Brain activation pattern induced by stimulation of L-type Ca2+-channels: contribution of Ca(V)1.3 and Ca(V)1.2 isoforms

Ca(V)1.2 and Ca(V)1.3, are the main dihydropyridine-sensitive L-type calcium channel isoforms in the brain. To reveal the contribution of each isoform to the neuronal activation pattern elicited by the dihydropyridine L-type calcium channel activator BayK 8644, we utilized Fos expression as a marker of neuronal activation in mutant mice (Ca(V)1.2(DHP-/-) mice) expressing dihydropyridine-insensitive Ca(V)1.2 L-type calcium channels. BayK 8644-treated wildtype mice displayed intense and widespread Fos expression throughout the neuroaxis in 77 of 80 brain regions quantified. The Fos response in Ca(V)1.2(DHP-/-) mice was greatly attenuated or absent in most of these areas, suggesting that a major part of the widespread Fos induction including most cortical areas was mediated by Ca(V)1.2 L-type calcium channels. BayK 8644-induced Fos expression in Ca(V)1.2(DHP-/-) mice indicating predominantly Ca(V)1.3 L-type calcium channel-mediated activation was noted in more restricted neuronal populations (20 of 80), in particular in the central amygdala, the bed nucleus of the stria terminalis, paraventricular hypothalamic nucleus, lateral preoptic area, locus coeruleus, lateral parabrachial nucleus, central nucleus of the inferior colliculus, and nucleus of the solitary tract. Our data indicate that selective stimulation of other than Ca(V)1.2 L-type calcium channels, mostly Ca(V)1.3, causes neuronal activation in a specific set of mainly limbic, hypothalamic and brainstem areas, which are associated with functions including integration of emotion-related behavior. Hence, selective modulation of Ca(V)1.3 L-type calcium channels could represent a novel (pharmacotherapeutic) tool to influence these CNS functions.     

Activity of cardiac L-type Ca2+ channels is sensitive to cytoplasmic calcium

Ca²⁺ -induced inactivation of L-type Ca²⁺ channels is proposed as an important negative feedback mechanism regulating Ca²⁺ entry. Here, for the first time, evidence for modification of heart L-type Ca²⁺ channel activity by cytoplasmic calcium is provided from excised insideout membrane patches. Ba²⁺ currents through cardiac L-type Ca²⁺ channels exhibited only modest inactivation in the absence of cytoplasmic Ca²⁺. Elevation of cytoplasmic Ca²⁺ to micromolar concentrations strikingly affected L-type Ca²⁺ channel activity as evaluated from ensemble average Ba²⁺ currents. Inactivation was markedly increased concomitant with a reduction of peak inward current, which was almost completely eliminated at about 15 M cytoplasmic Ca²⁺ concentration. Half maximal suppression of Ba²⁺ currents was observed at 2.3 M Ca²⁺. The observed modifications of L-type Ca²⁺ channel activity show that cytoplasmic Ca²⁺ induces channel closure. Below 4 M Ca²⁺, channels can be reversibly reactivated during repetitive depolarizations, while at high Ca²⁺ concentrations (15 M) most Ca²⁺ channels reside in a closed state. This may allow for a delicate regulation of Ca²⁺ entry, and consequently of heart contraction.     

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.     

T-type Ca2+ channels mediate propagation of odor-induced Ca2+ transients in rat olfactory receptor neurons

