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PubMed1992

Calcium channels in embryonic chick skeletal muscle cells after cultivation with calcium channel blocker.

The effects of chronic treatment with calcium channel blockers were studied on the expression of voltage-dependent calcium channels (VDCCs) in chick skeletal muscle cells developing in culture. Myotubes were treated after 2 days in culture with either 20 microM D600 or 10 microM nifedipine, and measurements were made of the maximum rate of rise (M.R.R.) of the two components of action potential, operated by T- and L-type VDCCs, respectively. Treatment with either blocker reduced the M.R.R. of the action potential component operated by the L-type VDCC throughout the culture period examined. The M.R.R. of the T-type VDCC component, on the other hand, was unaffected by either treatment. The reduction in the M.R.R. of the L-type component in blocker-treated cells is thought to be due to the down-regulation of the expression of L-type VDCC. Thus, it appears that the expression of L-type VDCC in the chick skeletal muscle cells can be regulated by a function of L-type VDCC, which mediate the entry of Ca2+ into the cells. The physiological significance of the L-type VDCC, which expressed prominently early in the development of skeletal muscle cells, for the differentiation of excitability is discussed.

Action Potentials↗

PubMed1992

Whole-cell currents in two subpopulations of cultured rat petrosal neurons with different tetrodotoxin sensitivities.

In this study we use whole-cell recording to characterize at least two distinct populations of cultured neurons from perinatal rat petrosal or petrosal/jugular ganglia based on differential sensitivity of the transient inward Na+ current to tetrodotoxin. These ganglia supply chemoreceptor and baroreceptor afferents which mediate several cardiovascular reflexes. Approximately 50% of the neurons sampled had Na+ currents that were virtually unaffected by bath addition of tetrodotoxin (0.5-2.0 microM) but were abolished by choline substitution for external Na+. The majority of the remaining neurons had Na+ currents that were rapidly and reversibly blocked by 500 nM tetrodotoxin. A few cells had both tetrodotoxin-resistant and tetrodotoxin-sensitive Na+ currents. All neurons had similar voltage-activated Ca2+ and K+ currents. The inward Ca2+ current had no obvious fast transient or T-type component and appeared to be due mainly to the presence of long-lasting L-type Ca2+ channels. The outward currents consisted largely of a delayed rectifying K+ current (IKdr) and a Ca(2+)-activated K+ current (IKca), but no obvious fast transient K+ current (IA) was observed. Exposure to a chemosensory stimulus, hypoxia (PO2 approximately 20 Torr), had no effect on these neurons, in contrast to the pronounced decrease in K+ current it produces in cultured glomus cells, the presumed chemoreceptors and normal targets for a subset of petrosal neurons in vivo. Current-clamp recordings indicated that some neurons gave single spikes while others gave multiple spikes in response to long-depolarizing stimuli. No correlation between spiking behaviour and tetrodotoxin-sensitivity was observed. Thus, cultures enriched in petrosal neurons contain subpopulations with differential sensitivities to tetrodotoxin. Since many of these neurons innervate a single chemosensory target organ, the carotid body, it is of interest to know whether one or both subtypes can form functional synapses with glomus cells of the carotid body and mediate a chemoreceptor reflex.

Animals↗

PubMed1992

P-type calcium channels in rat central and peripheral neurons.

The peptide toxin omega-Aga-IVA blocked P-type Ca2+ channel current in rat Purkinje neurons (KD approximately 2 nM) but had no effect on identified T-type, L-type, or N-type currents in a variety of central and peripheral neurons. omega-Aga-IVA blocked a substantial fraction of high threshold Ca2+ channel current in neurons from the hippocampal CA1 region (mean 26%), visual cortex (32%), spinal cord (45%), and dorsal root ganglia (23%), but less in hippocampal CA3 neurons (14%) and none in sympathetic neurons. In all cases, omega-Aga-IVA block could be reversed by a brief train of strong depolarizations. There was no overlap between current blocked by omega-Aga-IVA and the fractions blocked by dihydropyridines and omega-conotoxin GVIA, but not all current resistant to dihydropyridines and omega-conotoxin was blocked by omega-Aga-IVA. The results suggest that omega-Aga-IVA is highly selective for P-type channels and that many central neurons and some peripheral neurons possess substantial P-type current.

