Alpha 2-adrenoceptor Binding Sites Research Articles

Identification of a single α1‐adrenoceptor corresponding to the α1A‐subtype in rat submaxillary gland

1. The alpha 1-adrenoceptors present in membranes of rat liver, cortex and submaxillary gland were labelled with [3H]-prazosin and the affinity of 15 ligands for these receptors was determined. 2. In saturation studies, [3H]-prazosin bound with high affinity (Kd = 30-39 pM) to a single population of sites in all three preparations. 3. In competition studies using rat cortex, evidence for heterogeneity of the alpha 1-adrenoceptor binding sites was obtained. Displacement isotherms for amidephrine, benoxathian, oxymetazoline, phentolamine and WB 4101 were biphasic and were consistent with the presence of both alpha 1A- and alpha 1B-adrenoceptor subtypes as described by Morrow & Creese (1986) and Han et al. (1987). 4. The rat liver and submaxillary gland membrane preparations both possessed homogeneous populations of alpha 1-adrenoceptors. However, there were pharmacological differences between the receptors in these two preparations. Rat submaxillary gland alpha 1-adrenoceptors displayed high affinity for amidephrine, benoxathian, oxymetazoline, phentolamine and WB 4101 and therefore appeared to represent alpha 1A-adrenoceptors. Rat liver alpha 1-adrenoceptors possessed lower affinity for these ligands (6-65 fold) suggesting that these receptors were of the alpha 1B-subtype. 5. Spiperone exhibited 12.9 fold higher affinity for rat liver alpha 1B-adrenoceptors than for rat submaxillary gland alpha 1A-adrenoceptor and may therefore represent the first alpha 1B-adrenoceptor selective ligand.

Inhibition of Lipolysis by Agents Acting Via Adenylate Cyclase in Fat Cells from Infants and Adults

The in vitro lipolytic effect of catecholamines is poor during infancy because of enhanced alpha 2-adrenoceptor activity. The mechanisms behind this were investigated in isolated fat cells obtained from 1- to 4-mo-old infants and from adults. The cells were incubated with agents that inhibit lipolysis through distinct receptors coupled to adenylate cyclase via the inhibitory GTP binding coupling protein, Gi. The sensitivity to the alpha 2-adrenoceptor agonist clonidine was 14 times higher in the infant group as compared to the adults, whereas that to an adenosine analogue was 14 times lower. The sensitivities to prostaglandin E2 and nicotinic acid were similar in both age groups. Preincubation of the adipocytes with pertussis toxin abolished the antilipolytic effects of all agents. The density of alpha 2-adrenoceptor binding sites determined with [3H]yohimbine was increased by about 25% in the infants. In conclusion, the antilipolytic sensitivity of adenosine and alpha 2-adrenoceptors develops separately and may play different roles in the regulation of lipolysis in man. Furthermore, the enhanced alpha 2-adrenoceptor sensitivity during infancy seems at least in part to be due to an increase in the number of receptors.

Subclassification of α1‐adrenoceptor recognition sites by urapidil derivatives and other selective antagonists

