Porcine brain natriuretic peptide, another modulator of bovine adrenocortical steroidogenesis

Porcine brain natriuretic peptide (pBNP) significantly inhibited aldosterone production stimulated by an angiotensin II analog and ACTH-stimulated cortisol secretion, together with simultaneously increasing the formation of cGMP in dispersed bovine adrenocortical cells. Receptors for pBNP were identified in bovine adrenal gland using an in vitro receptor autoradiographic technique and studies of 125I-pBNP binding. In vitro receptor autoradiography demonstrated specific binding sites for 125I-pBNP in bovine adrenal cortex. Complete displacement of 125I-pBNP by unlabeled pBNP or human atrial natriuretic peptide (hANP) can take place at these sites. Analysis of 125I-pBNP binding to bovine adrenocortical membrane fractions showed that the adrenal cortex had high-affinity, low-capacity pBNP-binding sites, with a dissociation constant (Kd) of 2.32 +/- 0.33 x 10(-10) M (mean +/- SE) and a maximal binding capacity (Bmax) of 36.7 +/- 1.6 fmol/mg protein. Moreover, the specific binding sites for 125I-pBNP were completely displaced not only by unlabeled pBNP but also by unlabeled hANP. The hANP dose required for 50% inhibition of specific 125I-pBNP binding was almost identical to that for pBNP (IC50 values for hANP and pBNP: 8.5 x 10(-10) and 6.5 x 10(-10) M, respectively). These results suggest that pBNP exerts a suppressive effect on bovine adrenocortical steroidogenesis via a receptor which may be shared with ANP.     

Alpha-human natriuretic polypeptide (alpha-hANP) specific binding sites in bovine adrenal gland

The effects of synthetic alpha-human atrial natriuretic polypeptide (alpha-hANP) on steroidogenesis in bovine adrenocortical cells in primary monolayer culture were investigated. alpha-hANP did not inhibit basal aldosterone secretion. alpha-hANP induced a significant dose-dependent inhibition of basal levels of cortisol and dehydroepiandrosterone (DHEA) secretion and also of ACTH (10(-8) M)-stimulated increases in aldosterone, cortisol and DHEA secretion. Visualization of [125I]alpha-hANP binding sites in bovine adrenal gland by an in vitro autoradiographic technique demonstrated that these sites were highly localized in the adrenal cortex, especially the zona glomerulosa. These results suggest that the adrenal cortex may be a target organ for direct receptor-mediated actions of alpha-hANP.     

Atrial and brain natriuretic peptide in adrenal steroidogenesis.

We elucidated the role of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in human and bovine adrenocortical steroidogenesis. The urinary volume, sodium excretion and cyclic GMP (cGMP) excretion and plasma cGMP were markedly increased by the synthetic alpha-human ANP (alpha-hANP) infusion in healthy volunteers. Plasma arginine vasopressin (AVP) and aldosterone levels were significantly suppressed. Both ANP and BNP inhibited aldosterone, 19-OH-androstenedione, cortisol and DHEA secretion dose-dependently and increased the accumulation of intracellular cGMP in cultured human and bovine adrenal cells. alpha-hANP significantly suppressed P450scc-mRNA in cultured bovine adrenal cells stimulated by ACTH. Autoradiography and affinity labeling of [125I]hANP, and Scatchard plot demonstrated a specific ANP receptor in bovine and human adrenal glands. Purified ANP receptor from bovine adrenal glands identified two distinct types of ANP receptors, one is biologically active, the other is silent. A specific BNP receptor was also identified on the human and bovine adrenocortical cell membranes. The binding sites were displaced by unlabelled ANP as well as BNP. BNP showed an effect possibly via a receptor which may be shared with ANP. The mean basal plasma alpha-hANP level was 25 +/- 5 pg/ml in young men. We confirmed the presence of ANP and BNP in bovine and porcine adrenal medulla. Plasma or medullary ANP or BNP may directly modulate the adrenocortical steroidogenesis. We demonstrated that the lack of inhibitory effect of alpha-hANP on cultured aldosterone-producing adenoma (APA) cells was due to the decrease of ANP-specific receptor, which caused the loss of suppression of aldosterone and an increase in intracellular cGMP.     

