Rauwolscine Binding Sites Research Articles

Role of α 2-adrenoceptors in enhancement of antinociceptive effect in diabetic mice

The present studies investigated behavioral and neurochemical aspects of the noradrenergic and serotonergic nervous systems in streptozotocin-induced diabetic mice. We previously reported that intrathecal (i.t.) injection of norepinephrine significantly potentiated antinociception in diabetic mice compared to that in non-diabetic mice, and that antinociception due to norepinephrine injection was completely abolished by pretreatment with yohimbine, an α 2-adrenoceptor antagonist. The present studies demonstrated that i.t. injection of clonidine also showed more-potent antinociceptive activity in diabetic mice than in non-diabetic mice, but that i.t. methoxamine injection did not affect diabetic or non-diabetic mice. The antinociceptive potency due to i.t. injection of 5-HT was significantly lower in diabetic than in non-diabetic mice. In a neurochemical study, we found that the density of [ 3H]-rauwolscine binding sites in spinal α 2-adrenoceptors was significantly higher in diabetic than in non-diabetic mice, but that the binding affinity was unchanged. Spinal norepinephrine turnover was determined by measuring the decline in tissue norepinephrine concentration at 3 h after injection of the tyrosine hydroxylase inhibitor α-methyl- p-tyrosine. The spinal norepinephrine concentration decreased to 43.7% from the baseline in non-diabetic mice, while it was 21.0% in diabetic mice. These results suggest that, based on the decrease of norepinephrine release in the spinal cord, up-regulation of spinal α 2-adrenoceptors caused the increase of antinociception due to i.t. injection of an α 2-adrenoceptor agonist in streptozotocin-induced diabetic mice, and it seemed that the stimulation of α 2-adrenoceptors potentiated the antinociceptive effect. Thus, the spinal noradrenergic systems play an important moderating role in diabetes-induced neuropathic pain.

Passive avoidance learning involves alpha2-noradrenergic receptors in a day old chick.

Day-old chicks were trained on one-trial passive avoidance task, using methyl-anthranilate (MeA) as an aversive substance. Bilateral pharmacological manipulation of the intermediate hyperstriatum ventrale was performed by intracerebral application of an alpha2-noradrenergic agonist, clonidine (5 microM), or an antagonist, rauwolscine (300 microM). Only rauwolscine application (pre- or post-training) induced significant memory impairment. Quantitative receptor autoradiography was used to determine the kinetic properties of the binding sites for [3H]clonidine or [3H]rauwolscine in MeA-trained or water-trained (control) chicks, in forebrain areas known to be involved in avoidance learning. Scatchard analysis revealed that MeA-training resulted in a significant bilateral upregulation in the number of [3H]rauwolscine binding sites (Bmax) in the area of hyperstriatum ventrale. These findings suggest the importance of activation of alpha2-noradrenergic receptors in aversive learning in chicks.

Expression of α2 adrenoceptors during rat brain development—II. α2C messenger RNA expression and [ 3H]rauwolscine binding

The distributions of α 2C adrenoceptor messenger RNA and high-affinity [ 3H]rauwolscine binding sites were characterized in developing rat brain. Using in situ hybridization with 35S-labeled riboprobes directed against the third intracellular loop, α 2C messenger RNA expression appeared in an adult-like pattern during the first and second postnatal weeks, in the anterior olfactory nucleus, caudate–putamen, olfactory tubercles, islands of Calleja and hippocampus, following the time-course of maturation of these structures. Only in the cerebellum was α 2C messenger RNA transiently expressed during the critical period of granule cell development. High-affinity [ 3H]rauwolscine binding sites were detected using receptor autoradiography and revealed a similar spatial and temporal time-course of appearance during rat brain development. The highest numbers of binding sites were detected in the olfactory tubercles and islands of Calleja, and moderate numbers in the anterior olfactory nucleus, caudate–putamen and hippocampus. Like α 2C messenger RNA expression, high-affinity [ 3H]rauwolscine binding sites were transiently expressed in the cerebellum. In some areas (e.g., the substantia nigra), [ 3H]rauwolscine binding sites were detected even though α C2 messenger RNA expression was absent. The strong spatial and temporal correspondence between messenger RNA expression and radioligand binding supports the conclusion that [ 3H]rauwolscine selectively labels α 2C adrenoceptors in the rat brain. The developmental pattern which was observed is in marked contrast to the early, transient expression of the α 2A adrenoceptor. Thus, the α 2A and α 2C receptor types may serve distinct functional roles in the developing brain.

