Rauwolscine Binding Research Articles

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.

The alpha 2-adrenoceptors of the human retinoblastoma cell line (Y79) may represent an additional example of the alpha 2C-adrenoceptor.

1. In agreement with the literature, correlation of the ability of a series of agonists and antagonists to displace [3H]-rauwolscine binding shows the alpha 2-adrenoceptors of HT29 cells, NG108-15 cells, OK cells and homogenates of rat sublingual gland to represent four distinct subtypes. 2. [3H]-rauwolscine also bound with high affinity (KD = 0.30 +/- 0.10 mM) to a human retinoblastoma cell line (Y79). Specific binding represents 73% of total binding, and a Bmax of 38 +/- 1 fmol mg-1 protein was determined. 3. Correlation of antagonist affinities against [3H]-rauwolscine with corresponding values in the other four tissue sources showed the Y79 cells to resemble most closely the OK cells, the prototype example of an alpha 2C-adrenoceptor, with a correlation coefficient of 0.90 and a regression slope of 1.01 being obtained for 10 antagonists in these two systems. 4. Comparison of KD values for [3H]-rauwolscine also showed a similarity between the OK cells (0.19 +/- 0.07 nM) and Y79 cells. 5. These data suggest that the human retinoblastoma cell line may represent an additional example of the alpha 2C-adrenoceptor subtype.

Characterization of [ 3H]atipamezole as a radioligand for α2-adrenoceptors

Atipamezole (MPV-1248, 4-(2-ethyl-2,3-dihydro-1H-inden-2-yl)-1H-imidazole), a potent α 2-adrenoceptor antagonist, was tritiated to high specific activity. We then compared [ 3H]atipamezole and [ 3H]rauwolscine as radioligands for α 2-adrenoceptors in rat cerebral cortex, neonatal rat lungm, and human platelets. (−)-Noradrenaline and phentolamine were used to define specific α 2-adrenergic binding. Unlabelled atipamezole was used in a similar manner to define saturable, high-affinity non-adrenergic binding. [ 3H]Atipamezole binding to human platelets (K d 1.3 nM) and rat brain membranes (K d 0.5 nM) equilibrated rapidly and was displaced in the expected manner by α 2-adrenergic ligands. In contrast, [ 3H]atipamezole binding in neonatal rat lung membranes was only effectively inhibited by unlabelled atipamezole, and by high concentrations of idazoxan. The total density of binding sites for [ 3H]atipamezole was clearly in excess of the density of α 2-adrenoceptors in this tissue, as defined by [ 3H]rauwolscine binding. We conclude that [ 3H]atipamezole binds with high affinity to α 2-adrenoceptors in human platelets and rat cerebral cortex, and that the compound can be used to investigate α 2-adrenoceptor properties and drug actions in these tissues. In neonatal rat lung, [ 3H]atipamezole identified an additional population of binding sites, distinct from both classical α 2-adrenoceptors and idazoxan-defined imidazoline receptors. The pharmacological identity of these binding sites remains to be elucidated. This non-adrenergic component in the binding characteristics of [ 3H]atipamezole complicates its use as a general α 2-adrenoceptor radioligand.

Stable expression of recombinant human α2-adrenoceptor subtypes in two mammalian cell lines: characterization with [ 3H]rauwolscine binding, inhibition of adenylate cyclase and RNase protection assay

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

Differential localization of α2-adrenergic receptor subtypes in brain

The pharmacological identification and characterization of subtypes of α 2-adrenergic receptors have been confirmed by molecular biological investigations. Using receptor autoradiographic techniques, it has been possible to show regions of the brain where α 2 agonist binding ([ 3H]para-aminoclonidine) is preferentially labeling the presumed guaninenucleotide-sensitive, high-affinity conformations of the α 2 receptor. Careful examination of autoradiograms generated using the tritiated antagonists yohimbine, idazoxan, and rauwolscine also indicates some disparity in the regions occupied by these radiolabeled ligands. Inhibition of [ 3H]rauwolscine binding with the subtype selective compounds, ARC-239, or oxymetazoline demonstrates that there are discrete regions of the brain where one receptor subtype predominates over the other. These studies indicate that previous investigations utilizing the agonist para-aminoclonidine as the ligand for obtaining labeling of α 2 receptors have overlooked some regions of binding due to the subtype selectively of this ligand. A more complete localization of α 2-adrenergic receptors can be obtained using the triated antagonist rauwolscine, and the differential distribution of at least two subtypes of the α 2 receptor can be obtained by selective inhibition of this binding.

