Tamás Balla

Possible role of calcium uptake and calmodulin in adrenal glomerulosa cells: Effects of verapamil and trifluoperazine

The effects of verapamil and trifluoperazine were examined on isolated rat adrenal glomerulosa cells so as to assess the role of calcium ion influx and calmodulin in the function of this cell population. Verapamil (10(-5) and 10(-4) moles/1) slightly reduced the basal production rate of aldosterone and strongly inhibited the response to angiotensin II, potassium ions, corticotrophin (ACTH) and dibutyryl cyclic AMP (db-cAMP). The concentration of verapamil required to reduce the response to these agonists by 50% varied between 2 and 6 mumoles/1. Trifluoperazine (30 mumoles/1) slightly increased the basal production rate of aldosterone. The response to angiotensin and potassium was variably antagonized by 3 mumoles/1 trifluoperazine and completely inhibited by the drug at 30 mumoles/1. The antagonist at a concentration of 3 mumoles/1 exerted either a facilitatory or inhibitory effect on the response to ACTH and db-cAMP, depending on the concentration of the agonist. Trifluoperazine at a concentration of 30 mumoles/1 reduced the response to both agonists to a level which was 2-3 fold higher than that observed in appropriate control samples. The present results indicate that (1) calcium influx is an essential event in the aldosterone stimulating action of angiotensin II, potassium ions, ACTH and cyclic AMP; (2) stimulation by angiotensin II and potassium ions are completely dependent on calmodulin; (3) stimulation by ACTH and cyclic AMP is mediated by calmodulin-dependent and independent mechanisms.

Effects of lithium on angiotensin-stimulated phosphatidylinositol turnover and aldosterone production in adrenal glomerulosa cells: a possible causal relationship

Turnover of 32P‐labelled phosphatidylinositol (PI) was examined in isolated adrenal glomerulosa cells. Increased incorporation of [32P] phosphate into PI in response to angiotensin II was completely prevented by Li+. A simultaneous accumulation of 32P activity in phosphatidic acid (PA) was also observed. Angiotensin II increased the breakdown of PI despite the presence of Li+. These results suggest that Li is a suitable tool to interrupt the accelerated PI cycle in angiotensin‐stimulated cells. Aldosterone production of superfused cells was inhibited by Li+ when the cells were stimulated with angiotensin II. On the other hand, Li+ did not inhibit the aldosterone response of the cells to ACTH, a hormone which acts via cyclic AMP and does not enhance PI turnover in these cells. On the basis of these results, we assume that the inhibitory effect of Li+ on aldosterone production is related to its effect on PI turnover.

Control of phosphatidylinositol turnover in adrenal glomerulosa cells

The purpose of the present experiments was to compare the effects on phosphatidylinositol metabolism of agents stimulating aldosterone secretion. Glomerulosa cells, isolated from rat adrenals, were incubated in the presence of one of the following stimuli: angiotensin II, elevated potassium concentration, corticotropin, dibutyryl cyclic AMP and prostaglandin E2. Of all these substances, only angiotensin II stimulated the incorporation of [32P]phosphate into phosphatidylinositol. The effect was already detected 2.5 min and was still maintained 60 min after the onset of stimulation. A slight enhancement of the incorporation into other phospholipids was observed in the first minutes of stimulation. Cycloheximide abolished the effect of angiotensin II on aldosterone production, but not on phosphatidylinositol synthesis. In cells prelabelled with [32P]phosphate, radioactivity in phosphatidylinositol relative to that in other phospholipids decreased in response to angiotensin II within 5 min. This indicates that angiotensin II induces a specific breakdown of phosphatidylinositol. Corticotropin failed to enhance the incorporation of [32P]phosphate into phosphatidylinositol and other phospholipids in isolated fasciculate-reticularis cells. The results suggests that although both angiotensin II and potassium are presumed to act through changes in calcium metabolism, angiotensin alone generates the calcium signal by increased phosphatidylinositol turnover.

