Characterization of Binding Sites for the Angiotensin II Antagonist 125I-[Sarc1,Ile8]-Angiotensin II on Murine Neuroblastoma N1E-115 Cells

The murine neuroblastoma N1E-115 cell line contains binding sites for the angiotensin II (Ang II) receptor antagonist 125I-[Sarc1,Ile8]-Ang II (125I-SARILE). Binding of 125I-SARILE to N1E-115 membranes was rapid, reversible, and specific for Ang II-related peptides. The rank order potency of 125I-SARILE binding was the following: [Sarc1]-Ang II = [Sarc1,Ile8]-Ang II greater than Ang II greater than Ang III = [Sarc1,Thr8]-Ang II much greater than Ang I. Scatchard analysis of membranes prepared from confluent monolayers revealed a homogenous population of high affinity (KD = 383 +/- 60 pM) binding sites with a Bmax of 25.4 +/- 1.6 fmol/mg of protein. Moreover, the density, but not the affinity, of the binding sites increased as the cells progressed from logarithmic to stationary growth in culture. Finally, agonist, but not antagonist, binding to N1E-115 cells was regulated by guanine nucleotides. Collectively, these results suggest that the murine neuroblastoma N1E-115 cell line may provide a useful model in which to investigate the signal transduction mechanisms utilized by neuronal Ang II receptors.     

Angiotensin-(1-7) binds at the type 1 angiotensin II receptors in rat renal cortex.

Significant angiotensin (Ang) (1-7) production occurs in kidney and effects on renal function have been observed. The present study was undertaken to investigate binding characteristics of the heptapeptide to Ang II receptors present in rat renal cortex. [125I]-Ang II binding to rat glomeruli membranes was analyzed in the presence of increasing concentrations of Ang II, Ang-(1-7), DUP 753 and PD 123319. Linearity of the Scatchard plot of the [125I]-Ang II specific binding to rat glomeruli membranes indicated a single population of receptors, with a Kd value of 0.7 +/- 0.1 nM and a Bmax of 198 +/- 0.04 fmol/mg protein. DUP 753, an specific AT1 receptor antagonist, totally displaced the specific binding of [125I]-radiolabelled hormone with a Ki of 15.8 +/- 0.9 nM, while no changes were observed in the presence of the selective AT2 receptor antagonist, PD 123319. The specific [125I]-Ang II binding to rat glomerular membranes was displaced by Ang-(1-7) with high affinity (Ki = 8.0 +/- 3.2 nM). We conclude that radioligand binding assays in the presence of selective Ang II antagonists DUP 753 and PD 123319 suggest the unique presence of AT1, receptors in rat glomeruli and a possible role in the control of the biological renal effects of Ang-(1-7).     

Solubilization and Partial Characterization of Angiotensin II Receptors from Rat Brain

Rat brain angiotensin II (Ang II) receptors were solubilized with a yield of 30-40% using the synthetic detergent 3[(3-cholamidopropyl)dimethylammonio)]-1-propanesulfonate. Kinetic analysis employing the high-affinity antagonist 125I-Sar1,Ile8-Ang II indicated that the solubilized receptors exhibited the same properties as receptors present within intact brain membranes. Furthermore, there was a positive correlation (r = 0.99) between the respective pIC50 values of a series of agonist and antagonists competing for 125I-Sar1,Ile8-Ang II labeled binding sites in either solubilized or intact membranes. Moreover, covalent labeling of 125I-Ang II to solubilized receptors with the homo-bifunctional cross-linker disuccinimidyl suberate, followed by gel filtration, revealed one major and one minor binding peak with apparent molecular weights of 64,000 and 115,000, respectively. Two binding proteins of comparable molecular weights (i.e., 112,000 and 60,000) were also identified by covalent cross-linking of 125I-Ang II to solubilized brain membranes followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. In contrast, only the smaller molecular mass binding protein was observed when solubilized membranes were labeled with the antagonist 125I-Sar1,Ile8-Ang II prior to gel filtration, and chromatofocusing of antagonist labeled sites revealed only one peak with an isoelectric point of 6.2. The successful solubilization of these binding sites should facilitate continued investigation of Ang II receptors in the brain.     