Binding sites of atrial natriuretic peptide in tree shrew adrenal gland

Adrenal gland binding sites for atrial natriuretic peptide-(99-126) (ANP) were quantitated in tree shrew (Tupaia belangeri) by incubation of adrenal sections with (3-[125I]-iodotyrosyl28) atrial natriuretic peptide-(99-126), followed by autoradiography with computerized microdensitometry. In the adrenal glands, there are three types of ANP binding sites. One is located in the zona glomerulosa (BMax 84 +/- 6 fmol/mg protein; Kd 122 +/- 9 pM); the second in the zona fasciculata and reticularis (BMax 29 +/- 2 fmol/mg protein; Kd 153 +/- 6 pM) and the third in the adrenal medulla (BMax 179 +/- 1 fmol/mg protein; Kd 70 +/- 2 pM). Besides the influence of ANP on the regulation of adrenocortical mineralcorticoid and glucocorticoid secretion our findings raise the possibility for a local site of action of atrial natriuretic peptide in the regulation of adrenomedullary catecholamines in the tree shrew, primates and man.     

Studies on lipoprotein and adrenal steroidogenesis: I. Roles of low density lipoprotein- and high density lipoprotein-cholesterol in steroid production in cultured human adrenocortical cells

The roles of human low density lipoprotein (LDL)- cholesterol and high density lipoprotein (HDL)- cholesterol on adrenal steroidogenesis were investigated using cultured human adult and fetal adrenocortical cells and the findings were then compared to those obtained with bovine adrenocortical cells. The secretion of cortisol in both human and bovine adrenocortical cells was dose-dependently increased by the administration of LDL- or HDL-cholesterol in the presence of adrenocorticotropin (ACTH). LDL-cholesterol was utilized to a greater extent than HDL-cholesterol in both human and bovine adrenal steroidogenesis in the presence of ACTH. Exogenous lipoprotein-derived cholesterol was less utilized in human adrenal steroidogenesis than in bovine adrenal steroidogenesis, compared to the endogenous cholesterol. An increase in the secretion of cortisol and dehydroepi androsterone sulfate (DHEA-S) continued for the 5-day culture period, in the presence of lipoprotein cholesterol and ACTH in both human adult and fetal adrenocortical cells. The secretion of aldosterone increased on the first day of the culture period, then gradually decreased for the 5-day culture period in human adult adrenocortical cells, but not in human fetal adrenocortical cells in the presence of lipoprotein cholesterol and ACTH. These findings demonstrate that exogenous cholesterol utilized in the biosynthesis of steroids is mainly from LDL-cholesterol in both human adult and fetal adrenals and bovine adrenal and the proportion of cholesterol synthesized de novo is significantly larger in the human adult adrenal than in the bovine adrenal.     

Ultracytochemical localization of particulate guanylate cyclase in rat adrenal gland exposed to stimulation by porcine brain natriuretic peptide

Summary We studied the cytochemical localization of particulate guanylate cyclase (GC) in rat adrenal gland after stimulation with porcine brain natriuretic peptide (pBNP) by electron microscopy. In the adrenal cortex, GC activity, as demonstrated by the presence of reaction product, was prevalently localized to the zona glomerulosa and zona fasciculata, while the zona reticularis showed little GC reaction product. In the adrenal medulla, GC reaction product was present only in adrenalin-containing cells. All GC positivity was associated with intracellular membranes. No GC reaction product was detected in specimens incubated in media devoid of pBNP. In parallel samples incubated in the presence of rat atrial natriuretic factor (rANF), the distribution of rANF-stimulated GC activity was similar to that of pBNP-stimulated GC activity.     

Development of p-benzoylbenzoylated [N,C,rANP(1-28)]pBNP32 (pBNP1) derivatives and affinity photolabeling of the bovine NPR-A receptor

Two Nalpha-benzophenone-substituted photoprobes, derived from the high affinity NPR-A chimeric agonist [N, C, rANP(1-28)]pBNP32 (pBNP1) were assembled by solid-phase peptide synthesis. [Nalpha-p-benzoylbenzoyl, Tyr2]pBNP1 (probe A), and [Nalpha-p-benzoylbenzoyl, Tyr18]pBNP1 (probe B) were synthesized and their affinity was tested on bovine zona glomerulosa membrane preparations. Both were found to exert ANP-type high affinities (Kd = 20 pM) with Kd of 10 pM and 30 pM for probe A, and probe B, respectively. Photolabeling of NPR-A with both analogs cross-linked specifically the 130 kDa monomeric NPR-A. The maximal irreversible ligand incorporations were estimated at 18% and 41% for probe A, and probe B, respectively. These results show that the N-terminus of the chimeric compound can be acylated with a large chemical function, such as the benzophenone moiety, without loosing its affinity for the NPR-A receptor. Furthermore, Leu2 or Leu18 can be substituted with tyrosine without disturbing the binding capacity of the ligand. Finally, it appears that the pBNP1 N-terminus is close to the receptor structure as irreversible incorporation is observed after photolabeling.     

