Alpha 2-adrenoceptor Binding Sites Research Articles

Chronic hypobaric hypoxia induces tolerance to acute hypoxia and up-regulation in alpha-2 adrenoceptor in rat locus coeruleus

Hypoxia preconditioning has been shown to produce tolerance against brain injuries. The hypothesis of this study is that chronic hypobaric hypoxia may also induce acute hypoxia tolerance. We used intracellular recording in slices from rats exposed to chronic hypobaric hypoxia (exposed) and control to investigate the effects of chronic hypobaric hypoxia on the physiology of locus coeruleus (LC) including neuronal excitability. The results showed 35.7% reduced spontaneous firing rate and no change for membrane potential and input resistance in exposed neurons. In response to the alpha-2 adrenoceptor (A2R) agonist clonidine, both the hyperpolarizing potency and efficacy were increased indicated by a decreased EC50 (control: 30.9 nM and exposed: 19.7 nM) and a 50.5% increase in maximum hyperpolarized potential, respectively. A2R binding sites were also increased 21% in exposed neurons measured by radioligand [3H]rauwolscine binding assay. When treated with acute N2-hypoxia, the cell survival time (ST) was longer in exposed neurons, suggesting that a tolerance was induced. In addition, the ST for both groups of LC neurons was decreased by the A2R antagonist yohimbine and increased by the glutamate receptor antagonist kynurenic acid but not by MK-801; the decreased percentage of ST by yohimbine was larger and the increased percentage by kynurenic acid was smaller in exposed neurons. The results suggested that up-regulation of A2R and altered non-NMDA glutamate receptor function induced by chronic hypobaric hypoxia may underlie, in part, the decreased LC neuronal excitability and acute hypoxia tolerance.

Pancreatic beta-cell alpha2A adrenoceptor and phospholipid changes in hyperlipidemic rats.

We previously showed that a 48-h intravenous lipid infusion in rats induces pancreatic beta-cell hypersensitivity to catecholamines. Our aim was to study the lipid-related changes that may account for such hypersensitivity in pancreatic islets. We show here that a 48-h increase in plasma FFA alters the binding characteristics of beta-cell alpha2 adrenoceptors in rats. Lipid infusion decreases pancreatic norepinephrine (NE) turnover rate by 28%, reflecting a reduction of pancreatic NE stores. Following lipid infusion, the density of alpha2 adrenoceptor binding sites is significantly lower and receptor affinity higher, both in islet homogenates (by three- and fivefold, respectively) and isolated whole beta-cells (by two- and sixfold, respectively). These changes correlate with the elevated insulin response to glucose found in lipid-infused rats. We also found a modification of islet phospholipid content, particularly in phosphoethanolamine species containing infused FA such as palmitate, oleate, stearate, and linoleate. This may account for the modifications in receptor affinity. These results suggest that hyperlipidemia-associated pathologies such as diabetes and obesity not only may result from alterations of metabolic pathways but also may be a consequence of early modifications in nervous firing rates and signal transduction pathways.

Contractile properties of urethral smooth muscles of young and aged female dogs: morphological and pharmacological aspects.

Alpha1-adrenoceptors are highly concentrated in the urethral smooth muscles and may play an important role in the contraction of this area. However, detailed examinations of age-related changes of the properties of urethral smooth muscle have rarely been undertaken. The contractile properties of urethras from young non-parous and old parous female beagles were determined with a urethral function study, macroscopic autoradiography for urethras using [3H]-labeled tamsulosin and morphometry of the urethral muscles. The antagonistic effect (pA2) of prazosin for norepinephrine was 7.76+/-0.13 in young dogs and 7.62+/-0.06 in aged dogs. The specific binding of [3H]-tamsulosin (a relatively selective alpha1A-adrenoceptor antagonist) was recognized diffusely in proximal urethras with in vitro autoradiography. The density of binding in smooth muscles was approximately 60 and 40% in circular longitudinal layers, respectively, for both dogs. The female canine urethra had alpha1A, and alpha1L-adrenoceptors. No age-related changes were seen in the function of the proximal urethra, distribution of alpha1-adrenoceptor binding sites and smooth muscle densities.

Evidence for direct interaction of ketamine with alpha 1- and beta 2-adrenoceptors.

