Ca2+ dependence of small Ca2+-activated K+ channels in cultured N1E-115 mouse neuroblastoma cells

Single-channel properties of Ca²⁺-activated K⁺ channels have been investigated in excised membrane patches of N1E-115 mouse neuroblastoma cells under asymmetric K⁺ concentrations at 0 mV. The SK channels are blocked by 3 nM external apamin, are unaffected by 20 mM external tetraethylammonium (TEA) and have a single-channel conductance of 5.4 pS. The half-maximum open probability and opening frequency of SK channels are observed at 1 M internal Ca²⁺. Concentration/effect curves of these parameters are very steep with exponential slope factors between 7 and 13. Opentime distributions demonstrate the existence of at least two open states. The mean short open time increases with [Ca²⁺]_i, whereas the mean long open time is independent of [Ca²⁺]_i. At low [Ca²⁺]_i the short-lived open state predominates. At saturating [Ca²⁺]_i the number of longlived openings is more enhanced than the number of short-lived openings and both open states occur equally frequently. The opening frequency as well as the open times of SK channels are independent of the membrane potential in the range of –16 to +40 mV. The results indicate that activation of K⁺ current through SK channels is mainly determined by the Ca²⁺-dependent single-channel opening frequency. BK channels in N1E-115 cells are insensitive to 100 nM external apamin, are sensitive to external TEA in the millimolar range and have a single-channel conductance of 98 pS. Half-maximum open probability and opening frequency of the BK channel are observed at 7.5–21 M internal Ca²⁺. The slope factors of concentration/effect curves range between 1.7 and 2.9. As the BK channel open time is markedly enhanced at raised [Ca²⁺]_i, the Ca²⁺ dependence of the current through BK channels is determined by the single-channel opening frequency as well as the open time. SK as well as BK channels appear to be clustered and interact in a negative cooperative manner in multiple channel patches. The differences in Ca²⁺ dependence suggest that BK channels are activated by a local high [Ca²⁺]_i associated with Ca²⁺ influx, whereas SK channels may be activated by Ca²⁺ released from internal stores as well.     

Neuropeptide Y2-type receptor-mediated activation of large-conductance Ca2+-sensitive K+ channels in a human neuroblastoma cell line

We have proposed recently that a pertussistoxin-insensitive Ca²⁺ influx stimulated by Y₂-type receptor activation in CHP-234 human neuroblastoma cells underlies increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) induced by neuropeptide Y (NPY), which were strictly dependent on extracellular Ca²⁺ and independent of internal Ca²⁺ stores. We describe here the actions of NPY in these same cells, using the activity of Ca²⁺-activated K⁺ channels as an indicator of [Ca²⁺]_i. The elementary slope conductance of these channels was 110±3 pS (with an asymmetrical K⁺gradient), their activity was greatly increased by application of ionomycin, and they were reversibly blocked by 1 mM tetraethylammonium (TEA) and 100 nM charybdotoxin. Application of 100 nM NPY, in the presence but not in the absence of extracellular Ca²⁺, increased the channel open probability. ATP applied in the absence of external Ca²⁺ caused rises both in channel open probability and [Ca²⁺]_i. Inositol trisphosphate production was stimulated by ATP but not by NPY. In outside-out patches, NPY increased channel open probability, indicating that NPY-associated Ca²⁺ influx does not require all the intracellular machinery present in intact cells. Channel activation by NPY was unaffected by the replacement of guanosine 5-triphosphate (GTP) by (guanosine 5-O-(2-thiodiphosphate) (GDP[S]), a non-hydrolysable GDP analogue, in the pipette internal solution, consistent with the lack of involvement of G-proteins in the coupling of Y₂-type receptors to Ca²⁺ influx in CHP-234 cells.     

