Clonidine-induced inhibition of sympathetic nerve activity: No indication for a central presynaptic or an indirect sympathomimetic mode of action

Summary The effects of clonidine on blood pressure, heart rate, contractile state of the nictitating membranes, spontaneous sympathetic nerve activity and response of sympathetic nerves to hypothalamic stimulation were compared in normal anaesthetized cats and in anaesthetized cats pretreated with reserpine and -methyl-p-tyrosine. The pretreatment lowered the noradrenaline content of various parts of the brain to less than 5 ng/g, i.e. to less than 1–3% of that of the controls. Under the conditions of this severe noradrenaline depletion, blood pressure and heart rate were low and spontaneous sympathetic nerve activity consisted of continous, high-amplitude discharges which contrasted with the low-amplitude bursts of activity—synchronous with the respiration—of the controls. In contrast to the controls, clonidine did not lower blood pressure and heart rate in the cats with noradrenaline depletion; however, the clonidine-induced contractions of the nictitating membranes were of similar magnitude and duration in both groups of animals. The efficacy of clonidine in reducing or abolishing spontaneous sympathetic nerve activity and in inhibiting the response of sympathetic nerves to hypothalamic stimulation was equal in controls and in cats with noradrenaline depletion, its potency being 3-fold higher in the former. The results indicate a direct stimulation of -adrenoceptors by clonidine both in the periphery and in the central nervous system and make it unlikely that the central effect of clonidine on blood pressure is due to a release of noradrenaline from central adrenergic neurones. It is further concluded that clonidine activates an adrenergic mechanism in the central nervous system by stimulation of postsynaptic -adrenoceptors. The inhibition of such a mechanism as a consequence of a diminished noradrenaline release due to stimulation of presynaptic -adrenoceptors—as proposed from in vitro experiments—seems to be of no importance for the central effect of clonidine on sympathetic nerve activity and blood pressure.Preliminary results have been presented at the 15th Spring Meeting of the German Pharmacological Society in Mainz (Haeusler, 1974a).     

The effect of yohimbine on sympathetic responsiveness in essential hypertension

Summary We have studied the sympathetic response to blockade of presynaptic ₂-adrenoceptors in essential hypertension by measuring plasma concentrations of noradrenaline after a single oral dose of yohimbine, an ₂-adrenoceptor antagonist.Mean baseline plasma noradrenaline and adrenaline concentrations were similar in the hypertensive and normotensive groups. Yohimbine (0.2 mg×kg^–1 orally) caused a lesser increase in the plasma concentrations of noradrenaline in hypertensive patients (+67%) than in normotensive subjects (+178%) and a pressor response in hypertensive (but not in normotensive) patients.These results are consistent with an alteration in the balance of -adrenoceptors (for example presynaptic ₂-adrenoceptor desensitization and post-synaptic ₁-adrenoceptor hyper-responsiveness) which would help to develop and/or maintain arterial hypertension.     

Effects of the novel dopamine DA2-receptor agonist carmoxirole (EMD 45609) on noradrenergic and purinergic neurotransmission in rat isolated kidney