1. Ketamine has a number of effects that suggest that it may interact with alpha- and beta-adrenoceptors. To date, the experimental evidence for this has been indirect and has been based on physiological studies using competitive blocking agents. In the present study we sought to determine from receptor binding studies whether ketamine binds directly to alpha- and beta-adrenoceptors. 2. Membrane preparations of alpha 1- and beta 2-adrenergic binding sites were obtained from urinary bladder and urethrae of sheep. These binding sites were characterized by saturation analyses using [3H]-prazosin for alpha 1-adrenoceptor binding sites and [125I]-cyanopindolol (CYP) for the beta 2-adrenoceptor binding sites. The receptors were further characterized by displacement studies using selective and non-selective antagonists. 3. Studies in which ketamine was used to displace [3H]-prazosin revealed a Kd of 3.40 +/- 1.23 x 10(-3) mol/L for ketamine binding to alpha 1-adrenoceptors. Displacement studies of [125I]-CYP by ketamine showed a Kd of 0.35 +/- 0.03 x 10(-3) mol/L for ketamine binding to beta 2-adrenoceptors. 4. We conclude that ketamine interacts directly with both alpha 1- and beta 2-adrenoceptors and that such interactions probably explain the reported effects of this agent on the vasculature and the bronchial tree.

Alpha 2C-adrenoceptors mediate contractile responses to noradrenaline in the human saphenous vein.

We have investigated the subtype of alpha 2-adrenoceptor mediating isometric contractions of human saphenous vein in comparison with alpha 2-adrenoceptor ligand binding sites. Postjunctional alpha 2-adrenoceptors in the human saphenous vein were investigated in terms of the ability of alpha 2-adrenoceptor antagonists to shift the contractile potency of noradrenaline. The following antagonists were employed (potencies, pKB, in human saphenous vein in parentheses): chlorpromazine (6.98 +/- 0.24), BDF 8933 (7.60 +/- 0.06), prazosin (6.62 +/- 0.15), ARC 239 (7.19 +/- 0.15), yohimbine (7.23 +/- 0.09), HV 723 (7.52 +/- 0.14), WB 4101 (7.90 +/- 0.06), SKF 104078 (6.55 +/- 0.08), BRL 44408 (5.72 +/- 0.21). Antagonist potency at postjunctional alpha 2-adrenoceptors was correlated with antagonist affinity at alpha 2-adrenoceptor ligand binding sites in membranes of human platelet (alpha 2A), rat kidney (alpha 2B) and Sf9 cells expressing human recombinant receptors (alpha 2C), labelled with [3H]yohimbine. The correlation with the postjunctional alpha 2-adrenoceptor mediating contraction of the human saphenous vein was best for the human recombinant alpha 2C-adrenoceptor ligand binding site (r = 0.92, n = 8, P < 0.001), as compared to correlations with the alpha 2B-adrenoceptor ligand binding site of rat kidney (r = 0.62, n = 8, n.s.) and with the alpha 2A-adrenoceptor ligand binding site of human platelet (r = 0.23, n = 8, n.s.). It is concluded that the functional postjunctional alpha 2-adrenoceptor mediating contractions of the human saphenous vein closely resembles the human recombinant alpha 2C-adrenoceptor ligand binding site.

Investigation of the subtype of alpha 2-adrenoceptor mediating pressor responses in the pithed rat.

