Angiotensin-induced steroidogenesis in rabbit adrenal: effects of pH and calcium.

The effect of variations in pH and Ca2+ on angiotensin II (A-II)-induced steroidogenesis was tested on isolated adrenal glomerulosa cell suspensions. The results show that a reduction in pH from 7.4 to 6.5 produces both a shift to the left of the A-II dose-response curve as well as an increase in maximum steroid production. In contrast, removal of Ca2+ from the incubation medium virtually abolished steroidogenesis to A-II (5 X 10(-9)M(, KCl(10mM) and ACTH (250 microU/ml). The Ca2+ antagonist D-600, however, was less effective than simple removal of Ca2+ as 10(-4) M was required to block the steroidogenic response to these same agonists. The results indicate that the response characteristics of this system to A-II resemble most closely those seen with isolated arterial smooth muscle - especially rabbit aortic strips.     

Ovine adrenal fasciculata cells contain angiotensin-II receptors coupled to intracellular effectors but are resistant to the steroidogenic effects of this hormone

Ovine adrenal fasciculata cells (OAC) responded to ACTH but were resistant to the steroidogenic action of angiotensin-II (A-II), while bovine adrenal fasciculata cells (BAC) responded to this hormone as well as to ACTH. However both cell types contained specific A-II binding sites (120,000 +/- 14,000 and 85,000 +/- 10,000 sites per cell for OAC and BAC, respectively) of similar high affinity [dissociation constant (KD) congruent to 2 x 10(-9) M]. Moreover, in both cell types, A-II receptors were coupled to intracellular effectors since A-II: 1) stimulated the accumulation of inositol phosphates, although the effects in BAC were higher than in OAC; 2) enhanced the influx and the efflux of 45Ca2+; 3) increased cytosolic free Ca2+ concentration ([Ca2+]i); 4) potentiated ACTH-induced cAMP production; and 5) induced A-II receptor loss. Both cell types appear to have an active protein kinase C since the phorbol ester 4 beta-phorbol 12-myristate-13-acetate potentiates ACTH-induced cAMP production and caused A-II receptor loss. In addition, 4 beta-phorbol 12-myristate-13-acetate and Ca2+ ionophore enhanced the steroid production by BAC but had no effect on OAC. These results indicated that the steroidogenic refractoriness of OAC to A-II might involve some step(s) beyond the initial activation of the two branches of the phosphoinositide pathway, activation of protein kinase C and increase of [Ca2+]i, and before conversion of cholesterol to pregnenolone.     

Interferon-inducible genes in the rat adrenal gland and vascular smooth muscle cells

Chronic stimulation of the renin-angiotensin system results in increased zona glomerulosa cells and in cells expressing the final enzyme in the synthesis of aldosterone, the cytochrome P-450 aldosterone synthase. The genes activated during adrenal remodeling are not well defined. We have reported that the expression of interferon-inducible genes, 9–27, 1–8D and 1–8U in H295R cells is stimulated by A-II. The 9–27 gene is expressed mainly in leukocytes and is associated with cell proliferation. In this study, we searched for similar genes in a rat zona glomerulosa cDNA library, and examined the regulation of the expression of these genes. We found the Rat8 gene, which has been reported to be similar to human interferon-inducible genes, as well as two similar genes, No. 10 (1096 bp), and No. 16 (630 bp). Rat8 gene and No. 16 were mainly expressed in zona glomerulosa. The product of No. 10 is thought to be a secreted protein, unlike those of 8 and 16, and its expression in the adrenal was weak in comparison. The control of the expression of rat8 or No. 16 genes differs depending on the tissue. Expression in A10 cells (derived from rat embryo thoracic aorta) was not stimulated by A-II, nor was it influenced by salt intake in the adrenal gland, but it was reduced in vascular smooth muscle cells (VSMC) of rats on a low sodium diet. These results show that genes similar to the human 1–8 gene family are expressed in rat adrenal glomerulosa cells and VSMC, but their expression is not regulated by A-II. The function of these genes in VSMC and adrenal is unknown.     