Animals↗

PubMed1992

Sustained and transient calcium currents in horizontal cells of the white bass retina.

Calcium currents were recorded from cultured horizontal cells (HCs) isolated from adult white bass retinas, using the whole-cell patch-clamp technique. Ca2+ currents were enhanced using 10 mM extracellular Ca2+, while Na+ and K+ currents were pharmacologically suppressed. Two components of the Ca2+ current, one transient, the other sustained, were found. The large transient component of the Ca2+ current, which has not been seen before in HCs, is similar, but not identical, to the T-type Ca2+ current described previously in a variety of preparations. The sustained component of the Ca2+ current is similar, but not identical, to the L-type current described in other preparations. FTX, a factor isolated from the venom of the funnel-web spider, Agelenopsis aperta, preferentially and irreversibly blocks the sustained component of the Ca2+ current at very dilute concentrations. The sustained component of the Ca2+ current inactivates slowly, over the course of 15-60 s, in some HCs. This inactivation of the sustained Ca2+ current, when present, is primarily voltage dependent rather than Ca2+ dependent.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1992

Effects of intracellular ATP on calcium current in freshly dispersed single cells of guinea-pig portal vein.

In smooth muscle cells freshly dispersed from the guinea-pig portal vein, inward currents were recorded with the whole-cell clamp method in the presence of 2.4 mM Ca2+ under the condition in which outward K+ currents were suppressed and the effects of ATP in patch pipettes were studied. There was a large variation of the rate of current decay, the half-decay time ranging from 20 to 80 ms, but it was difficult to demonstrate clearly the presence of fast (T-type) and slow (L-type) Ca2+ channels based on threshold, the effects of changing holding potential, and of nicardipine, a dihydropyridine Ca2+ channel blocker. Intrapipette ATP had a weak potentiating effect on inward current, but did not seem to influence the correlation between peak current amplitude and current configuration. Cyanide (0.5 mM) reduced inward currents, particularly when ATP concentration was low and only limited recovery was observed. It was concluded that the amplitude and the rate of current decay of inward currents are not only determined by intracellular ATP, but also significantly influenced by some other factor, such as that involved in 'run-down' of Ca2+ currents.

Action Potentials↗

PubMed1992

Ionic currents in cultured rat hypothalamic neurones.

1. Dissociated neurones from embryonic rat hypothalamus were grown for several weeks in culture where they formed complex networks. These synaptically coupled networks were capable of generating synchronized bursting activity. Voltage-activated membrane currents were studied in these neurones using a patch clamp in the whole-cell configuration. 2. Outward currents were carried by K+ ions and consisted of an inactivating and a non-inactivating component. These components were similar to the transient K+ current (IA) and the delayed rectifier current (IK) reported in neurones from the postnatal rat hypothalamus. Application of Zn2+ (1 mM) blocked the transient component completely while reducing the non-inactivating component by only approximately 20%. 3. Inward currents were carried by Na+ and Ca2+ ions. Rapidly activating transient Na+ currents were activated at approximately -25 mV. TTX entirely blocked these currents at low concentration (300 nM). Voltage sensitivity of the Na+ conductance was 5.8 mV per e-fold change with half-maximal activation occurring at -8 mV. Na+ current kinetics could be well described by the Hodgkin-Huxley model (m3h). 4. With depolarizing pulses from a holding potential of -80 mV two Ca2+ current components with different ranges of activation were identified. Low voltage-activated (LVA, T-type) Ca2+ currents were activated at approximately -50 mV. High voltage-activated (HVA; also called L- or N-type) Ca2+ currents were observed at membrane potentials more positive to approximately -30 mV. LVA Ca2+ currents were observed in hypothalamic neurones that had developed a network of dendritic processes in the course of several weeks in culture. Activation and inactivation time constants of LVA Ca2+ currents were 15-25 ms and 30-100 ms (-30 to -45 mV). In contrast to HVA Ca2+ currents, no LVA Ca2+ currents were seen in neuronal somata obtained from the network cultures by mechanical dissociation. This suggests that most of the LVA Ca2+ channels are located on the dendritic tree rather than on the soma membrane. 5. HVA Ca2+ currents were maximal between 0 and +10 mV (external [Ca2+] = 5 mM). The time-to-peak was in the range of 1.7-5.4 ms (+30 to -10 mV). Tail currents following repolarization decayed monoexponentially with a time constant of approximately 210 microseconds. During 500 ms depolarizations, 90% of the current inactivated. The time course of inactivation showed two time constants of approximately 40 and approximately 700 ms.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Dopamine D2 receptor stimulation differentially affects voltage-activated calcium channels in rat pituitary melanotropic cells.

