Large-conductance calcium-activated potassium channels in neonatal rat intracardiac ganglion neurons

The properties of single Ca2+-activated K+ (BK) channels in neonatal rat intracardiac neurons were investigated using the patch-clamp recording technique. In symmetrical 140 mM K+, the single-channel slope conductance was linear in the voltage range -60/+60 mV, and was 207+/-19 pS. Na+ ions were not measurably permeant through the open channel. Channel activity increased with the cytoplasmic free Ca2+ concentration ([Ca2+]i) with a Hill plot giving a half-saturating [Ca2+] (K0.5) of 1.35 microM and slope of approximately equals 3. The BK channel was inhibited reversibly by external tetraethylammonium (TEA) ions, charybdotoxin, and quinine and was resistant to block by 4-aminopyridine and apamin. Ionomycin (1-10 microM) increased BK channel activity in the cell-attached recording configuration. The resting activity was consistent with a [Ca2+]i <100 nM and the increased channel activity evoked by ionomycin was consistent with a rise in [Ca2+]i to > or =0.3 microM. TEA (0.2-1 mM) increased the action potential duration approximately equals 1.5-fold and reduced the amplitude and duration of the afterhyperpolarization (AHP) by 26%. Charybdotoxin (100 nM) did not significantly alter the action potential duration or AHP amplitude but reduced the AHP duration by approximately equals 40%. Taken together, these data indicate that BK channel activation contributes to the action potential and AHP duration in rat intracardiac neurons.     

Two pathways for the activation of small-conductance potassium channels in neurons of substantia nigra pars reticulata

Neurons in substantia nigra pars reticulata express the messenger RNA for SK2 but not for SK3 subunits that form small-conductance, Ca2+-dependent K+ channels in dopamine neurons. To determine pathways for the activation of small-conductance, Ca2+-dependent K+ channels in substantia nigra pars reticulata neurons of rats and mice, we studied effects of the selective blocker of small-conductance, Ca2+-dependent K+ channels, apamin (0.01 or 0.3 microM). Apamin diminished the afterhyperpolarization following each action potential and induced burst discharges in substantia nigra pars reticulata neurons. Apamin had a robust effect already at a low (10 nM) concentration consistent with the expression of the SK2 subunit. Afterhyperpolarizations were also reduced by the Ca2+ channel blockers Ni2+ (100 microM) and omega-conotoxin GVIA (1 microM). Depletion of intracellular Ca2+ stores did not change the afterhyperpolarization. However, we observed outward current pulses that occurred independently from action potentials and were abrogated by apamin. Apart from a faster time course, they shared all properties with spontaneous hyperpolarizations or outward currents that ryanodine receptor-mediated Ca2+ release from intracellular stores induces in juvenile dopamine neurons. Sensitization of ryanodine receptors by caffeine silenced substantia nigra pars reticulata neurons. This effect was abolished by the depletion of intracellular Ca2+ stores. We conclude that SK2 channels in substantia nigra pars reticulata neurons are activated by Ca2+ influx through at least two types of Ca2+ channels in the membrane and by ryanodine receptor-mediated Ca2+ release from intracellular stores. Ryanodine receptors do not amplify small-conductance, Ca2+-dependent K+ channel activation by the Ca2+ influx during a single spike. Yet, ryanodine receptor-mediated Ca2+ release and, thereby, an activation of small-conductance, Ca2+-dependent K+ channels by intracellular Ca2+ are available for excitability modulation in these output neurons of the basal ganglia system.     