We have investigated the subtype of alpha 2-adrenoceptor mediating postjunctional pressor responses in the pithed rat in comparison with alpha 2-adrenoceptor ligand binding sites. In pithed rats, postjunctional alpha 2-adrenoceptors were investigated in terms of the ability of alpha 2-adrenoceptor antagonists to shift the pressor potency of the alpha 2-adrenoceptor agonist xylazine. Antagonist potency at postjunctional alpha 2-adrenoceptors in the pithed rat was correlated with antagonist affinity at alpha 2-adrenoceptor ligand binding sites in membranes of rat kidney (alpha 2B), Sf9 cells expressing human recombinant receptors (alpha 2C) and rat submandibular gland (alpha 2D) labelled with [3H]yohimbine. The correlation with the postjunctional alpha 2-adrenoceptor mediating pressor responses in the pithed rat was better for the alpha 2D-adrenoceptor ligand binding site of rat submandibular gland (r = 0.95, n = 9, P < 0.0001) and the alpha 2B-adrenoceptor ligand binding site of rat kidney (r = 0.90, n = 9, P < 0.001) than with the human recombinant alpha 2C-adrenoceptor ligand binding site (r = 0.81, n = 9, P < 0.01). When the pressor potencies of three additional antagonists were included in the correlations for alpha 2B- and alpha 2D-sites only, the correlation with alpha 2D-adrenoceptor ligand binding site of rat submandibular gland (r = 0.91, n = 12, P < 0.0001) was much better than with the alpha 2B-adrenoceptor ligand binding site of rat kidney (r = 0.77, n = 12, P < 0.01). It is concluded that the functional postjunctional alpha 2-adrenoceptors mediating pressor responses in the pithed rat most closely resemble the alpha 2D-adrenoceptors subtype.

Rat pineal alpha 1-adrenoceptor subtypes: studies using radioligand binding and reverse transcription-polymerase chain reaction analysis.

1. The pharmacological characteristics of alpha 1-adrenoceptor binding sites in rat pineal gland membranes, detected by use of a selective alpha 1-adrenoceptor antagonist ([125I]-iodo-2-[beta-(4-hydroxyphenyl) ethylaminomethyl]tetralone, [125I]-HEAT), were investigated with the alkylating agent, chloroethylclonidine (CEC), and in competition experiments with a number of adrenoceptor agonists and antagonists. 2. Chloroethylclonidine (CEC) treatment (10 microM, 10 min) of rat pineal membranes inactivated approximately 70% of specific [125I]-HEAT binding sites. Higher concentrations of CEC (up to 100 microM) or longer treatment periods (up to 40 min) were no more effective. 3. Adrenoceptor agonists and antagonists competitively inhibited [125I]-HEAT binding with Hill coefficients close to unity indicating a single alpha 1-adrenoceptor subtype is present. The affinity (Ki) of subtype selective agonists (oxymetazoline, SDZ NVI-085) and antagonists (5-methylurapidil, WB4101, benoxathian, phentolamine) was consistent with binding to an alpha 1B-adrenoceptor subtype. 4. The (-)- and (+)-enantiomers of niguldipine had an equal and low affinity for alpha 1-adrenoceptor binding sites both in untreated (log Ki-6.66 and -6.90 respectively) and CEC-treated membranes in which approximately 70% of sites had been inactivated (log Ki-6.41 and -6.86 respectively). This indicates that the small proportion of alpha 1-adrenoceptors insensitive to CEC are not alpha 1A-adrenoceptors. 5. mRNA was isolated from rat pinealocytes, cDNA was synthesized and then amplified by the polymerase chain reaction with alpha 1-adrenoceptor subtype specific primers. These experiments identified both alpha 1A- and alpha 1B-adrenoceptor mRNA, but not alpha 1D-mRNA in rat pinealocytes, although all three adrenoceptor subtypes were readily identified in rat brain cortex. 6. These data indicate that although both alpha 1A- and alpha 1B-adrenoceptor mRNAs are present in the pineal the major subtype of alpha 1-adrenoceptor expressed is the alpha 1B.

