Influence of the alpha-2 agonist oxaminozoline (S3341) on firing rate of central noradrenergic and serotonergic neurons in the rat. Comparison with clonidine

The firing rate of central locus coeruleus (LC) noradrenergic neurons and dorsal raphe (DR) serotonergic neurons was recorded in rats anaesthetized with chloral hydrate. The iontophoretic application or the i.v. perfusion of S3341, a new antihypertensive drug or clonidine decreased the frequency of discharge of LC neurons. Depending on the mode of administration clonidine was 54-63 times more potent than S3341. The selectivity of action of both drugs on alpha-2 vs. alpha-1 adrenoceptors was confirmed using yohimbine and prazosin: yohimbine completely blocked the inhibitory effect of S3341 or clonidine while prazosin did not prevent this effect. S3341 and clonidine regularly reduced the firing rate of DR neurons during i.v. perfusion but not during iontophoretic application. From these experiments is it concluded that S3341 and clonidine have a direct inhibitory effect on LC neurons via stimulation of alpha-2 autoreceptors and that both drugs have an indirect inhibitory effect on DR neurons, probably via impairment of noradrenergic transmission. Clinical studies show that S3341 induces much less sedative side effects than clonidine. In view of the great difference in the potency of these drugs to inhibit the firing rate of monoaminergic neurons which are known to be involved in sleep mechanisms, it is possible that the electrophysiological effects reported here relate to the sedative effects of these drugs.     

Prejunctional alpha 2-adrenoceptor mediated inhibitory action of clonidine and B-HT 920, but not urapidil in guinea pig ileum

Isolated guinea pig ileal longitudinal muscle was stimulated transmurally with a frequency of 0.1 Hz, duration of 0.5 msec, and supramaximal voltage (80-100 V). Transmural stimulation induces ileal contractions via activation of cholinergic neurons. alpha 2-Adrenergic agonists block the response to transmural stimulation via activation of prejunctional alpha 2 receptors which inhibit release of acetylcholine from cholinergic nerve terminals. Urapidil has been reported to have alpha 2-agonistic actions, and therefore was compared to the prototypic alpha 2 agonists, clonidine and B-HT 920. Clonidine and B-HT 920 depressed responses to transmural stimulation in the guinea pig ileum. Clonidine was the most potent inhibitor of the contractions, followed closely by B-HT 920. Very high concentrations of urapidil were necessary to suppress nerve-induced contractions of the ileum. The effects of clonidine and B-HT 920, but not urapidil, were antagonized by the selective alpha 2 antagonist, yohimbine. In unstimulated preparations, in which exogenous acetylcholine was used to elicit contractions of the ileum, urapidil depressed the response while clonidine and B-HT 920 had no effect. When PGF1 alpha was used to contract the ileum, no inhibitory effects were noted for urapidil, clonidine, or B-HT 920. Therefore urapidil, only in high concentrations, inhibits the contraction to transmural stimulation by depressing the response at a postjunctional cholinergic site. No evidence was found that urapidil can act as an agonist at a prejunctional alpha 2-receptor site.     

Presynaptic alpha-adrenoceptor mediated inhibition of the neurogenic cholinergic contraction in the isolated intestinal bulb of the carp (Cyprinus carpio)

The effects of norepinephrine, epinephrine and clonidine on neurogenic cholinergic contraction were examined in the presence of a beta-adrenoceptor blocking agent, carteolol (5 X 10(-6) M), in the isolated intestinal bulb of the carp. Norepinephrine, epinephrine (10(-9)-10(-6) M) and clonidine (10(-8)-10(-5) M) inhibited the contraction induced by low frequency (2 or 5 Hz) transmural stimulation (TMS) without inhibiting the contraction induced by acetylcholine (ACh, 6 X 10(-8)-4 X 10(-7) M). Methoxamine (10(-4) M) and phenylephrine (10(-4) M) showed no such inhibitory effect on the TMS-induced contraction. The inhibitory effects of catecholamines and clonidine were decreased by phentolamine (5.4 X 10(-6) M) and yohimbine (10(-7)-10(-6) M) but not by prazosin (7 X 10(-7)-10(-6) M). Nicotine (10(-6)-10(-4) M) and serotonin (3 X 10(-8)-3 X 10(-6) M) caused contraction of the intestinal bulb indirectly by releasing endogenous ACh. This contraction was inhibited by norepinephrine, epinephrine and clonidine in a concentration-dependent manner. The present results suggest that catecholamines and clonidine inhibit cholinergic transmission via the activation of a presynaptic alpha-adrenoceptor (presumably of alpha-2 type) located on the cholinergic nerve terminals innervating the smooth muscle of the intestinal bulb of the carp.     

