Dietary intake of sodium chloride in the rat influences [3H]nitrendipine binding to adrenal glomerulosa cell membranes but does not alter binding to vascular smooth muscle membranes.

Angiotensin II-stimulated secretion by adrenal glomerulosa cells and contraction by vascular smooth muscle (VSM) are dependent on calcium influx through membrane calcium channels. We have examined the hypothesis that the altered responsiveness of adrenal glomerulosa cells and VSM to angiotensin II during NaCl restriction may be associated with a change in membrane calcium channel number. To test this hypothesis, female rats were placed on a high or low NaCl diet. On the 14th day, membranes were prepared from the zona glomerulosa, aorta, mesenteric artery, and uterus. [3H]Nitrendipine binding was used to monitor calcium channel number. The [3H]nitrendipine binding capacity was observed to be higher in the zona glomerulosa during NaCl restriction than during high NaCl intake (83 +/- 18 vs. 49 +/- 9 fmol/mg protein, P less than 0.025, n = 6 paired experiments). The binding capacities of [3H]nitrendipine on the low and high NaCl diet were similar in the mesenteric artery (10 +/- 1 vs. 9 +/- 1 fmol/mg protein, n = 8), aorta (33 +/- 5 vs. 35 +/- 8 fmol/mg protein, n = 5), or uterus (87 +/- 15 vs. 85 +/- 16 fmol/mg protein, n = 4), respectively. The dissociation constants of [3H]nitrendipine binding did not differ on a low or high NaCl intake in the zona glomerulosa (0.84 +/- .12 vs. 0.79 +/- .10 nM), mesenteric artery (0.82 +/- .06 vs. 83 +/- .05 nM), aorta (0.90 +/- .11 vs. 0.92 +/- .12 nM), or uterus (0.55 +/- .12 vs. 0.56 +/- .10 nM), respectively. We conclude that the blunted response of VSM to angiotensin II during NaCl restriction is best explained by the previously reported lower number of angiotensin II receptors since calcium channel number does not change. In the adrenal glomerulosa cell, NaCl restriction is associated with a higher number of membrane calcium channels and angiotensin II receptors. The increase in calcium channel number may reflect the influence of an unknown factor(s) believed to be necessary for the full expression of the adrenal glomerulosa cell response to NaCl restriction.

Effect of adrenergic and muscarinic cholinergic agonists on atrial natriuretic peptide secretion by isolated rat atria. Potential role of the autonomic nervous system in modulating atrial natriuretic peptide secretion.

Stretching of the atrial wall is a known stimulant for atrial natriuretic peptide (ANP) secretion. Little is known about other factors that may influence ANP secretion. We examined the effects of the neurotransmitters of the autonomic nervous system on ANP secretion from isolated rat left atria. Superfusion with 10 muM norepinephrine produced a biphasic rise in ANP secretion with a peak response 2.5-fold above baseline secretion. To determine whether the response to norepinephrine primarily reflected alpha- or beta-adrenergic receptor stimulation, atria were superfused with 0.1 muM isoproterenol or 10 muM phenylephrine and 1 muM propranolol. ANP secretion in response to isoproterenol was biphasic, similar to the response to norepinephrine. Phenylephrine evoked a monophasic ANP secretory response, which was delayed in onset relative to that of isoproterenol or norepinephrine. Superfusion with 10 muM methacholine alone had no effect on ANP secretion, but rapidly attenuated norepinephrine-stimulated secretion by 67%. From these observations we conclude: (a) Both alpha- and beta-adrenergic agonists directly and distinctively stimulate ANP secretion; (b) Norepinephrine stimulates ANP secretion by both alpha- and beta-adrenergic mechanisms, however the secretory response pattern of norepinephrine reflects a predominence of beta-adrenergic activity; (c) Under basal conditions, methacholine does not influence ANP secretion; and (d) Methacholine inhibits norepinephrine-stimulated ANP secretion. Thus, in vivo, activation of the sympathetic nervous system may enhance ANP secretion, whereas a rise in parasympathetic tone may lower ANP secretion.

The developmental changes in plasma adrenal androgens during infancy and adrenarche are associated with changing activities of adrenal microsomal 17-hydroxylase and 17,20-desmolase.