Intracellular receptors for inositol 1,4,5-trisphosphate in angiotensin II target tissues

Many cells (including angiotensin II target cells) respond to external stimuli with accelerated hydrolysis of phosphatidylinositol 4,5-bisphosphate, generating 1,2-diacylglycerol and inositol 1,4,5-trisphosphate, a rapidly diffusible and potent Ca2+-mobilizing factor. Following its production at the plasma membrane level, inositol 1,4,5-trisphosphate is believed to interact with specific sites in the endoplasmic reticulum and triggers the release of stored Ca2+. Specific receptor sites for inositol 1,4,5-trisphosphate were recently identified in the bovine adrenal cortex (Baukal, A. J., Guillemette, G., Rubin, R., Sp?t, A., and Catt, K. J. (1985) Biochem. Biophys. Res. Commun. 133, 532-538) and have been further characterized in the adrenal cortex and other target tissues. The inositol 1,4,5-trisphosphate-binding sites are saturable and present in low concentration (104 +/- 48 fmol/mg protein) and exhibit high affinity for inositol 1,4,5-trisphosphate (Kd 1.7 +/- 0.6 nM). Their ligand specificity is illustrated by their low affinity for inositol 1,4-bisphosphate (Kd approximately 10(-7) M), inositol 1-phosphate and phytic acid (Kd approximately 10(-4) M), fructose 1,6-bisphosphate and 2,3-bisphosphoglycerate (Kd approximately 10(-3) M), with no detectable affinity for inositol 1-phosphate and myo-inositol. These binding sites are distinct from the degradative enzyme, inositol trisphosphate phosphatase, which has a much lower affinity for inositol trisphosphate (Km = 17 microM). Furthermore, submicromolar concentrations of inositol 1,4,5-trisphosphate evoked a rapid release of Ca2+ from nonmitochondrial ATP-dependent storage sites in the adrenal cortex. Specific and saturable binding sites for inositol 1,4,5-trisphosphate were also observed in the anterior pituitary (Kd = 0.87 +/- 0.31 nM, Bmax = 14.8 +/- 9.0 fmol/mg protein) and in the liver (Kd = 1.66 +/- 0.7 nM, Bmax = 147 +/- 24 fmol/mg protein). These data suggest that the binding sites described in this study are specific receptors through which inositol 1,4,5-trisphosphate mobilizes Ca2+ in target tissues for angiotensin II and other calcium-dependent hormones.     

Synthetic peptide KKRR corresponding to the human ACTH fragment 15-18 is an antagonist of the ACTH receptor

Tritium-labeled synthetic fragments of human adrenocorticotropic hormone (ACTH) [3H]ACTH (11-24) and [3H]ACTH (15-18) with a specific activity of 22 and 26 Ci/mmol, respectively, were obtained. It was found that [3H]ACTH (11-24) binds to membranes of the rat adrenal cortex with high affinity and high specificity (Kd 1.8 +/- 0.1 nM). Twenty nine fragments of ACTH (11-24) were synthesized, and their ability to inhibit the specific binding of [3H]ACTH (11-24) to adrenocortical membranes was investigated. The shortest active peptide was found to be an ACTH fragment (15-18) (KKRR) (Ki 2.3 +/- 0.2 nM), whose [3H] labeled derivative binds to rat adrenocortical membranes (Kd 2.1 +/- 0.1 nM) with a high affinity. The specific binding of [3H]ACTH-(15-18) was inhibited by 100% by unlabeled ACTH (11-24) (Ki 2.0 +/- 0.1 nM). ACTH (15-18) in the concentration range of 1-1000 nM did not affect the adenylate cyclase activity of adrenocortical membranes and, therefore, is an antagonist of the ACTH receptor.     