Endogenous and Expressed Angiotensin II Receptors on Xenopus Oocytes

Intact Xenopus oocytes contain a homogeneous population of binding sites for the angiotensin II (Ang II) receptor antagonist 125I-[Sarc1,Ile8]-Ang II (125I-SARILE). Binding of 125I-SARILE to intact oocytes was saturable and of high affinity with an apparent KD of 0.7 nM and maximal density of 0.12 fmol/oocyte. Binding of 125I-SARILE to oocytes also was specific for Ang II-related peptides with a rank order potency of: [Sarc1]-Ang II greater than Ang II greater than Ang III much greater than Ang I. However, these endogenous binding sites were present only in follicle-enclosed oocytes and within the follicular layer itself. On the other hand, injection of poly(A)+ RNA isolated from murine N1E-115 neuroblastoma cells into oocytes resulted in the appearance of 125I-SARILE binding sites even in defolliculated oocytes. These expressed receptors exhibited pharmacological properties similar to those endogenously present in the follicular layer, although their levels were much less. Collectively, these results suggest that endogenous Ang II receptors are present on Xenopus oocyte follicle cells, whereas Ang II receptors expressed from exogenous N1E-115 RNA are found on the oocytes themselves. In addition, the high density of Ang II receptors on the follicle cells emphasizes the necessity for care in using Xenopus oocytes for the expression of receptors encoded by exogenous RNAs.     

Sarcosine1,glycine8 angiotensin II is an AT1 angiotensin II receptor subtype selective antagonist

Studies predating the discovery of the two major subtypes of angiotensin II (Ang II) receptors, AT1 and AT2, revealed anomalous characteristics of sarcosine1,glycine8 Ang II (Sar1,Gly8 Ang II). It competed poorly for 125I-Ang II binding in bovine brain but potently antagonized dipsogenic responses to intracerebroventricularly administered Ang II. Subsequent recognition that bovine brain contains AT(2) receptors, while dipsogenic responses to Ang II are mediated by AT1 receptors, suggests that Sar1,Gly(8) Ang II is AT1 selective. Sar1,Gly8 Ang II competed for 125I-sarcosine1,isoleucine8 Ang II binding to AT1 receptors in pituitary, liver and adrenal (the latter with the AT2 selective antagonist PD 123,319) with Ki's of 0.66, 1.40 and 1.36 nM, respectively. In contrast, the Ki of Sar1,Gly8 Ang II for AT2 receptors in rat adrenal (with the selective AT1 antagonist losartan) was 52 nM. 125I-Sar1,Gly8 Ang II (0.5-3 nM) bound to AT1 receptors in pituitary, liver, heart, adrenal, and hypothalamic membranes with high affinity (Kd=0.43, 1.6, 2.3, 0.96 and 1.8 nM, respectively), but showed no saturable binding to the adrenal AT2 receptor. 125I-Sar1,Gly8 Ang II selectively labeled AT1 receptors in sections of adrenal using receptor autoradiography. Thus, binding studies reveal Sar1,Gly8 Ang II to be the first angiotensin peptide analog to show AT1 receptor selectivity. 125I-Sar1,Gly8 Ang II offers a new means to selectively radiolabel AT1 receptors and may help to characterize ligand docking sites and agonist switches for AT1 versus AT2 receptors.     

Receptors for Arg8-vasopressin, angiotensin II, and atrial natriuretic peptide in the mesenteric vasculature of pregnant rats.