The plasma concentrations of dehydroepiandrosterone, androstenedione, and dehydroepiandrosterone sulfate decrease during the first year of life, remain low during childhood, and then increase during adrenarche. To determine whether alterations in adrenal enzyme activity might explain the changing secretory pattern of the adrenal androgens, we measured human adrenal microsomal 3 beta-hydroxysteroid dehydrogenase-isomerase, 17,20-desmolase, 17-hydroxylase, and 21-hydroxylase activities. 12 adrenals from individuals aged 3 mo to 60 yr were studied. The patients were divided into three groups based upon the age of the patient when the adrenal glands were obtained: group 1, infants aged 3--8 mo (n = 3); group 2, preadrenarchal or early adrenarchal children aged 2--9 yr (n = 4); and group 3, adults aged 20--60 yr (n = 5). The mean activity of the 17,20-desmolase, 17-hydroxylase, and 21-hydroxylase fell by 50% and that of 3 beta-hydroxysteroid dehydrogenase-isomerase activity rose 80% from group 1 to 2. A fourfold increase in 17,20-desmolase (P less than 0.002) and 17-hydroxylase (P less than 0.001) activity and a doubling in 21-hydroxylase activity (P less than 0.005) occurred between groups 2 and 3. We conclude that the decline in plasma adrenal androgens after birth appears to be associated with a rise in 3 beta-hydroxysteroid dehydrogenase-isomerase and a fall in 17,20-desmolase and 17-hydroxylase activity. The subsequent increase in plasma adrenal androgen concentration during adrenarche is coincident with a rise in 17,20-desmolase and 17-hydroxylase activity.

Stimulation by calcitonin gene-related peptide of atrial natriuretic peptide secretion in vitro and its mechanism of action.

Calcitonin gene-related peptide (CGRP) is present in nerve fibers within atrial tissue, raising the possibility that CGRP release may influence atrial natriuretic peptide (ANP) secretion. We, therefore, examined the effect of CGRP on immunoreactive ANP (ANP-IR) secretion. Isolated rat left atria paced at 2 Hz were superfused with 0.1 microM CGRP. A biphasic 2-fold increase in ANP-IR secretion occurred in response to CGRP. We next examined the mechanism of CGRP-stimulated secretion. The biphasic ANP-IR secretory response to CGRP was similar to that induced by superfusion with the beta-adrenergic agonist isoproterenol and (Bu)2cAMP, but distinct from that of the non-cAMP dependent stimuli phenylephrine, ouabain, and Bay K 8644. Superfusion with 0.1 microM CGRP for 4 min with 100 microM isobutylmethylxanthine increased atrial cAMP content from 4.29 +/- 1.21 to 10.32 +/- 2.14 pmol/mg atrial weight (P less than 0.001). Atria were next superfused with methacholine, an inhibitor of adenylyl cyclase activation. The addition of 0.1 microM isoproterenol or 0.1 microM CGRP to the superfusate containing 10 microM methacholine failed to stimulate ANP-IR secretion and lowered cAMP accumulation by 70%. Superfusion with 10 microM atropine negated the inhibitory effects of methacholine. We conclude that 1) CGRP stimulates ANP-IR secretion in vitro; and 2) CGRP-stimulated secretion appears to be mediated by cAMP. Thus, ANP-IR secretion may be modulated by atrial nerve fibers containing CGRP in vivo.

Endothelin: a potent stimulus of atrial natriuretic peptide secretion by superfused rat atria and its dependency on calcium.