Unusual αadrenoceptor subtype in canine saphenous vein: comparison to mesenteric vein

1. We investigated the nature of the adrenoceptors in the dog saphenous vein (DSV) and dog mesenteric vein (DMV) to determine the nature of the unexpected interactions of phenylephrine and methoxamine with rauwolscine in the DSV, i.e. the ability of the putative alpha 2-adrenoceptor antagonist to inhibit competitively contractions to these alpha 1-agonists. Radioligand binding studies were performed in parallel with contractility studies. 2. Functionally, in the DSV, phenylephrine and methoxamine-induced, contractions were antagonized by rauwolscine with Schild slopes of -0.52 and -0.46, respectively and apparent pA2 values of 8.5 and 9.2, respectively. Such antagonism was not observed in the DMV. In the DSV, prazosin competes for [3H]-rauwolscine binding sites with a high and a low affinity binding site (Ki of 1.49 +/- 0.65 and 94.7 +/- 51 microM, n = 6, respectively). 3. Pretreatment with 100 microM chloroethylclonidine (CEC) for 15 min abolished [3H]-prazosin binding in microsomes from both veins and reduced binding (Bmax) of [3H]-rauwolscine in microsomes by 55.1 +/- 0.8% (n = 3) in the DSV but did not affect the Bmax in the DMV. CEC pretreatment in the venular rings denuded of endothelium caused persistent contraction in the DSV but not in the DMV. In the DSV, CEC appeared to interact with a single [3H]-rauwolscine binding site. In both the DSV and the DMV, CEC (100 microM) caused a significant shift in the EC50 values for phenylephrine and methoxamine. Maximum responses in the DMV were significantly attenuated while those in the DSV were unaffected when total tension was considered. 4. Studies of the functional interactions of the DSV and the DMV with WB 4101 or 5-methylurapidil (5-MU) suggested the presence of alpha 1D-adrenoceptors in the DSV and alpha 1A-adrenoceptors in the DMV. The receptors inactivated by CEC in the DMV and DSV may represent some or all of the receptors with properties of alpha 1D and alpha 1A-receptors present in the two veins. Studies of radioligand binding interactions of these two antagonists with [3H]-prazosin, were consistent with the presence of some alpha 1D-receptors in DSV and alpha 1A-receptors in DMV. These findings raise questions about the selectivity of CEC in differentiating alpha 1-adrenoceptor subtypes. 5. B-HT 920 caused contractions in the DSV smaller than those to the alpha 1-agonists but the maximum was not affected by CEC pretreatment. The EC50 values were shifted to the left after CEC. In radioligand binding studies, B-HT 920 competition for [3H]-rauwolscine binding was not significantly affected by CEC pretreatment. 6. These results suggest the presence of unusual alpha-adrenoceptors in the DSV. In addition to alpha 2-adrenoceptors, receptors recognizing rauwolscine as well as prazosin, WB 4101, phenylephrine and methoxamine and susceptible to inactivation by CEC are present. They appear to be, in part, unusual alpha 1D-adrenoceptors.

α 2-Adrenoceptors in the guinea-pig uterus: heterogeneity in the circular and longitudinal smooth muscle layers

Homogenate binding and functional studies have been undertaken to investigate the role of α 2-adrenoceptors on the circular and longitudinal myometrial layers of the dioestrous guinea-pig uterus. Each myometrial layer contained a single population of [ 3H]rauwolscine binding sites ( K Dvalues ≈ 3 nM) for which yohimbine exhibited a higher affinity than prazosin, and xylazine a higher affinity than phenylephrine, indicating the presence of α 2-adrenoceptor binding sites. In circular myometrium, xylazine enhanced contractile responses, was more potent than either noradrenaline or phenylephrine in inhibiting forskolin-stimulated accumulation of cyclic AMP, and reduced the inhibitory effect of forskolin on phenylephrine-induced phosphatidyl inositol hydrolysis. In longitudinal myometrium xylazine enhanced contractile responses to phenylephrine but did not inhibit forskolin-stimulated accumulation of cyclic AMP. We conclude that α 2-adrenoceptor binding sites are present in both uterine layers and mediate uterine contractility possibly through different mechanisms.