Alpha 2-adrenoceptor changes during cerebral ageing. The effect of Ginkgo biloba extract.

[3H]Rauwolscine binding to alpha 2-adrenoceptors in cerebral cortex and hippocampus membranes of young (4 months) and aged (24 months) Wistar rats has been investigated. In aged rats, Bmax values of [3H]rauwolscine binding were significantly reduced (25-32%) in the cerebral cortex and hippocampus, as compared with the number of alpha 2-adrenoceptors found in young rats. Chronic treatment with Ginkgo biloba extract did not alter [3H]rauwolscine binding in the hippocampus of young rats, but significantly increased (28%) the [3H]rauwolscine binding density in aged rats. These data confirm the previously described age-related noradrenergic alteration and suggest that noradrenergic activity in aged rats is more susceptible to Ginkgo biloba extract treatment.

Binding characteristics of naftopidil and α1-adrenoceptor antagonists to prostatic α-adrenoceptors in benign prostatic hypertrophy

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

Renal .ALPHA.2-Adrenoceptor Overexpression in Dahl Salt-Sensitive Rats. Regulation by Dietary NaCl and Dissociation from Salt-Insensitive Hypertension.

Dietary NaCl-induced changes in blood pressure and agonist and antagonist binding properties to renal α2-adrenoceptors were studied in inbred (Rapp) strains of Dahl salt-sensitive (S) and salt-resistant (R) rats. S and R rats were placed on either salt-deficient (0.13% NaCl in chow) or high salt (1% NaCl solution to drink plus 0.13% NaCl chow) diets for 1 to 8 weeks. Saturation binding studies with [3H]-rauwolscine and [3H]-p-aminoclonidine and competition studies with [3H]-rauwolscine and epinephrine were then performed in renal plasma membranes from each group. Systolic blood pressure rose significantly in S rats on the salt-deficient diet for 2 or more weeks, and was further increased by high salt intake, indicating that there are salt-sensitive and salt-insensitive components to the hypertension in inbred S rats. The blood pressure of R rats was unaffected by dietary NaCl content. Two or more weeks of high salt feeding significantly elevated renal α2-adrenoceptor density ([3H]-rauwolscine binding) in S but not R rats. Interestingly, NaCl restriction in weanling S rats completely prevented this increase in α2-adrenoceptor density over the course of the study. The number of high affinity agonist binding sites (assessed with [3H]-p-aminoclonidine and epinephrine displacement of [3H]-rauwolscine) was increased in S rats after high salt feeding. Removal of the high salt diet failed to reverse the NaCl-induced elevations in total and high affinity α2-adrenoceptor density in S rats. We conclude that: 1) the initiation of renal α2-adrenoceptor overexpression in Dahl S rats is entirely dependent on dietary NaCl content; 2) the overexpression of renal α2-adrenoceptors was independent of the salt-insensitive component of hypertension in S rats, precluding a causal linkage between these parameters; 3) the rise in renal α2-adrenoceptors was closely paralleled by salt-sensitive increments in blood pressure, suggesting a functional relationship between these two variables; 4) In contrast to reported changes in renal α2-adrenoceptor properties following renal denervation, dietary NaCl augments the number of high affinity renal α2-adrenoceptor agonist binding sites in S rats but does not increase the proportion of these sites relative to total receptor number. (Hypertens Res 1992; 15: 85-92)

Characterization of prazosin-sensitive alpha 2 B-adrenoceptors expressed by cultured rat IMCD cells