Blood pressure and sensitivity of blood vessels to vasoconstrictors are decreased in term-pregnant rats (20-21 days). To determine if changes in receptors for vasoactive peptides could account for these observations, receptor kinetics were measured for Arg8-vasopressin (AVP), angiotensin II (Ang II), and atrial natriuretic peptide (ANP) in the mesenteric vascular bed of the rat throughout pregnancy. Receptors for AVP were statistically similar in the five groups of animals (nonpregnant; pregnant 9, 15, and 21 days; and postpartum). The dissociation constant (KD) for [3H]AVP varied from 0.41 to 0.52 nmol/L (NS), while receptor density (Bmax) varied from 310 +/- 110 to 455 +/- 135 fmol/mg protein for six experimental measurements. Similar observations were made for Ang II receptors where KD of 125I-labelled Sar1, Ile8-Ang II was between 0.60 and 0.97 nmol/L and Bmax between 215 +/- 30 and 250 +/- 40 fmol/mg protein in the different groups. 125I-labelled ANP (101-126) receptors were markedly modified in terms of number of sites. Bmax was significantly increased during pregnancy (9 days, 429 +/- 86; 15 days, 541 +/- 54; 20 days, 438 +/- 72) and decreased in the postpartum period (133 +/- 21) by comparison with the nonpregnant group (245 +/- 35 fmol/mg protein), while KD was similar in the different experimental groups (57 to 82 pmol/L). Despite these increases in receptor density, the vasorelaxant effects of ANP was only increased at 9 days of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of 125I-Angiotensin III and 125I-Angiotensin II Binding to Rat Brain Membranes

The binding of 125I-angiotensin III (125I-ANG III) to rat brain membranes was examined and compared with that of 125I-angiotensin II (125I-ANG II). Degradation of each ligand, as monitored by HPLC, was effectively inhibited using fragments of ANG III and ANG II known to have little affinity for angiotensin binding sites. Three classes of 125I-ANG III-binding sites were observed based on affinity (KD = 0.13, 1.83, and 10.16 nM) and capacity (Bmax = 1.30, 18.41, and 67.2 fmol/mg protein, respectively). Two classes of 125I-ANG II-binding sites of high affinity (KD = 0.11 and 1.76 nM) and low capacity (Bmax = 1.03 and 18.86 fmol/mg protein, respectively) were also identified. Cross-displacement studies confirmed that the two highest-affinity 125I-ANG III-binding sites and the 125I-ANG II-binding sites were the same. On the other hand, the binding of 125I-ANG III to the low-affinity 125I-ANG III-binding site could not be inhibited with ANG II. These data imply that previously measured differences in the biological potency of cerebroventricularly applied ANG III and ANG II probably do not result from differential binding of these peptides to central angiotensin receptors.     

Renal nuclear angiotensin II receptors in normal and hypertensive rats

Accumulation of Angiotensin II (Ang II) in the kidneys of hypertensive rats infused chronically with Ang II occurs by AT1 receptor mediated internalization of Ang II, which may interact with intracellular targets, including nuclear binding sites. The aims of this study were to determine if kidney cell nuclei have specific Ang II binding sites and if chronic infusion of Ang II (70 ng/min; n=9) influences the nuclear Ang II binding capacity. Kidneys were harvested from control and Ang II infused rats and the renal cortexes were homogenized to obtain crude membrane preparations and nuclear fractions. Ang II binding sites were measured with a single point assay by incubating each fraction with 10 nM 125I-Sar-Ile-Ang II in the absence (total binding sites) or presence of either 2.5 M Sar-Leu-Ang II or 25 microM losartan to detect specific AT or AT1 binding sites. Both fractions exhibited specific Ang II binding sites that were displaced by both saralasin and losartan. In control rats, crude membrane preparations had 792 +/- 218 and the nuclear fraction had 543 +/- 222 fmol/mg protein AT1 receptors. AT1 receptor levels in membrane (885 +/- 170 fmol/mg protein) and nuclear fractions (610 +/- 198 fmol/mg protein) were not significantly different in Ang II infused rats. These data support the presence of nuclear Ang II receptors predominantly of the AT1 subtype in renal cells. Chronic Ang II infusion did not alter overall Ang II receptor densities.     