1. Whole-cell voltage clamp recordings were made from 141 rat pituitary melanotropic cells in short-term, serum-free, primary culture. The effects of the dopamine D2 receptor agonist, LY 171555, on sodium, potassium and barium currents were investigated. 2. Application of 1 microM-LY 171555 did not affect the inward sodium and outward potassium currents. 3. Application of LY 171555 reversibly inhibited barium currents, with the strongest inhibition on the early inward current. The effect was dose dependent (IC50 = 4 x 10(-8) M), maximal inhibition of the total current was 30% and the LY 171555-induced block (1 microM) was reversibly antagonized by (+/-)sulpiride (4 microM). 4. Using barium-selective saline solutions, different types of barium current (T, N, and two L components) were identified on the basis of their voltage-dependent kinetics. Their relative amplitudes differed between cells. 5. The T-type current activated at potentials positive to -60 mV, reaching peak amplitude between -20 and -10 mV. At -30 mV, this current was inhibited up to 30% by 1 microM-LY 171555. The time constants of activation (10-3 ms) and inactivation (50-20 ms) as well as the voltage dependence of inactivation (potential of half-maximal inactivation (H), -61 mV; slope factor (S), 4.9 mV) were not affected by LY 171555 application. 6. A rapidly inactivating (time constants 100-50 ms), high threshold current component was identified as an N-type current. This current activated at command potentials positive to -30 mV and reached a maximal amplitude at +10 mV. The steady-state inactivation was described by a single Boltzmann equation with H = -65 mV and S = 11.7 mV. Application of 1 microM-LY 171555 completely suppressed this current. 7. The slowly inactivating (time constants > 1500 ms), high-threshold, L-type current displayed the same voltage dependence of activation as the N current. The voltage dependence of inactivation was modelled by the sum of two Boltzmann equations (L1: H1 = -45 mV, S1 = 13.0 mV; L2:H2 = -11 mV, S2 = 6.0 mV), indicating the existence of two L channel populations. Neither time course, nor voltage dependence of inactivation were influenced by LY 171555. However, LY 171555 induced a slow-down in the time course of activation, which necessitated the use of two time constants to model the activation kinetics. One of these (approximately 2 ms) was also observed under control conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Novel isoform of Ca2+ channel in rat fetal cardiomyocytes.

1. Single cardiomyocytes of 18-day-old rat fetuses were isolated to characterize the cardiac Ca2+ channels in the fetal period, using whole-cell voltage clamp (Na+, K(+)-free external solution and K(+)-free internal solution), and depolarizing test pulses from a holding potential (HP) of -87 mV were applied. 2. The Ca2+ current was completely blocked by 2 mM-CO2+, but not completely blocked by the dihydropyridine (DHP) Ca2+ antagonist nifedipine. Nifedipine (3 microM) decreased the amplitude of the current (at -7 mV) by 65.9 +/- 3.4% (n = 20). At a HP of -47 mV, nifedipine decreased the Ca2+ current to about the same degree. Diltiazem (1 microM) did not block the nifedipine-resistant current which remained. 3. Nitrendipine, another DHP Ca2+ antagonist, had effects on the Ca2+ current similar to those of nifedipine. 4. The DHP-resistant current was not blocked by T-type channel blockers (Ni2+, tetramethrine) or an N-type blocker (omega-conotoxin). 5. In conclusion, rat fetal cardiomyocytes may have a unique type of Ca2+ channel (ICa(fe)), which decreases in amplitude and becomes less prominent during subsequent development.

Animals↗

PubMed1992

Postnatal changes in T-type calcium current density in rat atrial myocytes.

