The mechanism of the effect of K+ on the steroidogenesis of rat zona glomerulosa cells of the adrenal cortex: role of cyclic AMP

The effects of various concentrations of extracellular K+ (3.6-13 mM) on the steroid (corticosterone and aldosterone) and cyclic AMP outputs of capsular cells (95% zona glomerulosa) of the rat adrenal cortex were studied at different concentrations of extracellular Ca2+. Small amounts of EGTA (50 microM) were added to reduce the free Ca2+ concentrations effectively to zero at the lowest possible total Ca2+ concentration. At a total extracellular concentration of 2.5 mM Ca2+, in 27 experiments the mean values of the steroid and cAMP outputs showed a maximum at 8.4 mM K+. The increase in steroid and cAMP outputs at 5.9, 8.4 and 13 mM K+ compared with that at 3.6 mM were highly significant (p less than 0.01). The overall correlation of either corticosterone or aldosterone with cAMP outputs was also highly significant and was even better from 3.6 to 8.4 mM K+. Lowering the effective free concentration of Ca2+ to zero decreased the steroid and cAMP outputs significantly at all K+ concentrations, and no output was then significantly higher than at 3.6 mM. With the pooled data on outputs at all total Ca2+ (2.5, 0.5, 0.25, 0.10, 0.05 and 0.0 mM) and K+ (3.6, 5.9, 8.4 and 13 mM) concentrations, the correlation of either steroid with cAMP outputs was highly significant (but again optimally from 3.6 to 8.4 mM K+). Nifedipine (10(-6) to 10(-4) M) was added to the incubations with the aim of specifically inhibiting Ca2+ influx at total extracellular Ca2+ concentrations of 2.5, 1.25 and 0.25 mM and with the usual K+ concentrations. The cAMP outputs were reduced at all K+ concentrations above 3.6 mM K+. The effect was highly significant at 10(-4) M nifedipine and a total Ca2+ of 1.25 mM, which with the incubation conditions used, corresponds to the free Ca2+ concentrations in vivo. These results indicate that cAMP plays a significant role in the stimulation of steroid output by K+ particularly between 3.6 and 8.4 mM K+. In this range of K+ concentrations the stimulation of cAMP seems to be controlled by increases in Ca2+ influx. The correlation of steroid and cAMP output at the higher K+ concentrations (between 8.4 and 13 mM K) and at the various total Ca2+ concentrations is less significant. Also, with all concentrations of added nifedipine there is an 'anomalous' increase in steroid output at 13 mM K+ and at total Ca2+ concentrations of 2.5 and 1.25 mM. However, at the same K+ concentrations and at 0.25 mM Ca2+, nifedipine decreases steroid outputs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Potassium intake and aldosterone biosynthesis: the role of cytochrome P-450

K+ Repletion for 48 h of rats previously kept on a low K+ diet for 2 weeks specifically increased the conversion of corticosterone into aldosterone and 18-hydroxycorticosterone by incubated capsular fractions of rat adrenal tissue. This increase in the activity of the final steps of aldosterone biosynthesis was not accompanied by an increase in capsular adrenal mitochondrial cytochrome P-450 concentration. By contrast, an increased corticosterone-induced absorbance change (BI) was consistently found in capsular adrenal mitochondria upon K+ repletion. In addition, a type I-like absorbance change was induced with 18-hydroxy-11-deoxycorticosterone but not with 18-hydroxycorticosterone. Therefore, K+ repletion of K+ depleted rats specifically increased the binding of corticosterone and possibly 18-hydroxy-11-deoxycorticosterone to the 18-methyl oxidase enzyme complex. Whether this increased binding was due to an increase in enzyme protein concentration or due to a better availability of the substrate to the enzyme, could not be decided from these experiments.     

