Calcium regulates L-Type Ca2+ channel expression in rat skeletal muscle cells

Primary skeletal muscle cells were cultured in a normal- (1.8 mM) or high- (4.8 mM) Ca2+ culture medium to determine whether Ca2+ modulates the number of L-type Ca2+ channels. Skeletal myoballs cultured in a normal medium showed, when exposed to a high extracellular [Ca2+], ([Ca2+]e) a transient increase in intracellular [Ca2+] ([Ca2+]i) from a resting concentration of 60 to 160 nM. By day 3, however, when the experiments were made, [Ca2+]i no longer differed from control (pre-exposure to high Ca2+). The maximum charge movements in myoballs incubated in 1.8 and 4.8 mM were 16.4+/-1.05 (n=56) and 24.1+/-1.18 nC/microF (n=58; P<0.01), respectively, and peak Ca2+ currents at 20 mV were -10.8+/-1.09 (n=46) and -12.8+/-0.75 nA/microF (n=82), respectively (P>0.05). The tail current amplitudes in 1.8 and 4.8 mM Ca2+-treated cells were -9.3+/-1.23 and -14.2+/-1.37 nA/microF (P<0.05), respectively, at 10 mV and -15.3+/-1.76 and -23.6+/-2.02 nA/microF (P<0.05), respectively at 60 mV. The maximum binding of [3H]PN200-110 (a radioligand specific for L-type Ca2+ channel alpha1 subunits) in myoballs cultured in 1.8 and 4.8 mM [Ca2+]e was 1.34+/-0.23 and 3.2+/-0.63 pmol/mg protein (n=8; P<0.02), respectively. The increase in [Ca2+]i associated with the increases in charge movements, tail currents and the number of L-type Ca2+ channel alpha1 subunits in skeletal muscle cells cultured in high [Ca2+]e support the concept that extracellular Ca2+ influx modulates the expression of L-type Ca2+ channels in skeletal muscle cells.     

Mechanisms of intrinsic force in small human airways

We quantified the magnitude and investigated mechanisms regulating intrinsic force (IF) in human airway smooth muscle (hASM). IF was identified by reducing extracellular calcium (Ca2+) concentration to nominally zero in freshly isolated isometrically mounted 2mm human bronchi. Our results show: (1) the magnitude of IF is ～50% of the maximal total force elicited by acetylcholine (10(-5) M) and is epithelial independent, (2) IF can also be revealed by β-adrenergic activation (isoproterenol), non-specific cationic channel blockade (La3+) or L-type voltage gated Ca2+ channel blockade (nifedipine), (3) atropine, indomethacin, AA-861, or pyrilamine did not affect IF, (4) IF was reduced by the intracellular Ca2+ ([Ca2+]i) chelating agent BAPTA-AM, (5) ω-conotoxin had no effect on IF. In studies in cultured hASM cells nominally zero Ca2+ buffer and BAPTA-AM reduced [Ca2+]i but isoproterenol and nifedipine did not. Taken together these results indicate that rapid reduction of [Ca2+]i reveals a permissive relationship between extracellular Ca2+, [Ca2+]i and IF. However IF can be dissipated by mechanisms effecting Ca2+ sensitivity. We speculate that an increase of IF, a fundamental property of ASM, could be related to human airway clinical hyperresponsiveness and must be accounted for in in vitro studies of hASM.     

L-type Ca2+ channels are not involved in coronary endothelial Ca2+ influx mechanism responsible for endothelium-dependent relaxation

