Selective agonists reveal alpha(1A)- and alpha(1B)-adrenoceptor subtypes in caudal artery of the young rat

Multiple alpha(1)-adrenoceptors were evaluated in caudal artery of the young Wistar rat using selective agonists and antagonists. Arteries were exposed to the selective alpha(1A)-adrenoceptor agonist, A-61603 (N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl] methanesulfonamide) or to phenylephrine and to prazosin (alpha(1)-adrenoceptor antagonist), or the selective alpha(1A)-adrenoceptor antagonists 5-methylurapidil, RS 100329 (5-methyl-3-[3-[4-[2-(2,2,2,-trifluoroethoxy)phenyl]-1-piperazinyl]propyl]-2,4-(1H)-pyrimidinedione), RS 17053 (N-[2(2-cyclopropylmethoxy) ethyl]-5-chloro-alpha, alpha-dimethyl-1H-indole-3-ethanamide), and the selective alpha(1D)-adrenoceptor antagonist BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5] decane-7,9-dione). Results showed a 100-fold higher potency of A-61603 for the alpha(1)-adrenoceptor present in the artery, compared with phenylephrine. Prazosin displaced both agonists with high affinity, whereas 5-methylurapidil, RS 100329 and RS 17053 displaced A-61603 with high affinity, indicating the presence of alpha(1A)-adrenoceptors. The selective alpha(1A)-adrenoceptor antagonists blocked phenylephrine responses with low affinity, suggesting that phenylephrine activated a second receptor population in caudal artery. BMY 7378 antagonized with low affinity both A-61603 and phenylephrine-induced contractions, indicating absence of alpha(1D)-adrenoceptors in the vessel. The results suggest that functional alpha(1B)-adrenoceptors are present in caudal arteries of the young Wistar rat.     

The relationship between density of alpha-adrenoceptor binding sites and contractile responses in several porcine isolated blood vessels.

1. The aim of this study was to investigate constrictor alpha-adrenoceptors in three isolated blood vessels of the pig, the thoracic aorta (TA), the splenic artery (SA) and marginal ear vein (MEV) and then compare the functional response with the densities of alpha 1- and alpha 2-adrenoceptor binding sites in these and several other porcine vascular tissues, palmar common digital artery (PCDA), palmar lateral vein (PLV) and ear artery (EA). 2. Noradrenaline (NA), phenylephrine (PE) and UK14304 (all at 0.03-10 microM) elicited concentration-dependent contractions in the TA and MEV, with a rank order of potency of UK14304 > NA > PE. UK14304 produced maximal responses which were 58% (TA) and 65% (MEV) of that of NA. In the SA, UK14304 and PE produced maximal responses which were less than 10% and 50% of the NA-induced maximal response respectively, with an order of potency of NA > PE. In the SA, NA-induced contractions were competitively antagonized by prazosin (pA2 = 8.60 +/- 0.15). Further, rauwolscine (1-10 microM) antagonized NA-induced contractions with an apparent pKB of 6.09 +/- 0.11 (n = 6), indicating an action at alpha 1-adrenoceptors. The combination of the two antagonists at concentrations selective for alpha 1- (0.1 microM) and alpha 2-adrenoceptors (1 microM) had no greater effect than either antagonist alone. This suggests that the SA expresses only post-junctional alpha 1-adrenoceptors. 3. In the TA, prazosin produced non-parallel shifts in the NA-induced CRC and this was also observed with rauwolscine, where reductions in the maximal responses were also observed. In the MEV, prazosin was largely inactive in antagonizing NA-induced contractions. In both these vessels a combination of these two antagonists had a greater effect than either alone, indicating the presence of functional alpha 1- and alpha 2-adrenoceptors. The post-junctional alpha 2-adrenoceptors in all of these vessels were resistant to prazosin, suggesting the alpha 2-adrenoceptor to be of the alpha 2A/2D subtype. The expression of functional alpha 2-adrenoceptors was MEV > TA > PLV > PCDA > SA. 4. In radioligand binding studies using TA P2 pellet membranes, [3H]-prazosin and [3H]-RX821002 ([1,4-[6,7(n)-3H] benzodioxan-2-methoxy-2-yl)-2-imidazole) labelled different high affinity sites, and in competition studies using identical membranes corynanthine displaced [3H]-prazosin with 10 fold higher affinity than rauwolscine, indicating that [3H]-prazosin was selectively binding to alpha 1-adrenoceptor sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Specific binding of [3H]prazosin to myocardial cells isolated from adult rats