Muscarinic and nicotinic ACh receptor activation differentially mobilize Ca2+ in rat intracardiac ganglion neurons

The origin of intracellular Ca2+ concentration ([Ca2+]i) transients stimulated by nicotinic (nAChR) and muscarinic (mAChR) receptor activation was investigated in fura-2-loaded neonatal rat intracardiac neurons. ACh evoked [Ca2+]i increases that were reduced to approximately 60% of control in the presence of either atropine (1 microM) or mecamylamine (3 microM) and to <20% in the presence of both antagonists. Removal of external Ca2+ reduced ACh-induced responses to 58% of control, which was unchanged in the presence of mecamylamine but reduced to 5% of control by atropine. The nAChR-induced [Ca2+]i response was reduced to 50% by 10 microM ryanodine, whereas the mAChR-induced response was unaffected by ryanodine, suggesting that Ca2+ release from ryanodine-sensitive Ca2+ stores may only contribute to the nAChR-induced [Ca2+]i responses. Perforated-patch whole cell recording at -60 mV shows that the rise in [Ca2+]i is concomitant with slow outward currents on mAChR activation and with rapid inward currents after nAChR activation. In conclusion, different signaling pathways mediate the rise in [Ca2+]i and membrane currents evoked by ACh binding to nicotinic and muscarinic receptors in rat intracardiac neurons.     

BK channels in human glioma cells

Ion channels in inexcitable cells are involved in proliferation and volume regulation. Glioma cells robustly proliferate and undergo shape and volume changes during invasive migration. We investigated ion channel expression in two human glioma cell lines (D54MG and STTG-1). With low [Ca2+]i, both cell types displayed voltage-dependent currents that activated at positive voltages (more than +50 mV). Current density was sensitive to intracellular cation replacement with the following rank order; K+ > Cs+ approximately = Li+ > Na+. Currents were >80% inhibited by iberiotoxin (33 nM), charybdotoxin (50 nM), quinine (1 mM), tetrandrine (30 microM), and tetraethylammonium ion (TEA; 1 mM). Extracellular phloretin (100 microM), an activator of BK(Ca2+) channels, and elevated intracellular Ca2+ negatively shifted the I-V curve of whole cell currents. With 0, 0.1, and 1 microM [Ca2+]i, the half-maximal voltages, V(0.5), for whole cell current activation were +150, +65, and +12 mV, respectively. Elevating [K+]o potentiated whole cell currents in a fashion proportional to the square-root of [K+]o. Recording from cell-attached patches revealed large conductance channels (150-200 pS) with similar voltage dependence and activation kinetics as whole cell currents. These data indicate that human glioma cells express large-conductance, Ca2+ activated K+ (BK) channels. In amphotericin-perforated patches bradykinin (1 microM) activated TEA-sensitive currents that were abolished by preincubation with bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM). The BK channels described here may influence the responses of glioma cells to stimuli that increase [Ca2+]i.     

Ca(2+)-induced Ca(2+) release activates spontaneous miniature outward currents (SMOCs) in parasympathetic cardiac neurons.

Mudpuppy parasympathetic cardiac neurons exhibit spontaneous miniature outward currents (SMOCs) that are thought to be due to the activation of clusters of large conductance Ca(2+)-activated K(+) channels (BK channels) by localized release of Ca(2+) from internal stores close to the plasma membrane. Perforated-patch whole cell recordings were used to determine whether Ca(2+)-induced Ca(2+) release (CICR) is involved in SMOC generation. We confirmed that BK channels are involved by showing that SMOCs are inhibited by 100 nM iberiotoxin or 500 microM tetraethylammonium (TEA), but not by 100 nM apamin. SMOC frequency is decreased in solutions that contain 0 Ca(2+)/3.6 mM Mg(2+), and also in the presence of 1 microM nifedipine and 3 microM omega-conotoxin GVIA, suggesting that SMOC activation is dependent on calcium influx. However, Ca(2+) influx alone is not sufficient; SMOC activation is also dependent on Ca(2+) release from the caffeine- and ryanodine-sensitive Ca(2+) store, because exposure to 2 mM caffeine consistently caused an increase in SMOC frequency, and 10-100 microM ryanodine altered the configuration of SMOCs and eventually inhibited SMOC activity. Depletion of intracellular Ca(2+) stores by the Ca-ATPase inhibitor cyclopiazonic acid (10 microM) inhibited SMOC activity, even when Ca(2+) influx was not compromised. We also tested the effects of the membrane-permeable Ca(2+) chelators, bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid-AM (BAPTA-AM) and EGTA-AM. EGTA-AM (10 microM) caused no inhibition of SMOC activation, whereas 10 microM BAPTA-AM consistently inhibited SMOCs. After SMOCs were completely inhibited by BAPTA, 3 mM caffeine caused SMOC activity to resume. This effect was reversible on removal of caffeine and suggests that the source of Ca(2+) that triggers the internal Ca(2+) release channel is different from the source of Ca(2+) that activates clusters of BK channels. We propose that influx of Ca(2+) through voltage-dependent Ca(2+) channels is required for SMOC generation, but that the influx of Ca(2+) triggers CICR from intracellular stores, which then activates the BK channels responsible for SMOC generation.     

