Atrial natriuretic peptide inhibits protein phosphorylation stimulated by angiotensin II in bovine adrenal glomerulosa cells

Bovine adrenal glomerulosa cells were incubated with 32PO4 and either angiotensin II, atrial natriuretic peptide, or both. Solubilized cells were subjected to one-dimensional gel electrophoresis. Autoradiography and scintillation counting of gels showed that angiotensin increased labeling of one band, with an apparent molecular weight of 17,600. Atrial natriuretic peptide inhibited the angiotensin effect. Together with earlier results, this observation suggests that atrial natriuretic peptide affects aldosteronogenesis at the level of protein phosphorylation, but not by altering angiotensin receptors, calcium fluxes or phosphoinositide metabolism.     

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.     

Differential regulation of natriuretic peptide biosynthesis in bovine adrenal chromaffin cells

Chromaffin cells synthesize and secrete two forms of natriuretic peptides which are also found in the heart and in the central nervous system. While atrial tissue predominantly contains atrial natriuretic factor (ANF), brain tissue appears to produce relatively larger amounts of brain natriuretic peptide (BNP) also identified as aldosterone secretion inhibitory factor (ASIF), suggesting tissue-specific differential regulation of these two peptides. This report compares the modulation of the biosynthesis and secretion of ASIF with that of ANF using cultured chromaffin cells as a model system. Cholinergic nicotinic activation and KCl depolarization induce a 5-fold increase of the corelease of ASIF and pro-ASIF in cell culture medium concomitantly with a 3-fold stimulation of ANF and pro-ANF cosecretion. While the combined treatment with phorbol ester and forskolin produces a 2-fold increase in total ANF level, it induces a synergistic 20-fold elevation of total ASIF level. These results indicate that chromaffin cell secretagogues induce the cosecretion of both the precursor and mature forms of ASIF and ANF. The preferential stimulation of ASIF production is revealed by the combined treatment rendering the ASIF to ANF proportion similar to that in brain.     

Atrial natriuretic factor inhibits the early pathway of steroid biosynthesis in bovine adrenal cortex

We have previously determined that atrial natriuretic factor (ANF) is a potent inhibitor of steroid secretion in cultured bovine zona glomerulosa and fasciculata cells. The present report describes a comparison of the effect produced by ANF on aldosterone, deoxycorticosterone and progesterone secretions by zona glomerulosa cells and on cortisol, corticosterone and progesterone secretions by zona fasciculata cells. The equipotent inhibitory action of ANF on the stimulated secretion of these steroids in both cell types indicates a common site of action prior to progesterone synthesis at which ANF inhibits the steroidogenic pathway.     

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.     

Sites of action of angiotensin II, atrial natriuretic factor and guanabenz, on aldosterone biosynthesis.

It is well known that atrial natriuretic factor (ANF) inhibits aldosterone biosynthesis. Recent studies showed that amiloride can also inhibit adrenal steroidogenesis. Since the antihypertensive agent, guanabenz, is structurally related to amiloride, we have examined its action on aldosterone biosynthesis. The aim of this work was to localize the sites of action of angiotensin II (AII) and of ANF on steroidogenesis and to compare the effects of guanabenz to ANF. Trilostane, an inhibitor of 3 beta-hydroxysteroid dehydrogenase was used to separately study the early and late pathways of aldosterone biosynthesis. The different steps of steroidogenesis are stimulated by AII. ANF inhibits the formation of pregnenolone, the steps between progesterone and deoxycorticosterone, deoxycorticosterone and corticosterone and finally, corticosterone and aldosterone with ED50 of 114 +/- 17, 199 +/- 90, 14 +/- 3 and 92 +/- 34 pM of ANF, respectively, and around 70% of inhibition. These steps are also inhibited by guanabenz with ED50 of 66 +/- 17 microM for the formation of pregnenolone, 1.6 +/- 1.3, 3.3 +/- 1.7 and 29 +/- 4 microM for the last 3 steps. The percentage of inhibition by guanabenz was at least 80% for all the steps except for progesterone to deoxycorticosterone which is less than 35%. These results indicate that the major site of action of both AII and ANF could be at the level of intracellular signal transduction for the activation of mitochondrial steroidogenic enzymes or for the transport of steroids to mitochondria. We also showed that guanabenz mimics the inhibitory effects of ANF. This study with guanabenz suggests that it might be a prototype for a new family of antihypertensive agents.     

