Affinity cross-linking of atrial natriuretic factor to its receptor in bovine adrenal zona glomerulosa

125I-Labeled atrial natriuretic factor (ANF) was covalently cross-linked to its binding sites in bovine adrenal zona glomerulosa membranes using the hydrophilic cross-linker bis(sulfosuccinimidyl) suberate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol revealed that two protein bands with apparent Mr 68,000 and 114,000 were specifically labeled. The labeling of the two bands was prevented in a dose-dependent fashion by unlabeled ANF with a significant inhibition observed at 10(-10) M. High concentrations of angiotensin II and adrenocorticotropic hormone did not compete with 125I-ANF for binding and cross-linking. The dose-response curve for inhibition of covalent cross-linking of 125I-ANF by unlabeled ANF coincided with the dose-response curve for inhibition of binding to the receptor. No radioactive bands were observed in liver membranes. Experiments in which adrenal membranes were prepared and incubated in the presence of protease inhibitors showed no difference in the labeling pattern. Electrophoresis in the absence of reductant showed that the affinity-labeled species are not derived from larger disulfide-linked components. The apparent molecular weight of the two labeled species was not affected by a 100-fold variation in cross-linker concentration, and the labeling of both species increased in parallel. Possible relationships between the two labeled species are discussed.     

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.     

Functional heterogeneity of atrial natriuretic factor receptor in bovine adrenal zona glomerulosa is explained by an amiloride-sensitive high affinity molecular complex

The effects of amiloride on the molecular characteristics of the atrial natriuretic factor (ANF) receptor from bovine adrenal zona glomerulosa were studied by computer modeling of competitive binding data, by affinity labeling experiments, and by steric exclusion high performance liquid chromatography of solubilized receptor. The order of potency of a series of truncated ANF analogs in competing for 125I-ANF binding to bovine adrenal zona glomerulosa membranes was the same as that obtained for inhibition of aldosterone secretion. Deletion of amino acids at the COOH-terminal end drastically reduced the affinities of the peptides. Computer analysis of competition curves revealed that all ANF analogs tested show similar binding characteristics: shallow competition curves, discrimination of varying proportions of high and low affinity binding states, and sensitivity to amiloride which increases the proportion of the high affinity binding component. These results from binding studies are suggestive of potential heterogeneity of ANF binding sites. In contrast, results from affinity cross-linking experiments are consistent with the notion of a single receptor protein. Incubation of membranes with increasing concentrations of 125I-ANF-(99-126) up to 3 nM resulted in the labeling of a single band of Mr 130,000. The ability of ANF analogs to compete for the labeling of the Mr 130,000 band by 125I-ANF-(99-126) agreed well with their potency as inhibitors of 125I-ANF binding to intact membranes. Addition of amiloride caused a dose-dependent increase in the labeling of the Mr 130,000 band. A single Mr 130,000 band was also labeled in bovine aorta and LLC-PK1 cell membranes. In order to further investigate the molecular basis for the apparent heterogeneity of ANF binding we have prelabeled the membrane receptor with 125I-ANF-(99-126) prior to solubilization with octyl-beta-D-glucoside and chromatography on a Superose 6 steric exclusion column. The elution profile of the prelabeled receptor consistently showed two peaks of radioactivity with mean Stokes radii of 70 and 50 A. When amiloride was added to the incubation medium, the elution profile consisted almost exclusively of the 70-A peak. Quantitative analysis of the chromatographic profiles revealed that amiloride increases by 2-3 times the area of the 70-A peak. We conclude that the 70-A form represents a ternary complex of the receptor with an amiloride-sensitive effector protein.     

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.     

Rat atrial natriuretic polypeptide stimulation of adrenal zona glomerulosa cell growth

Rat atrial natriuretic polypeptide (rANP) was found to stimulate [3H] thymidine incorporation into the DNA of bovine adrenal glomerulosa cells in a primary culture in a dose-dependent manner. The minimum effective dose was a very low concentration (10(-12) M of ANP), suggesting that ANP had a physiological effect. These findings are the first indication that ANP possesses growth-stimulating activity with regard to adrenal zona glomerulosa cells.     

