Effect of dietary salt and cholesterol loading on vascular adrenergic receptors

To evaluate the effects of salt and cholesterol intake on vascular responses to catecholamines, alpha 1- and beta-adrenergic receptor densities were determined in control, cholesterol-loaded, salt-loaded with desoxycorticosterone acetate (DOCA) and furosemide-loaded male rabbits, using [3H]-prazosin and (-)-[125I]-cyanopindolol as ligands, respectively. In the aortic membrane, the density of alpha 1-adrenergic receptors (Bmax = 120 +/- 14 fmol/mg protein, Kd = 0.48 +/- 0.05 nM) was higher than that of beta-adrenergic receptors (Bmax = 10.5 +/- 1.7 fmol/mg protein, Kd = 47.1 +/- 8.6 pM). Salt loading and depletion did not alter the density or affinity of either the alpha 1- or beta-adrenergic receptors. By contrast, cholesterol loading significantly decreased alpha 1-adrenergic receptor affinity to a Kd value of 0.81 +/- 0.11 nM from the control level of 0.48 +/- 0.05 and increased the beta-adrenergic receptor density to a Bmax of 18.7 +/- 1.9 fmol/mg protein from the control level of 10.5 +/- 1.7. These results showed that the density of alpha 1-adrenergic receptors was higher than that of beta-adrenergic receptors in the rabbit aortic membrane preparation, and suggested that the sensitivity of aortic membrane to catecholamines was changed by cholesterol loading.     

Stimulation of high-affinity adenosine A2 receptors decreases the affinity of dopamine D2 receptors in rat striatal membranes.

Since high-affinity adenosine A2 receptors (A2a) are localized exclusively in dopamine-rich regions in the central nervous system and mediate inhibition of locomotor activity, we have examined the effect of A2a receptor activation on D1 and D2 receptor binding in membrane preparations of the rat striatum. The A2a agonist 2-[p-(2-carboxyethyl)phenethylamino]-5'- N-ethylcarboxamidoadenosine (CGS 21680) increased the Kd of the dopamine D2 agonist L-(-)-N-[3H]propylnorapomorphine without affecting the Bmax. The increase in Kd was maximal (40%) at 30 nM CGS 21680. CGS 21680 (30 nM) decreased the dopamine-induced inhibition of [3H]raclopride (a D2 antagonist) binding due to an increase (about 3-fold) in KH and KL, the dissociation constants of high- and low-affinity binding sites. The effects of CGS 21680 were antagonized by the adenosine antagonist 8-phenyltheophylline (10 microM). (-)-N6-(2-Phenylisopropyl)adenosine produced an effect similar to that of CGS 21680, provided the concentration used was high enough to stimulate A2a receptors (300 nM). GTP (50 microM) also decreased the dopamine-induced inhibition of [3H]raclopride binding but, in contrast to CGS 21680, GTP decreased the proportion of D2 receptors in the high-affinity state. CGS 21680 (30 nM) did not affect the Kd or Bmax of [3H]raclopride and failed to affect ligand binding to D1 receptors. Thus, stimulation of A2a receptors potently reduces the affinity of D2 agonist binding sites within the plasma membrane of striatal neurons. This A2a-D2 interaction may underlie the neuroleptic-like actions of adenosine agonists and the enhancing effects of adenosine antagonists, such as caffeine, on locomotor activity.     

Characterization of alpha 2-adrenergic receptors on rat pinealocytes.