Effects of L-type Ca2+ channel blockers on intracellular Ca2+ concentration ([Ca2+]i) changes evoked by the stimulations which cause endothelium-dependent relaxation were examined in freshly isolated pig coronary endothelial cells using fura-2 fluorescent analysis. Substance P and bradykinin produced endothelium-dependent relaxations of pig coronary arteries. The relaxations were inhibited significantly but not completely by N(omega)-nitro-L-arginine (L-NNA) or aspirin, suggesting that nitric oxide (NO), prostacyclin (PGI2) and endothelium-derived hyperpolarizing factor (EDHF) were involved in the responses. Both substance P and bradykinin elevated coronary endothelial [Ca2+]i in a biphasic manner: An initial transient increase was observed within a minute, which was followed by the subsequent sustained increase declining with time. In the medium without Ca2+, substance P-induced elevation of [Ca2+]i was markedly reduced. L-type Ca2+ channel blockers (nicardipine, diltiazem and verapamil) did not affect substance P-induced increase in endothelial [Ca2+]i. In consistent with this finding, Bay k 8644 failed to increase [Ca2+]i in partially depolarized endothelial cells. In contrast, substance P-induced elevation of endothelial [Ca2+]i was suppressed in high K+ solutions. These findings indicate that: (1) Substance P and bradykinin relax pig coronary artery via production/release of NO, PGI2 and EDHF from the endothelium; (2) The synthesis and release of these endothelium-derived factors are accompanied by an increase in endothelial [Ca2+]i; (3) Activation of L-type Ca2+ channels is not involved in coronary endothelial elevation of [Ca2+]i responsible for the production/release of these endothelium-derived factors. L-type Ca2+ channel blockers seem to be advantageous in the application for the disorders of coronary circulation with respect to that they do not prevent endothelial function to produce/release of endogenous vasorelaxants.     

Membrane stretch increases the activity of Ca(2+)-activated K+ channels in rabbit coronary vascular smooth muscles

It has been proposed that Ca(2+)-activated K+ channels play an essential role in maintaining vascular tone during stretch of blood vessel. However, the underlying mechanism of stretch-induced change of Ca(2+)-activated K+ channel activities are still unknown. The present experiment was designed to investigate the effect of membrane stretch on these channels whose activity was measured from rabbit coronary smooth muscle cells using a patch clamp technique. Ca(2+)-activated K+ channel were identified by their Ca2+ and voltage dependencies and its large conductances as in other preparations. Perfusion of cells with a hypotonic solution, which mimics stretching the cell membrane by making a cell swelling, produced an increase in channel activity in cell-attached patch mode. The similar increase was observed when negative pressure was applied into the patch pipette for stretching the cell membrane within a patch area. In inside-out patch, stretch still increased channel activity even under the conditions which exclude the possible involvement of secondary messengers, or of transmembrane Ca2+ influx via stretch-activated cation channels. Pretreatment of arachidonic acid or albumin showed no effect on stretch-induced channel activation, excluding the possibility of fatty acids mediated channel activation during membrane stretch. These results indicate that the stretch may directly increase the activity of Ca(2+)-activated K+ channels in our experimental condition.     

Characterisation of explanted endothelial cells from mouse aorta: electrophysiology and Ca2+ signalling

We describe here the isolation and primary culture of endothelial cells from mouse aorta ("primary explant technique"). These cells provide an excellent model for functional studies in transgenic mice. The primary explant method delivers cells that grow out from small pieces of mouse aorta placed on Matrigel enriched with endothelial growth factors. Cells can be studied on the Matrigel after removing the pieces of aorta or after passages by using dispase and reseeding the cells on gelatine-coated cover-slips. Cells on Matrigel or from the first and second passages were characterised using the combined patch-clamp and fura-2 fluorescence methods. Cells had a mean membrane resting potential of -19+/-3 mV (n=21), a membrane capacitance of 49+/-5 pF (n=37) and a resting cytosolic free [Ca2+] ([Ca2+]i) of 103+/-8 nM (n=30). Adenosine 5'-triphosphate (ATP), acetylcholine and bradykinin, but not histamine, induced fast release of intracellular Ca2+ followed by a sustained rise in [Ca2+]i. Oscillations in [Ca2+]i were observed at lower agonist concentrations. In nearly all cells (93%, n=30), these agonists activated charybdotoxin-sensitive, Ca2+-activated K+ channels and induced hyperpolarisation. In 84% of the cells (n=32), an increase in [Ca2+]i also activated strongly outwards-rectifying Cl- channels. These activated slowly at positive potentials and inactivated rapidly at negative potentials. Increasing [Ca2+]i to 1 microM activated a non-selective cation channel in 86% of the cells (n=28). Each tested cell responded to a challenge with hypotonic solution by activating a Cl- current that was modestly outwards rectifying and inactivated at positive potentials. This current is similar to the well-described swelling-activated current through volume-regulated anion channels (VRAC) in endothelial cells. However, its activation is slower, its inactivation faster and the current density lower than in cultured endothelial cells. It is concluded that the primary explant technique provides a reliable cell model for studying mouse vascular endothelial cell function.     