Low potency inhibition of Ca channel currents in human neuroblastoma (SH-SY5Y) cells by [Ala]NPY, an l-alanine substituted analogue of neuropeptide Y

Whole-cell Ca²⁺ channel currents were recorded in human neuroblastoma (SH-SY5Y) cells, using the perforated-patch technique with 10 mM Ba²⁺ as charge carrier. Neuropeptide Y (NPY; 10 nM to 1 μM) caused concentration-dependent inhibition of Ca²⁺ channel currents which were associated with a slowing in current activation kinetics. [Ala³¹]NPY, a residue 31 l-alanine substituted analogue of NPY, also inhibited Ca²⁺ channel currents and caused slowing of activation kinetics, but with approximately 6-fold lower potency. In the presence of 100 nM [Ala³¹]NPY (which itself had little or no effect on currents), the actions of NPY were similar in magnitude to its effects in the absence of the analogue. Our results suggest that substitution of isoleucine for alanine at residue 31 results in a NPY analogue which is a full agonist but with lower affinity for Y₂ receptors.     

Capacitative Ca2+ influx and a Ca2u+-dependent nonselective cation pathway are discriminated by genistein in mouse pancreatic acinar cells

We have investigated the effect of genistein on the hormone-stimulated Ca²⁺ influx and on a 28 pS nonselective cation channel in mouse pancreatic acinar cells using the Ca²⁺ indicator fluo-3 and the patch-clamp technique. The identity of the Ca^2u+ influx pathway has not been established in this cell type so far. Therefore we have investigated the Ca²⁺-dependent nonselective cation channel as a potential pathway for Ca²⁺ influx. Capacitative Ca²⁺ entry was induced by depletion of intracellular Ca²⁺ stores with 500 nM acetylcholine or with the Ca²⁺ ATPase inhibitor 2,5-di-tert- butylhydroquinone. In the presence of 100 M genistein, Ca²⁺ release was unimpaired, whereas Ca²⁺ influx was reversibly suppressed. Patch-clamp experiments demonstrated that genistein had no effect on Ca²⁺-activated nonselective cation channels, the activity of which was measured in excised membrane patches (inside/out) or in the whole-cell configuration. Therefore we conclude that this 28 pS nonselective cation channel does not contribute to Ca²⁺ influx into mouse exocrine pancreatic cells. With the exception of genistein and tyrphostin 25, other tyrosine kinase inhibitors such as methyl-2,5-dihydroxycinnamate, lavendustin A, herbimycin A, and tyrphostin B56 were without effect on Ca²⁺ signalling. Thus, the involvement of tyrosine phosphorylation in the activation of the Ca²⁺ entry mechanism in mouse pancreatic acinar cells is unclear.     

The pathway for refilling intracellular Ca2+ stores passes through the cytosol in human leukaemia cells

The pathway for refilling the intracellular Ca²⁺ stores of HL60 and U937 human leukaemia cells loaded with fura-2 has been investigated. On addition of external Ca²⁺ to cells with empty stores there was an increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) which preceded the refilling of the stores. The increase in [Ca²⁺]_i was faster than the refilling, by 3-to 15-fold, depending on the cell type. In measurements in single HL60 cells we found that the refilling of the stores correlated with the extent of the [Ca²⁺]_i increase on addition of external Ca²⁺. The cells showing no [Ca²⁺]_i increase were unable to refill their stores. The addition of Ni²⁺ to the extracellular medium prevented both the [Ca²⁺]_i increase and the refilling of the stores. These results indicate that the limiting step for store refilling is the entry of Ca²⁺ from the extracellular medium to the cytosol. Hence, we conclude that extracellular Ca²⁺ cannot gain access directly to the intracellular Ca²⁺ stores in these cells, but must first enter the cytosol and be taken up from there into the stores.     