The characteristics of alpha-adrenoceptors in rat myocardium were investigated by specific binding of [3H]prazosin to cells isolated from adult rat heart by perfusion with collagenase and hyaluronidase. The cells were incubated in Krebs-Ringer bicarbonate buffer gassed with 95% O2 and 5% CO2 at 31 degrees with the appropriate concentrations of the different ligands. Non-specific binding was defined by the addition of 10(-5) mole/l. phentolamine. The binding of [3H]prazosin was saturable and reached equilibrium within 15 min. Scatchard analysis showed a straight line giving an apparent dissociation constant, Kd, equal to 155.9 +/- 8.0 pmole/l. and a maximal number of binding sites equal to 76.7 +/- 11.1 fmole/mg protein. Inhibition of specific [3H]prazosin binding by different adrenergic blockers showed the order of potency characteristic of alpha 1-adrenoceptors: prazosin much greater than phentolamine greater than yohimbine much greater than propranolol. Inhibition by adrenergic agonists showed the order of potency: adrenaline greater than noradrenaline = phenylephrine greater than isoprenaline. The same orders of potency were observed in the presence of propranolol. However, propranolol slightly decreased the affinity for noradrenaline and phenylephrine. Hofstee analyses of the inhibition curves showed two binding components for all ordinary alpha-adrenoceptor blockers and agonists including unlabelled prazosin. In contrast, [3H]prazosin showed only one binding component. Both binding components were of the alpha 1-adrenoceptor subtype according to the order of potency of blockers. The different ligands had different affinity ratios for the two binding components giving them different profiles. Trifluoperazine, a phenothiazine compound, also had high affinity for the [3H]prazosin binding sites. This drug, however, apparently detected one class of binding sites only, as interpreted from the Hofstee analysis. Hill analyses of the inhibition data consistently yielded Hill constants, nH, in the range 0.75-0.85 except for [3H]prazosin, where nH = 1.02 and for trifluoperazine, where nH = 1.07. Although the two binding components may serve different functions, it seems impossible at present to relate the negative and the positive inotropic components, respectively, of the alpha-adrenergic inotropic response observed in functional studies only to one or the other binding component.     

Characterization of the alpha-adrenoceptors in the female rabbit urethra.

A radioligand binding technique was used to evaluate the proportions of alpha 1- and alpha 2-adrenoceptors in crude membrane preparations obtained from the female rabbit bladder base and urethra. In addition, urethral rings were studied in vitro in an attempt to determine if alpha 1- and/or alpha 2-adrenoceptors are located postjunctionally in the urethral smooth muscle. Studies of the inhibition of [3H]-dihydroergocryptine binding by the selective alpha 1-adrenoceptor antagonist prazosin or the selective alpha 2-adrenoceptor antagonist rauwolscine revealed the alpha-adrenoceptor population to consist of approximately 25% alpha 1-adrenoceptors and 75% alpha 2-adrenoceptors. These proportions were confirmed in saturation studies with [3H]-prazosin and [3H]-rauwolscine. The sum of alpha 1- and alpha 2-adrenoceptors labelled by these selective alpha 1- and alpha 2-adrenoceptor antagonists was about equal to the number labelled by the non-selective alpha-adrenoceptor antagonist [3H]-dihydroergocryptine. Noradrenaline, as well as the selective alpha 1-adrenoceptor agonist phenylephrine and the selective alpha 2-adrenoceptor agonist clonidine, induced contractions of urethral ring preparations. Prazosin blocked contractions induced by phenylephrine to a greater extent than contractions induced by clonidine. The opposite was true for the inhibitory effect of rauwolscine. In addition to showing that both alpha 1- and alpha 2-adrenoceptor binding sites exist in membrane preparations of the rabbit bladder base and urethra, the results reveal the presence of both adrenoceptor subtypes postjunctionally in the rabbit urethra; and both mediate contraction of the smooth muscle.     

Alfuzosin, a selective alpha 1-adrenoceptor antagonist in the lower urinary tract.