Elucidation and Characteristics of Non-opioid β-Endorphin Receptors in Rat Adrenal Cortex

beta-Endorphin-like decapeptide immunorphin (SLTCLVKGFY), a selective agonist of non-opioid beta-endorphin receptor, was labeled with tritium to specific activity of 24 Ci/mmol. It was used for the detection and characterization of non-opioid beta-endorphin receptors on rat adrenal cortex membranes (Kd1 = 39.6 +/- 2.0 nM, Bmax1 = 40.7 +/- 2.3 pmol/mg protein; Kd2 = 0.25 +/- 0.01 micro M, Bmax2 = 187.8 +/- 9.4 pmol/mg protein). beta-Endorphin was found to inhibit the [3H]immunorphin specific binding to membranes (Ki = 70.0 +/- 9.2 nM); naloxone, [Met5]enkephalin, and alpha- and gamma-endorphins tested in parallel were inactive. Immunorphin at concentrations of 10(-9)-10(-6) M was found to inhibit the adenylate cyclase activity in adrenocortical membranes, while intramuscular injection of immunorphin at doses of 10-100 micro g/kg was found to reduce the secretion of 11-oxycorticosteroids from the adrenals to the bloodstream.     

Binding of Brain and Atrial Natriuretic Peptides to Cultured Mouse Astrocytes and Effect on Cyclic GMP

125I-Porcine brain natriuretic peptide (125I-pBNP) bound to mouse astrocytes in primary culture in a time-dependent manner (t1/2 = 4.5 min), similar to 125I-human atrial natriuretic peptide (125I-hANP) (t1/2 = 5 min). Binding was saturable and reached equilibrium after 90 min at 22 degrees C for both radioligands. Scatchard analysis suggested a single class of binding sites for pBNP with a binding affinity and capacity (KD = 0.08 nM; Bmax = 78.3 fmol/mg of protein) similar to those of hANP1-28 (KD = 0.1 nM; Bmax = 90.3 fmol/mg of protein). In competition binding studies, pBNP or human/rat atrial natriuretic peptide (ANP) analogues [hANP1-28, rat ANP1-28 (rANP1-28), and rANP5-28] displaced 125I-hANP, 125I-pBNP, and 125I-rANP1-28 completely, all with IC50 values of less than nM (0.14-0.83 nM). All four peptides maximally stimulated cyclic GMP (cGMP) production by 10 min at 22 degrees C at concentrations of 1 microM with EC50 values ranging from 50 to 100 nM. However, maximal cGMP induction by brain natriuretic peptide (BNP) (25.9 +/- 2.1 pmol/mg of protein) was significantly greater than that by hANP1-28 (11.5 +/- 2.2 pmol/mg of protein), rANP1-28 (16.5 +/- 2.0 pmol/mg of protein), and rANP5-28 (15.8 +/- 2.2 pmol/mg of protein). These studies indicate that BNP and ANPs act on the same binding sites and with similar affinities in cultured mouse astrocytes. BNP, however, exerts a greater effect on cGMP production. The difference in both affinity and selectivity between binding and cGMP production may indicate the existence of receptor subtypes that respond differentially to natriuretic peptides despite similar binding characteristics.     

Characterisation of heterologous and homologous low-density lipoprotein binding to apolipoprotein B,E receptors on porcine adrenal cortex membranes: enhanced binding of trypsin-modified human low-density lipoprotein