Atrial natriuretic factor R1 receptor from bovine adrenal zona glomerulosa: purification, characterization, and modulation by amiloride

The atrial natriuretic factor (ANF) R1 receptor from bovine adrenal zona glomerulosa was solubilized with Triton X-100 and purified 13,000-fold, to apparent homogeneity, by sequential affinity chromatography on ANF-agarose and steric exclusion high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the purified receptor preparation in the absence or presence of dithiothreitol revealed a single protein band of Mr 130,000. Affinity cross-linking of 125I-ANF to the purified receptor resulted in the labeling of the Mr 130,000 band. The purified receptor bound ANF with a specific activity of 6.8 nmol/mg of protein, corresponding to a stoichiometry of 0.9 mol of ANF bound/mol of Mr 130,000 polypeptide. Starting with 500 g of adrenal zona glomerulosa tissue, we obtained more than 500 pmol of purified receptor with an overall yield of 9%. The purified receptor showed a typical ANF-R1 pharmacological specificity similar to that of the membrane-bound receptor. The homogeneous Mr 130,000 receptor protein displayed high guanylate cyclase activity [1.4 mumol of cyclic GMP formed min-1 (mg of protein)-1] which was not stimulated by ANF. This finding supports the notion that the ANF binding and the guanylate cyclase activities are intrinsic components of the same polypeptide. Finally, the purified ANF-R1 receptor retained its sensitivity to modulation by amiloride, suggesting the presence of an allosteric binding site for amiloride on the receptor protein.     

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.     

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.     

Isolation of bovine adrenal cortex mitochondria from the zona glomerulosa

Isolation procedures for the bovine adrenal cortex mitochondria from the zona glomerulosa are described. Special care was taken to avoid contamination of the mitochondria from the zona fasciculata.     

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.     

Solubilization and molecular weight estimation of atrial natriuretic factor receptor from bovine adrenal cortex

Receptors for atrial natriuretic factor (ANF) have been solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate from bovine adrenal cortex and characterized. The detergent extract retained specific high-affinity binding sites for 125I-ANF. Scatchard analysis of the equilibrium binding data revealed a single class of binding site with a K-d of 1.8 nM and a maximum binding capacity of 2.5 pmol/mg of protein. The size of the 125I-ANF X receptor complexes was estimated to be 140,000 daltons by gel filtration on TSK gel G3000SW. Affinity labeling followed by electrophoresis under nonreducing conditions and autoradiography also revealed a single band of a similar size (Mr = 130,000); this band, however, migrated as a Mr = 70,000 species under reducing electrophoretic conditions. These results indicate that the ANF receptor, having a Mr of 130,000 - 140,000, is composed of disulfide-linked subunits and the ANF-binding site is located on the 70-kDa component.     

Steroidogenesis in the zona glomerulosa of the adrenal cortex II. Distribution of cytochrome P-450 in the zona glomerulosa of the bovine adrenal cortex

Microsomes were obtained from the zona glomerulosa of the bovine adrenal cortex. Contamination of microsomes with other cellular organelles was examined using various marker enzymes and the electron microscope. Distribution of cytochrome P-450 in the zona glomerulosa was studied using various fractions including microsomes, described above, and mitochondria. The amount of cytochrome P-450 in mitochondria and in microsomes was determined to be 0.73 and 0.32 nmol/mg protein, respectively. The CO difference spectrum was affected not only by the concentration of added deoxycholate but also by the incubation time after addition. Approximately 40–50% of cytochrome P-450 in the samples was converted to cytochrome P-420 within 20–30 sec of incubation with deoxycholate.The content of RNA, phospholipids, and cytochromeb ₅ in microsomes obtained from the zona glomerulosa is also evaluated in comparison to that in microsomes obtained from the zona fasciculoreticularis.     

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.     

Topographical characterization of the domain structure of the bovine adrenal atrial natriuretic factor R1 receptor

We have studied the structure and function of the membrane atrial natriuretic factor R1 (ANF-R1) receptor using limited proteolysis and exoglycosidase treatment. Limited digestion with trypsin of the receptor from bovine adrenal zona glomerulosa membranes resulted in the conversion of the native 130-kDa receptor into a single membrane-associated ANF-binding proteolytic fragment of 70 kDa. The 70-kDa fragment bound ANF with enhanced binding affinity but retained intact ANF-R1 pharmacological specificity and was still sensitive to modulation by amiloride. Trypsin treatment of the membranes produced a dual effect on ANF binding. Low concentrations of trypsin (less than or equal to 25 micrograms/mg of protein) increased ANF binding while higher concentrations dose dependently reduced the binding of the hormone. The increase of ANF-binding activity was associated with the formation of the 70-kDa fragment while the loss of ANF binding paralleled the degradation of the 70-kDa fragment. Low concentrations of trypsin drastically decreased the ANF-sensitive guanylate cyclase activity of the membrane fraction. This loss of catalytic activity strongly correlated with the formation of the 70-kDa tryptic fragment. We also evaluated the effect of ANF binding on the susceptibility of the receptor to proteolytic cleavage. The occupied receptor exhibited a greater sensitivity to trypsin digestion than the unoccupied protein, consistent with the hypothesis that hormone binding induces an important conformational change in the receptor structure. On the other hand, the 70-kDa fragment was much more resistant to proteolysis when occupied by ANF, suggesting that the ANF-binding domain forms a very compact structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further investigations on the atrial natriuretic factor (ANF)-induced inhibition of the growth and steroidogenic capacity of rat adrenal zona glomerulosa in vivo

