Bovine Adrenal Glomerulosa Cells Research Articles

Mechanism of fatty acid inhibition of aldosterone synthesis by bovine adrenal glomerulosa cells.

Previous work from this laboratory has suggested that the adrenal glomerulosa is under tonic inhibition by fatty acids. The purpose of the present work was to define the mechanism by which fatty acids inhibit aldosterone synthesis. Experiments with isolated bovine adrenocortical cells showed the following. 1) Fatty acids inhibited angiotensin-II-stimulated and (Bu)2cAMP-stimulated aldosterone synthesis with similar potencies. 2) Inhibition of aldosterone synthesis was highly dependent on fatty acid chain length and degree of unsaturation as well as on configuration of double bonds. Oleic acid was the most potent inhibitor among fatty acids prominent in plasma. 3) Cortisol synthesis was less sensitive to oleic acid inhibition than was aldosterone synthesis. 4) Pregnenolone synthesis by angiotensin-II-stimulated adrenal glomerulosa cells was relatively insensitive to oleic acid. 5) For both glomerulosa and fasciculata cells, cortisol synthesis from 21-deoxycortisol, which requires the participation of P450(21), was relatively insensitive to fatty acids. Cortisol synthesis from corticosterone by fasciculata cells, which requires the participation of P450(17) alpha, was also insensitive to oleic acid. These are microsomal enzymes. 6) In glomerulosa cells, aldosterone synthesis from added corticosterone, which requires the 18-oxidase function of P450(11) beta, a mitochondrial enzyme, was potently inhibited by fatty acids; cortisol synthesis from 11-deoxycortisol by glomerulosa cells, which requires P450(11) beta, was less sensitive to inhibition, and cortisol synthesis from 11-deoxycortisol by fasciculata cells was even less sensitive. 7) Aldosterone synthesis from exogenous 18-hydroxycorticosterone was potently inhibited by oleic acid. Thus, fatty acids are potent inhibitors of the 18-oxidase function of the mitochondrial enzyme P450(11) beta, whereas nonmitochondrial steroidogenic enzymes and the 11-hydroxylase function of P450(11) beta are relatively insensitive to fatty acids. The special sensitivity of aldosterone synthesis to fatty acid inhibition appears to result from the unusual susceptibility of the 18-oxidase function of the mitochondrial steroidogenic enzyme P450(11) beta. This mechanism would allow differential regulation of aldosterone vs. cortisol production by unesterified fatty acids.

Early and late effects of angiotensin-II on Ca2+ fluxes in bovine adrenal zona glomerulosa cells.

Previous studies have yielded conflicting results concerning the effects of angiotensin-II (Ang II) on Ca2+ fluxes in adrenal zona glomerulosa (ZG) cells. The present study was designed to investigate the kinetics and dose-dependency of Ang II-mediated changes in Ca2+ influx and efflux in cultured bovine ZG cells. At a high (10 nM) Ang II concentration, cytosolic Ca2+ (Cai) shows a peak-plateau response for the first 15 min, with small Cai transients commonly observed with longer stimulations. At 50 pM Ang II, more sustained Cai changes were elicited, typically consisting of Cai oscillations. The underlying changes in Ca2+ influx and efflux were studied. The early modifications of Ca2+ influx after 2 min of agonist stimulation were biphasic, with uptake increased by 90% between 1-100 pM Ang II and inhibited by 30% at 10 nM Ang II. Furthermore, high (10 nM) Ang II doses inhibited extracellular K(+)-stimulated Ca2+ influx. After 30 min of Ang II stimulation, the later dose response of Ca2+ influx was of similar magnitude but shifted to the left, showing a maximal influx at 10 pM Ang II and a modest enhancement at 10 nM. Basal Ca2+ efflux followed a two-compartment exponential decay, reflecting rapid Ca2+ displacement from extracellular sites (k1) and active Ca2+ transport (k2). A high (10 nM) Ang II concentration induced a transient large increase (130%) in k2 during the initial phase of Ang II stimulation, which returned to basal values within 10 min. A low (50 pM) Ang II concentration induced a small sustained increase (30%) in k2. A 10-nM Ang II concentration markedly reduced the exchangeable Ca2+ pool, as Ca2+ mobilized from intracellular stores into the cytosol was rapidly extruded, while Ca2+ influx was inhibited. A more physiological (50 pM) concentration of Ang II did not significantly alter the total exchangeable Ca2+ pool due to modest stimulation of both Ca2+ efflux and influx. In summary, the initial transient Cai response to high Ang II results from a large Ca2+ mobilization combined with inhibition of Ca2+ influx, which does not allow for the refilling of Ca2+ stores. At later times, small increases in Ca2+ influx allow for the eventual recovery of exchangeable cell Ca2+ and an enhanced elevation in Cai. At low Ang II concentrations, stimulation of both Ca2+ influx and efflux are concurrent and maintained, allowing for a sustained increase in Cai with little change in exchangeable cell Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

