T-type Ca2+ channel lowers the threshold of spike generation in the newt olfactory receptor cell

Mechanisms underlying action potential generation in the newt olfactory receptor cell were investigated by using the whole-cell version of the patch-clamp technique. Isolated olfactory cells had a resting membrane potential of -70 +/- 9 mV. Injection of a depolarizing current step triggered action potentials under current clamp condition. The amplitude of the action potential was reduced by lowering external Na+ concentration. After a complete removal of Na+, however, cells still showed action potentials which was abolished either by Ca2+ removal or by an application of Ca2+ channel blocker (Co2+ or Ni2+), indicating an involvement of Ca2+ current in spike generation of newt olfactory receptor cells. Under the voltage clamp condition, depolarization of the cell to -40 mV from the holding voltage of -100 mV induced a fast transient inward current, which consisted of Na+ (INa) and T-type Ca2+ (ICa.T) currents. The amplitude of ICa,T was about one fourth of that of INa. Depolarization to more positive voltages also induced L-type Ca2+ current (ICa,L). ICa,L was as small as a few pA in normal Ringer solution. The activating voltage of ICa,T was approximately 10 mV more negative than that of INa. Under current clamp, action potentials generated by a least effective depolarization was almost completely blocked by 0.1 mM Ni2+ (a specific T-type Ca2+ channel blocker) even in the presence of Na+. These results suggest that ICa,T contributes to action potential in the newt olfactory receptor cell and lowers the threshold of spike generation.     

Generation of action potentials in a mathematical model of corticotrophs.

Corticotropin-releasing hormone (CRH) is an important regulator of adrenocorticotropin (ACTH) secretion from pituitary corticotroph cells. The intracellular signaling system that underlies this process involves modulation of voltage-sensitive Ca2+ channel activity, which leads to the generation of Ca2+ action potentials and influx of Ca2+. However, the mechanisms by which Ca2+ channel activity is modulated in corticotrophs are not currently known. We investigated this process in a Hodgkin-Huxley-type mathematical model of corticotroph plasma membrane electrical responses. We found that an increase in the L-type Ca2+ current was sufficient to generate action potentials from a previously resting state of the model. The increase in the L-type current could be elicited by either a shift in the voltage dependence of the current toward more negative potentials, or by an increase in the conductance of the current. Although either of these mechanisms is potentially responsible for the generation of action potentials, previous experimental evidence favors the former mechanism, with the magnitude of the shift required being consistent with the experimental findings. The model also shows that the T-type Ca2+ current plays a role in setting the excitability of the plasma membrane, but does not appear to contribute in a dynamic manner to action potential generation. Inhibition of a K+ conductance that is active at rest also affects the excitability of the plasma membrane.     

Frequency-dependent blockade of T-type Ca2+ current by efonidipine in cardiomyocytes

Efonidipine is a dihydropyridine Ca2+ antagonist with inhibitory effects on both L-type and T-type Ca2+ channels and potent bradycardiac activity especially in patients with high heart rate. In the present study, we examined the frequency dependence of efonidipine action on the T-type Ca2+ channel in isolated guinea-pig ventricular myocytes. The potency of efonidipine to inhibit the T-type Ca2+ current was higher under higher stimulation frequencies. The IC50 values were 1.3 x 10(-8), 2.0 x 10(-6) and 6.3 x 10(-6) M under stimulation frequencies of 1, 0.2 and 0.05 Hz, respectively. The reduction of T-type Ca2+ current amplitude was not accompanied by change in the time course of current decay. Efonidipine (10 microM) inhibited T-type Ca2+ current elicited by depolarization from holding potentials ranging from -90 to -30 mV by about 30%; the voltage-dependence of steady-state inactivation was not changed by the drug. Efonidipine slowed the recovery from inactivation following an inactivating prepulse. In conclusion, efonidipine was shown to have frequency-dependent inhibitory effects on the T-type Ca2+ channel, which could be explained by slow dissociation of the drug from the inactivated state of the channel.     

Inactivation properties of T-type calcium current in canine cardiac Purkinje cells.