Okadaic acid inhibits angiotensin II, adrenocorticotropin and potassium-dependent aldosterone secretion

The present study was designed to assess the effect of okadaic acid (OA), a protein phosphatase inhibitor, on aldosterone secretion in response to angiotensin II (AII), adrenocorticotropin (ACTH) and rises in external potassium concentration (K+). AII (10nM) caused a 20-fold increase in aldosterone production and OA reduced this response by 45%. ACTH (10nM) caused an 8.6-fold increase in aldosterone secretion and OA reduced this by 83%. Increasing K+ concentration from 3 to 12mM caused a 13-fold increase in aldosterone production, which OA inhibited by 36%. These results suggest that protein phosphatases participate in the control of adrenal steroid production, even though ACTH, AII and K+ act via different intracellular messenger systems.     

Intracellular mediators of potassium-induced aldosterone secretion

We have investigated the intracellular messengers of potassium in eliciting aldosterone secretion in calf adrenal glomerulosa cells since there were unresolved issues relating to the role of phosphoinositides, cAMP and protein kinases. We observed no evidence of hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in 3H-inositol labeled alf adrenal cells or increase of cAMP in response to potassium. Addition of calcium channel blocker, nitrendipine after stimulating adrenal glomerulosa cells with potassium, markedly inhibited aldosterone secretion. A calmodulin inhibitor (W-7) produced greater reduction of aldosterone secretion than an inhibitor of protein kinase C (H-7). These results suggest that a rise in cytosolic free calcium concentration through voltage-dependent calcium channel and calmodulin are the critical determinants of aldosterone secretion stimulated by potassium.     

Inhibitory actions of somatostatin on cyclic AMP and aldosterone production in agonist-stimulated adrenal glomerulosa cells

Somatostatin (SRIF) is a potent inhibitor of angiotensin II (AII)-stimulated aldosterone production in rat adrenal glomerulosa cells. This inhibition can be prevented by pretreatment of the cells with pertussis toxin, but little else is known about either the specificity or the biochemical bases of SRIF action in this tissue. We therefore conducted detailed studies of the influence of SRIF on steroidogenesis elicited by AII and the other two physiological stimuli of aldosterone production, K+ and adrenocorticotropic hormone (ACTH), in rat adrenal glomerulosa cells. We also determined the effects of SRIF on cytosolic calcium concentration ([Ca2+]i) and cellular cAMP levels. In these studies, SRIF was found to inhibit the aldosterone responses elicited by low concentrations of all three stimuli, which are believed to promote steroid secretion via discrete but interacting cellular signalling mechanisms. In addition, SRIF consistently lowered cellular cAMP levels in the presence of each of the three agents. However, SRIF caused a small and transient increase rather than a decrease in basal ([Ca2+]i), and had no effect on the subsequent elevation of ([Ca2+]i) by AII and K+. These data indicate that activation of a Gi-like protein by SRIF influences steroid responses to all three major regulators of glomerulosa-cell function, and suggest that basal levels of cAMP play a facilitatory or permissive role in the control of aldosterone production by predominantly calcium-mobilizing regulators of mineralocorticoid secretion.     

Atrial natriuretic factor inhibits angiotensin-induced aldosterone secretion: not through cGMP or interference with phospholipase C

ANF did not prevent the formation of [3H] inositol trisphosphate in response to AII but inhibited aldosterone secretion in calf adrenal glomerulosa cells. 8-bromo cGMP did not affect either inositol phosphate formation or aldosterone secretion. Changes in cytosolic Ca++ concentration induced by AII, as measured by Quin 2 fluorescence, were also unaffected by ANF. No difference in adrenal cell protein phosphorylation with AII or AII + ANF was observed. The results suggest that ANF may inhibit aldosterone secretion through a non-guanyl cyclase linked receptor system not involving the formation of phosphoinositide-derived second messengers. Interference with protein kinase C activity cannot be ruled out.     

Greater importance of Ca(2+)-calmodulin in maintenance of ang II- and K(+)-mediated aldosterone secretion: lesser role of protein kinase C.