1. The affinities of urapidil derivatives and other antagonists for alpha 1-adrenoceptors labelled by [3H]-prazosin were determined on membranes of six different rat tissues. 2. Urapidil and its 5-acetyl-, 5-formyl- and 5-methyl-derivative displaced [3H]-prazosin from alpha 1-adrenoceptor binding sites in a concentration-dependent manner which varied with tissue. IC50 values were lower in vas deferens, hippocampus and cerebral cortex than in heart, liver and spleen. For 5-methyl-urapidil, binding to two distinct sites could be demonstrated with mean K1 values of about 0.6 and 45 nM. Saturation binding studies with [3H]-prazosin in the presence of 5-methyl-urapidil indicated a competitive type of interaction between 5-methyl-urapidil and [3H]-prazosin. 3. The proportion of [3H]-prazosin binding sites with high affinity for 5-methyl-urapidil was 58% in vas deferens, 69% in hippocampus, 41% in cerebral cortex and 23% in myocardium. In liver and spleen virtually no high affinity sites were found. These values were in good agreement with the percentages of binding sites with high affinities for WB-4101 and phentolamine, indicating that all these antagonists bind to the same subtype of alpha 1-recognition sites, whereas other alpha-antagonists like BE 2254, yohimbine and unlabelled prazosin did not discriminate between two binding sites. 4. Preincubating membranes of the cerebral cortex with chloroethylclonidine preferentially inactivated [3H]-prazosin binding sites with low affinity for 5-methyl-urapidil. 5. The antagonist potencies of 5-methyl-urapidil and WB-4101 against alpha 1- adrenoceptor-mediated contractile responses were higher in vas deferens than in myocardium. The alpha 1-mediated effects in vas deferens but not in the heart were highly susceptible to nitrendipine. 6. Using 5-methyl-urapidil, the existence of two distinct alpha 1-adrenoceptor recognition sites could be demonstrated which correspond to the proposed alpha 1A- and alpha 1B-subtypes. Since 5-methyl-urapidil is one of the ligands with most selectivity between these subtypes in binding studies it may serve as a valuable tool for such investigations.

The stereospecificity of LY253352 for alpha 1-adrenoceptor binding sites in the brain and prostate.

1. The stereospecificity of the enantiomers of LY253352, a potent and selective alpha 1-adrenoceptor antagonist, were studied in the human prostate and canine brain using radioligand receptor binding methods. 2. The mean equilibrium dissociation constant (KD) in the canine brain and human prostatic adenoma was 84.4 pM and 65.4 pM, respectively. 3. The alpha 1-adrenoceptor density in the canine brain was approximately eight fold greater than in the human prostatic adenoma. 4. The mean Ki values of (-)-LY253352 and (+)-LY253352 in the prostate were 0.19 nM and 5.79 nM, respectively. 5. The mean Ki values of (-)-LY253352 and (+)-LY253352 in the brain were 0.29 nM and 34.7 nM, respectively. 6. This study indicates that the stereochemical specificity of the optical isomers of LY253352 is a manifestation of differential affinities of the enantiomers for alpha 1-adrenoceptor binding sites. 7. The differential affinities of (+)-LY253352 in the brain and prostate are suggestive of subtle unique properties of adrenoceptor binding sites in these tissues.

2,4-Diamino-6,7-dimethoxyquinoline derivatives as alpha 1-adrenoceptor antagonists and antihypertensive agents.

A series of 2,4-diamino-6,7-dimethoxyquinoline derivatives (2), prepared by LDA- or ZnCl2-mediated intramolecular cyclization of an N-[1-(dialkylamino)ethylidene]-2-cyano-4,5-dimethoxyaniline (3), was evaluated for alpha-adrenoceptor affinity and antihypertensive activity. Most compounds displayed high in vitro binding affinities (Ki's, 10(-10) M) for alpha 1-adrenoceptors with alpha 1-/alpha 2-selectivity ratios of at least 10,000. 4-Amino-2-[4-(2-furoyl)piperazin-1-yl]-6,7-dimethoxyquinoline++ + (14) proved to be the most potent member (Ki = 1.4 X 10(-10) M) of series 2, and displayed no activity at alpha 2-adrenoceptor binding sites at concentrations up to 10(-6) M. In the rabbit pulmonary artery, 14 was a highly potent (pA2 = 9.76 +/- 0.26) competitive antagonist of the alpha 1-mediated vasoconstrictor action of noradrenaline and was some 20 times more active than prazosin. pKa measurements confirmed that, at physiological pH, N-1 protonation of series 2 would efficiently provide 1b, a key pharmacophore for alpha 1-adrenoceptor recognition. Antihypertensive activity for series 2 was evaluated after oral administration (3 mg/kg) to spontaneously hypertensive rats (SHR) and falls in blood pressure were determined at 1 and 4.5 h. Various quinoline derivatives (2) proved to be effective antihypertensive agents in SHR, with both efficacy and duration of action at least equivalent to prazosin, and 14 displayed the most favorable overall profile. These observations are consistent with the high affinity and selectivity displayed by series 2 for postjunctional alpha 1-adrenoceptors.