Postnatal development of central nervous α2-adrenergic binding sites: an in vitro autoradiography study in the tree shrew

The postnatal development of the α 2-adrenoceptor pattern was investigated by in vitro receptor autoradiography with the antagonist [ 3H]rauwolscine in the brains of tree shrews ( Tupaia belangeri). At birth, high numbers of [ 3H]rauwolscine binding sites are diffusely distributed in the whole brain with exception of the neocortex which is very weakly labeled at this time. While the number of [ 3H]rauwolscine binding sites in the cerebellum decreases to low levels during the first three postnatal weeks, several brain regions show a significant increase in binding sites which are progressively concentrated in distinct nuclei. In the medulla oblongata, the diffuse labeling pattern changes so that binding sites become centralized in the dorsomedial nuclei. In the pons, similar changes can be observed with a moderate labeling of the locus coeruleus on postnatal day 10 and a strong labeling in the adult. In the thalamus, a transient appearance of high numbers of [ 3H]rauwolscine binding sites can be observed during the second and third postnatal week in specific nuclei. In the preoptic area and hypothalamus, there are only minor postnatal changes but the numbers of [ 3H]rauwolscine binding sites decrease between postnatal day 5 and adulthood. The high number of binding sites in the limbic system does not significantly change after birth. In the neocortex and the superior colliculus, the [ 3H]rauwolscine labeling pattern which is characteristic for the adult is achieved not before the third postnatal week. Competition experiments demonstrate that [ 3H]RAUW binds with high affinity to α 2-adrenoceptors in the postnatal as well as in the adult brain. Therefore, this study demonstrates region specific developmental profiles of the pattern of α 2-adrenoceptors in the postnatal tree shrew brain.

Postnatal development of 3H-rauwolscine binding sites in the dorsal lateral geniculate nucleus and the striate cortex of the tree shrew (Tupaia belangeri).

Noradrenaline has been shown to play an important role within the visual system of the brain. To analyze the postnatal development of alpha2-noradrenergic receptors in the visual system of tree shrews, we localized and quantified binding sites for the antagonist [3H]-rauwolscine by in vitro-autoradiography in the dorsal lateral geniculate nucleus and the striate cortex at different postnatal ages. At birth, the dorsal lateral geniculate nucleus is only slightly labeled by [3H]-rauwolscine. During the postnatal period, the number of binding sites increases to reach a maximum around postnatal day 20. Since the young tree shrews open their eyes at approximately day 19, it appears that this high concentration of alpha2-adrenoceptors is related to eye opening. In the adult animal, [3H]-rauwolscine labeling shows a laminated pattern in the dorsal lateral geniculate nucleus. Laminae 1, 2, and 3 are more strongly labeled than laminae 4, 5, and 6. In the striate cortex, the pattern of [3H]-rauwolscine-binding sites changes dramatically during the early postnatal period. Immediately after birth, there is only one layer, located within the subplate zone, which is labeled. From postnatal day 5 onwards, all cortical layers which can be distinguished on histologically stained sections reveal [3H]-rauwolscine-binding sites, but in layer IV, which is known to receive major inputs from the dorsal lateral geniculate nucleus, there is very little labeling during the first two postnatal weeks. In this layer, a large number of [3H]-rauwolscine-binding sites occurs between postnatal day 15 and 20, that is slightly before and around the time of eye opening.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiographic localization of prazosin and rauwolscine binding sites in the human kidney

In the human kidney, binding sites for the alpha 1-adrenoceptor antagonist 3H-prazosin and the alpha 2-adrenoceptor antagonist 3H-rauwolscine were localized and quantified by in vitro autoradiography. 3H-prazosin binding was found predominantly in the renal cortex. In the medulla, tubular structures were also specifically labelled. No binding sites, however, were detected in association with glomeruli or large blood vessels. 3H-rauwolscine labelled the medullary vascular bundles intensively, but no binding sites were associated with glomeruli or other cortical structures. Thus, the binding pattern for 3H-prazosin is quite similar in both human and rat renal cortex. There are, however, distinct differences between human and rat kidneys in the distribution of the alpha 2-adrenergic binding sites visualized with the antagonist 3H-rauwolscine.