High-density lipoprotein increases intracellular calcium levels by releasing calcium from internal stores in human endothelial cells

Elevated levels of high-density lipoproteins (HDL) appear to delay or prevent the development of atherosclerosis. The intracellular signaling mechanisms activated by HDL in vascular cells are currently under active investigation. In this study the effects of HDL on endothelial intracellular Ca levels (EC Ca(i)) are investigated. We show that HDL, like low density lipoproteins (LDL), increases EC Ca(i) in a dose-dependent fashion by releasing Ca from internal stores. Neither apolipoprotein A-I (apo A-I) nor apolipoprotein A-II (apo A-II) was responsible for the increase in EC Ca(i). HDL appeared to release Ca from the same internal stores as did LDL, since preincubation of EC with LDL prevented subsequent responses to HDL but not to the vasodilator ATP. In addition, preincubation of EC with pertussis toxin, an inhibitor of specific G proteins, as well as U73122, an inhibitor of phospholipase C, prevented a rise in EC Ca(i) in response to HDL. These findings suggest that HDL, like LDL, can modulate EC Ca(i) and that this occurs via a pertussis toxin-sensitive G protein-mediated pathway which involves phospholipase C.     

Activation of dihydropyridine-sensitive calcium channels and biphasic cytosolic calcium responses by angiotensin II in rat adrenal glomerulosa cells

The steroidogenic actions of angiotensin II (AII) and increased extracellular K+ concentrations [( K+]) in rat adrenal glomerulosa cells are selectively enhanced by the voltage-sensitive calcium channel agonist Bay K 8644 (BK 8644). The relationship between these effects of the dihydropyridine agonist and cytosolic calcium concentration [( Ca2+]i) was investigated in rat and bovine glomerulosa cells. In the rat glomerulosa cells, AII and increased [K+] elicited rapid elevations of [Ca2+]i with distinctive temporal characteristics. Whereas the [Ca2+]i response to [K+] declined to basal over 2-3 min, addition of 10 nM AII caused a biphasic increase in [Ca2+]i, with a rapid transient rise followed by a lower plateau phase that remained above basal for several minutes. BK 8644 alone did not affect [Ca2+]i, but at low concentrations (30 nM) increased the magnitude and duration of the [Ca2+]i response elicited by progressive elevation of extracellular [K+] to 12 mM. In AII-stimulated glomerulosa cells, 30 nM BK 8644 enhanced both phases of the cytosolic calcium response, with a more marked effect on the sustained plateau phase. In contrast to its prominent actions in rat glomerulosa cells, BK 8644 had no effect on AII-stimulated rises in [Ca2+]i in bovine glomerulosa cells, and only slightly enhanced their minor [Ca2+]i responses to potassium. These studies provide evidence that AII activates dihydropyridine-sensitive voltage-sensitive calcium channels in rat, but not bovine, adrenal glomerulosa cells. They also suggest that enhancement by BK 8644 of agonist-stimulated [Ca2+]i changes is responsible for its synergistic effects on aldosterone responses to potassium and AII in rat glomerulosa cells and emphasize the importance of the sustained phase of the cytosolic calcium signal in the steroidogenic action of AII.     

Stimulatory effect of insulin and insulin-like growth factor I on Gi proteins and angiotensin-II-induced phosphoinositide breakdown in cultured bovine adrenal cells

