine > terbutaline. Inhibition with INPEA and propranolol was competitive, as confirmed by Arunlakshana-Schild plots, showing that the dilatory response was a beta-receptor effect. The values for pA 2 (8.78 and 9.17) and K H (2.31 x 10 -9 M and 1.77 x 10 -9 M) for propranolol were indistinguishable in tests with terbutaline and isoproterenol, respectively. Comparison of the relative potencies of norepinephrine and epinephrine as well as isoproterenol and terbutaline suggested that the receptors were of the beta 1 type.

Buzzers and shocks were presented in random sequence and with no temporal association to rats in a shuttle-box, so that they performed pseudoconditioned barrier-crossing responses to the buzzer. Performance of these responses was enhanced by amphetamine and tyramine. The adrenergic blocking agents yohimbine and dibenamine, though ineffective on their own, antagonized the action of amphetamine and tyramine. Hexamethonium and methylatropine, also ineffective by themselves, potentiated the effect of amphetamine but not that of tyramine. Eserine depressed the performance of pseudoconditioned responses when given alone, and it antagonized the stimulant action of both amphetamine and tyramine. The results suggest that amphetamine and tyramine enhance pseudoconditioning through activation of a peripheral adrenergic mechanism and that amphetamine, in addition, triggers an opposing cholinergic mechanism, also peripheral, from which it can be released by the anticholinergic agents hexamethonium and methylatropine. The depressant action of eserine can also be explained by activation of the cholinergic mechanism.

1. The positive inotropic responses of rat paced left atria to adrenaline and noradrenaline were recorded. Desmethylimipramine (DMI, 1 microM) and metanephrine (10 microM) were initially present throughout. 2. The positive chronotropic responses of spontaneously beating right atria to adrenaline were used as a reference. In these, pindolol, in increasing concentrations, caused progressive shift of the concentration-response curves to the right, which yielded a pA2 value (8.15) compatible with antagonism of beta-adrenoceptors. 3. The left atrial tension responses to adrenaline showed an initial progressive displacement by pindolol (up to 3 microM) which gave an unexpectedly low pA2 value (6.48). However, with further increases in pindolol concentration there was no additional shift of the curve. In the presence of pindolol (3 microM), prazosin (0.1 microM) displaced the curve to the right but the pA2 value derived from this shift (7.75) was less than expected for alpha 1-adrenoceptor antagonism. 4. When the experiments in the presence of pindolol (3 microM) were repeated in the absence of DMI, prazosin displaced the concentration-response curves for adrenaline-induced left atrial tension to a greater extent and the pA2 value (8.76) was now compatible with adrenaline stimulating typical alpha 1-adrenoceptors. 5. The concentration-response curves for noradrenaline-induced left atrial tension were also progressively displaced to the right by pindolol (0.1, 0.3 and 1.0 microM). These concentrations yielded a Schild plot of unity slope and a pA2 value of 7.94 +/- 0.04. This was not significantly different from the pA2 value of 8.02 +/- 0.07 determined for pindolol against isoprenaline in the left atria, which indicates a normal interaction of noradrenaline with beta-adrenoceptors in the absence and presence of low concentrations of pindolol. 6. A further increase in the concentration of pindolol to 3 microM failed to induce an additional shift of the noradrenaline curves, whether a 'before and after' antagonist or a 'naive tissue' design was adopted. Similarly, the rightwards shift of the concentration-response curves by timolol reached a limit as the concentration was increased. In all cases the limit of shift occurred at a noradrenaline EC50 value of 5-10 microM. 7. At the limit of beta-adrenoceptor antagonism, prazosin and dibenamine did not displace the noradrenaline curves further. The residual inotropic response to noradrenaline therefore appeared to be mediated via neither alpha- nor beta-adrenoceptors. 8. DMI, in the absence of beta-blockade, produced the potentiation of adrenaline and noradrenaline expected of a neuronal uptake inhibitor. However, in the presence of pindolol, there was no potentiation of the right atrial rate response to adrenaline while its left atrial tension responses were antagonized. This suggested that DMI was acting as an alpha-adrenoceptor antagonist. It also explained the less-than-expected shift by prazosin of the adrenaline responses in the presence of both pindolol and DMI, the latter drug already exerting some alpha-blocking activity. In contrast, the left atrial tension responses to noradrenaline in the presence of pindolol (1 microM) were neither potentiated nor antagonized by DMI. 9. When the effects of prazosin upon left atrial tension responses to noradrenaline in the presence of pindolol (10 microM) were examined in the presence of a lower concentration of DMI (O.1 microM) or cocaine (1O microM), again there was no further shift of the curve. However, when the effect of prazosin) The Macmillan Press Ltd 1989 598 K.L. WILLIAMSON & K.J. BROADLEY was examined in the absence of DMI, but in the presence of pindolol (1 and 1O microM) or timolol (3 microM), there was a small shift of the curves by prazosin (0.1 microM). This yielded pA2 values of 7.19, 7.34 +/- 0.1 and 7.66 +/- 0.09, which were at least one order of magnitude less than literature values and that obtained with adrenaline (8.76 +/- 0.18), and are not consistent with noradrenaline stimulating an alpha 1-adrenoreceptor in the presence of beta-adrenoceptor blockade, the increase in left atrial tension by noradrenaline does not appear to be mediated by beta l- or typical alpha-adrenoceptors. This is in contrast to adrenaline which in these conditions stimulates typical alpha 1-adrenoceptors.