alpha 2-Adrenoceptor subtype expression was investigated in cultured rat inner medullary collecting duct (IMCD) cells using radioligand binding studies, Northern blot analysis, and adenosine 3',5'-cyclic monophosphate (cAMP) assays. [3H]rauwolscine bound to a single class of alpha 2-adrenoceptors with high affinity [Kd = 1.7 +/- 0.3 nM, maximum binding (Bmax) = 45.2 +/- 10.8 fmol/mg protein]. alpha 2-Adrenoceptor ligands inhibited [3H]rauwolscine binding with a rank order of potency characteristic of interaction with the alpha 2B-adrenoceptor [inhibitory constant (Ki) values (in nM) rauwolscine (1.95) greater than ARC-239 (8.52) greater than prazosin (237) greater than oxymetazoline (30,000)]. Northern blot analysis was performed using poly(A)+ RNA isolated from 90% confluent rat IMCD cells and probes derived from alpha 2-adrenoceptor DNA sequences from the rat nonglycosylated alpha 2B-adrenoceptor and the human alpha 2A-adrenoceptor. The alpha 2B probe hybridized to a 4.2-kb band under high stringency conditions, but the alpha 2A-adrenoceptor probe did not hybridize to this band. In functional studies, the full alpha 2-adrenoceptor agonists epinephrine and UK-14,304 potently inhibited vasopressin-stimulated cAMP accumulation by 50 to 70% [half-maximal response (EC50) (in nM) epinephrine = 11.2, UK-14,304 = 6.4]. Guanabenz and clonidine were partial agonists, inhibiting cAMP accumulation by 30 to 40% and were less potent than the full agonists [EC50 (in nM) 56.0 guanabenz and 94.5 clonidine]. Epinephrine-induced inhibition of cAMP accumulation was blocked by rauwolscine, prazosin, and ARC-239 but not by the alpha 1-adrenoceptor antagonist corynanthine. We conclude that rat IMCD cells in primary culture express functional alpha 2-adrenoceptors of the alpha 2B-subtype.

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)

Vascular effects of tetramethylpyrazuie: direct interaction with smooth muscle α-adrenoceptors

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

Down-regulation of α 2 adrenoceptors in ventrolateral medulla of spontaneously hypertensive rats

The binding of [ 3H]idaxazon to imidazole sites and [ 3H]rauwolscine to α adrenoceptors of neuronal membranes prepared from cerebral cortex and ventrolateral medulla of 10 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. [ 3H]idaxazon bound to the membranes of cerebral cortex and ventrolateral medulla at a single high affinity site. The binding of [ 3H]idaxazon in ventrolateral medulla and cerebral cortex was found to be similar in SHR and WKY rats. [ 3H]Rauwolscine bound to the membranes of cerebral cortex and ventrolateral medulla at a single high affinity site. The binding of [ 3H]rauwolscine in the cerebral cortex was found to be similar in SHR and WKY rats. However, in the ventrolateral medulla [ 3H]rauwolscine binding was found to be significantly lower in SHR as compared to WKY rats. The decreased binding was due a decrease (32%) in the B max value in SHR rats as compared to WKY rats. The K d values were similar in SHR and WKY rats. It is concluded that imidazole binding sites are not affected while, α 2 adrenergic binding sites are decreased in the ventrolateral medulla of SHR rats and may be contributing to the regulation of blood pressure.

Imidazoline-guanidinium and α2-adrenergic binding sites in basolateral membranes from human kidney

In the present study, we used [ 3H]idazoxan and [ 3H]rauwolscine to characterize the imidazoline-guanidinium receptive site (IGRS) and α 2-adrenoceptors in the human renal proximal tubule, respectively. In purified basolateral membranes, 11-fold enriched in Na +K + ATPase, [ 3H]idazoxan and [ 3H]rauwolscine binding was twofold higher than in homogenates ([ 3H]idazoxan: 87 ± 19 vs. 45 ± 23.3 fmol/mg protein. P < 0.05: [ 3H]rauwolscine: 56.4 ± 21.4 vs 25.2 ± 7.3 fmol/mg protein. P < 0.01. In competition studies performed at saturating concentration of [ 3H]idazoxan (15 nM), specific binding was competed for by epinephrine and rauwolscine only by 10–15% but was completely inhibited by imidazoline and guanidinium compounds. Thus, in human renal proximal tubule. [ 3H]idazoxan mainly binds to an IGRS. The highest density of α 2-adrenoceptors in basolateral membranes and of IGRS in partially purified membrane preparations, suggests that these two binding sites have a different subcellular localization. When compared to the rabbit renal IGRS, the human [ 3H]idazoxan binding site displays different affinities for guanabenz, rilmenidine, clonidine, amiloride and its derivatives that persist after membrane solubilization. In contrast, the human and rabbit renal IGRS share similar regulatory properties such as the sensitivity to K − and the insensitivity to Na +, divalent cations and 5′-guanylylimidodiphosphate (Gpp(NH)p). In conclusion, we demonstrated that, in the human renal proximal tubule, α 2-adrenoceptors are mainly located in basolateral membranes while IGRS appear to be associated with another cell compartment. As indicated by their common interaction with imidazoline and guanidinium derivatives and by similar regulatory properties, human and rabbit IGRS belong to the same family of membrane proteins. However, the differences in ligand-recognition properties between the two species propose a possible heterogeneity of IGRS.