Endothelin, a hormone secreted by endothelial cells, has potent vasoconstrictive properties. Due to its potential paracrine nature, we examined the effect of endothelin-I on atrial natriuretic peptide (ANP) secretion in vitro. Isolated superfused rat left atria, paced at 2 Hz, were used for study. Endothelin (1-100 nM) increased ANP secretion in a dose-dependent manner from 1.6- to 6.7-fold above baseline. Spontaneously beating right atria increased ANP secretion by 2.3-fold in response to 10 nM endothelin without a change in beat frequency. However, the right atrial ANP secretory response was less than the 3.8-fold increase seen by left atria, and the time to peak response was slower. The calcium dependency of endothelin-stimulated ANP secretion was examined using paced left atria. The dependency of endothelin-stimulated secretion on calcium influx was examined by lowering the superfusate calcium from 1.8 to 0.2 mM. The ANP secretory response to 10 nM endothelin was reduced by 65% with 0.2 mM calcium. Influx of calcium through voltage-dependent calcium channels was examined by superfusion with 50 microM nitrendipine. Nitrendipine decreased endothelin-stimulated ANP secretion by 51% without affecting endothelin binding. The role of intracellular calcium release from the sarcoplasmic reticulum (SR) was examined by superfusion with 1 microM ryanodine, an inhibitor of SR calcium release. Ryanodine had no effect on endothelin-stimulated ANP secretion. We conclude: 1) Endothelin is a potent stimulus of ANP secretion in vitro. 2) The relative secretory response of right atria to endothelin expressed as a function of basal secretion is less and the time to peak secretion delayed relative to left atria. 3) Enhanced calcium influx, primarily through voltage-dependent calcium channels, plays a significant role in endothelin-stimulated secretion. 4) Release of intracellular calcium from the SR does not participate in the secretory response. 5) Part of the stimulatory signal appears to be independent of calcium influx or intracellular calcium release. Thus, endothelin may be an important secretagogue or modulator of ANP secretion in vivo; however, its physiological role in regulating ANP secretion in vivo remains to be determined.

Calcium: its role in alpha 1-adrenergic stimulation of atrial natriuretic peptide secretion.

alpha 1-Adrenergic agonists increase atrial natriuretic peptide (ANP) secretion. The mechanism of alpha 1-adrenergic-stimulated secretion is not known. In this study we examine the calcium dependency of alpha 1-agonist-stimulated ANP secretion. Isolated superfused rat left atria paced at 2 Hz were used for study. Superfusion with 10 microM phenylephrine increased ANP secretion by 2-fold. Lowering the superfusate calcium concentration from 1.8 to 0.2 mM totally negated the secretory response to phenylephrine. To determine whether this reflected a reduction in calcium influx, reduced calcium release from the sarcoplasmic reticulum (SR), or both, atria were superfused with 1 microM ryanodine, an inhibitor of SR calcium release. Ryanodine had no effect on phenylephrine-stimulated ANP secretion. Atria were superfused with 10 microM nitrendipine to determine whether calcium influx through voltage-dependent calcium channels was a mechanism of calcium entry for stimulation. Nitrendipine inhibited phenylephrine-stimulated ANP secretion by 49% without interfering with alpha 1-adrenergic antagonist receptor binding. This finding was supported by the observation that phenylephrine-stimulated secretion was 52% lower in nonbeating atria. alpha 1-Adrenergic agonists have been reported to enhance Na-H antiporter activity. To determine whether the resulting rise in intracellular sodium may alter Na-Ca exchange to raise intracellular calcium levels, atria were superfused with the Na-H antiporter inhibitor, 5-(N,N-hexamethylene)amiloride. Superfusion with 25 microM 5-(N,N-hexamethylene)amiloride did not inhibit phenylephrine-stimulated ANP secretion. Lastly, the calcium dependency of the maintenance of an established response to phenylephrine was examined. Atria were superfused with phenylephrine in buffer containing 1.8 mM calcium for 45 min, followed by superfusion with phenylephrine in 0.2 mM calcium for 30 min. There was no fall in phenylephrine-stimulated secretion by atria superfused in 0.2 mM calcium. In contrast, addition of the alpha 1-adrenergic antagonist phentolamine induced an immediate fall in phenylephrine-stimulated ANP secretion. We conclude that 1) calcium influx is necessary to initiate alpha 1-agonist-stimulated ANP secretion; 2) calcium release from the SR does not play a role in alpha 1-agonist-stimulated secretion; 3) calcium entry through L-type calcium channels is responsible for half of the calcium influx; 4) enhanced Na-H antiporter activity does not play a role in alpha 1-agonist-stimulated secretion; and 5) maintenance of alpha 1-agonist-stimulated secretion is not dependent on calcium influx.

Atrial natriuretic peptides: the role of phenylalanine on biological activity.