1. Phenylephrine-induced contractions of rabbit isolated trigone and urethra were antagonized in a competitive manner by alfuzosin (pA2 7.44 and 7.30, respectively) and prazosin. 2. The characteristics of [3H]-prazosin binding to human prostatic adenoma tissue were evaluated. [3H]-prazosin was potently displaced by alpha 1-adrenoceptor specific agents including alfuzosin, its (+)- and (-)-enantiomers and prazosin (IC50 0.035, 0.023, 0.019 and 0.004 microM, respectively), but only weakly by alpha 2-adrenoceptor selective agents, for example, yohimbine (IC50 = 6.0 microM). 3. In the pithed rat, alfuzosin (0.03-0.3 mg kg-1, i.v.) markedly inhibited pressor responses produced by the alpha 1-selective agonist, cirazoline but inhibited only slightly responses to the alpha 2-selective agonist, UK 14,304. Alfuzosin (1 mg kg-1, i.v.) had minimal effects against responses mediated by stimulation of prejunctional alpha 2-receptors (UK 14,304-induced inhibition of sympathetic tachycardia). 4. In the anaesthetized cat, alfuzosin (0.001-1 mg kg-1, i.v.) and prazosin (0.001-0.3 mg kg-1, i.v.) produced dose-related inhibition of the increases in urethral pressure caused by stimulation of sympathetic hypogastric nerves. Prazosin was approximately 5 fold more potent than alfuzosin. When phenylephrine was employed to induce urethral and vascular alpha 1-mediated tone simultaneously, prazosin inhibited both stimuli with similar potency whereas alfusozin was 3-5 fold more potent against elevated urethral pressure. This functional uroselectivity of alfuzosin was more evident by the intraduodenal route, since doses of 0.03 and 0.1 mg kg-1 alfuzosin inhibited urethral pressure with minimal effects on arterial blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS)     

An examination of the postjunctional alpha-adrenoceptor subtypes for (-)-noradrenaline in several isolated blood vessels from the rabbit.

1. Postjunctional alpha-adrenoceptors in several isolated blood vessels from the rabbit have been characterized on the basis of the relative potency of the agonists noradrenaline (NA, non-selective), phenylephrine (alpha 1-selective) and UK-14304 (alpha 2-selective), and the potency of antagonists rauwolscine (alpha 2-selective) and corynanthine (alpha 1-selective) against contractions elicited by NA. In addition, the potency of prazosin against NA was also assessed in the venous preparations. 2. The thoracic aorta, ear artery and left renal vein appear to possess alpha 1-adrenoceptors since the agonist potency order was NA greater than phenylephrine greater than UK-14304, while corynanthine was 3-10 fold more potent than rauwolscine. 3. The ear vein appears to possess alpha 2-adrenoceptors. The rank order of agonist potency was UK-14304 greater than NA much greater than phenylephrine and all three agonists elicited responses of similar magnitude. Furthermore, rauwolscine was 30 fold more potent than corynanthine while prazosin failed to produce a concentration-dependent inhibition. 4. The saphenous vein and the plantaris vein appear to possess a mixture of both subtypes since the rank order of agonist potency was UK-14304 greater than NA much greater than phenylephrine, while responses elicited by UK-14304 were smaller than those to the other agonists. However, although rauwolscine was 20 to 100 fold more potent than corynanthine in both preparations, suggestive of predominantly alpha 2-adrenoceptors, prazosin was either potent (saphenous vein) or relatively inactive (plantaris vein). 5. The characteristics of postjunctional alpha 1- and alpha 2-adrenoceptors on isolated blood vessels from the rabbit are discussed in relation to the value of both the agonists, particularly NA, and the antagonists used in this study.     

Functional supersensitivity to the alpha 1-adrenoceptor agonist after repeated treatment with antidepressant drugs is not conditioned by beta-down-regulation

The effect of repeated administration of desipramine, and the (+)- and (-)-enantiomers of oxaprotiline (10 mg/kg i.p., twice daily for 14 days) on the binding of beta- and alpha 1-adrenoceptors in the cortex of the rat brain were studied. The functional consequences of such treatment were measured in a behavioural model, involving the exploratory activity of rats in response to administration of the alpha 1-agonist phenylephrine. Desipramine and (+)-oxaprotiline decreased the binding of [3H]dihydroalprenolol ([3H]DHA) to beta-adrenoceptors in the cortex, did not change the binding of [3H]prazosin to alpha 1-adrenoceptors, but enhanced behavioural responses to phenylephrine. A behavioural facilitation was also observed after administration of (-)-oxaprotiline, a substance which does not change the binding of [3H]DHA. These results indicate that a functional supersensitivity to the alpha 1-adrenoceptor agonist, after repeated treatment with antidepressants is not conditioned by beta-down-regulation.     