The relationship between density of α‐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. Further, rauwolscine displaced [3H]-RX821002 with approximately 100 fold greater affinity compared to corynanthine, which is indicative of selective alpha2-adrenoceptor binding.5. Separation of the P2 pellet into plasma membrane and mitochondrial fractions was carried out using a differential sucrose density gradient. [3H]-prazosin and [3H]-RX821002 binding sites were found in both the plasma membrane and mitochondrial fractions.6. In saturation studies all tissues produced single site saturation curves with no difference in the Kd(range 0.13-0.20nM) of the alpha1-adrenoceptor sites for [3H]-prazosin. However, there was considerable variation in Bmax of alpha 1-adrenoceptor sites; the highest density was found in the TA (397.9 =/- 52.7 fmol mg-1, n = 4), followed by the PCDA (256.7 +/- 22.7 fmol mg-1, n = 4), the PLV and SA having approximately equal density (143.6 +/- 3.9 and 159.1 +/- 7.0 fmol mg-1 respectively, n = 4 for both), followed bythe EA (91.3 +/- 10.5 fmol mg-1, n = 3) and the MEV had the lowest density (48.9 +/- 11.4 fmol mg-1,n = 3).7. In saturation studies using [3H]-RX821002, all tissues produced single site saturation curves with no differences in the Kd values (range 1.31 +/- 2.16 nM) but the highest densities were found in the TA and MEV (545.3 +/- 36.2 and 531.0 +/- 40.9 fmol mg-1 respectively), followed by the PLV (418.4 +/- 39.4 fmol mg-1), then the EA (266.3 +/- 40.0 fmol mg-1), and low densities of [3H]-RX821002 binding being found in the PCDA and SA (155.9 +/- 18.1 and 117.5 +/- 19.3 fmol mg-1 respectively).8. The pattern of binding site distribution for alpha l- and alpha 2-adrenoceptors is in reasonable agreement with functional studies carried out in these porcine vascular tissues; the TA has the highest densities of alpha 1-and alpha2-adrenoceptors; in the SA and PCDA there is a predominance (although small) of alpha l-adrenoceptor binding sites, the reverse of which is observed both in the PLV and MEV (i.e. greater density of alpha2-adrenoceptor sites). Thus, it would appear that alpha 1- and alpha2-adrenoceptor densities play a role in the expression of functional responses via these receptor subtypes; although it is interesting to note that the SA did have a small density of alpha 2-adrenoceptor binding sites, no functional response was observed after alpha2-adrenoceptor activation.

Agmatine recognizes ?2-adrenoceptor binding sites but neither activates nor inhibits ?2-adrenoceptors

It has been suggested that agmatine (decarboxylated arginine) is an endogenous clonidine-displacing substance (CDS) which recognizes alpha 2-adrenoceptor and non-adrenoceptor, imidazoline binding sites. We have examined the effect of agmatine at alpha 2-adrenoceptor binding sites and pre- and postjunctional alpha 2-adrenoceptors. Agmatine produced a concentration-dependent inhibition of 1 nmol/l 3H-clonidine binding to both rat (pKi-5.10 +/- 0.05) and bovine (pKi-4.77 +/- 0.38) cerebral cortex membranes. However, agmatine (0.1-100 microM) failed to activate pre-junctional alpha 2-adrenoceptors regulating transmitter release in the guinea-pig isolated ileum and rat isolated vas deferens, nor did it activate postjunctional alpha 2-adrenoceptors of the porcine isolated palmar lateral vein which mediate contraction or inhibition of forskolin-stimulated cyclic AMP formation. High concentrations of agmatine (10-30-fold the pKi at alpha 2-adrenoceptor binding sites) failed to influence alpha 2-adrenoceptor activation by either clonidine or UK-14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline bitartrate) in any of the peripheral preparations examined. Moreover, even in a preparation where an interaction with alpha 2-adrenoceptor binding sites on cell membranes can be demonstrated, the rat cerebral cortex, agmatine failed to inhibit forskolin-stimulated cyclic AMP in the intact tissue or affect the inhibition produced by the selective alpha 2-adrenoceptor agonist UK-14304. Agmatine was also devoid of agonist activity in two preparations, the rat isolated thoracic aorta and the rat isolated gastric fundus, in which CDS has been reported to produce non-adrenoceptor effects. Thus, we have confirmed that agmatine recognizes alpha 2-adrenoceptor binding sites and, therefore, is a CDS.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for the presence of a non-catecholamine, clonidine-displacing substance in crude, methanolic extracts of bovine brain and lung