Previous studies have shown that atrial natriuretic peptides (ANPs) inhibit the secretion of aldosterone by isolated rat adrenal glomerulosa cells stimulated by angiotensin II, ACTH, and potassium. Structure-function studies have concentrated on the significance of the C- and N-terminal chains for the biological activity of the peptide. We investigated the role of phenylalanine at positions 8 and 26 by using [Ala8]human (h) ANP or [Ala26]hANP analogs to inhibit potassium-stimulated aldosterone secretion in granulosa cells. hANP-(1-28) inhibited potassium-stimulated aldosterone secretion with an IC50 of 0.48 nM. Synthetic [Ala26]hANP inhibited the aldosterone response to potassium with an inhibitory curve relative to hANP-(1-28) (rIC50) of 6.0 nM, which was significantly greater than that for hANP (P less than 0.001). Synthetic [Ala8]hANP was markedly less effective as an inhibitor, with an estimated rIC50 of 3.0 microM (P less than 0.0001). To determine whether the analogs act as competitive antagonists to hANP-(1-28), experiments were performed in which a fixed concentration (0.1 microM) of the analog was incubated in the presence of increasing concentrations of hANP-(1-28). When hANP-(1-28) was incubated with [Ala8]hANP, the rIC50 (0.2 nM) was significantly less than that for hANP-(1-28) alone (P less than 0.02). When hANP-(1-28) was incubated with [Ala26]hANP, the rIC50 was 0.1 nM. In summary, [Ala8]hANP and [Ala26]hANP were significantly less potent than hANP-(1-28) as inhibitors of aldosterone production from granulosa cells. Both analogs shifted the hANP-(1-28) dose-response curve to the left. Neither analog functioned as a competitive antagonist to hANP-(1-28). Our results indicate that the hydrophobic phenyl groups at these two positions are required for full biological potency of ANP as an inhibitor of aldosterone production.

The adrenal capsule alters the response of zona glomerulosa cells to atrial natriuretic peptide.

Atrial natriuretic peptide (ANP) is a potent inhibitor of potassium-stimulated aldosterone secretion. In the present study, we observed rat alpha ANP to inhibit aldosterone secretion stimulated by 10 mM potassium with an IC50 of 0.15 +/- 0.02 nM (mean +/- SE) in dispersed rat adrenal glomerulosa cells. However, when rat adrenal capsules, which contain the zona glomerulosa, were superfused in vitro, ANP had no effect on aldosterone secretion. Superfusion with 10 mM potassium increased aldosterone secretion 3- to 4-fold above baseline. Addition of 10 nM ANP to the superfusate did not lower potassium-stimulated aldosterone secretion. When this same ANP-containing superfusate was incubated with dispersed adrenal glomerulosa cells, potassium-stimulated aldosterone secretion was inhibited by 90%, proving sustained biological potency of the superfused ANP. Incubation of [125I]iodo-ANP with adrenal capsules for 60 min resulted in 83% degradation of [125I]iodo-ANP, whereas no detectable degradation was observed with dispersed adrenal glomerulosa cells. Removal of blood from the adrenal capsules or culturing the capsules for 48 h did not render them responsive to superfused ANP. In contrast, superfusion of 0.1 mM cycloheximide inhibited potassium-stimulated aldosterone secretion by 90%. These results suggest that the adrenal capsule contains an ANP-degrading enzyme(s). This enzyme may be produced by adrenal glomerulosa cells. The local existence of a degrading enzyme for ANP may allow the zone glomerulosa to regulate its response to ANP.

Rapid regression of fetal adrenal zone and absence of adrenal reticular zone in the marmoset.

Developmental changes in plasma dehydropiandrosterone (DHA) and in adrenal histology were studied in several marmoset species (Callithrix jacchus and Saguinus labiatus, nigricollis, and fuscicollis) to evaluate these primates as experimental models for the study of fetal adrenal zone regression. Newborn marmosets had a prominent fetal adrenal zone, plasma DHA levels above 1000 ng/dl, and plasma DHA sulfate (DHAS) levels of 140 micrograms/dl. The fetal zone regressed dramatically during the first week of life, paralleled by a marked decline in plasma DHA, the plasma DHA to cortisol ratio, and plasma DHAS. The adult marmoset, however, had no adrenal reticular zone and no evidence of adrenal DHA secretion; DHA levels in castrate adults were undetectable (less than 25 ng/dl). Thus, the marmoset represents the first example of a primate that has a regressing, DHA- and DHAS-secreting fetal adrenal zone but that does not subsequently develop a DHA-secreting adrenal reticular zone.