Pharmacological characterization of angiotensin II binding sites in the canine pancreas.

High affinity 125I-angiotensin II (Ang II) binding sites were characterized in the canine pancreas. Total binding increased with protein concentration and equilibrium was reached within 60-90 min at 22 degrees C. Specific binding was saturable and averaged 70% of total. Scatchard analysis of binding yielded a KD of 0.48 +/- 0.18 nM with a Bmax of 32.8 +/- 6.5 fmol/mg protein (mean +/- SEM, n = 6). The addition of the reducing agent dithiothreitol increased specific binding two-fold. The rank order of displacement of 125I-Ang II binding by native angiotensin peptides was Ang II greater than or equal to Ang III greater than AngI greater than Ang(1-7) much greater than Ang(1-6). The use of the specific Ang II antagonists CGP 42112A, PD 123177, and DuP 753 revealed that the pancreas expresses two receptor subtypes. The majority of Ang II binding sites in the pancreas could be classified as type 2 (AT2), although type 1 (AT1) sites were also detected. In vitro autoradiography revealed binding sites localized over islet cells, acinar and duct cells, as well as the pancreatic vasculature. In addition, the autoradiographic studies confirmed the predominance of the AT2 receptor subtype throughout the pancreas.     

Down-regulation of mt1 melatonin receptors in rat ovary following estrogen exposure.

In this study, we have demonstrated that 2-[125I]-iodomelatonin binds specifically to rat ovarian granulosa cell (GC) membranes with high affinity (KD=83 pM; Bmax=3.28 fmol/mg protein). Using immunoblot analysis and an anti-mt1 melatonin receptor antibody, we have also detected mt1 melatonin receptors in rat ovary. Because melatonin has been reported to alter the steroidogenic responses of ovarian tissues to gonadotropins, a physiological role for intra-ovarian melatonin may exist. Thus, in order to investigate a possible intra-ovarian role for melatonin, we have used both an in vivo and in vitro model of follicular development. Treatment of immature (day 21) female rats with estradiol (E; 0.2 mg/d x 3 d; subcutaneous) was used to induce follicular growth. Membranes from both untreated (U) and E-treated animals' ovaries contained high-affinity 2-[125I]-iodomelatonin (I-MEL) binding sites (Kd=83 and 23 pM, respectively). Estradiol treatment in vivo caused a significant decrease (P<0.05) in binding of 2-[125I]-iodomelatonin to ovarian membranes with untreated animals' ovaries having a Bmax=3.28 fmol/mg protein vs. estradiol-treated animals' ovaries having a Bmax=0.92 fmol/mg protein. In addition, following Estradiol treatment, mt1 melatonin receptors in rat ovary were down-regulated (approximately 95%) using immunoblot analysis. Granulosa cells isolated from E-treated rats were further matured in vitro with testosterone (T) and the pituitary gonadotropin follicle-stimulating hormone (FSH). Granulosa cells were cultured with either T (10 ng/ml) or FSH (5.71 ng ovine FSH-20/ml) alone, or both FSH and T for 48 h. There was no statistically significant specific binding of 2-[125I]-iodomelatonin to GC membranes cultured with T or FSH alone. However, following a 48-h exposure to FSH and T in vitro specific 2-[125I]-iodomelatonin binding occurred with total 2-[125I]-iodomelatonin binding =3.15 [corrected] fmol/mg protein. Therefore, the existence of hormonally-regulated expression of high-affinity melatonin binding sites suggests that melatonin may have an important intra-ovarian physiological role.     

Effect of vasopressin administration and deficiency upon 3H-AVP binding sites in the CNS and periphery during development.