The characteristics of the binding of homologous and heterologous (human) LDL to membrane preparations from porcine adrenal cortex have been determined. The membranes displayed a single class of high-affinity, saturable binding site for both 125I-labelled porcine and human LDL, which was dependent on divalent cations, in addition to a low-affinity, non-saturable component(s). Porcine LDL displaced both 125I-labelled porcine and 125I-labelled human LDLs from the high-affinity binding site more effectively than human LDL, reflecting the lower Kd, (13.2 micrograms/ml) for porcine than human (Kd 19.2 micrograms/ml) LDL. These values are comparable to those obtained for half-maximal binding of human and bovine LDLs in a bovine adrenocortical membrane system (Kovanen, P.T., Basu, S.K., Goldstein, J.L. and Brown, M.S. (1979) Endocrinology 104, 610-616). Tryptic modification of porcine LDL (T-LDL) diminished its ability to compete with 125I-labelled native LDL for the high-affinity binding site; in contrast, 125I-labelled porcine T-LDL showed an elevated receptor affinity (Kd 9.7 micrograms/ml) and was more efficiently displaced by its unlabelled counterpart than by native porcine LDL. Tryptic treatment of human LDL similarly increased its binding affinity (Kd 8.3 micrograms/ml), although in this case, the unlabelled T-LDL displaced not only 125I-labelled human T-LDL but also 125I-labelled human LDL from the high-affinity site more effectively than native LDL. We conclude that (i) porcine adrenocortical membranes possess binding sites specific for LDL and resembling the apolipoprotein B,E receptors already demonstrated in murine, bovine and human adrenal cortex; (ii) tryptic modification of porcine LDL may remove or destroy segments of apolipoprotein B100 which contribute to receptor recognition sites on the surface of the particle; (iii) trypsinised porcine LDL may interact with the membrane binding site by a mechanism differing from that by which native LDL binds, and (iv) trypsinisation of human LDL may cleave or remove species-specific segments of the B100 protein at or close to the receptor recognition site(s) on the particle, thus decreasing structural differences between porcine and human LDL, and thereby enhancing its binding affinity for the porcine receptor.     

Gastric inhibitory polypeptide stimulates glucocorticoid secretion in rats, acting through specific receptors coupled with the adenylate cyclase-dependent signaling pathway.

Gastric inhibitory polypeptide (GIP) is a 42-amino acid peptide, belonging to the VIP-secretin-glucagon superfamily, some members of this group are able to regulate adrenocortical function. GIP-receptor mRNA has been detected in the rat adrenal cortex, but investigations on the effect of GIP on steroid-hormone secretion in this species are lacking. Hence, we have investigated the distribution of GIP binding sites in the rat adrenal gland and the effect of their activation in vivo and in vitro. Autoradiography evidenced abundant [125I]GIP binding sites exclusively in the inner adrenocortical layers, and the computer-assisted densitometric analysis of autoradiograms demonstrated that binding was displaced by cold GIP, but not by either ACTH or the selective ACTH-receptor antagonist corticotropin-inhibiting peptide (CIP). The intraperitoneal (IP) injection of GIP dose-dependently raised corticosterone, but not aldosterone plasma concentration: the maximal effective dose (10 nmol/rat) elicited a twofold increase. GIP did not affect aldosterone and cyclic-AMP release by dispersed zona glomerulosa cells. In contrast, GIP enhanced basal corticosterone secretion and cyclic-AMP release by dispersed inner adrenocortical cells in a concentration-dependent manner, and the maximal effective concentration (10(-7) M) evoked 1.5- and 2.4-fold rises in corticosterone and cyclic-AMP production, respectively. GIP (10(-7) M) did not display any additive or potentiating effect on corticosterone and cyclic-AMP responses to submaximal or maximal effective concentrations of ACTH. The corticosterone secretagogue action of 10(-7) M GIP was abolished by the protein kinase A (PKA) inhibitor H-89 (10(-5)M), and unaffected by CIP (10(-6)M). Collectively, these findings indicate that GIP exerts a moderate but statistically significant stimulatory effect on basal glucocorticoid secretion in rats, acting through specific receptors coupled with the adenylate cyclase/PKA-dependent signaling pathway.     

Brain natriuretic peptide interacts with atrial natriuretic peptide receptor in cultured rat vascular smooth muscle cells

The effect of synthetic porcine brain natriuretic peptide (pBNP), a novel brain peptide with sequence homology to alpha-human atrial natriuretic peptide (hANP), on receptor binding and cGMP generation, was studied in cultured rat vascular smooth muscle cells (VSMC) and compared with that of alpha-hANP. 125I-pBNP bound to the cells in a time-dependent manner similar to that of 125I-alpha-hANP. Scatchard analysis indicated a single class of binding sites for pBNP with affinity and capacity identical to those of alpha-hANP. pBNP and alpha-hANP were almost equipotent in inhibiting the binding of either radioligand and stimulating intracellular cGMP generation. These data indicate that BNP and ANP interact with the same receptor sites to activate guanylate cyclase in rat VSMC.     