Calcium dependency of frequency-stimulated atrial natriuretic peptide secretion.

In this study we examined the mechanism whereby atrial natriuretic peptide secretion is increased when the frequency of contraction is raised from 2 to 5 Hz. We tested the hypothesis that calcium plays a significant role in the frequency-stimulated response. Using superfused rat left atria, we found that lowering the superfusate calcium concentration from 1.8 to 0.2 mmol/L abolished the frequency-stimulated atrial natriuretic peptide secretory response. Superfusion with ryanodine (1 mumol/L), an inhibitor of sarcoplasmic reticulum calcium release, resulted in a minimal inhibitory effect. Superfusion with 50 mumol/L nitrendipine or 10 mumol/L diltiazem inhibited the frequency-stimulated response by 46% to 48%. The lack of total inhibition suggested that an additional mechanism of calcium influx was involved, namely, inward calcium movement carried by Na(+)-Ca2+ exchange. As intracellular sodium has been reported to rise with an increase in beat frequency, a fall in the sodium gradient would favor inward calcium movement by Na(+)-Ca2+ exchange. Because we could not directly assess the role of Na(+)-Ca2+ exchange in this experimental paradigm, we examined the effect of lowering the transmembrane sodium gradient on atrial natriuretic peptide secretion by superfusion with the sodium channel activator veratridine or the sodium ionophore monensin. Superfusion with 1 mumol/L veratridine increased atrial natriuretic peptide secretion by 2.3-fold, and 1, 5, and 10 mumol/L monensin increased secretion by 1.1-, 2.1-, and 15.7-fold, respectively. In addition, we examined the possibility that the reported rise in intracellular sodium associated with increased beat frequency was due to enhanced Na(+)-H+ antiporter activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of serum prolactin on plasma adrenal androgens and the production and metabolic clearance rate of dehydroepiandrosterone sulfate in normal and hyperprolactinemic subjects.

Hyperprolactinemic patients may have increases in plasma dehydroepiandrosterone (DHA) and dehydroepiandrosterone sulfate (DHAS). We examined the effect of lowering serum PRL with bromocriptine or pituitary surgery on the serum concentrations of adrenal androgens and on the production rate (PR) and MCR of DHAS in eight hyperprolactinemic women (HP). We also examined the effect of bromocriptine therapy on adrenal androgens in five normal men. Serum DHAS was elevated in HP compared to normal women (mean +/- SEM, 254 +/- 28 vs. 182 +/- 13 microgram/dl; P less than 0.04). Serum DHA and androstenedione (delta 4) in HP were not significantly different from normal. Serum PRL fell from 160 +/- 16 to 37 +/- 9 ng/ml during or after treatment. Mean 24-h serum DHAS fell from 198 +/- 30 to 106 +/- 17 micrograms/dl (P less than 0.001) with treatment, without a change in the mean 24-h serum cortisol concentration (6.2 +/- 0.4 vs. 6.6 +/- 0.4 micrograms/dl). Thus, the DHAS to cortisol (DHAS/F) ratio fell significantly (32 +/- 5 to 17 +/- 4; P less than 0.001). This was also true of the DHAS/F ratio during ACTH stimulation (8 +/- 1 to 6 +/- 1; P less than 0.02). Similar changes were found in basal and ACTH-stimulated DHA/F ratios, whereas the basal and ACTH-stimulated delta 4/F ratios did not change significantly with treatment. Treatment lowered the PR of DHAS from 27 +/- 5 to 17 +/- 3 mg/24 h (P less than 0.03) and increased the DHAS MCR from 16 +/- 2 to 21 +/- 3 liters/24 h (P less than 0.01). Bromocriptine treatment of normal men lowered serum PRL from 15 +/- 2 to less than 2.5 ng/ml. There were no significant changes in the basal and ACTH-stimulated serum DHAS/F, DHA/F, or delta 4/F ratios or DHAS PR and MCR during bromocriptine therapy. The failure of bromocriptine to significantly alter these steroids in normal men suggests that bromocriptine was not directly responsible for the changes in HP treated with this drug. A mechanism for the increased PR of DHAS in HP was sought by examining the serum concentrations of the steroid biosynthetic intermediates relevant to DHAS production. Lowering serum PRL was associated with a decrease in basal and ACTH-stimulated 17-hydroxypregnenolone/17-hydroxyprogesterone and DHA/delta 4 ratios, suggesting an increase in 3 beta-hydroxysteroid dehydrogenase/delta 4,5-isomerase activity. However, increased gonadal secretion of the delta 4-steroids may have occurred with the fall in serum PRL.(ABSTRACT TRUNCATED AT 400 WORDS)