Agonist-induced intracellular Ca2+ transients in HT29 cells

In the present study we have investigated the mechanism of intracellular Ca²⁺ activity ([Ca²⁺]_i) changes in HT₂₉ cells induced by adenosine triphosphate (ATP), carbachol (CCH), and neurotensin (NT). [Ca²⁺]_i was measured with the fluorescent Ca²⁺ indicator fura-2 at the single-cell level or in small cell plaques with high time resolution (1–40Hz). ATP and CCH induced not only a dose-dependent [Ca²⁺]_i peak response, but also changes of the plateau phase. The [Ca²⁺]_i plateau was inversely dependent on the ATP concentration, whereas the CCH-induced [Ca²⁺]_i plateau increased at higher CCH concentrations. NT showed (from 10^–10 to 10^–7 mol/l) in most cases only a [Ca²⁺]_i spike lasting 2–3 min. The [Ca²⁺]_i plateau induced by ATP (10^–6 mol/l) and CCH (10^–5 mol/l) was abolished by reducing the Ca²⁺ activity in the bath from 10^–3 to 10^–4 mol/l (n=7). In Ca²⁺-free bathing solution the [Ca²⁺]_i peak value for all three agonists was not altered. Using fura-2 quenching by Mn²⁺ as an indicator of Ca²⁺ influx the [Ca²⁺]_i peak was always reached before Mn²⁺ influx started. Every agonist showed this delayed stimulation of the Ca²⁺ influx with a lag time of 23±1.5 s (n=15) indicating a similar mechanism in each case. Verapamil (10^–6–10^–4 mol/l) blocked dose dependently both phases (peak and plateau) of the CCH-induced [Ca²⁺]_i increase. Short pre-incubation with verapamil augmented the effect on the [Ca²⁺]_i peak, whereas no further influence on the plateau was observed. Ni²⁺ (10^–3 mol/l) reduced the plateau value by 70%.     

Enhancement of Ca2+ channel currents in human neuroblastoma (SH-SY5Y) cells by phorbol esters with and without activation of protein kinase C

The effects of phorbol esters on Ca²⁺ channel currents in human neuroblastoma SH-SY5Y cells were studied using whole-cell patch-clamp recordings. Bath application of 12-O-tetradecanoylphorbol-13-acetate (TPA) or phorbol 12,13-dibutyrate (PDBu; 100 nM to 1 M), known activators of protein kinase C (PKC), enhanced Ca²⁺ channel currents in a voltage-dependent manner similar to that of Bay K 8644. TPA also enhanced Ca²⁺ channel currents during cell dialysis with the PKC pseudosubstrate, PKC(19–36), and in cells which had been pre-incubated with 500 nM staurosporine, and which were exposed to staurosporine during recordings. Application of 4-phorbol12, 13-didecanoate (4-PDD; 100 nM), which does not activate PKC, caused current enhancement similar to the effects of TPA. However, intracellular dialysis of TPA was without effect on Ca²⁺ channel currents. Residual Ca²⁺ channel currents recorded after exposure to 1 M -conotoxin GVIA were still enhanced by TPA, but in the presence of either Bay K 8644 (5 M) or nifedipine (5 M), TPA was without effect. When cells were pre-incubated for 10 min at 37° C with 100 nM TPA, currents subsequently recorded in its absence were enhanced as compared to untreated cells; 5 M nifedipine still inhibited currents to the same degree. This enhancement was not mimicked by 4PDD, and was inhibited by staurosporine. Our results indicate that acute applications of phorbol esters (at concentrations commonly used to activate PKC) enhance L-type Ca²⁺ channel currents in SH-SY5Y cells via a PKC-independent mechanism which appears similar to that induced by Bay K 8644. By contrast, pre-incubation with TPA enhances both L- and N-type currents via activation of PKC.     

Caffeine-induced oscillations of cytosolic Ca2+ in GH3 pituitary cells are not due to Ca2+ release from intracellular stores but to enhanced Ca2+ influx through voltage-gated Ca2+ channels

