Angiotensin II (AngII) plays a crucial role in the control of aldosterone biosynthesis in adrenal glomerulosa cells through the stimulation of two distinct Ca2+ entry pathways: (1) opening of voltage-operated calcium channels, and (2) activation of a capacitative Ca2+ entry that is dependent on calcium release from intracellular pools. Adrenocorticotrophic hormone (ACTH), on the other hand, a major hormonal regulator of steroidogenesis, induces an increase in intracellular cAMP through the activation of a G-protein-coupled adenylyl cyclase. Recent studies have demonstrated that the rise in cAMP induced by ACTH can be potentiated by AngII in bovine glomerulosa cells. The aim of the present study was to investigate the mechanism of AngII action on ACTH-induced cAMP production. In primary cultures of bovine glomerulosa cells, we found that AngII (100 nM), which had no effect by itself on cAMP production, significantly potentiated maximal ACTH-induced cAMP formation in the presence of extracellular calcium (1.2 mM). In contrast, in the absence of extracellular calcium, AngII did not affect ACTH-induced cAMP production. These results suggest that calcium entry into the cell plays an important role in the activation of the cyclase by AngII. The inhibition of voltage-operated calcium channels by nicardipine, a dihydropyridine calcium antagonist blocking both low-threshold (T-type) and high-threshold (L-type) Ca2+ channels, did not significantly affect the potentiating effect of AngII. Moreover, the cAMP response to ACTH was insensitive to activation of these Ca2+ channels induced by potassium ions and, even when cytosolic free-calcium concentration ([Ca2+]c) was kept elevated with the Ca2+ ionophore, ionomycin, no stimulation of adenylyl cyclase was observed at concentrations of [Ca2+]c up to 640 nM. In contrast, thapsigargin, an activator of capacitative Ca2+ influx, mimicked the potentiating effect of AngII on ACTH-induced cAMP formation. In agreement with the characteristics of cAMP modulation by Ca2+ in these cells, the presence of type III adenylyl cyclase was observed by immunodetection in bovine glomerulosa cell membranes. In conclusion, these data suggest a tight coupling between the capacitative Ca2+ influx induced upon stimulation by either AngII or thapsigargin and a calcium-sensitive isoform of adenylyl cyclase, probably type III, in bovine glomerulosa cells.

No abstract

In adrenal glomerulosa cells, low threshold voltage-activated (T-type) calcium channels play a crucial role in coupling physiological variations of extracellular potassium to aldosterone biosynthesis. Angiotensin II markedly reduced the activity of these channels by shifting their activation curve toward positive voltage values. This inhibition of the channels resulted in a marked decrease of the cytosolic free calcium concentration maintained by potassium. This effect was abolished by losartan, a specific antagonist of the angiotensin II AT1 receptor. Hormone action on T-type channels appeared to be mediated by protein kinase C because 1) it was mimicked by phorbol ester and diacylglycerol, and 2) it was significantly reduced by decreasing protein kinase C activity with specific inhibitors such as chelerythrine chloride or a pseudosubstrate of the enzyme, as well as by protein kinase C down-regulation. Similarly, protein kinase C activation reduced the cytosolic calcium response to potassium and the steroidogenic action of this agonist. Low threshold T-type calcium channels therefore appear as potential sites for the modulation of steroidogenesis by protein kinase C in adrenal glomerulosa cells.

Angiotensin-II (AngII)-induced Ca2+ influx in adrenal glomerulosa cells, a signal necessary for the stimulation of steroidogenesis by the hormone, is believed to involve two distinct mechanisms: 1) opening of voltage-operated Ca2+ channels, and 2) activation of a capacitative Ca2+ entry pathway that is dependent on calcium release from intracellular stores. Nicardipine, a dihydropyridine calcium antagonist, has been used to investigate the role of these Ca2+ entry mechanisms in the steroidogenic response to AngII. As demonstrated with the patch-clamp technique, micromolar concentrations of nicardipine completely blocked voltage-operated Ca2+ channel activity of both T- and L-types. This agent similarly inhibited the rise of cytosolic free calcium concentration induced by potassium, but did not significantly affect the response to thapsigargin, an activator of the capacitative pathway. Nicardipine reduced by only 22% the calcium influx stimulated by AngII, and the nicardipine-insensitive part of this response was abolished after exhausting the intracellular Ca2+ stores with thapsigargin. Similarly, aldosterone secretion induced by AngII was only partially inhibited (40%) by nicardipine at concentrations that completely abolished the steroidogenic response to potassium. Thapsigargin by itself was able to stimulate aldosterone production, an action highly potentiated by physiological concentrations of extracellular potassium. These data strongly suggest that the major part of the calcium influx response to AngII, leading to aldosterone formation, involves a capacitative calcium entry pathway activated by the release of calcium from intracellular stores. This mechanism of calcium influx could be responsible for some features of aldosterone response to the hormone, such as its poor sensitivity to dihydropyridines or its potentiation by potassium.