3H]Rauwolscine binding sites in the brains of male tree shrews are related to social status

The aim of the present study was to investigate the influence of social status on central nervous α 2-adrenoceptors. Using the specific α 2-adrenoceptor antagonist [ 3H]rauwolscine, binding sites in the brains of dominant and subordinate male tree shrews were quantified by in vitro autoradiography. In 5 of the 14 brain structures investigated, subordinates had significantly lower numbers of binding sites than dominants. These structures were the solitary tract nucleus, the dorsal motor nucleus of the vagus, the periaqueductal gray, the perifornical region of the hypothalamus and the medial nucleus of the amygdala. These brain areas are all intimately involved in the regulation of autonomic functions and of emotional behavior. Also the affinities for [ 3H]rauwolscine differed between the groups. In 3 nuclei, the solitary tract nucleus, the periaqueductal gray and the medial nucleus of the amygdala, dominants had significantly higher K d-values than subordinates. This demonstrates the presence of low affinity binding sites in dominants which do not exist in subordinates. It is suggested that the low number of [ 3H]rauwolscine binding sites in subordinates results from down-regulation of α 2-adrenoceptors by high levels of noradrenaline and/or adrenaline. The disappearance of low affinity [ 3H]rauwolscine binding sites may play an important role in the etiology of psychosocial stress.

Alpha 2-adrenergic receptors on the circular smooth muscle of canine small intestine.

We have studied the distribution and properties of alpha 2-adrenergic receptors in the circular muscle layer (containing deep muscular plexus) of canine small intestine. Using radioactivity labelled rauwolscine, we located the binding sites to the neuronal membranes supporting the prejunctional action of alpha 2-adrenergic agents in the gut. Moreover, although the functional data to suggest the existence of postjunctional alpha 2-adrenergic receptors coupled to contraction are not available so far, we measured a substantial number of rauwolscine binding sites on the smooth muscle plasma membranes. Scatchard and Hill analyses of the saturation data were indicative of the presence of a single high affinity site (Hill coefficient 0.996) with a KD value of 8.8 nM and the maximum number of binding sites (Bmax) of 313 fmol/mg of protein. Competition studies suggested the presence of multiple subtypes of alpha 2-adrenoceptors.

3H]Rauwolscine behaves as an agonist for the 5-HT a receptors in human frontal cortex membranes

The alpha 2 adrenergic antagonist [3H]rauwolscine binds with comparable nanomolar affinity to alpha 2 adrenoceptors and the nonadrenergic 5-HT1A receptors sites in human frontal cortex membranes. Addition of 0.5 mM GTP into the incubation medium produces a significant decrease in the amount of [3H]rauwolscine binding sites (Bmax = 230 +/- 16 and 115 +/- 11 fmol/mg protein in the absence and presence of GTP, respectively). The affinity for [3H]rauwolscine remains unchanged (i.e. KD = 40 +/- 0.9 nM and 4.1 +/- 1 nM). This effect of GTP can be attributed to decreased binding of the radioligand to the 5-HT1A receptors. GTP decreases binding of [3H]rauwolscine to nearly the same level as the one corresponding to the alpha 2 adrenoceptors in membranes from both the human frontal cortex and hippocampus. The venom of the marine cone snail, Conus tessulatus, preferentially inhibits [3H]rauwolscine binding to 5-HT1A receptors as compared with the alpha 2 adrenoceptors. Following complete masking of the 5-HT1A receptors by this venom. GTP no longer affects the saturation binding characteristics of [3H]rauwolscine for the remaining alpha 2 adrenoceptors. Nucleotides decrease the binding of [3H]rauwolscine to the 5-HT1A receptors with an order of potencies (i.e. GTP gamma S greater than GPP(NH)P much greater than GDP greater than GTP much greater than ATP) that is typical for nucleotide-mediated receptor-G protein dissociation. This suggests that [3H]rauwolscine is a 5-HT1A receptor agonist and this conclusion is compatible with earlier functional studies, indicating that rauwolscine (as well as yohimbine) has agonistic properties at the level of 5-HT autoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of subcellular localization of α 2-adrenoceptors in dog splenic nerve

Two weeks after denervation of the dog spleen, α 2-adrenoceptors labelled by [ 3H]-rauwolscine were decreased to 44% of control. In splenic nerve, high-affinity, specific, stereoselective [ 3H]rauwolscine binding was observed, with a B max of 211 ± 49.8fmol/mg protein and a K d of 4.33 ± 0.87nM. The [ 3H]rauwolscine binding sites in the splenic nerve were not co-localized with noradrenaline (NA) in the NA-storage vesicles, as revealed by sucrose density gradients. Moreover, unlike NA and its storage vesicles, no accumulation of α 2-adrenoceptors against a ligature could be observed. The results demonstrate the presence of α 2-receptors in the splenic nerve, and their localization in the axon, distinct from the large dense-cored vesicles.