In the present study we have prepared crude, methanolic extracts of bovine lung and bovine brain and, using radioligand binding assays in conjunction with a number of simple chromatographic techniques, provided evidence for the presence of a non-catecholamine 'clonidine-displacing substance' (CDS). The level of CDS in lung extracts (9 units/g wet weight n = 11) is approximately 3 times that in the brain extracts. Furthermore, the effect of the crude, methanolic extracts are selective for non-adrenoceptor, imidazoline (labelled by [3H]-idazoxan) and alpha 2-adrenoceptor binding sites (labelled by [3H]-clonidine); both extracts are 5-10-fold more potent displacers of ligand binding to alpha 2-adrenoceptors compared with binding to opiate receptors (labelled by [3H]-etorphine) and practically inactive against alpha 1-adrenoceptor and muscarinic binding sites (labelled by [3H]-prazosin and [3H]-quinuclidinyl benzilate, respectively). With the exception of the non-adrenoceptor, imidazoline binding assay, which used rat kidney membranes labelled by [3H]-idazoxan in the presence of the alpha 2-adrenoceptor antagonist RS-15385-197, all radioreceptor assays involved bovine cerebral cortex membranes. Although the extracts contain catecholamines (brain only), histamine (lung only) and monovalent cations (both), which have the potential to interfere with the radioligand binding assays, their concentrations were too low to account for the effects observed. Preliminary attempts at purification of the extracts revealed that CDS activities from the two tissues are similar, i.e., practically insoluble in organic solvents at room temperature, not affected by either Sep-Pak C18 column or anion exchange resins but retained (along with the monovalent cations) by cation exchange resin. However, following chromatographic separation on a Biogel P2 column, the CDS-containing eluates are cation-free and exhibit qualitatively similar elution profiles. Future experiments will involve further purification of 'clonidine-displacing substance' to characterize its interaction with alpha 2-adrenoceptor binding sites in greater detail and establish whether it has biological activity consistent with the properties implied by its effects in radioligand binding assays.

Potential antidepressant properties of SR 57746A, a novel compound with selectivity and high affinity for 5-HT1A receptors.

SR 57746A, 4-(3-trifluoromethylphenyl)-N-[2-(naphth-2-yl)ethyl]-1,2,3,6- tetrahydropyridine HCl, was studied for its specific 5-HT1A receptor agonist action and antidepressant-like effects in the rat. The compound showed a high affinity for 5-HT1A specific binding sites in the rat hippocampus (IC50 3 nM), moderate affinity (10(-7)-10(-6) M) for dopamine D2 receptor, 5-HT uptake, 5-HT2 and alpha 1-adrenoceptor binding sites and practically no effect on binding sites of monoamine, GABAA, benzodiazepine and histamine receptors. It inhibited forskolin-stimulated adenylate cyclase activity in rat hippocampal membranes at concentrations of 10(-6) and 10(-5) M. The effect of 10(-6) M SR 57746A on forskolin-stimulated adenylate cyclase activity was completely antagonized by 10(-6) M (-)-propranolol. Administered per os as a three-dose course to rats, SR 57746A significantly increased struggling in the forced swimming test at doses from 0.3 to 3 mg/kg. Single doses had no such effect. The effect of a three-dose course with 1 mg/kg SR 57746A on rats' struggling was antagonized by pretreatment with 5 mg/kg i.p. metergoline, a non-selective 5-HT receptor antagonist, and by 20 mg/kg i.p. (-)-propranolol, an antagonist at 5-HT1 receptors. Three oral doses of 100 mg/kg parachlorophenylalanine, an inhibitor of 5-HT synthesis, and 100 mg/kg i.p. (+/-)-sulpiride, an antagonist at dopamine D2 receptors, also antagonized the effect of SR 57746A in the forced swimming test. The results show that SR 57746A has selectivity and high affinity for 5-HT1A receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and Localization of Prostatic Alpha1 Adrenoceptors Using Radioligand Receptor Binding on Slide-Mounted Tissue Section

