Effect of progesterone on aldosterone secretion in rats.

In both human and animal studies high progesterone states are associated with elevated aldosterone production but variable changes in PRA. These experiments were designed to test the hypothesis that progesterone has an effect similar to a low sodium diet on the glomerulosa cell: increasing aldosterone synthase messenger RNA activity and aldosterone production. Ovariectomized (OVX) rats were injected with progesterone (1 mg/100 g) or vehicle (SHAM) for 5 days. In a separate study, intact rats were placed on a low (0.02%) or high (1.6%) sodium diet for 5 days. On the day of death, rats were decapitated and blood collected for serum hormone determinations. Isolated adrenal glomerulosa cells were incubated +/- 10 nM angiotensin II (A II), after which aldosterone and corticosterone were measured. Early (conversion of cholesterol to pregnenolone) and late (conversion of corticosterone to aldosterone) aldosterone pathway activity was assessed in parallel incubates by adding cyanoketone and excess corticosterone with subsequent measurement of pregnenolone and aldosterone. In vivo, progesterone administration, like dietary sodium restriction, caused a significant increase in PRA (p < or = 0.043) and plasma aldosterone (p < or = 0.009), with no change in plasma corticosterone. Additionally, both treatments caused a significant increase in baseline (P < or = 0.01) and A II-stimulated (p < or = 0.027) aldosterone secretion in vitro. This increased responsiveness was secondary to activation of late pathway activity (p < or = 0.022) as determined by both an increased conversion of corticosterone to aldosterone and by an increase in messenger RNA levels of the late pathway enzyme aldosterone synthase. Thus, chronic progesterone administration apparently does not directly influence aldosterone secretion, but rather acts indirectly to increase aldosterone by mechanisms similar to sodium restriction.

Sodium restriction increases aldosterone biosynthesis by increasing late pathway, but not early pathway, messenger ribonucleic acid levels and enzyme activity in normotensive rats.

To determine whether changes in dietary sodium intake modify the early and/or late pathways of aldosterone biosynthesis, we studied in Sprague-Dawley rats the effect of sodium restriction on early (conversion of cholesterol to pregnenolone) and late (conversion of corticosterone to aldosterone) pathway activity and on the mRNA levels for the enzymes regulating these steps. Sodium restriction increased basal and angiotensin-II-stimulated aldosterone output from isolated zona glomerulosa cells by 5- to 9-fold. This increase in aldosterone output did not appear to be due to changes in the conversion of cholesterol to pregnenolone or in the mRNA levels of the early pathway enzyme, cholesterol side-chain cleavage cytochrome P-450. In contrast, sodium restriction increased the conversion of corticosterone to aldosterone 10-fold and increased by over 10-fold the mRNA levels of the late pathway enzyme aldosterone synthase. Sodium restriction had no effect on zona glomerulosa levels of 11 beta-hydroxylase mRNA. In two other normotensive rats, Dahl salt-resistant and Wistar Kyoto, sodium restriction again specifically increased aldosterone synthase mRNA without altering 11 beta-hydroxylase or cholesterol side-chain cleavage cytochrome P-450 mRNA levels. Thus, it appears that sodium restriction specifically increases late pathway aldosterone synthase mRNA levels, resulting in an increase in enzyme levels, followed by an increase in late pathway activity and an increase in aldosterone output.

Abnormal norepinephrine and aldosterone responses to upright posture in nonmodulating hypertension.

