When is high-Ca+ microdomain required for mitochondrial Ca+ uptake?

Ca(2+) release from IP(3)-sensitive stores in the endoplasmic reticulum (ER) induced by Ca(2+)-mobilizing agonists generates high-Ca(2+) microdomains between ER vesicles and neighbouring mitochondria. Here we present a model that describes when such microdomains are required and when submicromolar [Ca(2+)] is sufficient for mitochondrial Ca(2+) uptake. Mitochondrial Ca(2+) uptake rate in angiotensin II-stimulated H295R adrenocortical cells correlates with the proximity between ER vesicles and the mitochondrion, reflecting the uptake promoting effect of high-Ca(2+) peri-mitochondrial microdomains. Silencing or inhibition of p38 mitogen-activated protein kinase (MAPK) or inhibition of the novel isoforms of protein kinase C enhances mitochondrial Ca(2+) uptake and abolishes the positive correlation between Ca(2+) uptake and ER-mitochondrion proximity. Inhibition of protein phosphatases attenuates mitochondrial Ca(2+) uptake and also abolishes its positive correlation with ER-mitochondrion proximity. We postulate that during IP(3)-induced Ca(2+) release, Ca(2+) uptake is confined to ER-close mitochondria, because of the simultaneous activation of the protein kinases. Attenuation of Ca(2+) uptake prevents Ca(2+) overload of mitochondria and thus protects the cell against apoptosis. On the other hand, all the mitochondria accumulate Ca(2+) at a non-inhibited rate during physiological Ca(2+) influx through the plasma membrane. Membrane potential is higher in ER-distant mitochondria, providing a bigger driving force for Ca(2+) uptake. Our model explains why comparable mitochondrial Ca(2+) signals are formed in response to K(+) and angiotensin II (equipotent in respect to global cytosolic Ca(2+) signals), although only the latter generates high-Ca(2+) microdomains.

Mitochondria respond to Ca2+ already in the submicromolar range: correlation with redox state.

Rapid formation of high-Ca2+ perimitochondrial cytoplasmic microdomains has been shown to evoke mitochondrial Ca2+ signal and activate mitochondrial dehydrogenases, however, the significance of submicromolar cytoplasmic Ca2+ concentrations in the control of mitochondrial metabolism has not been sufficiently elucidated. Here we studied the mitochondrial response to application of Ca2+ at buffered concentrations in permeabilized rat adrenal glomerulosa cells, in an insulin-producing cell line (INS-1/EK-3) and in an osteosarcoma cell line (143BmA-13). Mitochondrial Ca2+ concentration was measured with the fluorescent dye rhod-2 and, using an in situ calibration method, with the mitochondrially targeted luminescent protein mt-aequorin. In both endocrine cell types, mitochondrial Ca2+ concentration increased in response to elevated cytoplasmic Ca2+ concentration (between 60 and 740 nM) and an increase in mitochondrial Ca2+ concentration could be revealed already at a cytoplasmic Ca2+ concentration step from 60-140 nM. Similar responses were observed in the osteosarcoma cell line, although a clearcut response was first observed at 280 nM extramitochondrial Ca2+ only. As examined in glomerulosa cells, graded increases in cytoplasmic Ca2+ concentration were associated with graded increases in the reduction of mitochondrial pyridine nucleotides, consistent with Ca2+-dependent activation of mitochondrial dehydrogenases. Our data indicate that in addition to the recognized role of high-Ca2+ cytoplasmic microdomains, also small Ca2+ signals may influence mitochondrial metabolism.

Stimulus-secretion coupling and mitochondrial metabolism in steroid-secreting cells.

Ca(2+) signal in high-Ca(2+) perimitochondrial microdomains is amplified within the mitochondrial matrix and activates Ca(2+)-dependent dehydrogenases. In steroid-secreting cells, small cytoplasmic Ca(2+) signals may also augment mitochondrial Ca(2+) concentration. The ensuing formation of NADH and NADPH may have an essential role in supporting the increased steroid secretion.