Summary The effects of the classical dopamine DA₂-receptor agonist quinpirole (LY 171555) and the recently characterized DA₂-receptor agonist, carmoxirole (EMD 45609), on neurotransmission in rat isolated kidney were investigated. After preincubation with ³H-noradrenaline, the renal nerves were electrically stimulated. The stimulation induced (S-I) outflow of radioactivity was taken as an index of noradrenaline release. Quinpirole (0.3 mol/l) inhibited S-I outflow of radioactivity and pressor responses to renal nerve stimulation (RNS) at 1 Hz. Both effects of quinpirole were blocked by the DA₂-receptor antagonist S(–)-sulpiride (10 mol/l). The ₁, ₂-adrenoceptor antagonist phentolamine (1 mol/l) did not block the inhibitory effect of quinpirole. Carmoxirole (0.003 and 0.03 mol/l) did not alter and carmoxirole (0.3 mol/l) even enhanced S-I outflow of radioactivity, however, pressor responses to RNS were markedly reduced by carmoxirole (0.003–0.3 mol/l). Pressor responses to RNS were also markedly reduced by the ₁-adrenoceptor antagonist prazosin (0.1 mol/l). Carmoxirole (0.3 mol/l), prazosin (0.1 mol/l) and phentolamine (1 mol/l) totally abolished pressor responses to exogenous noradrenaline (0.05 mol/l). In contrast, quinpirole (0.3 mol/l) did not alter pressor responses to exogenous noradrenaline (0.05 mol/l). Furthermore, carmoxirole (0.003–0.3 mol/l) markedly reduced pressor responses induced by the ₁-adrenoceptor agonist methoxamine (1 mol/l) but even the highest concentration of carmoxirole (0.3 mol/l) had no effect on pressor responses induced by bolus injections of either neuropeptide Y (1.5 ng) or angiotensin II (1 ng). Phentolamine (1 mol/l) by itself markedly enhanced S-1 outflow of radioactivity and pressor responses to RNS were virtually unchanged. In the presence of phentolamine carmoxirole (0.03 and 0.3 mol/l) and quinpirole inhibited S-I outflow of radioactivity and pressor responses to RNS. Phentolamine resistant pressor responses to RNS were also inhibited by the P_2X-receptor desensitizing agent , -methylene adenosine triphosphate (mATP, 1 mol/l), which by itself in the presence of phentolamine did not alter S-I outflow of radioactivity. The inhibitory effects of carmoxirole (0.3 mol/l) in the presence of phentolame (1mol/l) were antagonized by S(–)-sulpiride (10 mol/l). The data suggest that activation of prejunctional DA₂-receptors by quinpirole inhibits noradrenaline release and thereby reduces pressor response to RNS at 1 Hz in rat isolated kidney. Carmoxirole activates prejunctional inhibitory DA₂-receptors, but this effect is masked by simultaneous blockade of inhibitory prejunctional -adrenoceptors. Pressor responses to RNS at 1 Hz in rat isolated kidney are largely due to neuronally released noradrenaline whereas phentolamine resistant pressor responses to RNS at 1 Hz are most likely due to ATP, which is co-released with noradrenaline. Carmoxirole inhibits pressor responses to RNS at 1 Hz as well as pressor responses induced by either exogenous noradrenaline or methoxamine by blocking postjunctional ₁-adrenoceptors. In addition carmoxirole and quinpirole seem to block phentolamine resistant pressor responses by inhibiting ATP release through activation of prejunctional DA₂-receptors. Send offprint requests to L. C. Rump at the above address     

Inhibitory effect of dopamine on canine gastric fundus

Summary The mechanism of the inhibitory effect of dopamine on canine stomach fundus was studied in longitudinal and circular muscle fundus strips, contracted by transmural electrical stimulation or by methacholine.Results obtained for longitudinal and circular strips were similar. Dopamine (1 · 10^–6–1 · 10^–4 M) concentration-dependently inhibited frequency-response curves to electrical stimulation; these concentrations did not change the resting tone of the strips. Dopamine (1 · 10^–4 M), tested on contractions of similar amplitude induced in the same strips by electrical stimulation at 0.5 Hz and by methacholine, inhibited the electrically induced contractions but had little influence on the contractions induced by methacholine. The inhibition of the electrically induced contractions by dopamine 1 · 10^–4 M was not influenced by the presence of cocaine 3 · 10^–5 M or hydrocortisone 3 · 10^–5 M.The ₁- and ₂-adrenoceptor antagonist phentolamine and the ₂-adrenoceptor antagonist rauwolscine markedly antagonized the inhibitory effect of dopamine on the response to electrical stimulation at 0.5 Hz. The ₁-adrenoceptor antagonist prazosin and the dopamine receptor antagonists haloperidol and domperidone had no effect. The dopamine receptor antagonist metoclopramide decreased the inhibitory effect of dopamine but had a similar effect on the inhibition caused by noradrenaline.These results indicate that the inhibitory effect of dopamine in the dog gastric fundus is mainly mediated by an interaction with ₂-adrenoceptors on the intramural cholinergic neurons; this effect is largely direct since it was not influenced by cocaine. These results are different from those obtained in the rat gastric fundus, where the inhibitory effect of dopamine is mainly indirect, and due to an interaction with -adrenoceptors on the intramural cholinergic neurons and with -adrenoceptors on the smooth muscle cells. Dedicated to Prof. Dr. E. J. Ariëns (Department of Pharmacology, University of Nijmegen, The Netherlands) on the occasion of his retirement     