The subgroup of patients with nonmodulating hypertension demonstrates a number of abnormalities of the renin-angiotensin-aldosterone axis. We previously identified abnormalities in plasma and urinary dopamine in nonmodulators and posited that this may be in part due to a generalized defect in sympathetic nervous system activity. In the present study we assessed the state of activation of the renin-angiotensin system and the sympathetic nervous system in normal subjects and patients with modulating, nonmodulating, and low renin essential hypertension during sodium depletion and change from supine to upright posture. Levels of plasma norepinephrine were higher in non-modulators during the posture study (P < 0.05). PRA rose with upright posture in all groups, but low renin subjects had a blunted response. Nonmodulators and low renin subjects had lower aldosterone levels both supine (P< 0.05) and upright (P< 0.01). However, the aldosterone/PRA increment ratio was increased in low renin subjects (P< 0.01), whereas it was decreased in nonmodulators. Twenty-four-hour urine collections for catecholamine determinations were obtained in a subgroup of the subjects, with nonmodulators showing higher levels of norepinephrine excretion which approached significance (P = 0.08). In vitro experiments using rat and human adrenal glomerulosa cells showed that norepinephrine does not affect aldosterone secretion per se. These observations extend the series of abnormalities observed in nonmodulating hypertension. However, it is likely that the alterations in norepinephrine levels during sodium depetion and upright posture are a secondary event and not linked to the altered aldosterone production in these patients.

Dose effect of adrenocorticotropin on aldosterone and cortisol biosynthesis in cultured bovine adrenal glomerulosa cells: in vitro correlate of hyperreninemic hypoaldosteronism.

In some critically ill patients, aldosterone secretion is diminished despite hyperreninemia. These same patients demonstrate appropriately elevated plasma ACTH and cortisol levels. In addition, infusion of ACTH or angiotensin-II (AII) fails to elicit the normal aldosterone response, implying that the defect is at the level of the zona glomerulosa (ZG) cell. To test the hypothesis that elevated ACTH levels induce this defect, Percoll-purified bovine ZG cells were plated in serum-free defined medium and cultured for 5 days. On days 1-4, cells were exposed to various concentrations of ACTH for 1 h. On the fifth day of culture, half of the wells pretreated with ACTH were treated for 1 h with AII (10(-7) M); the other half of the wells received another dose of ACTH for 1 h. Additionally, cells were exposed to daily 1-h pulses of AII (10(-7) M) alone or in combination with ACTH (10(-8) M) for 5 days. Acutely dispersed bovine ZG cells showed dose-dependent increases in aldosterone when incubated with ACTH, potassium, or AII, with minimal cortisol production. Acutely dispersed bovine fasciculata cells produced no aldosterone, but demonstrated a dose-dependent cortisol response to ACTH and AII, but not potassium. On day 1 of culture, the ZG cells demonstrated a significant (P less than 0.001 in all cases) dose-related increase in aldosterone secretion in response to ACTH. However, continued daily pulsation with ACTH resulted in a dose-dependent decrease in aldosterone secretion, with a concomitant dose- and time-related rise in cortisol production. Indeed, ACTH-induced cortisol production in ZG became similar to ACTH-induced cortisol production in zona fasciculata cells. The addition of AII to the daily ACTH pulse did not significantly alter the aldosterone or cortisol response patterns to ACTH alone. In contrast, ZG cells treated with AII alone for 5 days showed a minimal change in cortisol production and no reduction in aldosterone production until day 4. Northern blot analysis of total RNA isolated from ZG cells pulsed with ACTH for 5 days demonstrated a parallel dose-dependent increase in 17 alpha-hydroxylase mRNA, which did not occur in cells pulsed with AII alone. These in vitro results suggest that elevated ACTH levels over time induce 17 alpha-hydroxylase activity in ZG cells, thereby shifting steroid biosynthesis from an aldosterone-producing to a cortisol-producing pathway. It is likely that the chronically elevated ACTH levels in critically ill patients induce a similar change in ZG cell biosynthesis, resulting in their hyperreninemic hypoaldosterone state.

Effect of sodium intake on phosphoinositides and inositol trisphosphate response to angiotensin II, K+ and ACTH in rat glomerulosa cells.