T-type calcium channels in adrenal glomerulosa cells: GTP-dependent modulation by angiotensin II.

With the use of whole-cell and single-channel current recordings, we have examined in more detail the site of action of angiotensin II (AII) on multiple populations of voltage-gated calcium channels in bovine adrenal glomerulosa cells. AII (10 nM) enhances whole-cell T-type calcium channel current and increases the activity of single T-type calcium channels in cell-attached patch recordings. The AII-induced enhancement of whole-cell calcium channel currents is dependent on the presence of internal GTP and can be inhibited by the competitive AII-receptor antagonist saralasin (1 microM). These results show that AII augments the T-type calcium channel current in bovine adrenal glomerulosa cells.

Blocking T-type calcium channels with tetrandrine inhibits steroidogenesis in bovine adrenal glomerulosa cells.

Tetrandrine, an alkaloid extracted from a Chinese medicinal herb traditionally used in hypertension treatment, inhibited aldosterone production induced in bovine adrenal glomerulosa cells by either potassium ion, angiotensin II, or ACTH in a concentration-dependent manner (IC50 = 10 microM). The inhibition of the response to potassium by tetrandrine had a pattern very similar to that of nickel, a blocker of T-type calcium channels. In addition, tetrandrine prevented calcium influx induced by potassium or angiotensin II without affecting the calcium release phase stimulated by the hormone. The effect of tetrandrine on voltage-activated barium currents was investigated using the whole cell configuration of the patch clamp technique. T-type currents were isolated by recording the slowly deactivating currents elicited during repolarization of the cell to -65 mV after various depolarizing pulses. These currents were blocked by micromolar concentrations of the drug. The voltage sensitivity of channel activation was not affected by tetrandrine; nevertheless, the drug significantly slowed the deactivation of the current. The action of tetrandrine did not require the activation of the channel. Tetrandrine also affected L-type currents, as assessed after inactivating T channels for 100 msec, but at higher concentrations of the drug. Thus, tetrandrine affects with a similar potency aldosterone production, calcium influx, and T-type calcium channel activity. This finding strongly suggests a role for these channels in calcium signaling and control of steroidogenesis in adrenal glomerulosa cells.

Stimulation of early gene expression by angiotensin II in bovine adrenal glomerulosa cells: roles of calcium and protein kinase C

Stimulation of early gene expression by angiotensin II in bovine adrenal glomerulosa cells: roles of calcium and protein kinase C

Stimulation of early gene expression by angiotensin II in bovine adrenal glomerulosa cells: roles of calcium and protein kinase C.