Photoaffinity labeling of atrial natriuretic factor receptor in bovine and rat adrenal cortical membranes

Radioiodinated synthetic atrial natriuretic factor (ANF) bound to a single class of high affinity binding sites in the plasma membrane from bovine adrenal cortex with a KD of 7.4 X 10(-10) M. The binding affinities of related peptides showed close parallelism to their potencies in natriuretic and vasorelaxant activities. Incubation of adrenal membranes with radioiodinated 4-azidobenzoyl ANF or a similar derivative of its analogue followed by photolysis resulted in specific radiolabeling of a protein band in SDS gel electrophoresis with an apparent Mr of 124,000 in bovine or Mr of 126,000 in rat, which was abolished by inclusion of unmodified ANF in the incubation. Prevention of the labeling was dependent on the concentration of ANF and was not observed with atriopeptin I or with unrelated peptides, angiotensin II, ACTH or [Arg8] vasopressin. These results indicate specific covalent labeling of ANF-receptor or its subunit by the photoaffinity ligands.     

Atrial natriuretic peptide binding, cross-linking, and stimulation of cyclic GMP accumulation and particulate guanylate cyclase activity in cultured cells

The stimulation of cyclic GMP accumulation and particulate guanylate cyclase activity by atrial natriuretic peptide (ANP) was compared to the affinity and number of ANP receptors in eight cultured cell types. At 100 nM, ANP increased cyclic GMP by 13-fold in bovine adrenal cortical, 35-fold in human lung fibroblast, 58-fold in canine kidney epithelial, 60-fold in bovine aortic smooth muscle, 120-fold in rat mammary epithelial, 260-fold in rat Leydig, 300-fold in bovine kidney epithelial, and 475-fold in bovine aortic endothelial cells. ANP (1 microM) increased particulate guanylate cyclase activity by 1.5-, 2.5-, 3.1-, 3.2-, 5.0-, 7.0-, 7.8-, and 8.0-fold in bovine adrenal cortical, bovine aortic smooth muscle, human lung fibroblast, canine kidney epithelial, rat mammary epithelial, rat Leydig, bovine kidney epithelial, and bovine aortic endothelial cells, respectively. Specific 125I-ANP binding to intact rat Leydig (3,000 sites/cell; Kd = 0.11 nM), bovine aortic endothelial (14,000 sites/cell; Kd = 0.09 nM), bovine adrenal cortical (50,000 sites/cell; Kd = 0.12 nM), human lung fibroblast (80,000 sites/cell; Kd = 0.32 nM), and bovine aortic smooth muscle (310,000 sites/cell; Kd = 0.82 nM) cells was saturable and high affinity. No specific and saturable ANP binding was detected in bovine and canine kidney epithelial and rat mammary epithelial cells. Two ANP-binding sites of 66,000 and 130,000 daltons were specifically labeled by 125I-ANP after cross-linking with disuccinimidyl suberate. The 130,000-dalton ANP-binding sites bound to a GTP-agarose affinity column, and the specific activity of guanylate cyclase was increased by 90-fold in this fraction. Our results demonstrate that the increase in cyclic GMP accumulation and particulate guanylate cyclase activity by ANP does not correlate with the affinity and number of ANP-binding sites. These results suggest that multiple populations of ANP receptors exist in these cells and that only one receptor subtype (130,000 daltons) is associated with particulate guanylate cyclase activity.     

Differential response to adrenocorticotropic hormone analogs of bovine adrenal plasma membranes and cells.

The ability of three analogs of ACTH1-24 ([Gln5, Phe9] ACTH1-24, [Gln5, Ala9[Acth1-24, and [Gln5, Lys8, Phe9[ ACTH1-24) embodying tryptophan substitutions to activate the adenylate cyclase system of a bovine adrenal plasma membrane preparation was compared to the effect of the analogs on adenosine 3':5'-monophosphate (cyclic AMP) accumulation and steroidogenesis in viable bovine adrenocortical cells. The results were not comparable. Whereas the analogs antagonized the ACTH1-24-activated membrane cyclase they stimulated cyclic AMP accumulation as well as steroid production of the cells. None of the analogs inhibited steroidogenesis of ACTH1-24-stimulated cells, but two of them, at very high dose levels, inhibited cyclic AMP production. The ability of the analogs to stimulate steroidogenesis of the adrenal cells half-maximally decreased in the order tryptophan greater than phenylalanine greater than alanine, indicating that the aromaticity of the indole ring of tryptophan is necessary for maximal interaction between hormone and receptor. Both the absolute and relative steroidogenic potencies were the same for several analogs when assayed with rat adrenal cells. Although only a small fraction of the cell's potential to produce cyclic AMP was necessary to induce maximum steroid production, the relative activities of a series of analogs were the same for steroidogenesis as for cyclic AMP accumulation. Furthermore, the concentration of cyclic AMP necessary for full steroidogenesis was practically identical for a series of peptides that differed widely in potency. These findings support the postulate that cyclic AMP accumulation and steroidogenesis in adrenocortical cells are coupled processes. The differential behavior of bovine adrenal plasma membranes and bovine adrenocortical cells toward ACTH analogs indicates that structure-function studies using cyclase assays may not reflect events that take place in the intact adrenal or in cell preparations derived therefrom.     

