No abstract

Angiotensin-II (AII) receptors have been classified as AT1 and AT2 subtypes based on selective antagonists. AII binding sites in bovine ovary membranes were characterized using the radiolabeled AII antagonist, [125I]Sar1,Ile8-AII ([125I]SIA). The binding was specific and saturable with dissociation constant (Kd) and maximum binding (Bmax) of 0.18 +/- 0.08 nmol/l and 32.5 +/- 1.3 fmol/mg, respectively. Pretreatment of ovarian membranes with dithiothreitol (10 mumol/l) doubled the specific binding of [125I]SIA twofold to 63.5 +/- 2.8 fmol/mg. Guanine nucleotide had no significant effect on the affinity of agonist (AII) to compete for [125I]SIA binding. AII and a series of AII-related analogs were used in competition binding experiments, and the data were compared with those obtained with membranes prepared from bovine adrenal cortex and bovine cerebellum. The membranes from ovary and cerebellum showed similar binding characteristics, but they differed from those of adrenal cortex. CGP42112A and WL-19, AT2-subtype selective antagonists, inhibited [125I]SIA binding to ovarian membrane with IC50 values of 28 +/- 4 and 26.7 +/- 2.8 nmol/l, respectively. SK&F 108566 and DuP 753, AT1-subtype-selective antagonists, had very little effect on [125I]SIA binding to ovarian membranes. These data directly demonstrate that bovine ovary membranes have predominantly AT2-subtype AII receptors.

Membranes prepared from the medullary region of the porcine kidney displayed high affinity, high density (Kd, 0.12 nM; binding capacity, 127 fmol/mg protein) receptors for calcitonin gene-related peptide (CGRP). Human CGRP (hCGRP), rat CGRP (rCGRP), and the hCGRP analog [hCGRP-(8-37)] competed for the binding of [125I]hCGRP, whereas salmon calcitonin (sCT) and CGRP-(22-37) were very weak in displacing [125I]hCGRP binding. In accordance with these binding data, CGRP stimulated adenylate cyclase activity in these membrane preparations in a concentration-dependent manner, with an EC50 similar to that of the Kd for binding. In the same preparations, sCT was ineffective in stimulating adenylate cyclase activity, suggesting that porcine kidney medullary membranes possess receptors specific for CGRP. Further hCGRP-(8-37), a CGRP antagonist, inhibited CGRP-stimulated adenylate cyclase activity in a competitive manner. Covalent cross-linking of [125I]hCGRP to these membranes resulted in the specific labeling of one major band at approximately 30,000 mol wt and two minor bands at about 58,000 and 78,000 mol wt. The presence of CGRP receptors and their coupling to adenylate cyclase suggest a role for CGRP in kidney function, such as local regulation of the microcirculation, electrolyte transport, or water homeostasis in the porcine kidney.

Rat aortic smooth muscle cells were used as a model system to characterize the binding properties of [3H]SK&F 108566, an angiotensin type-1 (AT1) receptor antagonist. The binding was specific, saturable and reversible. The association and dissociation rates of [3H]SK&F 108566 binding to smooth muscle cells were monophasic and Scatchard analysis of equilibrium binding data yielded a linear plot indicating a homogenous population of binding sites. The maximum binding (Bmax) and apparent dissociation constant (Kd) were 22,000 +/- 6000 sites/cell and 0.83 +/- 0.08nM respectively. The pharmacological specificity of [3H]SK&F 108566 binding to smooth muscle cells is consistent with that observed for AT1 and confirms AT1 receptor specificity of this radioligand. High affinity binding was observed in membranes prepared from bovine adrenal cortex, rat liver and rat kidney glomeruli. COS cells transfected with cDNA encoding human AT1 angiotensin II receptors also displayed high affinity binding site for [3H]SK&F 108566. No specific binding could be detected on membranes prepared from bovine cerebellum, a tissue rich in the angiotensin type-2 (AT2) receptor. These observations indicate that [3H]SK&F 108566 binds to sites which have pharmacological characteristics of angiotensin II AT1 subtype receptors and can be used as a subtype-selective radioligand to characterize AII receptors in various systems.

Addition of calcitonin gene-related peptide (CGRP) to rat glomerular mesangial cells in culture resulted in an increase in cyclic adenosine monophosphate (cAMP) accumulation in a concentration-dependent fashion with an EC50 of approximately 3 nM. Inclusion of CGRP8-37, a CGRP1 selective antagonist shifted CGRP concentration-response curve to the right, suggesting the presence of CGRP1 subtype receptors in these cells. Pretreatment of these cells with CGRP followed by washing and rechallenge with CGRP or isoproterenol or forskolin resulted in selective attenuation of CGRP-mediated cAMP accumulation by 55-60% without affecting isoproterenol or forskolin-mediated accumulation, suggesting that CGRP pretreatment of mesangial cells induced homologous desensitization. CGRP-mediated desensitization of cAMP accumulation was found to be both concentration- and time-dependent. Desensitization did not affect the EC50 of CGRP for stimulation of cAMP accumulation, but resulted in a 55-60% reduction in maximal response. CGRP-mediated desensitization was fast, with half-maximal desensitization occurring as early as 5 min after pretreatment with CGRP. In addition, CGRP-mediated desensitization was blocked by the CGRP receptor antagonist, CGRP8-37, when included in the pretreatment protocol. These data suggest that rat mesangial cells display CGRP1 subtype receptors and that prolonged pretreatment of these cells with CGRP resulted in homologous desensitization.