Effects of ring fluorination on the adrenergic properties of phenylephrine

The adrenergic properties of 2-, 4- and 6-fluorophenylephrine (2-FPE, 4-FPE, 6-FPE) were compared to those of phenylephrine (PE). The order of affinities of these compounds for alpha 1-adrenoceptors as determined by displacement of [3H]prazosin and [3H]WB-4101 binding to brain membranes was the same as the order of potencies for eliciting two alpha 1-adrenergic metabolic responses in guinea-pig cerebral cortical synaptoneurosomes, namely the stimulation of phosphatidylinositol turnover and the potentiation of 2-chloroadenosine-induced accumulation of cyclic AMP. In all cases the order was 6-FPE greater than PE greater than 4-FPE greater than 2-FPE. The order of affinities for alpha 2-adrenoceptors as determined by displacement of binding of [3H]clonidine to brain membrane was 6-FPE greater than PE greater than or equal to 4-FPE = 2-FPE. In contrast, the order of potencies for inhibition of forskolin-stimulated adenylate cyclase activity in human platelet membranes via an alpha 2-adrenoceptor was 6-FPE approximately equal to PE greater than 4-FPE much greater than 2-FPE. The FPEs and PE were partial agonists compared to epinephrine in human platelets. The affinities of these compounds for beta-adrenoceptors as determined by displacement of binding of [3H]dihydroalprenolol to brain membranes are 2-FPE greater than PE greater than or equal to 4-FPE much greater than 6-FPE. The FPEs and PE had positive chronotropic and inotropic effects in isolated guinea-pig atria apparently through the activation of a beta-adrenoceptor, since pindolol blocked the response while prazosin did not. 6-FPE appeared less active than the other PEs in atria. In fat cell membranes, 2-FPE was more potent than PE in stimulating adenylate cyclase via a beta-adrenoceptor, while 4-FPE and 6-FPE were inactive. Both, 2-FPE and PE were partial agonists in fat cells compared to isoproterenol. Of the three FPEs, 6-FPE represents a more potent and more selective agonist for alpha-adrenoceptors compared to beta-adrenoceptors than PE, while 4-FPE and, in particular, 2-FPE are less potent and selective as alpha-agonists.     

Vascular effects of tetramethylpyrazine: direct interaction with smooth muscle alpha-adrenoceptors.

The interaction of tetramethylpyrazine, a vasoactive ingredient of a Chinese traditional medicinal plant, with the vascular muscle alpha 1-adrenoceptors was investigated by a direct radioligand binding technique using [3H]prazosin and vascular smooth muscle microsomes isolated from dog aorta and mesenteric artery. Tetramethylpyrazine inhibited the binding of [3H]prazosin to vascular muscle membranes in a concentration-dependent manner at a suboptimal concentration of prazosin. Scatchard analysis of the effect of tetramethylpyrazine on the saturation profile of [3H]prazosin binding to vascular muscle microsomes of either arterial muscle indicated a substantial increase of Kd values (the affinity for prazosin) without a change in Bmax (maximal binding sites for prazosin). Thus, the present results provide supporting evidence that the inhibitory effect of tetramethylpyrazine on the vasoconstriction of dog mesenteric artery induced by phenylephrine in the earlier studies may be, at least, in part due to a direct action at the recognition sites of alpha 1-adrenoceptors. Amiloride and amiloride-related compounds, which shares a common pyrazine ring structure with tetramethylpyrazine and other related derivatives, also inhibits the binding of [3H]prazosin to aortic muscle microsomal membranes. Functional studies of dog saphenous vein also indicated that both tetramethylpyrazine and its ethyl derivatives inhibited the responses induced by phenylephrine and B-HT 920 in the presence and absence of extracellular Ca2+. Together with our earlier findings that amiloride also inhibits [3H]prazosin and [3H]rauwolscine binding to vascular muscle alpha 1- and alpha 2-adrenoceptors, the present radioligand binding study in canine arteries and functional study in saphenous veins suggest that the above compounds containing the pyrazine nucleus indeed interacted at the alpha-adrenoceptor sites.     