Arginine8-vasopressin (AVP, 40 micrograms/100 g b.wt., SC) was administered to male Long-Evans (LE) pups from day 1 to 7 of life and the pups were sacrificed on day 8 or 60. 3H-AVP binding was performed on membranes prepared from the liver, kidney, and septum. No significant changes were observed in the kidney or septum of animals 8 or 60 days old. However, the chronic AVP treatment did result in a significant increase in the density of 3H-AVP binding sites in the liver when compared to control day 8 pups (control 44 +/- 2 vs. AVP 56 +/- 3 fmol/mg protein), with no change in affinity. This effect was maintained into adulthood, as the day 60 AVP-treated LE rats also showed a significant increase in liver 3H-AVP binding sites compared to control (control 186 +/- 9 vs. AVP 239 +/- 14 fmol/mg protein), with no change in affinity. A comparison of 3H-AVP binding sites in 8-day-old LE, heterozygous Brattleboro (HET-BB), and homozygous Brattleboro rats (HOM-BB) was performed to assess the effect of complete (HOM-BB) and partial (HET-BB) VP deficiency on binding sites in the CNS and periphery. The liver again was the only tissue in which a change in 3H-AVP binding characteristics was noted. The HOM-BB rat (Bmax 144 +/- 6 fmol/mg protein) displayed a significant increase in AVP binding sites from the LE rat (Bmax 100 +/- 7 fmol/mg protein), while the 3H-AVP binding sites in the HET-BB rat liver (Bmax 69.8 +/- 9 fmol/mg protein) were significantly lower than LE rats. Thus hepatic AVP receptors appear most sensitive to the presence or absence of vasopressin during the early postnatal period.     

High affinity angiotensin II receptors in myocardial sarcolemmal membranes. Characterization of receptors and covalent linkage of 125I-angiotensin II to a membrane component of 116,000 daltons

High affinity receptors for angiotensin II have been identified on purified cardiac sarcolemmal membranes. Equilibrium binding studies were performed with 125I-labeled angiotensin II and purified sarcolemmal vesicles from calf ventricle. The curvilinear Scatchard plots were evaluated by nonlinear regression analysis using a two-site model which identified a high affinity site Kd1 = 1.08 +/- 0.3 nM and N1 = 52 +/- 10 fmol/mg of protein and a low affinity site Kd2 = 52 +/- 16 nM and N2 = 988 +/- 170 fmol/mg of protein. Monovalent and divalent cations inhibited the binding of 125I-angiotensin II by 50%. The affinity of angiotensin II analogs for the receptor was determined using competitive binding assays; sarcosine, leucine-angiotensin II (Sar,Leu-angiotensin II), Kd = 0.53 nM; angiotensin II, Kd = 2.5 nM; des-aspartic acid-angiotensin II, Kd = 4.81 nM; angiotensin I, Kd = 77.6 nM. There is a positive correlation between potency in inducing positive inotropic response in myocardial preparations reported by others and potency for the hormone receptor observed in the binding assays. Pseudo-Hill plots of the binding data showed that agonists display biphasic binding with Hill numbers around 0.65 while antagonists recognized a single class of high affinity receptors with Hill numbers close to unity. These data were confirmed using 125I-Sar,Leu-angiotensin II in equilibrium binding studies which showed that this antagonist bound to a single class of receptor sites; Kd = 0.42 +/- 0.04 nM and N = 1050 +/- 110 fmol/mg of protein. Competition-binding experiments with this 125I-peptide yielded monophasic curves with Hill numbers close to unity for both agonists and antagonists. Membrane-bound 125I-angiotensin II was covalently linked to its receptor by the use of bifunctional cross-linking reagents such as dithiobis(succinimidyl propionate) and bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone. Analysis of the membranes showed the labeling of a component with an apparent Mr = 116,000. The affinity labeled species showed characteristics expected of a functional component of the high affinity receptor. The affinity labeling of this membrane component was inhibited by nanomolar angiotensin II or Sar,Leu-angiotensin II. Together these data indicate that high affinity receptors exist for angiotensin II that most likely mediate the positive inotropic effects of this hormone on myocardial cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nodose ganglionectomy reduces angiotensin II receptor binding in the rat brainstem