Recent data have shown that pretreatment of bovine adrenal fasciculata cells with insulin-like growth factor I (IGF-I) or insulin enhances the steroidogenic response to angiotensin II (A-II). In the present work we have studied the effects of both peptides on the first steps of the mechanism of action of A-II and on the amounts of pertussis toxin (PT)-sensitive guanine nucleotide binding proteins (Gi proteins). Both peptides increased A-II-induced phosphoinositide breakdown without modification of either A-II-induced Ca2+ uptake or the A-II-potentiating effect on ACTH-induced cAMP production. The effects of IGF-I at a nanomolar concentration were higher than those induced by insulin at a micromolar concentration, which in turn was higher than those induced by a nanomolar concentration of this peptide. Treatment of cells with pertussis toxin (0.5 microgram/ml) for 24 h reduced by 25% of the A-II-induced phosphoinositide breakdown in control cells and 32% and 28% in cells pretreated with insulin at nanomolar and micromolar concentrations, respectively, but had no significant effect in cells pretreated with IGF-I. No effect of pertussis toxin was observed on A-II-induced Ca2+ uptake or on its potentiating action on ACTH-induced cAMP production. Moreover, both IGF-I and insulin enhanced the amounts of Gi protein(s) evaluated by pertussis toxin ADP-ribosylation or immunoblotting. Again, the effects of insulin at nanomolar concentrations were lower than those induced by the same concentrations of IGF-I or insulin at micromolar concentrations. These results suggest that, in bovine adrenal fasciculata cells, A-II receptors are coupled to the phosphoinositide pathway through pertussis toxin sensitive and insensitive Gp protein(s). Moreover, the findings also indicate that the enhanced A-II responsiveness of IGF-I or insulin treated cells is in part mediated through an increase in the amount of G protein(s).     

Altered turnover of polyphosphoinositides in the erythrocyte membrane of the spontaneously hypertensive rat

The metabolism of inositol phospholipids of the erythrocyte membrane was compared in normotensive Wistar-Kyoto (WKY), spontaneously hypertensive (SHR), and stroke-prone SHR (SHR-SP) rats. This was performed on isolated ghost membranes by measuring the incorporation of 32P from [ gamma-32P ] adenosine triphosphate (ATP) into the diphosphoinositides (DPI) and the triphosphoinositides (TPI) which were the only 32P-labeled phospholipids. 32P-labeling of TPI was altered in adult and 3-week-old SHR as well as in SHR-SP compared to WKY controls; the radioactivity associated with TPI in hypertensive rats was about 30% lower than that associated with TPI in age-matched normotensive controls. By contrast, the radioactivity associated with DPI was similar in both hypertensive and normotensive rats. Measurement of the phosphoinositide distribution in both SHR and WKY indicates that the change observed in 32P-TPI could not be accounted for by a reduced phosphatidylinositol content in SHR membrane. Measurement of the Mg2+-activated TPI-phosphomonoesterase and of the Ca2+-activated polyphosphoinositide phosphodiesterase activities did not show any significant difference between SHR and WKY. It thus appears that the altered phosphoinositide metabolism observed in hypertensive rats was a consequence of some alteration in the activity of kinases which are responsible for the conversion of phosphatidylinositol into DPI and TPI. These results also suggest that the defect in phosphoinositide metabolism observed in genetically hypertensive rats was not a consequence of the blood pressure elevation and could be related to the pathogenesis of hypertension.     

Angiotensin-II inhibits Na+/K+ pump in rat adrenal glomerulosa cells: possible contribution to stimulation of aldosterone production.

The control of Na+/K+ pump activity was studied in rat adrenal glomerulosa cells. Ninety percent of K+/86Rb accumulation was blocked by ouabain, and the dose-response curve of inhibition by ouabain was monophasic (IC50, approximately 80 microM), suggesting the role of a single type of Na+/K+ pump (alpha-isoenzyme) in 86Rb accumulation by rat glomerulosa cells. The basal activity of the Na+/K+ pump was much higher in glomerulosa cells than in adrenal fasciculata cells or hepatocytes, as judged by the ouabain-sensitive uptake of 86Rb. In contrast to the two other cell types, increasing Na+ influx with the Na+ ionophore monensin failed to significantly affect ouabain-sensitive 86Rb uptake in glomerulosa cells, suggesting that in glomerulosa cells even the resting intracellular Na+ concentration is sufficient for maximal activity of the Na+/K+ pump. Angiotensin-II (AII) inhibited the ouabain-sensitive 86Rb uptake by glomerulosa cells. The effect of AII was abolished by the selective antagonist of the AT1 type of AII receptors (DuP 753), while PD 123177, an AT2 antagonist was ineffective. AT1 receptors of glomerulosa cells coupled to phospholipase-C activation and, thus, to Ca2+ signal. The inhibitory effect of AII was dependent on the extracellular Ca2+ concentration, but an elevation of cytoplasmic Ca2+ by Ca2+ ionophore ionomycin failed to mimic the effect of AII. These data suggest that Ca2+ is required for but does not mediate the inhibitory effect of AII on the Na+/K+pump. Pharmacological activation of protein kinase-C by phorbol ester did not modify 86Rb accumulation by the cells. Ouabain induced a nifedipine-sensitive elevation in the cytoplasmic Ca2+ concentration and exerted a stimulatory effect on aldosterone production, suggesting participation of the inhibition of the Na+/K+ pump in the aldosterone stimulatory action of AII.     