The kinetic behavior of T-type Ca2+ current (ICa-T) was studied in canine cardiac Purkinje cells using a single suction-pipette whole-cell voltage clamp method. ICa-T was studied without contamination of conventional L-type Ca2+ current (ICa-L). Ca2+, Sr2+, or Ba2+ were used as the charge carrier. During maintained depolarization ICa-T decayed rapidly, and under most conditions the decay showed a voltage-dependent single exponential time course that did not depend on the species of charge carrier. The development of inactivation did not depend on Ca2+, but the time course required more than a single exponential process. Just negative to the threshold voltage for activating ICa-T, inactivation slowly developed and there was a delay in its onset. The time course of recovery from inactivation was dependent on the protocol used to measure it. As the duration of an inactivating voltage step was increased, recovery slowed markedly and there was a delay in its onset. The time course of recovery could be fit as a biexponential. The fast and slow time constants of recovery were relatively constant, however, the relative amplitudes were dependent on the duration of the inactivating voltage step. Recovery was not dependent on Ca2+, and it was slower at a less negative voltage. These results suggest that the T-type Ca2+ channel in cardiac Purkinje cells follows a complex kinetic scheme dependent only on voltage. This behavior can be accounted for by incorporating into a Markovian model several inactivated and closed states.     

Block of T-type Ca channels in guinea pig atrial cells by antiarrhythmic agents and Ca channel antagonists

Myocardial cells have two types of Ca channels commonly called T-type and L-type. Whole cell Ca channel currents in guinea pig atrial myocytes can be separated and quantitated by analyzing channel closing kinetics after a brief depolarization (tail current analysis). L-type Ca channels deactivate rapidly when the membrane is repolarized and T-type Ca channels deactivate relatively slowly. Ca channel block by the therapeutically useful Ca channel antagonists is voltage dependent, so it is desirable to study block of both channel types over an extended voltage range. Tail current analysis allows this and was used to study block of both types of Ca channels under identical conditions. Amiodarone, bepridil, and cinnarizine block T-type Ca channels more potently than L-type Ca channels when binding equilibrates at normal diastolic potentials (approximately -90 mV). None of these drugs is a selective blocker of T-type Ca channels because block of L-type Ca channels is enhanced when cells are almost completely depolarized. Although weak block of T-type Ca channels by 1,4-dihydropyridines has usually been reported, we found that felodipine blocks these channels with high affinity. When most T-type Ca channels are inactivated, the apparent dissociation constant (KI) is 13 nM. Felodipine also blocks T-type Ca channels in GH3 cells (a cell line derived from rat anterior pituitary), but KI = 700 nM. Thus, T-type Ca channels in different cell types are pharmacologically distinct. Felodipine can block L-type Ca channels in atrial cells more potently than T-type Ca channels, but block of L-type Ca channels is potent only at depolarized potentials; block of both channel types is comparable at normal diastolic membrane potentials. Felodipine and the 1,4-dihydropyridines isradipine and (-)-202-791 are approximately equipotent at blocking T-type Ca channels, but differ substantially in potency for block of L-type Ca channels. Block of T-type Ca channels may account for some of the pharmacological effects of 1,4-dihydropyridines and for the antiarrhythmic activity of amiodarone and bepridil.     

Membrane currents in taste cells of the rat fungiform papilla. Evidence for two types of Ca currents and inhibition of K currents by saccharin