Specific receptors for atrial natriuretic factor (ANF) in cultured vascular smooth muscle cells of rat aorta

Specific binding site for atrial natriuretic factor (ANF), a potent natriuretic and vasorelaxant polypeptide recently isolated from mammalian atria, was studied in cultured vascular smooth muscle cells (VSMC) of the rat aorta. Binding studies of 125I-labeled-synthetic alpha-human natriuretic peptide (alpha-hANP) revealed the presence of a non-interacting, single class of high affinity binding sites for alpha-hANP on VSMC in culture: the apparent dissociation constant (Kd) was approximately 1-2 X 10(-9)M and the number of maximal binding sites was approximately 200,000-300,000 sites/cell. A variety of vasoactive substances and other polypeptide hormones did not affect the binding of 125I-labeled-alpha-hANP to its binding sites. alpha-hANP significantly increased the concentrations of intracellular cyclic GMP in VSMC in a dose-dependent manner (3.2 X 10(-9)-1.6 X 10(-7)M). These data indicate that the specific receptor for ANF is present in VSMC and suggest that intracellular cyclic GMP may be involved in its vasorelaxant effect.     

Characterization of specific receptors for atrial natriuretic factor in bovine adrenal zona glomerulosa

We have recently shown that synthetic rat atrial natriuretic factor (ANF) directly inhibits mineralocorticoid and glucocorticoid secretion in cultured bovine adrenal cells with a potency of 100 pM. [125I]iodo-ANF was used in the present study to characterize potential receptor sites in bovine zona glomerulosa membranes. ANF binds to a class of high affinity binding sites with a pK of 10.2 and a density of 1.3 pmol/mg protein. Detailed competition curves with ANF document a class of high affinity sites with a pK of 10.2 and also a second class of lower affinity sites with a pK of 8.5. Nonspecific binding amounts to less than 10% of [125I]iodo-ANF binding at concentrations less than 100 pM. High affinity binding of [125I]iodo-ANF is reversible with a half-time of association of 15 minutes at 25 pM and a half-time of dissociation of 140 minutes. Monovalent cations Na, Li and K equipotently enhance [125I]iodo-ANF specific binding. Divalent cations Mg, Ca and Mn also increase [125I]iodo-ANF specific binding, with Mn being the most active cation. No effect of guanine nucleotide could be detected on ANF binding. The binding of [125I]iodo-ANF is very specific and is not inhibited by 1 microM angiotensin II, ACTH, VIP, somatostatin, Leu-enkephalin, dynorphin or by the N-terminal of POMC. The N-terminal fragment ANF-(1-16) is also completely inactive. Reduction of the disulfide bridge of ANF inactivates the peptide. This enabled the development of a highly specific radio-receptor assay for ANF with a minimum detectable dose of 2 femtomoles. The results document the specific receptor involved in the potent inhibitory effect of ANF on adrenal steroidogenesis and indicate that bovine adrenal zonal glomerulosa provide a highly sensitive system for studying the recently discovered atrial natriuretic factor.     

Binding sites for inositol trisphosphate in the bovine adrenal cortex

Binding sites for inositol trisphosphate (IP3) have been identified in bovine adrenal cortex, employing [32P]IP3 prepared from human erythrocytes radiolabeled with [32P]ATP. IP3 was bound to adrenal microsomes with high affinity (Kd = 5 nM) and low capacity (186 fmol/mg protein). During kinetic studies, half-maximal binding was reached in less than one min at 4 degrees C, and dissociation was even more rapid with t1/2 of about 10 sec. [32P]IP2 showed no binding to the microsomal sites, which represent putative receptors at which IP3 acts to elevate intracellular calcium concentration during the actions of peptide hormones such as angiotensin II.