pA pF-1 Research Articles

Comparison of ouabain-sensitive and -insensitive Na/K pumps in HEK293 cells

The Na/K pump current I p of single HEK293 cells either untransfected (endogenous I p) or transfected with the α1 subunit of the rat Na/K pump (exogenous I p) was investigated in Na-containing solution by means of whole-cell recording at 30°C. The endogenous I p was irreversibly blocked by 10 −4 M ouabain or 2·10 −4 M dihydro-ouabain (DHO). Its density amounted to 0.33 pA pF −1 at 0 mV and 5.4 mM K o. It was half maximally activated at 1.5 mM K o and increased linearly with depolarization over the entire voltage range studied (−80 to +60 mV). In contrast, HEK293 cells stably transfected with cDNA for the cardiac glycoside-resistant α1 subunit of the rat Na/K pump showed an I p in the presence of 10 −4 M ouabain and 2·10 −4 M DHO, respectively. This exogenous I p was reversibly blocked by 10 −2 M ouabain. Half maximal activation of the exogenous I p occurred at 1.7 mM K o. Its amplitude increased linearly with depolarization at negative voltages but remained almost constant at positive membrane potentials. Comparison with the I p of isolated rat cardiac ventricular myocytes strongly suggests that the exogenous I p in HEK293 cells is generated by the α1 subunit of the rat Na/K pump since it displays identical properties. Therefore, HEK293 cells represent an expression system well suited for the electrophysiological analysis of recombinant, cardiac glycoside-resistant Na/K pumps by means of whole-cell recording.

A slowly activating voltage-dependent K+ current in rat pituitary nerve terminals.

1. A novel slowly activating voltage-dependent K+ current was observed in isolated nerve terminals from rat neurohypophysis using the whole-cell configuration of the patch-clamp technique. 2. The activation kinetics of the slow current could be fitted assuming Hodgkin--Huxley-type kinetics, an exponential, n, of 1.3 and activation time constants decreasing from 4 s at -50 mV to 0.7s at +40 mV. 3. A positive shift of reversal potential was observed when [K+] was increased in the bath solution. The current is carried mainly but not exclusively by K+ ions. 4. When intracellular free [Mg2+] was low (approximately 60 microM), average current density was 74 pA pF-1 at membrane potentials around 0 mV. In 83% of nerve terminals current amplitude was > 10 pA pF-1. 5. The slow current was never observed when the pipette contained 4.6 mM free Mg2+. At a physiological level of free Mg2+ (0.5 mM) the average current density was 16 pA pF-1. 6. When nerve terminals were analysed after patch-clamp experiments for vasopressin content by immunodetection, no difference in current amplitude was found between the terminals containing vasopressin and all analysed terminals. 7. The voltage dependence of activation was fitted by a Boltzmann equation giving a half-activation potential of -37 mV and a slope factor of about 9 mV. 8. Tail current deactivation kinetics was biexponential with time constants of 0.12 and 1.5s. Kinetics was dependent on the duration of the activating pulse. 9. Noise analysis of the slow current indicated a single-channel current of 0.33 pA at +6 mV, corresponding to a single-channel conductance of 4.3 pS. 10. This is the first demonstration of a current similar to the slow K+ current, IKs, in a neurone, suggesting that a protein similar to the IKs-inducing channel protein IsK (minK) may be present in peptidergic nerve terminals. 11. The activation properties are consistent with a role of the slow current in inhibition of excitability, at least at the level of the nerve terminal.

Formula omitted] exchange current: lack of effect of taurine

The intracellular taurine content is very high in some types of cells, including myocardial cells, and taurine becomes depleted in failing hearts. The effect of taurine on inward (forward) and outward (reversal) Na + Ca 2+ exchange current ( I Na Ca ) was examined in freshly isolated rat (11–20-day-old) ventricular cells using whole-cell voltage clamp. I Na Ca was evoked by a 300 ms ramp hyperpolarizing pulse at a rate of 0.67 V/s (from + 60 to −140 mV) from a holding potential of −40 mV. Under the condition of 500 nM Ca 2+ in the pipette, when the bath solution was changed from 150 mM Li + to 150 mM Na +, an inward I Na Ca was initiated. On the other hand, under the condition of 50 mM Na + and 56 nM Ca 2+ in the pipette, when the bath solution was changed from Na +-free, Ca 2+-free solution to Na +-free, 2 mM Ca 2+, an outward I Na Ca was initiated. Both inward and outward I Na Ca were completely blocked by 5 mM Ni 2+, helping to identify the current as I Na Ca . Forward and reversal I Na Ca was measured in the absence (control) and presence of 30 mM taurine added to the bath. The current densities ( pA pF ) of inward I Na Ca at −50 mV were 0.75 ± 0.12 in control and 0.73 ± 0.11 in presence of 30 mM taurine. Outward I Na Ca at +50 mV were 0.73 ± 0.08 in control and 0.65 ± 0.02 in 30 mM taurine. There were no significant differences between control and taurine for both inward and outward I Na Ca . Therefore, the various effects of taurine reported on the heart are not caused by a direct effect of taurine on Na Ca exchange.