Number of K(Ca) channels underlying spontaneous miniature outward currents (SMOCs) in mudpuppy cardiac neurons

Spontaneous miniature outward currents (SMOCs) in parasympathetic neurons from mudpuppy cardiac ganglia are caused by activation of TEA- and iberiotoxin-sensitive, Ca(2+)-dependent K(+) (BK) channels. Previously we reported that SMOCs are activated by Ca(2+)-induced Ca(2+) release (CICR) from caffeine- and ryanodine-sensitive intracellular Ca(2+) stores. In the present study, we analyzed the single channel currents that contribute to SMOC generation in mudpuppy cardiac neurons. The slope conductance of BK channels, determined from the I-V relationship of single-channel currents recorded with cell-attached patches in physiological K(+) concentrations, was 84 pS. The evidence supporting the identity of this channel as the channel involved in SMOC generation was its sensitivity to internal Ca(2+), external TEA, and caffeine. In cell-attached patch recordings, 166 microM TEA applied in the pipette reduced single-channel current amplitude by 32%, and bath-applied caffeine increased BK channel activity. The ratio between the averaged SMOC amplitude and the single-channel current amplitude was used to estimate the average number of channels involved in SMOC generation. The estimated number of channels involved in generation of an averaged SMOC ranged from 18 to 23 channels. We also determined that the Po of the BK channels at the peak of a SMOC remains constant at voltages more positive than -20 mV, suggesting that the transient rise in intracellular Ca(2+) from ryanodine-sensitive intracellular stores in the vicinity of the BK channel reached concentrations most likely exceeding 40 microM.     

A comparison between the distinct inward currents activated in rat cultured dorsal root ganglion neurones by intracellular flash photolysis of two forms of caged cyclic guanosine monophosphate

Whole cell inward currents activated by intracellular photorelease of cyclic guanosine monophosphate (cGMP) were investigated in cultured dorsal root ganglion (DRG) neurones. The actions of two distinct types of caged cGMP (NPE-caged cGMP and a highly water-soluble caged cGMP) were compared. Rapidly activating inward currents were evoked by cGMP in a subpopulation (12.5%) of neurones and these currents may be due to activity of cyclic nucleotide-gated channels. In contrast in 52% of DRG neurones intracellular photorelease of cGMP activated a delayed Ca(2+)-dependent inward current through the generation of cyclic ADPribose and mobilisation of Ca(2+) from ryanodine sensitive intracellular stores. Similar delayed inward currents were activated by both caged compounds but only NPE-caged cGMP evoked rapidly activating currents. Cyclic GMP appears to increase excitability in some DRG neurones by diverse mechanisms.     