Non-Adrenergic Binding Sites for the “α-Antagonist” [3H] Idazoxan in Rabbit Urethral Smooth Muscle

In the present study, we have provided evidence that [3H] rauwolscine and [3H] idazoxan bind to different sites in rabbit urethra. The [3H] idazoxan capacity and affinity was 215 +/- 14 fmol/mg protein and 1.59 +/- 0.16 nM while [3H] rauwolscine binding parameters were 45.9 +/- 3.4 fmol/mg protein and 2.39 +/- 0.27 nM. [3H] idazoxan specific binding was inhibited only by compounds possessing an imidazoli(di)ne or a guanidinium moiety, while [3H] rauwolscine specific binding was inhibited by phenylethanolamines and classical alpha 2-antagonists. [3H] idazoxan was inhibited by KCl in a competitive and by MnCl2 in a non-competitive way, while other cations such as Na+, Li+ and Mg2+ did not inhibit [3H] idazoxan binding. Moreover, we investigated the regional distribution of [3H] idazoxan and [3H] rauwolscine along the rabbit urethra using quantitative autoradiography. Analysis of the films revealed a different distribution of these two binding sites on the urethral sections.

Influence of steroid hormone treatments or pregnancy on [3H]prazosin and [3H]rauwolscine binding to myometrial α-adrenoceptors in the ewe

The adrenergic antagonists [3H]prazosin and [3H] rauwolscine were used to identify alpha 1- and alpha 2-adrenoceptors respectively in the ovine myometrium. Ewes were allocated to four groups according to steroid hormone treatments or physiological status, namely ovariectomized ewes either as untreated controls, treated with oestradiol-17 beta or progestagen plus oestradiol-17 beta, and pregnant ewes at mid-gestation. Binding of both [3H]prazosin and [3H]rauwolscine to membrane preparations from the ovine myometrium was saturable, of high affinity and rapidly reversed by phentolamine (10 mumol/l). Based on the relative order of potency of selected adrenergic agonists and antagonists, the myometrial binding sites labelled by [3H]prazosin and [3H]rauwolscine were characterized as alpha 1- and alpha 2-adrenoceptors respectively. Saturation binding studies with [3H]prazosin showed that the number of alpha 1-adrenoceptors was low (maximal binding capacity, Bmax, between 19 and 24 fmol/mg protein) and there were no noticeable differences between the animal groups. Moreover, the equilibrium dissociation constant (Kd) did not vary significantly between groups (Kd between 0.10 and 0.17 nmol/l). In contrast, saturation binding studies with [3H]rauwolscine revealed the presence of a high number of alpha 2-adrenoceptors. Values of Bmax were far higher in the pregnant ewes (1096 +/- 241 fmol/mg protein; means +/- S.D.) than in any of the non-pregnant ovariectomized ewes. For these latter groups, the highest Bmax values were found in the group treated with both progestagen and oestrogen (382 +/- 77 fmol/mg protein) compared with treatment with oestrogen alone (101 +/- 8 fmol/mg protein) or with controls (82 +/- 12 fmol/mg protein.(ABSTRACT TRUNCATED AT 250 WORDS)

α2-adrenoceptors in homogenates of rat nasal mucosa

The specific binding of [ 3H]rauwolscine to rat nasal mucosa membranes was saturable, stereoselective and of high affinity. The Scatchard plot pointed to a homogeneous population of binding sites (K d = 3.6 ± 0.6 nM; B max = 5.1 ± 0.7 pmol/g). The non-specific binding appeared to be non-linear, probably due to filter binding. Inhibition of [ 3H]rauwolscine binding with the subtype-selective antagonist, prazosin, suggested the presence of α 2-adrenoceptor subclasses in rat nasal mucosa. The (−)-epinephrine inhibition curves demonstrated high- and low-affinity agonist binding sites. A monophasic (−)-epinephrine inhibition curve was obtained in the presence of guanine nucleotides.

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.