Lack of stereoselectivity in the inotropic and phosphodiesterase inhibitory effects of saterinone enantiomers

The enantiomers of the positive inotropic and a1-adrenoceptor blocking agent saterinone (+/-)-1,2-dihydro-5-[4-[2-hydroxy-3- [4-(2-methoxyphenyl)-1-piperazinyl]propoxy] phenyl]-6-methyl-2-oxo-3-pyridine-carbonitrile, BDF 8634) have been investigated with in vitro and in vivo models in laboratory animals. In the guinea pig papillary muscle, saterinone enantiomers had equipotent inotropic activity and were also as potent as racemic saterinone; the (R)-enantiomer, however, exhibited a greater efficacy than the related compounds. Saterinone and its enantiomers were equipotent in the inhibition of phosphodiesterase PDE III activity in the guinea pig myocardium. The equipotent inotropic effects were also observed after parenteral and enteral administration in cats. In receptor binding studies, (S)-saterinone was 10-fold more potent than (R)-saterinone by inhibiting [3H]-prazosin binding to specific alpha 1-adrenoceptor sites in rat brain cortex membranes. However, in the isolated thoracic aorta of the rabbit, (S)-saterinone was only 3-fold more potent than (R)-saterinone at preventing the pressor effects of phenylephrine. When the enantiomers were tested in vivo against the pressor effects of phenylephrine in the pithed rat, (S)-saterinone was only 2-fold more potent than (R)-saterinone in its alpha 1-adrenoceptor blocking potency. Thus the enantiomers of saterinone do not display enantio-selectivity in their inotropic and PDE III inhibitory effects in vitro, nor in their cardiotonic effects in vivo. There is a slight enantio-selectivity at alpha 1-adrenoceptors in receptor binding studies, but this is reduced to biologically irrelevant magnitude in functional studies in vitro and in vivo.     

Effect of (+)-niguldipine on myocardial alpha 1-adrenoceptors in the rabbit.

The influence of the alpha 1A-adrenoceptor subtype-selective antagonist (+)-niguldipine on the alpha 1-mediated positive inotropic effect was assessed in the isolated rabbit ventricular myocardium. (+)-Niguldipine displaced the specific binding of [3H]prazosin to a membrane fraction derived from rabbit ventricular muscle with high (Ki = 64.6 pmol/l; RH = 23%) and low (Ki = 7.08 nmol/l) affinity. (+)-Niguldipine displaced specific [3H]CGP-121177 binding only at very high concentrations (Ki = 118 nmol/l). (+)-Niguldipine at 0.1 pmol/l and higher shifted the concentration-response curve for the alpha 1-mediated positive inotropic effect downwards, but at higher concentrations (up to 10 and 100 nmol/l) it did not cause a further shift of the curve. (+)-Niguldipine (1-100 nmol/l) did not affect the beta-mediated positive inotropic effect and the basal force of concentration. (-)-Niguldipine also showed a selective inhibitory action on the alpha 1-adrenoceptor-mediated positive inotropic effect, but its affinity and potency were approximately 1-2 log units lower than those of (+)-niguldipine. The present results indicate that the alpha 1A-adrenoceptor subtype is involved in the alpha 1-mediated positive inotropic effect. (+)-Niguldipine (or (-)-niguldipine with lower affinity) is able to antagonize selectively the cardiac alpha 1A-adrenoceptor-mediated positive inotropic effect. The magnitude of the alpha 1A-mediated inotropic effect, however, may be much less than that mediated by the alpha 1B-subtype in the rabbit ventricular myocardium.     

Studies on YM-12617: A selective and potent antagonist of postsynaptic α1-adrenoceptors

YM-12617, 5-[2-[[2-(2-ethoxyphenoxy)ethyl]-amino]propyl]-2 -methoxybenzenesulfonamide HCl is a structurally new type of extremely potent alpha 1-adrenoceptor antagonist. Its alpha-adrenoceptor blocking properties have been compared with those of prazosin, phentolamine and yohimbine using both pharmacological and 3H-ligand binding techniques in vitro and in vivo. In the isolated rabbit aorta, a tissue known to contain mainly alpha 1-adrenoceptors at postjunctional sites, YM-12617 competitively antagonized noradrenaline-induced contraction with a pA2 value of 10.11. Although YM-12617 was also a competitive antagonist toward clonidine at prejunctional alpha 2-adrenoceptors in the isolated rat vas deferens, its affinity for these receptors (pA2 = 6.41) was 5,000 times lower than that displayed for the postjunctional alpha 1-adrenoceptors in the isolated rabbit aorta. YM-12617 displaced both 3H-WB 4101 and 3H-clonidine binding to rat brain membranes; however, the affinity of YM-12617 for alpha 1-adrenoceptors (pKi = 9.64) was 3800 times higher than that for alpha 2-adrenoceptors (pKi = 6.06). Based on pA2 values obtained in the isolated tissues and pKi values in the binding assays, YM-12617 was 2-18, 36-117 and 1,740-5,750 times more potent than prazosin, phentolamine and yohimbine in antagonizing alpha 1-adrenoceptors, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute effects of phenoxybenzamine on alpha-adrenoceptor responses in vivo and in vitro: relation of in vivo pressor responses to the number of specific adrenoceptor binding sites