Purinergic activation of BK channels in clonal kidney cells (Vero cells)

We have studied the activation of a high-conductance channel in clonal kidney cells from African green monkey (Vero cells) using patch-clamp recordings and microfluorometric (fura-2) measurements of cytosolic Ca2+. The single-channel conductance in excised patches is 170 pS in symmetrical 140 mM KCl. The channel is highly selective for K+ and activated by membrane depolarization and application of Ca2+ to the cytoplasmatic side of the patch. The channel is, thus, a large-conductance Ca2+-activated K+ channel (BK channel). Cell-attached recordings revealed that the channel is inactive in unstimulated cells. Extracellular application of less than 0.1 microM ATP transiently increased the cytosolic Ca2+ concentration ([Ca2+]i) to about 550 nM, and induced membrane hyperpolarization caused by Ca2+-activated K+ currents. ATP stimulation also activated BK channels in cell-attached patches at both the normal-resting potential and during membrane hyperpolarization. The increase in [Ca2+]i was owing to Ca2+ release from internal stores, suggesting that Vero cells express G-protein-coupled purinergic receptors (P2Y) mediating IP3-induced release of Ca2+. The P2Y receptors were sensitive to both uracil triphosphate (UTP) and adenosine diphosphate (ADP), and the rank of agonist potency was ATP > UTP >/= ADP. This result indicates the presence of both P2Y1 and P2Y2 receptors or a receptor subtype with untypical agonist sensitivity. It has previously been shown that hypotonic challenge activates BK channels in both normal and clonal kidney cells. The subsequent loss of KCl may be an important factor in cellular volume regulation. Our results support the idea of an autocrine role of ATP in this process. A minute release of ATP induced by hypotonically evoked membrane stretch may activate the P2Y receptors, subsequently increasing [Ca2+]i and thus causing K+ efflux through BK channels.     

Ca2+ influx, but not Ca2+ release from internal stores, is required for the PACAP-induced increase in excitability in guinea pig intracardiac neurons

Mechanisms modulating the pituitary adenylate cyclase activating polypeptide (PACAP)-induced increase in excitability have been studied using dissociated guinea pig intrinsic cardiac neurons and intact ganglion preparations. Measurements of intracellular calcium (Ca2+) with the fluorescent Ca2+ indicator dye fluo-3 indicated that neither PACAP nor vasoactive intestinal polypeptide (VIP) at either 100 nM or 1 microM produced a discernible elevation of intracellular Ca2+ in dissociated intracardiac neurons. For neurons in ganglion whole mount preparations kept in control bath solution, local application of PACAP significantly increased excitability, as indicated by the number of action potentials generated by long depolarizing current pulses. However, in a Ca2+ -deficient solution in which external Ca2+ was replaced by Mg2+ or when cells were bathed in control solution containing 200 microM Cd2+, PACAP did not enhance action potential firing. In contrast, in a Ca2+ -deficient solution with Ca2+ replaced by strontium (Sr2+), PACAP increased excitability. PACAP increased excitability in cells treated with a combination of 20 microM ryanodine and 10 mM caffeine to interrupt release of Ca2+ from internal stores. Experiments using fluo-3 showed that ryanodine/caffeine pretreatment eliminated subsequent caffeine-induced Ca2+ release from intracellular stores, whereas exposure to the Ca2+ -deficient solution did not. In dissociated intracardiac neurons voltage clamped with the perforated patch recording technique, 100 nM PACAP decreased the voltage-dependent barium current (IBa). These results show that, in the guinea pig intracardiac neurons, the PACAP-induced increase in excitability apparently requires Ca2+ influx through Cd2+ -sensitive calcium permeable channels other than voltage-dependent Ca2+ channels, but not Ca2+ release from internal stores.     

K(+)-evoked dopamine release depends on a cytosolic Ca2+ pool regulated by N-type Ca2+ channels.