In vivo studies on alpha-adrenergic receptor subtypes in human veins

Summary We studied in vivo responsiveness of venous ₁ and ₂-adrenoceptors, measuring the diameter changes in superficial veins in response to -adrenergic agonists and antagonists in healthy human volunteers. The dorsal hand vein technique was used because it permits complete dose-response studies of venous constriction without confounding reflex alterations.Local infusions of all agonists studied induced dose-dependent contraction of the hand vein; the maximal effects (Emax) were: norepinephrine (88% ± 10%), methox amine (97% ± 5%), phenylephrine (95% ± 6%), clonidine (54% ± 12%), and azepexole (68% ± 26%). Clonidine reduced the norepinephrine-induced venoconstriction by 11% ± 10%. Oral doses of 1 mg prazosin antagonized the venoconstriction induced by norepinephrine, methoxamine, and clonidine, but not by azepexole. Yohimbineantagonism was observed against all agonists studied. Inhibition by yohimbine of clonidine-induced venoconstriction was irreversible over 60–180 min.Results show that the in vivo effects on veins of -adrenergic agonists are in good agreement with results from in vitro experiments. Agonists with ₁- and ₂-adrenoceptor subtype selectivity cause venoconstriction in vivo, but ₂-receptor mediated constriction is intrinsically weaker. Clonidine acts as a partial antagonist against norepinephrine, presumably on postsynaptic ₂-receptors. At high doses, ₂-adrenoceptor subtype selectivity of clonidine and yohimbine appear to be partially lost in vivo. Send offprint requests to H. G. Eichler at the above address     

Discontinuation of chronic clonidine treatment: Evidence for facilitated brain noradrenergic neurotransmission

Summary Clonidine, 0.1 mg/kg i.p., or saline was administered to mice twice daily for 12 days. After withdrawal of the drug (14–18.5 h after the last injection), the accumulation of Dopa during 30 min after inhibition of central aromatic amino acid decarboxylase by NSD 1015 (3-hydroxy-benzyl hydrazine, 150 mg/kg i.p.) was significantly increased in the noradrenaline (NA)-rich, but dopamine (DA)-poor, brain stem but not in the DA-rich, but NA-poor corpus striatum. An increased Dopa accumulation was also found in the limbic system and, probably, in the hemispheres. The central accumulation of 5-hydroxytryptophan (5-HTP) was significantly increased in the limbic system. Clonidine, 0.1 mg/kg i.p., administered to mice during the withdrawal phase caused reduction of the accumulation of Dopa and 5-HTP (during 30 min after NSD 1015, 150 mg/kg i.p.) in all brain regions studied to approximately the same levels, as when given to saline-pretreated controls. The disappearance rates of brain NA and DA after synthesis inhibition (-methyl-p-tyrosine methylester HCl 250 mg/kg i.p., 4 h) were probably not altered by clonidine withdrawal.The locomotor stimulation by the central catecholamine receptor agonists apomorphine plus clonidine (1.5 mg/kg i.p. both) after reserpine (10 mg/kg, 4 h) pretreatment was significantly enhanced in clonidine withdrawal mice. However, the motor stimulation by apomorphine (1.5 mg/kg i.p.) alone after reserpine pretreatment was not affected. The spontaneous motor activity was not significantly altered by the withdrawal of clonidine.The data show that brain NA and 5-HT systems are affected by clonidine withdrawal in the opposite direction to that seen after acute administration of low doses of the drug, when they are inhibited. Since postsynaptic central -adrenoceptors, which are stimulated by high but not by low doses of clonidine, showed an enhanced response to high doses of clonidine following discontinuation of a chronic low dose regimen, supersensitivity due to functional noradrenergic denervation might be implicated.     

Chronic clorgyline treatment enhances release of norepinephrine following sympathetic stimulation in the rat

Summary Plasma catecholamine levels were determined in pithed rats during electrical stimulation of the entire sympathetic nervous system. In animals treated chronically with clorgyline (1 mg/kg daily for 21 days) the increment in plasma norepinephrine concentration during stimulation was greather than in control animals, whereas a single dose of clorgyline (2 mg/kg 2 h before pithing), which produced the same degree of inhibition of arterial MAO type A and a similar increase in arterial norepinephrine content, had no effect on the plasma norepinephrine response to stimulation. Injection of yohimbine (1 mg/kg) produced the same degree of enhancement of plasma norepinephrine response to stimulation in chronically treated and control animals, showing that the overall gain of the ₂-adrenoceptor inhibitory loop in vascular sympathetic nerves was not affected. Plasma epinephrine concentration during electrical stimulation was also increased by chronic but not by acute clorgyline treatment. Chronic clorgyline treatment did not significantly affect the total systemic metabolic clearance rate of infused norepinephrine, thus the increased plasma norepinephrine response to stimulation reflects an increased release rate from sympathetic neurons.In rats treated chronically with clorgyline, the pressor response to norepinephrine in the presence of yohimbine (0.3 mg/kg) was significantly reduced, whereas the pressor response to guanabenz was unchanged. There was also no change in the guanabenz-induced inhibition of the tachycardic response to electrical stimulation. These results show that the enhanced norepinephrine release produced by chronic clorgyline treatment leads to down-regulation of post-synaptic ₁-adrenoreceptors with no change in post-synaptic ₂-adrenoceptors or in cardiac presynaptic ₂-adrenoreceptors, and are in agreement with an intrasynaptic location of ₁-adrenoceptors and an extra-synaptic location of ₂-adrenoceptors in the rat vasculature.     