The adrenal glomerulosa cell is a major site of action of angiotensin II (AII), which binds to AT1 receptors to stimulate phosphoinositide hydrolysis and Ca2+ mobilization, and the subsequent production of aldosterone. All also influences adrenal growth and proliferation and promotes thymidine incorporation in adrenocortical cells. In primary cultures of bovine glomerulosa cells, AII was found to induce the expression of several early growth response genes (c-fos, c-jun, JunB, and Krox 24). This effect of AII was dose-dependent and was blocked by [Sar1,IIe8] AII and the nonpeptide antagonist DuP 753, indicating that it is mediated by the AT1 subtype of the AII receptor. ACTH, which elevates cAMP in glomerulosa cells, was a relatively weak inducer of c-fos expression but was as potent as AII in stimulating the expression of JunB. ACTH did not further enhance the maximal effect of AII on c-fos expression. The role of the AII-induced cytoplasmic Ca2+ increase in generating the c-fos response was suggested by the ability of the Ca2+ ionophore ionomycin to induce c-fos expression. However, mobilization of intracellular Ca2+ by the Ca2+ ATPase inhibitor thapsigargin, as well as the stimulation of Ca2+ influx by depolarization with potassium, were less potent stimuli of c-fos expression. Omission of Ca2+ from the extracellular medium, which abolishes the plateau phase of the AII-induced Ca2+ signal without affecting the early increase due to Ca2+ mobilization, enhanced the early phase of the AII-induced c-fos response, indicating that Ca2+ also has an inhibitory effect on the early gene response. Activation of protein kinase C by phorbol 12-myristate, 13-acetate (PMA) also stimulated c-fos expression, but the combination of PMA and ionomycin did not further increase the c-fos response. Inhibition of protein kinase C by staurosporine, or its depletion by prolonged exposure to PMA, prevented the c-fos response to PMA but only partially inhibited the response to AII, suggesting the involvement of other factors in stimulus-transcription coupling from the AT1 receptor.

ANP-(7-23) stimulates a DHP-sensitive Ca2+ conductance and reduces cellular cAMP via a cGMP-independent mechanism.

Atrial natriuretic peptide (ANP) potently inhibits aldosterone secretion from the adrenal glomerulosa cell. In many tissues ANP action is associated with an increase in cellular guanosine 3',5'-cyclic monophosphate (cGMP) mediated through binding of the peptide to one of its receptors [ANP-A(R1)]. However, in the adrenal glomerulosa cell, the physiological significance of this rise in cGMP content has been contested. In an effort to determine whether non-cyclase-containing ANP receptors, such as ANP-C(R2), are linked to any of the events triggered by ANP binding, we utilized a truncated ANP analogue, ANP-(7-23), which at low doses exhibits selectivity for the ANP-C(R2) receptor. With the use of bovine adrenal glomerulosa cells, low concentrations (1 nM) of ANP-(7-23) failed to stimulate cGMP production, did not lower cytosolic calcium in the presence of low K+, and did not inhibit aldosterone secretion. At 1 nM, however, the analogue decreased cellular adenosine 3',5'-cyclic monophosphate content [8.27 +/- 0.51 vs. 6.74 +/- 0.09 (SE) pmol/10(6) cells; P less than 0.02] and, only in the presence of high extracellular [K+], increased cytosolic calcium. This ANP-induced rise in cytosolic calcium was abolished by the addition of a low dose (30 nM) of the dihydropyridine nitrendipine. ANP-(7-23) when utilized at a higher concentration (500 nM) lost its selectivity for the ANP-R2 receptor and increased cellular cGMP content (control, 0.27 +/- 0.02 vs. 500 nM ANP-(7-23), 0.448 +/- 0.02 pmol/10(6) cells; P less than 0.01). At 500 nM, ANP-(7-23) also inhibited aldosterone secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II receptor-mediated calcium influx in bovine adrenal glomerulosa cells

