External calcium is required for activation of phospholipase C by angiotensin II in adrenal glomerulosa cells

Previous studies have shown that external calcium (Ca²⁺) is required for the effects of angiotensin II (AII) on aldosterone secretion in adrenal glomerulosa zone. Using bovine adrenal glomerulosa cells prepared by collagenase dispersion, we examined whether external Ca²⁺ is required for the activation of phospholipase C by AII. Adrenal glomerulosa cells were exposed to Ca-EGTA buffered media to provide accurate estimates of external free Ca²⁺ concentrations. Phospholipase C activation was evaluated by measurement of inositol phosphates production. At 0.1 M Ca²⁺ and less, sustained AII effects on inositol monophosphate (IP), inositol bisphosphate (IP₂) and inositol trisphosphate (IP₃) were markedly inhibited. Increasing the Ca²⁺ concentration to 50kM or greater fully restored All-induced inositol phosphates production. AII-induced increases in cytosolic Ca²⁺ measured by Quin-2 fluorescence, were diminished at lower external Ca²⁺ concentrations. Treating adrenal glomerulosa cells with Chelex-100, a strong Ca²⁺ binding resin, blocked early activation of phospholipase C by AII. Inhibition of IP₃ production was also observed when inhibitors of Ca²⁺ movement across the plasma membrane were used, viz., La²⁺, TMB-8 and nifedipine. The requirement for Ca²⁺ during AII-induced activation of phospholipase C may be explained, at least partly by a requirement for Ca²⁺ at a site between the AII receptor and Phospholipase C.     

Control of calcium homeostasis by angiotensin II in adrenal glomerulosa cells through activation of p38 MAPK

Angiotensin II-induced activation of aldosterone secretion in adrenal glomerulosa cells is mediated by an increase of intracellular calcium. We describe here a new Ca2+-regulatory pathway involving the inhibition by angiotensin II of calcium extrusion through the Na+/Ca2+ exchanger. Caffeine reduced both the angiotensin II-induced calcium signal and aldosterone production in bovine glomerulosa cells. These effects were independent of cAMP or calcium release from intracellular stores. The calcium response to angiotensin II was more sensitive to caffeine than the response to potassium, suggesting that the drug interacts with a pathway specifically elicited by the hormone. In calcium-free medium, calcium returned more rapidly to basal levels after angiotensin II stimulation in the presence of caffeine. Thapsigargin had no effect on these kinetics, but diltiazem, which inhibits the Na+/Ca2+ exchanger, markedly reduced the rate of calcium decrease and abolished caffeine action. The involvement of this exchanger was supported by the effect of cell depolarization and of a reduction of extracellular sodium on the rate of calcium extrusion. We also determined the mechanism of angiotensin II action on the exchanger. Phorbol esters reduced the rate of calcium extrusion, which was increased by baicalein, an inhibitor of lipoxygenases, and by SB 203580, an inhibitor of the p38 MAPK. Finally, we showed that angiotensin II acutely activates, in a caffeine-sensitive manner, p38 MAPK in glomerulosa cells. In conclusion, in bovine glomerulosa cells, the Na+/Ca2+ exchanger plays a crucial role in extruding calcium, and, by reducing its activity, angiotensin II influences the amplitude of the calcium signal. The hormone exerts its action on the exchanger through a caffeine-sensitive pathway involving the p38 MAPK and lipoxygenase products.     