Existence of two types of postjunctional alpha-adrenoceptors in the isolated canine internal carotid artery

The pharmacological characteristics of postjunctional alpha-adrenoceptors in isolated canine internal carotid arteries were investigated by the use of selective agonists and antagonists for alpha 1- and alpha 2-adrenoceptors. Norepinephrine, phenylephrine, and xylazine caused concentration-dependent contractions in the helical strips. The contraction induced by 10(-4)M xylazine was significantly smaller than that produced by 10(-4)M norepinephrine or 10(-4)M phenylephrine. The contraction induced by 10(-4)M phenylephrine was almost the same value as that induced by 10(-4)M norepinephrine. Phentolamine (10(-8) and 10(-7)M) caused a parallel shift to the right of the concentration-response curve to norepinephrine. The contractile responses to low concentrations of norepinephrine were significantly suppressed by pretreatment with an alpha 2-antagonist such as yohimbine (10(-9) and 10(-8)M) or DG5128 (10(-7) and 10(-6)M). On the other hand, the responses to higher concentrations of norepinephrine were mainly reduced by low concentrations of an alpha 1-antagonist, prazosin (3 x 10(-10) and 3 x 10(-9)M). These results suggest that both alpha 1- and alpha 2-adrenoceptors are located on the plasma membrane of smooth muscle cells in canine internal carotid arteries and that the norepinephrine-induced contractions at low and high concentrations are mainly mediated by activation of alpha 2- and alpha 1-adrenoceptors, respectively.     

Alpha1 and alpha2 adrenoceptor mediated melanosome aggregatory responses in vitro in Oreochromis mossambica (Peters) melanophores

Effects of specific and non-specific adrenoceptor agonists and antagonists were examined on the isolated scale melanophores of O. mossambica in physiological Ringer solution. The responses were recorded as melanophore size index. It was observed that adrenaline, nor-adrenaline, phenylpropanolamine, clonidine and phenylepherine induced melanosome aggregation in a dose-dependent manner. Denervation of the fish melanophores increased the sensitivity of the melanophores to adrenaline but not to nor-adrenaline. Phentolamine (3.55 x 10(-5) M), prazosin (2.38 x 10(-5) M) and yohimbine (2.821 x 10(-5) M) significantly inhibited the aggregatory responses of the fish melanophores to adrenaline, nor-adrenaline, clonidine and phenylepherine. The blocking effect of yohimbine was significantly higher than that of prazosin. It is concluded that the effect of adrenaline is directly mediated through the receptors and alpha2 adrenoceptors are predominantly involved in the aggregatory responses of this fish melanophores, while alpha1 adrenoceptors presence has been indicated.     

Neurogenic dilatation and constriction of rat superior mesenteric artery in vitro: mechanisms and mediators

In the rat superior mesenteric arteries, the mechanical responses to perivascular nerve stimulation were characterized. The predominant response was contraction mediated by the release of norepinephrine, acting postjunctionally on alpha 1-adrenoceptors. These frequency-dependent contractions were unaffected by the alpha 2-selective adrenoceptor antagonist yohimbine, but were markedly attenuated by clonidine, the alpha 2-selective adrenoceptor agonist. In the presence of prazosin, the alpha 1-selective antagonist, a significant component of the nerve-mediated contraction was still present. At the concentrations used, prazosin, yohimbine, as well as clonidine acted as competitive antagonists of response to exogenous norepinephrine. This differential inhibition of norepinephrine- and nerve-mediated responses suggested the presence of distinct postjunctional adrenoceptors. The effects of clonidine and yohimbine are interpreted to arise from prejunctional modulation of norepinephrine release. In 30 of the 100 vessels studied, there was spontaneous myogenic tone. In these arteries, field stimulation caused frequency- and voltage-dependent relaxations. These responses were neural in origin, dependent on sympathetic nerve activity, but were nonadrenergic and noncholinergic in nature. Naloxone, indomethacin, and substance P inhibited these relaxations with no significant effect on the tone. The opioid agonist, 1-13 dynorphin relaxed these vessels and only naloxone inhibited this response. The effects of these agents were selective against field-stimulated responses since they did not alter the relaxation to the nonspecific agent sodium nitroprusside. These results provide circumstantial evidence for opioid-mediated vascular relaxation that is presynaptically modulated by prostanoids and substance P.     