Calcium influx into adrenal glomerulosa cells is a key event during the stimulation of aldosterone secretion by physiological increases in extracellular potassium concentrations. Two types of voltage-operated calcium channels, T- and L-types, are present on bovine glomerulosa cells, but their respective functions are not yet clearly defined. Using the patch-clamp method in the perforated patch configuration combined with microfluorimetry of cytosolic calcium, we demonstrate that L-type channels are exclusively responsible for the sustained elevation of cytosolic calcium observed upon stimulation with extracellular potassium, even at low, physiological concentrations of this agonist. In contrast, aldosterone secretion appears closely related to T-type channel activity. Moreover, when the activity of each channel type is selectively modulated by pharmacological agents, such as dihydropyridines or zonisamide, the cytosolic calcium response can be clearly dissociated from the steroidogenic response. Similarly, modulation of T channel activation by protein kinase C results in a parallel inhibition of aldosterone secretion, without any effect on the levels of cytosolic free calcium. This direct functional link between T-type calcium channel activity and steroidogenesis suggests a model in which calcium entering the cell through these channels bypasses the cytosol to activate intramitochondrial steps of aldosterone biosynthesis.

Thapsigargin, an inhibitor of the microsomal Ca2+ pumps, has been extensively used to study the intracellular Ca2+ pool participating in the generation of the agonist-induced Ca2+ signal in various cell types. A dual effect of this agent was observed in bovine adrenal zona glomerulosa cells. At nanomolar concentrations, thapsigargin stimulated a sustained Ca2+ influx, probably resulting from Ca(2+)-store depletion. In contrast, when added at micromolar concentrations, thapsigargin prevented the rise in cytosolic free Ca2+ concentration ([Ca2+]c) induced by K+. This inhibitory effect of thapsigargin on voltage-activated Ca2+ channels was confirmed by measuring Ba2+ currents by the patch-clamp technique. Both low-threshold (T-type) and high-threshold (L-type) Ca2+ channels were affected by micromolar concentrations of thapsigargin. Analysis of the current-voltage relationship for T-type channels revealed that thapsigargin did not modify the sensitivity of these channels to the voltage, but decreased the maximal current flowing through the channels. In conclusion, thapsigargin appears to exert a dual effect on adrenal glomerulosa cells. At lower concentrations, this agent induces a sustained Ca2+ entry, whereas at higher concentrations it decreases [Ca2+]c by blocking voltage-activated Ca2+ channels.

Angiotensin-II (AngII)-induced Ca2+ influx in adrenal glomerulosa cells, a signal necessary for the stimulation of steroidogenesis by the hormone, is believed to involve two distinct mechanisms: 1) opening of voltage-operated Ca2+ channels, and 2) activation of a capacitative Ca2+ entry pathway that is dependent on calcium release from intracellular stores. Nicardipine, a dihydropyridine calcium antagonist, has been used to investigate the role of these Ca2+ entry mechanisms in the steroidogenic response to AngII. As demonstrated with the patch-clamp technique, micromolar concentrations of nicardipine completely blocked voltage-operated Ca2+ channel activity of both T- and L-types. This agent similarly inhibited the rise of cytosolic free calcium concentration induced by potassium, but did not significantly affect the response to thapsigargin, an activator of the capacitative pathway. Nicardipine reduced by only 22% the calcium influx stimulated by AngII, and the nicardipine-insensitive part of this response was abolished after exhausting the intracellular Ca2+ stores with thapsigargin. Similarly, aldosterone secretion induced by AngII was only partially inhibited (40%) by nicardipine at concentrations that completely abolished the steroidogenic response to potassium. Thapsigargin by itself was able to stimulate aldosterone production, an action highly potentiated by physiological concentrations of extracellular potassium. These data strongly suggest that the major part of the calcium influx response to AngII, leading to aldosterone formation, involves a capacitative calcium entry pathway activated by the release of calcium from intracellular stores. This mechanism of calcium influx could be responsible for some features of aldosterone response to the hormone, such as its poor sensitivity to dihydropyridines or its potentiation by potassium.

Bufo marinus bladder H-K-ATPase belongs to the Na-K-ATPase and H-K-ATPase subfamily of oligomeric P-type ATPases and is closely related to rat and human nongastric H-K-ATPases. It has been demonstrated that this ATPase transports K(+) into the cell in exchange for protons and sodium ions, but the stoichiometry of this cation exchange is not yet known. We studied the electrogenic properties of B. marinus bladder H-K-ATPase expressed in Xenopus laevis oocytes. In a HEPES-buffered solution, K(+) activation of the H-K-ATPase induced a slow-onset inward current that reached an amplitude of approximately 20 nA after 1-2 min. When measurements were performed in a solution containing 25 mM HCO at a PCO(2) of 40 Torr, the negative current activated by K(+) was reduced. In noninjected oocytes, intracellular alkalization activated an inward current similar to that due to B. marinus H-K-ATPase. We conclude that the transport activity of the nongastric B. marinus H-K-ATPase is not intrinsically electrogenic but that the inward current observed in oocytes expressing this ion pump is secondary to intracellular alkalization induced by proton transport.