Single channel properties and regulated expression of Ca(2+) release-activated Ca(2+) (CRAC) channels in human T cells.

Although the crucial role of Ca(2+) influx in lymphocyte activation has been well documented, little is known about the properties or expression levels of Ca(2+) channels in normal human T lymphocytes. The use of Na(+) as the permeant ion in divalent-free solution permitted Ca(2+) release-activated Ca(2+) (CRAC) channel activation, kinetic properties, and functional expression levels to be investigated with single channel resolution in resting and phytohemagglutinin (PHA)-activated human T cells. Passive Ca(2+) store depletion resulted in the opening of 41-pS CRAC channels characterized by high open probabilities, voltage-dependent block by extracellular Ca(2+) in the micromolar range, selective Ca(2+) permeation in the millimolar range, and inactivation that depended upon intracellular Mg(2+) ions. The number of CRAC channels per cell increased greatly from approximately 15 in resting T cells to approximately 140 in activated T cells. Treatment with the phorbol ester PMA also increased CRAC channel expression to approximately 60 channels per cell, whereas the immunosuppressive drug cyclosporin A (1 microM) suppressed the PHA-induced increase in functional channel expression. Capacitative Ca(2+) influx induced by thapsigargin was also significantly enhanced in activated T cells. We conclude that a surprisingly low number of CRAC channels are sufficient to mediate Ca(2+) influx in human resting T cells, and that the expression of CRAC channels increases approximately 10-fold during activation, resulting in enhanced Ca(2+) signaling.

A current activated on depletion of intracellular Ca2+ stores can regulate exocytosis in adrenal chromaffin cells.

Exocytosis in excitable cells is strongly coupled to Ca2+ entry through voltage-gated channels but can be evoked by activation of membrane receptors that release Ca2+ from inositol 1,4, 5-trisphosphate-sensitive internal stores. In many cell types, depletion of Ca2+ stores activates Ca2+ influx across the plasma membrane, a process known as capacitative or store-operated Ca2+ entry. This influx is mediated by a number of voltage-independent, Ca2+-selective currents. In addition to replenishing Ca2+ stores, these currents are hypothesized to play an important role in agonist-evoked secretion in nonexcitable cells, although this has not been confirmed experimentally. The existence and physiological function of such currents in excitable cells is not known. Using the capacitance detection technique to monitor exocytosis, we provide direct experimental evidence that a similar mechanism exists in bovine adrenal chromaffin cells. Depletion of intracellular Ca2+ stores with thapsigargin, a SERCA pump inhibitor, or with BAPTA, an exogenous Ca2+ chelator, activates a small-amplitude, voltage-independent current that is carried by Ca2+ and Na+ ions. Ca2+ entry through this pathway is sufficient to stimulate exocytosis at negative membrane potentials. In addition, depolarization-evoked exocytosis is markedly facilitated on activation of the current. These data suggest that excitable cells possess a store-operated Ca2+ influx mechanism that may both directly trigger exocytosis and modulate excitation-secretion coupling.

Control of Ca2+ channel current and exocytosis in rat lactotrophs by basally active protein kinase C and calcineurin.

Modulation of voltage-activated Ca2+ channel activity by phosphorylation was studied in metabolically intact voltage-clamped rat lactotrophs. Experiments using Ba2+ as a charge carrier indicated that a phorbol ester protein kinase C activator stimulates high-voltage-activated Ca2+ channel currents, but has no effect on low-voltage-activated currents. Extracellular application of structurally and mechanistically distinct protein kinase C inhibitors (staurosporin, H7, calphostin C, chelerythrine and Ro 31-8220) preferentially inhibited the high-voltage-activated Ba2+ current. This suggests that protein kinase C is required for maintainance of Ca2+ channel activity even in the absence of modulators. Cyclosporin A, an inhibitor of the Ca2+/calmodulin-dependent protein phosphatase calcineurin, increased the high-voltage-activated Ca2+ channel current, and staurosporin reversed this effect. Thus, dephosphosphorylation by calcineurin may limit basal Ca2+ channel activity. Time-domain monitoring of cellular capacitance changes demonstrated that cyclosporin A and 12-O-tetradecanoyl-phorbol-13-acetate do not affect exocytosis at a hyperpolarized potential, but each enhances depolarization-induced exocytosis. Facilitation of exocytosis by cyclosporin A differed from 12-O-tetradecanoyl-phorbol-13-acetate in that it was biphasic. The delayed facilitation induced by cyclosporin A could be accounted for by stimulation of the voltage-gated Ca2+ current. These results suggest that the high-voltage activated Ca2+ channel current in rat lactotrophs is determined by the opposing basal activities of protein kinase C and calcineurin. Furthermore, it is concluded that the regulation of Ca2+ channels by protein kinase C and calcineurin affects depolarization-induced exocytosis.

Dexamethasone rapidly increases calcium channel subunit messenger RNA expression and high voltage-activated calcium current in clonal pituitary cells.