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

Lithium (Li) at a concentration, which exerts prophylactic effects in affective disorders is known to alter noradrenaline turnover and the beta-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 beta-adrenoceptor binding sites by 10% (3H-dihydroalprenolol binding) leaving the number of alpha 1- and alpha 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 beta-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 beta-adrenoceptor binding induced by 6-OH-dopamine, nor did it alter the beta-adrenoceptor down-regulation following chronic administration of desipramine. The up-regulation of alpha 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 beta-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.

Neuropeptide y- and alpha-adrenergic receptors in pig spleen: Localization, binding characteristics, cyclic amp effects and functional responses in control and denervated animals

The localization of neuropeptide Y binding sites in the pig spleen, as revealed by [ 125I]Bolton-Hunter-labelled porcine neuropeptide Y and alpha 1-adrenergic receptor binding sites, as revealed by [ 125I](2-beta/4-hydroxy-phenyl/-ethylaminomethyl)-tetralone as radioligand, was compared with the distribution of neuropeptide Y and noradrenaline nerves, the latter revealed by tyrosine hydroxylase and dopamine-beta-hydroxylase, using immunohistochemistry. A large degree of codistribution was obtained between [ 125I]neuropeptide Y and alpha 1-binding sites in the capsule, trabeculae, blood vessels and the red pulp of the spleen. Neuropeptide Y and tyrosine hydroxylase as well as dopamine-beta-hydroxylase-positive nerves were identical in the spleen and had a similar gross distribution pattern as the [ 125I]neuropeptide Y and alpha 1 binding sites. In functional studies using the isolated blood-perfused spleen from pentobarbital-anaesthetized pigs, neuropeptide Y, noradrenaline and the alpha 1-selective agonist phenylephrine contracted the capsule and induced vasoconstriction in the spleen in vivo. However, the selective alpha 2-adrenoceptor agonists clonidine and azepexole had no effects on blood flow or perfusion pressure, suggesting that postjunctional alpha-receptors were of the alpha 1 type. Neuropeptide Y inhibited the forskolin-evoked, cyclic adenosine monophosphate formation in vitro. The [ 125I]neuropeptide Y binding, with an equilibrium-dissociation constant of 503 ± 73pM and a maximal number of specific binding sites of 23 ± 3fmol/mg protein, the neuropeptide Y-induced perfusion-pressure increase in vivo and the inhibition of forskolin-evoked cyclic adenosine monophosphate formation in vitro were dependent on the amidation of the C-terminal portion of the peptide molecule. Furthermore, the effects of neuropeptide Y were not changed by alpha- and beta-adrenoceptor blockade using prazosin and propranolol. Two weeks after postganglionic denervation the neuropeptide Y and the noradrenaline contents of the pig spleen were reduced by 97% and 99%, respectively. These changes were associated with a selective supersensitivity for the noradrenaline-induced perfusion-pressure increase in vivo compared with the effect of neuropeptide Y. However, a similar potentiation of the noradrenaline effect was induced by the monoamine-uptake blocker desipramine in the absence of denervation and there was no change in the functional response to phenylephrine after denervation. This indicated that the denervation supersensitivity for noradrenaline was of prejunctional rather than of postjunctional origin, although a significant increase in the maximal number of specific binding sites of the [ 3H]prazosin binding (75%) was observed in vitro. No significant changes in the equilibrium-dissociation constant or in the maximal number of specific binding sites were observed after denervation for the [ 125I]neuropeptide Y binding sites. Furthermore, the neuropeptide Y-induced inhibition of forskolin-induced cyclic adenosine monophosphate formation was not changed after denervation. It is concluded that alpha 1-adrenoceptor and neuropeptide Y binding sites are co-distributed and can be activated in parallel to induce muscle contraction in support of a co-transmitter role for noradrenaline and neuropeptide Y in the sympathetic nervous control of the pig spleen. Denervation was associated with a selective up-regulation of the alpha 1-adrenoceptor binding sites, but no changes in the binding characteristics were observed for neuropeptide Y, suggesting the absence of homeostatic responses in the regulation of neuropeptide Y receptors of the spleen.