alpha 2-Adrenergic receptors in rat pineal membranes were characterized using p-[125I]iodoclonidine, a highly selective, high specific activity ligand. Binding was rapid (association constant rate = 0.0462 nM/min-1) and reversible after the addition of phentolamine (apparent dissociation rate constant = 0.04 min-1). Saturation experiments indicate the presence of a single class of noncooperative binding sites, with an equilibrium binding constant (Kd) of 1.1 +/- 0.3 nM and a binding capacity (Bmax) of 69 +/- 9 fmol/mg protein. Analysis of the relative potency of selected adrenoreceptor agonists and antagonists in competition studies with p-[125I]iodoclonidine indicates that the ligand is binding to a member of the family of alpha 2-adrenergic receptors that has a high affinity for oxymetazoline, phentolamine, and (-)norepinephrine and a low affinity for prazosin, similar to the recently described alpha 2-adrenergic receptor present in the bovine pineal gland, classified as belonging to the newly described alpha 2D-adrenergic receptor subtype. Rat pineal alpha 2-adrenergic receptors were unaltered after nerve endings degenerated. This observation and the recent finding that alpha 2-adrenergic agonists potentiate N6,2'-O-dibutyryl-cAMP or isobutylmethylxanthine stimulation of arylalkylamine N-acetyltransferase in the rat pineal gland establish that alpha 2D-like adrenergic receptors are located on pinealocytes.     

[3H]xanthine amine congener of 1,3-dipropyl-8-phenylxanthine: an antagonist radioligand for adenosine receptors.

An amine-functionalized derivative of 1,3-dipropyl-8-phenylxanthine has been prepared in tritiated form as a xanthine amine congener ([3H]XAC) for use as an antagonist radioligand for adenosine receptors. [3H]XAC has higher receptor affinity, higher specific activity, lower nonspecific membrane binding, and more favorable hydrophilicity than 1,3-diethyl-8-[3H]phenylxanthine, the xanthine commonly used for adenosine receptor binding. In rat cerebral cortical membranes, [3H]XAC exhibits saturable, specific binding with a Kd of 1.23 nM and a Bmax of 580 fmol/mg of protein at 37 degrees C. N6-(R-Phenylisopropyl)adenosine is a more potent inhibitor of [3H]XAC binding than is 5'-N-ethylcarboxamidoadenosine, indicating that binding is to an A1-adenosine receptor. In the absence of GTP, the inhibition curves for adenosine agonists versus [3H]XAC binding are biphasic, indicating that [3H]XAC is binding to low- and high-affinity agonist states of the A1 receptor. In the presence of GTP, adenosine analogs exhibit monophasic, low-affinity inhibition of binding of [3H]XAC. Inhibition of [3H]XAC binding by theophylline or by various 8-phenylxanthines is monophasic, and the potencies are commensurate with the potencies of these xanthines as adenosine receptor antagonists. The receptor sites in calf brain membranes exhibit a higher affinity (Kd = 0.17 nM) for [3H]XAC, whereas sites in guinea pig exhibit a slightly lower affinity (Kd = 3.0 nM). Densities of [3H]XAC binding sites are similar in brain membranes from all species.     

Guanine nucleotide-binding protein regulation of melatonin receptors in lizard brain.

Melatonin receptors were identified and characterized in crude membrane preparations from lizard brain by using 125I-labeled melatonin (125I-Mel), a potent melatonin agonist. 125I-Mel binding sites were saturable; Scatchard analysis revealed high-affinity and lower affinity binding sites, with apparent Kd of 2.3 +/- 1.0 x 10(-11) M and 2.06 +/- 0.43 x 10(-10) M, respectively. Binding was reversible and inhibited by melatonin and closely related analogs but not by serotonin or norepinephrine. Treatment of crude membranes with the nonhydrolyzable GTP analog guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), significantly reduced the number of high-affinity receptors and increased the dissociation rate of 125I-Mel from its receptor. Furthermore, GTP[gamma S] treatment of ligand-receptor complexes solubilized by Triton X-100 also led to a rapid dissociation of 125I-Mel from solubilized ligand-receptor complexes. Gel filtration chromatography of solubilized ligand-receptor complexes revealed two major peaks of radio-activity corresponding to Mr greater than 400,000 and Mr ca. 110,000. This elution profile was markedly altered by pretreatment with GTP[gamma S] before solubilization; only the Mr 110,000 peak was present in GTP[gamma S]-pretreated membranes. The results strongly suggest that 125I-Mel binding sites in lizard brain are melatonin receptors, with agonist-promoted guanine nucleotide-binding protein (G protein) coupling and that the apparent molecular size of receptors uncoupled from G proteins is about 110,000.     

