Interaction of neuronal calcium sensor-1 (NCS-1) with phosphatidylinositol 4-kinase beta stimulates lipid kinase activity and affects membrane trafficking in COS-7 cells.

Phosphatidylinositol 4-kinases (PI4K) catalyze the first step in the synthesis of phosphatidylinositol 4,5-bisphosphate, an important lipid regulator of several cellular functions. Here we show that the Ca(2+)-binding protein, neuronal calcium sensor-1 (NCS-1), can physically associate with the type III PI4Kbeta with functional consequences affecting the kinase. Recombinant PI4Kbeta, but not its glutathione S-transferase-fused form, showed enhanced PI kinase activity when incubated with recombinant NCS-1, but only if the latter was myristoylated. Similarly, in vitro translated NCS-1, but not its myristoylation-defective mutant, was found associated with recombinant- or in vitro translated PI4Kbeta in PI4Kbeta-immunoprecipitates. When expressed in COS-7 cells, PI4Kbeta and NCS-1 formed a complex that could be immunoprecipitated with antibodies against either proteins, and PI 4-kinase activity was present in anti-NCS-1 immunoprecipitates. Expressed NCS-1-YFP showed co-localization with endogenous PI4Kbeta primarily in the Golgi, but it was also present in the walls of numerous large perinuclear vesicles. Co-expression of a catalytically inactive PI4Kbeta inhibited the development of this vesicular phenotype. Transfection of PI4Kbeta and NCS-1 had no effect on basal PIP synthesis in permeabilized COS-7 cells, but it increased the wortmannin-sensitive [(32)P]phosphate incorporation into phosphatidylinositol 4-phosphate during Ca(2+)-induced phospholipase C activation. These results together indicate that NCS-1 is able to interact with PI4Kbeta also in mammalian cells and may play a role in the regulation of this enzyme in specific cellular compartments affecting vesicular trafficking.

Monitoring agonist-induced phospholipase C activation in live cells by fluorescence resonance energy transfer.

Agonist-induced intracellular Ca(2+) signals following phospholipase C (PLC) activation display a variety of patterns, including transient, sustained, and oscillatory behavior. These Ca(2+) changes have been well characterized, but detailed kinetic analyses of PLC activation in single living cells is lacking, due to the absence of suitable indicators for use in vivo. Recently, green fluorescent protein-tagged pleckstrin homology domains have been employed to monitor PLC activation in single cells, based on (confocal) imaging of their fluorescence translocation from the membrane to the cytosol that occurs upon hydrolysis of phosphatidylinositol bisphosphate. Here we describe fluorescence resonance energy transfer between pleckstrin homology domains of PLCdelta1 tagged with cyan and yellow fluorescent proteins as a sensitive readout of phosphatidylinositol bisphosphate metabolism for use both in cell populations and in single cells. Fluorescence resonance energy transfer requires significantly less excitation intensity, enabling prolonged and fast data acquisition without the cell damage that limits confocal experiments. It also allows experiments on motile or extremely flat cells, and can be scaled to record from cell populations as well as single neurites. Characterization of responses to various agonists by this method reveals that stimuli that elicit very similar Ca(2+) mobilization responses can exhibit widely different kinetics of PLC activation, and that the latter appears to follow receptor activation more faithfully than the cytosolic Ca(2+) transient.

Energetic and conformational aspects of A:T base-pair opening within the DNA double helix.

Free energy profiles of opening of a centrally placed A:T pair within a DNA oligomer exhibits two regimes: Elastic deformation due to hydrogen bond rupture and a roughly linear region due to loss of stacking and solvation. Thymine opens equally easily into the minor and major grooves, while adenine favors the major groove direction. No significant variations from canonical backbone conformations were observed; however base opening induces considerable changes in surrounding solvent distribution, leading finally to a water channel which passes through the double helix.

Identification of renox, an NAD(P)H oxidase in kidney.