Pressor responses to intravenous phenylephrine, an adrenoceptor agonist, and to the alpha 2-adrenoceptor-selective agonist guanabenz were examined in conscious rabbits 30 min after treatment with a range of doses of phenoxybenzamine (10(-5) to 5 mg/kg). The maximum number of specific prazosin- and clonidine-binding sites in the spleen of rabbits sacrificed 30 min after receiving phenoxybenzamine were measured using radioligand binding techniques. Treatment with phenoxybenzamine resulted in a dose-dependent reduction in the maximum pressor response to both phenylephrine and guanabenz, although phenoxybenzamine was a more potent antagonist at postsynaptic alpha 1- than at postsynaptic alpha 2-adrenoceptors. Phenoxybenzamine treatment also caused a dose-dependent reduction in specific [3H]prazosin and [3H]clonidine binding. The maximum in vivo pressor response to guanabenz was observed only when all specific clonidine-binding sites were present. There was a close correlation between in vitro receptor number and in vivo pressor responses for alpha 2-adrenoceptor stimulation but not for alpha 1-adrenoceptor-mediated responses. The maximum pressor response to phenylephrine could be obtained in rabbits in which the number of specific prazosin-binding sites was reduced by 60%. These experiments provide an approach to relating in vitro receptor number to in vivo responses.     

Characterization of the selectivity, specificity and potency of medetomidine as an alpha 2-adrenoceptor agonist

Medetomidine (4-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole) was tested for alpha 2-adrenoceptor agonist activity and compared to several reference agents. In binding studies carried out with rat brain membrane preparations, medetomidine showed high affinity for alpha 2-adrenoceptors, as measured by the displacement of [3H]clonidine (Ki 1.08 nM compared to 1.62, 3.20, 6.22 and 194 nM for detomidine, clonidine, UK 14,304 and xylazine, respectively). The affinity of medetomidine for alpha 1-adrenoceptors, as measured by [3H]prazosin displacement, was much weaker, yielding a relative alpha 2/alpha 1 selectivity ratio of 1620 which is 5-10 times higher than that of the reference compounds. Medetomidine caused a concentration-dependent inhibition of the twitch response in electrically stimulated mouse vas deferens with a pD2 value of 9.0 compared to that of 8.6, 8.5, 8.2 and 7.1 for detomidine, clonidine, UK 14,304 and xylazine, respectively. The effect of medetomidine was antagonized by idazoxan. In anaesthetized rats, medetomidine caused a dose-dependent mydriasis which could be reversed by alpha 2-adrenoceptor blockade. In receptor binding experiments and isolated organs medetomidine had no affinity or effects on beta 1-, beta 2-, H1, H2, 5-HT1, 5-HT2, muscarine, dopamine, tryptamine, GABA, opiate and benzodiazepine receptors. Based on these results, medetomidine can be classified as a potent, selective and specific alpha 2-adrenoceptor agonist.     

Alpha 1-adrenoceptor-mediated inositol phospholipid hydrolysis in rat cerebral cortex: relationship between receptor occupancy and response and effects of denervation