Loss of high-affinity prostacyclin receptors in platelets and the lack of prostaglandin-induced inhibition of platelet-stimulated thrombin generation in subjects with spinal cord injury.

Coronary artery disease is a leading cause of death in individuals with chronic spinal cord injury (SCI). However, platelets of those with SCI (n = 30) showed neither increased aggregation nor resistance to the antiaggregatory effects of prostacyclin when compared with normal controls (n = 30). Prostanoid-induced cAMP synthesis was similar in both groups. In contrast, prostacyclin, which completely inhibited the platelet-stimulated thrombin generation in normal controls, failed to do so in those with SCI. Scatchard analysis of the binding of [3H]prostaglandin E1, used as a prostacyclin receptor probe, showed the presence of one high-affinity (Kd1 = 8.11 +/- 2.80 nM; n1 = 172 +/- 32 sites per cell) and one low-affinity (Kd2 = 1.01 +/- 0.3 microM; n2 = 1772 +/- 226 sites per cell) prostacyclin receptor in normal platelets. In contrast, the same analysis in subjects with SCI showed significant loss (P < 0.001) of high-affinity receptor sites (Kd1 = 6.34 +/- 1.91 nM; n1 = 43 +/- 10 sites per cell) with no significant change in the low affinity-receptors (Kd2 = 1.22 +/- 0.23; n2 = 1820 +/- 421). Treatment of these platelets with insulin, which has been demonstrated to restore both of the high- and low-affinity prostaglandin receptor numbers to within normal ranges in coronary artery disease, increased high-affinity receptor numbers and restored the prostacyclin effect on thrombin generation. These results demonstrate that the loss of the inhibitory effect of prostacyclin on the stimulation of thrombin generation was due to the loss of platelet high-affinity prostanoid receptors, which may contribute to atherogenesis in individuals with chronic SCI.     

Guanine nucleotide modulation of high affinity gonadotropin-releasing hormone receptors in rat mammary tumors.

We previously suggested that gonadotropin-releasing-hormone (GnRH) analogues activate the phosphoinositide pathway in rat mammary tumor membranes. In the present study we analyzed the binding of GnRH analogues to these membranes and assessed its modulation by guanine nucleotides. [125I]Buserelin (a GnRH superagonist) binding is specific because it is displaced only by GnRH analogues. Scatchard plot analysis reveals high affinity binding sites (Kd = 2.5 +/- 0.8 nM, Bmax = 250 +/- 120 fmol/mg membrane protein) and low affinity binding sites (Kd 1.1 +/- 0.3 microM, Bmax = 200 +/- 105 pmol/mg membrane protein). Guanine nucleotides increased the ED50 of [125I]buserelin displacement, and almost completely eliminated the high affinity binding. Similar results were obtained with [125I]D-Trp6-GnRH--another GnRH superagonist. The inhibition of buserelin binding by guanine nucleotides was specific for nucleotides that interact with G-binding proteins and was dose-dependent with a maximal effect at 10 microM GTP gamma S. Kinetic analysis of buserelin binding revealed that the dissociation rate increased at least 4-fold in the presence of 10 microM GTP gamma S. These results support the hypothesis that GnRH analogues interact directly with mammary tumors and activate a G-protein-dependent transducing mechanism.     

Beta-adrenergic receptors in catfish liver membranes: characterization and coupling to adenylate cyclase.

beta-Adrenergic binding sites in catfish liver membranes have been characterized by centrifugal assay, using a beta-adrenergic receptor antagonist, (-)-[3H]dihydroalprenolol ([3H]DHA). Binding of the radioligand was saturable and reversible. At 22 degrees equilibrium conditions were established in 15 min and the half-time for dissociation of bound [3H]DHA was approximately 4 min. Analysis of binding data was compatible with the existence of two classes of binding sites: a low-affinity site had a Kd of 62.3 nM and a Bmax of 452.0 fmol/mg protein, while the high-affinity site had a Kd of 2.04 nM and a Bmax of 46.7 fmol/mg protein. The dissociation constant of (-)-alprenolol for the beta-adrenergic receptors was about 2 nM as determined independently by direct kinetic studies and by inhibition of isoproterenol-stimulated adenylate cyclase activity. Phenylephrine was as potent as other catecholamines in inhibiting [3H]DHA binding, indicating that fish adrenoceptor subtyping is different from that of mammals.     

