Maitotoxin induces a calcium-dependent membrane depolarization in GH4C1 pituitary cells via activation of type L voltage-dependent calcium channels.

Maitotoxin (MTX) is a water-soluble polyether, isolated from the marine dinoflagellate Gambierdiscus toxicus, that stimulates hormone release and Ca2+ influx. We have investigated the action by which MTX induces Ca2+ influx and stimulates prolactin (PRL) release from GH4C1 rat pituitary cells. PRL release elicited by MTX is abolished in a concentration-dependent manner by nimodipine, a dihydropyridine (DHP) antagonist of type L voltage-dependent calcium channels (L-VDCC), indicating that MTX-enhanced PRL release occurs via activation of type L-VDCC. As an initial approach to determine whether MTX interacts directly with VDCC, we examined whether MTX affects the binding of [3H]PN 200-110, a DHP class antagonist, in intact GH4C1 cells. MTX increased the Bmax of [3H]PN 200-110 binding to intact GH4C1 cells from 4.6 +/- 0.03 to 12.5 +/- 2.2 fmol/10(6) cells, without changing the Kd. This indicates that MTX does not bind to the DHP site, but rather suggests that MTX may have an allosteric interaction with the DHP binding site. The effect of MTX on DHP binding was largely (65%) calcium-dependent. We next examined whether MTX alters the membrane potential of GH4C1 cells using the potential sensitive fluorescent dye bisoxonol. Addition of 100 ng/ml MTX to GH4C1 cells caused a membrane depolarization within 2.5 min which reached a plateau at 5 min. The MTX-induced depolarization was not prevented by substitution of impermeant choline ions for Na+. It was similarly unaffected by K+ channel blockers or by depleting the K+ chemical concentration gradient with gramicidin, a monovalent cation pore-forming agent. By contrast, low extracellular Ca2+ totally abolished the depolarization response, and nimodipine at 100 nM substantially reduced the MTX-induced membrane depolarization. These results indicate that the predominant effect of MTX on depolarization is Ca2+ influx through L-VDCC. Taken together, our results indicate that MTX-enhanced PRL release occurs exclusively via activation of type L-VDCC in GH4C1 cells. We suggest that MTX induces an initial slow calcium conductance, possibly via an allosteric interaction with a component of the VDCC complex, which, in turn, initiates a positive feedback mechanism involving calcium-dependent membrane depolarization and voltage-dependent activation of calcium channels.     

Calcium-dependent inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells

The inactivation of calcium channels in mammalian pituitary tumor cells (GH3) was studied with patch electrodes under voltage clamp in cell-free membrane patches and in dialyzed cells. The calcium current elicited by depolarization from a holding potential of -40 mV passed predominantly through one class of channels previously shown to be modulated by dihydropyridines and cAMP-dependent phosphorylation (Armstrong and Eckert, 1987). When exogenous calcium buffers were omitted from the pipette solution, the macroscopic calcium current through those channels inactivated with a half time of approximately 10 ms to a steady state level 40-75% smaller than the peak. Inactivation was also measured as the reduction in peak current during a test pulse that closely followed a prepulse. Inactivation was largely reduced or eliminated by (a) buffering free calcium in the pipette solution to less than 10(-8) M; (b) replacing extracellular calcium with barium; (c) increasing the prepulse voltage from +10 to +60 mV; or (d) increasing the intracellular concentration of cAMP, either 'directly' with dibutyryl-cAMP or indirectly by activating adenylate cyclase with forskolin or vasoactive intestinal peptide. Thus, inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells only occurs when membrane depolarization leads to calcium ion entry and intracellular accumulation.     

Regulation of L-type calcium channels in pituitary GH(4)C(1) cells by depolarization.