Angiotensin II (Ang II) receptor binding sites in the dorsomedial medulla of intact and unilaterally nodose ganglionectomized rats were identified and characterized using 125I-sarcosine,isoleucine Ang II. This radioligand bound saturably and with high affinity to rat brain homogenates and to sections of rat brainstem. Specific (1 microM angiotensin II displaceable) binding of 125I-sarcosine,isoleucine Ang II was displaced by angiotensin analogues with a potency order similar to that described for angiotensin II receptors. Unilateral nodose ganglionectomy caused a reduction in Ang II receptor binding in the medial solitary tract nucleus, dorsal motor nucleus of the vagus, and area postrema ipsilateral to the lesioned ganglion. This observation suggests that Ang II receptors in the dorsomedial medulla may be located on axon terminals of vagal afferents and cell bodies of vagal efferents.     

Characterization of angiotensin II receptor subtypes in rat liver

Radioligand binding studies identified two classes of 125I-angiotensin II-binding sites in rat liver membranes. High affinity binding sites (Kd = 0.35 +/- 0.13 nM, N = 372 +/- 69 fmol/mg of protein) were inactivated by dithiothreitol (0.1-10 mM) without any apparent change in low affinity binding sites (Kd = 3.1 +/- 0.8 nM, N = 658 +/- 112 fmol/mg of protein). Dithiothreitol inactivation was readily reversible but could be made permanent by alkylation of membrane proteins with iodoacetamide. Angiotensin II stimulation of glycogen phosphorylase in isolated rat hepatocytes (maximal stimulation 780%, EC50 = 0.4 nM) was completely inhibited by 10 mM dithiothreitol, a concentration which also abolished high affinity site binding; phosphorylase stimulation by glucagon and norepinephrine under these conditions was unaltered. Angiotensin II inhibition of glucagon-stimulated adenylate cyclase activity in hepatocytes required higher angiotensin II concentrations (EC50 = 3 nM) than phosphorylase stimulation and was not affected by dithiothreitol. Fractional occupancy of high affinity binding sites by 125I-angiotensin II correlated closely with angiotensin II-mediated phosphorylase stimulation, whereas occupancy of low affinity sites paralleled inhibition of adenylate cyclase activity. These data indicate that the physiologic effects of angiotensin II in rat liver are mediated by two distinct receptors, apparently not interconvertible, and provide the first evidence for angiotensin II receptor subtypes with differing biochemical features and mechanisms of action.     

Rat ovarian angiotensin II receptors. Characterization and coupling to estrogen secretion

Angiotensin II receptor agonist (125I-angiotensin II) and antagonist (125I-[Sar1,Ile8]angiotensin II) bind in a specific and saturable manner to rat ovarian membranes. Agonist and antagonist binding affinity (KD approximately 0.5 nM) and the number of sites estimated (Bmax approximately 60 fmol/mg of protein) were similar. Dissociation of receptor-bound agonist was more rapid than the dissociation of receptor-bound antagonist, and agonist, but not antagonist, dissociation from the receptor was accelerated by GTP gamma S. A 0-150 mM increase in Na+ produced a 27% increase in the KD of agonist binding. Antagonist binding was not modified by Na+. These studies suggest that both agonist and antagonist identify putative angiotensin II receptors in the ovary but that the properties of agonist and antagonist binding are distinct. Angiotensin II antagonist binding sites are present on the granulosa cell layer of rat ovarian follicles (Speth, R. C., Bumpus, F. M., and Husain, A. (1986) Eur. J. Pharmacol. 130, 351-352). To determine the role of angiotensin II in ovarian function, we examined angiotensin II receptors and function during the onset of puberty. High affinity and low capacity angiotensin II receptors were present in ovaries from immature rats. After pregnant mare's serum gonadotropin induced ovulation in immature rats, antagonist binding to total ovarian membranes increased over 3-fold. In vitro incubation of peripubertal ovaries with 1 microM angiotensin II produced a stimulation of estrogen, but not progesterone, secretion. This steroidogenic effect of angiotensin II was most pronounced in the luteal phase of the estrus cycle. These studies point toward the involvement of angiotensin II in the regulation of ovarian function, possibly through modulation of follicular estrogen levels.     