Propagation of odor-induced Ca(2+) transients from the cilia/knob to the soma in mammalian olfactory receptor neurons (ORNs) is thought to be mediated exclusively by high-voltage-activated Ca(2+) channels. However, using confocal Ca(2+) imaging and immunocytochemistry we identified functional T-type Ca(2+) channels in rat ORNs. Here we show that T-type Ca(2+) channels in ORNs also mediate propagation of odor-induced Ca(2+) transients from the knob to the soma. In the presence of the selective inhibitor of T-type Ca(2+) channels mibefradil (10-15 microM) or Ni(2+) (100 microM), odor- and forskolin/3-isobutyl-1-methyl-xanthine (IBMX)-induced Ca(2+) transients in the soma and dendrite were either strongly inhibited or abolished. The percentage of inhibition of the Ca(2+) transients in the knob, however, was 40-50% less than that in the soma. Ca(2+) transients induced by 30 mM K(+) were partially inhibited by mibefradil, but without a significant difference in the extent of inhibition between the knob and soma. Furthermore, an increase of as little as 2.5 mM in the extracellular K(+) concentration (7.5 mM K(+)) was found to induce Ca(2+) transients in ORNs, and such responses were completely inhibited by mibefradil or Ni(2+). Total replacement of extracellular Na(+) with N-methyl-d-glutamate inhibited none of the odor-, forskolin/IBMX- or 7.5 mM K(+)-induced Ca(2+) transients. Positive immunoreactivity to the Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 subunits of the T-type Ca(2+) channel was observed throughout the soma, dendrite and knob. These data suggest that involvement of T-type Ca(2+) channels in the propagation of odor-induced Ca(2+) transients in ORNs may contribute to signal transduction and odor sensitivity.     

Role of L-type Ca2+ channels in iron transport and iron-overload cardiomyopathy

Excessive body iron or iron overload occurs under conditions such as primary (hereditary) hemochromatosis and secondary iron overload (hemosiderosis), which are reaching epidemic levels worldwide. Primary hemochromatosis is the most common genetic disorder with an allele frequency greater than 10% in individuals of European ancestry, while hemosiderosis is less common but associated with a much higher morbidity and mortality. Iron overload leads to iron deposition in many tissues especially the liver, brain, heart and endocrine tissues. Elevated cardiac iron leads to diastolic dysfunction, arrhythmias and dilated cardiomyopathy, and is the primary determinant of survival in patients with secondary iron overload as well as a leading cause of morbidity and mortality in primary hemochromatosis patients. In addition, iron-induced cardiac injury plays a role in acute iron toxicosis (iron poisoning), myocardial ischemia–reperfusion injury, Friedreich ataxia and neurodegenerative diseases. Patients with iron overload also routinely suffer from a range of endocrinopathies, including diabetes mellitus and anterior pituitary dysfunction. Despite clear connections between elevated iron and clinical disease, iron transport remains poorly understood. While low-capacity divalent metal and transferrin-bound transporters are critical under normal physiological conditions, L-type Ca²⁺ channels (LTCC) are high-capacity pathways of ferrous iron (Fe²⁺) uptake into cardiomyocytes especially under iron overload conditions. Fe²⁺ uptake through L-type Ca²⁺ channels may also be crucial in other excitable cells such as pancreatic beta cells, anterior pituitary cells and neurons. Consequently, LTCC blockers represent a potential new therapy to reduce the toxic effects of excess iron.     

Cyclic AMP-dependent phosphorylation modifies the gating properties of L-type Ca2+ channels in bovine adrenal chromaffin cells

We investigated the effects of cAMP-dependent phosphorylation on the voltage- and time-dependent gating properties of Ca²⁺ channel currents recorded from bovine adrenal chromaffin cells under whole-cell voltage clamp. Extracellular perfusion with the membrane-permeant activator of cAMP-dependent protein kinase, 8-bromo(8-Br)-cAMP (1 mM), caused a 49%, 29%, and 21% increase in Ca²⁺ current (I _Ca) amplitudes evoked by voltage steps to 0, +10, and +20 mV respectively (mean values from eight cells, p0.05). Analysis of voltage-dependent steady-state activation (m ) curves revealed a 0.70±0.27 charge increase in the activation-gate valency (z _m) following 8-Br-cAMP perfusion. Similar responses were observed when Ba²⁺ was the charge carrier, where z _m was increased by 1.33±0.34 charges (n=8). The membrane potential for half activation (V _1/2) was also significantly shifted 6 mV more negative for I _Ba (mean, n=8). The time course for I _Ba (and I _Ca) activation was well described by second-order m ² kinetics. The derived time constant for activation (_m) was voltage-dependent, and the _m/V relation shifted negatively after 8-Br-cAMP treatment. Ca²⁺ channel gating rates were derived from the (_m) and m ² values according to a Hodgkin-Huxley type m ² activation process. The forward rate ( _m) for channel activation was increased by 8-Br-cAMP at membrane potentials 0 mV, and the backward rate (_m) decreased at potentials +10 mV. Time-dependent inactivation of I _Ca consisted of a slowly decaying component (_h 300 ms) and a non-inactivating steady-state component. The currents contributed by the two inactivation processes displayed different voltage dependences, the effects of 8-Br-cAMP being exclusively on the slowly inactivating L-type component.     