Vasopressin increases cytosolic free [Ca2+] in the neonatal rat cardiomyocyte. Evidence for V1 subtype receptors

Radioligand binding studies of the cardiac arginine vasopressin (AVP) receptor, together with studies on the AVP-evoked alterations in the [Ca2+]i levels, were undertaken using primary cultures of neonatal rat cardiomyocytes. Rapid, reversible, specific, high-affinity and low-capacity binding sites were detected for the agonist, [3H]AVP, and the V1 selective antagonist, d(CH2)5 Tyr (Me)-[3H]AVP (V1 antagonist), radioligands. The V2 selective antagonist radioligand, d(CH2)5 D-Ile des-Gly NH2-[3H]AVP, showed very little binding even at very high concentrations. [3H]AVP and [3H]V1 antagonist specific binding attained equilibrium in 10 minutes at 37 degrees C. The Kd and Bmax values (mean +/- SEM) were [3H]AVP: Kd 1.44 +/- 0.18 nM; Bmax 5,253 +/- 590 sites/cell; [3H]V1 antagonist: Kd 0.96 +/- 0.10 nM; Bmax 6,869 +/- 485 sites/cell. Ki values for a series of AVP-related peptide analogues and antagonists determined by competitive inhibition of [3H]AVP binding were consistent with the saturation data. The results suggest that these cells possess a homogeneous population of V1 subtype AVP receptors. AVP increased [Ca2+]i in a concentration-dependent manner as judged by fura-2 fluorescence. This was completely attenuated by inclusion of the V1 antagonist. The maximal increase in [Ca2+]i evoked by AVP from a resting level of 60 +/- 5 nM was less (250 +/- 35 nM) in comparison to the maximal response evoked by angiotensin II (2,337 +/- 640 nM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Acinar cholecystokinin (CCK) receptors in the exocrine pancreas of the rat: effect of adrenalectomy

The binding of [125I]BH-CCK-8 to membranes of acinar cells from rats at 6 and 21 days after adrenalectomy was studied. The optimum conditions of time and temperature were previously established as being 120 min and 30 degrees C. Under these conditions, the membranes of the adrenalectomized animals of both groups (6 and 21 days) bound more radioligand than those from control rats. However, a qualitative study of the binding showed that the affinities of binding were much lower; in particular, the high affinity receptors had a Kd of 0.94 +/- 0.33 nM in the controls and this was 14.9 +/- 1.29 nM in the 6-day adrenalectomized animals, although the maximum binding capacity did not vary significantly. However, in the case of the low affinity receptors, there was a gradual increase in the maximum binding capacity as the time after adrenalectomy progressed: 717 +/- 121, 1,987 +/- 183, and 10,175 +/- 862 fmol/mg for the control, 6-day, and 21-day adrenalectomized rats, respectively. In the latter situation, the high affinity receptors completely disappeared. These results, which coincide with a marked deficit in protein secretion already described in adrenalectomized rats, can be accounted for in terms of the possible negative cooperativity exerted by the low affinity receptors on the high affinity hormone-receptor complex, the protein secretion of the acinar cells normally mediated by the high affinity receptors becoming paralyzed.     