A pH-sensitive chloride current in the chemoreceptor cell of rat carotid body.

1. Cardiorespiratory response to acidosis is initiated by the carotid body. 2. The direct effect of extracellular pH (pH(o)) on the chloride currents of isolated chemoreceptor cells of the rat carotid body was investigated using the whole-cell patch-clamp technique. 3. On applying intra- and extracellular solutions with a symmetrical high-Cl(-) content and with the monovalent cations replaced with membrane-impermeant ones, an inwardly rectifying Cl(-) current was found. 4. The current activated slowly and did not display any time-dependent inactivation. Current activation was present at membrane potentials negative to 0 mV (pH(o) = 7.0). 5. The current was activated by extracellular acidosis and inhibited by alkalosis in the physiologically relevant pH range of 7.0-7.8. 6. The current was reduced by 0.1 mM Cd2+ to the level of the leak current and by 1 mM anthracene-9-carboxylic acid (9-AC) to about 40 %, while 0.1 mM Ba2+ had no effect. 7. Application of 1 mM 9-AC caused a slow but statistically significant increase in the resting pH(i) (from a mean of 7.29 to 7.37 in 5 min) in clusters of chemoreceptor cells in CO(2)/HCO3(-)-buffered media as measured with carboxy-SNARF-1. 8. When membrane potential changes were estimated in the cell-attached mode, 1 mM 9-AC hyperpolarized three out of five tested cells (by 14 mV in average) incubated in CO(2)/HCO3(-)-buffered media. 9. In summary, chemoreceptor cells express an inwardly rectifying Cl(-) current, which is directly regulated by pH(o). The current may participate in intracellular acidification and membrane depolarization during acidic challenge.

Inhibition of TASK-1 potassium channel by phospholipase C.

The two-pore-domain K(+) channel, TASK-1, was recently shown to be a target of receptor-mediated regulation in neurons and in adrenal glomerulosa cells. Here, we demonstrate that TASK-1 expressed in Xenopus laevis oocytes is inhibited by different Ca(2+)-mobilizing agonists. Lysophosphatidic acid, via its endogenous receptor, and ANG II and carbachol, via their heterologously expressed ANG II type 1a and M(1) muscarinic receptors, respectively, inhibit TASK-1. This effect can be mimicked by guanosine 5'-O-(3-thiotriphosphate), indicating the involvement of GTP-binding protein(s). The phospholipase C inhibitor U-73122 reduced the receptor-mediated inhibition of TASK-1. Downstream signals of phospholipase C action (inositol 1,4,5-trisphosphate, cytoplasmic Ca(2+) concentration, and diacylglycerol) do not mediate the inhibition. Unlike the G(q)-coupled receptors, stimulation of the G(i)-activating M(2) muscarinic receptor coexpressed with TASK-1 results in an only minimal decrease of the TASK-1 current. However, additional coexpression of phospholipase C-beta(2) (which is responsive also to G(i) beta gamma-subunits) renders M(2) receptor activation effective. This indicates the significance of phospholipase C activity in the receptor-mediated inhibition of TASK-1.

pH-sensitive inwardly rectifying chloride current in cultured rat cortical astrocytes.

The effect of pH(o) on plasma membrane chloride current of cultured rat cortical astrocytes was investigated using the whole-cell patch-clamp technique. In the presence of intra- and extracellular solutions with symmetrical high Cl(-) content and K(+) channel inhibitors, the cells exhibited an inwardly rectifying current. The current activated slowly at potentials negative to -40 mV and did not display time-dependent inactivation. The current was inhibited by 0.1 mM Cd(2+), 0.1 mM Zn(2+), 1 mM 9-anthracene-carboxylic acid, and 0.2 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid, but not by 10 mM Ba(2+) or 3 mM Cs(+). Reversal potential of the current followed the chloride equilibrium potential and was not influenced by changes in K(+) or Na(+) concentration. The inwardly rectifying chloride current was augmented by extracellular acidosis and reduced by alkalosis. The pH sensitivity was most pronounced in the physiologically relevant pH(o) range of 6.9--7.9. Lowering pH to 6.4 induced no additional increase in steady-state current amplitude compared with pH(o) 6.9, but it substantially slowed the activation kinetics. According to its kinetic and pharmacological properties this chloride current is similar to that found in cultured rat astrocytes after long-term treatment with dibutyryl-cAMP, however, in our cultures it was consistently expressed without any treatment with the drug. Considering that astrocytes possess carbonic anhydrase and Cl(-)/HCO3(-) antiporter, this current may participate in the regulation of the interstitial and astrocyte pH.