Effect of osmolarity on aldosterone production by rat adrenal glomerulosa cells

The effect of osmotic changes on aldosterone production, [Ca2+]i and voltage-gated Ca2+ currents, was studied in cultured rat glomerulosa cells. Alteration of osmolarity by sucrose addition in the 250-330 mosM range did not influence aldosterone production per se, but it substantially affected K+-stimulated aldosterone production. Hyposmosis markedly increased the hormone response evoked by raising [K+] from 3.6 to 5 mM, whereas hyperosmosis had a mild decreasing effect. Cytoplasmic [Ca2+]i, measured in single glomerulosa cells, did not show detectable change in response to either hyposmotic or hyperosmotic exposure, but the [Ca2+]i signal evoked by elevation of [K+] to 5 mM was augmented in hyposmotic solution. The osmosensitivity of the transient (T)-type and long-lasting (L)-type voltage-gated Ca2+ currents was studied using the nystatin-perforated voltage-clamp technique. Lowering osmolarity to 250 mosM significantly increased the amplitude of the T-type current, and it had a transient augmenting effect on L-type current amplitude. Hyperosmotic solution (330 mosM) reduced L-type current amplitude but did not evoke significant change in T-type current. These results indicate that the responsiveness of rat glomerulosa cells to physiological elevation of [K+] is remarkably influenced by changes in osmolarity by means of modulating the function of voltage-gated Ca2+ channels.     

Triose phosphate isomerase deficiency in 3 French families: two novel null alleles, a frameshift mutation (TPI Alfortville) and an alteration in the initiation codon (TPI Paris)

Three French families with triose phosphate isomerase (TPI) deficiency were studied, and 2 new mutations giving rise to null alleles were observed: a frameshift mutation with deletion of the 86-87 TG dinucleotide in codon 29 (TPI Alfortville) and a T-->A transversion in nucleotide 2 of the initiation codon (TPI Paris). The first mutation occurred in compound heterozygosity with the frequent E105D mutation. The second mutation occurred in association with the 2-nucleotide promoter variant (-43G,-46A). In a third family, the propositus was an E105D homozygote. In the TPI Paris family, the coinheritance of the -43,-46 promoter variant appeared to exert little, if any, effect on TPI enzyme activity, a finding consistent with 2 previous reports that questioned the putative role of the promoter polymorphism as a true deficiency variant. Similarly, the further coinheritance of glucose-6-phosphate dehydrogenase (G6PD) A- (202 G-->A/376 A-->G) appeared to have little effect on the observed phenotype. Compound heterozygosity for the E105D mutation with the null allele TPI Alfortville appeared to lead to a more severe clinical syndrome than did E105D homozygosity, suggesting that compound heterozygosity with null alleles may lead to more profound clinical abnormalities than homozygosity with missense alleles. A simple, rapid polymerase chain reaction and restriction enzyme procedure for the E105D mutation was developed for prenatal diagnosis in one family and subsequently used for screening in the other families.     