In this study we have investigated various components of the stimulus-secretion coupling process leading to aldosterone secretion from the calf adrenal glomerulosa cells as evoked by angiotensin II (AII) and potassium (K+). The roles of Ca2+, calmodulin and protein kinase C in the sustained phase rather than initiation of aldosterone secretion were of special interest. Our investigations revealed that the reduction of extracellular Ca2+ by EGTA or interruption of Ca2+ influx by nitrendipine at various time points after stimulation with either AII or K+ markedly compromised aldosterone secretion. Calmodulin inhibitors, calmidazolium and W-7 reduced aldosterone secretion profoundly. Agonists of protein kinase C, phorbol ester or diacylglycerol analogues failed to stimulate aldosterone secretion while the protein kinase C inhibitor, H-7, only partially inhibited aldosterone secretion at a concentration which completely inhibited protein kinase C activity. Calmodulin inhibitors produced significantly greater inhibition of aldosterone secretion than inhibitors of protein kinase C.     

Effect of K+ concentration on the rate of synthesis of fast-labeling proteins in the adrenal cortex

The effect of K+ concentration on protein biosynthesis and 32P-incorporation into the acid-insoluble fraction of dog and guinea pig adrenal cortex slices was studied. An increase in K+ concentration in the incubation medium from 3 to 8-11 mM induced after 15-20 min of incubation a significant stimulation of 14C-leucine incorporation into the acid-insoluble fraction of post-mitochondrial supernatant. More extensive labelling of this fraction with 32P was observed. Addition of valinomycin caused a shift in the maximum of 14C-leucine incorporation towards lower K+ concentrations. The Na+,K+-ATPase inhibitors--ouabain and strophantin K--reduced the K+-stimulated protein synthesis. These data suggest that K+ transport into the cell is necessary for the stimulating effect to be manifested. Chelation of Ca2+ strongly decreased the incorporation of 14C-leucine into proteins in the presence of 5 mM K+. However, protein labelling increased with a gradual rise in K+ concentration up to 25 mM.     

Forskolin activation of apical Cl- channel and Na+/K+/2Cl- cotransporter via a PTK-dependent pathway in renal epithelium

Forskolin induced the transepithelial Cl- transport (secretion) by activating the apical Cl- channel and basolateral Na+/K+/2Cl- cotransporter in renal epithelial A6 cells via an increase in cytosolic cAMP concentration. The cAMP activation of apical Cl- channel and Na+/K+/2Cl- cotransporter was partially mediated through a protein kinase A (PKA)-dependent pathway, but a PKA-independent pathway was also suggested to be involved in the cAMP activation. Therefore, we assessed a possibility of involvement of protein tyrosine kinase (PTK)-dependent pathway as a PKA-independent pathway in the cAMP activation by applying a PTK inhibitor, tyrphostin A23 (AG18). Tyrphostin A23 abolished the forskolin-induced transepithelial Cl- secretion by partially diminishing the activity of the Cl- channel and completely inhibiting the Na+/K+/2Cl- cotransporter. Further, forskolin increased phosphorylation of protein tyrosine, suggesting that cAMP activates PTK. These observations suggest that cAMP activates the Cl- channel and the Na+/K+/2Cl- cotransporter by activating PTK.     

Tyrosine Hydroxylase Regulation: Apparent Kinetic Alterations Following Incubation of Brain Slices in a Sodium-free Medium