Membrane depolarization evoked by 25-40 mM K+ elicited an immediate increase of somatic and neuritic [Ca2+]i in cultured dopaminergic neurons as measured by digital fluorescence microscope imaging. The rise of neuritic [Ca2+]i was inhibited by N-type but not L-type Ca2+ channel blockers, while the rise of somatic [Ca2+]i was prevented by both L- and N-type Ca2+ channel blockers. Similarly, depolarization-induced [3H]dopamine release was selectively attenuated by N-type Ca2+ channel blockers. The present results suggest that [3H]dopamine release from mesencephalic neuronal cell cultures relates to a Ca(2+)-dependent mechanism regulated by N-type channels located in the vicinity of the exocytotic sites within neuritic processes.     

Modulation of action potential firing by iberiotoxin and NS1619 in rat dorsal root ganglion neurons

The present study investigated the effects of iberiotoxin (IbTx), a peptide toxin blocker of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels and NS1619, a BK(Ca) channel opener, on action potential firing of small and medium size afferent neurons from L6 and S1 dorsal root ganglia of adult rats. Application of IbTx (100 nM) reduced whole-cell outward currents in 67% of small and medium size neurons. Analysis of action potential profile revealed that IbTx significantly prolonged the duration of action potential and increased firing frequency of afferent neurons. IbTx did not significantly alter the resting membrane potential, threshold for action potential activation and action potential amplitude. The benzimidazolone NS1619 (10 microM) increased opening activity of a Ca(2+)-dependent channel as assessed by single channel measurements. In contrast to IbTx, NS1619 reversibly suppressed action potential firing, attributable to increases in threshold for evoking action potential, reduction in action potential amplitude and increases in amplitude of afterhyperpolarization. The effect of NS1619 on neuronal firing was sensitive to IbTx, indicating the attenuation of neuronal firing by NS1619 was mediated by opening BK(Ca) channels. NS1619 also reduced neuronal hyperexcitability evoked by 4-aminopyridine (4-AP), a transient-inactivated K(+) channel (A-current) blocker, in an IbTx-sensitive manner.These results indicate that IbTx-sensitive BK(Ca) channels exist in both small and medium diameter dorsal root ganglion (DRG) neurons and play important roles in the repolarization of action potential and firing frequency. NS1619 modulates action potential firing and suppresses 4-AP-evoked hyperexcitability in DRG neurons, in part, by opening BK(Ca) channels. These results suggest that opening BK(Ca) channels might be sufficient to suppress hyperexcitability of afferent neurons as those evoked by stimulants or by disease states.     

Large-conductance Ca2+-activated potassium channels in secretory neurons.

Large-conductance Ca2+-activated K+ channels (BK) are believed to underlie interburst intervals and contribute to the control of hormone release in several secretory cells. In crustacean neurosecretory cells, Ca2+ entry associated with electrical activity could act as a modulator of membrane K+ conductance. Therefore we studied the contribution of BK channels to the macroscopic outward current in the X-organ of crayfish, and their participation in electrophysiological activity, as well as their sensitivity toward intracellular Ca2+, ATP, and voltage, by using the patch-clamp technique. The BK channels had a conductance of 223 pS and rectified inwardly in symmetrical K+. These channels were highly selective to K+ ions; potassium permeability (PK) value was 2.3 x 10(-13) cm(3) s(-1). The BK channels were sensitive to internal Ca2+ concentration, voltage dependent, and activated by intracellular MgATP. Voltage sensitivity (k) was approximately 13 mV, and the half-activation membrane potentials depended on the internal Ca2+ concentration. Calcium ions (0.3-3 microM) applied to the internal membrane surface caused an enhancement of the channel activity. This activation of BK channels by internal calcium had a KD(0) of 0.22 microM and was probably due to the binding of only one or two Ca2+ ions to the channel. Addition of MgATP (0.01-3 mM) to the internal solution increased steady state-open probability. The dissociation constant for MgATP (KD) was 119 microM, and the Hill coefficient (h) was 0.6, according to the Hill analysis. Ca2+-activated K+ currents recorded from whole cells were suppressed by either adding Cd2+ (0.4 mM) or removing Ca2+ ions from the external solution. TEA (1 mM) or charybdotoxin (100 nM) blocked these currents. Our results showed that both BK and K(ATP) channels are present in the same cell. Even when BK and K(ATP) channels were voltage dependent and modulated by internal Ca2+ and ATP, the profile of sensitivity was quite different for each kind of channel. It is tempting to suggest that BK and KATP channels contribute independently to the regulation of spontaneous discharge patterns in crayfish neurosecretory cells.     

