Internalization of atrial natriuretic peptide by adrenal glomerulosa cells

Internalization of 125I-labelled atrial natriuretic peptide ([ 125I]ANP) by rat adrenal glomerulosa cells in vivo was investigated by means of an ultrastructural autoradiographic approach. One to 30 min after IV injection of [125I]ANP, silver grains were found, at the light microscope level, over all glomerulosa cells; coinjection of 20 micrograms of unlabelled ANP inhibited this binding by 64%. At the electron microscope level, the time-course study indicated maximal silver grain densities in plasma membranes 1 min after IV injection; grains were detected in mitochondria (external membranes and matrix) 2 min after injection, with maximal labelling at 15 min. The cytoplasmic matrix was labelled only 30 min after injection. During the time-course, labelling of nuclei, Golgi apparatus, and lysosomes was minimal. The data suggest that after binding to plasma membranes ANP is rapidly internalized and distributed within glomerulosa cells. The association of radioactivity with mitochondria suggests that ANP may have intracellular sites of action complementary to those on plasma membranes.     

Effect of atrial natriuretic peptide on ACTH, dibutyryl cAMP, angiotensin II and potassium-stimulated aldosterone secretion by rat adrenal glomerulosa cells

We examined the effect of rat atrial natriuretic peptide (ANP) on ACTH, dibutyryl cAMP, angiotensin II and potassium-stimulated aldosterone secretion by dispersed rat adrenal glomerulosa cells. ANP inhibited ACTH, angiotensin II and potassium-stimulated aldosterone secretion with IC50's between 0.15-0.20 nM. Inhibition by 10 nM ANP could not be overcome with higher concentrations of these stimuli. ANP shifted the dibutyryl cAMP dose-response curve slightly to the right but did not blunt the maximal aldosterone secretory response. The sites of ANP inhibition in the aldosterone biosynthetic pathway for these stimuli were also examined. ANP inhibited activation of the cholesterol desmolase (CD) enzyme complex by ACTH, angiotensin II and potassium. Activation of the corticosterone methyl oxidase (CMO) enzyme complex by potassium was inhibited by ANP, however, activation by ACTH was not blocked. We concluded that: 1) ANP is a potent inhibitor of ACTH, angiotensin II and potassium-stimulated aldosterone secretion; 2) inhibition of ACTH stimulation is primarily due to lower cAMP levels and; 3) inhibition of angiotensin II and potassium stimulation reflects a block in the activating mechanism of the CMO and/or CD enzyme complexes, whereas CD but not CMO activation by ACTH is inhibited by ANP.     

Effects of atrial natriuretic peptide, angiotensin II and III on norepinephrine uptake in the rat adrenal medulla.

The effects of atrial natriuretic peptide (ANP), angiotensin II (ANG II) and angiotensin III (ANG III) on norepinephrine (NE) uptake were studied in the adrenal medulla of the rat. One microM ANG II and 10 microM ANG III decreased NE uptake while 10 nM and 100 nM ANP increased it. Subthreshold concentrations of ANP (1 nM) blunted the inhibitory effect of 1 microM ANG II but did not modify the inhibitory effect of 10 microM ANG III. The increasing effects of 100 nM ANP on NE uptake were partially reversed by subthreshold concentrations of ANG II (1 nM) and blunted by 1 nM ANG III. The interaction between ANP and the renin-angiotensin system could contribute to modulate the sympathetic function in the adrenal medulla.     

Actions of synthetic atrial natriuretic factor on bovine adrenal glomerulosa

Synthetic atrial natriuretic factor (ANF) inhibited aldosterone production by suspensions of bovine adrenal glomerulosa cells. Inhibition by ANF was most pronounced when basal aldosterone production was measured. The effects of angiotensin II (AII), N6,O2'-dibutyryl-adenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP), and elevated potassium were also inhibited by ANF. Inhibition could be partially overcome by high doses of agonist. Inhibition was localized to the early pathway of aldosteronogenesis, to a step before cholesterol side-chain cleavage. ANF had no effect on binding of AII to receptors, on the stimulation by AII of phospholipid turnover, or on the alteration by AII of calcium fluxes.     