The neurosecretory anterior pituitary GH(4)C(1) cells exhibit the high voltage-activated dihydropyridine-sensitive L-type and the low voltage-activated T-type calcium currents. The activity of L-type calcium channels is tightly coupled to secretion of prolactin and other hormones in these cells. Depolarization induced by elevated extracellular K(+) reduces the dihydropyridine (+)-[(3)H]PN200-110 binding site density and (45)Ca(2+) uptake in these cells (). This study presents a functional analysis by electrophysiological techniques of short term regulation of L-type Ca(2+) channels in GH(4)C(1) cells by membrane depolarization. Depolarization of GH(4)C(1) cells by 50 mm K(+) rapidly reduced the barium currents through L-type calcium channels by approximately 70% and shifted the voltage dependence of activation by 10 mV to more depolarized potentials. Down-regulation depended on the strength of the depolarizing stimuli and was reversible. The currents recovered to near control levels on repolarization. Down-regulation of the calcium channel currents was calcium-dependent but may not have been due to excessive accumulation of intracellular calcium. Membrane depolarization by voltage clamping and by veratridine also produced a down-regulation of calcium channel currents. The down-regulation of the currents had an autocrine component. This study reveals a calcium-dependent down-regulation of the L-type calcium channel currents by depolarization.     

Evidence that potassium channels regulate prolactin secretion in GH4C1 cells by causing extracellular calcium influx

Tetraethylammonium (TEA), a K+ channel blocker, induced prolactin (PRL) secretion in GH4C1 cells in a dose-dependent manner when applied at a concentration from 1-20 mM. During continuous exposure to TEA, a significant increase in PRL secretion occurred by 20 min and the response was sustained until the end of a 60-min exposure. Blocking Ca2+ influx by employing a Ca(2+)-depleted medium or the Ca2+ channel blocker, nifedipine, prevented induction of PRL secretion by 20 mM TEA. Preincubation of the cells for 10 min with 20 mM TEA did not inhibit PRL secretion induced by thyrotropin-releasing hormone (TRH), phorbol 12-myristate 13-acetate (TPA) or by cell swelling produced by 30% medium hyposmolarity, but significantly depressed that induced by depolarizing 30 mM K+. BaCl2, another K+ channel blocker, had the same effect on PRL secretion as TEA. The data suggest that blocking K+ channels may cause membrane depolarization, thereby inducing Ca2+ influx which is a potent stimulus for PRL secretion in GH4C1 cells.     

Steady-state currents through voltage-dependent, dihydropyridine-sensitive Ca2+ channels in GH3 pituitary cells.

Ca2+ influx via voltage-dependent Ca2+ channels is known to be elicited during action potentials but possibly also occurs at the resting potential. The steady-state current through voltage-dependent Ca2+ channels and its role for the electrical activity was, therefore, investigated in pituitary GH3 cells. Applying the recently developed 'nystatin-modification' of the patch-clamp technique, most GH3 cells (18 out of 23 cells) fired spontaneous action potentials from a baseline membrane potential of 43.7 +/- 4.6 mV (mean +/- s.d., n = 23). The frequency of action potentials was stimulated about twofold by Bay K 8644 (100 nM), a Ca(2+)-channel stimulator, and action potentials were completely suppressed by the Ca(2+)-channel blocker PN 200-110 (100 nM). Voltage clamping GH3 cells at fixed potentials for several minutes and with 1 mM Ba2+ as divalent charge carrier, we observed steady-state Ca(2+)-channel currents that were dihydropyridine-sensitive and displayed a U-shaped current-voltage relation. The results strongly suggest that the observed long lasting, dihydropyridine-sensitive Ca(2+)-channel currents provide a steady-state conductivity for Ca2+ at the resting potential and are essential for the generation of action potentials in GH3 pituitary cells.     