Comparative behavioral characterization of the neuroactive steroids 3α-OH,5α-pregnan-20-one and 3α-OH,5β-pregnan-20-one in rodents

Pregnan steroids have been shown to possess anesthetic, hypnotic, anticonvulsant and anxiolytic properties. In this study, two endogenous neuroactive steroid isomers, 3-hydroxy-5-pregnan-20-one (3,5-P) and 3-hydroxy-5-pregnan-20-one 3,5-P), were studied for differences in their pharmacological properties using behavioral assays. 3,5-P and 3,5-P were similar in their potencies and efficacies in blocking pentylenetetrazol-induced seizures in mice (ED₅₀: 3,5-P=2.8 mg/kg and 3,5-P=3.0 mg/kg). Similarly, both neuroactive steroids produced roto-rod deficits within the same range of potency (TD₅₀:3,5-P=18.8 mg/kg and 3,5-P=21.2 mg/kg). However, in animal models of anxiety, subtle differences were observed between the two isomers. In both the light/dark transition test and elevated plus-maze, 3,5-P was more efficacious than 3,5-P, though both compounds had similar potencies. In the Geller-Seifter test, 3,5-P was more potent and efficacious than 3,5-P. Neither compound had significant effects on unpunished responding within the dose range tested. Both compounds produced similar biphasic curves in the locomotor test. All together, the data indicate that 3,5-P and 3,5-P have similar anticonvulsant activity, but the 5-isomer possesses more potent and efficacious anxiolytic properties than the 5-isomer.     

In vitro studies on 6-fluoronoradrenaline at several peripheral sympathetic neuroeffector junctions

Summary A comparison of the effects of noradrenaline and 6-fluoronoradrenaline has been made at several peripheral sympathetic neuroeffector junctions. In the rat vas deferens preparation in the presence of 1 M cocaine, 6-fluoronor-adrenaline was found to be about 9 times more potent than noradrenaline as an agonist at presynaptic inhibitory ₂-adrenoceptors. In the rabbit aorta, 6-fluoronoradrenaline had approximately one tenth of the potency of noradrenaline in stimulating the postsynaptic ₁-adrenoceptors. Furthermore 6-fluoronoradrenaline, in contrast to previous reports, appears to be a substrate for the neuronal uptake process since exposure to cocaine potentiated the inhibition of the twitch response of the vas deferens by 6-fluoronoradrenaline. In addition, 6-fluoronoradrenaline increased the spontaneous outflow of radioactivity from rabbit pulmonary artery strips prelabelled with ³H-noradrenaline and this increase was blocked by cocaine (30M).These results demonstrate that 6-fluoronoradrenaline is a preferential ₂-adrenoceptor agonist which is a substrate for the neuronal uptake process in peripheral sympathetically innervated smooth muscle preparations.     

Inhibitory dopamine receptors on sympathetic neurons innervating the cardiovascular system of the pithed rat