1. Postnatal changes in Ca2+ current were studied in voltage clamped atrial myocytes isolated from Sprague-Dawley rats. T- and L-type Ca2+ currents were identified using standard electrophysiological and pharmacological techniques. Cells were studied from seven groups of male and six groups of female rats ranging in age from 3 to 14 weeks. 2. The density of atrial T-type Ca2+ current showed significant variation during postnatal development, with a maximum density reached at 4.5-5 weeks. At this age, T-current density was 1.44 +/- 0.11 pA/pF (n = 23) for cells isolated from male and 1.25 +/- 0.09 pA/pF (n = 25) for cells isolated from female animals in bathing solutions containing 2 mM-Ca2+. T-current density in atrial cells isolated from younger animals (3.5 weeks postnatal) averaged 1.22 +/- 0.06 (n = 18) and 1.00 +/- 0.05 pA/pF (n = 22) or 85 and 80% of the maximum seen at 4.5-5 weeks for male and female rats, respectively. For rats older than 13 weeks, the average T-current density in atrial cells was 0.50 +/- 0.03 (n = 18) and 0.51 +/- 0.02 pA/pF (n = 35) or 35 and 41% of the maximum seen at 4.5-5 weeks for male and female rats, respectively. 3. In contrast to the T-type current, the density of atrial L-type Ca2+ current remained unchanged in rats from 3 to 14 weeks old. L-type current averaged 8.2 +/- 0.2 (n = 134) in male and 7.9 +/- 0.2 pA/pF (n = 102) in female rats. 4. Fluctuation analysis was used to estimate single T-channel current levels in 4.5- and 7.5-week-old male rats. While the T-current density differed by 70% at these two postnatal ages, no significant difference (P > 0.2) in single channel current was found. Single channel current was 0.12 +/- 0.01 pA (n = 9) for cells from 4.5-week-old and 0.13 +/- 0.01 pA (n = 7) for cells from 7.5-week-old rats. Currents were stimulated by test pulses from -80 to -30 mV at 5 mM-Ca2+. 5. No postnatal changes were seen in either the kinetics of activation or inactivation of macroscopic T-current.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Macroscopic and unitary properties of physiological ion flux through L-type Ca2+ channels in guinea-pig heart cells.

1. We investigated the currents through L-type Ca2+ channels when Ca2+ (1-10 mM) was the charge carrier, as is the case physiologically. 2. Na+ was removed from both the external and internal solutions to eliminate currents through Na+ channels and Na(+)-Ca2+ exchange. 3. From a holding potential of -50 mV only L-type channels were available to open with depolarization. Macroscopic L-type currents were maximal during depolarizing pulses to +10 mV (peak current density of 4.7 +/- 0.3 nA nF-1). 4. During depolarizing steps as long as 180 ms, the decay of current through L-type channels was incomplete, in contrast to that of T-type current. 5. Unitary currents recorded with comparable ionic conditions and voltage protocols exhibited a single-channel conductance of 6.9 pS in 10 mM Ca2+. Ensemble average currents reproduced accurately the features of whole-cell L-type current, including the maintained component. 6. Convolution analysis was employed to clarify the single-channel basis of the complex current waveform of L-type channels. First openings underlie the peak, while the maintained pedestal is generated by multiple re-openings. As with T-type channels, single openings are brief and contribute little to the time course of the average current. 7. The prominent maintained component of macroscopic and ensemble average L-type current cannot be explained by simple Markov models in which current decay reflects the progressive entry of channels into an absorbing inactivated state. 8. We considered the possibility that the maintained component of current arises from the existence of multiple distinct gating patterns, one of which lacks inactivation. Individual sweeps were sorted among three patterns of gating (no openings, active-early and active-late). Patterns of activity are not randomly distributed; instead, they tend to cluster over time. 9. Most of the maintained current is attributable to the 'active-late' pattern of gating. Considered separately, this pattern can be well described by a simple Markov chain lacking an inactivated state. The 'active-early' gating pattern accounts entirely for the initial current transient, and for about one-third of the maintained component; thus, inactivation, even when present, must be reversible rather than absorbing. 10. The unitary current amplitudes and peak open probabilities measured for single L-type channels, when compared to the average macroscopic L-type current density, predict 170 functional channels per picofarad, or 28,000 L-type channels per typical ventricular myocyte.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Ca2+ currents in single myocytes from human mesenteric arteries: evidence for a physiological role of L-type channels.

