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Sustained phospholipase D activation in response to angiotensin II but not carbachol in bovine adrenal glomerulosa cells.

ABSTRACT: We have demonstrated previously that in bovine adrenal glomerulosa cells, phospholipase D (PLD) activity can indirectly result in the generation of sn-1,2-diacylglycerol (DAG) through its production of phosphatidic acid (PA) and the subsequent action of PA phosphohydrolase. Furthermore, the PLD-generated DAG can trigger aldosterone secretion. Therefore, we characterized PLD activation by two agonists, angiotensin II (Ang II) and carbachol, to determine if the activity of the enzyme might underlie sustained aldosterone secretion. We determined that Ang II-induced PLD activation occurred via the angiotensin-1 receptor (AT1), and that a specific AT1 antagonist, losartan, inhibited this activation, whereas the same concentration of the AT2-specific antagonist, PD 123319, had no effect. Ang II activated PLD with a dose dependence similar to that observed for aldosterone secretion, with slight increases in activity induced by 0.1 nM Ang II and maximal activation at 10 nM. We also found that Ang II induced a sustained activation of PLD, but that the effect of carbachol, a stable analogue of acetylcholine, was transient; PLD activity increased within 5 min of exposure to carbachol but then ceased by 15 min. Higher carbachol concentrations were also unable to sustain PLD activation. These results suggest that the Ang II-elicited activation of PLD is associated with a sustained increase in aldosterone secretion from glomerulosa cells and further provide the first evidence, to our knowledge, of differences in the kinetics of PLD activation in response to two physiologically relevant agonists. Finally, we speculate that this disparity correlates with different functional responses induced by the two agents.

SUBMITTER: Jung E

PROVIDER: S-EPMC1219159 | biostudies-other | 1998 Feb

REPOSITORIES: biostudies-other

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Project description: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.

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Sustained phospholipase D activation in response to angiotensin II but not carbachol in bovine adrenal glomerulosa cells.

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