Taste buds were isolated from the fungiform papilla of the rat tongue and the receptor cells (TRCs) were patch clamped. Seals were obtained on the basolateral membrane of 281 TRCs, protruding from the intact taste buds or isolated by micro-dissection. In whole-cell configuration 72% of the cells had a TTX blockable transient Na inward current (mean peak amplitude 0.74 nA). All cells had outward K currents. Their activation was slower than for the Na current and a slow inactivation was also noticeable. The K currents were blocked by tetraethylammonium, Ba, and 4-aminopyridine, and were absent when the pipette contained Cs instead of K. With 100 mM Ba or 100 mM Ca in the bath, two types of inward current were observed. An L-type Ca current (ICaL) activated at -20 mV had a mean peak amplitude of 440 pA and inactivated very slowly. At 3 mM Ca the activation threshold of ICaL was near -40 mV. A transient T-type current (ICaT) activated at -50 mV had an average peak amplitude of 53 pA and inactivated with a time constant of 36 ms at -30 mV. ICaL was blocked more efficiently by Cd and D600 than ICaT. ICaT was blocked by 0.2 mM Ni and half blocked by 200 microM amiloride. In whole-cell voltage clamp, Na-saccharin caused (in 34% of 55 cells tested) a decrease in outward K currents by 21%, which may be expected to depolarize the TRCs. Also, Na-saccharin caused some taste cells to fire action potentials (on-cell, 7 out of 24 cells; whole-cell, 2 out of 38 cells responding to saccharin) of amplitudes sufficient to activate ICaL. Thus the action potentials will cause Ca inflow, which may trigger release of transmitter.     

Ionic currents in cardiac myocytes of squid, Alloteuthis subulata

This paper provides the first study of voltage-sensitive membrane currents present in heart myocytes from cephalopods. Whole cell patch clamp recordings have revealed six different ionic currents in myocytes freshly dissociated from squid cardiac tissues (branchial and systemic hearts). Three types of outward potassium currents were identified: first, a transient outward voltage-activated A-current (I_A), blocked by 4-aminopyridine, and inactivated by holding the cells at a potential of −40 mV; second, an outward, voltage-activated, delayed rectifier current with a sustained time course (I_K); and third, an outward, calcium-dependent, potassium current (I_K(Ca)) sensitive to Co²⁺ and apamin, and with the characteristic N-shaped current voltage relationship. Three inward voltage-activated currents were also identified. First, a rapidly activating and inactivating, sodium current (I_Na), blocked by tetrodotoxin, inactivated at holding potentials more positive than −40 mV, and abolished when external sodium was replaced by choline. Second, an L-type calcium current (I_Ca,L) with a sustained time course, suppressed by nifedipine or Co²⁺, and enhanced by substituting Ca²⁺ for Ba²⁺ in the external medium. The third inward current was also carried by calcium ions, but could be distinguished from the L-type current by differences in its voltage dependence. It also had a more transient time course, was activated at more negative potentials, and resembled the previously described low-voltage-activated, T-type calcium current. Accepted: 24 September 1999     

Both T- and L-type Ca2+ channels can contribute to excitation-contraction coupling in cardiac Purkinje cells.

Although L-type Ca2+ channels have been shown to play a central role in cardiac excitation-contraction (E-C) coupling, little is known about the role of T-type Ca2+ channels in this process. We used the amphotericin B perforated patch method to study the possible role of T-type Ca2+ current in E-C coupling in isolated canine Purkinje myocytes where both Ca2+ currents are large. T-type Ca2+ current was separated from L-type Ca2+ current using protocols employing the different voltage dependencies of the channel types and their different sensitivities to pharmacological blockade. We showed that Ca2+ admitted through either T- or L-type Ca2+ channels is capable of initiating contraction and that the contractions depended on Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR). The contractions, however, had different properties. Those initiated by Ca2+ entry through T-type Ca2+ channels had a longer delay to the onset of shortening, slower rates of shortening and relaxation, lower peak shortening, and longer time to peak shortening. These differences were present even when L-type Ca2+ current amplitude, or charge entry, was less than that of T-type Ca2+ current, suggesting that Ca2+ entry through the T-type Ca2+ channel is a less effective signal transduction mechanism to the SR than is Ca2+ entry through the L-type Ca2+ channel. We conclude that under our experimental conditions in cardiac Purkinje cells Ca2+ entry through the T-type Ca2+ channel can activate cell contraction. However, Ca2+ entry through the L-type Ca2+ channel is a more effective signal transduction mechanism. Our findings support the concept that different structural relationships exist between these channel types and the SR Ca2+ release mechanism.     

Cyclic AMP-independent, dual regulation of voltage-dependent Ca2+ currents by LHRH and somatostatin in a pituitary cell line.