ATP suppresses activity of Ca2+ -activated K+ channels by Ca2+ chelation

Ca²⁺-activated maxi K⁺ channels were studied in inside-out patches from smooth muscle cells isolated from either porcine coronary arteries or guinea-pig urinary bladder. As described by Groschner et al. (Pflügers Arch 417:517, 1990), channel activity (NP _o) was stimulated by 3 M [Ca²⁺]_c (1 mM Ca-EGTA adjusted to a calculated pCa of 5.5) and was suppressed by the addition of 1 mM Na₂ATP. The following results suggest that suppression of NP _o by Na₂ATP is due to Ca²⁺ chelation and hence reduction of [Ca²⁺]_c and reduced Ca²⁺ activation of the channel. The effect was absent when Mg ATP was used instead of Na₂ATP. The effect was diminished by increasing the [EGTA] from 1 to 10 mM. The effect was absent when [Ca²⁺]_c was buffered with 10 mM HDTA (apparent pK _Ca 5.58) instead of EGTA (pK _Ca 6.8). A Ca²⁺-sensitive electrode system indicated that 1 mM Na₂ATP reduced [Ca²⁺]_c in 1 mM Ca-EGTA from 3 M to 1.4 M. Na₂ATP, Na₂GTP, Li₄AMP-PNP and NaADP reduced measured [Ca²⁺]_c in parallel with their suppression of NP _o. After the Na₂ATP-induced reduction of [Ca²⁺]_c was re-adjusted by adding either CaCl₂ or MgCl₂, the effect of Na₂ATP on NP _o disappeared. In vivo, intracellular [Mg²⁺] exceeds free [ATP^4–], hence ATP modulation of maxi K⁺ channels due to Ca²⁺ chelation is without biological relevance.     

Store-operated Ca2+ entry in platelets occurs independently of transient receptor potential (TRP) C1

Changes in [Ca²⁺]_i are a central step in platelet activation. In nonexcitable cells, receptor-mediated depletion of intracellular Ca²⁺ stores triggers Ca²⁺ entry through store-operated calcium (SOC) channels. Stromal interaction molecule 1 (STIM1) has been identified as an endoplasmic reticulum (ER)-resident Ca²⁺ sensor that regulates store-operated calcium entry (SOCE), but the identity of the SOC channel in platelets has been controversially debated. Some investigators proposed transient receptor potential (TRP) C1 to fulfil this function based on the observation that antibodies against the channel impaired SOCE in platelets. However, others could not detect TRPC1 in the plasma membrane of platelets and raised doubts about the specificity of the inhibiting anti-TRPC1 antibodies. To address the role of TRPC1 in SOCE in platelets, we analyzed mice lacking TRPC1. Platelets from these mice display fully intact SOCE and also otherwise unaltered calcium homeostasis compared to wild-type. Furthermore, platelet function in vitro and in vivo is not altered in the absence of TRPC1. Finally, studies on human platelets revealed that the presumably inhibitory anti-TRPC1 antibodies have no specific effect on SOCE and fail to bind to the protein. Together, these results provide evidence that SOCE in platelets is mediated by channels other than TRPC1. David Varga-Szabo and Kalwant S. Authi contributed equally to this article.     