Identification of alpha 1-adrenergic receptors on sarcolemma from normal subjects and patients with idiopathic dilated cardiomyopathy: characteristics and linkage to GTP-binding protein

Discontinuous density sucrose gradient centrifugation was used to isolate membrane vesicles from the left ventricle of three normal subjects (one prospective organ donor and two traffic victims whose hearts were obtained 1 hour after death) and nine patients undergoing cardiac transplantation as a consequence of idiopathic dilated cardiomyopathy. Sarcolemma-enriched subcellular fractions, detected in the interface between 8.55% and 25% sucrose, were identified by the increased activity of Na+,K+-ATPase and by enrichment in beta-adrenergic receptor density. The density of beta-adrenergic receptors was lower in vesicles from diseased hearts (610 +/- 71 fmol/mg protein) than in vesicles from normal hearts (1,410 +/- 226 fmol/mg protein; p less than 0.01). alpha 1-Adrenergic receptors were identified in these membrane vesicles by [3H]prazosin binding. Specific binding of [3H]prazosin was about 50% of the total binding at 1 nM, and alpha 1-adrenergic binding sites were saturable at approximately 3 nM. Scatchard analysis revealed 58 +/- 5 fmol/mg protein (KD = 0.90 +/- 0.08 nM) in pathological hearts and 30 +/- 5 fmol/mg protein (KD = 0.90 +/- 0.03 nM) in normal hearts (p less than 0.01). The displacement curve of (-)-norepinephrine in membrane vesicles from normal hearts delineated one subpopulation of alpha 1-adrenergic receptors; the addition of 0.1 mM GTP did not cause right shift. In membrane vesicles from diseased heart, the displacement curve of (-)-norepinephrine disclosed two subpopulations of alpha 1-adrenergic receptors. A right shift that occurred after addition of GTP showed that in this case alpha 1-adrenergic receptors were functionally coupled with GTP-binding protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and characterization of a homogeneous population of beta 2-adrenergic receptors on human alveolar macrophages

Human alveolar macrophages obtained by bronchoalveolar lavage were studied with the high specific activity beta-adrenergic ligand [125I]pindolol and found to possess a moderate density of beta-adrenergic receptors. Using macrophage membranes, the receptor density (Bmax) was 42 +/- 9 fmol/mg protein with an apparent equilibrium dissociation constant (Kd) of 44 +/- 9 pM (mean +/- SEM). With intact macrophages, the Bmax = 5,643 +/- 942 sites/cell with Kd = 29 +/- 9 pM. Competition binding studies with subtype-specific antagonists revealed an exclusive population of beta 2-adrenergic receptors. Incubation of intact macrophages with the beta-adrenergic agonist isoproterenol caused a 6-fold increase in intracellular cyclic AMP (cAMP). Prostaglandin E1 and forskolin, which activate adenylate cyclase via different mechanisms, afforded typical marked increases in macrophage cAMP. Saturation binding, competition binding, and cAMP accumulation studies may all be performed from a single sample of about 2 x 10(7) cells, which can be obtained by bronchoalveolar lavage. This should facilitate studies of in vivo regulation of human alveolar macrophage beta-adrenergic receptors with regard to immune function and mediator release, and as a possible reflection of lung parenchymal receptors.     

Characterization of melatonin receptors in the rat area postrema: modulation of affinity with cations and guanine nucleotides

We have localized and characterized the binding of the melatonin agonist, 2-[125I]iodomelatonin, in the rat area postrema (AP), by using quantitative autoradiography in vitro. At equilibrium conditions, Scatchard analysis revealed saturable high-affinity binding to a single class of sites (Kd 45.9 +/- (SE) 6.0 pM and Bmax 30.8 +/- 4.6 fmol/mg protein, n = 4 experiments with a total of 18 rats). Melatonin and 6-hydroxymelatonin were potent displacers of 2-[125I]iodomelatonin binding in the AP (IC50 20 and 500 pM, respectively) while N-acetylserotonin exhibited only a modest potency (IC50 25 nM). Micromolar concentrations of guanine nucleotides dose-dependently and specifically inhibited agonist binding at 22 degrees C. Saturation studies revealed that this was due to a decrease in binding affinity. Divalent cations (4 mM CaCl2 or 2 mM MgCl2) had no detectable effect on the affinity of the binding site, whereas physiological concentrations of NaCl significantly decreased the binding affinity. These results demonstrate specific high-affinity binding sites for 2-[125I]iodomelatonin in the rat AP and suggest coupling of these putative receptors to guanosine nucleotide binding regulatory protein(s).     