The characteristics of catecholamine-mediated breakdown of inositol phospholipids in rat cerebral cortex slices have been examined using a direct assay involving prelabeling with [3H]inositol and examining the production of labelled inositol phosphates in the presence of lithium. Noradrenaline produced a marked stimulation of inositol phosphate accumulation and this response could be potently and competitively antagonised by the alpha 1-adrenoceptor antagonist prazosin. The alpha 2-antagonist yohimbine was almost 1000-fold less potent at antagonising noradrenaline inositol phospholipid response. Noradrenaline and adrenaline were full agonists at alpha 1-adrenoceptors but phenylephrine and methoxamine were only partial agonists in their ability to stimulate inositol phospholipid metabolism. There was a significant correlation between the ability of a variety of agonists and antagonists to activate or inhibit [3H]inositol phosphate accumulation and their ability to displace the alpha 1-adrenoceptor selective ligand [3H]prazosin from specific binding sites when assays were performed on rat cerebral cortical slices under identical conditions. The similarity of EC50 values of agonists stimulating inositol phosphate accumulation and their IC50 values in [3H]prazosin binding experiments suggested a close relationship between receptor occupancy and alpha 1-mediated inositol phosphate accumulation. Further experiments were performed to examine this directly by inactivating alpha 1-adrenoceptors with the alkylating antagonist phenoxybenzamine. After washing out unbound antagonist, [3H]prazosin binding was reduced to a very similar proportion to that observed on the maximal noradrenaline-stimulated accumulation of [3H]inositol phosphates in the slices. The EC50 values for noradrenaline-stimulated inositol phosphate accumulation was unaltered and the affinity of [3H]prazosin for the remaining sites was equally unaffected. In rats treated 14 days previously with i.c.v. 6-hydroxydopamine (2 X 250 micrograms) there was a small increase in alpha 1-adrenoceptor binding sites but a parallel shift to the left in the noradrenaline [3H]inositol phosphate accumulation dose-response curve. On the other hand, the partial agonist phenylephrine induced a larger maximal response in denervated animals without a change in the EC50 values. When slices from 6-hydroxydopamine treated animals were preincubated with phenoxybenzamine, the loss in alpha 1-adrenoceptor binding sites was greater than the reduction in the maximal response to noradrenaline. This may indicate the development of a small receptor reserve after denervation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Involvement of the noradrenergic system in the seizures of epileptic El mice

We studied the role of the noradrenergic system in the seizures of epileptic El mice. To this end, the anticonvulsant activity of adrenergic drugs was tested with a scoring method, and the binding of [3H]dihydroalprenolol, [3H]prazosin and [3H]yohimbine was evaluated in whole brains and various brain regions from stimulated and unstimulated El mice, and their maternal ddy mice. The seizures of El mice were inhibited by noradrenaline, phenylephrine, oxymetazoline, clonidine and yohimbine in a dose-dependent manner. These preventive effects of alpha-adrenoceptor agonists were antagonized by pretreatment with alpha-adrenoceptor antagonists. The preventive effect of yohimbine was reversed by pretreatment with clonidine or alpha-methyl-p-tyrosine, although the latter drug did not affect the anticonvulsant effect of clonidine. The binding of [3H]dihydroalprenolol was the same in the three groups of mice. More [3H]prazosin was bound in the cerebellum and striatum, and there were more [3H]yohimbine binding sites in the whole brain, cerebral cortex, hippocampus and brainstem of stimulated and unstimulated El mice than in the same areas of ddy mice. These findings suggest that up-regulated alpha 1- and alpha 2-adrenoceptors are involved in the inhibition of the seizures of El mice.     

Pharmacological properties of the positive inotropic and alpha 1-adrenoceptor blocking agent saterinone

The pharmacological properties of saterinone [+/-)-1,2-dihydro-5-[4-[2-hydroxy-3-[4-(2-methoxyphenyl)-1-piperazinyl] propoxy]phenyl]-6-methyl-2-oxo-3-pyridine-carbonitrile, BDF 8634) were investigated in isolated organs of the guinea pig and in human platelets. Saterinone was found to be a potent antagonist at vascular alpha 1-adrenoceptors with a pA2-value of 8.46 +/- 0.12. Besides its affinity for alpha 1-adrenoceptors saterinone exerted a positive inotropic effect in the isolated papillary muscle at an EC50-value of 3.2 X 10(-6)mol/l indicating 10-fold greater potency than milrinone. Comparable EC50-values were also found for the inotropic, chronotropic and bronchodilatory actions of the drug, indicating a common mechanism for these effects. The inotropic effects were not mediated by beta-adrenergic or H2-histaminergic receptors, but were shown to involve an elevation of myocardial cyclic adenosine monophosphate (cAMP) content. Saterinone also inhibited crude cAMP phosphodiesterase (PDE) activity in homogenates obtained from guinea pig right ventricles. The IC50-value for PDE-inhibition was 2.3 X 10(-5) mol/l and thus at a higher concentration than the inotropic effect. Saterinone was a potent inhibitor of human platelet aggregation induced by adenosine diphosphate, collagen and arachidonate. Against the latter agonist, saterinone was about 40-fold more effective than acetylsalicylic acid. In conclusion, saterinone exhibited a dual mechanism of action--direct inotropic effects in the myocardium and alpha 1-receptor blockade in the guinea pig vasculature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis, alpha-adrenoceptors affinity and alpha 1-adrenoceptor antagonistic properties of some 1,4-substituted piperazine derivatives