Summary Additional experimental evidence was obtained for an inhibitory function of prejunctional ₂-adrenoceptors and/or dopamine receptors located on noradrenergic neurons innervating the heart and resistance vessels of the pithed normotensive rat. Mixed ₂-adrenoceptor receptor agonists, differing in selectivity towards either receptor type, i.e. N,N-di-n-propyldopamine (DPDA), 2-N, N-di-n-propylamino-6, 7-dihydroxy-1,2,3,4-tetrahydronaphthalene (DP-6,7-ADTN), B-HT 920 and B-HT 933 (azepexole) were used.In pithed normotensive rats, DPDA (30 and 100 g/kg/min) dose-dependently inhibited the electrical stimulation-induced increase in diastolic pressure, but did not significantly affect the stimulation-evoked increase in heart rate. The inhibition exerted by DPDA was blocked by haloperidol and sulpiride (0.3 mg/kg of each), but not by yohimbine (1 mg/kg), indicating the involvement of dopamine receptors. In this respect, sulpiride and haloperidol were found approximately equipotent.DP-6,7-ADTN (10 and 30 g/kg/min) impaired both tachycardic and vasoconstrictor responses in a dose-dependent manner. Sulpiride (0.3 mg/kg) only partially restored the DP-6,7-ADTN-depressed stimulation-evoked increase in diastolic pressure, whereas yohimbine (1 mg/kg) alone was without effect. The combination of both antagonists completely prevented the inhibition caused by DP-6,7-ADTN. On the other hand, yohimbine (1 mg/kg), but not sulpiride (0.3 mg/kg), selectively antagonized the DP-6,7-ADTN-induced inhibition of stimulation-evoked tachycardia.B-HT 920 (1, 3 and 10 g/kg/min) very effectively reduced the increase in diastolic pressure and heart rate caused by electrical stimulation. Inhibitory dopamine as well as ₂-adrenoceptors participated in the vascular effects of B-HT 920, whereas ₂-adrenoceptors were only involved in the cardioinhibitory response to this agonist.B-HT 933 (0.6 and 1 mg/kg/min) dose-dependently reduced the stimulation-evoked increase in arterial pressure through selective stimulation of inhibitory ₂-adrenoceptors, dopamine receptors not taking a part.The results confirm and extend the observations that in addition to ₂-adrenoceptors inhibitory dopamine receptors are located on the sympathetic neurons connected with the arterial vasculature of the pithed normotensive rat. The sympathetic nerves innervating the rat heart do not contain inhibitory dopamine receptors; their activity only can be modulated by ₂-adrenoceptor stimulation. In the pithed normotensive rat, activation of prejunctionally located ₂-adrenoceptors more effectively inhibits the sympathetic activity directed to the heart than that to the resistance vessels.     

Studies on alpha adrenoceptors in guinea-pig peripheral lung strips

Summary Contractile responses of guinea-pig peripheral lung strips to noradrenaline were determined in the presence of propranolol (2.5 × 10^–6 mol/l). All strips (n = 44) contracted to noradrenaline and a geometric mean EC₅₀ of 1.4 × 10^–6 mol/l (1.1 × 10^–6 mol/l, 1.8 x 10^–6 mol/l 95% confidence limits) was obtained. Contractions were antagonised by phentolamine (5 × 10^–7–10^–5 mol/l) and by prazosin (10–10^–7 mol/l). Dose-ratios (DR) were calculated and log (DR-1) was plotted against log concentration of antagonist to yield slopes (± SE) of 0.84 ± 0.14 and 0.73 ± 0.22 respectively which were not significantly different from unity. A pA₂ value (± SE) of 6.7 ± 0.2 was obtained for phentolamine and 7.5 ± 0.1 for prazosin. Yohimbine (10^–7–10^–5 mol/l) did not significantly affect the maximal tension generated or the EC₅₀ values for noradrenaline. These results suggest that ₁ adrenoceptors are mediating the contractile responses to noradrenaline. In the presence of cocaine (10^–5 mol/l, n=18), normetanephrine (2 × 10^–5 mol/l, n = 15), hydrocortisone (2.5 × 10^–5 mol/l, n = 15) and normetanephrine (2 × 10^–5-5 mol/l) plus cocaine (10^–6 5 mol/l, n=15) pA₂ values for phentolamine of 6.9, 6.7, 6.6, and 6.3 respectively were obtained. Since these pA₂ values are not significantly different from 6.7, it is unlikely that this original pA₂ value, which is lower than values obtained with phentolamine at -adrenoceptors in other tissues, may be explained by neuronal or extraneuronal uptake of noradrenaline. A possible explanation may be that more than one population of -adrenoceptors contribute to changes in tension in peripheral lung strips. Send offprint requests to J. P. Seale at the above address     

α1B- but not α1A-adrenoceptors mediate inositol phosphate generation

Summary We used novel highly subtype-selective antagonists to study whether _1A- and/or _1B-adrenoceptors mediate the stimulation of inositol phosphate generation by noradrenaline in rat cerebral cortex. Phentolamine (10 M) and prazosin (100 nM) completely abolished the stimulated inositol phosphate generation. The _1A-selective antagonists 5-methyl-urapidil (100 nM) and (+)– and (–)-niguldipine (10 nM) caused only weak inhibition or none at all although these concentrations occupied _1A-adrenoceptors almost completely. In contrast, pretreatment with the irreversible _1B-selective chloroethylclonidine reduced the noradrenaline-stimulated inositol phosphate generation by 76 ± 8%. These data demonstrate that _1B-adrenoceptors couple to inositol phosphate generation; the signal transduction system of _1A-adrenoceptors remains unclear. Send offprint requests to G. Gross at the above address     