In an attempt to determine if alterations in intraneuronal Ca2+ may regulate tyrosine hydroxylase activity, brain slices were subjected to experimental manipulations known to increase the intraneuronal concentration of free Ca2+ ions. Incubation of either striatal or olfactory tubercle slices in a Na+-free medium for 15 min at 37 degrees resulted in a marked increase in the activity of tyrosine hydroxylase present in the 20,000 g supernatant fraction of homogenates prepared from the slices. Tyrosine hydroxylase isolated from slices previously incubated in a Na+-free, choline-enriched medium or in a Na+-free, sucrose-enriched medium exhibited maximal activities when assayed at pH 6.0 and 7.0, respectively. However, the percentage stimulation of enzyme activity induced by incubation of the slices in a Na+-free medium was maximal when the enzyme assays were performed at pH 7.0. The observed increase in enzyme activity seems to be mediated by a decrease in the apparent Km of the enzyme for pteridine cofactor, regardless of whether the kinetic enzyme analyses were conducted at pH 6.0 or 7.0, and by an increase in the Ki of the enzyme for end-product inhibitor dopamine. The apparent kinetic changes in the enzyme do not seem to result from alterations in the endogenous dopamine content of the slices, and they are independent of any increase in dopamine release that might have occurred as a response to the augmented intraneuronal Ca2+ concentration. Furthermore, the activation of tyrosine hydroxylase produced by incubating slices in a Na+-free medium is observed even in slices depleted of dopamine by pretreatment of rats with reserpine 90 min before preparation of brain slices. The activation of tyrosine hydroxylase observed under these experimental conditions does not seem to be mediated by cAMP or by a cAMP-dependent phosphorylation process. It is suggested that the changes in tyrosine hydroxylase reported are mediated primarily by a rise in the free Ca2+ concentration within the nerve tissue. These observations are consistent with the hypothesis that the kinetic activation of tyrosine hydroxylase produced after depolarization of central dopaminergic neurons may occur through a Ca2+-dependent even other than transmitter release.     

Regulation of aldosterone production from zona glomerulosa cells by ANG II and cAMP: evidence for PKA-independent activation of CaMK by cAMP

Aldosterone production in zona glomerulosa (ZG) cells of adrenal glands is regulated by various extracellular stimuli (K(+), ANG II, ACTH) that all converge on two major intracellular signaling pathways: an increase in cAMP production and calcium (Ca(2+)) mobilization. However, molecular events downstream of the increase in intracellular cAMP and Ca(2+) content are controversial and far from being completely resolved. Here, we found that Ca(2+)/calmodulin-dependent protein kinases (CaMKs) play a predominant role in the regulation of aldosterone production stimulated by ANG II, ACTH, and cAMP. The specific CaMK inhibitor KN93 strongly reduced ANG II-, ACTH-, and cAMP-stimulated aldosterone production. In in vitro kinase assays and intact cells, we could show that cAMP-induced activation of CaMK, using the adenylate cyclase activator forskolin or the cAMP-analog Sp-5,6-DCI-cBIMPS (cBIMPS), was not mediated by PKA. Activation of the recently identified cAMP target protein Epac (exchange protein directly activated by cAMP) by 8-pCPT-2'-O-Me-cAMP had no effect on CaMK activity and aldosterone production. Furthermore, we provide evidence that cAMP effects in ZG cells do not involve Ca(2+) or MAPK signaling. Our results suggest that ZG cells, in addition to PKA and Epac/Rap proteins, contain other as yet unidentified cAMP mediator(s) involved in regulating CaMK activity and aldosterone secretion.     

Regulation of hormone biosynthesis in guinea pig adrenal glands by potassium ions as affected by dihydropyridines. 1. Analysis of changes in steroidogenesis evoked by dihydropyridines]

Influence of Ca2+ channel modulators BAY K 8644, nitrendipine and newly synthesized derivative of 1,4-dihydropyridine: 4-(3', 4'-dimethoxyphenyl)-2,6-dimethyl-3,5- diethoxy-carbonyl-1,4-dihydropyridine-(DHP-51) on aldosterone production by adrenocortical slices depends on K+ content in the incubation medium. The modulators only slightly influence the hormone output at low K+ level in the medium. Intensive synthesis of aldosterone at high level of potassium in the medium was prevented in the presence of DHP-51 and low concentration of BAY K 8644. DHP-51 inhibited [3H]-cholesterol incorporation into all main corticosteroids in the high-potassium medium.     