Identification of two calcium channel receptor sites for [3H]nitrendipine in mammalian cardiac and smooth muscle membrane.

Various Ca-channel blockers differ in cardiovascular action despite common effects at the Ca channel. Many investigators have reported only a single high-affinity receptor for binding of [3H]nitrendipine, a dihydropyridine Ca-channel blocker. Its equilibrium dissociation constant (Kd) does not match the concentration of nitrendipine needed for a physiological effect on the mammalian cardiac Ca channel. The purpose of these studies was to clarify the existing discrepancy between pharmacological properties of nitrendipine receptors and the physiological effects of the dihydropyridine blockers. Of particular importance in this regard was to provide a pharmacological correlate for electrophysiological studies demonstrating multiple voltage-dependent conformational states of the Ca channel, which show differing affinities for the dihydropyridine Ca-channel blockers. By use of an improved ligand binding assay, our studies demonstrate both "high-affinity" and "low-affinity" [3H]nitrendipine receptors with Kd values corresponding well with observed physiologically effective nitrendipine concentrations. We detected two distinct populations of nitrendipine receptors in rat heart and bovine aortic membrane. A high-affinity Kd value of 0.2-0.3 nM was found, which seems to correspond to the physiologically functional state of the Ca channel in smooth muscle, since the Kd value is similar to the concentration at which nitrendipine inhibits contraction. However, in contrast to numerous other studies, we observed that the predominant component of [3H]nitrendipine binding (95-99%) had a low-affinity Kd value (235 nM). This putative low-affinity [3H]nitrendipine receptor may correspond to the physiologically functional state of the Ca channel in cardiac muscle.     

Prostaglandin E receptors in cardiac sarcolemma. Identification and coupling to adenylate cyclase

Purified cardiac sarcolemmal membrane vesicles were used to determine if specific prostaglandin (PG) receptors are present on the myocyte. Two binding sites for PGE2 were identified in isolated bovine sarcolemmal membranes: a high-affinity site with a dissociation constant (Kd) of 0.32 nM and a maximum binding (Bmax) of 376 fmol/mg of protein and a lower-affinity site with a Kd of 3.41 nM and a Bmax of 2,112 fmol/mg of protein. In competition experiments, unlabeled PGE1 displaced [3H]PGE2 from its membrane receptor at concentrations similar to those of unlabeled PGE2. Both PGF2 alpha and PGD2 displaced [3H]PGE2 from the membrane, but only at high concentrations (greater than 10(-6) M and greater than 10(-5)M, respectively). Digestion of sarcolemmal membrane with trypsin resulted in a threefold decrease in specific [3H]PGE2 binding. Phosphorylation of the membrane with protein kinase A also decreased specific [3H]PGE2 binding. At concentrations of PGE2 that occupy the high-affinity site, sarcolemmal adenylate cyclase activity was inhibited in the presence of 5'-guanylylimidodiphosphate [Gpp(NH)p]. We conclude that the isolated cardiac sarcolemmal membrane contains a high-affinity binding site for PGE2 that is functionally coupled to adenylate cyclase. The binding site is stereospecific and probably recognizes the 9-keto,11-hydroxyl portion of the ring structure of these prostaglandins.     