Alpha1 adrenoceptor binding sites have been characterized in prostatic tissue homogenates using radioligand receptor binding studies. The objective of the present study was to characterize and localize prostatic alpha1 adrenoceptor binding sites using slide-mounted tissue sections and the ligand 3H-prazosin. The present study demonstrated that preincubation is not required; the optimal incubation interval is 40 minutes; and a 1-minute wash (once or twice) maximizes the proportion of specific 3H-prazosin binding. Saturation studies were performed at 8 different concentrations of 3H-prazosin ranging between 0.0625nM. to 8.0nM. The binding of 3H-prazosin was consistently saturable and of high affinity. The mean Kd and Bmax determined from 6 saturation studies was 4.16 × 10−10M. and 1.30fmol./mg. wet weight, respectively. The pharmacology of these 3H-prazosin binding sites was characterized using competitive displacement experiments. The mean IC50 corrected for prazosin, phentolamine and yohimbine was 7.8 × 10−10M., 6.0 × 10−10M. and 2.1 × 10−10M. The rank order of the IC50 corrected values indicates that alpha1 binding sites were measured under the assay conditions. In the present study, the mean values for Kd, Bmax and IC50 corrected are similar to values previously reported using prostatic tissue homogenates. Prostatic tissue sections were apposed to x-ray film after being incubated with 3nM. 3H-prazosin (total prazosin binding) and 3nM. 3H-prazosin + 8 μM. prazosin (nonspecific prazosin binding). The autoradiograms were analyzed using a computerized analyzing system. The specific radioactive densities of 3H-prazosin in the stroma and glandular epithelium were 1099 ± 48pCi/mg. and 163 ± 42pCi/mg. The present study validates the technique of assaying alpha1 adrenoceptor binding sites on slide-mounted prostatic tissue sections and provides further evidence that alpha1 adrenoceptor binding sites are localized primarily to the stromal elements of the prostate.

Alpha 1-adrenoceptor subtypes in the rat ventricular muscle.

Scatchard analyses of [3H]prazosin binding in rat ventricular muscle membranes showed biphasic curves, which identified alpha 1High- and alpha 1Low-affinity sites. The alpha 1High-affinity site was completely inhibited by 1 microM phenoxybenzamine. The displacement potencies of alpha 1-adrenergic antagonists were characterized by [3H]prazosin binding to alpha 1High- and alpha 1Low-affinity sites in the absence and presence of 1 microM phenoxybenzamine. The affinities of most chemicals for alpha 1Low-affinity sites were significantly lower than those for alpha 1High-affinity sites, but WB-4101 (2-(2,6-dimethoxy-phenoxyethyl)aminomethyl-1,4-benzodioxane), arotinolol, cinanserin, nifedipine, and p-aminoclonidine had the same affinities for both alpha 1Low- and alpha 1High-affinity sites. These results show that two alpha 1-adrenoceptor subtypes, alpha 1High- and alpha 1Low-affinity, are present in the rat heart, and that there are physical variations in alpha 1-adrenoceptor binding sites, based on their selectivity to antagonists.

Actions of 8-hydroxy-2-(N-dipropylamino) tetralin (8-OH-DPAT) at alpha 2-adrenoceptors.

1 We have examined the actions of the 5-HT1A-receptor ligand 8-OH-DPAT at alpha 2-adrenoceptor ligand binding sites in human platelet and rat kidney membranes and at functional pre- and postjunctional alpha 2-adrenoceptors in rat atrium and human saphenous vein, respectively. 2 8-OH-DPAT had low affinity for the alpha 2A ligand binding site of human platelet but showed 10 times higher affinity for the alpha 2B ligand binding site of rat kidney (K1 of 6.41, -log M). 3 In functional studies, 8-OH-DPAT had low potency as an antagonist of noradrenaline-induced contractions in human saphenous vein, with a KB of 5.40 (-log M). 4 In rat atria preincubated with [3H]-noradrenaline, 8-OH-DPAT potentiated stimulation-evoked overflow of tritium with an EC30 (concentration producing 30% increase in the stimulation-evoked overflow) of 6.37 (-log M). 5 It is concluded that 8-OH-DPAT shows selectively for the alpha 4B ligand binding site of rat kidney and for the functional prejunctional alpha 2-adrenoceptor of rat atrium, which resembles the alpha 2B ligand binding site.