High-performance reversed-phase ion-pair chromatographic study of myo-inositol phosphates: Separation of myo-inositol phosphates, some common nucleotides and sugar phosphates

A detailed study of all the major chromatographic variables affecting the retention behaviour and separation of myo-inositol phosphates in reversed-phase ion-pair chromatographic systems was carried out. The parameters studied included the eluent concentration of the pairing ion, the eluent concentration of the organic modifier and the buffer salt, the pH of the eluent, the minimum column plate count necessary for the separation of the inositol trisphosphate isomers and isocratic and gradient modes of separation. The retention behaviour of some common nucleotides and sugar phosphates was also investigated as these phosphates present chromatographic interference problems in biochemical studies based on the cellular incorporation of [32P]Pi. The separation methods developed appear to be superior to established anion-exchange separation techniques in terms of separation speed and mildness of the chromatographic conditions.

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.

The effect of angiotensin II on arachidonate metabolism in adrenal glomerulosa cells

The effect of angiotensin II on arachidonate metabolism was examined in rat adrenal glomerulosa cells. Incorporation of both [3H]arachidonate and [32P]phosphate into phosphatidylinositol (PI) were significantly stimulated by angiotensin II. These effects were abolished by lithium, a cation, which was found suitable to prevent increased synthesis of PI in our previous study (T. Balla et al., FEBS Letters 171, 179, 1984). On the other hand, the phospholipase A2 inhibitor mepacrine failed to inhibit the increased labelling of PI. These observations suggest that the increased 3H labelling of PI occurs via CDP-diacylglycerol, and not via enhanced deacylation-reacylation cycle. The validity of this assumption was further supported, since angiotensin II failed to stimulate the formation of lyso-PI, as examined by both [32P]phosphate incorporation and pulse-chase techniques. Angiotensin II decreased the incorporation of [3H]arachidonate into phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Considering that we did not find arachidonate release either from phospholipids or from other possible arachidonate sources this decrease may not be due to dilution of the tracer. Thus we assume that angiotensin II may induce a shift in phospholipid synthesis from PC and PE to phosphoinositides. These observations indicate that the enhanced hydrolysis and synthesis of PI in response to angiotensin II is not associated with increased phospholipase A2 activity in adrenal glomerulosa cells.

Angiotensin II stimulates phosphatidylinositol turnover in adrenal glomerulosa cells by a calcium-independent mechanism.

Angiotensin II enhances phosphatidylinositol turnover in isolated adrenal glomerulosa cells. In the present experiments we examined whether this effect required the presence of extracellular Ca2+. It was found that neither the stimulation of phosphatidylinositol breakdown nor the stimulation of incorporation of [32P]phosphate into phosphatidic acid and phosphatidylinositol required the presence of extracellular Ca2+. These observations suggest that the enhancement of phosphatidylinositol turnover may precede, but does not depend on, angiotensin-induced Ca2+ influx.

Role of calcium ions and calmodulin in the aldosterone stimulating action of prostaglandin E2.

Verapamil (10−6-10−4). an antagonist of calcium influx inhibited prostaglandin E2 (PGE2)-induced aldosterone production by isolated rat glomerulosa cells. The concentration required to inhibit the response of aldosterone production by 50% was 2.2 μM on average and did not depend on the concentration of PGE2. The stimulatory effect of PGE2 was almost completely antagonized by 30 μM trifluoperazine. an inhibitor of calmodulin.

The effect of endogenous angiotensin on the biosynthesis of aldosterone in the rat.

Abstract Modification of the site of action of angiotensin II in adrenal cortex by the duration of stimulation and by changes in salt-water balance was examined in three different forms of sodium depletion. Increased formation of angiotensin was induced in conscious rats by peritoneal dialysis with glucose solution, administration of the diuretic drug furosemide or dietary sodium restriction. The conversion of corticosterone to aldosterone by incubated adrenal capsular tissue was estimated by conversion techniques using tracer amounts and excess amounts of substrate. Acute peritoneal or diuretic sodium depletion failed to affect the conversion rate of tracer amount of [ 3 H]-corticosterone to [ 3 H]-aldosterone but decreased the specific activity of [ 3 H]-aldosterone. Dietary sodium restriction increased, peritoneal dialysis did not change and administration of furosemide decreased the formation of aldosterone from excess amounts of exogenous corticosterone in the presence of cyanoketone. These results, together with the results of our previous superfusion experiments, suggest that increased conversion of corticosterone to aldosterone is a phenomenon specific for chronic sodium depletion. In acute sodium deficiency the action of angiotensin II is confined to a step before the formation of corticosterone. The importance of discrepancies between tracer and excess substrate conversion experiments is discussed.