Cytoplasmic Ca2+ at low submicromolar concentration stimulates mitochondrial metabolism in rat luteal cells.

The cytoplasmic Ca2+ signal is transferred to the mitochondrial matrix and activates mitochondrial dehydrogenases. The requirement for supramicromolar cytoplasmic [Ca2+] ([Ca2+]i) in perimitochondrial microdomains in this response has been suggested. We studied the correlation between [Ca2+]i, mitochondrial [Ca2+] ([Ca2+]m) and mitochondrial formation of reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] in the presence of submicromolar [Ca2+]i in cultured rat "large" luteal cells. [Ca2+]i was monitored fluorimetrically with fura-PE3, [Ca2+]m with rhod-2 and NAD(P)H with autofluorescence. In intact cells, prostaglandin F2alpha, which induces both intracellular Ca2+ release and Ca2+ entry, stimulated mitochondrial NAD(P)H formation. Thapsigargin-induced Ca2+ release and subsequent capacitative Ca2+ entry, both resulting in Ca2+ responses not exceeding 150-200 nM, also enhanced the reduction of pyridine nucleotides. As shown in inhibitor studies, the increased steady-state NAD(P)H level was due to activation of Ca2+-dependent dehydrogenases. [Ca2+]m, measured in permeabilized cells, increased moderately, but significantly, following elevation of [Ca2+]i from 50 to 180 nM, showed a further gradual increase at higher submicromolar [Ca2+]i values and rose steeply at supramicromolar [Ca2+]i. In summary, our results demonstrate that, in a steroid-producing cell type, net mitochondrial Ca2+ uptake and mitochondrial dehydrogenation can be activated even by low submicromolar increases of [Ca2+]i.

TASK (TWIK-related acid-sensitive K+ channel) is expressed in glomerulosa cells of rat adrenal cortex and inhibited by angiotensin II.

The present study was conducted to explore the possible contribution of a recently described leak K+ channel, TASK (TWIK-related acid-sensitive K+ channel), to the high resting K+ conductance of adrenal glomerulosa cells. Northern blot analysis showed the strongest TASK message in adrenal glomerulosa (capsular) tissue among the examined tissues including heart and brain. Single-cell PCR demonstrated TASK expression in glomerulosa cells. In patch-clamp experiments performed on isolated glomerulosa cells the inward current at -100 mV in 30 mM [K+] (reflecting mainly potassium conductance) was pH sensitive (17+/-2% reduction when the pH changed from 7.4 to 6.7). In Xenopus oocytes injected with mRNA prepared from adrenal glomerulosa tissue the expressed K+ current at -100 mV was virtually insensitive to tetraethylammonium (3 mM) and 4-aminopyridine (3 mM). Ba2+ (300 microM) and Cs+ (3 mM) induced voltage-dependent block. Lidocaine (1 mM) and extracellular acidification from pH 7.5 to 6.7 inhibited the current (by 28% and 16%, respectively). This inhibitory profile is similar (although it is not identical) to that of TASK expressed by injecting its cRNA. In oocytes injected with adrenal glomerulosa mRNA, TASK antisense oligonucleotide reduced significantly the expression of K+ current at -100 mV, while the sense oligonucleotide failed to have inhibitory effect. Application of angiotensin II (10 nM) both in isolated glomerulosa cells and in oocytes injected with adrenal glomerulosa mRNA inhibited the K+ current at -100 mV. Similarly, in oocytes coexpressing TASK and ATla angiotensin II receptor, angiotensin II inhibited the TASK current. These data together indicate that TASK contributes to the generation of high resting potassium permeability of glomerulosa cells, and this background K+ channel may be a target of hormonal regulation.