Characterization of voltage-gated calcium currents in freshly isolated smooth muscle cells from rat tail main artery.

The aim of the present study was to characterize voltage-gated Ca2+ currents in smooth muscle cells freshly isolated from rat tail main artery in the presence of 5 mmol L(-1) external Ca2+. Calcium currents were identified on the basis of their voltage dependencies and sensitivity to nifedipine, Ni2+ and cinnarizine. In the majority of the cells studied, T- and L-type currents were observed, while the remaining cells showed predominantly L-type currents. In the latter group of cells, holding potential change from -50 to either -70 or -90 mV increased the corresponding inward current amplitude while its voltage activation threshold remained unchanged. The steady state inactivation of L-type Ca2+ channels showed half-maximal inactivation at -38 mV. A Ca2+-dependent inactivation was also evident. Nifedipine (3 micromol L(-1)) blocked L-type but not T-type Ca2+ currents. Ni2+ (50 micromol L(-1)) as well as cinnarizine (1 micromol L(-1)) suppressed the nifedipine-resistant, T-type component of the currents. At higher concentrations, both Ni2+ (0.3-1 mmol L(-1)) and cinnarizine (10 micromol L(-1)) blocked the net inward current. Replacement of Ca2+ with 10 mmol L(-)1 Ba2+ significantly increased the amplitude of L-type Ca2+ currents. These results demonstrate that smooth muscle cells freshly isolated from rat tail main artery may be divided into two populations, one expressing both L- and T-type and the other only L-type Ca2+ channels. Furthermore, this report shows that in arterial smooth muscle cells cinnarizine potently inhibited T-type currents at low concentrations (1 micromol L(-1)) but also blocked L-type Ca2+ currents at higher concentrations (10 micromol L(-1)).     

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.     

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.     

Effects of magnesium on prostacyclin synthesis and intracellular free calcium concentration in vascular cells.

This study investigated the effects of extracellular magnesium concentration ([Mg2+]e; 0.3-3 mM) on intracellular free calcium concentration ([Ca2+]i) and prostacyclin (PGI2) production in cultured human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells from rats (VSMC) under basal and agonist-stimulated conditions. We used histamine as agonist which increases [Ca2+]i and PGI2 production in HUVEC, norepinephrine in VSMC. [Mg2+]e dose-dependently increased basal and agonist-stimulated PGI2 production in both cells. [Mg2+]e dose-dependently reduced basal [Ca2+]i in VSMC, but did not influence in HUVEC. In both cells, increasing [Mg2+]e reduced agonist-stimulated [Ca2+]i responses. Furthermore, [Mg2+]e dose-dependently reduced agonist-stimulated [Ca2+]i in Ca(2+)-free buffer, indicating intracellular Ca2+ release. In VSMC, 10(-6) M diltiazem and 10(-7) M nifedipine, Ca2+ channel blockers, reduced agonist-stimulated [Ca2+]i as well as 3 mM Mg2+, but did not affect PGI2 production. [Mg2+]e amplified dose-dependently arachidonic acid-induced PGI2 production in both cells, suggesting the activation of cyclooxygenase and/or PGI2 synthetase. Our results suggest that [Mg2+]e influences intracellular Ca2+ mobilization of not only vascular smooth muscle cells but also endothelial cells by inhibiting both Ca2+ influx and intracellular Ca2+ release. [Mg2+]e enhances PGI2 production in both types of cells, although the mechanism is likely to be independent from Ca2+ mobilization.     

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.     