The cytoplasmic calcium ([Ca2+]i) response to angiotensin II (AII) in bovine adrenal glomerulosa cells is characterized by an initial transient peak due to intracellular Ca2+ mobilization, followed by a sustained plateau phase that is dependent on Ca2+ entry from the extracellular fluid. In Fura-2 loaded cells, the calcium channel antagonists, nifedipine (1 microM) and verapamil (20 microM), only partially reduced the cytosolic calcium profile induced by AII. The dihydropyridine agonist, Bay K 8644, caused a moderate increase in [Ca2+]i when added in concentrations of 50-100 nM, but did not enhance the AII-induced rise in [Ca2+]i. These results indicate that most of the AII-stimulated Ca2+ influx is through channels that are insensitive to dihydropyridines and verapamil. In contrast, the inorganic Ca2+ channel blocker, LaCl3 (10 microM), inhibited the AII-induced plateau phase by more than 50%. The AII-induced Ca2+ signal was not affected by treatment with pertussis toxin (100-300 ng/ml for 12 h). The prior addition of specific AII-antagonists (DuP 753, a nonpeptide antagonist, and three peptide analogs, [Sar1,Thr8]AII, [Sar1,Ala8]AII, and [Sar1,Ile8]AII) completely inhibited the AII-induced Ca2+ signal. However, addition of up to 25,000 molar excess of these antagonists at intervals from 10 sec to 5 min after AII caused progressively less attenuation of the sustained Ca2+ signal. After 5 min, addition of antagonists did not inhibit the agonist-induced Ca2+ response for up to 20 min. The progressive loss of ability of the antagonists to inhibit the sustained elevation of [Ca2+]i could reflect prolonged activation of the receptor or of a subsequent process that maintains Ca2+ influx despite receptor blockade. It is possible that sequestration and/or endocytosis of the AII-receptor complex is accompanied by continued generation of intracellular signals that contribute to the maintenance of the [Ca2+]i response.

Angiotensin II receptor-mediated calcium influx in bovine adrenal glomerulosa cells.

The cytoplasmic calcium ([Ca2+]i) response to angiotensin II (AII) in bovine adrenal glomerulosa cells is characterized by an initial transient peak due to intracellular Ca2+ mobilization, followed by a sustained plateau phase that is dependent on Ca2+ entry from the extracellular fluid. In Fura-2 loaded cells, the calcium channel antagonists, nifedipine (1 microM) and verapamil (20 microM), only partially reduced the cytosolic calcium profile induced by AII. The dihydropyridine agonist, Bay K 8644, caused a moderate increase in [Ca2+]i when added in concentrations of 50-100 nM, but did not enhance the AII-induced rise in [Ca2+]i. These results indicate that most of the AII-stimulated Ca2+ influx is through channels that are insensitive to dihydropyridines and verapamil. In contrast, the inorganic Ca2+ channel blocker, LaCl3 (10 microM), inhibited the AII-induced plateau phase by more than 50%. The AII-induced Ca2+ signal was not affected by treatment with pertussis toxin (100-300 ng/ml for 12 h). The prior addition of specific AII-antagonists (DuP 753, a nonpeptide antagonist, and three peptide analogs, [Sar1,Thr8]AII, [Sar1,Ala8]AII, and [Sar1,Ile8]AII) completely inhibited the AII-induced Ca2+ signal. However, addition of up to 25,000 molar excess of these antagonists at intervals from 10 sec to 5 min after AII caused progressively less attenuation of the sustained Ca2+ signal. After 5 min, addition of antagonists did not inhibit the agonist-induced Ca2+ response for up to 20 min. The progressive loss of ability of the antagonists to inhibit the sustained elevation of [Ca2+]i could reflect prolonged activation of the receptor or of a subsequent process that maintains Ca2+ influx despite receptor blockade. It is possible that sequestration and/or endocytosis of the AII-receptor complex is accompanied by continued generation of intracellular signals that contribute to the maintenance of the [Ca2+]i response.

Signal transduction mechanisms involved in carbachol-induced aldosterone secretion from bovine adrenal glomerulosa cells

In cultured bovine adrenal glomerulosa cells, diacylglycerol content remains elevated for up to 75 min following the removal of angiotensin II. This maintained increase could provide a mechanism by which angiotensin II pretreatment may prime cells to secrete aldosterone in response to the calcium channel agonist Bay K 8644. In the present study we find that carbachol failed both to produce this persistent diacylglycerol elevation and to exert a priming effect. In addition, because carbachol was also a less potent activator of phospholipase D than angiotensin II, our results implicate phospholipase D in the maintained increase in diacylglycerol content observed following stimulation with and removal of angiotensin II. Carbachol also elicited changes in the radiolabeled levels of both myristate- and arachidonate-containing diacylglycerol. However, the rapid decline in diacylglycerol content following carbachol removal resembled the rapid fall in arachidonate-diacylglycerol; we therefore proposed that the diacylglycerol species generated with carbachol stimulation contains predominantly arachidonic acid. In summary, our results suggest that prolonged elevations in diacylglycerol content following removal of hormones such as angiotensin II, as well as the identity of the diacylglycerol species itself, may be important in the regulation of cellular responses.