Inhibitory actions of somatostatin on cyclic AMP and aldosterone production in agonist-stimulated adrenal glomerulosa cells

Somatostatin (SRIF) is a potent inhibitor of angiotensin II (AII)-stimulated aldosterone production in rat adrenal glomerulosa cells. This inhibition can be prevented by pretreatment of the cells with pertussis toxin, but little else is known about either the specificity or the biochemical bases of SRIF action in this tissue. We therefore conducted detailed studies of the influence of SRIF on steroidogenesis elicited by AII and the other two physiological stimuli of aldosterone production, K+ and adrenocorticotropic hormone (ACTH), in rat adrenal glomerulosa cells. We also determined the effects of SRIF on cytosolic calcium concentration ([Ca2+]i) and cellular cAMP levels. In these studies, SRIF was found to inhibit the aldosterone responses elicited by low concentrations of all three stimuli, which are believed to promote steroid secretion via discrete but interacting cellular signalling mechanisms. In addition, SRIF consistently lowered cellular cAMP levels in the presence of each of the three agents. However, SRIF caused a small and transient increase rather than a decrease in basal ([Ca2+]i), and had no effect on the subsequent elevation of ([Ca2+]i) by AII and K+. These data indicate that activation of a Gi-like protein by SRIF influences steroid responses to all three major regulators of glomerulosa-cell function, and suggest that basal levels of cAMP play a facilitatory or permissive role in the control of aldosterone production by predominantly calcium-mobilizing regulators of mineralocorticoid secretion.     

Hormonal modulation of atrial natriuretic factor receptors and effects on adrenal glomerulosa cells of female rats

This study was done to determine if a decrease in the aldosterone-suppressant effect of atrial natriuretic factor (ANF) by progesterone and an increase by estrogen was caused by modulation of adrenal zona glomerulosa ANF receptors. Freshly dispersed glomerulosa cells from virgin, 13–15 day pregnant, ovariectomized (OVX) estradiol-17β-treated and OVX progesterone-treated rats were used. Competitive displacement of specifically bound [¹²⁵I]ANF_1–8 with unlabelled ANF_1–28 yielded concentrations of guanylate cyclase-linked ANF-R1 plus ANF-R2 (clearance) receptors and the displacement with unlabelled ANF_4–23 yielded ANF-R2 receptors; the difference between the two was treated as the concentration of ANF-R1 receptors. Pregnancy and progesterone decreased and estrogen increased the number of glomerulosa ANF-R1 receptors. ANF produced a significantly greater suppression of potassium-induced aldosterone secretion in cells from OVX estradiol-treated rats than in cells from OVX progesterone-treated animals. These data suggest that the inhibition of the aldosterone-suppressant activity of ANF by progesterone is the result of a downregulation of ANF-R1 receptors.     

Stimulation of protein phosphorylation by atrial natriuretic factor in plasma membranes of bovine adrenal cortical cells

Atrial natriuretic factor (ANF) rapidly enhanced phosphorylation of plasma membrane proteins of bovine adrenal cortical cells. Pretreatment of the membranes with ANF (1 x 10(-8)M to 1 x 10(-7)M) resulted two- to four-fold in an incorporation of 32p-radioactivity from [gamma -32p]ATP as compared to the controls. The guanosine 3', 5' monophosphate (cGMP) which has been considered a second messenger of ANF also enhanced the phosphorylation of several proteins which were stimulated by ANF. However, the phosphorylation of certain proteins was stimulated differentially only by either ANF or cGMP. These results suggest that ANF-induced protein phosphorylation may play a role in transmembrane signalling pathway involving other second messenger(s) in addition to cGMP during the biological action of ANF.     

Human alpha atrial natriuretic peptide inhibits angiotensin stimulated aldosterone biosynthesis in bovine adrenal glands

The behaviour of aldosterone output was evaluated in isolated and superfused bovine adrenal glands during superfusion with human alpha atrial natriuretic peptide on its own or with angiotensin II or a antagonist dopaminergic drug: metoclopramide. H alpha-ANP even in high concentrations did not reduce the basal amount of aldosterone released from bovine adrenal glands, nor did it modify aldosterone response to metoclopramide, but it partially inhibited aldosterone stimulation by angiotensin II. These data suggest that atrial natriuretic factor may affect sodium secretion through the modulation of aldosterone secretion.     