Inhibitory effect of ginger (Zingiber officinale) on rat ileal motility in vitro

Ginger (Zingiber officinale rhizome) is a widespread herbal medicine mainly used for the treatment of gastrointestinal diseases, including dyspepsia, nausea and diarrhoea. In the present study we evaluated the effect of this herbal remedy on the contractions induced by electrical stimulation (EFS) or acetylcholine in the isolated rat ileum. Ginger (0.01-1000 microg/ml) inhibited both EFS- and acetylcholine-evoked contractions, being more potent in inhibiting the contractions induced by EFS. The depressant effect of ginger on EFS-induced contractions was reduced by the vanilloid receptor antagonist capsazepine (10(-5) M), but unaffected by the alpha(2)-adrenergic antagonist yohimbine (10(-7) M), the CB(1) receptor antagonist SR141716A (10(-6) M), the opioid antagonist naloxone (10(-6) M) or by the NO synthase inhibitor L-NAME (3 x 10(-4) M). Zingerone (up to 3 x 10(-4) M), one of the active ingredients of ginger, did not possess inhibitory effects. It is concluded that ginger possesses both prejunctional and postjunctional inhibitory effects on ileal contractility; the prejunctional inhibitory effect of ginger on enteric excitatory transmission could involve a capsazepine-sensible site (possibly vanilloid receptors).     

Clonidine-induced coronary vasodilatation in isolated guinea pig heart is not mediated by endothelial alpha2 adrenoceptors.

Functional role of endothelial alpha(2)-adrenoceptor in coronary circulation remains unclear. Clonidine, an agonist of alpha(2)-adrenoceptors, was reported to induce coronary vasodilatation via stimulation of endothelial alpha(2)-adrenoceptors or coronary vasoconstriction involving vascular smooth muscle alpha(2)-adrenoceptors. Moreover, H(2) receptor-dependent responses to clonidine were described. Here, we reassess the contribution of endothelial alpha(2)-adrenoceptor and H(2) receptors to coronary flow and contractility responses induced by clonidine in the isolated guinea pig heart. We found that clonidine (10(-9) - 10(-6) M) produced concentration-dependent coronary vasoconstriction without a significant change in contractility. This response was inhibited by the alpha(1)/alpha(2)-adrenoceptor antagonist - phentolamine (10(-5) M) and the selective alpha(2)-adrenoceptor antagonist yohimbine (10(-6) M), but it was not changed by the selective alpha(1)-adrenoceptor antagonist prazosin (10(-6) M). In the presence of nitric oxide synthase inhibitor, L-NAME (10(-4) M) the clonidine-induced vasoconstriction was potentiated. Clonidine at high concentrations of 10(-5) - 3 x 10(-5) M produced coronary vasodilatation, and an increase in myocardial contractility. These responses were abolished by a selective H(2)-receptor antagonist, ranitidine (10(-5) M), but not by phentolamine (10(-5) M). We conclude that in the isolated guinea pig heart, clonidine-induced vasoconstriction is mediated by activation of smooth muscle alpha(2)-adrenoceptors whereas clonidine-induced coronary vasodilatation is mediated by activation of vascular H(2) histaminergic receptors. Accordingly, endothelial alpha(2)-adrenoceptors does not seem to play a major role in coronary flow response induced by clonidine.     