Cholinergic Receptor-Mediated Phosphorylation and Activation of Tyrosine Hydroxylase in Cultured Bovine Adrenal Chromaffin Cells

We have identified a 56-kilodalton protein in cultured bovine adrenal chromaffin cells that is phosphorylated when catecholamine secretion is stimulated. Immunodetection on Western blots from both one- and two-dimensional polyacrylamide gels indicated that this protein was tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis. Two-dimensional polyacrylamide gel electrophoresis of proteins from unstimulated cells revealed small amounts of phosphorylated protein with a molecular weight of 56K and pI values of 6.37 and 6.27 which were subunits of tyrosine hydroxylase. Nicotinic stimulation of chromaffin cells caused the phosphorylation of three proteins of 56 kilodaltons with pI values of approximately 6.37, 6.27, and 6.15 which were tyrosine hydroxylase. The immunochemical analysis also revealed that there was unphosphorylated tyrosine hydroxylase 56 kilodaltons with a pI of 6.5 which may have decreased on nicotinic stimulation. The phosphorylation of tyrosine hydroxylase was associated with an increase in in situ conversion of [3H]tyrosine to [3H]dihydroxyphenylalanine ([3H]DOPA). Muscarinic stimulation also caused phosphorylation of tyrosine hydroxylase, but to a smaller extent than did nicotinic stimulation. The secretagogues, elevated K+ and Ba2+, stimulated phosphorylation of tyrosine hydroxylase and [3H]DOPA production. The effects of nicotinic stimulation and elevated K+ on tyrosine hydroxylase phosphorylation and [3H]DOPA production were Ca2+-dependent. Nicotinic agonists also raised cyclic AMP levels in chromaffin cells after 2 min. Dibutyryl cyclic AMP and forskolin, which have little effect on catecholamine secretion, also caused phosphorylation of tyrosine hydroxylase. These stimulators of cyclic AMP-dependent processes caused the appearance of two phosphorylated subunits of tyrosine hydroxylase with pI values of 6.37 and 6.27. There was also a small amount of phosphorylated subunit with a pI of 6.15. Both agents stimulated [3H]DOPA production. The experiments indicate that tyrosine hydroxylase is phosphorylated and activated when chromaffin cells are stimulated to secrete. The data suggest that the earliest phosphorylation of tyrosine hydroxylase induced by a nicotinic agonist occurs through stimulation of a Ca2+-dependent protein kinase. After 2 min phosphorylation by a cyclic AMP-dependent protein kinase may also occur. Phosphorylation of tyrosine hydroxylase is associated with an increase in in situ tyrosine hydroxylase activity.     

Cyclic AMP-independent effects of ACTH on glomerulosa cells of the rat adrenal cortex

The aim of the present paper is to point out the complexity of ACTH action in glomerulosa cells of the adrenal cortex. We demonstrate that the increase in cAMP production induced by ACTH is the result of a balance between activation of adenylyl cyclase and direct modulation of a PDE2 phosphodiestease activity, an effect mediated by inhibition of cGMP content. Moreover, Ca²⁺ is essential for cAMP production and aldosterone secretion, but its exact primary action is not clearly determined. We recently described that ACTH activated a chloride channel, via the Ras protein, which can be involved in steroidogenesis. ACTH also increases tyrosine phosphorylation of several proteins. These data, together with those of phospholipase C activation, indicate that ACTH action in the adrenal is complex, and most certainly not limited to cAMP production, in particular for the low concentrations of the hormone.

Some years ago, cAMP was considered to be the unique second messenger of ACTH action; now it becomes more and more evident that ACTH triggers complex signaling pathways using several second messengers in a closely interacting way. The most predominant point is that these signals are observed for low concentrations of ACTH.     

Evidence for a paracrine role of adrenomedullin in the physiological resetting of aldosterone secretion by rat adrenal zona glomerulosa