To assess the impact of sodium intake on the adrenal phosphoinositide system, rats were maintained on a low or normal salt diet for 5 days, and glomerulosa cell preparations (2 x 10(5) cells) were stimulated by angiotensin II (AII; 10 nmol/l), potassium (K+; 8.7 mmol/l) or ACTH (0.1 nmol/l) for 0, 2, 4, 6, 12, 15 and 60 s. Levels of phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns 4-P), phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) and inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) + inositol 1,3,4-trisphosphate (Ins 1,3,4-P3) were assayed by a microspectrophotometric procedure. Non-stimulated levels of PtdIns, PtdIns 4-P, PtdIns 4,5-P2 and Ins 1,4,5-P3 (+ Ins 1,3,4-P3) (means +/- S.E.M.; n = 36) in cells from rats on the low Na+ intake were 580 +/- 6.5, 187 +/- 2.6, 82 +/- 3 and 95 +/- 1.2 pmol per incubate respectively, indistinguishable from those observed in rats on a normal Na+ intake, except for the significantly (P less than 0.025) greater PtdIns 4,5-P2 level. In response to AII stimulation, all four compounds showed an earlier and greater peak response when cells were obtained from animals on a low rather than a high sodium intake. All values has returned to control levels by 12-15 s, regardless of the level of sodium intake. In contrast, with K+ stimulation there were no differences in the peak response of cells from rats on the two dietary intakes, but there was a shift of the peak to a longer time-interval (6 versus 8 s) in animals maintained on a low sodium intake.(ABSTRACT TRUNCATED AT 250 WORDS)

Mass determination of polyphosphoinositides and inositol triphosphate in rat adrenal glomerulosa cells with a microspectrophotometric method.

A method is described for the assay of subnanogram amounts of phosphorus in phospholipids and organic phosphates. The formation of a complex with a high molar absorption coefficient at 600 nm when malachite green is added to phosphomolybdate at low pH and the adaptation of a microspectrophotometer to quantify the color in 10 microliters solution have made it possible for a dose-response curve from 0.1-1.2 ng phosphorus to be developed. The method has been applied to the assay of phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns 4-P), phosphatidylinositol-4,5-diphosphate (PtdIns 4,5-P2), and inositol-1,4,5-triphosphate (Ins 1,4,5-P3) in rat adrenal glomerulosa cells after stimulation with angiotensin II (AII), K+, and ACTH for 0, 2, 4, 6, 8, 10, 12, 15, and 60 sec. A control (nonstimulated) sample was incubated concomitantly for every time period. Nonstimulated cell levels (mean +/- SEM; n = 216) were: PtdIns, 577 +/- 6.4; PtdIns 4-P, 183 +/- 3.1; PtdIns 4,5-P2, 59 +/- 1.8; and Ins 1,4,5-P3, 94 +/- 1.3 pmol/incubate. Maximum increase in levels of PtdIns, PtdIns 4-P, PtdIns 4,5-P2, and Ins 1,4,5-P3 above control values was obtained after 8 sec with AII (10(-8) M) and after 6 sec with K+ (8.7 mM) stimulation. The values (picomoles per 2 X 10(5) cell incubate; n = 4) were: PtdIns, 808 +/- 28; PtdIns 4-P, 263 +/- 20; PtdIns 4,5-P2, 112 +/- 10; and Ins 1,4,5-P3, 136 +/- 4 for AII stimulation, and PtdIns, 925 +/- 76, PtdIns 4-P, 308 +/- 11; PtdIns 4,5-P2 146 +/- 28; and Ins 1,4,5-P3, 149 +/- 5 for K+ stimulation. No increase above control levels could be found at any incubation time after ACTH stimulation. Thus, both AII and K+ stimulate a short-lived increase in the mass of several elements of the phosphatidylinositol pathway. The discrepancy between these mass determinations and isotope study suggests that only some, but not all, pools are labeled by currently available techniques.

Comparative effect of angiotensin II, potassium, adrenocorticotropin, and cyclic adenosine 3',5'-monophosphate on cytosolic calcium in rat adrenal cells.