Porcine brain natriuretic peptide receptor in bovine adrenal cortex

The action of porcine brain natriuretic peptide (pBNP) on the steroidogenesis was investigated in cultured bovine adrenocortical cells. Porcine BNP induced a significant dose-dependent inhibition of both ACTH- and A II-stimulated aldosterone secretion. 10(-8) M and 10(-7) M pBNP also significantly inhibited ACTH-stimulated cortisol and dehydroepiandrosterone (DHEA) secretions. Binding studies of [125I]-pBNP to bovine adrenocortical membrane fractions showed that adrenal cortex had high-affinity and low-capacity pBNP binding sites, with a dissociation constant (Kd) of 1.70 x 10(-10) M and a maximal binding capacity (Bmax) of 19.9 fmol/mg protein. Finally, the 135 Kd radioactive band was specially visualized in the affinity labeling of bovine adrenal cortex with disuccinimidyl suberate (DSS). These results suggest that pBNP may have receptor-mediated suppressive actions on bovine adrenal steroidogenesis, similar to that in atrial natriuretic peptide (ANP).     

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.     

Inhibition of aldosterone secretion by atrial natriuretic peptide in chicken adrenocortical cells

Dispersed chicken adrenocortical cells were preincubated with atrial natriuretic peptide (rANP), sodium nitroprusside (SNP) or 8-bromo cyclic GMP, followed by incubations with ACTH, chicken PTH, cholera toxin or various steroid intermediates of aldosterone production. Cyclic AMP production and aldosterone secretion were evaluated, in order to determine the sites of ANP inhibition in the sequence of events leading to aldosterone secretion. Dose-dependent inhibitory effects on ACTH-stimulated aldosterone secretion by rANP and SNP were observed. Both agents appeared to stimulate cGMP production by the particulate fraction of the avian adrenocortical cells. Aldosterone production, stimulated by cyclic AMP agonists such as ACTH, chicken PTH and cholera toxin, was significantly inhibited by ANP. On the other hand, ANP did not interfere with production or degradation of cAMP. Each of the aldosterone intermediates--pregnenolone, progesterone, 11-deoxycorticosterone and corticosterone--promoted aldosterone production when included in the incubation media. Atrial natriuretic peptide and SNP inhibited aldosterone secretion when enhanced by the intermediates, by about 40-60%, but the ACTH-stimulated secretion was inhibited by over 90%. The results suggest two sites of inhibition by ANP in the pathway of aldosterone synthesis and secretion: synthesis of cholesterol or pregnenolone, and conversion of corticosterone to aldosterone. The inhibition by 8-bromo cGMP of aldosterone secretion and the similar sites of inhibition for ANP and SNP suggest that cyclic GMP mediates the inhibition in both cases.     

Dopamine inhibition of potassium-stimulated aldosterone biosynthesis in bovine adrenal zona glomerulosa cells