Oxygen sensing is essential for homeostasis in all aerobic organisms, but its mechanism is poorly understood. Data suggest that a phagocytic-like NAD(P)H oxidase producing reactive oxygen species serves as a primary sensor for oxygen. We have characterized a source of superoxide anions in the kidney that we refer to as a renal NAD(P)H oxidase or Renox. Renox is homologous to gp91(phox) (91-kDa subunit of the phagocyte oxidase), the electron-transporting subunit of phagocytic NADPH oxidase, and contains all of the structural motifs considered essential for binding of heme, flavin, and nucleotide. In situ RNA hybridization revealed that renox is highly expressed at the site of erythropoietin production in the renal cortex, showing the greatest accumulation of renox mRNA in proximal convoluted tubule epithelial cells. NIH 3T3 fibroblasts overexpressing transfected Renox show increased production of superoxide and develop signs of cellular senescence. Our data suggest that Renox, as a renal source of reactive oxygen species, is a likely candidate for the oxygen sensor function regulating oxygen-dependent gene expression and may also have a role in the development of inflammatory processes in the kidney.

Modelling the catalytic reaction in human aldose reductase.

Aldose reductase (ALR2) has received considerable attention due to its possible link to long-term diabetic complications. Although crystal structures and kinetic data reveal important aspects of the reaction mechanism, details of the catalytic step are still unclear. In this paper a computer simulation study is presented that utilizes the hybrid quantum mechanical and molecular mechanical (QM-MM) potential to elucidate the nature of the hydride and proton transfer steps in the reduction of D-glyceraldehyde by ALR2. Several reaction pathways were investigated in two models with either Tyr48 or protonated His110+ acting as the potential proton donor in the active site. Calculations show that the substrate binds to ALR2 through hydrogen bonds in an orientation that facilitates the stereospecific catalytic step in both models. It is established that in the case that His110 is present in the protonated form in the native complex, it is the energetically favored proton donor compared with Tyr48 in the active pocket with neutral His110. The reaction mechanisms in the different models are discussed based on structural and energetic considerations.

Phosphatidylinositol 3-kinase-dependent membrane association of the Bruton's tyrosine kinase pleckstrin homology domain visualized in single living cells.

Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) has been proposed to act as a second messenger to recruit regulatory proteins to the plasma membrane via their pleckstrin homology (PH) domains. The PH domain of Bruton's tyrosine kinase (Btk), which is mutated in the human disease X-linked agammaglobulinemia, has been shown to interact with PI(3,4,5)P3 in vitro. In this study, a fusion protein containing the PH domain of Btk and the enhanced green fluorescent protein (BtkPH-GFP) was constructed and utilized to study the ability of this PH domain to interact with membrane inositol phospholipids inside living cells. The localization of expressed BtkPH-GFP in quiescent NIH 3T3 cells was indistinguishable from that of GFP alone, both being cytosolic as assessed by confocal microscopy. In NIH 3T3 cells coexpressing BtkPH-GFP and the epidermal growth factor receptor, activation of epidermal growth factor or endogenous platelet-derived growth factor receptors caused a rapid (<3 min) translocation of the cytosolic fluorescence to ruffle-like membrane structures. This response was not observed in cells expressing GFP only and was completely inhibited by treatment with the PI 3-kinase inhibitors wortmannin and LY 292004. Membrane-targeted PI 3-kinase also caused membrane localization of BtkPH-GFP that was slowly reversed by wortmannin. When the R28C mutation of the Btk PH domain, which causes X-linked agammaglobulinemia, was introduced into the fluorescent construct, no translocation was observed after stimulation. In contrast, the E41K mutation, which confers transforming activity to native Btk, caused significant membrane localization of BtkPH-GFP with characteristics indicating its possible binding to PI(4,5)P2. This mutant, but not wild-type BtkPH-GFP, interfered with agonist-induced PI(4,5)P2 hydrolysis in COS-7 cells. These results show in intact cells that the PH domain of Btk binds selectively to 3-phosphorylated lipids after activation of PI 3-kinase enzymes and that losing such binding ability or specificity results in gross abnormalities in the function of the enzyme. Therefore, the interaction with PI(3,4,5)P3 is likely to be an important determinant of the physiological regulation of Btk and can be utilized to visualize the dynamics and spatiotemporal organization of changes in this phospholipid in living cells.

Selective inhibition of potassium-stimulated rat adrenal glomerulosa cells by ruthenium red.