Bradykinin blocks angiotensin II-induced hypertrophy in the presence of endothelial cells.

Angiotensin-converting enzyme inhibitors block left ventricular hypertrophy in vivo. A component of this effect has been attributed to tissue accumulation of bradykinin. Little is known regarding the effect of bradykinin on cardiomyocytes. The objectives of the present study were to define the effects of bradykinin on isolated ventricular cardiomyocytes (from adult and neonatal rat hearts) and to determine the extent to which bradykinin blocks hypertrophy in vitro. Bradykinin was found to be a hypertrophic agonist, as defined by increased protein synthesis and atrial natriuretic peptide secretion and expression. Bradykinin (10 micromol/L) increased [3H]phenylalanine incorporation by 23+/-3% in adult and by 36+/-10% in neonatal cardiomyocytes. Constitutive atrial natriuretic peptide secretion by neonatal myocytes was increased 357+/-103%. All effects of bradykinin were abolished by the B2-kinin receptor antagonist Hoe 140. These increases were similar in magnitude to those observed with phenylephrine (20 micromol/L) and angiotensin II (1 micromol/L). However, in cardiomyocytes cocultured with endothelial cells, bradykinin did not increase protein synthesis. Angiotensin II increased [3H]phenylalanine incorporation by 24+/-3% in adult cardiomyocytes in monoculture and by 22+/-2% in adult rat cardiomyocytes cocultured with endothelial cells. Bradykinin abolished this angiotensin II-induced hypertrophy in myocytes cultured with endothelial cells but not in myocytes studied in the absence of endothelial cells. In conclusion, bradykinin has a direct hypertrophic effect on ventricular myocytes. The presence of endothelial cells is required for the antihypertrophic effects of bradykinin. The results suggest that the increase in local concentration of bradykinin associated with angiotensin-converting enzyme inhibition is an important mechanism by which hypertrophy can be blocked. Manifestation of this mechanism appears to require bradykinin-stimulated release of paracrine factor(s) from endothelial cells, which are also able to block the hypertrophic effects of Ang II.

Isosexual precocious pseudopuberty secondary to a feminizing adrenal tumor.

We report a 2 10/12-yr-old girl with precocious pseudopuberty due to a feminizing adrenal carcinoma without Cushing's syndrome. The patient had marked elevation of plasma concentrations of the delta 5 adrenal steroids dehydroepiandosterone and dehydroepiandrosterone sulfate and increased levels of androstenedione, estrone, estradiol, and testosterone. Adrenal microsomal 3 beta-hydroxysteroid dehydrogenase-isomerase 17-hydroxylase, 17,20-desmolase, and 21-hydroxylase activities in the tumor and adjacent normal adrenal gland were measured. The tumor had approximately normal levels of 17-hydroxylase and 17,20-desmolase activity, with low levels of 21-hydroxylase and 3 beta-hydroxysteroid dehydrogenase-isomerase activities. This combination of enzyme activity may explain the absence of Cushing's syndrome and the high levels of delta 5 adrenal steroids. This patient demonstrates that adrenal neoplasms arising in girls may mimic isosexual true precocious puberty and should be included in the differential diagnosis of precocious puberty.

Dissociation of cortisol and adrenal androgen secretion in the hypophysectomized, adrenocorticotropin-replaced chimpanzee.