Angiotensin II differentially affects cyclic AMP formation in intact adrenal glomerulosa cells and in purified membrane preparations

In the present study we have investigated the cyclic AMP (cAMP) responses to angiotensin II (AII) in isolated rat adrenal glomerulosa cells and in purified membrane preparations. When cells were incubated with 10 nM AII cAMP cellular content increased 2-fold at 5 min and 3-fold at 10 min, then rapidly declined. The effect of AII was dose-dependent with EC50 of 4 nM and was mediated by AII receptors as shown by the pharmacological characterization with AII analogs and AII receptor antagonists. Since AII inhibited cAMP formation in purified adrenal cortical membrane preparations, the stimulatory effect observed in intact cells could be indirect and mediated by other intracellular events.     

Temporal patterns of protein phosphorylation after angiotensin II, A23187 and/or 12-O-tetradecanoylphorbol 13-acetate in adrenal glomerulosa cells.

The temporal patterns of protein phosphorylation in the adrenal glomerulosa cell were analysed by two-dimensional electrophoresis after stimulation with 10 nM-angiotensin II or various agents [10 nM-12-O-tetradecanoylphorbol 13-acetate (TPA), 50 nM-A23187, 1 microM-nitrendipine], administered singly or in combination. These patterns were compared with the temporal patterns of aldosterone secretion induced by the same agonists and antagonists. After 1 and 30 min of stimulation with angiotensin II, different patterns of protein phosphorylation were observed. A comparison of these patterns reveals that: the phosphorylation of only one protein was persistently enhanced during the continuous incubation with angiotensin II; the phosphorylation of five proteins was transiently enhanced (at 1 min but not 30 min); and the phosphorylation of three proteins did not occur at 1 min but was seen at 30 min. Addition of the phorbol ester TPA alone, which at 30 min is without effect in enhancing aldosterone production, has no effect on protein phosphorylation. The combined addition of TPA and the Ca2+ ionophore, A23187, which, like angiotensin II, evokes a sustained increase in aldosterone production, reproduced the temporal patterns of protein phosphorylation seen after angiotensin II action. Manipulations (A23187 alone, angiotensin II plus nitrendipine) which evoke only a transient rise in aldosterone production rate induce a transient rise in cellular protein phosphorylation. The 1 min patterns of phosphorylation seen after A23187 or combined angiotensin II and nitrendipine (a Ca2+ channel antagonist) are similar to those observed after 1 min of angiotensin II stimulation. These results suggest that, when angiotensin II acts, the initial cellular response is mediated by a different mechanism than that responsible for the sustained response.     

Effect of atrial natriuretic peptide on adrenal renin and aldosterone

The effect of atrial natriuretic peptide (ANP) on adrenal renin and aldosterone was investigated in anesthetized rats. Under pentobarbital anesthesia 40 mg/kg), intravenous infusion of ANP (0.25 micrograms/kg/min) for 45 min failed to alter the adrenal renin, adrenal aldosterone, and plasma aldosterone (PA). In this condition, intraperitoneal injection of ACTH (10 micrograms/kg) significantly increased the adrenal renin (from 2.4 +/- 0.1 to 5.0 +/- 0.08 ng/mg protein/h, P less than 0.05), adrenal aldosterone (from 13.6 +/- 1.3 to 22.7 +/- 2.3 ng/mg protein, P less than 0.01) and PA (from 59.8 +/- 5.8 to 75.5 +/- 7.4 ng/dl, P less than 0.05), respectively. Under ACTH stimulation, ANP infusion induced significant decreases in adrenal renin (from 5.0 +/- 0.08 to 2.8 +/- 0.2 ng/mg protein/h, P less than 0.05), adrenal aldosterone (from 22.7 +/- 2.3 to 16.2 +/- 1.8 ng/mg protein, P less than 0.05) and PA (from 75.5 +/- 7.4 to 61.6 +/- 4.9 ng/dl). These results suggest a possible role for adrenal renin in the mechanism underlying the inhibitory effect of ANP on aldosterone production in vivo.     

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.     

Phosphorylation of adrenal histone H3 is affected by angiotensin, ACTH, dibutyryl cAMP, and atrial natriuretic peptide

Angiotensin (AII) and atrial natriuretic peptide (ANP) exert opposite effects on phosphorylation of a 17.6 kDa nuclear protein from bovine adrenal glomerulosa cells. The protein was separated by sodium dodecyl sulfate electrophoresis and blotted onto polyvinylidene difluoride, and the N-terminal sequence was obtained. This sequence corresponded to histone H3. Another polyacrylamide gel electrophoresis system was used to confirm that AII stimulated the phosphorylation of histone H3. ACTH[1-24] stimulated phosphorylation of the same protein. Dibutyryl cAMP stimulated phosphorylation of a 17.6-kDa protein, and two gel electrophoresis systems confirmed that the protein affected was histone H3. In situ peptide mapping using papain, of either purified standard histone H3 or of the adrenal 17.6-kDa protein, produced the same major fragment as observed by silver staining. Therefore, the 17.6-kDa protein that is affected by AII, ANP, ACTH, and dibutyryl cAMP is histone H3. This finding suggests that in addition to their mutually antagonistic effects on acute steroidogenesis, AII and ANP may exert opposite effects on adrenal cell functions involving the nucleus.     