Adrenomedullin (ADM) has been recently found to directly inhibit agonist-stimulated aldosterone secretion by dispersed zona glomerulosa (ZG) cells and to stimulate basal catecholamine release by adrenomedullary fragments. In light of the fact that catecholamines enhance aldosterone secretion acting in a paracrine manner, we have investigated whether these two effects of ADM may interact when the integrity of the adrenal gland is preserved. ADM increased basal aldosterone output by adrenal slices containing a core of adrenal medulla, and the effect was blocked by the beta-adrenoceptor antagonist l-alprenolol. In contrast, ADM evoked a moderate inhibition of K(+)-stimulated aldosterone production, and the blockade was complete in the presence of l-alprenolol. The in vivo bolus injection of ADM did not affect plasma aldosterone concentration (PAC) in rats under basal conditions. Conversely, when rat ZG secretory function was enhanced (by sodium restriction or infusion with angiotensin-II [ANG-II]) or depressed (by sodium loading or infusion with the angiotensin-converting enzyme inhibitor captopril), ADM evoked a sizeable decrease or increase in PAC, respectively. The prolonged infusion with the ADM receptor antagonist ADM(22-52) caused a further enhancement of PAC in sodium-restricted or ANG-II-treated rats, and a further moderate decrease of it in sodium-loaded or captopril-administered animals. RIA showed that ADM plasma concentration did not exceed a concentration of 10(-11) M in any group of animals. Under basal conditions, ADM adrenal content was 1.2-2.0 pmol/g, which may give rise to local concentrations higher than 10(-8) M (i.e. well above the minimal effective ones in vitro). ADM adrenal concentration was markedly increased (from two-fold to three-fold) by both ZG stimulatory and suppressive treatments. Collectively, our findings suggest that in vivo 1) ADM, in addition to directly inhibit aldosterone secretion, may enhance it indirectly by eliciting catecholamine release, the two actions annulling each other under basal conditions; 2) under conditions leading to enhanced aldosterone secretion, the direct inhibitory effect of ADM prevails over the indirect stimulatory one, and the reverse occurs when aldosterone secretion is decreased; and 3) the modulatory action of ADM on the aldosterone secretion has a physiological relevance, endogenous ADM being locally synthesized in adrenals.     

Swelling and ion uptake in cat cerebrocortical slices:

Cat cerebrocortical slices incubating in medium containing normal K+ concentrations were exposed to a number of different transmitters. Norepinephrine, histamine and adenosine or 2-chloroadenosine caused increased swelling of the slices associated with an increased Na+ and Cl- content. These effects were seen only when both Cl- and HCO3- were present in the medium, and were inhibited by a number of anion transport inhibitors. These characteristics were identical to those of the HCO3(-)-dependent component of the swelling induced by high K+ levels in the medium. Other transmitters, namely 5-hydroxytryptamine, dopamine, and gamma-amino butyric acid, were ineffective. The effects of norepinephrine, histamine and 2-chloroadenosine were antagonised by propranolol and phentolamine, chlorpheniramine and diphenhydramine, and theophylline respectively. These antagonists also inhibited HCO3(-)-dependent, K+-stimulated swelling. The transmitters which induced swelling also stimulated the carbonic anhydrase activity of cerebrocortical slices. We conclude from these data that the HCO3(-)-dependent component of K+-stimulated swelling may be due to K+-stimulated release of transmitters. Furthermore, the fact that the transmitters which induce swelling have also been reported to be most effective in increasing cAMP content in both brain slices or cultured astrocytes is consistent with the swelling response being mediated via cAMP-induced changes and being predominantly localized to astrocytes.     

Electrolyte excretion in alcohol preferring and alcohol avoiding rats

Water and electrolyte metabolism was studied in alcohol preferring (AA) and alcohol avoiding (ANA) rats. During water diuresis AA rats had higher Mg, cAMP, creatinine and inorganic phosphate excretion, but lower urine and urinary protein output. During ethanol diuresis AA rats had lower Na, K, Ca, protein and urine output, but higher cAMP and inorganic phosphate excretion. Ethanol increased K, Ca and urine output in ANA rats only. A slight increase of blood pH was observed only in AA rats. Before ethanol ANA rats had higher plasma Ca concentration. Plasma aldosterone level was higher in AA rats. High salt excretion of ANA rats may lead them to prefer salt containing energy sources and therefore to avoid ethanol. On the other hand, renal salt conservation in AA rats may lead them to prefer ethanol.     