1. Voltage-gated Ca2+ currents (ICa) in isolated human mesenteric arterial cells were characterized in solutions containing normal (1.5 mM) Ca2+ and elevated concentrations of divalent cations using the conventional whole-cell patch clamp technique. 2. In normal Ca2+ solution, depolarization beyond -40 mV elicited a slowly decaying ICa which reached a maximum at +10 mV and appeared to reverse between +40 and +50 mV. The amplitude of this current in a group of cells correlated with cell membrane capacitance. 3. In two of thirty-three cells a small transient component of inward current was detected in the voltage range between -40 and -10 mV when cells were held at -80 mV. This current was abolished at a holding potential of -40 mV, while the current at 10 mV was not affected. These currents were referred to as T- and L-type Ca2+ respectively. 4. Elevation of the extracellular Ca2+ concentration to 20 mM shifted the voltage dependencies of Ca2+ current activation and inactivation by approximately +20 mV; a small T-current component was then observed in seven of nine cells held at -60 mV. 5. Replacement of 1.5 mM Ca2+ with 10 mM Ba2+ increased the amplitude of the current elicited at +10 mV by a factor of 3.7 and a small barium current (IBa) through T-type Ca2+ channels was also observed in most cells studied. Activation and steady-state inactivation curves for L-type current were found to be almost identical in both solutions. The steady-state inactivation for the T-type IBa was, however, more than 30 mV more negative (half-inactivation potential of -62.6 mV) of that for L-current in 1.5 mM Ca2+ and 10 mM Ba2+ solutions (-30.4 and -24.9 mV respectively). 6. A sustained inward Ca2+ channel current was recorded in the presence of normal Ca2+ and high divalent cation concentrations during 30 s depolarizations. The amplitude of this sustained current was found to be similar to the theoretical 'window current' predicted by the overlap of the activation and inactivation functions in these solutions. 7. Examination of the inactivation of the L-type current using a two-pulse protocol with a 240 ms prepulse revealed a U-shaped potential dependency for ICa, but not for IBa, suggesting the presence of a Ca(2+)-dependent component of the inactivation process. 8. These cells resemble other arterial smooth muscle cells previously studied in that they demonstrate both T- and L-components of ICa.(ABSTRACT TRUNCATED AT 400 WORDS)

Barium↗

PubMed1992

Voltage-dependent ionic currents in dissociated paratracheal ganglion cells of the rat.

1. Conventional whole-cell voltage-clamp technique was used to study the electrophysiological and pharmacological properties of voltage-dependent Na+, K+ and Ca2+ channels in parasympathetic neurones enzymatically dissociated from the paratracheal ganglia of rat trachea. The voltage-dependent Na+, K+ and Ca2+ currents (INa, IK and ICa) were separated by the use of ion subtraction and pharmacological treatments. 2. INa was activated by a step depolarization more positive than -50 mV and fully activated at positive potentials more than +10 mV. The inactivation phase of INa consisted of fast and slow exponential components (tau if and tau is, respectively). The tau if and tau is were voltage dependent and decreased with a more positive step pulse. 3. The time course for recovery of INa from the complete inactivation exhibited two exponential processes. 4. The reversal potential of INa was equal to the Na+ equilibrium potential (ENa) and resembled a simple Na+ electrode depending only on external Na+ concentration. 5. Tetrodotoxin (TTX) reduced INa without affecting the current kinetics in a concentration-dependent manner, and the concentration of half-maximal inhibition (IC50) was 6 x 10(-9) M. There was no TTX-resistant component of INa in any of the cells tested. 6. Scorpion toxin increased the peak amplitude of INa and prolonged the inactivation phase in a time- and concentration-dependent manner. In the presence of toxin, both tau is and the fractional contribution of the slow current component to total INa increased concentration dependently. 7. High-threshold (L-type) ICa was activated by a step depolarization more positive than -30 mV and reached a peak at near 0 mV in the external solution with 2.5 mM Ca2+. The current was inactivated to only a small extent (< 10%) during 100 ms of depolarizing step pulse. There was no low-threshold (T-type) ICa in this preparation. 8. The maximum ICa in individual current-voltage (I-V) relationships was saturated by an increase in extracellular Ca2+ concentration ([Ca2+]o). The I-V relationships were also shifted along the voltage axis to the more positive potential with increasing [Ca2+]o. 9. The inactivation process of the L-type ICa was dependent on Ca2+ influxes (ICa-dependent inactivation). 10. Relative maximum peak currents of divalent cations passing through the L-type Ca2+ channels were in the order of IBa > ICa > ISr. 11. Organic and inorganic Ca2+ antagonists blocked the ICa in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Multiple calcium currents in a thyroid C-cell line: biophysical properties and pharmacology.