Dopamine inhibits angiotensin II-stimulated aldosterone production by an effect on the late phase of biosynthesis. This study was undertaken to investigate the effect of dopamine on potassium-stimulated aldosterone biosynthesis in adrenal glomerulosa cells in vitro. As potassium concentrations were increased from 0 to 12 mM, aldosterone production increased up to 6 mM potassium, but not beyond this concentration. Dopamine (10(-5)M) inhibited the aldosterone response to potassium. The effect of potassium on pregnenolone accumulation (the early phase of aldosterone biosynthesis) was assessed in cells treated with trilostane which inhibits the conversion of pregnenolone onward to aldosterone. Increasing potassium concentrations up to 12 mM gave increasing pregnenolone accumulation; however dopamine did not influence this effect. The potassium stimulated conversion of corticosterone to aldosterone, an index of activity in the late phase of aldosterone biosynthesis, was assessed using aminoglutethimide to prevent cholesterol side-chain cleavage. Significantly more corticosterone was converted to aldosterone at 6 mM potassium than at 0 or 12 mM; dopamine inhibited the conversion of corticosterone to aldosterone at 6 mM potassium. These data indicate that dopamine inhibits potassium-stimulated aldosterone production by an effect restricted to the late phase of the aldosterone biosynthetic pathway similar to its previously established effect on angiotensin II-stimulated aldosterone biosynthesis.     

Modulation of Ca2+ channels by atrial natriuretic peptide in the bovine adrenal glomerulosa cell.

In the bovine adrenal glomerulosa cell, calcium influx through voltage-dependent calcium channels is critical to maintaining an aldosterone secretory response. In patch clamp, atrial natriuretic peptide (ANP) inhibits T-type calcium channel current yet stimulates L-type calcium channel current. In the present study the channel effects of ANP observed in the patch-clamp configuration were extended and related to populations of cells. We observed the following. (i) The effect of ANP on T-channel current resulted in the reduction in the open state probability. ANP decreased the mean open state duration from 14.2 to 1.8 ms/sweep. (ii) In the weakly depolarized cell stimulated by 8 mM K+, ANP reduced the level of aequorin luminescence (a measure of cytosolic calcium) and completely inhibited the stimulated rate of aldosterone secretion, returning it to prestimulation values. These effects are consistent with a decrease in net calcium channel influx and the reported inhibition of T-channel current. In contrast, the calcium channel blocker, nitrendipine, which at low dose selectively blocks L-type calcium channel flux, only slightly reduced luminescence, and partially inhibited the sustained secretory response. (iii) In the strongly depolarized cell, stimulated by 60 mM K+, ANP increased the level of aequorin luminescence consistent with an increase in net calcium channel influx and the reported stimulation of L-channel current. These results indicate that under physiological conditions the inhibition of T-type calcium channels may be involved in the inhibition of the aldosterone secretion induced by ANP.     

Characterisation of atrial natriuretic peptide receptors in bovine ventricular sarcolemma

Analysis of [125I]-ANP binding data in an isolated bovine ventricular sarcolemmal membrane fraction revealed a single high affinity binding site (Kd approximately 5 x 10(-11) M). The ring deleted ANP analogue des [QSGLG]-ANP (4-23)-NH2 bound with a 1000-fold lower affinity indicating the absence of C-type receptors in this preparation. ANP stimulated guanylate cyclase activity by up to 2-fold with half-maximal activation at approximately 10(-9) M. Crosslinking [125I]-ANP to its receptor with disuccinimidyl suberate (DSS) revealed two radiolabelled bands of 120 kDa and 65 kDa on non-denaturing SDS-PAGE. Radioactive signals from both bands were lost by reducing the sample with beta-mercaptoethanol prior to electrophoresis, in which case a radioactive fragment of less than 5 kDa migrated with the dye front. These results suggest that the binding of ANP to both high and low molecular weight "receptor" proteins may be associated with the hydrolysis of the peptide.     

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.     

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.     

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.     