Effect of calcium entry blockers on blood pressure and vasoconstrictor responses to alpha-1 adrenoceptor stimulation in conscious spontaneously hypertensive rats.

In order to investigate whether vascular alpha-1 adrenoceptor antagonism plays a role in the antihypertensive effect of verapamil, tiapamil, and nifedipine, we studied their potencies to inhibit K(+)-induced 45Ca2+ influx in rat isolated aorta and [3H]prazosin binding in rat brain membranes in vitro as well as their antihypertensive effect and functional alpha-1 adrenoceptor blockade in conscious spontaneously hypertensive rats (SHR) in vivo. Tiapamil proved 70 times less potent than verapamil in inhibiting calcium influx, but was equipotent in displacing [3H]prazosin. Nifedipine proved 10 times more potent than verapamil as calcium channel blocker but displayed negligible affinity for alpha-1 adrenoceptors in vitro. In conscious SHR, the three calcium channel blockers dose-dependently reduced mean arterial pressure after oral administration. Only at maximal anti-hypertensive doses, the increases in diastolic pressure to intravenous injection of the selective alpha-1 adrenoceptor agonist cirazoline were temporarily suppressed by nifedipine, verapamil, and tiapamil. No relationship existed between the relative potencies as calcium channel blocker and affinities for alpha-1 adrenoceptor binding sites in vitro with functional vascular alpha-1 adrenoceptor blockade in vivo. The data do not support the hypothesis that vascular alpha-1 adrenoceptor blockade plays a significant role in the anti-hypertensive effect of verapamil and related calcium channel blockers.

Two renal alpha 2-adrenergic receptor sites revealed by p-aminoclonidine binding

[3H]p-aminoclonidine [3H]PAC, a specific alpha 2-adrenergic agonist, was used to characterize alpha 2-adrenoceptor binding in rat renal membranes. Rosenthal plots demonstrated two binding sites with Kds of approximately 1.7 and 14.2 nM and Bmaxs (maximum binding) of 47.3 and 218.8 fmol/mg protein for the high- and low-affinity sites, respectively. These characteristics were confirmed by estimate of Kd parameters based on association and dissociation experiments. Pseudo-Hill coefficients generated from drug inhibition experiments were all less than unity, suggesting differential binding at two alpha 2-adrenoceptor binding sites. Specific alpha 2-adrenergic agonists exhibited greater binding affinity to both sites than did nonspecific drugs, and all drugs displayed greater affinity for the high- than the low-affinity binding site. Both guanyl nucleotides and sodium chloride inhibited [3H]PAC binding more at the high-affinity than at the low-affinity site. Renal denervation resulted in significant upregulation of receptor density only at the high-affinity sites with no change in receptor affinity at either site, suggesting that a majority of the alpha 2-adrenoceptors in the kidney are postsynaptic. Thus all lines of evidence in this study indicate that two alpha 2-adrenoceptor binding sites exist in the rat kidney.

Influence of oestradiol on alpha 2-adrenoceptor binding sites on intact platelets of young male volunteers.

The specific binding (Bmax) of (3H-methyl)yohimbine to alpha 2-adrenoceptor binding sites on intact platelets was increased by 43% in 6 out of 7 young male volunteers 4 days after a single i.m. injection of 10 mg sustained release oestradiol, and it had returned to the starting value 4 weeks after drug administration. Mean plasma oestradiol was 331 pg/ml on Day 4 and it was within the pre-treatment control range of 19 pg/ml 4 weeks after the injection. A close correlation between the post-injection plasma oestradiol concentration and the increase in alpha 2-adrenoceptor binding sites on intact platelets was not found.