The whole cell version of the patch-clamp technique was used to characterize voltage-gated Ca2+ channels in the calcitonin-secreting rat thyroid C-cell line 6-23 (clone 6). Three types of Ca2+ channels could be distinguished based on differences in voltage dependence, kinetics, and pharmacological sensitivity. T-type current was half-maximal at -31 mV, showed steady-state voltage-dependent inactivation that was half-maximal at -57 mV, inactivated with a voltage-dependent time constant that reached a minimum of 20 ms at potentials positive to -20 mV, and deactivated with a single time constant of approximately 2 ms at -80 mV. Reactivation of inactivated channels occurred with a time constant of 1.26 s at -90 mV. T current was selectively blocked by Ni2+ at concentrations between 5 and 50 microM. La3+ and Y3+ blocked the T current at 10- to 20-fold lower concentrations. Dihydropyridine-sensitive L-type current was half-maximal at a test potential of -3 mV and was approximately doubled in size when Ba2+ replaced Ca2+ as the charge carrier. Unlike L-type Ca2+ current in many cells, this current in C-cells displayed little Ca(2+)-dependent inactivation. N-type current was composed of inactivating and sustained components that were inhibited by omega-conotoxin. The inactivating component was half-maximal at +9 mV and could be fitted by two exponentials with time constants of 22 and 142 ms. A slow inactivation of N current with a time constant of 24.9 s was observed upon switching the holding potential from -80 to -40 mV. These results demonstrate that, similar to other neural crest derived cells, thyroid C-cells express multiple Ca2+ channels, including one previously observed only in neurons.

Animals↗

PubMed1991

Ca2+ current and Ca2+ transients under action potential clamp in guinea pig ventricular myocytes.

Precise characterization of the magnitude and kinetics of transsarcolemmal Ca2+ influx during an action potential (AP) is essential for a complete understanding of excitation-contraction coupling in heart. Using a voltage-clamp protocol that simulated a physiological AP (AP clamp), we characterized the properties of the Ca2+ current (ICa) in guinea pig ventricular myocytes. The AP-generated ICa showed a complex time course that was different from ICa generated by a square pulse. ICa activated rapidly during the upstroke of the AP and then partially inactivated during the plateau. The fast component of ICa reached a peak value of -7.6 +/- 1.0 pA/pF at 2.40 +/- 0.30 ms after depolarization, followed by a slow component with a peak value of -2.9 +/- 0.4 pA/pF during the plateau. ICa generated by an AP was composed of both L- and T-type Ca2+ channels. T-type Ca2+ current contributed to the fast component of ICa and L-type Ca2+ current contributed to both fast and slow components of ICa. Activation of beta-adrenoceptors enhanced ICa with a maximal effect lasting throughout the entire plateau of the AP. Measurements of cytosolic Ca2+ transients using fura-2 indicated that the ICa was responsible for triggering Ca2+ release from the sarcoplasmic reticulum. The AP clamp provides a new approach for investigation of the relationship between ICa and Ca2+ transients under more physiological conditions.

Action Potentials↗

PubMed1992

Apamin, a highly potent fetal L-type Ca2+ current blocker in single heart cells.

Apamin, a bee venom polypeptide, was reported to block the naturally occurring Ca2+ slow action potentials (APs) in cultured cell reaggregates from old chick hearts [Bkaily, G. et al. Am. J. Physiol. 248 (Heart Circ. Physiol. 17): H961-H965, 1985] as well as the tetrodotoxin (TTX)- and Mn(2+)-insensitive slow Na+ current in young embryonic chick heart cells (Bkaily, G. In Vitro Toxicology. Academic, In press; Bkaily et al. J. Mol. Cell. Cardiol. 23: 25-39, 1991). With the use of the whole cell voltage-clamp technique in single ventricular cells from 10-day-old chick embryos and 17- to 20-wk-old human fetuses, two types of Ca2+ currents (ICa), T and L, were found. These two types of slow inward current in both heart preparations were nearly similar in their voltage, kinetics, and pharmacology. Apamin, a slow Ca2+ action potential blocker in old embryonic chick heart, was found to block the L-type ICa (IL) in a dose-dependent manner without affecting the T-type ICa in both heart cell preparations. The blockade of the IL by apamin was completely reversible upon washout with apamin-free solution. Therefore, when compared with nifedipine or to PN 200-110, apamin seems to be a highly potent L-type Ca2+ channel blocker in heart cells.