Angiotensin II receptors and mechanisms of action in adrenal glomerulosa cells

The plasma-membrane receptors, coupling mechanisms, and effector enzymes that mediate target-cell activation by angiotensin II (AII) have been characterized in rat and bovine adrenal glomerulosa cells. The AII holoreceptor is a glycoprotein of Mr approximately 125,000 under non-denaturing conditions. Photoaffinity labeling of AII receptors with azido-AII derivatives has shown size heterogeneity among the AII binding sites between species and target tissues, with Mr values of 55,000 to 79,000. Such variations in molecular size probably reflect differences in carbohydrate content of the individual receptor sites. The adrenal AII receptor, like that in other tissues, is coupled to the inhibitory guanine nucleotide inhibitory protein (Ni). However, studies with pertussis toxin have shown that stimulation of aldosterone production by AII is not mediated by Ni but by a pertussis-insensitive nucleotide regulatory protein of unidentified nature. Although Ni is not involved in the stimulatory action of AII on steroidogenesis, it does mediate the inhibitory effects of high concentrations of AII upon aldosterone production. The actions of AII on adrenal cortical function are thus regulated by at least two guanine nucleotide regulatory proteins that are selectively activated by increasing AII concentrations. The principal effector enzyme in AII action is phospholipase C, which is rapidly stimulated in rat and bovine glomerulosa after AII receptor activation. AII-induced breakdown of phosphatidylinositol bisphosphate (PIP2) and phosphatidylinositol phosphate (PIP) leads to formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2). These are metabolized predominantly to inositol-4-monophosphate, which serves as a marker of polyphosphoinositide breakdown, whereas inositol-1-phosphate is largely derived from phosphatidylinositol hydrolysis. The AII-stimulated glomerulosa cell also produces inositol 1,3,4-trisphosphate, a biologically inactive IP3 isomer formed from Ins-1,4,5-trisphosphate via inositol tetrakisphosphate (IP4) during ligand activation in several calcium-dependent target cells. The Ins-1,4,5-P3 formed during AII action binds with high affinity to specific intracellular receptors that have been characterized in the bovine adrenal gland and other AII target tissues, and may represent the sites through which IP3 causes calcium mobilization during the initiation of cellular responses.     

Inositol polyphosphate production and regulation of cytosolic calcium during the biphasic activation of adrenal glomerulosa cells by angiotensin II

Stimulation of aldosterone production by angiotensin II in the adrenal glomerulosa cell is mediated by increased phosphoinositide turnover and elevation of intracellular Ca2+ concentration. In cultured bovine glomerulosa cells, angiotensin II caused rapid increases in inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) levels and cytosolic Ca2+ during the first minute of stimulation, when both responses peaked between 5 and 10 s and subsequently declined to above-baseline levels. In addition to this temporal correlation, the dose-response relationships of the angiotensin-induced peak increases in cytosolic Ca2+ concentrations and Ins-1,4,5-P3 levels measured at 10 s were closely similar. However, at later times (greater than 1 min) there was a secondary elevation of Ins-1,4,5-P3, paralleled by increased formation of inositol 1,3,4,5-tetrakisphosphate that was associated with cytosolic Ca2+ concentrations only slightly above the resting value. These results are consistent with the primary role of Ins-1,4,5-P3 in calcium mobilization during activation of the glomerulosa cell by angiotensin II. They also suggest that Ins-1,4,5-P3 participates in the later phase of the target-cell response, possibly by acting alone or in conjunction with its phosphorylated metabolites to promote calcium entry and elevation of cytosolic Ca2+ during the sustained phase of aldosterone secretion.     

Role of calcium fluxes in the sustained phase of angiotensin II-mediated aldosterone secretion from adrenal glomerulosa cells