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.

Inhibition of voltage-gated calcium channels by fluoxetine in rat hippocampal pyramidal cells.

Fluoxetine, an antidepressant which is used world-wide, is a prominent member of the class of selective serotonin re-uptake inhibitors. Recently, inhibition of voltage-gated Na(+) and K(+) channels by fluoxetine has also been reported. We examined the effect of fluoxetine on voltage-gated calcium channels using the patch-clamp technique in the whole-cell configuration. In hippocampal pyramidal cells, fluoxetine inhibited the low-voltage-activated (T-type) calcium current with an IC(50) of 6.8 microM. Fluoxetine decreased the high-voltage-activated (HVA) calcium current with an IC(50) between 1 and 2 microM. Nifedipine and omega-conotoxin GVIA inhibited the HVA current by 24% and 43%, respectively. Fluoxetine (3 microM), applied in addition to nifedipine or omega-conotoxin, further reduced the current. When fluoxetine (3 microM) was applied first neither nifedipine nor omega-conotoxin attenuated the remaining component of the HVA current. This observation indicates that fluoxetine inhibits both L- and N-type currents. In addition, fluoxetine inhibited the HVA calcium current in carotid body type I chemoreceptor cells and pyramidal neurons prepared from prefrontal cortex. In hippocampal pyramidal cells high K(+)-induced seizure-like activity was inhibited by 1 microM fluoxetine; the mean burst duration was shortened by an average of 44%. These results provide evidence for inhibition of T-, N- and L-type voltage-gated calcium channels by fluoxetine at therapeutically relevant concentrations.

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.

Cytoplasmic Ca2+ signalling and reduction of mitochondrial pyridine nucleotides in adrenal glomerulosa cells in response to K+, angiotensin II and vasopressin.

We have examined the mitochondrial formation of NAD(P)H in rat adrenal glomerulosa cells. A short-term elevation of the K+ concentration from 3.6 to 8.4 mM induced a reversible increase in the formation of reduced pyridine nucleotides. Potassium applied after the addition of rotenone had no further effect, confirming that the redox signal was of mitochondrial origin. Inhibition of aldosterone synthesis by aminoglutethimide in K+-stimulated cells decreased the rate of decay of the NAD(P)H signal upon the termination of stimulation, indicating that the NADPH formed was consumed in aldosterone synthesis. When the NAD(P)H signal was measured simultaneously with the cytoplasmic free Ca2+ concentration ([Ca2+]i), elevation of the K+ concentration to 6.6 or 8.4 mM induced parallel increases in [Ca2+]i and NAD(P)H formation. The rates of increase and decrease of NAD(P)H were lower than for [Ca2+]i, confirming that the redox signal was secondary to the Ca2+ signal. Angiotensin II (100 pM-1 nM) induced an oscillatory NAD(P)H signal which usually returned to a lower baseline concentration, while a sustained signal with superimposed oscillations was observed at higher concentrations. Simultaneous measurements showed that NAD(P)H levels followed the [Ca2+]i pattern evoked by angiotensin II. Vasopressin (100 nM) also induced parallel oscillations of [Ca2+]i and NAD(P)H. A sustained rise in the extramitochondrial Ca2+ concentration to 1 microM induced a sustained elevation of the intramitochondrial Ca2+ concentration in permeabilized cells, as measured with rhod-2. A sustained rise in [Ca2+]i evoked by long-term stimulation with 8.4 mM K+ or 2.5 nM angiotensin II resulted in sustained NAD(P)H production. These Ca2+-dependent changes in the mitochondrial redox state support the biological response, i.e. aldosterone secretion by glomerulosa cells.