Animals↗

PubMed1992

Calcium currents in hearts with persistent truncus arteriosus.

We have used the neural crest model of defective heart development to characterize both L- and T-type Ca2+ currents (ICa,L and ICa,T) in ventricular myocytes from embryonic chick hearts with a severe outflow tract anomaly known as persistent truncus arteriosus (PTA). Because of smaller whole embryo weights but no significant change in the weights of ventricles with PTA, the ventricle to whole embryo weight ratios from hearts with PTA were 61% larger than normal at day 11 of incubation. There was a 51% reduction in the peak magnitude of ICa,L at a test potential of +10 mV (-1.4 vs. -0.7 microA/cm2), whereas ICa,T, a proportionately large fraction of the total Ca2+ current in the embryonic chick ventricle, was unaffected. In comparison to sham-operated controls, ICa,L was otherwise not different. Half-activation occurred at about -1 and -41 mV, whereas half-inactivation occurred at -19 and -61 mV for ICa,L and ICa,T, respectively. The time for half-recovery from inactivation were not different and were 200 and 230 ms for ICa,L and ICa,T, respectively. The time for half-decay of the currents and their responses to BAY K 8644 were also similar in both sham-operated and experimental hearts. Although the dihydropyridine receptor binding experiments suggest that the total number of L-type Ca2+ channels was not different, the results from the physiological experiments indicate that the number of functional L-type channels available for opening and/or the single-channel conductance may be reduced in hearts with PTA. Finally, our results with the neural crest model indicate that it is unlikely that the development of Ca2+ currents is influenced by the onset of cholinergic innervation in the heart.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1992

Cholesterol increases the L-type voltage-sensitive calcium channel current in arterial smooth muscle cells.

To determine whether membrane free cholesterol affects calcium currents in vascular smooth muscle cells, whole-cell patch clamp recordings were made before and after cholesterol enrichment of cells by exposure to cholesterol-rich liposomes. Exposure to cholesterol-rich liposomes resulted in a gradual increase in the L-type current over 20 hours and a plateau (73 +/- 7% increase over basal) between 20 and 32 hours. This effect was associated with a rightward shift in the inactivation potential and a decrease in the sensitivity to (-)-PN-202-791, a dihydropyridine antagonist. There was no change in the maximum L-type current stimulated by (+)-PN-202-791, a dihydropyridine agonist. Liposome exposure caused a small, transient increase in the T-type current (peak effect, 20 minutes). We conclude that membrane cholesterol has important effects on the L-type calcium current in vascular smooth muscle cells, which is most likely due to an alteration in channel functional state rather than an increase in channel expression.

Animals↗

PubMed1992

Fast Na+ channels and slow Ca2+ current in smooth muscle from pregnant rat uterus.

Smooth muscle cells normally do not possess fast Na+ channels, but inward current is carried through two types of Ca2+ channels: slow (L-type) Ca2+ channels and fast (T-type) Ca2+ channels. Using whole-cell voltage clamp of single smooth muscle cells isolated from the longitudinal layer of 18-day pregnant rat uterus, depolarizing pulses, applied from a holding potential of -90 mV, evoked two types of inward current, fast and slow [8]. The fast inward current decayed within 30 ms, depended on [Na]o, and was inhibited by TTX (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]o, and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na+ channel current, and that the slow inward current is a Ca2+ slow channel current. A fast-inactivating Ca2+ channel current was not evident. Thus, the ion channels which generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells which possess fast Na+ channels, and it is suggested that the fast Na+ current may be involved in spread of excitation. The Ca2+ channel current density also was higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and may facilitate parturition. Isoproterenol (beta-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 of 12 mM) and nifedipine (K0.5 of 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) to a small extent. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions is not due to stimulation of ICa(s). Figure 11 summarizes the possible mechanisms by which uterine contractility can be modulated. In contrast to vascular smooth muscle, neither ISO nor adenosine, which produce elevation of cyclic AMP, affected ICa and INa. Therefore, no arrow can be drawn between cA-PK/cG-PK and the Ca2+ slow channel.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine↗