Ca2+ uptake by the sarcoplasmic reticulum decreases the amplitude of depolarization-dependent [Ca2+]i transients in rat gastric myocytes

Gastric myocytes loaded with fura-2 were voltage-clamped at -60 mV. Depolarizations to 0 mV evoked nifedipine-sensitive (5 microM) inward currents and Ca2+ transients. Cyclopiazonic acid (5 microM) elevated steady-state [Ca2+]i and reduced Ca current (ICa), but when divalent cations were omitted from the extracellular solution, cyclopiazonic acid had no effect on either the amplitude or the current-voltage relationship of the nifedipine-sensitive current. This suggests that the reduction in ICa was caused by the rise in steady-state [Ca2+]i. The relationship between the total Ca2+ influx carried by the Ca2+ current (sigmaI(Ca).dt) and the amplitude of the Ca2+ transient (delta[Ca2+]i) was analysed for experiments using physiological Ca2+ solutions by calculating the ratio delta[Ca2+]i/sigmaI(Ca).dt. Cyclopiazonic acid (5 microM) and ryanodine (10 microM) both increased this ratio, indicating a decrease in the buffering power of the cell. Mimicking the increase in steady-state [Ca2+]i produced by these agents by changing the holding potential to -40 mV, however, did not affect delta[Ca2+]i/sigmaI(Ca).dt. It was concluded that up-take by a ryanodine-sensitive store normally limits Ca2+ distribution to the bulk cytoplasm following entry to the cell through dihydropyridine-sensitive channels.     

The neoglycoprotein mannose-bovine serum albumin, but not progesterone, activates T-type calcium channels in human spermatozoa.

The neoglycoproteins alpha-D-mannose-bovine serum albumin (mannose-BSA) and N-acetyl-alpha-D-glucosamine-BSA (glucNAc-BSA) were shown to rapidly increase intracellular free calcium ([Ca2+]i) in human spermatozoa. The increase in [Ca2+]i induced by these neoglycoproteins accounts for the known ability of these compounds to induce the acrosome reaction in human spermatozoa. Our data support the hypothesis that mannose-BSA, but not progesterone, activates T-type Ca2+ channels in human spermatozoa for the following reasons: (i) the capacity of mannose-BSA to increase [Ca2+]i was inhibited by the specific T-type Ca2+ channel blocker mibefradil (IC50 = 10(-6) mol/l) while progesterone was not inhibited by 10(-5) M mibefradil; (ii) the effect of mannose-BSA to elevate [Ca2+]i was inhibited more potently by Ni2+ (IC50 = 0.1 mmol/l) than Cd2+ (IC50 = 0.5 mmol/l), whereas the effect of progesterone to elevate [Ca2+]i was inhibited equally by Ni2+ and Cd2+ (IC50 = 0.25 mmol/l); (iii) the effects of mannose-BSA and progesterone to increase [Ca2+]i were greater than additive. These data support the idea that mannose-BSA and progesterone were activating distinct Ca2+ channels, one of which was a T-type Ca2+ channel activated by mannose-BSA whereas the Ca2+ channel that was activated by progesterone has yet to be defined at the molecular level.     