Several alpha-adrenergic antagonists inhibited the activation of calmodulin-stimulated phosphodiesterase at concentrations that had little or no effect on basal phosphodiesterase activity. The most potent of these compounds were phenoxybenzamine and dibenamine (IC50 values of about 1 microM); the amino acid ergot alkaloids ergocryptine, ergocristine, ergotamine and their dihydrogenated derivatives were less potent calmodulin-inhibitors (IC50 values of 35-80 microM). The amino ergot alkaloids ergonovine and methysergide were essentially devoid of inhibitory activity. A variety of other alpha 1-antagonists (phentolamine, tolazoline and prazosin), an alpha 2-antagonist (yohimbine), alpha-agonists (norepinephrine, phenylephrine and clonidine), beta-adrenergic antagonists (propranolol and practolol) and the beta-adrenergic agonist methoxyphenamine displayed little or no anti-calmodulin activity (IC50 values greater than 300 microM). Similarly, the alkylating agents chlorambucil and mechlorethamine also failed to inhibit calmodulin activity. Phenoxybenzamine and dibenamine inhibited calmodulin activity irreversibly, whereas the inhibition caused by other alpha adrenergic blocking agents was reversible. Phenoxybenzamine inhibited calmodulin activity by binding directly to it. This binding was calcium-dependent and irreversible. The irreversible binding and inhibition of calmodulin activity by phenoxybenzamine (or dibenamine) may serve as a useful tool for studying the sites at which drugs bind to calmodulin and may also be useful for studying the distribution and turnover of calmodulin.

Two beta-carbolines, methyl beta-carboline-3-carboxylate (beta-CCM) and ethyl beta-carboline-3-carboxylate (beta-CCE), caused the parallel shift of the dose-response curve for cholecystokinin (CCK) in isolated guinea-pig gallbladder muscle. The Schild plot regarding the parallel shift in the dose-response curves had a regression line with a slope of 1.03 and a pA2 value of 5.17 for beta-CCE, while the method of van Rossum gave a pA2 value of 5.24 for beta-CCE and 5.53 for beta-CCM. Both the beta-carbolines protected CCK receptors in the gallbladder muscle from alkylation by dibenamine, but beta-CCM did not protect acetylcholine receptors from dibenamine alkylation. These results suggest that beta-CCM and beta-CCE, so-called inverse agonists of benzodiazepines (BZP), antagonize the CCK action in the gallbladder muscle in a competitive manner, and the antagonism takes place at CCK receptor sites. No spare receptors for CCK were found in the guinea-pig gallbladder muscle.

Crystalline perchlorate salts of aziridinium ions derived from phenoxybenzamine and dibenamine were prepared. Both aziridinium ions were tested on the rat vas deferens and found to possess alpha-adrenergic potencies which were nearly identical with those of the parent compounds. The hydrolysis rates of phenoxybenzamine and dibenamine aziridinium ions (2a,b) in physiological medium were found to be 6.0 4 x 10(-4) and 8.35 x 10(-4) sec-1, respectively. The rates of cyclization of the parent amines to 2a and 2b in aqueous medium were 1.9 x 10(-2) and 7.2 x 10(-3) sec-1, respectively. The potencies and kinetic profiles indicate that the aziridinium ion is the only active species in alpha-adrenergic blockade. Moreover, differences in potency between phenoxybenzamine and dibenamine appear to be exclusively to a difference in receptor affinity rather than to a difference in intrinsic alkylating ability.

Serum glucose was elevated immediately after ip administration of a single large dose of fluoride (NaF 35 mg/kg) to rats. Moreover, elevation of serum glucose following ip administration of 35 mg/kg of fluoride to rats was suppressed by adrenalectomy, dibenamine, or propranolol, but not by thyroid-parathyroidectomy. The elevation of serum glucose was associated with enhancement of glucose-6-phosphatase activities in liver and kidney in fluoride-treated rats.