Effects of angiotensin II and dibutyryl cyclic adenosine monophosphate on phosphatidylinositol metabolism, 45Ca2+ fluxes, and aldosterone synthesis in bovine adrenal glomerulosa cells

Angiotensin II (AII) and N6,O2'-dibutyryladenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP) both stimulated aldosterone synthesis in bovine adrenal glomerulosa cells. AII altered 45Ca2+ fluxes and increased 32PO4 incorporation into phosphatidylinositol in these cells, whereas dibutyryl cyclic AMP did not affect either process. Neither AII nor dibutyryl cyclic AMP increased the mass of phosphatidylinositol. Both agents are known to stimulate pregnenolone synthesis. Thus, although dibutyryl cyclic AMP and AII may increase aldosterone synthesis at a common site (pregnenolone synthesis), they do so by different mechanisms. AII stimulation of phosphatidylinositol labeling by 32PO4 (the "PI effect") was blocked when cells were incubated in a medium containing both EGTA and the calcium antagonist, 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxy-benzoate hydrochloride (TMB-8), suggesting a calcium requirement for the PI effect.     

Modulation by NaCl of atrial natriuretic peptide receptor levels and cyclic GMP responsiveness to atrial natriuretic peptide of cultured vascular endothelial cells.

Type C atrial natriuretic peptide (ANP) receptor levels in cultured vascular endothelial cells were found to be very sensitive to NaCl and shown to be inversely related to the magnitude of ANP-induced cGMP response of the cells. Endothelial cells from bovine carotid artery were subcultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum (MEM-FBS) and in MEM-FBS plus 25 and 50 mM NaCl. Determination, after several passages, of ANP receptor levels in these cells by 125I-ANP binding assay and affinity labeling revealed a marked reduction in the number of type C receptor in the NaCl-treated cells, whereas type A receptor density was not affected. RNase protection assay to estimate the levels of type C receptor mRNA indicated that the reduction occurred at a pre-translational level. In spite of the decrease in type C receptor number and no significant change in type A receptor (i.e. particulate guanylate cyclase) levels, cGMP response of the NaCl-treated cells to ANP was greatly exaggerated; this sensitization was also observed in membrane preparations. Simple masking of type C ANP receptor with C-ANF (des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP), a ring-deleted ANP analog, did not produce any sensitization of the cGMP response to ANP; therefore, the above phenomenon cannot simply be explained by the clearance function of the type C receptor. Although whether the type C receptor depletion is directly related to the sensitization of the type A receptor/cyclase is not known, the phenomenon reported and characterized here will serve as a useful basis for elucidating ANP receptor regulation and activation.     

Role of cyclic AMP and protein kinase on the steroidogenic action of ACTH, prostaglandin E1 and dibutyryl cyclic AMP in normal adrenal cells and adrenal tumor cells from humans.

The role of the cyclic AMP-protein kinase system in mediating the steroidogenic effect of ACTH, prostaglandin E1 and dibutyryl cyclic AMP, induced similar stimulations of protein kinase activity, cyclic AMP was studied using human adrenal cells isolated from normal and adrenocortical secreting tumors. At high concentrations of ACTH, complete activation of protein kinase of normal adrenal cells was observed within 3 min, at the time when cyclic AMP production was slightly increased and there was still no stimulation of steroidogenesis. At supramaximal concentrations, ACTH, PGE1 and dibutyryl cyclic AMP and cortisol productions in adrenal cells isolated from normal and from one adrenocortical tumor. In one tumor in which the adenylate cyclase activity was insensitive to ACTH, the hormone was unable to stimulate protein kinase or steroidogenesis, but the cells responded to both PGE1 and dibutyryl cyclic AMP. In another tumor in which the adenylate cyclase was insensitive to PGE1, this compound also did not increase protein kinase activity or steroidogenesis, but both parameters were stimulated by ACTH and dibutyryl cyclic AMP. After incubation of normal adrenal cells with increasing concentrations of ACTH (0.01-100 nM) marked differences were found between cyclic AMP formation and cortisol production. However at the lowest concentrations of ACTH exerting an effect on steroid production a close linked correlation was found between protein kinase activation and cortisol production, but half-maximal and maximal cortisol production occurs at lower concentration of ACTH than was necessary to induce the same stimulation of protein kinase. Similar findings were found after incubating the adrenal cells with dibutyryl cyclic AMP (0.01-10 mM). The results implicate an important role of the cyclic AMP-protein kinase system during activation of adrenal cell steroidogenesis by low concentrations of steroidogenic compounds.     