Caffeine, a well known facilitator of Ca²⁺-induced Ca²⁺ release, induced oscillations of cytosolic free Ca²⁺ ([Ca²⁺]_i) in GH₃ pituitary cells. These oscillations were dependent on the presence of extracellular Ca²⁺ and blocked by dihydropyridines, suggesting that they are due to Ca²⁺ entry through L-type Ca²⁺ channels, rather than to Ca²⁺ release from the intracellular Ca²⁺ stores. Emptying the stores by treatment with ionomycin or thapsigargin did not prevent the caffeine-induced [Ca²⁺]_i oscillations. Treatment with caffeine occluded phase 2 ([Ca²⁺]_i oscillations) of the action of thyrotropin-releasing hormone (TRH) without modifying phase 1 (Ca²⁺ release from the intracellular stores). Caffeine also inhibited the [Ca²⁺]_i increase induced by depolarization with high-K⁺ solutions (56% at 20 mM), suggesting direct inhibition of the Ca²⁺ entry through voltage-gated Ca²⁺ channels. We propose that the [Ca²⁺]_i increase induced by caffeine in GH₃ cells takes place by a mechanism similar to that of TRH, i.e. membrane depolarization that increases the firing frequency of action potentials. The increase of the electrical activity overcomes the direct inhibitory effect on voltage-gated Ca²⁺ channels with the result of increased Ca²⁺ entry and a rise in [Ca²⁺]_i. Consideration of this action cautions interpretation of previous experiments in which caffeine was assumed to increase [Ca²⁺]_i only by facilitating the release of Ca²⁺ from intracellular Ca²⁺ stores.     

Capacitative Ca2+ entry contributes to the Ca2+ influx induced by thyrotropin-releasing hormone (TRH) in GH3 pituitary cells

Treatment of GH₃ cells with either hypothalamic peptide thyrotropin-releasing hormone (TRH), the endomembrane Ca²⁺-ATPase inhibitor thapsigargin or the Ca²⁺ ionophore ionomycin mobilized, with different kinetics, essentially all of the Ca²⁺ pool from the intracellular Ca²⁺ stores. Any of the above-described treatments induced a sustained increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), which was dependent on extracellular Ca²⁺ and was prevented by Ni²⁺ but not by dihydropyridines (DHPs), suggesting that it was due to capacitative Ca²⁺ entry via activation of a plasma membrane pathway which opened upon the emptying of the intracellular Ca²⁺ stores. The increase of the plasma membrane permeability to Ca²⁺ correlated negatively with the filling degree of the intracellular Ca²⁺ stores and was reversed by refilling of the stores. The mechanism of capacitative Ca²⁺ entry into GH₃ cells differed from similar mechanisms described in several types of blood cells in that the pathway was poorly permeable to Mn²⁺ and not sensitive to cytochrome P₄₅₀ inhibitors. In GH₃ cells, TRH induced a transient [Ca²⁺]_i increase due to Ca²⁺ release from the stores (phase 1) followed by a sustained [Ca²⁺]_i increase due to Ca²⁺ entry (phase 2). At the single-cell level, phase 2 was composed of a DHP-insensitive sustained [Ca²⁺]_i increase, due to activation of capacitative Ca²⁺ entry, superimposed upon which DHP-sensitive [Ca²⁺]_i oscillations took place. The two components of the TRH-induced Ca²⁺ entry differed also in that [Ca²⁺]_i oscillations remained for several minutes after TRH removal, whereas the sustained [Ca²⁺]_i increase dropped quickly to prestimulatory levels, following the same time course as the refilling of the stores. The drop was prevented when the refilling was inhibited by thapsigargin. It is concluded that, even though the mechanisms of capacitative Ca²⁺ entry may show differences from cell to cell, it is also present and may contribute to the regulation of physiological functions in excitable cells such as GH₃. There, capacitative Ca²⁺ entry cooperates with voltage-gated Ca²⁺ channels to generate the [Ca²⁺]_i increase seen during phase 2 of TRH action. This contribution of capacitative Ca²⁺ entry may be relevant to the enhancement of prolactin secretion induced by TRH.     