When angiotensin II stimulates aldosterone secretion, it causes a rapid but transient mobilization of calcium from an intracellular pool and a sustained increase in the influx of calcium in adrenal glomerulosa cells. The present studies were undertaken to determine the respective roles of the two angiotensin II-induced changes in cellular calcium metabolism in modulating events during the sustained phase of cellular response which is thought to be mediated by the C-kinase branch of the calcium messenger system. The sustained response to angiotensin II is only 50% of maximal in cells pretreated with dantrolene in a concentration sufficient to inhibit the angiotensin II-induced mobilization of intracellular calcium. Also, if A23187 is added to cells simultaneously with 1-oleoyl-2-acetylglycerol (OAG), the aldosterone secretory response is similar to that seen after angiotensin II. However, if A23187 is added first and the transient aldosterone secretory response allowed to decay, and OAG then added, the sustained aldosterone secretory response is only 45-50% of maximal. Addition of the calcium channel agonist, BAY K 8644, with OAG leads to an aldosterone secretory response which is only 50% of maximal, but if upon addition of OAG and BAY K 8644 the cells are also exposed for 5 min to media containing 8 mM K+, then the sustained secretory response is maximal. These data imply that the initial transient rise in the [Ca2+] of the cell cytosol plays a role in determining the extent to which C-kinase is shifted from its calcium-insensitive to its calcium-sensitive form. The second group of experiments examined the relationship between the sustained angiotensin II-induced increase in plasma membrane calcium influx and the sustained aldosterone secretory response. The results show that in the presence of 1 microM nitrendipine or 2 mM extracellular K+, angiotensin II causes no increase in calcium influx and only a transient rather than a sustained increase in the rate of aldosterone secretion indicating that the sustained phase of the response is dependent upon a continued high rate of Ca2+ influx which regulates the rate of turnover of the activated C-kinase.     

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

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

Effects of ACTH and angiotensin II on cytosolic calcium in cultured adrenal glomerulosa cells. Role of cAMP production in the ACTH effect

We have used microspectrofluorometry and video imaging techniques in order to study and compare the changes in intracellular calcium concentrations [( Ca2+]i) of individual Fura-2 loaded glomerulosa cells cultured for three days and stimulated either with angiotensin II (AT), K+, or adrenocorticotropin (ACTH). As previously demonstrated for freshly isolated cells, K+ ion induces an immediate increase in [Ca2+]i, although AT induces a biphasic response, characterized by an initial transient spike, followed by a sustained plateau. In this study, we demonstrate, for the first time, that ACTH is able to induce a [Ca2+]i increase in cultured glomerulosa cells from rat and bovine sources. Moreover, it is clear that the pattern of [Ca2+]i increase elicited by ACTH is different from that observed with AT. In most cases, addition of ACTH leads to a slow increase in [Ca2+]i after a long latency period ranging from 10-15 min, which could be correlated to cAMP time-production. The present results show that: (a) in the absence of extracellular Ca2+, ACTH does not increase [Ca2+]i; (b) the response develops slowly and cases immediately after [Ca2+]e depletion or addition of calcium channel blockers, such as nifedipine or omega-conotoxin; (c) the addition of the calcium channel agonist Bay K 8644 enhances the ACTH response; (d) the cAMP analog, 8-Br-cAMP, induces an increase in [Ca2+]i similar to that observed with ACTH, which is also dependent of the presence of calcium in the extracellular medium; (e) time-production of ACTH-induced cAMP follows quite well the increase in [Ca2+]i; (f) Bay K 8644 also enhances the 8-Br-cAMP induced increase in [Ca2+]i; and (g) ACTH-induced Cai response is inhibited by the specific protein kinase A blocker, HA1004. These observations, combined with previous results obtained on the effects of ACTH on calcium currents and action potentials, suggest that the [Ca2+]i increase induced by ACTH results from a calcium influx through dihydropyridine and omega-conotoxin sensitive calcium channels, which need to be phosphorylated by cAMP for full activation. The use of video-imaging techniques has allowed us to examine the spatial distribution of changes in [Ca2+]i in single cells. The ability to simultaneously record images of a number of cells confirm the heterogeneity of cellular responses, and corroborate results obtained through photocounting only. Our results indicate that ACTH initially increases [Ca2+]i locally beneath the cell membrane and throughout the cell thereafter, whereas angiotensin II elicits a more prominent effect in certain regions of the cell and eventually extends to the entire cell surface.     