Voltage-dependent Ca2+ currents appear to be involved in the actions of hormones that regulate pituitary secretion. In order to investigate modulation of Ca2+ currents by release-inducing and release-inhibiting hormones, we performed whole-cell clamp experiments in the pituitary cell line GH3. The resting potential was approximately -40 mV; spontaneous action potentials were observed in the majority of cells. Superfusion of cells with the stimulatory hormone, LHRH, depolarized the plasma membrane to approximately -10 mV, whereas the inhibitory hormone, somatostatin, caused hyperpolarization to approximately -60 mV; both hormones suppressed spontaneous action potentials. Under voltage clamp conditions, GH3 cells exhibited slowly and fast inactivating Ca2+ currents. LHRH increased whereas somatostatin decreased the slowly inactivating currents; fast inactivating currents were not affected by these hormones. The stimulatory effect of LHRH was not mimicked by intracellularly applied cAMP. In contrast to vasoactive intestinal peptide and forskolin, LHRH did not activate adenylate cyclase in membranes of GH3 cells, but rather appeared to cause inhibition of the enzyme. Hormonal stimulation and inhibition of inward currents were abolished by pretreatment of the cells with pertussis toxin. In membranes of GH3 cells, we identified a pertussis toxin-sensitive G-protein of the Gi-type and Go. We conclude that LHRH and somatostatin modulate voltage-dependent Ca2+ currents via cAMP-independent mechanisms involving pertussis toxin-sensitive G-proteins. The occurrence of both pertussis toxin-sensitive hormonal stimulation and inhibition of voltage-dependent Ca2+ currents in one cell type suggest that these opposite regulations are mediated by distinct G-proteins.     

Functional significance of voltage-dependent conductances in Limulus ventral photoreceptors

The influence of voltage-dependent conductances on the receptor potential of Limulus ventral photoreceptors was investigated. During prolonged, bright illumination, the receptor potential consists of an initial transient phase followed by a smaller plateau phase. Generally, a spike appears on the rising edge of the transient phase, and often a dip occurs between the transient and plateau. Block of the rapidly inactivating outward current, iA, by 4-aminopyridine eliminates the dip under some conditions. Block of maintained outward current by internal tetraethylammonium increases the height of the plateau phase, but does not eliminate the dip. Block of the voltage-dependent Na+ and Ca2+ current by external Ni2+ eliminates the spike. The voltage-dependent Ca2+ conductance also influences the sensitivity of the photoreceptor to light as indicated by the following evidence: depolarizing voltage- clamp pulses reduce sensitivity to light. This reduction is blocked by removal of external Ca2+ or by block of inward Ca2+ current with Ni2+. The reduction of sensitivity depends on the amplitude of the pulse, reaching a maximum at or approximately +15 mV. The voltage dependence is consistent with the hypothesis that the desensitization results from passive Ca2+ entry through a voltage-dependent conductance.     

L-type Ca2+ currents in ascidian eggs.

We have studied Ca2+ currents in ascidian eggs using the whole-cell clamp technique. T and L components, as observed in somatic cells, are present and the L-type current predominates. Since the IV relationship for these inward currents overlap at -30 mV, separation of the two components using different voltage regimes is not feasible. Increasing external Ca2+ results in larger currents. The L-type current decreases in a dose-dependent fashion in the presence of Mn2+ and Nifedipine, while the T-type current is inhibited in Ni2+. When Ba2+ was used as the carrier ion, channel kinetics and conductance were completely altered. Considering the density and kinetics of L-type channels in unfertilized eggs it is probable they play an important role in regulating cytosolic Ca2+ during early developmental processes.     

Spontaneous electrical and calcium oscillations in unstimulated pituitary gonadotrophs.