Inhibitory effect of tyramine-induced release of catecholamines on renin secretion

Summary The effect of the indirect sympathomimetic agent tyramine on the isoprenaline-induced increase in plasma renin concentration was investigated in conscious rats. Tyramine caused a dose-dependent decrease in the isoprenaline-induced elevation of plasma renin concentration. Pretreatment of the rats with reserpine abolished this effect of tyramine, indicating that tyramine released catecholamines which acted on the inhibitory adrenoceptors. Pretreatment with phenoxybenzamine, an -adrenoceptor antagonist, also abolished the inhibitory effect of tyramine on renin release, indicating that -adrenoceptors mediated the observed inhibition of renin release.In rats with chronically denervated kidneys tyramine did not inhibit renin release. It is concluded that catecholamines which are released from renal sympathetic nerve endings can suppress renin release by activating -adrenoceptors.Supported by DFG Me 541/1     

The effect of microelectrophoretically applied clonidine on single cerebral cortical neurones in the rat

Summary The technique of microelectrophoresis was used in order to examine the effects of clonidine on single neurones in the somatosensory cortex of the rat, and to compare its actions with those of noradrenaline and phenylephrine. Clonidine evoked only excitatory responses on cortical neurones. The clonidine-sensitive neurones were also excited by noradrenaline and phenylephrine. Clonidine had a consistently lower apparent potency than either noradrenaline or phenylcphrine. Responses to clonidine had a slower time-course than responses to the other two adrenoceptor agonists, both the latencies to onset and the recovery times being longer for responses to clonidine than for responses to noradrenaline and phenylephrine. When the mobilities of clonidine and phenylephrine were compared using an in vitro method, no significant difference was found between the mobilities of the two ionic species, suggesting that they have similar transport numbers. Thus the difference between the potencies and time-courses of responses to clonidine and phenylephrine are presumably of biological origin. Responses to clonidine were antagonised by microelectrophoretically applied prazosin; responses to phenylephrine were equally antagonised, while responses to acetylcholine were not affected. Clonidine could reversibly antagonise excitatory responses to both noradrenaline and phenylephrine, whithout affecting responses to acetylcholine. The results suggest that clonidine may act as a partial agonist at excitatory ₁-adrenoceptors on cortical neurones.     

Trifluoperazine and calmidazolium have multiple actions on the release of noradrenaline from sympathetic nerves of mouse atria

The aim of this study was to determine whether the calmodulin inhibitors trifluoperazine (TFP) and calmidazolium (CMZ) could decrease the action-potential-evoked release of noradrenaline from mouse isolated atria incubated with [³H]-noradrenaline in support of the hypothesis that calmodulin is involved in neurotransmitter release.TFP (10 M and 30 M) significantly enhanced stimulation-induced (S-1) outflow of radioactivity from mouse atria but had no effect at 1.0 M or 70 M. TFP (70 M) also significantly increased the spontaneous outflow of radioactivity. The facilitatory effect of TFP (10 M) on S-I outflow of radioactivity persisted in either the presence of 3-isobutyl-1-methylxanthine (100 M) or atropine (0.3 M) indicating that this effect of TFP was not mediated through either inhibition of phosphodiesterases or through interference with presynaptic muscarinic receptors, respectively. In the presence of phentolamine, the facilitatory effect of TFP (10 M) on S-I outflow was reduced but there was no effect on S-I outflow at 70 M. However, in the presence of a combination of both phentolamine (l.0 M) and the neuronal uptake blocker desipramine (1.0 M) a significant inhibitory effect of TFP (70 M) on the S-I outflow of radioactivity was observed, indicating that effects of TFP on presynaptic a-adrenoceptors and neuronal uptake had disguised an inhibitory effect on S-1 noradrenaline release. Another inhibitor of the Ca²⁺-calmodulin complex, calmidazolium (CMZ, 10 M) inhibited the S-1 outflow of radioactivity but had no effect at 1.0 M. However, CMZ (10 M) also induced a concomitant increase in the spontaneous outflow of radioactivity. In the presence of both phentolamine (1.0 M) and desipramine (1.0 M), CMZ (10 M) also decreased S-1 outflow of radioactivity. The spontaneous outflow of radioactivity by calmidazolium (10 M) was mainly attributable to a rise in unmetabolized noradrenaline.Since concentrations of both TFP and CMZ, which inhibited S-1 noradrenaline release, also caused an increase in the spontaneous outflow of radioactivity, it is possible that the inhibitory effects on S-1 noradrenaline release may be secondary to changes in spontaneous outflow. This suggests that these drugs have complex effects on transmitter release dynamics which are perhaps due to multiple roles for calmodulin within the sympathetic nerve terminal. Correspondence to: M. Barrington at the above address     