Potassium-depolarization-induced cytoplasmic [Ca2+] transients in freshly dissociated pyramidal neurones of the rat dorsal cochlear nucleus

The significance of voltage-activated Ca2+ currents in eliciting cytoplasmic Ca2+ transients was studied in pyramidal neurones isolated from the rat dorsal cochlear nucleus using combined enzyme treatment/mechanical trituration. Increases in cytoplasmic Ca2+ concentration ([Ca2+]i) were evoked by K+-induced depolarizations (10-50 mM) and monitored by the Fura-2 fluorimetric technique. The acutely dissociated neurones had a resting [Ca2+]i of 17.2+/-0.5 nM. They possessed caffeine-sensitive Ca2+ stores which were empty at rest; these stores could be filled with Ca2+ entering from the extracellular space and were re-emptied quickly. The effects of various specific high-voltage-activated (HVA) Ca2+ channel antagonists (nifedipine, omega-agatoxin IVA and omega-conotoxin GVIA) on [Ca2+]i transients were tested. Analysis of the blocking effects of these agents on the [Ca2+]i, transients indicates that, in the pyramidal neurones of the dorsal cochlear nucleus, N-type Ca2+ channels are primarily responsible for producing the depolarization-induced increases in [Ca2+]i.     

Sphingosine-1-phosphate-induced intracellular Ca2+ mobilization in human endothelial cells.

The authors have studied the effect of sphingosine-1-phosphate (S1P) on Ca2+ release from intracellular stores in cultured human umbilical vein endothelial cells (HUVECs). In the presence of extracellular Ca2+, S1P increased intracellular Ca2+ concentration ([Ca2+]i) and this increase was partially inhibited by La3+ (1 microM), indicating that S1P induces Ca2+ influx from extracellular pool and Ca2+ release from intracellular stores. S1P increased [Ca2+]i concentration dependently in Ca2+-free extracellular solution. The Hill coefficient (1.7) and EC50 (420 nM) was obtained from the concentration-response relationship. When caffeine depleted Ca2+ store in the presence of ryanodine, S1P did not induce intracellular Ca2+ release. Furthermore, the Ca2+-induced Ca2+ release inhibitors ruthenium red or dantrolene completely inhibited S1P-induced intracellular Ca2+ release. S1P-induced intracellular Ca2+ release was inhibited by the phospholipase C (PLC) inhibitors neomycin and U73312, or the inositol 1,4,5-triphosphate (IP3)-gated Ca2+ channel blocker aminoethoxybiphenyl borane (2-APB). In contrast, S1P-induced intracellular Ca2+ release was not inhibited by the mitochondrial Ca2+ uptake inhibitor CCCP or the mitochondrial Ca2+ release inhibitor cyclosporin A. These results show that S1P mobilizes Ca2+ from intracellular stores primarily via Ca2+-induced and IP3-induced Ca2+ release and this Ca2+ mobilization is independent of mitochondrial Ca2+ stores.     

The relationship between acetylcholine-evoked Ca2+-dependent current and the Ca2+ concentrations in the cytosol and the lumen of the endoplasmic reticulum in pancreatic acinar cells

In a study of isolated mouse pancreatic acinar cells, we used the patch-clamp whole-cell recording configuration to monitor the Ca(2+)-dependent inward ionic current and simultaneously measured the Ca2+ concentration in either the cytosol ([Ca2+]i) or the lumen of the endoplasmic reticulum ([Ca2+]Lu), using appropriate Ca(2+)-sensitive fluorescent probes. A high concentration of acetylcholine (ACh, 10 microM) evoked an increase in [Ca2+]i, which resulted in the activation of Ca(2+)-dependent inward current. Continued ACh application for several minutes led to a marked reduction in both the current and the [Ca2+]i response and after about 4-10 min of sustained ACh stimulation, the inward current response had disappeared and [Ca2+]i was back to the pre-stimulation level. Repeated stimulation with shorter pulses of ACh (10 microM) resulted in responses of declining magnitude both in terms of inward current and [Ca2+]i rises. The ACh-activated inward current was entirely dependent on the elevation of [Ca2+]i, but at a relatively high [Ca2+]i the current was saturated. ACh caused a rapid release of Ca2+ from the lumen of the endoplasmic reticulum and after discontinuation of stimulation, [Ca2+]Lu was only very slowly (10-15 min) fully restored to the pre-stimulation level. Repeated applications of ACh did not change the relationships between the Ca(2+)-dependent current and [Ca2+]i or the current and [Ca2+]Lu. When [Ca2+]Lu was greater than 100 microM, the ACh-evoked Ca2+ release from the store was so large that the current response was initially saturated. We conclude that the ACh-evoked current response essentially depends on the release of stored Ca2+. Desensitization is mainly due to the relatively slow reloading of the intracellular stores with Ca2+.     