The present study compares changes in cytosolic calcium and steroidogenesis when rat adrenal cells are stimulated with potassium (K+), angiotensin II (AII), ACTH, and (Bu)2cAMP (cAMP). The calcium-sensitive fluorescent dye, quin 2, was used to determine cytosolic calcium concentrations. K+ and AII both induced parallel increases in cytosolic calcium and aldosterone output. Removal of external calcium from the incubation media or addition of nifedipine inhibited the rise in cytosolic calcium in response to these two secretagogues. Inhibition of release of intracellularly-bound calcium by incubating the cells with 8-(N-N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride or dantrolene sodium reduced the rise in cytosolic calcium in response to these two secretagogues by 40-50%. In contrast, neither ACTH nor cAMP altered cytosolic calcium levels in the glomerulosa cells, even though quin 2-loaded cells showed a normal steroidogenic response to these agents. Thus, there was a dissociation between the cytosolic calcium response and steroidogenesis during cAMP stimulation of glomerulosa cells. Fasciculata cells incubated in the presence of increasing concentrations of cAMP, ACTH, K+, or AII failed to demonstrate an increase in cytosolic calcium, although the cells had a normal steroidogenic response to ACTH and cAMP. These results suggest that the responses of fasciculata and glomerulosa cells to secretagogues have different dependencies on calcium. The fasciculata cell has little calcium dependency while the glomerulosa cell has a variable dependency. In the glomerulosa cell, both AII and K+ induced similar responses in steroid output and cytosolic calcium, suggesting an important role for cytosolic calcium as a mediator of the steroidogenic effect of these secretagogues. Furthermore, part of the increase in cytosolic calcium induced by these agents is due to release of intracellularly bound calcium and part from increased calcium flux across the cell membrane. The absence of such dependency with cAMP suggests that an increase in intracellular calcium levels is not required for increased steroidogenesis in glomerulosa cells.

Is the adrenal angiotensin receptor angiotensin II--or angiotensin III like?

In order to determine whether the adrenal receptor is primarily directed at angiotensin II (AII) or angiotensin III (AIII) the following in vitro experiments were performed examining aldosterone responsiveness in isolated glomerulosa cells. 1) Cells exposed to increasing doses (2.4 X 10(-10)M - 2.4 X 10(-6)M) of AII or AIII were found to be significantly more responsive to AII (AII's ED50 was 6.3 X 10(-10) M vs AIII's 4.6 X 10(-9)M P less than 0.001). 2) Octapeptide analogues (Sar1 Ala8 and Asn1 Ala8), while demonstrating different inhibitory potencies relative to each other, were equally effective in blocking AII vs AIII stimulation. 3) The heptapeptide analogues (des1 Ala8 and des1 Ile8) however, inhibited AIII stimulation preferentially (P less than 0.01). 4) The 8 alanine octapeptide analogues were better inhibitors of both AII and AIII stimulation than the 8 alanine heptapeptide analogue. 5) HPLC analysis indicated that AII and AIII were being degraded at the same rate during the incubation procedure. These results, taken together, strongly suggest that the angiotensin adrenal receptor is an AII receptor.

Metoclopramide inhibits aldosterone biosynthesis in vitro.

Metoclopramide, a dopaminergic antagonist, has consistently elevated plasma aldosterone levels in vivo. To determine whether this was a direct action of metoclopramide on adrenal steroidogenesis, we examined the response of collagenase-dispersed rat adrenal glomerulosa cells to metoclopramide in vitro. The effect of increasing concentrations of metoclopramide (3 X 10(-10) to 3 X 10(-4) M) on basal as well as angiotensin II (2.4 X 10(-10) to 2.4 X 10(-8) M)-, ACTH (3.5 X 10(-11) M)- and potassium (5.9 meq/liter)-stimulated aldosterone production was evaluated. Metoclopramide caused a dose-related decrease in basal and stimulated aldosterone production (P less than 0.01). In addition, metoclopramide also blocked basal and stimulated corticosterone production (P less than 0.01). This was not due to an irreversible toxic effect, since glomerulosa cells preincubated with 3 X 10(-4) M metoclopramide excluded trypan blue dye and responded to ACTH stimulation. Sodium metabisulfite, an antioxidant present in the metoclopramide preparation, did not contribute to the metoclopramide effect. These results indicate that metoclopramide is an aldosterone antagonist in vitro, contrary to reported data obtained in vivo. Thus, metoclopramide may be a partial dopaminergic agonist: in vitro where dopamine levels are negligible, it is an agonist, whereas in vivo where dopamine concentrations are greater, it is an antagonist.