Preponderance of β- over α-adrenoceptors in mediating the positive inotropic effect of phenylephrine in the ferret ventricular myocardium

Summary [³H]prazosin bound to the membrane fraction derived from the ferret ventricular muscle with high affinity in a saturable manner (K _d = 0.25 nmol/l and B _max = 27 fmol/mg protein in the right ventricle). [³H]CGP-12177, a -adrenoceptor ligand, bound to the membrane fraction with a _{K d} value of 0.29 nmol/l and a B _max of 42 fmol/mg protein. In the isolated ferret papillary muscle driven at 1 Hz at 37°C, phenylephrine elicited a concentration-dependent positive intropic effect. The maximal effect of phenylephrine was comparable to that of isoprenaline. Prazosin (0.3 ol/l) shifted the concentration-response curve for phenylephrine slightly but significantly to the right, the maximal response being unaffected. In contrast, bupranolol (0.3 gmol/l) shifted the curve for phenylephrine markedly downwards: the maximal response was depressed significantly to 40% and the curve became less steep. In the presence of prazosin and bupranolol the curve was shifted to the right, being essentially parallel to the control curve. These results indicate that in the ferret ventricular myocardium both - and -adrenoceptors mediate the positive inotropic effect of phenylephrine. The extent of contribution of the two classes of adrenoceptor is quite different from that in other mammalian species. In the ferret heart, -adrenoceptors predominate over -adrenoceptors in mediating the positive inotropic effect of phenylephrine, although the number of -adrenoceptors is not especially high when compared with other species. Send offprint requests to M. Endoh at the above address     

Effects of ring substitution on the pre- and postjunctional alpha-adrenergic activity of aryliminoimidazolidines

Summary The pre- and postjunctional -adrenergic agonist potency of a series of aryliminoimidazolidines was determined in the isolated rabbit ear artery. This series included clonidine, an antihypertensive agent thought to act by stimulating brainstem -receptors and known to be a preferentially prejunctional -adrenergic agonist. Although all of the compounds acted preferentially on the prejunctional -adrenoceptor, ring substitution had a dramatic effect on both potency and the degree of selectivity. 2-(3,4-Dihydroxyphenylimino) imidazolidine was both the most potent and most selective prejunctional -agonist in this series.     

Effect of chronic lithium administration on adrenoceptor binding and adrenoceptor regulation in rat cerebral cortex

Summary Lithium (Li) at a concentration, which exerts prophylactic effects in affective disorders is known to alter noradrenaline turnover and the -adrenoceptor-dependent cAMP accumulation. In the present study the action of chronic Li administration (at least 5 weeks) on agonist and antagonist binding to adrenoceptors and on the regulation of adrenoceptors was investigated in rat cerebral cortex. Li treatment caused a small but significant decrease in the number of -adrenoceptor binding sites by 10% (³H-dihydroalprenolol binding) leaving the number of ₁- and ₂-adrenoceptor binding sites (³H-prazosin and ³H-rauwolscine, respectively) unchanged. The affinity of the radioligands as well as the affinity of agonists to these binding sites were not altered. The up-regulation of -adrenoceptor binding sites produced by repeated reserpine injections was inhibited by 32% in rats treated concomitantly with Li, although the noradrenaline depleting effect of reserpine was not impaired. In contrast, Li treatment had no effect on the up-regulation of -adrenoceptor binding induced by 6-OH-dopamine, nor did it alter the -adrenoceptor down-regulation following chronic administration of desipramine. The up-regulation of ₁-adrenoceptor binding sites caused by reserpine or 6-OH-dopamine also remained unaffected by Li. It is concluded that chronic Li has limited effects on cortical adrenoceptors and their regulation. The inhibition of -adrenoceptor up-regulation caused by reserpine may reflect an action of Li on non-adrenergic systems rather than a general stabilizing effect on adrenoceptors proposed previously.Send offprint requests to: G. Gross     

Effect of phorbol esters and polymyxin B on modulation of noradrenaline release in mouse atria