Glucocorticoid hormones increase voltage-gated Ca(2)+ current density in clonal pituitary cells. To test whether these steroids might stimulate expression of Ca(2)+ channel genes, messenger RNase protection assays were used to measure alpha IC and alpha ID RNAs that encode pore-forming subunits of L-type Ca2+ channels. We show here that dexamethasone rapidly increases alpha IC messenger RNA expression without affecting alpha ID messenger RNA level. This up-regulation of channel messenger RNA is also produced by natural glucocorticoids and is blocked by the glucocorticoid antagonist Ru48386. The up-regulation of the channel subunit messenger RNA expression is associated with an increase in high voltage-activated Ca(2)+ current density. Thus, glucocorticoids may produce a long-term effect on Ca(2)+ homeostasis in clonal pituitary cells by differentially regulating expression of Ca(2)+ channel subunit genes.

Three phases of TRH-induced facilitation of exocytosis by single lactotrophs.

Membrane capacitance measurements were used to study neuropeptide modulation of exocytosis by perforated patch clamped rat lactotrophs. We report that depolarizing voltage-clamp pulses evoke exocytosis that is steeply dependent on Ca2+ influx through voltage-gated Ca2+ channels. Furthermore, we find that the neuropeptide TRH (thyrotropin-releasing hormone) acts in three phases to promote exocytosis. First, TRH transiently (within approximately 0.5 min) triggers depolarization- and extracellular Ca(2+)-independent exocytosis. Second, within 3 min of application, TRH facilitates depolarization-evoked exocytosis while inhibiting voltage-gated Ca2+ current. Finally, after 8 min, TRH further enhances depolarization-evoked exocytosis by increasing high-voltage-activated (HVA) Ca2+ channel current. Activation of protein kinase C (PKC) with a phorbol ester also stimulates depolarization-evoked exocytosis by increasing Ca2+ current. Therefore, PKC can only account for the last effect of TRH. Thus, a single neuromodulator may employ several temporally distinct mechanisms to stimulate peptide secretion.

Inhibition of voltage-gated K+ channel gene expression by the neuropeptide thyrotropin-releasing hormone.

Many neurotransmitters regulate action potential activity in neuronal, endocrine, and cardiac cells by rapidly modulating the gating of K+ channels. Neurotransmitters might also produce prolonged effects on excitability by regulating the expression of K+ channel genes. Here we show that the neuropeptide thyrotropin-releasing hormone (TRH) down-regulates Kv1.5 and Kv2.1 K+ channel mRNAs in clonal pituitary cells. The effect on Kv1.5 mRNA expression does not require protein synthesis and is due to decreased transcription. Immunoblots demonstrate that Kv1.5 and Kv2.1 immunoreactivities are significantly reduced by TRH within 12 hr. The change in channel protein expression is associated with a decrease in voltage-gated K+ currents. Thus, TRH enhances excitability by inhibiting K+ channel gene expression. Neuropeptide regulation of K+ channel gene expression may produce long-term changes in neuronal action potential activity and synaptic transmission.

Dexamethasone rapidly induces Kv1.5 K+ channel gene transcription and expression in clonal pituitary cells.

Glucocorticoids specifically increase Kv1.5 K+ channel mRNA in normal and clonal (GH3) rat pituitary cells. Here, we demonstrate that dexamethasone, a glucocorticoid agonist, rapidly induces Kv1.5 gene transcription, but does not affect Kv1.5 mRNA turnover (t1/2 approximately 0.5 hr) in GH3 cells. Immunoblots indicate that the steroid also increases the expression of the 76 kd Kv1.5 protein approximately 3-fold within 12 hr without altering its half-life (t1/2 approximately 4 hr). In contrast, Kv1.4 protein expression is unaffected. Finally, we find that the induction of Kv1.5 protein is associated with an increase in a noninactivating component of the voltage-gated K+ current. Our results indicate that hormones and neurotransmitters may act within hours to regulate excitability by controlling K+ channel gene expression.

Glucocorticoid modulation of calcium currents in growth hormone 3 cells.

Clonal malignant pituitary growth hormone 3 cells were used for the analysis of the influence of hydrocortisone and dexamethasone on voltage-gated calcium currents and hormone secretion. The whole-cell patch-clamp technique was used. The presence of low-threshold inactivating and high-threshold persisting components in the total calcium current was shown; they could be separated at less negative holding potential level. Some increase in current densities of both components was observed as early as 30 min after treatment with 10(-6) mol/l glucocorticoids. The increase was maximal for both types of currents after 2 h of incubation; however, the high-threshold component was affected much more strongly (current density increased by more than four-fold) than the low-threshold one (current density increased by about a three-fold). Potentiation of currents was blocked by actinomycine D (10(-4) M), suggesting that protein synthesis was required. A substantial increase in growth hormone secretion (measured by radioimmunoassay method) was observed in the same cells after 2 h of incubation with hydrocortisone, while the secretion of prolactin remained even slightly depressed.

[Action of omega-conotoxin on calcium currents of the rat pituitary GH3 cell line]. / Deistvie omega-konotoksina na kal'tsievye toki kletok linii GH3 gipofiza krysy.