Cholesteryl hemisuccinate alters membrane fluidity, angiotensin receptors, and responses in adrenal glomerulosa cells

We examined the effects of cholesteryl hemisuccinate on membrane fluidity and angiotensin II (AII) actions in bovine adrenal glomerulosa cells. Incubating cells with cholesteryl hemisuccinate decreased membrane fluidity and markedly inhibited AII binding. The effect on binding was characterized by a decrease in AII receptor number. The effects of AII on phosphatidyl inositol turnover and calcium fluxes, proposed intermediaries of AII actions on aldosterone secretion, were less impaired than AII binding by cholesteryl hemisccinate. AII stimulation of aldosterone secretion was preserved despite the decrease in AII binding after cholesteryl hemisuccinate treatment. These results indicate that AII binding can be dissociated from its effects on aldosteronogenesis by a reagent that alters membrane fluidity.     

Phosphorylation of high- and low-molecular-mass atrial natriuretic peptide analogs by cyclic AMP-dependent protein kinase

Synthetic high- and low-molecular-mass atrial peptides were phosphorylated in vitro by cyclic AMP-dependent protein kinase and [32P]ATP. From a series of atrial peptide analogs, it was deduced that the amino acid sequence, Arg101-Ser104 of atriopeptin was required for optimal phosphorylation. Phosphorylated AP(99-126) was less potent than the parent atriopeptin in vasorelaxant activity and receptor-binding properties. These results indicate that the presence of a phosphate group at the N-terminus of AP(99-126) decreases the interaction of the peptide with its receptor and, as a consequence, decreases bioactivity. These observations are in contrast to those of Rittenhouse et al. [(1986) J. Biol. Chem. 261, 7607-7610] who reported that phosphorylation of AP(101-126) enhanced the stimulation of Na/K/Cl cotransport in cultured vascular smooth muscle cells.     

Stimulation of cyclic GMP synthesis in human cultured glomerular cells by atrial natriuretic peptide

Recently a stimulatory effect of atrial natriuretic peptide (ANP) on the particulate guanylate cyclase system has been reported in the glomeruli from different species. Using cultures of homogeneous human glomerular cell lines, we found that rat and human ANP stimulated markedly cGMP formation in epithelial cells with a threshold dose of 1 nM. A 20-fold increase was obtained at 5 microM. Stimulation was also present but less substantial (2-fold at 5 microM) in mesangial cells. cGMP was formed rapidly and released in the medium. ANP and sodium nitroprusside, an activator of soluble guanylate cyclase, had additive effects on cGMP formation. ANP did not inhibit cAMP formation in both cell lines. These results demonstrate that, at least in the human species, epithelial cells represent the main target of ANP in the glomerulus. Synthesis of cGMP in the glomerular epithelial cells in response to ANP also suggests that the excess of urinary cGMP produced by the kidney which is observed after ANP administration is of glomerular rather than of tubular origin.     

Effects of ACTH and calcium on cyclic AMP production and steroid output by the zona glomerulosa of the adrenal cortex.

Effects of ACTH and calcium on cyclic AMP production and steroid output by the zona glomerulosa (the capsular fraction) from the rat adrenal cortex have been studied. Although high concentrations of extracellular calcium potentiated the stimulatory action of ACTH on cyclic AMP and aldosterone output, tetracaine or verapamil inhibited aldosterone output but not cyclic AMP production during ACTH-stimulation. Lanthanum reduced both aldosterone and cyclic AMP accumulation induced by ACTH. These results suggest that an extracellular calcium would be essential in stimulating the capsular steroidogenesis without involvement of the cyclic AMP system.