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.

Bradykinin stimulates aldosterone release from cultured bovine adrenocortical cells through bradykinin B2 receptors.

The adrenal cortex contains a kallikrein-like enzyme that may lead to bradykinin (BK) formation. This study was designed to determine whether BK acts on adrenocortical cells to stimulate steroid secretion. BK, Lys-BK, a specific BK 2 (B2) receptor agonist, and desArg9-BK, a specific BK 1 (B1) receptor agonist, all stimulated aldosterone secretion from cultured bovine adrenal zona glomerulosa cells. BK and Lys-BK were equipotent (EC50 = 2 x 10(-9) M), whereas desArg9-BK was 1000-fold less potent. The maximal effects of BK and BK analogs were comparable to the maximal effects of adrenocorticotropin or angiotensin II. A B2, but not a B1, receptor antagonist inhibited BK-stimulated aldosterone release. Verapamil and N,N-diethylamino-octyl-3,4,5-trimethoxybenzoate, which reduce intracellular calcium concentrations, reduced BK-stimulated aldosterone secretion. Although BK stimulated both prostacyclin and aldosterone production, indomethacin abolished prostacyclin production without affecting aldosterone secretion. In cultured adrenal fasciculata cells, high concentrations of BK stimulated cortisol release, but B1 or B2 receptor agonists were not effective. BK-stimulated cortisol secretion was reduced by N,N-diethylamino-octyl-3,4,5-trimethoxybenzoate but not by indomethacin. In summary, BK stimulates aldosterone release from cultured adrenal glomerulosa cells via high affinity B2 receptors. The effect is calcium-dependent and independent of prostaglandins. BK also increases cortisol release; however, this stimulation requires high concentrations of BK and may be mediated by an unknown receptor or by a receptor-independent mechanism.

Endothelin-1 potentiation of angiotensin II stimulation of aldosterone production.

Endothelin-1 (ET-1) binds to specific receptors in cultured bovine adrenal glomerulosa cells and stimulates aldosterone secretion with a 50% effective concentration (EC50) of 300 +/- 80 pM (mean +/- SE). The relative stimulatory potency for ET-1 is significantly less than that of angiotensin II (ANG II). The incubation of calf zona glomerulosa cells in primary culture with ET-1 and ANG II resulted in a significant potentiation of ANG II effect on aldosterone secretion. The EC50 of ET-1 potentiation of ANG II-induced stimulation of aldosterone secretion was 40 +/- 5 pM (mean +/- SE, n = 4), which is lower than the EC50 for ET-1 stimulation of aldosterone secretion. Adrenocorticotropic hormone (ACTH) stimulation of aldosterone secretion, but not that of potassium, was also potentiated by ET-1, but to a lesser degree. ET-1 and ET-1-mediated potentiation of ANG II-stimulated aldosterone biosynthesis increased both the early and late pathways of aldosterone biosynthesis, but the potentiation was greater for the early pathway. Preincubation with ET-1 for at least 15 min, followed by extensive washing to remove bound ET-1, also resulted in persistent potentiation of ANG II-mediated aldosterone secretion. ET-2, sarafotoxin, and vasoactive intestinal contractor potentiation of ANG II action were very similar to that of ET-1. ET-3 and Big-ET-1 potentiated ANG II stimulation only at the highest doses tested and the proendothelin-(110-130) fragment was inactive. ET-1 potentiation of ANG II action is likely to be mediated through an ETB receptor subtype.(ABSTRACT TRUNCATED AT 250 WORDS)

The conversion of corticosterone to aldosterone is the site of the oxygen sensitivity of the bovine adrenal zona glomerulosa