Hyperosmotic but not hyposmotic stress evokes a rise in cytosolic Ca2+ concentration in endothelium of intact rat aorta

The effects of osmotic stress on the cytosolic Ca2+ concentration ([Ca2+]i) in the endothelium of excised intact rat aorta were investigated using the indicator fura-2 and the patch clamp technique. Hyperosmotic stress evoked a reversible rise in endothelial [Ca2+]i in the presence but not absence of extracellular Ca2+, indicating that it evoked Ca2+ entry without release from intracellular stores. Hyposmotic stress was without significant effect. Cytochalasins B and D reduced the effect of hyperosmotic stress but not acetylcholine on endothelial [Ca2+]i. In endothelium isolated from underlying smooth muscle, hyperosmotic stress produced a rise in the [Ca2+]i and depolarisation of the endothelial membrane potential whereas hyposmotic stress was without effect. Mechanosensitive cationic channels recorded in cell-attached patches were activated by hyperosmotic solutions applied to the endothelium and inhibited by hyposmotic solutions. These data suggest that shrinkage of endothelial cells evokes an increase in [Ca2+]i by opening a pathway for Ca2+ entry from the extracellular space. The mechanosensitive ionic channels which we have previously described may be responsible for this response.     

Macrophage L-type Ca2+ channel antagonists alter Chlamydia pneumoniae MOMP and HSP-60 mRNA gene expression,and improve antibiotic susceptibility

Recent data have shown a unique relationship between Ca2+ signaling in macrophages through L-type channels and the outcome of C. pneumoniae infection of such cells. The present investigation seeks to provide insights into the manner in which macrophage L-type Ca2+ channel operation affects major outer membrane protein (MOMP) and heat shock protein-60 (HSP-60) mRNA gene expression (factors associated with Chlamydia chronicity), and the possible effect of this on antibiotic susceptibility. Intracellular calcium ([Ca2+]i) chelation using varying doses of 1,2-bis (o-aminophenoxy) ethane-N,N,N'N'--tetra acetic acid (acetoxymethyl) ester (BAPTA-AM) induced an increase in MOMP and a decrease in HSP-60 mRNA gene expression. L-type Ca2+ channel antagonists produced an identical but enhanced effect. Since these findings associate specialized Ca2+ channels to Chlamydia chronicity, it was important to determine Ca2+ channel effect on the usual antibiotic refractory form of C. pneumoniae in macrophages. Inhibition of macrophage L-type Ca2+ channel operation improved C. pneumoniae antibiotic susceptibility assessed by decreased inclusion counts or down-regulated MOMP and HSP-60 mRNA gene expression. These findings provide molecular insights into how specialized Ca2+ channels influence Chlamydia chronic course in macrophages and demonstrates a role for L-type Ca2+ channel inhibitors in enhanced C. pneumoniae susceptibility to antibiotic therapy.     

PKC-mediated modulation of L-type calcium channels may contribute to fat-induced insulin resistance

Increased intracellular free calcium [Ca2+]i has been noted in adipocytes, platelets, and leukocytes of subjects with insulin resistance syndrome or allied disorders. In rodent studies, measures which increase [Ca2+]i in adipocytes and skeletal muscle are associated with impaired insulin signaling, attributable at least in part to diminished ability of insulin to activate phosphoserine phosphatase-1 (PP-1). In fat-fed insulin resistant rats, pre-treatment with a drug that selectively chelates intracellular calcium eliminates about half of the decrement in insulin-stimulated glucose uptake induced by fat feeding; since this chelator does not influence the insulin sensitivity of chow-fed rats, it is reasonable to suspect that fat feeding boosts [Ca2+]i in skeletal muscle, and that this effect is partially responsible for the associated reduction in insulin sensitivity. Clinical insulin resistance is associated with increased levels of triglycerides and other fatty acid metabolites in muscle fibers; this can give rise to diacylglycerol-mediated activation of PKC, which in turn compromises insulin signaling by triggering kinase cascades that phosphorylate IRS-1 on key serine residues. Yet there is also evidence that, in skeletal muscle, PKC activity up-regulates the function of L-type calcium channels, increasing their maximal conductance while left-shifting their voltage dependence. Thus, the PKC activation associated with fat overexposure might be expected to boost basal [Ca2+]i in skeletal muscle, potentially impeding insulin-mediated activation of PP-1. This hypothesis is consistent with several clinical studies demonstrating that long-acting inhibitors of L-type calcium channels can improve insulin sensitivity in overweight hypertensives; it should be readily testable in rodent models of fat-induced insulin resistance. Since parathyroid hormone can act on adipocytes and muscle to boost [Ca2+]i, mild secondary hyperparathyroidism associated with low calcium intakes and poor vitamin D status may contribute to insulin resistance, consistent with certain clinical and epidemiological findings. Magnesium, often thought of as a mild calcium antagonist, appears to have favorable effects on insulin sensitivity and risk for diabetes, and recent evidence indicates that increases of intracellular magnesium within the physiological range can diminish calcium influx through phosphorylated L-type calcium channels. It will be of interest to determine whether calcium antagonism does indeed underlie the favorable influence of good magnesium status on insulin function. A report that chromium picolinate can induce the plasmalemmal Ca2+-ATPase in smooth muscle cells, raises the possibility that modulation of calcium transport might play a role in the insulin-sensitizing efficacy of bioactive chromium.     