The effect of the cationic dye, ruthenium red (RR), on ionic fluxes, Ca2+ signal generation, and stimulation of aldosterone production was studied in isolated rat adrenal glomerulosa cells. In these cells, increased extracellular [K+] as well as angiotensin II (Ang II) elevate cytoplasmic Ca2+ concentration and thereupon activate steroidogenesis. However, the mode of action of the two stimuli are different: while a dihidropyridine-sensitive mechanism contributes to the response to both agonists, Ang II induces Ca2+ release from intracellular stores and causes capacitative Ca2+ influx, whereas K+ was recently shown to activate a plasma membrane Ca2+ current (Igl) independently of membrane depolarization. The difference is reflected in the sensitivity of the response of the cells to RR. The Ang II-induced Ca2+ signal and aldosterone production were not inhibited, but rather slightly potentiated by the dye. This potentiation was probably the consequence of the membrane-depolarizing effect of RR, due to the observed inhibition of the resting K+ conductance. Conversely, Ca2+ signal and aldosterone production were significantly reduced by RR when the cells were stimulated by moderately elevated [K+] (6-8 mM). Our patch clamp studies suggest that this effect was related to the inhibition of different voltage-dependent and -independent inward Ca2+ currents and indicates the functional importance of the latter in the signal transduction of the potassium-stimulated glomerulosa cell.

Visualization of phosphoinositides that bind pleckstrin homology domains: calcium- and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools.

Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) pools that bind pleckstrin homology (PH) domains were visualized by cellular expression of a phospholipase C (PLC)delta PH domain-green fluorescent protein fusion construct and analysis of confocal images in living cells. Plasma membrane localization of the fluorescent probe required the presence of three basic residues within the PLCdelta PH domain known to form critical contacts with PtdIns(4, 5)P2. Activation of endogenous PLCs by ionophores or by receptor stimulation produced rapid redistribution of the fluorescent signal from the membrane to cytosol, which was reversed after Ca2+ chelation. In both ionomycin- and agonist-stimulated cells, fluorescent probe distribution closely correlated with changes in absolute mass of PtdIns(4,5)P2. Inhibition of PtdIns(4,5)P2 synthesis by quercetin or phenylarsine oxide prevented the relocalization of the fluorescent probe to the membranes after Ca2+ chelation in ionomycin-treated cells or during agonist stimulation. In contrast, the synthesis of the PtdIns(4,5)P2 imaged by the PH domain was not sensitive to concentrations of wortmannin that had been found inhibitory of the synthesis of myo-[3H]inositol- labeled PtdIns(4,5)P2. Identification and dynamic imaging of phosphoinositides that interact with PH domains will further our understanding of the regulation of such proteins by inositol phospholipids.

Calcium current activated by potassium ions in voltage-clamped rat hippocampal pyramidal neurones.

1. Neuronal activity results in local elevation of extracellular K+ concentration ([K+]o). 2. Using the patch-clamp technique in the whole-cell configuration, we investigated whether extracellular K+ activates non-voltage-operated Ca2+ channels in pyramidal cells cultured from rat embryonic hippocampi. 3. K+ (12 mM) reversibly activated a sustained inward current at a holding potential of -100 mV. Membrane conductance and variance of noise were significantly increased by K+. This current could be observed at membrane potentials negative to +60 mV. 4. Inhibitors of inward rectifier K+ channels and hyperpolarization-induced cation current reduced the current only at potentials negative to -50 mV. 5. The K+-induced current was activated in Na+-free but not in Ca2+-free medium, did not depend on cytosolic [Cl-], and was blocked by Cd2+ but not by organic channel inhibitors. 6. Half-maximal activation of the current (at -100 mV) was attained at [K+]o approximately 20 mM. 7. The current is similar to Igl, a K+-induced Ca2+ current described in glomerulosa cells. It was also present in pyramidal cells from prefrontal cortex but not in hippocampal bipolar and glial cells. 8. Activation of K+-induced Ca2+ current may elevate cytoplasmic [Ca2+] at [K+]o levels which are insufficent to activate voltage-dependent Ca2+ channels.

Voltage dependent calcium channels in adrenal glomerulosa cells and in insulin producing cells.