We tested the hypothesis that adrenal androgen production is supported by a pituitary factor distinct from ACTH. Six adult male chimpanzees who had completed adrenal maturation (adrenarche) were castrated and either hypophysectomized or sham hypophysectomized. Hypophysectomized animals received synthetic ACTH-(1-24) and T4 to prevent adrenal insufficiency and hypothyroidism. Adrenal function was evaluated with a 3-h ACTH infusion before and 7, 21, 40, 120, and 180 days after hypophysectomy. Plasma cortisol (F), dehydroepiandrosterone (DHA), DHA sulfate (DHAS), and androstenedione (delta 4A) were measured at six time points during the ACTH infusions. The mean ratios of DHA to F, DHAS to F and delta 4A to F during ACTH infusion were calculated as indices of the relative activity of the androgen pathway compared to that of the cortisol pathway. The DHA to F ratio during ACTH infusion was 31% of the pretreatment level 40 days after hypophysectomy (P less than 0.01 compared to the sham-hypophysectomized controls). The DHAS to F ratio during ACTH infusion, which paralleled the DHA to F ratio, also fell significantly (P less than 0.025). Hypophysectomy did not alter the delta 4A to F ratio. None of the ratios was altered by sham hypophysectomy. MCRs for F and DHA, which were measured before and 180 days after hypophysectomy or sham hypophysectomy, did not change significantly. Additionally, plasma corticosteroid-binding globulin levels remained unchanged throughout the study for both groups of chimpanzees. Thus, the changes in the DHA to F ratio cannot be explained by alterations in the MCRs of DHA or F or in the plasma transport protein for F. These data suggest that ACTH maintained normal F and delta 4A secretion after hypophysectomy but failed to maintain normal DHA and DHAS secretion. This is consistent with the hypothesis that normal delta 5-adrenal androgen secretion is dependent upon a non-ACTH pituitary factor or with the hypothesis of different ACTH requirements for the maintenance of F and delta 5-adrenal androgen secretion.

Unique calcium dependencies of the activating mechanism of the early and late aldosterone biosynthetic pathways in the rat.

This study compared the extracellular calcium dependency and the enzymatic locus of that dependency for N6 O2'-dibutyryl cyclic AMP (dbcAMP)-, angiotensin II- and potassium-stimulated aldosterone secretion in dispersed rat glomerulosa cells. The need for extracellular calcium, calcium influx, and specifically for calcium influx through the calcium channel was examined. dbcAMP, angiotensin II and potassium, in the presence of calcium (3.5 mmol/l), significantly (P less than 0.01) increased aldosterone output by at least 1.5-fold. Yet in the absence of extracellular calcium or in the presence of lanthanum (an inhibitor of calcium influx by most mechanisms) all three stimuli failed to increase aldosterone secretion. Nifedipine, a dihydropyridine calcium channel antagonist, significantly (P less than 0.01) reduced angiotensin II- and potassium-stimulated aldosterone secretion, but had no effect on dbcAMP-stimulated aldosterone secretion (100 +/- 14 vs 105 +/- 19 pmol/10(6) cells). Likewise nitrendipine failed to inhibit ACTH-stimulated aldosterone secretion. Angiotension II and potassium activation of both the early aldosterone biosynthetic pathway (as reflected by pregnenolone production in the presence of cyanoketone) and also its late pathway (as reflected by the conversion of exogenous corticosterone to aldosterone in the presence of cyanoketone) were significantly (P less than 0.01) inhibited by lanthanum, nifedipine and by reducing the extracellular calcium concentration. However, with dbcAMP stimulation, none of these manipulations modified pregnenolone production. Late pathway activation by dbcAMP was inhibited by lanthanum and a reduction in extracellular calcium, but not by nifedipine. These observations suggest that: the extracellular calcium dependency of dbcAMP-, angiotensin II- and potassium-stimulated aldosterone secretion reflects a need for calcium influx; with dbcAMP stimulation, activation of the late pathway is dependent on calcium influx by a calcium channel-independent mechanism, whereas activation of the early pathway is not dependent on extracellular calcium or calcium influx and activation of both the early and late pathway by angiotensin II and potassium is dependent on calcium influx by a calcium channel-dependent mechanism. Therefore, we conclude that the mechanism of activation of the early aldosterone biosynthetic pathway by dbcAMP is different from angiotensin II or potassium and early pathway activation is distinct from that of late pathway activation with dbcAMP stimulation.

Insights into the mechanism by which inhibition of Na,K-ATPase stimulates aldosterone production.