Ca2+ channel Ca2+-dependent inactivation in a mammalian central neuron involves the cytoskeleton

Ca²⁺ channel inactivation was investigated in acutely isolated hippocampal pyramidal neurons from adult rats and found to have a component dependent on intracellular Ca²⁺. Ca²⁺-dependent inactivation was identified as the additional inactivation of channel current observed when Ca²⁺ replaced Ba²⁺ as the current carrying ion, and was found to be an independent process from that of Ba²⁺ current inactivation based on three lines of evidence: (1) no correlation between Ca²⁺-dependent inactivation and Ba²⁺ current inactivation was found, (2) only Ca²⁺-dependent inactivation was reduced by intracellular application of Ca²⁺ chelators, and (3) only Ca²⁺-dependent inactivation was sensitive to compounds which alter the cytoskeleton. Drugs which stabilize (taxol and phalloidin) and destabilize (colchicine and cytochalasin B) the cytoskeleton altered the development and recovery from Ca²⁺-dependent inactivation, indicating that the neuronal cytoskeleton may mediate Ca²⁺ channel sensitivity to intracellular Ca²⁺. Ca²⁺-dependent inactivation was not associated with a particular subset of Ca²⁺ channels, suggesting that all Ca²⁺ channels in these neurons are inactivated by intracellular Ca²⁺.     

Blocking actions of Ca2+ antagonists on the Ca2+ channels in the smooth muscle cell membrane of rabbit small intestine

Actions of Ca²⁺ antagonists, verapamil, nicardipine and diltiazem, were investigated on the Ca²⁺ inward current in the fragmented smooth muscle cell membrane (smooth muscle ball; SMB) obtained from the longitudinal muscle layer of the rabbit ileum, by enzymatic dispersion. All Ca²⁺ antagonists inhibited the inward current, in a dose-dependent manner. The ID₅₀ value on the maximum amplitude of the inward current of nicardipine was 24 nM, and this value was roughly 50 times lower than values obtained with verapamil and diltiazem, when the inward current was provoked by 0 mV command pulse from the holding potential of –60 mV. Lowering the holding potential to –80 mV shifted the dose-response curve to the right. When depolarizing pulses (100 ms, stepped up to 0 mV from –60 mV or –80 mV) were applied every 20 s, the peak amplitude of the inward current remained unchanged, but nicardipine immediately, and diltiazem and verapamil slowly reduced the peak amplitude. These slow inhibitions by the latter two drugs depended on the frequency or number of stimulations. Nicardipine but not diltiazem and verapamil shifted the voltage-dependent inactivation curve to the left (3 s duration of the conditioning pulse). However, with a longer conditioning pulse (10 s) verapamil and diltiazem shifted the voltage-dependent inactivation curves to the left. Therefore, the inhibitory actions of these Ca²⁺ antagonists differ. Namely, diltiazem and verapamil inhibit the Ca²⁺ channels, mainly in a frequency-or use-dependent manner while nicardipine does so in a voltage-dependent manner.     

Mechanism of the ATP-induced rise in cytosolic Ca2+ in freshly isolated smooth muscle cells from human saphenous vein

Effects of exogenous adenosine 5-triphosphate (ATP) were studied by measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and membrane currents in myocytes freshly isolated from the human saphenous vein. At a holding potential of –60 mV, ATP (10 M) elicited a transient inward current and increased [Ca²⁺]_i. These effects of ATP were inhibited by ,-methylene adenosine 5-triphosphate (AMPCPP, 10 M). The ATP-gated current corresponded to a non-selective cation conductance allowing Ca²⁺ entry. The ATP-induced [Ca²⁺]_i rise was abolished in Ca²⁺-free solution and was reduced to 30.1±5.5% (n=14) of the control response when ATP was applied immediately after caffeine, and to 23.7±3.8% (n=11) in the presence of thapsigargin. The Ca²⁺-induced Ca²⁺ release blocker tetracaine inhibited the rise in [Ca²⁺]_i induced by both caffeine and ATP, with apparent inhibitory constants of 70 M and 100 M, respectively. Of the ATP-induced increase in [Ca²⁺]_i 29.3±3.9% (n=8) was tetracaine resistant. It is concluded that the effects of ATP in human saphenous vein myocytes are only mediated by activation of P_2x receptor channels. The ATP-induced [Ca²⁺]_i rise is due to both Ca²⁺ entry and Ca²⁺ release activated by Ca²⁺ ions that enter the cell through P_2x receptor channels.     