Mechanisms underlying pre‐ and postjunctional effects of neuropeptide Y in sympathetic vascular control

The effects of porcine neuropeptide Y (NPY) regarding sympathetic vascular control were studied in vitro on isolated rat blood vessels. The 10(-9)M NPY enhanced (about two-fold) the contractile responses to transmural nerve stimulation (TNS), noradrenaline (NA) and adrenaline (about two-fold) in the femoral artery. Higher concentrations of NPY (greater than 10(-8)M) caused an adrenoceptor-resistant contraction per se. The TNS-evoked [3H]NA efflux was significantly reduced by NPY in a concentration-dependent manner (threshold 10(-9)M). The calcium antagonist, nifedipine, abolished the contractile effects of NPY and the NPY-induced enhancement of NA contractions but did not influence the prejunctional inhibition of [3H]NA release. Receptor-binding studies showed that the ratio of alpha 1-to alpha 2-adrenoceptors in the femoral artery was 30:1. The NPY did not cause any detectable change in the number of alpha 1-or alpha 2-adrenoceptor binding sites or in the affinity of alpha 2-binding sites, as revealed by prazosin- and clonidine-binding, respectively. The NPY also inhibited the TNS-evoked [3H]NA release (by 42-86%) in the superior mesenteric and basilar arteries and in femoral and portal veins. The NPY still depressed TNS-evoked [3H]NA secretion from the portal vein in the presence of phentolamine. The NPY caused a clear-cut contraction in the basilar artery, increased the contractile force of spontaneous contractions in the portal vein, while only weak responses were observed in the superior mesenteric artery and femoral vein. The NA-induced contraction was markedly enhanced by NPY in the superior mesenteric artery, only slightly enhanced in the portal vein and uninfluenced in the femoral vein. In conclusion, in all blood vessels tested, NPY depresses the TNS-evoked [3H]NA secretion via a nifedipine-resistant action. Furthermore, NPY exerts a variable, Ca2+-dependent vasoconstrictor effect and enhancement of NA and TNS contractions.

Studies on alpha 2-adrenoceptors in rabbits with perinephritis hypertension.

Increased responses to intravenous injections of the full alpha 2-adrenoceptor agonists, alpha-methyl noradrenaline and noradrenaline, were observed in conscious rabbits with perinephritis hypertension. However, responses to the partial agonist, BHT 920, and the antagonist, idazoxan, were unchanged. These increased responses to noradrenaline and alpha-methyl noradrenaline did not appear to be related to the alpha 1- or beta-adrenoceptor activity or to altered clearance of the agonists. The pro-aggregatory response of platelets from hypertensive rabbits and normotensive rabbits to adenosine diphosphate (ADP) was similar, but the sensitivity of platelets from hypertensive rabbits to the adrenaline potentiation of the pro-aggregatory response was increased. This was not related to changes in the number of alpha 2-adrenoceptor binding sites or to changes at platelet beta-adrenoceptors. In contrast the aggregatory responses to the partial agonist, guanabenz, were similar in normotensive and hypertensive animals. In rabbits with perinephritis hypertension responses to phenylethanolamines (full agonists at the alpha 2-adrenoceptor) were increased in vivo in the conscious animal and in vitro in platelets, whereas responses to imidazolines which are partial agonists were similar in normotensive and hypertensive animals. These differences may be related to alterations in the coupling of alpha2-adrenoceptor activation to response in the hypertensive rabbits.

Chronic cobalt-induced epilepsy: noradrenaline ionophoresis and adrenoceptor binding studies in the rat cerebral cortex.

Several studies indicate that brain noradrenaline (NA) depletion facilitates the occurrence of epileptogenic syndromes in various animal models. In cobalt-induced epilepsy in the rat activity is associated with a cortical NA denervation. In order to search for cortical adrenoceptor modifications, inonophoretic studies and adrenoceptor binding assays were performed. At the period of maximal seizure activity, there was a significant supersensitivity of cortical neurons to the ionophoretic application of NA. An increase in the density of beta-adrenoceptor binding sites was observed. No modification in alpha 1- and alpha 2-adrenoceptor binding sites was found. This suggests that in cobalt-induced epilepsy there is a denervation supersensitivity which rests on a selective involvement of beta-adrenoceptors.

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.