Thapsigargin-induced increase in cytoplasmic Ca2+ concentration and aldosterone production in rat adrenal glomerulosa cells: interaction with potassium and angiotensin-II

Thapsigargin (Tg), a microsomal Ca2+ pump inhibitor, dose-dependently increases the cytoplasmic Ca2+ concentration and aldosterone production without having any striking effect on the formation of inositol phosphates in isolated rat adrenal glomerulosa cells. The interaction of Tg with the major Ca2(+)-mediated stimuli of glomerulosa cells on aldosterone production was also examined. The effects of Tg and the Ca2(+)-mobilizing angiotensin-II (AII) were additive. The aldosterone production stimulatory effect of potassium, which induces Ca2+ influx via voltage-operated Ca2+ channels, was potentiated by Tg. The positive interaction between Tg and potassium on aldosterone production raises the possibility that stimuli generating Ca2+ signal by depleting intracellular Ca2+ stores, such as Tg or AII, enhance the response of the cell to depolarization. Such an interaction between AII and potassium may have an important role in the physiological control of aldosterone production.     

Pyridine nucleotide redox state parallels production of aldosterone in potassium-stimulated adrenal glomerulosa cells.

Extracellular potassium ions (K+) raise the intracellular concentration of free Ca2+ ([Ca2+]i) by gating voltage-dependent Ca2+ channels and stimulate aldosterone production in adrenal glomerulosa cells. The pathway leading from calcium influx to increased steroid synthesis has not been completely elucidated. In the present study we demonstrate that the reduction of pyridine nucleotides known to be required for steroid hydroxylation is enhanced by K+ (4.1-8.4 mM) in single rat glomerulosa cells. The action of K+ was strictly dependent on the presence of extracellular Ca2+. Amytal, a blocker of site I of the mitochondrial respiratory chain, abolished the K+ effect, indicating a mitochondrial origin for the recorded changes. Supraphysiological K+ concentration (18 mM) resulted in a further increase in [Ca2+]i, while steroidogenesis was decreased as measured in cell suspensions. However, a possible explanation for this dichotomy is provided by the finding that the level of reduced pyridine nucleotides also decreased at supraphysiological K+ concentration.     

Angiotensin II and potassium activate different calcium entry mechanisms in rat adrenal glomerulosa cells

Initial 45Ca uptake was measured in isolated rat glomerulosa cells. A small reduction in membrane potential produced by increasing the K+ concentration from 2 to 3.6 mmol/l stimulated 45Ca uptake by about 35%, while a bigger depolarization induced by 18.5 mmol K+/l increased the uptake by about 100%. Since Ca2+ influx was already activated at a calculated membrane potential below -70 mV, and was found to be sensitive to the dihydropyridine antagonist nifedipine (1 mumol/l), but insensitive to nickel ions (100 mumol/l), it does not meet the criteria established for T- or L-type voltage-dependent Ca2+ channels. Exposure of glomerulosa cells to angiotensin II (AII) for 10 min also enhanced the rate of 45Ca influx. The effect of AII was not sensitive to 1 mumol nifedipine/l, but was strongly inhibited by 5-(N-4-chlorobenzyl)-N-(2',4'-dimethyl)benzamil (CBDMB, 30 mumol/l), an inhibitor of the Na+/Ca2+ antiporter. These observations suggest that during the sustained phase of stimulation with AII, a CBDMB-sensitive mechanism, rather than dihydropyridine-sensitive calcium channels, is involved in Ca2+ uptake in rat glomerulosa cells. The bulk Ca2+ influx did not correlate with aldosterone production; however, the maintained activity of different Ca2+ entry mechanisms seems to be essential for AII-induced aldosterone production.     