We have examined the structure and function of Ca2+ channels in excitable endocrine cell types, in rat adrenal glomerulosa cells and in two insulin producing cell types, the rat pancreatic beta cell and the INS-1 cell line. In previous studies on glomerulosa cells, we observed low (T-type) and high threshold (L-type) voltage dependent Ca2+ currents in addition to a K+ induced inward rectifying Ca2+ current (Igl). beta cells are known to exhibit T-, L- and N-type currents. We have now found that INS-1 cells also show low threshold (T-type) and high threshold Ca2+ currents. The latter was further resolved by organic inhibitors into L-type and P/Q-type currents and no Igl was detected. The expression of the pore-forming alpha 1 subunit of voltage dependent Ca2+ channels was studied by means of reverse transcription-polymerase chain reaction (RT-PCR), followed by restriction enzyme mapping and/or sequencing. Both in glomerulosa and pancreatic beta cells, the neuroendocrine (D) class of the alpha 1 subunit, known to be responsible for L-type current, represents the majority of the PCR product. Comparable amounts of the neuroendocrine (D) and the neuronal A-type alpha 1 subunits dominate the message in INS-1 cells. Different characteristics of Ca2+ currents in these cell types is discussed in view of the channel repertoire.

Electrophysiological study on the high K+ sensitivity of rat glomerulosa cells.

Elevation of extracellular potassium concentration by as little as some tenth of mM activates rat adrenal glomerulosa cells. In the present study some factors responsible for this high K+ sensitivity were examined. Using whole-cell voltage-clamp technique we found that both T-type and L-type voltage-dependent Ca2+ channels have very low threshold potential (-71 and -58 mV, resp.). By means of patch-clamp technique combined with single-cell fluorimetry we also provided evidence that the activation of Igl, a K+-activated inward rectifying current is associated with Ca2+ influx. Both the low activation threshold of voltage-dependent Ca2+ channels and the function of Igl contribute to the exceptional K+ sensitivity of the glomerulosa cells.

Signaling events activated by angiotensin II receptors: what goes before and after the calcium signals.

Angiotensin II (Ang II) receptors of the AT1 subtype are coupled to heterotrimeric G nucleotide-binding proteins, G(q/11), to activate phospholipase C-beta isoforms with production of inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol. The resultant release of intracellular Ca2+ and increased Ca2+ influx are major determinants of several acute cellular responses initiated by Ang II, including secretion of aldosterone from the adrenal cortex and smooth muscle contraction. However, cellular events related to more prolonged effects of Ang II, such as hypertrophic and hyperplastic responses, are triggered by intracellular signaling cascades that are less dependent on Ca2+ signals. The Ang II-induced activation of Raf-1 kinase, p42 MAP-kinase and c-fos expression in response to Ang II in adrenal glomerulosa cells does not require Ca2+ influx. Moreover, the dose-response relationships for Raf-1 activation, MAP-kinase activation and mitogenesis show significantly higher sensitivity to Ang II than the InsP3, Ca2+-release and aldosterone secretory responses. The sensitivities of both Raf-1 kinase and MAP-kinase stimulation by Ang II to the inhibitors of phosphoinositide kinases, wortmannin and LY 294002, suggest that inositol phospholipids may play a role in these activation events unrelated to their role in Ca2+ signaling. To investigate the changes of various inositides after stimulation at the single cell level, fluorescent probes were developed in which pleckstrin homology domains with distinct binding specificities to inositol phospholipids were fused to the green fluorescent protein and expressed in NIH 3T3 cells. The use of these probes revealed heterogeneity of the inositol lipid pools and their complex relationship to Ca2+ signals. The use of these tools will help to further clarify the complex role of these lipids in initiating Ca2+-dependent and -independent signaling responses.

Activation of calcium current in voltage-clamped rat glomerulosa cells by potassium ions.