Activation of Ca(2+)-dependent K+ channel and Cl- conductance in canine pancreatic acinar cells through a cyclic AMP pathway.

Effects of adenosine 3',5'-cyclic monophosphate (cAMP) on Ca(2+)-dependent K+ channel and Cl- conductance in the plasma membrane of isolated canine pancreatic acinar cells were studied by patch-clamp methods. In whole-cell current recordings on isolated cells dialyzed with K(+)-rich solution containing 0.5 mM EGTA, addition of 0.5 mM dibutyryl cAMP (dbcAMP), or 50 microM forskolin to the bath increased outward K+ and inward Cl- currents associated with depolarizing and hyperpolarizing voltage jumps, respectively. In intact cells (cell-attached configurations), addition of 0.5 mM dbcAMP or 50 microM forskolin to the bath increased the opening of single K+ channel. In Ca(2+)-free external solution (bath and pipette) 50 microM forskolin or 0.5 mM dbcAMP application evoked an increase in the opening of single K+ channel in intact cells. Addition of 0.5 mM dbcAMP to the bath solution containing 10 mM EGTA without Ca2+ increased the currents of whole-cell dialyzed with K(+)-rich solution containing 10 mM EGTA. When cell was dialyzed with 20 mM EGTA, dbcAMP, or forskolin application did not increase the whole-cell currents. In excised inside-out patches, addition of the catalytic subunit of cAMP-dependent protein kinase (16 U/ml) in the presence of 0.3 mM ATP to the cytoplasmic face of membrane activated the K+ channel, but 0.1 mM cAMP did not. These results suggest that cAMP-dependent phosphorylation can activate Ca(2+)-dependent K+ channels without increase in intracellular free Ca2+ and cAMP-dependent mechanism can activate Ca(2+)-dependent Cl- conductances without the increase in Ca2+ in canine pancreatic acinar cells.     

Capacitative Ca2+ influx and activation of the neutrophil respiratory burst. Different regulation of plasma membrane- and granule-localized NADPH-oxidase

The neutrophil NADPH-oxidase may be activated in the plasma membrane, resulting in release of oxygen metabolites extracellularly, or in the granule or phagosomal membranes, giving intracellular production of oxidants. An increase in [Ca2+]i mediated through binding of fMLF to its receptor is part of a signaling cascade that activates the plasma membrane-localized oxidase. In contrast, a rise in [Ca2+]i induced by a Ca2+ ionophore results in activation of the intracellular pool of oxidase. We mimicked fMLF-induced emptying of intracellular Ca2+ stores with thapsigargin. This induced a pronounced intracellular oxidase activity but no extracellular release of oxidants. The thapsigargin-induced effect was dependent on capacitative Ca2+ influx, because the effect was inhibited dose-dependently by EGTA and the Ca2+ channel blocker La3+. At La3+ concentrations between 200 and 400 microM, thapsigargin also induced a massive extracellular production of superoxide anion. No other channel blockers tested induced a similar effect. We conclude that elevation in [Ca2+]i by capacitative Ca2+ influx induces NADPH-oxidase activation at an intracellular site. Further, activation of the plasma membrane-localized NADPH-oxidase is regulated by a more complex Ca2+ signaling, involving capacitative Ca2+ influx and possibly the specific action of La3+-sensitive Ca2+ channels.