Regulation of angiotensin II receptors and steroidogenic responsiveness in cultured bovine fasciculata and glomerulosa adrenal cells

The aim of the present study was to investigate the effect of several effectors on angiotensin II (A-II) receptors and steroidogenic responsiveness in cultured bovine fasciculata cells. Treatment of adrenal cells for 24 h with A-II (0.1 microM), corticotropin (1 nM), phorbol ester (PMA 0.1 microM), calcium ionophore A23187 (0.1 microM) and cyclic 8-bromoAMP (1 mM) produced a loss of A-II receptors whereas the A-II antagonist [Sar1-Ala8]A-II (0.1 microM) led to a small but significant increase. The extent of the down-regulation of receptors following maximal concentrations of A-II was greater than that produced by the other agents. The effects of A-II were dose-dependent with a ID50 of 3 nM. Since cycloheximide and actinomycin blocked the down-regulation of receptors, it seems likely that the effectors lead to the synthesis of certain proteins which inhibit the recycling of internalized receptors. Pretreatment of adrenal cells with A-II induced both homologous (90% decrease) and heterologous (corticotropin 83, PMA and ionophore 76% decrease) steroidogenic desensitization. However, the cAMP response to corticotropin of A-II-pretreated cells was higher (P less than 0.001) than for control cells. Pretreatment with PMA and A23187 also resulted in both homologous and heterologous steroidogenic refractoriness but to a lesser degree than that induced by A-II. In contrast, corticotropin-pretreated cells responded normally to further stimulation with corticotropin or A-II. Similarly pretreatment of bovine adrenal glomerulosa cells with A-II (1 nM and 0.1 microM) and corticotropin (1 nM) also induced A-II receptor loss and steroidogenic refractoriness. The present findings indicate that, in contrast to the results reported in vivo in the rat, where A-II leads to up-regulation of its own receptors on glomerulosa cells and increases steroidogenic responsiveness, this peptide results in both down-regulation and desensitization in cultured bovine fasciculata and glomerulosa cells. Our results also emphasize the absence of correlation between A-II receptor loss and steroidogenic responsiveness.     

Background calcium permeable channels in glomerulosa cells from adrenal gland

The cell-attached recording mode of the patch-clamp technique was used to study Ca2+ permeable background currents of glomerulosa cells from rat and bovine adrenal gland. With a pipette filled with 110 mM BaCl2 or 90 mM CaCl2, three different types of unitary currents were detected. The B1 channel demonstrates a nonlinear I-V curve. The conductances are 4 and 7 pS at -40 and -70 mV, respectively. The curve of the opening probability vs. membrane potential is bell shaped with its maximum at -70 mV. The B2 channel has a conductance of 6 pS, while the B3 channel shows a nonlinear I-V relationship with conductances close to 17 and 10 pS at HPs of -60 and -20 mV. The three types of currents are insensitive to dihydropyridines. We suggest that these background currents could be responsible for the basal calcium influx and aldosterone secretion previously observed in nonstimulated glomerulosa cells.     

Angiotensin II and FCCP mobilizes calcium from different intracellular pools in adrenal glomerulosa cells; analysis of calcium fluxes

The aim of the present study was to examine the effect of angiotensin II on the different pools of exchangeable Ca2+ in isolated rat adrenal glomerulosa cells. On the basis of steady state analysis of 45Ca exchange curves at least three kinetically distinct Ca2+ compartments are present in these cells. The most rapidly exchangeable compartment was regarded as Ca2+ loosely bound to the glycocalyx and the other compartments were considered to be intracellular Ca2+ pools. The effect of angiotensin II on different intracellular compartments was examined by adding the hormone at different phases of Ca2+ washout. Angiotensin increased the rate of 45Ca efflux within 1.5 min when added at the beginning of the washout. This effect, however, could not be detected when the hormone was added at the 30th min of washout, indicating that at least one hormone sensitive pool had lost most of its radioactivity by this time. In contrast to angiotensin II, the mitochondrial uncoupler FCCP mobilized almost the same quantity of 45Ca irrespective of the time of its addition during the washout. This latter finding suggests that this presumably mitochondrial Ca2+ pool has a slow rate of exchange and thus differs from the pool initially mobilized by angiotensin II. The initial Ca2+ mobilizing effect of angiotensin II was also observed in a Ca2+-free media which contained EGTA, indicating that this effect is not triggered by increased Ca2+ influx. In the present study we demonstrate in the intact glomerulosa cell that angiotensin II mobilizes Ca2+ from an intracellular Ca2+ store which appears to be distinct from the FCCP-sensitive store.     