Inhibition of cholinergic neurotransmission in human airways by beta 2-adrenoceptors

The purpose of the study was to determine whether catecholamines modulate cholinergic neurotransmission in isolated human airway smooth muscle. Bronchial rings were suspended in organ baths for isometric measurement of tension, and contractions were induced by either electrical field stimulation (EFS) or exogenous acetylcholine (ACh). Isoproterenol, epinephrine, and norepinephrine in that order of potency produced concentration-dependent inhibition of comparable responses to EFS and ACh. However a potency difference of 100-fold for isoproterenol (IC50 = 4.80 X 10(-8) M for EFS and 3.70 X 10(-6) M for ACh) and 10-fold for both epinephrine and norepinephrine was observed for inhibition of responses to EFS compared with responses to ACh. The inhibitory effects of isoproterenol on responses to EFS were prevented by propranolol and ICI 118551 (a beta 2-antagonist) but not by betaxolol (a beta 1-antagonist). Tyramine had no effect on contractions elicited by EFS. These experiments demonstrate that beta-agonists inhibit cholinergic nerve-induced contractions of human bronchi more potently than contractions induced by exogenous ACh, suggesting modulation of cholinergic neurotransmission by prejunctional beta 2-receptors.     

Binding characteristics of naftopidil and alpha 1-adrenoceptor antagonists to prostatic alpha-adrenoceptors in benign prostatic hypertrophy.

Binding properties of naftopidil and alpha 1-adrenoceptor antagonists to alpha-adrenoceptors in prostates from benign prostatic hypertrophy (BPH) were characterized by radioreceptor assays using [3H]prazosin and [3H]rauwolscine. Specific binding of [3H]prazosin and [3H]rauwolscine in human prostatic membranes was saturable and of high affinity, and it showed a pharmacological specificity which characterized alpha 1 and alpha 2-adrenoceptors, respectively. Naftopidil and several alpha 1 antagonists competed for prostatic [3H]prazosin binding in order: R-(-)-YM-12617 greater than prazosin greater than bunazosin greater than terazosin greater than naftopidil greater than urapidil, and the inhibitory effect (Ki = 11.6 nM) of naftopidil was 10 to 45 times less potent than quinazoline derivatives such as prazosin, bunazosin and terazosin. The potencies of these antagonists in competing for [3H]prazosin binding sites in human prostates correlated well with their pharmacological potencies (pA2). Scatchard analysis indicated that the decrease of prostatic [3H]prazosin binding by naftopidil was due to a marked increase in the Kd value without a change in the Bmax value. The inhibition of prostatic [3H]prazosin binding by naftopidil was reversible. Naftopidil also inhibited prostatic [3H]rauwolscine binding (Ki = 70.0 nM). Thus, it is suggested that naftopidil antagonizes alpha 1-adrenoceptors in human prostates in a competitive and reversible manner.     

Alpha 2-adrenoceptor agonists stimulate growth hormone secretion but have no acute effects on plasma cortisol under basal conditions

Ten normal male subjects were administered clonidine (0.1 and 0.2 mg) or a highly-selective alpha 2-adrenoceptor agonist S 3341 (1 and 2 mg) on separate occasions in a double-blind randomised placebo-controlled study; blood samples were obtained for measurement of serum GH and plasma cortisol via an indwelling venous cannula for 4 h after each drug administration. Both clonidine and S 3341 were equally effective at lowering supine, and particularly standing BP; both also caused mild sedation, although this was slightly less marked for S 3341. High doses of both clonidine and S 3341 significantly increased serum GH (peak increment after clonidine: 18.8 +/- 5.5 mU/l (mean +/- SEM); peak increment after S 3341: 21.1 +/- 4.8 mU/l). Neither drug affected plasma cortisol. It is concluded that GH release may be stimulated by alpha 2-adrenoceptor agonism. As S 3341 has slightly less central activity than clonidine at the doses employed, but is at least equally potent at stimulating GH release, it is probable that the alpha 2-adrenoceptor stimulation of GH release occurs outside the blood-brain barrier. The pituitary-adrenal axis appears resistant to manipulation of alpha 2-adrenoceptors under basal conditions.     