A series of different 1,4-substituted piperazine derivatives (1-11) was synthesized. It comprised 1-(substituted-phenoxyalkyl)-4-(2-methoxyphenyl)piperazine derivatives (1-5); 1,4-bis(substituted-phenoxyethyl)piperazine derivatives (6-8) and 1-(substituted-phenoxy)-3-(substituted-phenoxyalkylpiperazin-1-yl)propan-2-ol derivatives (9-11). All compounds were evaluated for affinity toward alpha 1- and alpha 2-receptors by radioligand binding assays on rat cerebral cortex using [3H]prazosin and [3H]clonidine as specific radioligand, respectively. Furthermore alpha 1-antagonistic properties were checked for most promising compounds (1-5 and 10) by means of inhibition of phenylephrine induced contraction in isolated rat aorta. Antagonistic potency stayed in agreement with radioligand binding results. The most active compounds (1-5) displaced [3H]prazosin from cortical binding sites in low nanomolar range (Ki = 2.1-13.1 nM). Compound 10 showed slightly lower affinity for alpha 1-adrenoceptor (Ki = 781 nM). Compounds 2-5 displayed the strongest antagonistic activity with pA2 values ranging from 8.441 to 8.807. Compound 1 gave a pA2 value of 7.868, while compound 10 showed the weakest antagonistic potency, giving a pA2 value of 6.374. 1-[3-(2-Chloro-6-methylphenoxy)propyl]-4-(2-methoxyphenyl)piperazine hydrochloride (5) showed the best alpha 1- affinity properties with a Ki(alpha 1) value of 2.1 nM and it was 61.05 fold more selective toward alpha 1 than alpha 2-receptors. The best properties showed 1-[3-(2,6-dimethylphenoxy)propyl]-4-(2-methoxyphenyl)piperazine hydrochloride (4) with a Ki(alpha 1) value of 2.4 nM, a 142.13 fold better selectivity to alpha 1 - over alpha 2-adrenoceptors and the best antagonistic potency (pA2 = 8.807). It is worth to emphasized that all most promising compounds possessed an 1-(o-methoxyphenyl)piperazine moiety which probably plays an important role in the affinity to alpha-adrenoceptors.     

Characterization of alpha 2-adrenoceptor binding properties of imidazoline-like drugs, azoloazepine derivatives and beta-phenethylamine-like drugs in human platelet membranes

To characterize the agonist profile of alpha 2-adrenoceptor agonists (imidazoline-like drugs, azoloazepine derivatives, beta-phenethylamines-like drugs) on human platelets, the characteristics of alpha 2-adrenoceptors (KD, Bmax) have been evaluated and the affinity constants measured by displacement technique and computer-assisted analysis of the curves. Furthermore, since alpha 2-adrenoceptor agonists interact with the post-synaptic receptors in a calcium-operated channel, whether the effect of calcium-entry inhibitors (verapamil, nifedipine, diltiazem) is related to a competition with alpha 2-receptors has also been examined. By Scatchard analysis, it was calculated that in human platelets alpha 2-adrenoceptors have KD = 3.45 nM and Bmax = 247 fmol (mg protein)-1. As far as the potency is concerned, imidazoline-like drugs were the most potent agonists in human platelet alpha 2-adrenoceptors (guanabenz IC50 = 8.6 +/- 0.8 X 10(-8), B-HT 920 IC50 = 2.9 +/- 0.3 X 10(-7), (-)-adrenaline IC50 = 3.4 +/- 0.5 X 10(-7)). Among the calcium-entry inhibitors only verapamil antagonized [3H]rauwolscine binding: the effect was stereospecific, (-)-D 600 being more potent than (+)-D 600. Nifedipine and diltiazem did not affect alpha 2-receptor binding. It is concluded that human platelets alpha 2-receptors share the agonist potency profile of other tissues containing alpha 2-receptors (brain, pre-synaptic junction), and that among calcium-entry blockers only verapamil can antagonize alpha 2-agonists. Nifedipine and diltiazem do not appear to interact stereospecifically with alpha 2-adrenoceptors.