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.     

The acid test: the discovery of two-pore channels (TPCs) as NAADP-gated endolysosomal Ca2+ release channels

In this review, we describe the background and implications of our recent discovery that two-pore channels (TPCs) comprise a novel class of calcium release channels gated by the intracellular messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Their localisation to the endolysosomal system highlights a new function for these organelles as targets for NAADP-mediated Ca²⁺ mobilisation. In addition, we describe how TPCs may also trigger further Ca²⁺ release by coupling to the endoplasmic reticular stores through activation of IP₃ receptors and ryanodine receptors.     

Modulation of single slow (L-type) calcium channels by intracellular ATP in vascular smooth muscle cells

Involvement of ATP in the regulation of slow (L-type) Ca²⁺ channels of vascular smooth muscle cells was investigated by recording single Ca²⁺ channel currents (single-channel conductance of 18 pS) using a patch clamp technique. In the cell-attached configuration, intracellular composition was modified by permeabilizing the cell membrane with mechanical disruption at one end of the cell. Single cells were freshly isolated from guinea-pig portal vein by collagenase treatment. For the channel recordings, the pipette solution contained 100 mM Ba²⁺ and the bath contained K⁺-rich solution (with 5 mM EGTA) to depolarize the membrane to near 0 mV. The channel activity decreased usually within 3 min after permeabilizing the cell end and exposure to ATP-free bath solution. If ATP (1–5 mM) was applied to the bath (access to cell interior) before complete disappearance of channel activity, channel activity was partially recovered. ATP did not change the current amplitude (i) or the mean open time of the channels, whereas the number of channels available for opening and/or the probability of their being open (NP _o) were increased by ATP. A non-hydrolyzable analogue of ATP, AMP-PNP, did not exert an ATP-like effect; ATP--S had a weak effect. With 1 M Bay-K-8644 (Ca²⁺ channel agonist) in the pipette, the activity of the Ca²⁺ channel was high; such activity persisted for more than 10 min after permeabilizing the cell and exposting to ATP-free solution containing KCN (1 mM) and 2-deoxy-d-glucose (10 mM). These results indicate that activation of slow Ca²⁺ channels requires ATP. The effect of ATP may be exerted by phosphorylation and/or an energy-requiring step. Bay-K-8644 may change the nature of the slow Ca²⁺ channel, making it resistant to rundown.     

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⁺.     

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.     

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.     

L-alanine evokes opening of single Ca2+-activated K+ channels in rat liver cells

Cellular uptake of neutral amino acids via Na⁺ cotransporters is known to be associated with an increased membrane K⁺ conductance mediated by an unknown mechanism that is essential for avoiding excessive cell swelling. We now demonstrate by patch-clamp single-channel current recording that exposure of rat liver cells to L-alanine, but not the poorly transported D-stereoisomer, evokes opening of single K⁺ channels and that this effect is reversible upon removal of the amino acid. The nature of the conductance pathways opened in the intact cell by L-alanine has been investigated in cell-free excised membrane patches where it can be shown that the K⁺-selective channels are opened by Ca²⁺ acting from the inside of the membrane at a concentration as low as 0.1 M.     