Effects of dried bonito (katsuobushi) and captopril, an angiotensin I-converting enzyme inhibitor, on rat isolated aorta: a possible mechanism of antihypertensive action

In order to elucidate the mechanism of the antihypertensive action of dried bonito (katsuobushi), we compared the effects of dried bonito extracts with those of captopril, an angiotensin I-converting enzyme (ACE) inhibitor, on aorta preparations isolated from rats. Dried bonito extracts (3 x 10(-4) to 3 x 10(-3) g/ml) more potently relaxed contractions induced by norepinephrine (10(-7) M) than contractions induced by KCl (55.9 mM). Dried bonito extracts (3 x 10(-3) g/ml) slightly inhibited 10(-7) M angiotensin I-induced contractions. In contrast, captopril (10(-8) to 10(-7) M) did not affect 10(-7) M norepinephrine- or 55.9 mM KCl-induced contractions, but a higher concentration of captopril (10(-6) M) very slightly relaxed it. Captopril (10(-8) to 10(-6) M) markedly inhibited 10(-7) M angiotensin I-induced contractions in a concentration-dependent manner. These results suggest that antihypertensive mechanism of action induced by dried bonito involves direct action on vascular smooth muscle in addition to ACE-inhibitory activity.     

Differential cardiovascular effects of central clonidine and B-HT 920 in conscious rats

The effect of intracerebroventricular (i.c.v.) injection of the alpha 2-adrenoceptor agonists clonidine and B-HT 920 on mean arterial pressure (MAP), heart rate (HR), and plasma concentrations of noradrenaline and adrenaline was examined in conscious unrestrained rats. The injection of 1.0 microgram clonidine significantly decreased MAP and slightly decreased HR. Plasma noradrenaline and adrenaline levels were slightly but not significantly decreased after the injection of 1 microgram clonidine. In contrast, the injection of 0.1-10.0 micrograms B-HT 920 increased MAP and decreased HR. Plasma noradrenaline and adrenaline levels were slightly increased after the injection of the 1- and 10-micrograms doses. The i.c.v. injection of the alpha 2-antagonist rauwolscine slightly but not significantly increased MAP and plasma noradrenaline and adrenaline levels. The responses to i.c.v. injection of clonidine and B-HT 920 were not changed by prior administration of rauwolscine. Neither the pressor response to B-HT 920 nor the depressor response to clonidine was abolished by rauwolscine, suggesting that neither response was mediated by alpha 2-adrenoceptors.     

The effects of xylazine and alpha-adrenoreceptor antagonists on bovine uterine contractility in vitro

The effects of alpha-adrenoreceptor antagonists prazosin (alpha-1), yohimbine (alpha-2), and idazoxan (alpha-2) on xylazine-induced bovine uterine contractility were tested in vitro. Uterine strips from proestrous/estrous and diestrous cows were mounted in tissue baths containing Tyrode's solution. Changes in uterine contractility were measured by strain gauge. The following results were observed: 1) Xylazine increased uterine contractility in a dose dependent manner (cumulative concentrations: 10(-8), 3x10(-8), 10(-7), 3x10(-7) and 10(-6)M). 2) Idazoxan (10(-8), 10(-7) and 10(-6)M) and yohimbine (10(-6), 10(-5) and 10(-4)M) antagonized uterine contractility induced by xylazine in a dose-dependent manner. Idazoxan was approximately 50 to 100 times more potent than yohimbine. 3) Prazosin (10(-5)M) did not alter the effect of xylazine on uterine contractility. These results suggested that xylazine-induced uterine contractility in the cyclic cow is directly mediated by myometrial alpha-2 adrenoreceptors.     

Identification and characterization of (3H)-rauwolscine binding to alpha 2-adrenoceptors in the canine saphenous vein

The biochemical exploration of the alpha 2-adrenergic receptors was investigated in the canine saphenous vein using the highly selective alpha 2-adrenergic antagonist rauwolscine as a tritiated ligand. Following an enzymatic digestive pretreatment, we isolated a purified smooth muscle cell membrane fraction from saphenous veins in quantity sufficient to permit us to study the venous alpha 2-adrenoceptor content. The binding of tritiated rauwolscine was rapid, specific, saturable and reversible. The presence of high affinity binding sites (Kd = 1.53 +/- 0.71 nM) with a density of binding Bmax of 125.2 +/- 43.1 fmol/mg protein was demonstrated on a unique class of non interacting sites (nHill = 1.001 +/- 0.06). The kinetically derived Kd was 1.28 nM, in good agreement with the value obtained from saturation isotherms. The pharmacological profile of these sites was assessed by the comparison of the potency of alpha-adrenergic agonists and antagonists to inhibit 1 nM (3H)-rauwolscine. Their efficacy was respectively: rauwolscine greater than phentolamine greater than RX 781094 greater than clonidine much greater than prazosin greater than (-)-phenylephrine greater than (-)-noradrenaline. The results showed that (3H)-rauwolscine bound specifically to sites in our membranal preparation, which had the pharmacological characteristics of the alpha 2-adrenoceptors. The correlation between biochemical and pharmacological data revealed the usefulness of binding methods in the further study of adrenergic mechanisms in the canine saphenous vein.     