Somatostatin receptors on rat pancreatic acinar cells. Pharmacological and structural characterization and demonstration of down-regulation in streptozotocin diabetes

The binding of somatostatin-14 (S-14) to rat pancreatic acinar cell membranes was characterized using [125I-Tyr11]S-14 as the radioligand. Maximum binding was observed at pH 7.4 and was Ca2+-dependent. Such Ca2+ dependence of S-14 receptor binding was not observed in other tissues. Scatchard analysis of the competitive inhibition by S-14 of [125I-Tyr11]S-14 binding revealed a single class of high affinity sites (Kd = 0.5 +/- 0.07 nM) with a binding capacity (Bmax) of 266 +/- 22 fmol/mg of protein. [D-Trp8]S-14 and structural analogs with halogenated Trp moiety exhibited 2-32-fold greater binding affinity than S-14, [D-F5-Trp8]S-14 being the most potent. [Tyr11]S-14 was equipotent with S-14. The affinity of somatostatin-28 for binding to these receptors was 50% of that of S-14. Cholecystokinin octapeptide (CCK-8) inhibited the binding of [125I-Tyr11]S-14, but its inhibition curve was not parallel to that of S-14. In the presence of 1 nM CCK-8, the Bmax of S-14 receptors was reduced to 150 +/- 17 fmol/mg of protein. Dibutyryl cyclic GMP, a CCK receptor antagonist, partially reversed the inhibitory action of CCK-8, suggesting that CCK receptors mediate the inhibition of S-14 receptor binding. GDP, GTP, and guanyl-5'-yl imidodiphosphate inhibit S-14 receptor binding in this tissue. The inhibition was shown to be due to decrease in binding capacity and not due to change in affinity. Specifically bound [125I-Tyr11]S-14 cross-linked to the S-14 receptors was found associated with three proteins of approximate Mr = 200,000, 80,000, and 70,000 which could be detected under both reducing and nonreducing conditions. Finally, pancreatic acinar cell S-14 receptors were shown to be down-regulated by persistent hypersomatostatinemia 1 week after streptozotocin-induced diabetes characterized by decreased Bmax (105 +/- 13 fmol/mg of protein) without any change in affinity. We conclude that pancreatic acinar cell membrane S-14 receptors require Ca2+ for maximal binding and thus differ from S-14 receptors in other tissues, S-14 receptors in this tissue also exhibit selective ligand specificities, these receptors are regulated by CCK-8 and guanine nucleotides, three receptor proteins of apparent Mr = 200,000, 80,000, and 70,000 specifically bind S-14, and (v) these receptors are regulated by S-14 in vivo as evidenced by decreased binding in streptozotocin diabetic rats characterized by hypersomatostatinemia.     

Characterization of central melatonin receptors using 125I-melatonin

The binding of 125I-melatonin, a potent analog of melatonin, to rat brain synaptosomal preparations was investigated. 125I-melatonin bound with high affinity (Kd = 38 nM) to a single class of sites (Bmax = 81 fmol/mg protein). Kinetic studies indicated that binding was time-dependent and reversible. Specific 125I-melatonin binding was inhibited by melatonin, and was unaffected by other structurally related compounds including serotonin. Binding of 125I-melatonin was greatly reduced if the synaptosomal preparations were pretreated by heat or trypsin but was unaffected by freeze-thawing. These results suggest that 125I-melatonin may serve as a valuable probe for studying melatonin receptors.     