Initiation sites for Ca2+ signals in endothelial cells

Intracellular Ca²⁺ signals in response to inositol 1,4,5-trisphosphate-producing agents often present themselves as Ca²⁺ oscillations and propagating Ca²⁺ waves originating at discrete initiation sites. We studied the spatial organization of the Ca²⁺ signal in single CPAE endothelial cells stimulated with adenosine triphosphate. The long, thin processes presented a higher agonist sensitivity and, for the same agonist concentration, a faster rise in cytoplasmic Ca²⁺ concentration and rate of wave propagation than the cell body. Ca²⁺ waves originated preferentially in one of these processes and then invaded the cell body. Removal of external Ca²⁺ induced a progressive inhibition up to blockade of the response in the process but not in the cell body. These findings suggest that CPAE cells contain many individual store units, each of which has the inherent ability to set the stage for Ca²⁺ release. A diffusing messenger originating from the initiation zone then coordinates the events leading to Ca²⁺ release in the individual store units to produce a Ca²⁺ wave.     

The leading role of membrane Ca(2+)-ATPase in recovery of Ca(2+) homeostasis after glutamate shock

Combined blockade of Na⁺/Ca²⁺ exchange, Ca²⁺ uptake by mitochondria and endoplasmic reticulum usually does not prevent recovery of the basal level of intracellular Ca²⁺ after 1-min action of glutamate (100 M) or K⁺ (50 mM). However, replacement of Ca²⁺ with Ba²⁺, which cannot be transported by Ca²⁺-ATPase, considerably delayed the decrease in intracellular Ba²⁺ after its rise caused by glutamate or potassium application in all examined cells, which attest to an important role of Ca²⁺-ATPase in Ca²⁺ extrusion after the action of glutamate or K⁺.     

Effects of 2,5-di(tert-butyl)-1,4-hydroquinone on intracellular free Ca2+ levels and histamine secretion in RBL-2H3 cells

The effects of 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ) on the intracellular free Ca²⁺ level ([Ca²⁺]_i) and histamine secretion of rat basophilic leukemia (RBL-2H3) cells were examined. DTBHQ (0.1–10 µmol/l) alone induced rapid and sustained increases in [Ca²⁺]_i in a concentration-dependent manner. In cells sensitized with anti-dinitrophenyl IgE, DTBHQ (10 µmol/1) further increased the antigen (dinitrophenylated BSA)-induced Ca²⁺ response. In the absence of external Ca²⁺ with addition of 1 mmol/1 EGTA, both DTBHQ (10 µmol/l) and the antigen (10 µg/ml) induced transient increase in [Ca²⁺]_i. In sensitized cells, both DTBHQ (10 µmol/1) and antigen (10 µg/ml) elicited histamine secretion, although the response was far stronger in the latter case. The DTBHQ-induced histamine secretion was markedly enhanced by addition of the protein kinase C activator, phorbol 12-myristate 13-acetate (TPA) (10 ng/ml) whereas TPA alone did not cause any increase. Moreover, DTBHQ enhanced the antigen-induced histamine secretion. The results suggest that DTBHQ increases [Ca²⁺]_i and enhances antigeninduced histamine secretion while DTBHQ alone does not cause as much histamine secretion as antigen, which support the idea that calcium signals are necessary but are not sufficient for maximum histamine secretion in RBL-2H3 cells.     

Intracellular Ca2+ signals in human-derived pancreatic somatostatin-secreting cells (QGP-1N)