Selective alpha2-adrenergic properties of dexmedetomidine over clonidine in the human forearm.

We tested the hypothesis that dexmedetomidine (Dex) has greater alpha(2)- vs. alpha(1) selectivity than clonidine and causes more alpha(2)-selective vasoconstriction in the human forearm. After local beta-adrenergic blockade with propranolol, forearm blood flow (plethysmography) responses to brachial artery administration of Dex, clonidine, and phenylephrine (alpha(1)-agonist) were determined in healthy young adults before and after alpha(2)-blockade with yohimbine (n = 10) or alpha(1)-blockade with prazosin (n = 9). Yohimbine had no effect on phenylephrine-mediated vasoconstriction but blunted Dex-mediated vasoconstriction (mean +/- SE: -41 +/- 5 vs. -11 +/- 2%; before vs. after yohimbine) more than clonidine-mediated vasoconstriction (-39 +/- 5 vs. -28 +/- 4%; before vs. after yohimbine) (P < 0.02). Prazosin blunted phenylephrine-mediated vasoconstriction (-39 +/- 4 vs. -8 +/- 2%; before vs. after prazosin) but had similar effects on both Dex- (-30 +/- 4 vs. -39 +/- 6%; before vs. after prazosin) and clonidine-mediated vasoconstriction (-29 +/- 3 vs. -41 +/- 7%; before vs. after prazosin) (P > 0.7). Both Dex and clonidine reduced deep forearm venous norepinephrine concentrations to a similar extent (-59 +/- 12 vs. -55 +/- 10 pg/ml; Dex vs. clonidine, P > 0.6); this effect was abolished by yohimbine and blunted by prazosin. These results suggest that Dex causes more alpha(2)-selective vasoconstriction in the forearm than clonidine. The similar vasoconstrictor responses to both drugs after prazosin might be explained by the presynaptic effects on norepinephrine release.     

Pharmacological analysis of the mechanisms involved in the tachycardic and vasopressor responses to the antimigraine agent, isometheptene, in pithed rats

The present study set out to investigate the pharmacological profile of the cardiovascular responses induced by the antimigraine agent, isometheptene, in pithed rats. For this purpose, intravenous (i.v.) administration of blocking doses of the antagonists prazosin (alpha1; 100 microg/kg), rauwolscine (alpha2; 300 microg/kg), the combination of prazosin (100 microg/kg) plus rauwolscine (300 microg/kg), propranolol (beta; 1000 microg/kg), ritanserin (5-HT2; 100 microg/kg) or equivalent volumes of saline (1 ml/kg) were used. Isometheptene (0.03, 0.1, 0.3, 1 and 3 mg/kg, i.v.) produced dose-dependent increases in heart rate and diastolic blood pressure which were highly reproducible as they remained unaltered after saline. These tachycardic responses to isometheptene remained unaffected after prazosin, rauwolscine, ritanserin or the combination prazosin plus rauwolscine, but were abolished after propranolol. In contrast, the isometheptene-induced vasopressor responses were not significantly modified after the above doses of rauwolscine, ritanserin or propranolol, but were markedly blocked after prazosin or the combination of prazosin plus rauwolscine; the latter blockade did not significantly differ from that produced by prazosin alone. Interestingly, in rats pretreated intraperitoneally (i.p.) with reserpine (5 mg/kg; -24 h), isometheptene-induced tachycardic responses were abolished whereas the corresponding vasopressor responses were markedly attenuated and subsequently blocked by prazosin. It is concluded that isometheptene-induced tachycardic responses seem to involve only an indirect (tyramine-like action) mechanism mediated by beta-adrenoceptors, whilst the corresponding vasopressor responses are mediated by a predominantly indirect (tyramine-like action), as well as a minor direct (alpha1-adrenoceptors), sympathomimetic mechanism.     