The effect of epinephrine (Epi) on the microcirculation of the pulp and its neuropharmacological mechanism has been established, as well as the existence of alpha adrenergic receptors in the blood vessels of the pulp. In addition, our results showed that local injections of Epi at doses greater than 10-8 M produced an extreme drop in intrapulpal pressure (IPP). This effect was not obtained when Epi was injected intravenously.

Studies were made on tritiated histamine binding to the receptor-rich membrane fraction and solubilized sites and its displacement by various drugs. H1-Agonists and antagonists displaced histamine most effectively. A H2-agonist and atropine were less effective and propranolol, phentolamine and imidazole acetic acid had little effect. The solubilized binding sites showed the same specificity of binding as the membrane fraction. Membrane fragments had two binding constants, whereas solubilized sites had only one. Solubilized sites bound similar amounts of histamine and dibenamine: the latter was applied to intact tissue under conditions which would presumably cause specific binding to histamine receptors. These binding characteristics show that the method used was adequate for purification of histamine receptors from smooth muscle of cat small intestine.

Summary Rabbit aortic tissue was labeled with 3H-phenoxybenzamine or 14C-dibenamine and treated with collagenase and elastase to remove collagen and elastin. The residue consisting of numerous small membranous vesicles and disrupted aggregates of actomyosin was treated with 2% SDS-EtSH and chromatographed on a Sephadex G-200 column. The radioactivity was located in a protein peak of molecular weight approximately 64,000 thus providing a demonstration of a protein-2-halogenoethylamine complex in smooth muscle.

This study investigated the stimulus property of nicotine in the rat. The primary objectives of the study were 1. to determine the time course of the nicotine stimulus and its relationship to brain levels of the drug and 2. to determine whether the nicotine stimulus is dependent upon the integrity of specific neurotransmitter systems. A lever choice discrimination was used. After injection of nicotine, depression of one lever in an operant test chamber resulted in food reinforcement according to a variable interval schedule of 15 sec. When saline was administered, the opposite lever was reinforced. A high degree of discriminated responding was observed when either 400 Μg/kg or 200 Μg/kg of nicotine was used as a discriminative stimulus. The degree of discrimination decreased as the length of the time period between the injection of nicotine and the test of discrimination was increased. This decline in discrimination was similar to the decline in brain levels of nicotine suggesting that nicotine discrimination is directly related to the concentration of nicotine in the brain. Atropine, mecamylamine, dibenamine, propranolol and α-methyl-para-tyrosine (AMPT) were all tested, in a range of doses, for effects upon nicotine discrimination. Of these, only mecamylamine antagonized the nicotine stimulus. These results indicate that the stimulus effect of nicotine is mediated specifically through nicotinic-cholinergic receptors and not muscarinic-cholinergic or adrenergic receptors.

In previous papers, tyramine (5 mg/kg), LSD (0.3 mg/kg), LSD + dibenamine (10 mg/kg), and diazepam (5 mg/kg) have been found to have opposite effects on the performance of shuttle responses to a buzzer by rats in a so-called “pseudoconditioning” and in a typical “two-way avoidance” test. The main differences between the two tests are that in the former the stimuli are given at random (i.e., without a constant temporal relation or “pairing”) and shocks are not contingent upon responses. A state of shock-dependent drive may be assumed to be similarly present, however, in both situations. It was decided to test the effect of these drug treatments on two other experimental paradigms: one in which buzzers and shocks were “paired” (i.e., they partially overlapped) but in which shocks were given on all trials independently of responses (i.e., no “contingency”), and another one in which the buzzer-shock interval was varied at random but shocks were contingent upon shuttling to the buzzer. In the former test, LSD and diazepam increased shuttling to the buzzer, whereas tyramine and dibenamine had no effect and dibenamine partially blocked the action of LSD. In the latter test, tyramine and LSD + dibenamine depressed responding, diazepam increased it, and LSD and dibenamine on their own had no effect. Drug influences on the latter test were very similar to those previously observed on a two-way avoidance situation, which suggests that these drugs may affect two-way avoidance possibly by an action mainly on the “contingency” mechanism. Furthermore, the present data also suggest that drive and what here was called “pairing” and “contingency” are separable factors in the elicitation of shuttle responses to a buzzer in buzzer + shock paradigms.

SummaryAn interaction between sarcoma-180/TG cells and vesicular stomatitis virus in adult mice resulted in the rapid onset of extensive mortality. This interaction, termed lethal synergy, occurred only at early stages of ascites induction in animals with no prior virus contact. A significant sparing effect conferred by the serotonin antagonist dibenamine was reversed by the administration of serotonin. The cause of death was not determined, but a mechanism involving hypersensitivity is indicated.