Quantitative in vitro autoradiographic characterization of [125I]angiotensin III binding sites in rat adrenal gland

A single class of high-affinity binding sites for [125I]angiotensin III and [125I]angiotensin II were found in rat adrenal medulla and zona glomerulosa by quantitative autoradiography. In the medulla, Kd were 1.46 and 1.16 nM, and Bmax 1700 and 1700 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. In the zona glomerulosa, Kd were 0.86 and 0.90 nM, and Bmax 790 and 560 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. Unlabeled angiotensin III and angiotensin II displaced [125I]angiotensin III with similar potency in both adrenal zona glomerulosa and medulla. Our findings suggest that angiotensin III and angiotensin II might share the same binding sites in adrenal gland and support the hypothesis of a role for angiotensin III in the adrenal medulla and zona glomerulosa.     

Effects of Pro-Leu-Gly-NH2 and cyclo (Leu-Gly) on the binding of 3H-quinuclidinyl benzilate to striatal cholinergic muscarinic receptors

The effects of Pro-Leu-Gly-NH2 (melanotropin release inhibiting factor, MIF) and its analog, cyclo (Leu-Gly) on the mouse and rat striatal cholinergic muscarinic receptors labeled with 3H-quinuclidinyl benzilate (QNB) were investigated. 3H-QNB bound to the rat striatal muscarinic receptors at a single high affinity site with receptor density (Bmax value) of 1200 fmol per mg protein and an apparent dissociation constant (Kd value) of 53.5 pM. At 140 pM concentration of 3H-QNB, the specific binding to the receptors was 724 fmol per mg protein. MIF in a concentration range of 10(-9) to 10(-4) M did not alter the binding of 3H-QNB but at 10(-3) M decreased the binding by 25%. Cyclo (Leu-Gly), on the other hand, in the concentration range of 10(-9) to 10(-3) M had no effect on the binding of 3H-QNB. A single injection of MIF (3 or 10 mg/kg IP) to rats did not alter the Bmax or the Kd value of 3H-QNB to bind to the striatal membranes. 3H-QNB bound to the mouse striatal muscarinic receptors at a single high affinity site with a Bmax value of 991 fmol/per mg protein and a Kd value of 21 pM. Neither acute administration of MIF (3 or 10 mg/kg IP) nor chronic treatment of the peptide (2, 8 or 32 mg/kg IP, daily for 5 days) to mice could influence the binding of 3H-QNB to the striatal muscarinic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

A characterization of beta-adrenergic receptors on cellular and perigranular membranes of rat peritoneal mast cells

Beta-adrenergic receptors were characterized by measuring the specific binding of 3H-dihydroalprenolol (DHA) on intact isolated rat peritoneal mast cells (RPMC) and on perigranular membranes derived from purified RPMC granules. The specific binding of 3H-DHA reaches an equilibrium within 30 min at 5 degrees C and is linear with cell number. Scatchard analysis reveals two populations of binding sites on intact cells: with KD = 10.6 +/- 2.6 and 129 +/- 4.7 nM and Bmax of 186 +/- 38 and 1200 +/- 415 fmol/10(6) cells, respectively. Each cell contains 120 X 10(3) high-affinity binding sites and 720 X 10(3) low-affinity binding sites. There appears to be neither alpha-adrenergic nor muscarinic cholinergic receptors on the RPMC. Specific binding of 3H-DHA also occurred to isolated granules with perigranular membranes. The binding was saturable with a single population of binding sites with an affinity (KD) of 7.0 +/- 0.45 nM. Maximum binding (Bmax) was calculated at 56.6 +/- 1.9 fmol/10(9) granules. Subfractionation of granule components demonstrated that the specific binding sites appear to be localized exclusively on the perigranular membrane.