Na-Ca exchanger as a calcium influx pathway in adrenal glomerulosa cells

When aequorin-loaded glomerulosa cells were incubated in isotonic Na2+-free medium containing N-methyl-D-glucamine instead of NaCl, there was an increase in cytoplasmic free calcium concentration, [Ca2+] c, which was not observed when extracellular calcium concentration was reduced to 1 microM. Upon removal of extracellular sodium, there was nearly five-fold increase in fractional efflux ratio of calcium. The reduction of extracellular sodium resulted in a stimulation of calcium influx rate, the magnitude of which was dependent on extracellular sodium concentration. Similar stimulation of calcium influx was observed when extracellular sodium was replaced with lithium. Nitrendipine did not affect the calcium influx induced by the reduction of extracellular sodium while a derivative of amiloride 3',4'-dichlorobenzamil, which inhibits Na-Ca exchange, attenuated calcium influx observed in sodium-free medium. These results indicate that removal of extracellular sodium leads to an increase in [Ca2+] c by stimulating calcium influx and that calcium enters the cell via Na-Ca exchanger.     

Characteristics of angiotensin II-, K+- and ACTH-induced calcium influx in adrenal glomerulosa cells. Evidence that angiotensin II, K+, and ACTH may open a common calcium channel

The characteristics of angiotensin II-, K+-, and adrenocorticotropin (ACTH)-induced calcium influx were studied in isolated adrenal glomerulosa cells. Basal calcium influx rate is 0.64 +/- 0.09 nmol/min/mg of protein. Addition of angiotensin II (1 nM) causes a rapid 230% increase in calcium influx rate. This angiotensin II-induced calcium influx is sustained and is rapidly reversed by angiotensin II antagonist, [Sar1,Ala8]angiotensin II. Addition of either K+ or ACTH (1 nM) causes a 340 or 160% increase, respectively, in the rate of calcium influx. The effect of either angiotensin II, K+, or ACTH on calcium influx is dependent on extracellular calcium. The apparent Km for calcium is 0.46, 0.35, and 0.32 mM, respectively. When the extracellular concentration of K+ is 2 mM, neither angiotensin II nor ACTH stimulates calcium influx. Conversely, when extracellular K+ is increased to 6 mM, both angiotensin II and ACTH cause a greater stimulation of calcium influx than at 4 mM K+. When extracellular K+ is increased to 10 mM, calcium influx is 360% of the basal influx seen at 4 mM K+, and neither angiotensin II nor ACTH further stimulates the influx rate. Nitrendipine (1 microM) blocks both angiotensin II- and K+-induced calcium influx completely. In contrast, 10 microM nitrendipine does not completely block ACTH-induced calcium influx. The calcium channel agonist, BAY K 8644, also stimulates calcium influx; 10 nM BAY K 8644 leads to a rate of calcium influx which is 185% of basal. This BAY K 8644-induced increase in calcium influx and that caused by either angiotensin II or ACTH are additive. In contrast, BAY K 8644 has more than an additive effect on the calcium influx when paired with 6 mM K+. These results suggest that angiotensin II, K+, and ACTH stimulate calcium influx via a common calcium channel but act by different mechanisms to alter its function.     

ACTH-induced inhibition of the action of angiotensin II in bovine zona glomerulosa cells. A modulatory effect of cyclic AMP on the angiotensin II receptor.