Block of voltage-dependent calcium channels by aliphatic monoamines

We have recently identified farnesol, an intermediate in the mevalonate pathway, as a potent endogenous modulator and blocker of N-type calcium channels (Roullet, J. B., R. L. Spaetgens, T. Burlingame, and G. W. Zamponi. 1999. J. Biol. Chem. 274:25439-25446). Here, we investigate the action of structurally related compounds on various types of voltage-dependent Ca(2+) channels transiently expressed in human embryonic kidney cells. 1-Dodecanol, despite sharing the 12-carbon backbone and headgroup of farnesol, exhibited a significantly lower blocking affinity for N-type Ca(2+) channels. Among several additional 12-carbon compounds tested, dodecylamine (DDA) mediated the highest affinity inhibition of N-type channels, indicating that the functional headgroup is a critical determinant of blocking affinity. This inhibition was concentration-dependent and relatively non-discriminatory among N-, L-, P/Q-, and R-Ca(2+) channel subtypes. However, whereas L-type channels exhibited predominantly resting channel block, the non-L-type isoforms showed substantial rapid open channel block manifested by a speeding of the apparent time course of current decay and block of the inactivated state. Consistent with these findings, we observed significant frequency-dependence of block and dependence on external Ba(2+) concentration for N-type, but not L-type, channels. We also systematically investigated the drug structural requirements for N-type channel inhibition. Blocking affinity varied with carbon chain length and showed a clear maximum at C12 and C13, with shorter and longer molecules producing progressively weaker peak current block. Overall, our data indicate that aliphatic monoamines may constitute a novel class of potent inhibitors of voltage-dependent Ca(2+) channels, with block being governed by rigid structural requirements and channel-specific state dependencies.     

Stimulation of single L-type calcium channels in rat pituitary GH3 cells by thyrotropin-releasing hormone.

Hormonal stimulation of voltage-dependent Ca2+ channels in pituitary cells is thought to contribute to the sustained phase of Ca2+ entry and secretion induced by secretion stimulating hormones and has been suggested as a mechanism for refilling the Ca2+ stores. Using the cell-attached patch-clamp technique, we studied the stimulation of single Ca2+ channels by thyrotropin-releasing hormone (TRH) in rat GH3 cells. We show that TRH applied from the bath switched the activity of single L-type Ca2+ channels from a gating mode with very low open probability (po) to a gating mode with slightly smaller conductance but 10 times higher po. Interconversions between these two gating modes were also observed under basal conditions, where the equilibrium was shifted towards the low po mode. TRH applied from the pipette had no effect, indicating the involvement of a cytosolic compound in the stimulatory pathway. We show that TRH does not potentiate all the L-type Ca2+ channels in a given membrane patch and report evidence for co-expression of two functionally different L-type Ca2+ channels. Our results uncover the biophysical mechanism of hormonal stimulation of voltage-dependent Ca2+ channels in GH3 cells and are consistent with differential modulation of different subtypes of dihydropyridine-sensitive Ca2+ channels.     

Potentiation by stannous chloride of calcium entry into osteoblastic MC3T3-E1 cells through voltage-dependent L-type calcium channels

The present study was undertaken to confirm that L-type Ca(2+) channels are involved in Ca(2+) entry into osteoblastic MC3T3-E1 cells and to examine the effect of SnCl2, a Ca(2+)]-channel activator, on the intracellular Ca(2+)concentration ([Ca(2+)]i). High K(+)concentration-dependently raised the [Ca(2+)]i. All of the L-type Ca(2+)channel blockers used here, such as nifedipine, nicardipine, verapamil, and diltiazem, and CdCl2 (a non-selective blocker) inhibited the high K(+)-induced [Ca(2+)]i rise, but v-conotoxin GVIA (an N-type blocker) and NiCl2(a T-type blocker) had no effect. Application of SnCl2 alone did not change the [Ca(2+)]i. However, in the presence of high K(+), SnCl2 enhanced the high K(+)-induced [Ca(2+)]i rise, which was inhibited by Ca(2+)]-free medium or nifedipine. In the case where high K(+)was applied prior to SnCl2, SnCl2 alone raised the [Ca(2+)]i by itself. In conclusion, MC3T3-E1 cells possess the voltage-dependent L-type Ca(2+)] channels and SnCl2 facilitates the Ca(2+) entry through the L-type ones under the condition of the membrane depolarization. There is the possibility that Ca(2+) release from intracellular Ca(2+) stores is involved in the action of SnCl2.     