Subclassification of the presynaptic α2‐autoreceptors in rabbit brain cortex

1. alpha 2-Adrenoceptor binding sites have been subclassified into alpha 2A sites of which a main characteristic is very low affinity for prazosin, and alpha 2B sites with relatively high affinity for prazosin. The presynaptic alpha 2-autoreceptors in rabbit brain cortex were studied in order to classify them in terms of alpha 2A and alpha 2B. Release of [3H]-noradrenaline in cortical slices was elicited by trains of 4 pulses delivered at 100 Hz. 2. Clonidine caused concentration-dependent inhibition of the stimulation-evoked overflow of tritium, with an EC50 of 7.5 nM and a maximal inhibition by 96%. 3. The following alpha-adrenoceptor antagonists shifted the concentration-response curve of clonidine to the right (antagonist-receptor dissociation constants KD in brackets): yohimbine (14 nM), 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazo le (BRL 44408; 15 nM) and 1,2-dimethyl-2,3,9,13betetrahydro-1H-dibenzo[c,f]imidazo[1,5-a]aze pine (BRL 41992; 630 nM). Prazosin 1 microM and 2-[2-[4-(o-methoxyphenyl)piperazine-1-yl]-ethyl]-4,4-dimethyl-1,3 (2H,4H)-isoquinolinedione (AR-C 239) 1 microM failed to antagonize the effect of clonidine. Higher concentrations of prazosin and AR-C 239 greatly accelerated the basal efflux of tritium. 4. The method used permits the functional determination of antagonist affinities undistorted by endogenous alpha 2-autoinhibition. A comparison with affinities derived from radioligand binding experiments indicates that the presynaptic alpha 2-autoreceptors in rabbit brain cortex are markedly different from the alpha 2B-subtype and probably belong to the prazosin-insensitive alpha 2A-subtype.

Myocardial alpha 1-adrenoceptors mediate positive inotropic effect and changes in phosphatidylinositol metabolism. Species differences in receptor distribution and the intracellular coupling process in mammalian ventricular myocardium.

Species-dependent variations of myocardial alpha 1-adrenoceptor-mediated positive inotropic effects of epinephrine were assessed in relation to characteristics of alpha 1-receptor bindings and acceleration of phosphatidylinositol metabolism in the isolated rat, rabbit, and dog ventricular myocardium. Epinephrine in the presence of the beta-adrenoceptor antagonist bupranolol (10(-6) M) elicited a positive inotropic effect through activation of alpha 1-adrenoceptors in rat and rabbit, whereas in dog ventricular myocardium, bupranolol abolished the positive inotropic effect of epinephrine. [3H]Prazosin bound to membrane fractions derived from rat, rabbit, and dog ventricular muscle with high affinities in a saturable and reversible manner. In dog, Bmax and Kd values of alpha 1-adrenoceptor binding sites were identical to those in rabbit ventricular muscle. The Bmax value of alpha 1-adrenoceptors in rat ventricle was the highest, amounting to two to four times those in rabbit and dog. Epinephrine displacement curves for the specific binding of [3H]prazosin in the membrane fraction of these species showed high and low affinity sites with slope factors significantly less than unity, which were shifted to single low affinity sites with slope factors close to unity by addition of 5'-guanylylimidodiphosphate. Accumulation of [3H]inositol 1-phosphate [( 3H]IP1) in ventricular slices prelabeled with [3H]myo-inositol was increased by epinephrine in a time- and concentration-dependent manner in rat ventricular slices. [3H]IP1 accumulation likewise was facilitated by alpha 1-adrenoceptor stimulation in rabbit ventricular slices, whereas the extent of [3H]IP1 accumulation was much less than that in rat. In dog ventricular slices, [3H]IP1 was not accumulated by epinephrine. In rabbit papillary muscle, the time course of increase in contractile force induced by alpha-adrenoceptors coincided with the prolongation of the action potential duration with a similar time course, which is in strong contrast to previous findings in rat that the contractile response was dissociated from the electrophysiological response to alpha-adrenoceptor stimulation. The present results indicate that a wide range of variation of alpha 1-adrenoceptor-mediated regulation of myocardial contractility may be ascribed to different contributions of facilitatory as well as inhibitory regulatory processes that lead to intracellular Ca2+ mobilization subsequent to myocardial alpha 1-adrenoceptor activation among mammalian species.

α-Adrenoceptors in Dog Mesenteric Vessels–Subcellular Distribution and Number of [3H]Prazosin and [3H]Rauwolscine Binding Sites