Age-related changes in glucocorticoid and androgen receptors of cultured human pubic skin fibroblasts

In order to evaluate age-related changes in glucocorticoid receptor and androgen receptor of cultured human pubic skin fibroblasts in young and aged men, we determined both [3H]dexamethasone binding and [3H]methyltrienolone (R1881, potent androgenic steroid) binding, using dispersed whole cell assay. Scatchard analyses of specific [3H]dexamethasone binding to the fibroblasts of young and aged men showed a single class of high-affinity binding sites with a mean (+/- SD) binding site concentration (Bmax) of 12.69 +/- 2.36 X 10(4) and 12.87 +/- 12.21 X 10(4) sites/cell, respectively, and mean (+/- SD) dissociation constant (Kd) of 5.60 +/- 0.41 and 7.36 +/- 2.17 nM, respectively. Scatchard analyses of specific [3H]R1881 binding to the same cultured skin fibroblasts of young and aged men showed a single class of high-affinity binding sites with a mean Bmax of 5.77 +/- 1.02 X 10(3) and 2.82 +/- 0.97 X 10(3) sites/cell, respectively, and a mean Kd of 0.56 +/- 0.23 and 0.50 +/- 0.28 nM, respectively. These findings indicate that there were no significant age-related changes in binding site and affinity of glucocorticoid receptor in cultured human pubic skin fibroblasts, whereas binding sites of androgen receptor significantly decreased in those of aged men as compared to young men, without significant change in affinity.     

Oxytocin and vasopressin: distinct receptors in myometrium

The binding characteristics of [3H]oxytocin [( 3H]OT) and [3H]lysine vasopressin [( 3H]LVP) to nonpregnant human myometrium were investigated. Binding of both radioligands was saturable, time dependent, and reversible. Whereas [3H]OT was found to bind to a single class of sites with high affinity [Kd, 1.5 +/- 0.4 (+/- SEM) nM] and low capacity [maximum binding (Bmax), 34 +/- 6 fmol/mg protein], [3H]LVP bound to two classes of sites, one with high affinity (Kd, 2.2 +/- 0.1 nM) and low capacity (Bmax, 198 +/- 7 fmol/mg protein) and another with low affinity (Kd, 655 +/- 209 nM) and high capacity (Bmax, 5794 +/- 1616 fmol/mg protein). The binding of the labeled peptides also displayed a marked difference in sensitivity to Mg2+ and guanine nucleotides. These differences in binding characteristics as well as the differences in potency of analogs in competing for [3H]OT and [3H]LVP binding indicate the presence of distinct receptors for OT and vasopressin in human myometrium. Pharmacological characterization of the high affinity binding sites for [3H]LVP indicated that these are of the V1 subtype. Although, as suggested by others, vasopressin and OT can bind to the same sites, the presence of distinct receptors for both peptides provides an explanation for the previously reported difference in myometrial responsiveness to OT and vasopressin.     

Corticoid receptors in rat brain: evidence for an aldosterone receptor.

The existence of high and low affinity mineralocorticoid-binding macromolecules (receptors) has been demonstrated in vitro in cytosols derived from the adrenalectomized rat brain by the specific binding of [3H]aldosterone (3H-A). The high-affinity aldosterone sites can be distinguished from those sites which have a higher affinity for either [3H]dexamethasone (3H-DM) or [3H]corticosterone (3H-B) on the basis of selectivity for spirolactone SC-9420 or non-radioactive A, DM, and B. The binding of 3H-A to the receptors was maximal after 2 hours of incubation of 0-4C. No significant binding of 3H-A to the receptors could be demonstrated when incubations of the radioactive ligand were performed at either 20 or 37 C, indicating that the receptor is heat-liabile. Scatchard analysis of the 3H-A binding data over a 200-fold concentration range of 3H-A indicated that there are two binding sites for aldosterone, a high affinity component (a1) with a Kd approximately equal to 1.5 X 10(-9)M and a low-affinity component (a2) with a Kd approximately equal to 6.3 X 10(-8)M. A similar study using 3H-DM as the radioactive ligand demonstrated only one site for the 3H-DM binding with a Kd = 6.2 X 10(-9)M. The presence of specific aldosterone receptors in the brain with high affinity, limited capacity, and selectivity for aldosterone suggests a possible extra-renal mechanism of action of the hormone in or mediated through the CNS.     

Influence of steroid hormone treatments or pregnancy on [3H]prazosin and [3H]rauwolscine binding to myometrial alpha-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)