Single-cell microfluorimetry techniques have been used to examine the effects of acetylcholine (0.1–100 M) on the intracellular free calcium ion concentration ([Ca²⁺]_i) in a human-derived pancreatic somatostatin-secreting cell line, QGP-1N. When applied to the bath solution, acetylcholine was found to evoke a marked and rapid increase in [Ca²⁺]_i at all concentrations tested. These responses were either sustained, or associated with the generation of complex patterns of [Ca²⁺]_i transients. Overall, the pattern of response was concentration related. In general, 0.1–10 M acetylcholine initiated a series of repetitive oscillations in cytoplasmic Ca²⁺, whilst at higher concentrations the responses consisted of a rapid rise in [Ca²⁺]_i followed by a smaller more sustained increase. Without external Ca²⁺, 100 M acetylcholine caused only a transient rise in [Ca²⁺]_i, whereas lower concentrations of the agonist were able to initiate, but not maintain, [Ca²⁺]_i oscillations. Acetylcholine-evoked Ca²⁺ signals were abolished by atropine (1–10 M), verapamil (100 M) and caffeine (20 mM). Nifedipine failed to have any significant effect upon agonist-evoked increases in [Ca²⁺]_i, whilst 50 mM KCl, used to depolarise the cell membrane, only elicited a transient increase in [Ca²⁺]_i. Ryanodine (50–500 nM) and caffeine (1–20 mM) did not increase basal Ca²⁺ levels, but the Ca²⁺-ATPase inhibitors 2,5-di(tert-butyl)-hydroquinone (TBQ) and thapsigargin both elevated [Ca²⁺]_i levels. These data demonstrate for the first time cytosolic Ca²⁺ signals in single isolated somatostatin-secreting cells of the pancreas. We have demonstrated that acetylcholine will evoke both Ca²⁺ influx and Ca²⁺ mobilisation, and we have partially addressed the subcellular mechanism responsible for these events.     

The effect of L-type Ca2+ channel blockers on anoxia-induced increases in intracellular Ca2+ concentration in rabbit proximal tubule cells in primary culture

Ca²⁺ channel blockers (CCB) have been shown to be protective against ischaemic damage of the kidney, suggesting an important role for intracellular Ca²⁺ ([Ca²⁺]_i) in generating cell damage. To delineate the mechanism behind this protective effect, we studied [Ca²⁺]_i in cultured proximal tubule (PT) cells during anoxia in the absence of glycolysis and the effect of methoxyverapamil (D600) and felodipine on [Ca²⁺]_i during anoxia. A method was developed whereby [Ca²⁺]_i in cultured PT cells could be measured continuously with a fura-2 imaging technique during anoxic periods up to 60 min. Complete absence of O₂ was realised by inclusion of a mixture of oxygenases in an anoxic chamber. [Ca²⁺]_i in PT cells started to rise after 10 min of anoxia and reached maximal levels at 30 min, which remained stable up to 60 min. The onset of this increase and the maximal levels reached varied markedly among individual cells. The mean values for normoxic and anoxic [Ca²⁺]_i were 118±2 (n=98) and 662±22 (n=160) nM, respectively. D600 (1 M), but not felodipine (10 M), significantly reduced basal [Ca²⁺]_i in normoxic incubations. During anoxia 1 M and 100 M D 600 significantly decreased anoxic [Ca²⁺]_i levels by 22 and 63% respectively. Felodipine at 10 M was as effective as 1 M D600. Removal of extracellular Ca²⁺ and addition of 0.1 mM La³⁺ completely abolished anoxia-induced increases in [Ca²⁺]_i. We conclude that anoxia induces increases in [Ca²⁺]_i in rabbit PT cells in primary culture, which results from Ca²⁺ influx. Since this Ca²⁺ influx is partially inhibited by low doses of CCBs, Ltype Ca²⁺ channels may be involved.     

Glutamate receptor activation in cultured cerebellar granule cells increases cytosolic free Ca2+ by mobilization of cellular Ca2+ and activation of Ca2+ influx