A prolonged infusion with ANF (20 micrograms/kg/h for 7 days) induced atrophy of zona glomerulosa cells and lowering of basal plasma concentration of aldosterone in rats whose hypothalamo-hypophyseal-adrenal axis and renin-angiotensin system had been interrupted by the simultaneous administration of dexamethasone/captopril and maintenance doses of ACTH/angiotensin II. Chronic ANF treatment also caused comparable reductions in the aldosterone response of zona glomerulosa cells to the acute stimulation with angiotensin II, potassium and ACTH. These data are interpreted to indicate that ANF exerts an inhibitory effect on the growth and secretory activity of rat zona glomerulosa, and that the mechanism underlying this action of ANF does not involve blockade of renin release or ACTH secretion.     

Steroidogenesis in the zona glomerulosa of the adrenal cortex I. Isolation and some properties of mitochondria from the zona glomerulosa of the bovine adrenal cortex

Isolation procedures for mitochondria from the zona glomerulosa of the bovine adrenal cortex are described and the properties of the mitochondria thus prepared are compared with those isolated from the zona fasciculoreticularis. The cristal membranes of mitochondria in the zona glomerulosa in situ are tubular or tubulovesicular, whereas those of mitochondria in the zona fasciculoreticularis in situ are vesicular. When mitochondria are isolated from the former zone, they invariably showed the condensed configuration regardless of isolation media, whereas those isolated from the latter zone in an ST medium showed the orthodox configuration. When Ca²⁺ was added to mitochondria isolated either from the zona glomerulosa or the zona fasciculoreticularis in an STE medium in the condensed configuration, a transition from the condensed to the orthodox configuration took place; the cristal membranes of mitochondria from the zona glomerulosa became tubular or tubulovesicular and those of mitochondria from the zona fasciculoreticularis became vesicular. Contaminations of mitrochondria of the zona glomerulosa with other cellular organelles were examined using various marker enzymes. There was no difference in cytochrome content between mitochondria of the two zones specified above. The coupling efficiency of mitochondria of the zona glomerulosa was found to be remarkably effected by temperature during the isolation procedures. Effects of various substrates, isolation media, and bovine serum albumin on the coupling efficiency of mitochondria of the glomerulosa are also described.     

Intramitochondrial bodies in sheep adrenal zona glomerulosa cells

Summary Relatively large, mostly rounded, very electron dense intramitochondrial bodies in adrenal zona glomerulosa cells of sheep are described and their nature and connection to protein in the mitochondria discussed. The so called azocarmine granules seen in the light microscope may be identical with the intramitochondrial bodies in the zona glomerulosa cells.     

Ascorbate stimulates monooxygenase-dependent steroidogenesis in adrenal zona glomerulosa

It is well known that ascorbic acid (Asc) is highly concentrated in the adrenal gland, but its function in the gland is not thoroughly elucidated. We therefore examined the possibility that Asc participates in steroidogenic monooxygenase systems of the adrenal cortex with the aid of the regenerating system including outer mitochondrial membrane cytochrome b (OMb). When Asc availability was limited in rat mutants unable to synthesize Asc, the increase in plasma aldosterone concentration under Na-deficiency was suppressed without effect on plasma corticosterone concentration. Aldosterone formation in the isolated mitochondrial fraction of the zona glomerulosa (zG) of the adrenal cortex was stimulated by the addition of Asc and NADH, while corticosterone formation was not. Consistently zG showed a high level of Asc regeneration activity and was rich in OMb among adrenocortical zones. Taken together, the enhanced aldosterone formation that is catalyzed by one of the steroidogenic monooxygenases, P450aldo, may be supported by Asc with its regenerating system.     

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.