1. We examined Ca2+ influx mechanisms using the whole-cell patch-clamp technique in primary cultures of rat glomerulosa cells. 2. Depolarization of the plasma membrane, as studied by a stepwise or ramp depolarization technique, activated low-threshold, transient (T-type) and high-threshold, long-lasting (L-type) voltage-dependent calcium channels (VDCCs). 3. Extracellular K+ activated an inward current (Ig1), even in voltage-clamped cells. This phenomenon was observed within the physiological concentration range, beginning at 4.6 mM K+o (as opposed to the control level of 3.6 mM K+o). Increased cell conductance and increased background noise indicated that Ig1 is evoked by enhanced channel activity. Potassium induced no outward current in the voltage range examined (-120 to +60 mV). 4. When non-permeable anions were present only in the pipette and Na+ and Mg2+ were omitted from the bath, K+ still activated the current. Ig1 was blocked by 100 microM cadmium but was insensitive to 2 microM nifedipine or to 300 microM Ni2+. 5. In fluorimetric studies elevation of the cytoplasmic Ca2+ concentration in response to K+ (5.6-13.6 mM) was reduced only partially when VDCCs were blocked with Ni2+ (200 microM) and nifedipine (2 microM). 6. Elevation of the K+ concentration shifted the threshold potential of the T-type calcium channel in the negative direction. 7. In summary, K+ as a ligand activates Ca(2+)-permeable channels in rat glomerulosa cells. This current may contribute to the development of Ca2+ signals in response to stimulation with K+ in the physiological range. The reduction of the activation threshold of the T-type current by K+ may also be of physiological significance.

Dopamine blocks T-type calcium channels in cultured rat adrenal glomerulosa cells.

Dopamine is known to inhibit aldosterone secretion. In the present study using whole-cell voltage-clamp technique we found that dopamine, bromocriptine and quinpirole inhibit low-threshold (T-type) voltage dependent Ca2+ channels. The inhibiton was sustained and reversible, and it was prevented by sulpiride. These findings indicate that the effect of dopamine was mediated via DA2 receptors.

Highly co-operative Ca2+ activation of intermediate-conductance K+ channels in granulocytes from a human cell line.

1. To study Ca(2+)-activated K+ currents in dimethyl sulphoxide (DMSO)-differentiated HL-60 cells (HL-60 granulocytes), we have combined the patch clamp technique with microfluorimetric measurements of the cytosolic free Ca2+ concentration ([Ca2+]i). 2. Elevations of [Ca2+]i induced by the receptor agonist N-formyl-L-methionyl-L-phenylalanine (f-MLP), by cellular spreading or by the Ca2+ ionophore ionomycin, activated whole-cell currents. The kinetics of the current elevations closely paralleled the kinetics of the elevations in [Ca2+]i. Cellular spreading induced oscillations in [Ca2+]i and parallel oscillatory changes in the amplitude of the recorded currents. 3. The reversal potential of the Ca(2+)-activated current was a function of the extracellular K+ concentration (56.1 mV per log [K+]), demonstrating that the underlying conductance was selective for K+. 4. The current was blocked by charybdotoxin, but insensitive to apamin. 5. The whole-cell current was inwardly rectifying. No time-dependent activation or inactivation of the current could be observed within the range of voltages tested (-100 to +100 mV). 6. The dependence of the current amplitude on the measured [Ca2+]i revealed a half-maximal activation at approximately 350 nM [Ca2+]i, and a highly co-operative activation by [Ca2+]i with an apparent Hill coefficient of approximately 8. Neither the half-maximal activation by [Ca2+]i nor the apparent Hill coefficient depended on the voltage, and they were identical for Ca2+ elevations caused by the ionophore and the receptor agonist. 7. Analysis of Ca(2+)-activated single-channel events in cell-attached recordings revealed an inwardly rectifying K+ channel with a slope conductance of 35 pS. Fluctuation analysis of the Ca(2+)-activated whole-cell current suggested an underlying single-channel conductance of a similar size (28 pS). 8. In summary, we describe a charybdotoxin-sensitive, intermediate-conductance Ca(2+)-activated K+ channel in HL-60 granulocytes. The characteristics of the Ca2+ activation of this current (i.e. sensitivity to submicromolar [Ca2+]i, high co-operativity and voltage independence) are similar to the Ca2+ activation of the apamin-sensitive small-conductance K+ channel. Our results also suggest that [Ca2+]i elevations are the predominant, if not the only, activators of this channel during physiological stimulation of HL-60 granulocytes.

Regulation of Ca2+ influx in myeloid cells. Role of plasma membrane potential, inositol phosphates, cytosolic free [Ca2+], and filling state of intracellular Ca2+ stores.