Summary The Ca²⁺ sensitive fluorescent probe, fura-2 has been used to monitor cytosolic free calcium levels in mature primary cultures of cerebellar granule cells during exposure to L-glutamate and other excitatory amino acids: quisqualate (QA), kainate (KA) and N-methyl-D-aspartate (NMDA). Glutamate at micromolar concentrations produced a prompt and dose-related increase in the intracellular concentration of free Ca²⁺, ([Ca²⁺]_i), whereas QA, KA and NMDA had no effect. This increase was also seen in the absence of extracellular Ca²⁺, suggesting that L-glutamate promotes mobilization of Ca²⁺ from intracellular stores. In the presence of extracellular calcium, the elevation of [Ca²⁺]_i was, in part, mediated by an increase in the plasma membrane permeability to Ca²⁺. This Ca²⁺ influx was not affected by the Ca²⁺-channel antagonist 1-Verapamil. However, L-Verapamil did block the increase in [Ca²⁺]_i seen after depolarization of the cells with potassium. The Ca²⁺ response elicited by glutamate was partially blocked by the excitatory amino acid antagonist glutamate diethyl ester (GDEE). Furthermore, glutamate stimulated the formation of inositol mono-, bis-, tris and tetrakisphosphates (IP1, IP2, IP3, and IP4) suggesting a role for these compounds for the increase in [Ca²⁺]_i.     

Cut-off phenomenon in the protective effect of alcohols against lysophosphatidylcholine-induced calcium overload

We studied the effect of chain length on the protective effect of alcohols against lysophosphatidylcholine (LPC)-induced Ca²⁺ overload in neonatal rat cardiomyocytes. We previously found that ethanol retards Ca²⁺ elevation. Cells were loaded with the Ca²⁺-sensitive fluorophore fura-2, and changes in fluorescence were followed. The addition of 10 M LPC increased Ca²⁺, which reached a plateau after an 8–10 min delay. The presence of 88 mM n-propanol, n-butanol, tert-butanol, or 2,2-dimethylpropanol significantly increased the delay by 94–213%. However, n-pentanol at 2 mM or 88 mM had no protective effect. Among n-alcohols, the increase in lag time was inversely proportional to the length of the carbon chain. Chain length, rather than molecular weight determines the effect, because 2,2-dimethylpropanol had a protective effect. The influence of alcohols on LPC micelle formation was estimated from the increase in octadecyl rhodamine B fluorescence; the increase by n-alcohols was directly proportional to chain length, indicating that micelle formation was not involved in the extension of lag time. The absence of the protective effect when the alcohol aliphatic chain exceeds four carbons suggests that the effect of ethanol may be mediated via a small lipophilic pocket on a protein, or to lateral pressure perturbation in the membrane.     

GABA(B) receptor-mediated ERK1/2 phosphorylation via a direct interaction with Ca(V)1.3 channels

Neuronal L-type Ca(2+) channels play pivotal roles in regulating gene expression, cell survival, and synaptic plasticity. The Ca(V)1.2 and Ca(V)1.3 channels are 2 main subtypes of neuronal L-type Ca(2+) channels. However, the specific roles of Ca(V)1.2 and Ca(V)1.3 in L-type Ca(2+) channel-mediated neuronal responses and their cellular mechanisms are poorly elucidated. On the basis of our previous study demonstrating a physical interaction between the Ca(V)1.3 channel and GABA(B) receptor (GABA(B)R), we further examined the involvement of Ca(V)1.2 and Ca(V)1.3 in the GABA(B)R-mediated activation of ERK(1/2), a kinase involved in both CREB activation and synaptic plasticity. After confirming the involvement of L-type Ca(2+) channels in baclofen-induced ERK(1/2) phosphorylation, we examined a specific role of Ca(V)1.2 and Ca(V)1.3 channels in the baclofen effect. Using siRNA-mediated silencing of Ca(V)1.2 or Ca(V)1.3 messenger, we determined the relevance of each channel subtype to baclofen-induced ERK(1/2) phosphorylation in a mouse hippocampal cell line (HT-22) and primary cultured rat neurons. In the detailed characterization of each subtype using HEK293 cells transfected with Ca(V)1.2 or Ca(V)1.3, we found that GABA(B)R can increase ERK(1/2) phosphorylation and Ca(V)1.3 channel activity through direct interaction with Ca(V)1.3 channels. These results suggest a functional interaction between Ca(V)1.3 and GABA(B)R and important implications of Ca(V)1.3/GABA(B)R clusters for translating synaptic activity into gene expression alterations.