Involvement of the adrenal glands in the action of the atrial natriuretic factor

Adrenalectomized, medullectomized and sham operated rats were treated with either a chronic infusion or a bolus injection of the synthetic atrial natriuretic factor (ANF). ANF did not enhance natriuresis and diuresis in sham operated conscious animals during chronic infusion, but it had a potent action when injected as a bolus into anesthetized rats. The absence of the whole adrenal glands, but not adrenal medulla profoundly modified the renal response to ANF: a) following chronic administration of ANF, the baseline natriuresis paradoxically decreased in adrenalectomized rats, and b) in response to a bolus injection of ANF the natriuretic and diuretic actions of the peptide were attenuated in these animals. The medullectomy-induced decreased natriuresis and dopamine excretion were corrected by ANF infusion. Furthermore, ANF suppressed the compensatory increase of norepinephrine excretion secondary to adrenalectomy. The data suggest that the presence of the adrenal cortex is necessary for the natriuretic and diuretic actions of ANF. The decrease in urinary DA excretion may reflect diminished dopaminergic activity and contribute to the post-medullectomy antinatriuresis, a phenomenon which can be corrected by ANF infusion. ANF may also have a depressing activity on the increased sympathetic tone.     

Effects of atrial natriuretic peptide, angiotensin, cyclic AMP, and potassium on protein phosphorylation in adrenal glomerulosa cells

Bovine adrenal glomerulosa cells were incubated with 32PO4 and angiotensin II (AII), atrial natriuretic peptide (ANP) (rat[8-33]), N6,O2'-dibutyryl cyclic AMP, or elevated potassium (7.2 mM). Solubilized cells were analyzed by one-dimensional polyacrylamide gel electrophoresis, autoradiography, and laser densitometry. AII and dibutyryl cyclic AMP increased labeling of a 17.6 kd protein. Elevated potassium did not alter labeling of this protein. ANP inhibited labeling, whether basal or stimulated by AII, and to a lesser extent that stimulated by dibutyryl cyclic AMP. Similar dose-response curves were obtained for the effect of AII on labeling of the 17.6 Kd band and on aldosterone synthesis; ANP had a similar inhibitory effect on AII-stimulated phosphorylation and aldosterone synthesis. Effects of AII and ANP were apparent after 15 minutes of hormone treatment. Fractionation of labeled cells showed that the 17.6 Kd protein was not in cytosol, mitochondria, or endoplasmic reticulum, but was enriched in a crude nuclear fraction. These results suggest that AII and ANP affect aldosterone synthesis at the level of protein phosphorylation.     

Alpha-human atrial natriuretic polypeptide inhibits 19-hydroxy-androstenedione secretion by human adrenal cells

We investigated the effect of ACTH, angiotensin II (AII), and alpha-human atrial natriuretic polypeptide (alpha-hANP) which plays an important role of water-electrolytes balance, on 19-hydroxyandrostenedione (19-hydroxyandrost-4-ene-3,17-dione, 19-OH-A-dione) secretion by cultured human adrenal cells. 19-OH-A-dione in culture media was measured using a specific RIA. Basal 19-OH-A-dione secretion by adrenal cells was 0.69 +/- 0.08 ng/3h/10(6) cells and significantly rose to 1.17 +/- 0.14 ng/3h/10(6) cells in the presence of ACTH, but not in the presence of A II. These results demonstrate that 196-OH-A-dione is directly secreted from adrenal cells. alpha-hANP significantly inhibited both basal and ACTH-stimulated 19-OH-A-dione secretions, as well as aldosterone. These results demonstrate that alpha-hANP inhibits aldosterone activity by means of the inhibition of both aldosterone and 19-OH-A-dione (an aldosterone amplifier) secretion by adrenal cells.     

Immunohistochemical localization of atrial natriuretic peptide receptor in bovine kidney and lung

Receptors for atrial natriuretic peptide (ANP) were localized in the alveoli and bronchiolar smooth muscle cells of bovine lung and in podocytes of the kidney by immunofluorescence and immunoperoxidase methods. Two specific antisera were raised against the ANP receptor purified from bovine lung plasma membranes: anti-Rc 140 and anti-Rc 70. Anti-Rc 140 was raised against the 140 KD native receptor having a homodimeric structure, and anti-Rc 70 was elicited by immunizing a rabbit with the 70 KD reduced subunits. Essentially identical staining patterns were obtained with both antisera. Identification of ANP receptor sites would provide useful information in understanding the pulmonary and renal actions of ANP.