Intracellular calcium release is more efficient than calcium influx in stimulating mitochondrial NAD(P)H formation in adrenal glomerulosa cells.

We compared the effect on mitochondrial NAD(P)H formation of calcium release from intracellular stores with that of calcium influx from the extracellular space. Simultaneous measurements of cytoplasmic free calcium ion concentration and mitochondrial NAD(P)H were performed on fura-PE3-loaded single rat adrenal glomerulosa cells. The effects of equipotent stimuli in terms of the evoked Ca2+ response were compared. Angiotensin II (AII; 1 nM) induced a higher amplitude NAD(P)H response than K+ (5.6-7.6 mM). Vasopressin (1 microM) also induced a greater initial NAD(P)H formation than K+, although the Ca2+ signal evoked by the two agonists had similar amplitude. To examine the effect of Ca2+ release from internal stores we applied AII in Ca2+-free medium. We compared the effect on NAD(P)H formation of Ca2+ release with Ca2+ influx induced by K+, and with capacitative Ca2+ influx induced by AII. NAD(P)H formation in response to Ca2+ release was greater than that induced by Ca2+ influx, irrespective of whether induced by K+ or AII. Our results indicate that Ca2+, presumably released in the vicinity of mitochondria, activates mitochondrial dehydrogenases more efficiently than Ca2+ entering through the plasma membrane. These data confirm the biological significance of previous observations showing that Ca2+ released from inositol 1,4, 5-trisphosphate-sensitive internal stores increases mitochondrial matrix [Ca2+] to a greater extent than extracellular Ca2+.

The role of voltage-dependent calcium channels in angiotensin-stimulated glomerulosa cells.

The concept that voltage-dependent Ca2+ influx is essential in the aldosterone stimulating action of angiotensin II (AII) has been recently challenged by the demonstration of the dihydropyridine (DHP) insensitive 'capacitative' Ca2+ uptake mechanism. The DHP-sensitivity of AII-induced aldosterone secretion is still to be explained. In rat glomerulosa cells the lag phase of AII-induced depolarization is more than 30 s, and there is no enhanced Ca2+ influx within the first min of stimulation. Yet we observed that DHPs as well as diltiazem influenced also the peak of cytoplasmic Ca2+ signal, although the peak (approximately 12 s) is attributed to Ca2+ release alone. Nifedipine reduced the Ca2+ transient induced by AII even after complete inhibition of Ca2+ channel activity. Recalling the loose attachment of InsP3 receptors (IP3R) to the plasma membrane, and the homology between the cytosolic domain of IP3R and the Ca2+ release channel (ryanodine receptor) of skeletal muscle, we proposed that DHP-sensitive L-type Ca2+ channels (DHP receptors) influence InsP3-induced Ca2+ release rather than Ca2+ influx in AII-stimulated cells. Although the dominant isoform is the neuroendocrine (D) one, the skeletal muscle isoform of L-type voltage-dependent Ca2+ channel is also expressed in rat glomerulosa cells. This isoform may be a candidate for protein-protein interaction between DHPR and subplasmalemmal IP3R, similarly to that occurring between DHP receptors and ryanodine receptors in skeletal muscle.

Expression of inositol 1,4,5-trisphosphate receptors in rat adrenocortical zones.

Previously we demonstrated the presence of InsP3R-I, -II and -III subtypes in the zona glomerulosa. Now we have examined the expression of different subtypes of inositol 1,4,5-trisphosphate receptor (InsP3R) in the inner zones of rat adrenal cortex. RNA extracted from decapsulated adrenal tissue (zonae fasciculata-reticularis and the medulla) or from fasciculata-reticularis cells was reverse transcribed. Subsequent polymerase chain reaction revealed the presence of InsP3R-I, -II and -III subtypes in decapsulated tissue but failed to demonstrate the expression of any known subtypes of InsP3R in fasciculata-reticularis cells. Accordingly, InsP3 receptors expressed in the decapsulated tissue are of medullary origin.

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.