Both angiotensin II and adrenocorticotropic hormone (ACTH) are well known to play a crucial role on the regulation of aldosterone production in adrenal glomerulosa cells. Recent observations suggest that the steroidogenic action of ACTH is mediated via the cAMP messenger system, whereas angiotensin II acts mainly through the phosphoinositide pathway. However, there have been no reports concerning the interaction between the cAMP messenger system activated by ACTH and the Ca2+ messenger system induced by angiotensin II. Both ACTH and angiotensin II simultaneously act on adrenal cells for regulating steroidogenesis under physiological conditions. Thus the present experiments were performed to examine the effect of ACTH on the action of angiotensin II by measuring angiotensin II receptor activity, cytosolic Ca2+ movement, and aldosterone production. The major findings of the present study are that short-term exposure to a high dose of ACTH (10(-7) M) inhibited 125I-angiotensin II binding to bovine adrenal glomerulosa cells, decreased the initial spike phase of [Ca2+]i induced by angiotensin II, and inhibition of angiotensin II-induced aldosterone production. Low dose of ACTH (10(-10) M), which did not increase cAMP formation, did not affect angiotensin II receptor activity. These studies have shown that angiotensin II receptors of bovine adrenal glomerulosa cells can be down-regulated by 1 mM dibutyryl cyclic AMP, as well as by effectors which are able to activate cAMP formation (10(-7) M ACTH and 10(-5) M forskolin). The rapid decrease in angiotensin II receptors induced by 10(-7)M ACTH was associated with a decreased steroidogenic responsiveness and a decreased rise in the [Ca2+]i response induced by angiotensin II. These studies show that the cAMP-dependent processes activated by ACTH have the capacity to interfere with signal transduction mechanisms initiated by receptors for angiotensin II.     

The effects of extracellular K+ and angiotensin II on cytosolic Ca++ and steroidogenesis in adrenal glomerulosa cells

We evaluated changes in cytosolic calcium concentration (Ca++) and steroidogenesis in rat adrenal glomerulosa cells (GC) stimulated with potassium (K+) or angiotensin II (AII). Cytosolic Ca++ concentration was determined using the Ca++-sensitive, fluorescent dye QUIN 2. Raising extracellular K+ increased cytosolic Ca++ from 267 +/- 23 nM at 3.7 mM K+ to a maximum of 377 +/- 40 nM at 8.7 mM K+ (p less than 0.01, N = 23). AII also increased cytosolic Ca++ from 238 +/- 20 nM to a maximum of 427 +/- 42 nM at 10(-7) M (p less than 0.01, N = 16). In parallel studies, K+ and AII stimulated aldosterone secretion from QUIN 2-loaded GC at concentrations similar to those which raised cytosolic Ca++. QUIN 2-loaded cells were as responsive steroidogenically as unloaded cells and showed trypan blue exclusion of 98% suggesting that QUIN 2 did not compromise cellular viability. These results provide direct support for a role of cytosolic Ca++ as a second messenger during stimulation of aldosterone secretion by both K+ and AII.     

Serotonin stimulates calcium influx in isolated rat adrenal zona glomerulosa cells.

To investigate the role of calcium as a second messenger in serotonin-stimulated aldosterone secretion, radiolabelled calcium influx studies were carried out in purified rat adrenal zona glomerulosa cells using 45CaCl2. The results show that serotonin caused calcium influx within 45 seconds of addition and this continued for up to 105 seconds. Angiotensin II also caused calcium influx; however, the effect was significantly smaller than that of serotonin. Serotonin-stimulated calcium influx could be inhibited by the calcium antagonist verapamil and by methysergide, a selective serotonin receptor type-1/2 antagonist. The data indicate that serotonin directly stimulates calcium uptake in zona glomerulosa cells via calcium channels which are coupled to specific serotonin receptors.     

Temporal patterns of protein phosphorylation after angiotensin II, A23187 and/or 12-O-tetradecanoylphorbol 13-acetate in adrenal glomerulosa cells.