Single pituitary cells often fire spontaneous action potentials (APs), which are believed to underlie spiking fluctuations in cytosolic calcium concentration ([Ca2+]i). To address how these basal [Ca2+]i fluctuations depend on changes in plasma membrane voltage (V), simultaneous measurements of V and [Ca2+]i were performed in rat pituitary gonadotrophs. The data show that each [Ca2+]i spike is produced by the Ca2+ entry during a single AP. Using these and previously obtained patch-clamp data, we develop a quantitative mathematical model of this plasma membrane oscillator and the accompanying spatiotemporal [Ca2+]i oscillations. The model demonstrates that AP-induced [Ca2+]i spiking is prominent only in a thin shell layer neighboring the cell surface. This localized [Ca2+]i spike transiently activates the Ca2(+)- dependent K+ current resulting in a sharp afterhyperpolarization following each voltage spike. In accord with experimental observations, the model shows that the frequency and amplitude of the voltage spikes are highly sensitive to current injection and to the blocking of the Ca(2+)-sensitive current. Computations also predict that leaving the membrane channels intact, the firing rate can be modified by changing the Ca2+ handling parameters: the Ca2+ diffusion rate, the Ca2+ buffering capacity, and the plasma membrane Ca2+ pump rate. Finally, the model suggests reasons that spontaneous APs were seen in some gonadotrophs but not in others. This model provides a basis for further exploring how plasma membrane electrical activity is involved in the control of cytosolic calcium level in unstimulated as well as agonist-stimulated gonadotrophs.     

Overexpression of human beta2-adrenergic receptors increases gain of excitation-contraction coupling in mouse ventricular myocytes

This study investigated cardiac excitation-contraction coupling at 37 degrees C in transgenic mice with cardiac-specific overexpression of human beta2-adrenergic receptors (TG4 mice). In field-stimulated myocytes, contraction was significantly greater in TG4 compared with wild-type (WT) ventricular myocytes. In contrast, when duration of depolarization was controlled with rectangular voltage clamp steps, contraction amplitudes initiated by test steps were the same in WT and TG4 myocytes. When cells were voltage clamped with action potentials simulating TG4 and WT action potential configurations, contractions were greater with long TG4 action potentials and smaller with shorter WT action potentials, which suggests an important role for action potential configuration. Interestingly, peak amplitude of L-type Ca2+ current (I(Ca-L)) initiated by rectangular test steps was reduced, although the voltage dependencies of contractions and currents were not altered. To explore the basis for the altered relation between contraction and I(Ca-L), Ca2+ concentrations were measured in myocytes loaded with fura 2. Diastolic concentrations of free Ca2+ and amplitudes of Ca2+ transients were similar in voltage-clamped myocytes from WT and TG4 mice. However, sarcoplasmic reticulum (SR) Ca2+ content assessed with the rapid application of caffeine was elevated in TG4 cells. Increased SR Ca2+ was accompanied by increased frequency and amplitudes of spontaneous Ca2+ sparks measured at 37 degrees C with fluo 3. These observations suggest that the gain of Ca(2+)-induced Ca2+ release is increased in TG4 myocytes. Increased gain counteracts the effects of decreased amplitude of I(Ca-L) in voltage-clamped myocytes and likely contributes to increased contraction amplitudes in field-stimulated TG4 myocytes.     

Control of action potential-driven calcium influx in GT1 neurons by the activation status of sodium and calcium channels

An analysis of the relationship between electrical membrane activity and Ca2+ influx in differentiated GnRH-secreting (GT1) neurons revealed that most cells exhibited spontaneous, extracellular Ca(2+)-dependent action potentials (APs). Spiking was initiated by a slow pacemaker depolarization from a baseline potential between -75 and -50 mV, and AP frequency increased with membrane depolarization. More hyperpolarized cells fired sharp APs with limited capacity to promote Ca2+ influx, whereas more depolarized cells fired broad APs with enhanced capacity for Ca2+ influx. Characterization of the inward currents in GT1 cells revealed the presence of tetrodotoxin-sensitive Na+, Ni(2+)-sensitive T-type Ca2+, and dihydropyridine-sensitive L-type Ca2+ components. The availability of Na+ and T-type Ca2+ channels was dependent on the baseline potential, which determined the activation/inactivation status of these channels. Whereas all three channels were involved in the generation of sharp APs, L-type channels were solely responsible for the spike depolarization in cells exhibiting broad APs. Activation of GnRH receptors led to biphasic changes in cytosolic Ca2+ concentration ([Ca2+]i), with an early, extracellular Ca(2+)-independent peak and a sustained, extracellular Ca(2+)-dependent phase. During the peak [Ca2+]i response, electrical activity was abolished due to transient hyperpolarization. This was followed by sustained depolarization of cells and resumption of firing of increased frequency with a shift from sharp to broad APs. The GnRH-induced change in firing pattern accounted for about 50% of the elevated Ca2+ influx, the remainder being independent of spiking. Basal [Ca2+]i was also dependent on Ca2+ influx through AP-driven and voltage-insensitive pathways. Thus, in both resting and agonist-stimulated GT1 cells, membrane depolarization limits the participation of Na+ and T-type channels in firing, but facilitates AP-driven Ca2+ influx.     