Role of cell volume in K+-induced Ca2+ signaling by rat adrenal glomerulosa cells

The involvement of cell volume in the K+-evoked Ca2+ signaling was studied in cultured rat glomerulosa cells. Previously we reported that hyposmosis (250 mOsm) increased the amplitude of T-type Ca2+ current and, accordingly, enhanced the Ca2+ response of cultured rat glomerulosa cells to K+. In the present study we found that this enhancement is not influenced by the cytoskeleton-disrupting drugs cytochalasin-D (20 microM) and colchicine (100 microM). Elevation of extracellular potassium concentration ([K+]e) from 3.6 to 4.6-8.6 mM induced cell swelling, which had slower kinetics than the Ca2+ signal. Cytoplasmic Ca2+ signal measured in single glomerulosa cells in response to stimulation with 5 mm K+ for 2 min showed two phases: after a rapid rise reaching a plateau within 20-30 sec, [Ca2+]c increased further slowly by approximately one third. When 5 mM K+ was coapplied with elevation of extracellular osmolarity from 290 to 320 mOsm, the second phase was prevented. These results indicate that cell swelling evoked by physiological elevation of [K+]e may contribute to the generation of sustained Ca2+ signals by enhancing voltage-activated Ca2+ influx.     

Activation of 5-HT(7) receptor in rat glomerulosa cells is associated with an increase in adenylyl cyclase activity and calcium influx through T-type calcium channels

Serotonin (5-HT) stimulates aldosterone secretion from the rat adrenal gland through 5-HT(7) receptors. The aim of the present study was to investigate the transduction mechanisms associated with activation of 5-HT(7) receptors in rat glomerulosa cells. The stimulatory effect of 5-HT on aldosterone secretion and cAMP formation was significantly reduced by the 5-HT(7) receptor antagonist LY 215840. Pretreatment of cells with the adenylyl cyclase inhibitor SQ 22536 or the PKA inhibitor H-89 markedly attenuated the effect of 5-HT on aldosterone secretion. Conversely, type 2 and 4 phosphodiesterase inhibitors potentiated the 5-HT-induced stimulation of aldosterone secretion. Administration of 5-HT in the vicinity of cultured glomerulosa cells induced a slowly developing and robust increase in cytosolic calcium concentration ([Ca(2+)](i)). The effect of 5-HT on [Ca(2+)](i) was suppressed by mibefradil, a T-type calcium channel blocker. Patch-clamp studies confirmed that 5-HT activated a T-type calcium current. Mibefradil also induced a dose-dependent inhibition of 5-HT-induced aldosterone secretion. The sequence of events associated with activation of 5-HT(7) receptors was investigated. The PKA inhibitor H-89 markedly attenuated both the [Ca(2+)](i) response and the activation of T-type calcium current induced by 5-HT. In contrast, reduction of the calcium concentration in the incubation medium did not affect 5-HT- induced cAMP formation. Preincubation of glomerulosa cells with cholera toxin abolished the stimulatory effect of 5-HT on aldosterone secretion, but pertussis toxin had no effect. Taken together, these data demonstrate that, in rat glomerulosa cells, activation of native 5-HT(7) receptors stimulates cAMP formation through a G(salpha) protein, which in turn provokes calcium influx through T-type calcium channels. Both the adenylyl cyclase/PKA pathway and the calcium influx are involved in 5-HT-induced aldosterone secretion.     

Atrial natriuretic peptide inhibition of calcium ionophore A23187-stimulated aldosterone secretion in rat adrenal glomerulosa cells.

The effect of atrial natriuretic peptide (ANP) on calcium ionophore A23187-stimulated aldosterone secretion was investigated using collagenase-dispersed rat adrenal glomerulosa cell suspensions. A23187 treatment induced a dose-dependent stimulation of aldosterone secretion, exhibiting an EC50 of approximately 75 nM. In agreement with the presumed action of A23187 as a Ca2+ ionophore, stimulation was dependent on the extracellular Ca2+ concentration, being completely inhibited in nominally Ca(2+)-free medium. In such Ca(2+)-free medium, stimulation of aldosterone secretion by bath applied 25-hydroxycholesterol was not inhibited, indicating that cells and biosynthetic pathway enzymes were not inhibited by low extracellular Ca2+ levels. A23187-induced aldosterone secretion was also inhibited by more than 90% when cells were simultaneously treated with ANP. Maximal ANP inhibition of A23187-stimulated aldosterone secretion was not overcome by concentrations of A23187 up to 10 microM or by increasing the extracellular Ca2+ concentration from 1.25 to 5 mM in the presence of A23187 and ANP. Addition of A23187 to ACTH-, angiotensin II-, or K(+)-stimulated glomerulosa cells did not overcome ANP-induced inhibition of aldosterone secretion stimulated by these secretagogues. In contrast to ANP inhibition of Ca(2+)-dependent A23187 stimulation of aldosterone secretion, ANP inhibition of dBcAMP-stimulated aldosterone secretion was readily overcome by increasing the dBcAMP concentration. These results indicated that ANP selectively and noncompetitively inhibited an intracellular step necessary for Ca(2+)-dependent stimulation of the early pathway of aldosterone biosynthesis in rat adrenal glomerulosa cells.     