Effects of activin A on ionic channels in human FSH-secreting tumour cells.

1. Effects of activin A on ionic channels were examined in human FSH-secreting tumour cells using electrophysiological techniques. 2. Under voltage clamp with the conventional whole-cell clamp technique, the voltage-gated Na+ channel, the T- and L-type Ca2+ channels, the delayed K+ channel and the A-channel were observed. 3. With the nystatin-perforated whole-cell clamp technique, the same voltage-gated channels were recorded. Activin A (10(-7) M) increased the amplitude of the L-type Ca2+ current, whereas it decreased the amplitude of the delayed K+ current. 4. Under current clamp with the perforated whole-cell clamp technique, more than 80% of the cells exhibited spontaneous action potentials. Application of 10(-7) M activin A depolarized the membrane with a conductance increase and augmented action potential frequency. The reversal potential of the activin A-induced current was -20 to 0 mV. The activin A-induced current was abolished in a Na(+)-free extracellular solution, indicating that the membrane depolarization caused by activin A was due to the conductance increase to Na+ ions through non-selective cation channels.     

Effects of hypoxia on membrane potential and intracellular calcium in rat neonatal carotid body type I cells.

1. We have studied the effects of hypoxia on membrane potential and [Ca2+]i in enzymically isolated type I cells of the neonatal rat carotid body (the principal respiratory O2 chemosensor). Isolated cells were maintained in short term culture (3-36 h) before use. [Ca2+]i was measured using the Ca(2+)-sensitive fluoroprobe indo-1. Indo-1 was loaded into cells using the esterified form indo-1 AM. Membrane potential was measured (and clamped) in single isolated type I cells using the perforated-patch (amphotericin B) whole-cell recording technique. 2. Graded reductions in PO2 from 160 Torr to 38, 19, 8, 5 and 0 Torr induced a graded rise of [Ca2+]i in both single and clumps of type I cells. 3. The rise of [Ca2+]i in response to anoxia was 98% inhibited by removal of external Ca2+ (+1 mM EGTA), indicating the probable involvement of Ca2+ influx from the external medium in mediating the anoxic [Ca2+]i response. 4. The L-type Ca2+ channel antagonist nicardipine (10 microM) inhibited the anoxic [Ca2+]i response by 67%, and the non-selective Ca2+ channel antagonist Ni2+ (2 mM) inhibited the response by 77%. 5. Under voltage recording conditions, anoxia induced a reversible membrane depolarization (or receptor potential) accompanied, in many cases, by trains of action potentials. These electrical events were coincident with a rapid rise of [Ca2+]i. When cells were voltage clamped close to their resting potential (-40 to -60 mV), the [Ca2+]i response to anoxia was greatly reduced and its onset was much slower.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Calcium and fos involvement in brain-derived Ca(2+)-binding protein (S100)-dependent apoptosis in rat phaeochromocytoma cells