Inhibition of Na,K-adenosine triphosphatase (Na,K-ATPase) activity by ouabain has been shown to increase the release of aldosterone from rat glomerulosa cells, but the mechanism by which this elevation of aldosterone production occurs has not been established. Small changes in membrane potential can significantly affect aldosterone release. Consequently, inhibition of Na,K-ATPase in glomerulosa cells may stimulate aldosterone production by membrane depolarization. If so, ouabain-stimulated production should be dependent on calcium influx through voltage-gated calcium channels. It has previously been shown that ouabain induces a moderately rapid increase in cytosolic calcium in rat glomerulosa cells. Therefore, in this study, we test whether ouabain stimulates aldosterone production with a time course consistent with early membrane depolarization as suggested by the previously reported early increase in cytosolic calcium. To study the time course of aldosterone production, we developed a perfusion technique that allows an examination of the initial effects of ouabain on aldosterone production. The results show that ouabain rapidly stimulates aldosterone production. Continuous perfusion with 0.25 or 1 mmol/L ouabain induced a brisk, robust increase in aldosterone production, followed by a decrease to near baseline over 60 minutes. Ouabain-stimulated aldosterone production was dependent on the presence of extracellular calcium and calcium influx through voltage-gated calcium channels. Our results support the hypothesis that the inhibition of Na,K-ATPase in rat adrenal glomerulosa cells immediately depolarizes the membrane potential and opens voltage-gated calcium channels.

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.

Potential limitations of recrystallization for the definitive identification of radioactive steroids.

The usefulness of recrystallization in establishing the radiochemical purity of steroids is widely recognized, but the potential limitations of the technique have received little attention. The current study reports the failure of standard recrystallization procedures using methanol/water as the solvent pair to separate contaminating 14C-17-hydroxyprogesterone (17-hydroxy-4-pregnene-3, 20-dione) from 3H- and 14C-labeled 11-deoxycortisol (17,21-dihydroxy-4-pregnene-3,20-dione) despite ten serial crystallizations. The standard criteria of radiochemical purity were met despite gross impurity of the crystals as evidenced by thin layer chromatography. Thus, recrystallization may, under certain conditions, yield misleading results when employed as the only method for identifying radioactive steroids. These observations illustrate the importance of an optimal choice of solvent and crystallization conditions, and emphasize the need for confirmation by derivative formation and chromatography.

Calcium, its role in isoproterenol-stimulated atrial natriuretic peptide secretion by superfused rat atria.

The beta-adrenergic agonist isoproterenol stimulates immunoreactive atrial natriuretic peptide (IR-ANP) secretion by superfused rat atria in vitro. beta-Adrenergic agonists alter the cellular handling of calcium, which culminates in a rise in the systolic calcium concentration. This is achieved by increasing calcium influx through voltage-dependent calcium channels and by increasing the storage pool of calcium in the sarcoplasmic reticulum (SR). We therefore asked the question whether isoproterenol-stimulated IR-ANP secretion was dependent on the protein kinase A-induced rise in systolic calcium or was due to a direct effect of protein kinase A activation. Isolated rat left atria paced at 3 Hz were superfused in vitro. IR-ANP secretion was determined by radioimmunoassay of timed collections of the superfusate. Superfusion with 0.1 microM isoproterenol or 0.5 mM dibutyryl cyclic AMP increased IR-ANP secretion twofold. Stimulated IR-ANP secretion was lowered to near baseline by lowering the buffer calcium concentration from 1.8 to 0.2 mM or by adding to the superfusate 10 microM nitrendipine (a calcium-channel blocker) or 1 microM ryanodine (an inhibitor of SR calcium release). Superfusion of nonbeating, electrically quiescent left atria with 0.1 microM isoproterenol failed to stimulate IR-ANP secretion. We conclude: 1) Isoproterenol-stimulated IR-ANP secretion is dependent on calcium influx through voltage-dependent calcium channels and on the release of calcium from the SR. 2) Enhanced calcium influx alone is not adequate to maintain isoproterenol-stimulated IR-ANP secretion. 3) The SR appears to be the primary source of calcium for isoproterenol-stimulated IR-ANP secretion.(ABSTRACT TRUNCATED AT 250 WORDS)