Small-conductance Ca2+-activated K+ channels in bovine chromaffin cells

Simultaneous whole-cell patch-clamp and fura-2 fluorescence [Ca²⁺]_i measurements were used to characterize Ca²⁺-activated K⁺ currents in cultured bovine chromaffin cells. Extracellular application of histamine (10 M) induced a rise of [Ca²⁺]_i concomitantly with an outward current at holding potentials positive to –80 mV. The activation of the current reflected an increase in conductance, which did not depend on membrane potential in the range –80 mV to –40 mV. Increasing the extracellular K⁺ concentration to 20 mM at the holding potential of –78 mV was associated with inwardly directed currents during the [Ca²⁺]_i elevations induced either by histamine (10 M) or short voltage-clamp depolarizations. The current reversal potential was close to the K⁺ equilibrium potential, being a function of external K⁺ concentration. Current fluctuation analysis suggested a unit conductance of 3–5 pS for the channel that underlies this K⁺ current. The current could be blocked by apamin (1 M). Whole-cell current-clamp recordings snowed that histamine (10 M) application caused a transient hyperpolarization, which evolved in parallel with the [Ca²⁺]_i changes. It is proposed that a small-conductance Ca²⁺-activated K⁺ channel is present in the membrane of bovine chromaffin cells and may be involved in regulating catecholamine secretion by the adrenal glands of various species.     

Low Ca2+-sensitive maxi-K+ channels in human cultured fibroblasts

The patch clamp technique was used to reveal single channel activity in the membrane of human cultured fibroblasts. The most frequently detected ion channel type was a Ca²⁺-dependent K⁺ channel with a conductance of 287±38 pS in symmetrical 130 mM KCl. The channel showed a peculiar low Ca²⁺-sensitivity compared to that of similar channels in other preparations. In fact micromolar values of internal Ca²⁺ were not effective in the channel activation, except at high depolarizing membrane potentials. The activity was highly increased only when the channel was exposed to relatively high internal Ca²⁺ concentrations (0.2–2.0 mM).     

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.     

Critical role of L-type voltage-dependent Ca channels in neural progenitor cell proliferation induced by hypoxia

Hypoxia can promote proliferation of neural progenitor cells in vitro and in vivo, however, the mechanisms underlying this phenomenon remain largely unknown. Calcium ions are important for the proliferation of progenitor cells. In this study, we reported that Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channels mediated hypoxia-promoted proliferation of neural progenitor cells isolated from embryonic day 14.5 rat mesencephalon. Cell number was greatly increased in the cultured neural progenitor cells exposed to physiological hypoxia (3% O₂, 72 h) compared with normoxia exposure (20% O_2, 72 h). Increased intracellular Ca²⁺ concentration was also observed when the cells were exposed to hypoxia. Moreover, removal of extracellular Ca²⁺ or administration of nicardipine, an agent known to block L-type Ca²⁺ channels, resulted in suppression of the hypoxia-induced increase in intracellular Ca²⁺ and cell numbers. These results suggest that hypoxia promoted the proliferation of neural progenitor cells by increasing Ca²⁺ influx, which was likely a result of upregulation of L-type voltage-dependent Ca²⁺ channel function.     

High-frequency fatigue of skeletal muscle: role of extracellular Ca2+

The present study evaluated whether Ca(2+) entry operates during fatigue of skeletal muscle. The involvement of different skeletal muscle membrane calcium channels and of the Na(+)/Ca(2+) exchanger (NCX) has been examined. The decline of force was analysed in vitro in mouse soleus and EDL muscles submitted to 60 and 110 Hz continuous stimulation, respectively. Stimulation with this high-frequency fatigue (HFF) protocol, in Ca(2+)-free conditions, caused in soleus muscle a dramatic increase of fatigue, while in the presence of high Ca(2+) fatigue was reduced. In EDL muscle, HFF was not affected by external Ca(2+) levels either way, suggesting that external Ca(2+) plays a general protective role only in soleus. Calciseptine, a specific antagonist of the cardiac isoform (alpha1C) of the dihydropyridine receptor, gadolinium, a blocker of both stretch-activated and store-operated Ca(2+) channels, as well as inhibitors of P2X receptors did not affect the development of HFF. Conversely, the Ca(2+) ionophore A23187 increased the protective action of extracellular Ca(2+). KB-R7943, a selective inhibitor of the reverse mode of NCX, produced an effect similar to that of Ca(2+)-free solution. These results indicate that a transmembrane Ca(2+) influx, mainly through NCX, may play a protective role during HFF development in soleus muscle.