Summary Phorbol 12-myristate 13-acetate (PMA; 0.03, 0.1 and 1.0 mol/l), a protein kinase C activating phorbol ester, significantly enhanced the stimulation-induced (S-I) outflow of radioactivity at 5 Hz stimulation in mouse atria preincubated with [³H]-noradrenaline, whereas a phorbol ester which does not activate protein kinase C, phorbol 13-acetate (0.1 mol/l), had no effect. This suggests that protein kinase C may have a role in modulating sympathetic neurotransmission.Polymyxin B (7 and 21 mol/l), an inhibitor of protein kinase C, had no effect on the S-I outflow of radioactivity. However, it had a significant inhibitory effect in a concentration of 70 mol/l. Polymyxin B (21 mol/l) reduced the facilitation of the S-I outflow of radioactivity produced by PMA (0.03 mol/l), 8-bromo-cyclic AMP (90 mol/l), tetraethylammonium chloride (300 mol/l), and idazoxan (0.1 mol/l). Furthermore, when a higher frequency of stimulation was applied (10 Hz rather than 5 Hz), polymyxin B (21 pmol/1) by itself inhibited the S-I outflow of radioactivity.In the presence of a concentration of PMA (0.1 mol/l) that was maximally effective in enhancing the S-I outflow of radioactivity, both idazoxan (0.1 mol/l) and 8-bromocyclic AMP (90 mol/l) still enhanced the S-I outflow. This suggests that these agents are not operating through protein kinase C and further suggests that the inhibitory effect of polymyxin B on these agents cannot be due to inhibition of protein kinase C. The effects of clonidine on the S-I outflow were not affected by a maximally effective concentration of PMA (0.1 mol/l). These results suggest that protein kinase C is not involved in a ₂-adrenoceptor mediated modulation of noradrenaline release. Send offprint requests to I. F. Musgrave at the above address     

Release-inhibiting α2-adrenoceptors at serotonergic axons in rat and rabbit brain cortex: evidence for pharmacological identity with α2-autoreceptors

The pharmacological properties of the presynaptic ₂-adrenoceptors modulating the release of serotonin in rat and rabbit brain cortex (₂-heteroreceptors) were compared with the properties of presynaptic ₂-autoreceptors in the same brain area. Brain cortex slices were preincubated with [³H]-serotonin or [³H]-noradrenaline and then superfused and stimulated by brief high-frequency pulse trains.The ₂-adrenoceptor agonist bromoxidine reduced the electrically evoked overflow of tritium in experiments with both [³H]-noradrenaline and [³H]-serotonin and in brain slices from either species. The antagonists phentolamine, idazoxan, (+)-mianserin, rauwolscine, 5-chloro-4(1-butyl-1,2,5,6-tetrahydropyridin-3-yl)-thiazole-2-amine (ORG 20350), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), (–)-mianserin and corynanthine caused parallel shifts of the concentration-inhibition curves of bromoxidine to the right. Negative logarithms of antagonist dissociation constants pK_d were calculated from the shifts. In the rat, the ₂-autoreceptor pK_d value of each single antagonist was similar to its ₂-heteroreceptor pK_d value, maximal difference 0.4, giving a close correlation, r = 0.97 (P<0.001). In the rabbit equally, the ₂-autoreceptor pK_d value of each single antagonist was similar to its ₂-heteroreceptor pK_d value, maximal difference 0.4, again yielding a close correlation, r = 0.96 (P < 0.001). However, antagonist pK_d values at rat ₂-autoreceptors differed from those at rabbit ₂-autoreceptors, r = 0.70 (P > 0.05), and antagonist pK_d values at rat ₂-heteroreceptors differed from those at rabbit ₂-heteroreceptors, r = 0.64 (P > 0.05). Comparison with radioligand binding experiments from the literature indicated that, in the rat, both auto- and heteroreceptors conformed best to the _2D subtype (r 0.97, P < 0.01 for pK_d correlation with binding sites in rat submaxillary gland) whereas, in the rabbit, they conformed best to the _2A subtype (r 0.93, P < 0.01 for pK_d correlation with binding sites in HT29 cells).It is concluded that, in both the rat and the rabbit, the ₂-adrenoceptors modulating the release of serotonin are pharmacologically identical with the presynaptic ₂-autoreceptors. However, rat ₂-autoreceptors and -heteroreceptors differ pharmacologically from rabbit ₂-autoreceptors and -heteroreceptors. Presynaptic ₂-auto-as well as -heteroreceptors are _2D in the rat and _2A in the rabbit. Correspondence to: N. Limberger at the above address