The dissociation of renin and aldosterone observed during hypoxia in vivo has been attributed to a direct inhibition of low oxygen on adrenal zona glomerulosa function. We have demonstrated that the adrenal zona glomerulosa production of aldosterone in vitro is directly proportional to a wide range of oxygen concentrations in the physiological range but that cortisol production from coincubated fasciculata cells is not oxygen sensitive. The present study examined the hypothesis that the sensitivity to O2 is limited to the aldosteronogenic late pathway. In order to localize the site of oxygen sensitivity, we measured endogenous pregnenolone production (early pathway) and the conversion of exogenous corticosterone to aldosterone (ALDO) (i.e. 18-hydroxylase activity) in adrenal cells treated with cyanoketone (3-beta-hydroxy-steroid dehydrogenase inhibitor). Acutely dispersed bovine adrenal glomerulosa cells (four experiments in pentuplicate) were incubated under low (5%) vs. normal (21%) O2 in the presence of cyanoketone (CK; 1 microM) and/or the following: corticosterone (500 ng/ml), angiotensin II (ANG II; 10 nM), or dibutyryl cAMP (1 mM). Conversion of exogenous corticosterone to ALDO in the presence of CK was inhibited by 41 +/- 1% under low O2. This was similar to the inhibitory effect of low O2 on ANG II-stimulated aldosterone production from endogenous precursors in the absence of CK (52 +/- 11% inhibition). Basal, ANG II-, and cAMP-stimulated endogenous pregnenolone production was not significantly reduced by low O2. In another experiment, glomerulosa cells were incubated under 5, 13, or 50% vs. 21% O2 in the presence of CK (1 microM) and different concentrations of corticosterone (10-1000 ng/ml). ALDO production was significantly inhibited by low O2 when corticosterone was greater than or equal to 500 ng/ml and ALDO was significantly augmented by high O2 when added corticosterone was 1000 ng/ml. We conclude that the conversion of corticosterone to ALDO (i.e. 18-hydroxylase) appears to be the primary site of oxygen sensitivity since 1) pregnenolone production was unaffected and 2) the magnitude of the inhibition of the conversion of corticosterone to ALDO by low O2 in the presence of CK was similar to the inhibition of ALDO production from endogenous precursors in the absence of CK. These studies demonstrate that oxygen sensitivity of the steroidogenic pathway is a unique, constitutive property of 18-hydroxylase, the enzyme which catalyzes the conversion of corticosterone to ALDO. We propose that the sensitivity of 18-hydroxylase to oxygen accounts for the dissociation of renin and aldosterone during hypoxia in vivo.

The conversion of corticosterone to aldosterone is the site of the oxygen sensitivity of the bovine adrenal zona glomerulosa.

The dissociation of renin and aldosterone observed during hypoxia in vivo has been attributed to a direct inhibition of low oxygen on adrenal zona glomerulosa function. We have demonstrated that the adrenal zona glomerulosa production of aldosterone in vitro is directly proportional to a wide range of oxygen concentrations in the physiological range but that cortisol production from coincubated fasciculata cells is not oxygen sensitive. The present study examined the hypothesis that the sensitivity to O2 is limited to the aldosteronogenic late pathway. In order to localize the site of oxygen sensitivity, we measured endogenous pregnenolone production (early pathway) and the conversion of exogenous corticosterone to aldosterone (ALDO) (i.e. 18-hydroxylase activity) in adrenal cells treated with cyanoketone (3-beta-hydroxy-steroid dehydrogenase inhibitor). Acutely dispersed bovine adrenal glomerulosa cells (four experiments in pentuplicate) were incubated under low (5%) vs. normal (21%) O2 in the presence of cyanoketone (CK; 1 microM) and/or the following: corticosterone (500 ng/ml), angiotensin II (ANG II; 10 nM), or dibutyryl cAMP (1 mM). Conversion of exogenous corticosterone to ALDO in the presence of CK was inhibited by 41 +/- 1% under low O2. This was similar to the inhibitory effect of low O2 on ANG II-stimulated aldosterone production from endogenous precursors in the absence of CK (52 +/- 11% inhibition). Basal, ANG II-, and cAMP-stimulated endogenous pregnenolone production was not significantly reduced by low O2. In another experiment, glomerulosa cells were incubated under 5, 13, or 50% vs. 21% O2 in the presence of CK (1 microM) and different concentrations of corticosterone (10-1000 ng/ml). ALDO production was significantly inhibited by low O2 when corticosterone was greater than or equal to 500 ng/ml and ALDO was significantly augmented by high O2 when added corticosterone was 1000 ng/ml. We conclude that the conversion of corticosterone to ALDO (i.e. 18-hydroxylase) appears to be the primary site of oxygen sensitivity since 1) pregnenolone production was unaffected and 2) the magnitude of the inhibition of the conversion of corticosterone to ALDO by low O2 in the presence of CK was similar to the inhibition of ALDO production from endogenous precursors in the absence of CK. These studies demonstrate that oxygen sensitivity of the steroidogenic pathway is a unique, constitutive property of 18-hydroxylase, the enzyme which catalyzes the conversion of corticosterone to ALDO. We propose that the sensitivity of 18-hydroxylase to oxygen accounts for the dissociation of renin and aldosterone during hypoxia in vivo.