In vitro responsiveness of aldosterone-producing adenoma to angiotensin II, K and ACTH

In vitro responsiveness to various stimulators of aldosterone secretion was studied in a perifusion system using slices obtained from three aldosterone-producing adenomas (APAs), three adjacent nontumorous glands and three normal adrenal glands. All three APA tissues responded to angiotensin II, K and ACTH in vitro. Angiotensin II (10 nM), K (12 mM) and ACTH (10 nM) caused more than a 2-fold increase in aldosterone secretion. The sensitivity of APA tissues to angiotensin II was identical to that in normal adrenal cortex. In slices obtained from APA, angiotensin II induced rapid increases in [3H]inositol and [45Ca] efflux, both of which preceded the aldosterone response. These results suggest that APA cells have an almost normal transducing system to stimulators of aldosterone secretion.     

The pharmacologic control of adrenal steroidogenesis

The control of cortisol secretion by ACTH and of aldosterone secretion by angiotensin is exerted upon separate cell populations in the adrenal cortex. Cells of the zona faciculata and the zona glomerulosa, while sharing common steroidogenic pathways, are affected differently by hormones and drugs. Fasciculata cells demonstrate increased cAMP formation and cortisol output primarily in response to ACTH. ACTH receptors, when occupied by hormone, transmit an activating signal to membrane-bound adenylate cyclase by a mechanism that may require the translocation of Ca²⁺. Although the precise way in which increased intracellular cAMP leads to increased steroidogenesis is unknown, protein phosphorylation and new protein synthesis are probably involved. Glomerulosa cells also respond to ACTH, but are uniquely responsive to physiological concentrations of angiotensin II and K⁺. The responsiveness of these cells to angiotensin may be governed by alterations in receptor number. Whether occupied angiotensin receptors activate steroidogenesis via cAMP is uncertain, but alterations in Ca²⁺ distribution within the cell may again be involved. Dopamine probably exerts a tonic inhibitory effect on glomerulosa cell function. Competitive inhibitory analogs for both ACTH and angiotensin II are available, but thus far all inhibitors have retained weak agonist properties. Because the regulatory processes for both cortisol and aldosterone are complex, a wide variety of drugs can affect rates of steroidogenesis $\text{in}$$\text{vivo}$.     

Exposure to an Extremely-Low-Frequency Magnetic Field Stimulates Adrenal Steroidogenesis via Inhibition of Phosphodiesterase Activity in a Mouse Adrenal Cell Line

Extremely low-frequency magnetic fields (ELF-MFs) are generated by power lines and household electrical devices. In the last several decades, some evidence has shown an association between ELF-MF exposure and depression and/or anxiety in epidemiological and animal studies. The mechanism underlying ELF-MF-induced depression is considered to involve adrenal steroidogenesis, which is triggered by ELF-MF exposure. However, how ELF-MFs stimulate adrenal steroidogenesis is controversial. In the current study, we investigated the effect of ELF-MF exposure on the mouse adrenal cortex-derived Y-1 cell line and the human adrenal cortex-derived H295R cell line to clarify whether the ELF-MF stimulates adrenal steroidogenesis directly. ELF-MF exposure was found to significantly stimulate adrenal steroidogenesis (p < 0.01–0.05) and the expression of adrenal steroid synthetic enzymes (p < 0.05) in Y-1 cells, but the effect was weak in H295R cells. Y-1 cells exposed to an ELF-MF showed significant decreases in phosphodiesterase activity (p < 0.05) and intracellular Ca²⁺ concentration (p < 0.01) and significant increases in intracellular cyclic adenosine monophosphate (cAMP) concentration (p < 0.001–0.05) and cAMP response element-binding protein phosphorylation (p < 0.05). The increase in cAMP was not inhibited by treatment with NF449, an inhibitor of the Gs alpha subunit of G protein. Our results suggest that ELF-MF exposure stimulates adrenal steroidogenesis via an increase in intracellular cAMP caused by the inhibition of phosphodiesterase activity in Y-1 cells. The same mechanism may trigger the increase in adrenal steroid secretion in mice observed in our previous study.