Autocrine regulation of calcium influx and gonadotropin-releasing hormone secretion in hypothalamic neurons.

Gonadotropin-releasing hormone (GnRH) receptors are expressed in hypothalamic tissues from adult rats, cultured fetal hypothalamic cells, and immortalized GnRH-secreting neurons (GT1 cells). Their activation by GnRH agonists leads to an overall increase in the extracellular Ca2+-dependent pulsatile release of GnRH. Electrophysiological studies showed that GT1 cells exhibit spontaneous, extracellular Ca2+-dependent action potentials, and that their inward currents include Na+, T-type and L-type Ca2+ components. Several types of potassium channels, including apamin-sensitive Ca2+-controlled potassium (SK) channels, are also expressed in GT1 cells. Activation of GnRH receptors leads to biphasic changes in intracellular Ca2+ concentration ([Ca2+]i), with an early and extracellular Ca2+-independent peak and a sustained and extracellular Ca2+-dependent plateau phase. During the peak [Ca2+]i response, electrical activity is abolished due to transient hyperpolarization that is mediated by SK channels. This is followed by sustained depolarization and resumption of firing with increased spike frequency and duration. The agonist-induced depolarization and increased firing are independent of [Ca2+]i and are not mediated by inhibition of K+ currents, but by facilitation of a voltage-insensitive and store depletion-activated Ca2+-conducting inward current. The dual control of pacemaker activity by SK and store depletion-activated Ca2+ channels facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process accounts for the autoregulatory action of GnRH on its release from hypothalamic neurons.     

CRH-induced electrical activity and calcium signalling in pituitary corticotrophs

Pituitary corticotroph cells generate repetitive action potentials and associated Ca2+ transients in response to the agonist corticotropin releasing hormone (CRH). There is indirect evidence suggesting that the agonist, by way of complex intracellular mechanisms, modulates the voltage sensitivity of the L-type Ca2+ channels embedded in the plasma membrane. We have previously constructed a Hodgkin-Huxley-type model of this process, which indicated that an increase in the L-type Ca2+ current is sufficient to generate repetitive action potentials (LeBeau et al. (1997). Biophys. J.73, 1263-1275). CRH is also believed to inhibit an inwardly rectifying K+ current. In this paper, we have found that a CRH-induced inhibition of the inwardly rectifying K+ current increases the model action potential firing frequency, [Ca2+]i transients and membrane excitability. This dual modulatory action of CRH on inward rectifier and voltage-gated Ca2+ channels better describes the observed CRH-induced effects. This structural alteration to the model along with parameter changes bring the model firing frequency in line with experimental data. We also show that the model exhibits experimentally observed bursting behaviour, where the depolarization spike is followed by small oscillations in the membrane potential.     