α-adrenergic inhibition of cyclic AMP accumulation in hamster adipocytes similarity of receptor with α-2 adrenergic receptors

The present communication shows the effects of several α-adrenergic agonists and antagonists on cyclic AMP levels in hamster epididymal adipocytes. In response to ACTH (30 mU/ml) in combination with 1-methyl-3-isobutylxanthine (0.10 mM) or adenosine deaminase (1.0 μg/ml), cyclic AMP levels increased to a maximum by 10 min and this level was maintained for another 20 min. Elevated cyclic AMP levels were partially suppressed by the α-adrenergic agents clonidine, methoxamine, methyl norepinephrine and phenylephrine. The lowest effective concentration of each of these agonists required to suppress cyclic AMP levels was 10 nM clonidine; 3 μM methoxamine; 10 μM methyl norepinephrine; 10 μM phenylephrine. Clonidine and methoxamine suppressed cyclic AMP levels by nearly 65% while phenylephrine and methyl norepinephrine caused only a 30% decline. Studies of the relative potencies of α-adrenergic blocking drugs on prevention of the inhibitory effect of clonidine on cyclic AMP levels disclosed that phentolamine and yohimbine were more potent blockers of clonidine action than phenoxybenzamine and prazosin. The rank order of potencies of agonists at causing suppression of cyclic AMP levels and the rank order of potencies of antagonists of clonidine action suggest similarity of the α-adrenergic receptors present on hamster adipocytes, which affect cyclic AMP accumulation to α-2 adrenergic receptors.     

Stereoselective and nonstereoselective inhibition exhibited by the enantiomers of verapamil

The interaction of the isomers of verapamil with sites on the calcium channel and alpha 1-adrenergic receptor has been examined. The inhibitory potency of these enantiomers differ with respect to the agonist. KCl- or clonidine-induced contractions of rabbit aortic rings were inhibited in a stereoselective manner by the enantiomers of verapamil with the (-)-isomer being more potent than the (+)-isomer. Similarly, (-)-verapamil was also more potent at displacing (-)-[N-methyl-3H]desmethoxyverapamil than was the (+)-isomer. In contrast, the inhibition of norepinephrine- or phenylephrine-induced aortic contractions was not stereoselective. Differences in enantiomer potency were also observed in vivo. The ability of clonidine to increase blood pressure in the anesthetized rat was blocked in a stereoselective manner by the verapamil enantiomers, while inhibition of the pressor actions of phenylephrine was not. In summary, for agents that rely heavily on calcium channel function (KCl, clonidine), stereoselective inhibition was observed. Stereoselective inhibition was not observed against high efficacy alpha 1-agonists. This difference in stereochemistry argues that verapamil does not act at the same site when inhibiting clonidine or KCl action when compared with norepinephrine or phenylephrine.     

Stable expression of recombinant human alpha 2-adrenoceptor subtypes in two mammalian cell lines: characterization with [3H]rauwolscine binding, inhibition of adenylate cyclase and RNase protection assay.

Cloning of the genes encoding distinct subtypes of human alpha 2-adrenergic receptors (alpha 2-AR) allows the separate recombinant expression of each individual subtype in heterologous systems. We report here the transfection, selection and preliminary pharmacological characterization of two mammalian cell lines, adherent Shionogi S115 mouse mammary tumour cells and human B-lymphoblastoid IBW4 cells growing in suspension, expressing the human alpha 2-AR subtypes alpha 2-C4 and alpha 2-C10 at densities of approx. 2 x 10(5) receptors/cell. Transfection of the subtype genes was verified using a specific RNase protection assay. Pharmacological characterization was carried out with [3H]rauwolscine binding, which was inhibited by oxymetazoline and prazosin in a subtype-selective manner. The sensitivity of (-)-noradrenaline binding to the GTP-analogue 5'-guanylylimidodiphosphate suggested that the receptors are coupled to G-proteins. This was verified in S115 cells by efficient inhibition of forskolin-stimulated cAMP production by the alpha 2-AR agonists, (-)-noradrenaline and clonidine. These cell lines thus appear to be suitable for pharmacological studies on receptor function and ligand binding.