Influence of the external concentration of potassium ions on functioning of voltage-dependent potassium channels in GH3 cells

The patch-clamp technique in a whole-cell configuration was used to study the influence of the external concentration of potassium ions on the characteristics of the voltage-dependent potassium current in the plasma membrane of GH₃ cells (a cell line isolated from the rat pituitary body tumor). The [K⁺] _out shift from 5 to 100 mM induced a monotonic increase in potassium current with a constant difference between the testing potential and potassium equilibrium potential. The dependence of an activation time constant, _n , and a steady-state activation,n, of the potassium current on [K⁺] _out is of a distinct non-monotononic character with the extremum at 20 mM. Our experiments and theoretical speculations allow us to suppose that the interaction of potassium ions with slowly relaxing charged channel-forming protein groups induced the observed effects.Neirofiziologiya/Neurophysiology, Vol. 27, No. 2, pp. 110–115, March–April, 1995.     

Copper-induced intracellular calcium release requires extracellular calcium entry and activation of L-type voltage-dependent calcium channels in Ulva compressa

The marine alga Ulva compressa exposed to 10 µM copper showed a triphasic increase of intracellular calcium with maximal levels at 2, 3 and 12 h involving the activation of ryanodine-, Ins(1,4,5)P₃- and NAADP-sensitive calcium channels. In order to analyze the requirement of extracellular calcium entry for intracellular calcium release as well as the activation of voltage-dependent calcium channels (VDCC) and phospholipase C, U. compressa was treated with EGTA, a non-permeable calcium chelating agent, with verapamil, nipfedipine and diltiazem, inhibitors of L-type VDCC, and with neomycin and U731222, inhibitors of phospholipase C. The release of intracellular calcium was partially inhibited with EGTA at 2 and 3 h and completely inhibited at 12 h of copper exposure and decreased with inhibitors of L-type VDCC and phospholipase C. Thus, copper-induced intracellular calcium release depends on calcium entry and activation of L-type VDCC and phospholipase C. An integrative model of copper-induced cellular responses in U. compressa is presented.     

Homologous regulation of voltage-dependent calcium channels by 1,4-dihydropyridines

Chronic treatment of PC 12 cells with the 1,4-dihydropyridine Ca2+ channel antagonist nifedipine [5 x 10-8M/5 days] and the activator S Bay K 8644 [5 x 10-7 M/5 days] resulted in up- and down-regulation of 1,4-dihydropyridine binding site density by 29 and 24%, respectively, without change in affinity. These changes in binding site density represent functional changes as indicated by the corresponding changes in K+ depolarization-induced 45Ca2+ uptake and in whole cell currents carried by Ba2+ ions. This homologous regulation of voltage-dependent Ca2+ channels [VDCC] by potent and specific ligands parallels that observed for other classes of membrane receptors.     

Temperature-dependent modulation of CaV3 T-type calcium channels by protein kinases C and A in mammalian cells

Modulation of low voltage-activated Ca(V)3 T-type calcium channels remains poorly characterized compared with high voltage-activated Ca(V)1 and Ca(V)2 calcium channels. Notably, it is yet unresolved whether Ca(V)3 channels are modulated by protein kinases in mammalian cells. In this study, we demonstrate that protein kinase A (PKA) and PKC (but not PKG) activation induces a potent increase in Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3 currents in various mammalian cell lines. Notably, we show that protein kinase effects occur at physiological temperature ( approximately 30-37 degrees C) but not at room temperature ( approximately 22-27 degrees C). This temperature dependence could involve kinase translocation, which is impaired at room temperature. A similar temperature dependence was observed for PKC-mediated increase in high voltage-activated Ca(V)2.3 currents. We also report that neither Ca(V)3 surface expression nor T-current macroscopic properties are modified upon kinase activation. In addition, we provide evidence for the direct phosphorylation of Ca(V)3.2 channels by PKA in in vitro assays. Overall, our results clearly establish the role of PKA and PKC in the modulation of Ca(V)3 T-channels and further highlight the key role of the physiological temperature in the effects described.     