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.

Ca2+ channels and aldosterone secretion: modulation by K+ and atrial natriuretic peptide

Two populations of voltage-dependent Ca2+ channels, T-type and L-type, are present in bovine adrenal glomerulosa cells. Activation of these channels by cell depolarization with the resultant increase in Ca2+ influx may be one way in which agonists regulate aldosterone secretion. In addition, these channels may be the site of antagonist action. In the present study, we have demonstrated that atrial natriuretic peptide (ANP), an antagonist of aldosterone secretion, alters only the voltage dependence of inactivation of the T-type channel while enhancing the voltage dependence of activation of a subpopulation of L-type channels. These patch-clamp data, which demonstrated contrasting effects of ANP on the activity of T- and L-type Ca2+ channels correlated with changes induced in cytosolic calcium [( Ca2+]i). In the weakly depolarized cell, ANP (greater than 30 pM) lowered [Ca2+]i, in contrast to the strongly depolarized cell, in which ANP (greater than 10 pM) raised [Ca2+]i. Similar alterations in the level of [Ca2+]i in the stimulated cell were induced by the Ca(2+)-channel blocker nitrendipine and the L-type channel agonist, (-)BAY K 8644. With increasing concentrations of extracellular K+ (3.5-60 mM) the rate of aldosterone secretion rose nonmonotonically. ANP inhibited secretion over this broad range of K+ concentrations; however, its potency as an inhibitor of secretion was diminished in the strongly depolarized cell. These data are discussed in the context of a model that proposes a role for sustained Ca2+ influx in cell activation.

Relationship between agonist- and thapsigargin-sensitive calcium pools in adrenal glomerulosa cells. Thapsigargin-induced Ca2+ mobilization and entry.

The relationships between agonist-sensitive calcium pools and those discharged by the Ca(2+)-ATPase inhibitor thapsigargin were studied in intact bovine adrenal glomerulosa cells and a subcellular adrenocortical membrane fraction. In Fura-2-loaded glomerulosa cells, angiotensin II (AII) stimulated a rapid increase in cytoplasmic Ca2+ concentration ([Ca2+]i) followed by a smaller plateau phase that was dependent on extra-cellular Ca2+. In such cells thapsigargin caused a sustained and dose-dependent increase in [Ca2+]i which was diminished in Ca(2+)-deficient medium. The contribution of an influx component to the thapsigargin-induced [Ca2+]i response was demonstrated by measurement of 45Ca influx rate in glomerulosa cells. Thapsigargin-induced Ca2+ entry was significantly less than that evoked by AII, and its kinetics were similar to those of the concomitant increase in [Ca2+]i. The rate of emptying of the agonist-responsive Ca2+ pool after thapsigargin treatment, as indicated by the progressive decrease in the size of the AII-induced Ca2+ transient, showed a rapid initial (t1/2 = 1.7 min) component that accounted for about 80% of the response and a slowly decreasing phase with t1/2 = 112 min. The latter thapsigargin-resistant component was abolished by the removal of extracellular Ca2+. Pretreatment with AII dose-dependently attenuated but did not abolish the subsequent Ca2+ response to thapsigargin and also increased the rate of the Ca2+ rise induced by thapsigargin. In bovine adrenocortical microsomes, thapsigargin inhibited the ATP-dependent filling of Ca2+ pools and caused a dose-dependent rise in extravesicular Ca2+ levels when added to previously loaded microsomes. The thapsigargin-releasable Ca2+ pool in adrenal microsomes was larger than the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-sensitive Ca2+ pool but only slightly greater than the GTP-releasable pool. Ins(1,4,5)P3-induced Ca2+ release was reduced markedly when ATP-dependent Ca2+ loading of the microsomes was prevented by prior addition of thapsigargin. However, the subsequent Ca2+ response to Ins(1,4,5)P3 was consistently better preserved after the addition of thapsigargin to microsomes preloaded with Ca2+. This difference suggests that although Ca2+ uptake by the Ins(1,4,5)P3-responsive pool is also sensitive to thapsigargin, once filled, this pool shows a slower passive leakage than other thapsigargin-sensitive pools. These findings indicate that thapsigargin increases [Ca2+]i by inhibiting Ca2+ uptake into multiple intracellular Ca2+ pools and by also promoting entry of extracellular Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Contrasting effects of sn-1,2-dioctanoyl glycerol as compared to other protein kinase C activators in adrenal glomerulosa cells