Brain-derived calcium-binding protein S100 induces apoptosis in a significant fraction of rat phaeochromocytoma (PC12) cells. We used single cell techniques (patch clamp, videomicroscopy and immunocytochemistry) to clarify some of the specific aspects of S100-induced apoptosis, the modality(ies) of early intracellular Ca2+ concentration increase and the expression of some classes of genes (c-fos, c-jun, bax, bcl-x, p-15, p-21) known to be implicated in apoptosis of different cells. The results show that S100: (1) causes an increase of [Ca2+]i due to an increased conductance of L-type Ca2+ channels; (2) induces a sustained increase of the Fos levels which is evident since the first time point tested (3 h) and remains elevated until to the last time point (72 h). All these data suggest that S100-derived apoptosis in PC12 cells may be the consequence of a system involving an increase in L-type Ca2+ channel conductance with consequent [Ca2+]i increase which up-regulates, directly or indirectly, the expression of Fos.     

Spontaneous action potentials initiate rhythmic intercellular calcium waves in immortalized hypothalamic (GT1-1) neurons.

GT1-1 cells exhibit spontaneous action potentials and transient increases in intracellular calcium concentration ([Ca2+]i) that occur in individual cells and as spatially propagated intercellular Ca2+ waves. In this study, simultaneous cell-attached patch-clamp recording of action currents (indicative of action potentials) and fluorescence imaging of [Ca2+]i revealed that Ca2+ transients in GT1-1 cells were preceded by a single action current or a burst of action currents. Action currents preceded Ca2+ transients in a similar pattern regardless of whether the Ca2+ transients were limited to the individual cell or occurred as part of an intercellular Ca2+ wave. Both the action currents and Ca2+ transients were abolished by 1 microM tetrodotoxin. Removal of extracellular Ca2+ abolished all spontaneous Ca2+ transients without inhibiting the firing of action currents. Nimodipine, which blocks L-type Ca2+ currents in GT1-1 cells, also abolished all spontaneous Ca2+ signaling. Delivery of small voltage steps to the patch pipette in the cell-attached configuration elicited action currents the latency to firing of which decreased with increasing amplitude of the voltage step. These results indicate that spontaneous intercellular Ca2+ waves are generated by a propagated depolarization, the firing of action potentials in individual cells, and the resulting influx of Ca2+ through L-type Ca2+ channels. These patterns of spontaneous activity may be important in driving the pulsatile release of GnRH from networks of cells.     

Simultaneous recordings of cytosolic Ca2+ level and membrane potential and current during the response to thyroliberin in clonal rat anterior pituitary cells

The response to thyroliberin in prolactin-producing rat GH4C1 clonal cells was studied using fura-2 to monitor the cytosolic Ca2+ level ([Ca2+]i) in single cells, combined with recordings of membrane potential and current. The average value of [Ca2+]i was 109 nM (mean +/- SD, n = 112), and evoked action potentials caused transient elevations of about 100 nM. At higher firing frequencies these transients merged to a sustained elevation. In 100% of the cells thyroliberin caused an instant rise in [Ca2+]i, peaking at 795 +/- 300 nM (n = 112). This first phase of the thyroliberin response was associated with hyperpolarization in current clamp and outward current in voltage clamp, caused by the opening of Ca2(+)-activated K+ channels. In 75% of the cells the initial peak in [Ca2+]i was followed by a prolonged plateau phase at 247 +/- 76 nM (n = 84). In current clamp the second-phase elevation of [Ca2+]i was linked to either a modest depolarization in combination with enhanced firing frequency or a more pronounced depolarization in silent cells. This elevation of [Ca2+]i was reversed by hyperpolarizing current injection. No second-phase elevation of [Ca2+]i was observed during voltage clamp at a holding potential of -50 mV. Short exposure to Ca2(+)-free conditions eliminated the second-phase elevation in [Ca2+]i, whereas the first phase remained intact. Our experiments show a direct relationship between electrical activity and [Ca2+]i in the GH4C1 cells. The second-phase elevation of [Ca2+]i caused by thyroliberin is the result of influx through voltage-sensitive Ca2+ channels, without involving agonist-gated channels.