G protein-induced trafficking of voltage-dependent calcium channels

Calcium channels are well known targets for inhibition by G protein-coupled receptors, and multiple forms of inhibition have been described. Here we report a novel mechanism for G protein-mediated modulation of neuronal voltage-dependent calcium channels that involves the destabilization and subsequent removal of calcium channels from the plasma membrane. Imaging experiments in living sensory neurons show that, within seconds of receptor activation, calcium channels are cleared from the membrane and sequestered in clathrin-coated vesicles. Disruption of the L1-CAM-ankyrin B complex with the calcium channel mimics transmitter-induced trafficking of the channels, reduces calcium influx, and decreases exocytosis. Our results suggest that G protein-induced removal of plasma membrane calcium channels is a consequence of disrupting channel-cytoskeleton interactions and might represent a novel mechanism of presynaptic inhibition.     

Stimulation of calcium intake by growth-hormone-releasing-hormone in GH3 cells]

The effect of GH-RH in the intra-cytosolic free Ca2+ concentration was studied in GH3 cells. To this end, we have used microspectrofluorimetry performed on single cells. We show that 60% of cells respond to a brief application of 100 nM GH-RH by an increase of their [Ca2+]i (mean increase 100% over basal values). This response which is blocked by calcium channel inhibitors results from an increased influx of Ca2+ ions from the external medium.     

Barium ions enter chromaffin cells via voltage-dependent calcium channels and induce secretion by a mechanism independent of calcium

Barium ions enter chromaffin cells via voltage-sensitive calcium channels, although the intracellular site of barium action is distinct from that of calcium. The entry of barium primarily through voltage-sensitive channels was indicated by experiments showing inhibition of 133Ba2+ uptake by blockers of voltage-dependent calcium channels. In addition, 133Ba2+ uptake was stimulated by 50 mM KCl but not by nicotine. Furthermore, 133Ba2+ uptake was inhibited by hyperosmolarity, which specifically blocks the voltage-sensitive calcium channel but not the receptor-associated calcium channel. These conclusions from studies on barium uptake were also borne out by experiments measuring catecholamine secretion. Thus, blockers of voltage-dependent calcium channels which inhibited barium uptake also inhibited barium-induced catecholamine secretion. In other experiments, simultaneous stimulation with nicotine and barium in the presence of calcium evoked coincident and additive catecholamine secretion. By contrast, when 50 mM KCl was substituted for nicotine in the same experimental design, barium ions inhibited potassium-induced catecholamine secretion at low calcium concentrations. Only at high calcium concentrations were barium-induced and potassium-induced secretion additive. These data also indicate that barium and calcium compete at the voltage-sensitive pathway. Furthermore, these additivity data suggest that once inside the cell, barium and calcium have two distinct mechanisms of action. As predicted by this hypothesis, in digitonin-permeabilized chromaffin cells either calcium or barium stimulated catecholamine release, and in the presence of both cations catecholamine secretion was equivalent to the sum of secretion with either cation alone. Additional support of this concept comes from experiments showing that while calcium-mediated catecholamine secretion is sensitive to trifluoperazine and imipramine, barium-mediated secretion is not. Taken together, all these data indicate that there are two distinct intracellular sites of action for barium and calcium. In contrast to catecholamine secretion, non-exocytotic ascorbic acid secretion was induced by nicotine and potassium in the presence of calcium, but not by barium alone. These data provide additional evidence that barium acts by a different mechanism than calcium, in still another secretory system in chromaffin cells.