Angiotensin II acts on adrenal glomerulosa cells to induce the phospholipase C-mediated generation of inositol trisphosphate and sn-1,2-diacyglycerol as the major products of inositol phospholipid breakdown. This last product is known to activate protein kinase C, but its role in the action of angiotensin II on steroidogenesis has not been defined. We report herein that, in bovine adrenal glomerulosa cells, protein kinase C activators, such as phorbol 12,13-dibutyrate, 12-O-tetradecanoylphorbol-13-acetate, mezerein and sn 1,2 oleoyl acetoylglycerol, each failed to increase steroidogenesis. These results contrast with our recent report on the enhancement of aldosterone output by sn-1,2-dioctanoylglycerol (DiC8) [ J. Steroid Biochem. 35 (1990) 19–33]. In addition, the difference between DiC8 and the other protein kinase activators was also observed in the pattern of 86Rb efflux from preloaded glomerulosa cells; only DiC8 mimicked the effect of angiotensin II on ion fluxes. Furthermore, staurosporine, a potent inhibitor of protein kinase C, was capable of amplifying the aldosterone output induced by a maximally effective concentration of DiC8 or angiotensin II. These data suggest that the effect of the cell permeant DiC8 on aldosterone biosynthesis either is not mediated by protein kinase C activation, or is mediated by a phorbol ester-insensitive isoenzyme of protein kinase C.

Parathyroid hormone modulates angiotensin II-induced aldosterone secretion from the adrenal glomerulosa cell.

The effect of PTH on aldosterone secretion from isolated bovine adrenal glomerulosa cells was examined. PTH binding was autoradiographically localized to the adrenal cortex, suggesting a specific effect. This binding of PTH was displaceable by cold PTH, but not by ACTH. No binding was observed in the adrenal medulla. In addition, PTH was shown to stimulate aldosterone secretion in a dose-dependent manner and to potentiate aldosterone secretion in response to angiotensin-II, such that PTH (10(-9)M) elevated the secretory rate from 58.6 +/- 6.8 to 110.9 +/- 19 pg/min.million cells in the presence of 10 nM angiotensin-II. The magnitude of the synergism between the two hormones depended on the concentrations of PTH and angiotensin-II as well as the time during which aldosterone secretion was measured. Within the first 15 min of stimulation, PTH increased the sensitivity to angiotensin-II, shifting the Ka for activation from 1.0 to 0.3 nM. In contrast, between 30-45 min of angiotensin-II stimulation, PTH elevated the maximal secretory response to angiotensin-II from 109 +/- 3.4 to 219 +/- 13.3 pg/min.million cells. By itself PTH elicited only a small increase in the intracellular Ca2+ concentration, as measured by aequorin luminescence in glomerulosa cells. In cells pretreated with angiotensin-II or 15 mM potassium, the intracellular calcium response to PTH was markedly potentiated. PTH was also found to cause a small increase in the cellular cAMP content. Thus, PTH stimulates aldosterone secretion from adrenal glomerulosa cells, both alone and in combination with angiotensin-II.