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.

Characterization of α-adrenoceptor subtypes by [3H]prazosin and [3H]rauwolscine binding to canine venous smooth muscle membranes

Postsynaptic alpha-adrenoceptor subtypes were studied using [3H]prazosin and [3H]rauwolscine binding to plasmalemma-enriched microsomal fractions isolated from dog saphenous veins and mesenteric veins. Both radioligands showed saturable binding consistent with the presence of a single homogeneous binding site in each case, based on Scatchard analysis. The Kd values of [3H]prazosin and [3H]rauwolscine, calculated from kinetic studies were similar to those from equilibrium binding data in both venous muscle membranes. The microsomal membranes of dog saphenous vein and mesenteric vein contained about a fourfold higher density of the high affinity [3H]rauwolscine binding sites than those for [3H]prazosin binding. In competition studies, IC50 values for displacement of rauwolscine or prazosin suggested that the sites of interaction for the antagonists prazosin and rauwolscine were independent. Phenylephrine, a functionally selective alpha-adrenoceptor agonist, competed with a similar IC50 value for the specific binding sites of [3H]prazosin and [3H]rauwolscine; but B-HT 920, a functionally selective alpha 2-adrenoceptor agonist, competed for [3H]rauwolscine and [3H]prazosin binding with distinctly different IC50 values. Our data show the existence of two populations of alpha-adrenoceptor antagonist binding sites in the plasma membranes of dog saphenous vein and mesenteric vein, and raise the question whether agonist selectively depends on different affinities or on differential efficacies at one or two sites.

Characterization of intrarenal arterial adrenergic receptors in renovascular hypertension.

alpha-Adrenergic receptor subtypes were investigated using [3H]prazosin, an alpha 1 selective antagonist, and the alpha 2 selective antagonist [3H]rauwolscine in a smooth muscle plasma membrane enriched microsomal fraction prepared from rabbit intrarenal arterial vasculature. Both radioligands displayed single components on Scatchard analysis. The specific binding of [3H]prazosin was of high affinity (0.54 +/- 0.04 nM) with a maximum binding capacity (Bmax) of 212 +/- 15 fmol/mg protein. The maximum number of [3H]rauwolscine binding sites was 64 +/- 4 fmol/mg of protein with a dissociation constant (Kd) of 5.60 +2- 0.27 nM. Binding of both radioligands was rapid, saturable, and specific. alpha 1- and alpha 2-adrenergic receptors in the intrarenal arterial membrane preparation were also characterized at 2-, 4-6-, and 10-12-week intervals during the course of development and maintenance of chronic two-kidney, one clip (2K1C) Goldblatt hypertension and in age-matched sham-operated normotensive control rabbits. The alpha 1-adrenergic receptor affinity for [3H]prazosin binding in hypertensive rabbits was significantly increased in the stenotic, but not contralateral, kidney at 2 weeks; however, at 6 weeks the receptor affinity of both kidneys was significantly increased compared with those of the normotensive control group. No difference in alpha 1-adrenergic receptor affinity was seen at 12 weeks, and there were no changes in Bmax at any of the weekly intervals. Neither the Kd, nor Bmax, for [3H]rauwolscine in either kidney showed a significant difference between hypertensive rabbits and normotensive control rabbits. These studies demonstrate the existence in the rabbit intrarenal arterial vasculature of binding sites with alpha 1- and alpha 2-adrenergic receptor specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride interacts with [ 3H]idazoxan and [ 3H]rauwolscine binding sites in rabbit urethra

Amiloride and its analogues (N-ethylisopropylamiloride and banzamil) interact more potently with [ 3H]idazoxan binding sites (nM range) than with [ 3H]rauwolscine binding sites (μM range) in the rabbit urethra. The binding of both radioligands was competitivelly inhibited by amiloride, with increased K D values and no change in their binding capacity. Interestingly, amiloride was a potent inhibitor at [ 3H]idazoxan binding sites in rabbit urethral smooth muscle at concentrations far below those required to inhibit the Na +/H + exchanger or electrogenic pump.