Binding of the alpha-adrenergic antagonists [3H]prazosin and [3H]rauwolscine to well-characterized subcellular membrane fractions isolated from dog mesenteric arteries and veins was studied. Binding of both ligands was saturable with Kd values of 0.5 +/- 0.1 nM for [3H]prazosin and 5.85 +/- 0.85 nM for [3H]rauwolscine in arteries, and 0.87 +/- 0.4 nM for [3H]prazosin and 6.6 +/- 1.5 nM for [3H]rauwolscine in veins. In veins, the maximum number of binding sites for [3H]rauwolscine was higher than that for [3H]prazosin, whereas in arteries the maximum number of binding sites for each ligand was similar. In microsomes from dog aorta, the maximum number of bindings sites for [3H]prazosin was higher than that for [3H]rauwolscine. Neuronal membrane contamination in these studies was minimized by dissection procedures and evaluated by the comparison of [3H]saxitoxin binding in various preparations. Only mesenteric veins responded functionally to agonists acting on alpha 2 adrenoceptors. This study thus identified two distinct populations of [3H]prazosin and [3H]rauwolscine binding sites in the plasma membranes of dog mesenteric vessels and suggests that a much higher density of alpha 2-compared to alpha 1-adrenoceptor binding sites is required for a contractile response.

Assessment of imiloxan as a selective α2B‐adrenoceptor antagonist

1. The alpha 2-adrenoceptor binding sites of rabbit spleen and rat kidney, labelled with [3H]-rauwolscine, were characterized using a range of subtype selective ligands. 2. In rabbit spleen, the alpha-2-adrenoceptor binding sites displayed high affinity for oxymetazoline and WB 4101 and low affinity for prazosin and chlorpromazine suggesting the presence of an alpha 2A subtype. 3. There was evidence for heterogeneity of the alpha 2-adrenoceptor binding sites present in rabbit spleen. The results obtained with oxymetazoline and WB 4101 indicated that at least 75% of the [3H]-rauwolscine binding sites in this preparation displayed a pharmacology consistent with the presence of an alpha 2A subtype. 4. In rat kidney, the alpha 2-adrenoceptor binding sites displayed high affinity for prazosin and chlorpromazine and low affinity for oxymetazoline and WB 4101 suggesting the presence of an alpha 2B subtype. 5. The inclusion of guanylylimidodiphosphate (Gpp(NH)p, 0.1 mM) did not modify the pharmacology of the alpha 2-adrenoceptor binding sites present in the two preparations. Furthermore, when the two membrane preparations were combined, the resultant pharmacology was still consistent with the presence of two receptors that retained the characteristics of the alpha 2A and alpha 2B subtypes. 6. Imiloxan was identified as a selective alpha 2B ligand while benoxathian displayed a high degree of selectivity for the alpha 2A-adrenoceptor binding site. The selectivity of imiloxan for the alpha 2B-adrenoceptor binding site, coupled with its specificity for alpha 2-adrenoceptors, should make it a valuable tool in the classification of alpha 2-adrenoceptor subtypes.

Peripubertal Changes in the Nature of the GnRH Response to Alpha-Adrenoceptor Stimulation in vitro and their Modulation by Testosterone

Adrenergic mechanisms have been widely implicated in the regulation of GnRH secretion in adult rats but their role in young animals, in which the activity of the GnRH neurones is minimal, is unclear. These experiments were done to examine the effects of alpha-adrenoceptor stimulation on the secretion in vitro of GnRH by hypothalami from immature and adult male rats. The alpha 1-adrenoceptor agonist, phenylephrine (10(-9) - 10(-7) M), stimulated release of GnRH from hypothalami from adult (200 g) and peripubertal (150 g) rats but inhibited markedly the secretion of the releasing factor from the limited stores available in hypothalami from immature (50 or 100 g) rats. The stimulatory and inhibitory responses to phenylephrine, evident in adult and younger rats respectively, were concentration-dependent and antagonized readily by the selective alpha 1-adrenoceptor antagonist, alfuzosin (10(-6) M), but not by the beta-adrenoceptor antagonist, propranolol (10(-6) M). Hypothalami from 14-day castrated adult rats, in which the serum LH was elevated and hypothalamic GnRH content reduced, responded to alpha 1-adrenoceptor stimulation in vitro, like those from immature rats, with a marked reduction in GnRH release. In contrast, hypothalami from corresponding castrates bearing testosterone implants, which maintained the hypothalamic GnRH content and serum LH and testosterone concentrations at levels similar to those of intact controls, exhibited the normal 'adult' response to phenylephrine. Studies utilizing 3H-prazosin indicated that the number (Bmax) of hypothalamic alpha 1-adrenoceptor binding sites increases at puberty but that receptor affinity (KD) is unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

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.