Whole-cell modification of the patch clamp method was used to examine the action of omega-CgTX on calcium currents in GH3 pituitary cells. Two quite distinct components of inward calcium currents were observed in the presence of 15 mmol/l of calcium in the external solution. One was activated from the holding potential -80 mV by testing pulses more positive than -50 mV. The shift of the holding potential to -40 mV resulted in the stationary inactivation of this low voltage activated current component. It was found that omega-CgTX activated both low-threshold and high-threshold calcium currents at the first moment of application, but low-threshold current component increased more significantly. Full effect was developed for less than 30 s. Then time decay of currents was comparable with that of the "wash-out" process. Incubation of cells in the growth medium that contained 5 mumol/l omega-CgTX during 2 hour induced an increase in density of both types of calcium currents, then it fell after 2 hours of incubation in the same medium.

[Changes in the morphometric parameters of neuroblastoma cells cultured at an elevated pH]. / Izmenenie morfometricheskikh parametrov kletok neiroblastomy, kul'tiviruemykh v usloviiakh povyshennogo pH.

The phenomena arising during morphological differentiation of neuroblastoma cells under pH of culture medium 8.0-8.2 have been investigated. The initial cell population divided into two parts--proliferating and differentiating cells--on the third day of cultivation in the modified medium. Reliable correlation between somatic size and neurite length has been found in differentiating cells. Thus somatic size may be used as morphological criterion of differentiated neuroblastoma cells under present conditions. The established relations may be used for the further investigation of morphofunctional alterations under induced differentiation of neuroblastoma cells.

[Sodium and calcium channels of the somatic membrane of neuroblastoma cells during artificially induced differentiation]. / Issledovanie natrievykh i kal'tsievykh kanalov somaticheskoi membrany kletok neiroblastomy v protsesse ikh iskusstvenno vyzvannoi differentsirovki.

Sodium and calcium channels passing inward currents were studied by intracellular dialysis technique and voltage clamp in the somatic membrane of neuroblastoma cells during their morphological differentiation induced by increasing pH of the culture medium up to 8.0-8.2. Kinetic and voltage-dependent properties of sodium and calcium channels of differentiated cells and cells grown in the suspension culture were identical. Densities of sodium currents in the somatic membrane of neuroblastoma cells grown in suspension culture were about 7.3 +/- 0.8 microA/microF and from 37 +/- 5.2 microA/microF to 54.7 +/- 3.6 microA/microF of differentiated cells at different terms of cultivation. Densities of calcium currents in the membrane of cells grown in suspension were about 1.4 +/- 0.2 microA/microF, while in differentiated cells they ranged from 1.1 +/- 0.2 to 2.8 +/- 0.4 microA/microF at different terms of cultivation. Induction or reduction of differentiation by varying pH of the culture medium produced reciprocal changes in densities of sodium and calcium channels.

[Morphologic differentiation of neuroblastoma cells induced by dimethyl sulfoxide]. / Morfologicheskaia differentsirovka kletok neiroblastomy, indutsirovannaia dimetilsul'foksidom.

Morphological features of neuroblastoma cells cultured in the presence of 1.0% dimethyl sulfoxide (DMSO) were investigated. The induced differentiation was characterized by appearance of long axon-like processes (neurites), cell size enlargement and inhibition of cell growth. Quantitative criteria for cell differentiation depending on survival time in the modified medium were estimated. The increase of total length of neurites was linear with time, the rate of their extension being 20.0 +/- 3 micron/h. The area of differentiated cell soma is 6-7 times higher than that of control cells. Increase of the DMSO concentration to 2.0% did not intensify neurite growth and enlargement of cell size but suppressed mitosis. Morphological criteria of cell differentiation are compared with probable functional changes in these cells.

[Effect of changes in the pH of the medium on neuroblastoma cell differentiation in culture]. / Vliianie izmeneniia pH sredy na differentsirovku kletok neiroblastomy v kul'ture.

Morphological differentiation of neuroblastoma cells in culture was found to be depended on extracellular pH value. Increase of pH value up to 8.2 resulted in the appearance of both differentiated and dividing cell populations. The morphologically mature state of differentiated cells was observed at 7th-8th day and their population remained stable state for over 4 weeks. Increase of pH value to 8.5-8.9 caused retraction of cell processes of differentiated cells and inhibited multiplication of the dividing cells.

[Calcium currents of differentiated mouse neuroblastoma cells]. / Kal'tsievye toki differentsirovannykh kletok neiroblastomy myshi.

Differentiated neuroblastoma cells (clone N18TG2-A1) were investigated by means of intracellular dialysis technique. A slow component of the potential-dependent inward current was observed in these cells. Changes in current amplitude produced by changes in the ionic composition of intra- and extracellular media showed that this component is carried by calcium ions. The calcium inward current was observed in all investigated cells. It could be activated by membrane depolarization to the level of -70 - -65 mV and reached maximum amplitude at -30 - -40 mV. Kinetic parameters of this current were studied. A conclusion is made that calcium current in differentiated neuroblastoma cells is similar to the fast component of the calcium current in normal neurons of rat dorsal root ganglion.