Chronic steroid contraceptive treatment decreases renal α-adrenoceptor levels in the rat

Female Sprague-Dawley rats were injected s.c. with ethynyloestradiol (EE 2, 0.2 μg/day) and levonorgestrel (NG, 2.0 μg/day) separately and in combination (EE 2/NG). Binding of [ 3H]rauwolscine ( α 2-adrenoceptor specific) and [ 3H]prazosin ( α 1-adrenoceptor specific) was examined in crude membrane suspensions prepared from whole rat kidney after 3, 6 and 12 weeks of steroid administration. Receptor affinity was high for both ligands (K D, equilibrium dissociation constant [ 3H]rauwolscine, ⋍2.0 nM ; [ 3H]prazosin, ⋍0.2 nM ) and was not altered in rats chronically treated with steroid contraceptives. The B max (maximum density of binding sites) for [ 3H]prazosin binding was not altered, indicating no change in the number of renal α 1-adrenoceptors. NG administered alone did not affect the numbers of α 1- or α 2-adrenoceptors. Catechol metabolites of endogenous oestrogens did not displace the binding of either radioligand, suggesting that these metabolites do not directly interact with renal α-adrenoceptors. However, after 12 weeks treatment, the number of [ 3H]rauwolscine binding sites was reduced in both EE 2 (B max, 133 ± 7 fmol/mg protein)- and combined EE 2/NG (135 ± 11 fmol/mg protein)-treated rats, compared to controls (162 ± 9 fmol/mg protein). Since renal α 2-adrenoceptors inhibit renin release, this reduction in α 2-adrenoceptor number may contribute to increased renin levels associated with oestrogen-induced hypertension.

Evidence that epinephrine acts preferentially as an antilipolytic agent in abdominal human subcutaneous fat cells: assessment by analysis of beta and alpha 2 adrenoceptor properties.

Investigations were carried out to demonstrate the function and the possible advantage of the interplay between beta 1 and alpha 2 adrenoceptor sites in the regulation of human subcutaneous fat-cell lipolysis. alpha 2 and beta adrenoceptor binding studies were conducted with antagonist radioligands and revealed that alpha 2-adrenoceptors ([3H]yohimbine and [3H]rauwolscine binding sites) are more numerous than beta 1-adrenoceptors ([3H]dihydroalprenolol and [3H]CGP-12177 binding sites) in human fat-cell membranes. Physiological agonists epinephrine and norepinephrine competed with [3H]-ligand sites with a higher affinity for alpha 2 sites than for beta 1 sites. Epinephrine exhibited a higher affinity than norepinephrine for the alpha 2 sites; the two amines had the same affinity for beta 1 sites. In lipolysis studies conducted in the absence of adenosine deaminase the beta lipolytic action of the biological amines predominated; after alpha 2-adrenoceptor blockade by yohimbine or idazoxan, the amines exhibited an intrinsic activity similar to that of isoproterenol. When adenosine was prevented from accumulating in the incubation medium by inclusion of adenosine deaminase, low concentrations of epinephrine and norepinephrine preferentially exerted an antilipolytic action. We conclude that: he lipolytic response in abdominal human subcutaneous fat cells to physiological amines results from the interplay between beta 1-and alpha 2-adrenoceptor stimulation; alpha 2 adrenoceptors, with their higher number and higher affinity for the physiological amines, and the adrenoceptor population involved at the lowest (i.e. physiological) concentrations of the amines.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired Recovery of Alpha1-and Alpha2-adrenergic Receptors in Brain Tissue of Aged Rats

The influence of age on the rate of recovery of alpha 1- and alpha 2-adrenergic receptors was determined in rat brain following their irreversible inhibition by phenoxybenzamine (PBZ). Fischer 344 rats (3- or 24-months old) were administered two doses of vehicle or PBZ at 12-hour intervals. At various times after the last dose of PBZ, alpha-adrenergic receptors were quantified in washed membranes of cerebral cortex and hypothalamus, using [3H]prazosin and [3H]rauwolscine to label alpha 1- and alpha 2-adrenergic receptors, respectively. Recovery of prazosin binding sites in cortex and hypothalamus and of rauwolscine binding sites in cortex was significantly delayed in aged versus young rats. Administration of [3H]PBZ to young and aged rats revealed no apparent differences in the concentration or rate of disappearance of PBZ or its metabolites that could account for these age-related changes. These data suggest that the synthesis of both alpha 1- and alpha 2-adrenergic receptors is impaired in brain tissue from aged rats.