The role of L- and T-type Ca2+ currents during the in vitro aging of murine myogenic (i28) cells in culture

The age-related decline in skeletal muscle strength could, in part, result from alterations in the mechanism of excitation-contraction coupling, responsible for muscle contraction. In the present work, we used the in vitro aging of murine myogenic (i28) cells as a model, to investigate whether the inefficiency of aged satellite cells to generate functional skeletal muscle fibres could be partly due to defective voltage-dependent Ca2+ currents. The whole-cell patch clamp technique was employed to measure L- and T-type Ca2+ currents in myotubes derived from the differentiation and fusion of these cells reaching replicative senescence. Our data showed that the expression and the amplitude of these currents decreased significantly during in vitro aging. Moreover, the analysis of the L-type current evoked in young and old cells by positive voltage steps, revealed no differences in the kinetics of activation, but significant alterations in the rate of inactivation. These effects of in vitro aging on voltage-dependent Ca2+ currents could also be related to their inability to fuse into myotubes. Taken together, our data support the hypothesis that age-related effects on voltage-dependent L- and T-type currents could be one of the causes of the failure of satellite cells to efficiently counteract the impairment in muscle force.     

A slowly inactivating calcium current works as a calcium sensor in calcitonin-secreting cells.

Calcitonin (CT)-secreting cells (C-cells) are remarkably sensitive to changes in the extracellular Ca2+ concentration. In order to detect the mechanism by which C-cells monitor Ca2+, we compared a C-cell line responding to Ca2+ (rMTC cells) with another one known to have a defect in this Ca2+ signal transduction (TT cells). Rises of the Ca2+ concentration caused rMTC cells to depolarize and/or elicited spontaneous action potentials. Under voltage-clamp conditions, rMTC cells showed a slowly decaying Ca2+ inward current which was sensitive to dihydropyridines but not to Ni2+ at a low concentration. In contrast, the 'defective' TT cells neither depolarized nor fired action potentials with high Ca2+; they only exhibited an Ni2(+)-sensitive, transient Ca2+ current. The data strongly suggest that the slowly inactivating Ca2+ current is a prerequisite for Ca2(+)-sensitivity of C-cells and that fast inactivating channels are not sufficient to act as sensors of the extracellular Ca2+ concentration.     

Voltage-activated Ca2+ currents in insulin-secreting cells

Membrane voltage and voltage-clamped membrane currents have been investigated with the whole-cell patch clamp method in the insulin-secreting cell line RINm5F. The mean resting membrane potential of RINm5F cells was found to be -52 mV. Overshooting spike potentials could be evoked by depolarising voltage steps in the absence of a secretagogue. Inward membrane currents evoked by depolarising voltage steps were dependent upon extracellular Ca2+ and blocked by Co2+, nifedipine and verapamil. Outward membrane currents which were evoked by depolarising voltage steps to positive membrane potentials were reduced when Ca2+ entry was prevented. It is concluded that the voltage-activated Ca2+ currents underlie the voltage-activated spike potentials recorded from insulin-secreting cells.     

Sodium and calcium currents in action potentials of rat somatotrophs: their possible functions in growth hormone secretion

We report that both Na+ and Ca2+ currents are involved in the action potentials and in the hormone release from rat somatotrophs in primary culture. Single somatotrophs were identified by reverse hemolytic plaque assay (RHPA) and transmembrane voltage and currents were recorded using the whole-cell mode of the patch-clamp technique. Somatotrophs displayed a mean resting potential of -80mV and an average input resistance of 5.7G omega. Most of the cells showed spontaneous or evoked action potentials. Single action potentials or the initial spike in a burst were characterized by their high amplitude and short duration. Tetrodotoxin (TTX, 1 microM) blocked single action potentials and the initial spikes in a burst, whereas action potentials of long duration and low amplitude persisted. Cobalt (2 mM) plus TTX (1 microM) blocked all the action potentials. Voltage-clamp experiments confirmed the presence of both a TTX-sensitive Na+ current and Co2(+)-sensitive Ca2+ currents. TTX or Na(+)-free medium slightly decreased the basal release of GH but did not markedly modify hGRF-stimulated GH release. However, Co2+ (2 mM), which partially decreased the basal release, totally blocked hGRF-stimulated release. We conclude that (1) Na+ currents which initiate rapid action potentials may participate in spontaneous GH release; (2) Ca2+ currents, which give rise to long duration action potentials and membrane voltage fluctuation, are probably involved in both basal and hGRF-stimulated GH releases.