Proadrenomedullin N-terminal 20 peptide (PAMP), acting through PAMP(12-20)-sensitive receptors, inhibits Ca2+-dependent, agonist-stimulated secretion of human adrenal glands.

Proadrenomedullin N-terminal 20 peptide (PAMP) is a 20-amino acid hypotensive peptide expressed in the adrenal medulla. We investigated the localization and function of PAMP receptors in the human adrenal gland. Autoradiography showed the presence of [125I]PAMP-binding sites in both zona glomerulosa and adrenal medulla that were displaced by cold PAMP and PAMP(12-20) but not by other preproadrenomedullin-derived peptides. PAMP, but not PAMP(12-20), counteracted, in a concentration dependent manner, both aldosterone response of zona glomerulosa cells and catecholamine response of adrenal medulla cells to BAYK-8644, the selective agonist of voltage-activated Ca2+ channels, as well as to K+ and angiotensin II. PAMP(12-20) partially reversed this antisecretagogue effect of PAMP. Collectively, these findings suggest (1) that PAMP inhibits Ca2+-dependent, agonist-stimulated aldosterone and catecholamine secretion, acting via specific receptors and through a mechanism involving the impairment of Ca2+ influx; and (2) that PAMP(12-20) acts as a weak antagonist of PAMP receptors, thereby suggesting that both C- and N-terminal sequences of the PAMP molecule are required for this peptide to exert its antisecretagogue action on the human adrenal gland.     

The role of tyrosine kinases in capacitative calcium influx-mediated aldosterone production in bovine adrenal zona glomerulosa cells.

In adrenal glomerulosa cells, the stimulation of aldosterone biosynthesis by angiotensin II (Ang II) involves the activation of a capacitative Ca(2+) influx through calcium release-activated calcium (CRAC) channels. In various mammalian cell systems, it has been shown that CRAC channel activation and Ca(2+) entry require tyrosine kinase activity. We have therefore examined in this work whether similar mechanisms contribute to Ang II-induced mineralocorticoid biosynthesis. In fluo-3-loaded isolated bovine glomerulosa cells, two inhibitors of tyrosine kinases, genistein and methyl-2, 5-dihydroxycinnamate (MDHC) (100 microM) prevented capacitative Ca(2+) entry elicited by Ang II (by 54 and 62% respectively), while the inhibitor of epidermal growth factor (EGF) receptor tyrosine kinase, lavendustin A, was without effect. Similar results were observed on Ca(2+) influx triggered by thapsigargin, an inhibitor of microsomal Ca(2+) pumps. The inhibitors blocked Ang II-stimulated pregnenolone and aldosterone production in the same rank order. In addition to its specific effect on capacitative Ca(2+) influx, genistein also affected the late steps of the steroidogenic pathway, as shown by experiments in which the rate-limiting step (intramitochondrial cholesterol transfer) was bypassed with 25-OH-cholesterol (25-OH-Chol), cytosolic calcium was clamped at stimulated levels or precursors of the late enzymatic steps were supplied. In contrast, genistin, a structural analogue of genistein devoid of tyrosine kinase inhibitory activity, was almost without effect on pregnenolone or 11-deoxycorticosterone (DOC) conversion to aldosterone. These results suggest that, in bovine adrenal glomerulosa cells, Ang II promotes capacitative Ca(2+) influx and aldosterone biosynthesis through tyrosine kinase activation.