The temporal patterns of protein phosphorylation in the adrenal glomerulosa cell were analysed by two-dimensional electrophoresis after stimulation with 10 nM-angiotensin II or various agents [10 nM-12-O-tetradecanoylphorbol 13-acetate (TPA), 50 nM-A23187, 1 microM-nitrendipine], administered singly or in combination. These patterns were compared with the temporal patterns of aldosterone secretion induced by the same agonists and antagonists. After 1 and 30 min of stimulation with angiotensin II, different patterns of protein phosphorylation were observed. A comparison of these patterns reveals that: the phosphorylation of only one protein was persistently enhanced during the continuous incubation with angiotensin II; the phosphorylation of five proteins was transiently enhanced (at 1 min but not 30 min); and the phosphorylation of three proteins did not occur at 1 min but was seen at 30 min. Addition of the phorbol ester TPA alone, which at 30 min is without effect in enhancing aldosterone production, has no effect on protein phosphorylation. The combined addition of TPA and the Ca2+ ionophore, A23187, which, like angiotensin II, evokes a sustained increase in aldosterone production, reproduced the temporal patterns of protein phosphorylation seen after angiotensin II action. Manipulations (A23187 alone, angiotensin II plus nitrendipine) which evoke only a transient rise in aldosterone production rate induce a transient rise in cellular protein phosphorylation. The 1 min patterns of phosphorylation seen after A23187 or combined angiotensin II and nitrendipine (a Ca2+ channel antagonist) are similar to those observed after 1 min of angiotensin II stimulation. These results suggest that, when angiotensin II acts, the initial cellular response is mediated by a different mechanism than that responsible for the sustained response.     

Phorbol ester inhibits angiotensin-induced activation of phospholipase C in adrenal glomerulosa cells. Its implication in the sustained action of angiotensin.

The present study was undertaken to determine whether an agonist-induced activation of C-kinase leads to an inhibition of phospholipase C in adrenal glomerulosa cells. When cells are treated with 100 nM-TPA (12-O-tetradecanoylphorbol 13-acetate), subsequent angiotensin ('angiotensin II')-induced aldosterone secretion is greatly inhibited. Treatment with TPA completely inhibits the angiotensin-induced increase in both inositol trisphosphate and the cytosolic Ca2+ concentration. The dose-response curve for TPA-induced inhibition reveals that quite a high concentration of TPA is necessary to block angiotensin action compared with that needed to stimulate aldosterone secretion. 1-Oleoyl-2-acetylglycerol has a weak inhibitory effect, whereas neither 4 alpha-phorbol 12,13-didecanoate or 4 beta-phorbol inhibits angiotensin action. When the time course of changes in inositol trisphosphate and diacylglycerol is measured, angiotensin action is sustained for up to 30 min. In addition, 100 nM-TPA added after 20 min of angiotensin addition attenuates production of both inositol trisphosphate and diacylglycerol. These results suggest that high dose of TPA inhibits angiotensin-induced activation of phospholipase C by acting, at least partly, on C-kinase, but that an inhibitory effect of TPA may be a pharmacological effect with little physiological significance in this system.     

Evidence for two distinct voltage-gated calcium channel currents in bovine adrenal glomerulosa cells

We analyzed inward Ca2+ currents in single bovine adrenal glomerulosa cell using whole-cell patch clamp techniques. Two types of voltage-gated Ca2+ channel currents were identified. One was a transient (T) type which decayed within 100 ms, characterized by a low threshold voltage (about -70 mv) similar to that seen in rat adrenal glomerulosa cells (Matsunaga, H. et al. (1987) Pflügers Arch. 408, 351-355.) Another was a long-lasting (L) type which shows a more positive threshold potential. The present results suggest that while T type Ca2+ channels may explain initial calcium influx in response to an elevation in extracellular K+, L type Ca2+ channels may allow sustained calcium influx which is necessary for sustained aldosterone secretion.