To study the mediation of Ca2+ influx by second messengers in myeloid cells, we have combined the whole-cell patch clamp technique with microfluorimetric measurements of [Ca2+]i. Me2SO-differentiated HL-60 cells were loaded with the fluorescent Ca2+ indicator Indo-1, allowed to adhere to glass slides, and patch-clamped. Receptor agonists and Ca(2+)-ATPase inhibitors were applied by superfusion and inositol phosphates by microperfusion through the patch pipette. In voltage-clamped cells, [Ca2+]i elevations with a sustained phase could be induced by (a) the chemoattractant receptor agonist FMLP, (b) the Ca(2+)-releasing second messenger myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P3], as well as its nonmetabolizable analogues, and (c) the Ca(2+)-ATPase inhibitor cyclopiazonic acid, which depletes intracellular Ca2+ stores. In the absence of extracellular Ca2+, responses to all stimuli were short-lasting, monophasic transients; however, subsequent addition of Ca2+ to the extracellular medium led to an immediate [Ca2+]i increase. In all cases, the sustained phase of the [Ca2+]i elevations could be inhibited by millimolar concentrations of extracellular Ni2+, and its amplitude could be decreased by depolarization of the plasma membrane. Thus, the sustained phase of the Ca2+ elevations was due to Ca2+ influx through a pathway sensitive to the electrical driving force and to Ni2+. No Ca2+ influx could be observed after (a) plasma membrane depolarization in resting cells, (b) an imposed [Ca2+]i transient independent of receptor activation, or (c) microperfusion of myo-inositol(1,3,4,5)tetrahisphosphate (Ins(1,3,4,5)P4). Also, Ins(1,3,4,5)P4 did not have additive effects when co-perfused with a submaximal concentration of Ins(1,4,5)P3. Our results suggest that, in myeloid cells, activation of chemoattractant receptors induces an electrogenic, Ni(2+)-sensitive Ca2+ influx via generation of Ins(1,4,5)P3. Ins(1,4,5)P3 might activate Ca2+ influx directly, or by depletion of intracellular Ca2+ stores, but not via [Ca2+]i increase or Ins(1,3,4,5)P4 generation.

The role of protein kinase-C in control of aldosterone production by rat adrenal glomerulosa cells: activation of protein kinase-C by stimulation with potassium.

The role of protein kinase-C (PKC) in control of the function of rat adrenal glomerulosa cells was studied. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, inhibited the stimulation of aldosterone production induced by K+ (5.4 mM) or ACTH (5 pM) in a dose-dependent manner. Phorbol 12,13-dibutyrate, another phorbol ester that activates PKC, also exerted an inhibitory effect, while the inactive 4 alpha-phorbol 12,13-didecanoate failed to affect aldosterone production. The inhibitory effect of PMA (5 nM) was reversed by preincubation of the cells with staurosporine (ST; 50 nM), an inhibitor of PKC. These data suggest that pharmacological activation of PKC initiates an inhibitory mechanism in rat glomerulosa cells. To elucidate whether PKC is activated by physiological stimuli, the effects of ST and down-regulation of PKC by prolonged pretreatment with PMA on stimulation of aldosterone production were studied. The effects of angiotensin-II (AII) and K+, but not that of ACTH, were enhanced by ST pretreatment. This potentiation was prompt and transient in the case of AII (2.5 nM), while it developed gradually when the cells were stimulated with K+ (5.4 or 18 mM). Long term pretreatment (6 h) of glomerulosa cells with PMA also enhanced the stimulatory effect of AII (300 pM) and K+ (5.4 mM). These data together suggest that the actions of AII and K+ on aldosterone production involve a PKC-mediated inhibition. Activation of PKC by AII is probably due to formation of diacylglycerol via receptor-mediated activation of phosphoinositide-specific phospholipase-C. Stimulation with K+ caused a moderate accumulation of [3H]inositol phosphate in a concentration-dependent manner. Since this effect was abolished by nifedipine, activation of phospholipase-C may have been secondary to Ca2+ entry. The concomitant formation of diacylglycerol may contribute to activation of PKC in K+ stimulated cells. In conclusion, our data support the view that PKC participates in the physiological control of aldosterone production by rat adrenal glomerulosa cells. In addition to AII, K+ may activate PKC. Regardless of whether the enzyme is activated by phorbol esters or physiological stimuli, it exerts an inhibitory, rather than stimulatory, action on steroid 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.

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.