Angiotensin II activation of protein kinase C decreases delayed rectifier K+ current in rabbit vascular myocytes.

1. The effect of angiotension II (Ang) on delayed rectifier K+ current (IK(V)) was studied in isolated rabbit portal vein smooth muscle cells using standard whole-cell voltage clamp technique. The effect of 100 nM Ang on macroscopic, whole-cell IK(V) was assessed in myocytes dialysed with 10 mM BAPTA, 5 mM ATP and 1 mM GTP either at room temperature or at 30 degrees C. 2. Application of Ang caused a decline in IK(V) which was reversed upon washout of the drug. Tail current recorded after 250 ms pulses to +30 mV and repolarization to -40 mV was reduced from 3.9 +/- 0.7 to 2.5 +/- 0.5 pA pF-1 at 20 degrees C (n = 6) and from 4.5 +/- 0.5 to 3.13 +/- 0.4 pA pF-1 at 30 degrees C(n = 17). 3. Ang had no effect on outward current in the presence of an AT1 selective antagonist, losartan (1 microM), which alone had no direct effect on the amplitude of IK(V). Substitution of extracellular Ca2+ with Mg2+ in the presence of 10 microM intracellular BAPTA did not affect the suppression of IK(V) by Ang. 4. Ang induced a decrease in time constant for the rapid phase of inactivation of the macroscopic current (tau 1 reduced from 377 +/- 32 to 245 +/- 11 ms; tau 2 unchanged, n = 17). Neither the voltage dependence of activation nor inactivation were affected by Ang. 5. The inhibition of IK(V) by Ang was abolished by intracellular dialysis with the selective PKC inhibitors, calphostin C (1 microM) and chelerythrine (50 microM). These data provide strong evidence that the decline in IK(V) due to Ang treatment is due to PKC activation. 6. The pattern of expression of PKC isoforms was examined in rabbit portal vein using isoenzyme-specific antibodies: alpha, epsilon and zeta isoenzymes were detected, but beta, gamma, delta and eta isoenzymes were not. 7. The lack of requirement for Ca2+, as well as the sensitivity of the Ang response to chelerythrine, suggest the involvement of the Ca(2+)-independent PKC isoenzyme epsilon in the signal transduction pathway responsible for IK(V) inhibition by Ang.

The properties and distribution of inward rectifier potassium currents in pig coronary arterial smooth muscle.

1. Whole-cell potassium currents were studied in single smooth muscle cells enzymatically isolated from pig coronary arteries. 2. In cells isolated from small diameter branches of the left anterior descending coronary artery (LAD), an inward rectifier potassium current (IK(IR)) was identified, which was inhibited by extracellular barium ions, suggesting the presence of inward rectifier potassium (KIR) channels. 3. The conductance for IK(IR) measured in 6, 12, 60 and 140 mM extracellular potassium was a function of membrane potential and the extracellular potassium concentration. 4. On hyperpolarization, IK(IR) activated along an exponential time course with a time constant that was voltage dependent. 5. Inward rectifier current was compared in cells isolated from coronary vessels taken from different points along the vascular tree. Current density was greater in cells isolated from small diameter coronary arteries; at -140 mV it was -20.5 +/- 4.4 pA pF-1 (n = 23) in 4th order branches of the LAD, but -0.8 +/- 0.2 pA pF-1 (n = 11) in the LAD itself. 6. In contrast to IK(IR), there was little effect of arterial diameter on the density of voltage-dependent potassium current; densities at +30 mV were 12.8 +/- 1.3 pA pF-1 (n = 19) in 4th order branches and 17.4 +/- 3.1 pA pF-1 (n = 11) in the LAD. 7. We conclude that KIR channels are present in pig coronary arteries, and that they are expressed at a higher density in small diameter arteries. The presence of an enhanced IK(IR) may have functional consequences for the regulation of cell membrane potential and tone in small coronary arteries.

Failure of nimodipine to prevent or correct the long-term nerve conduction defect and increased neuronal Ca 2+-currents in the diabetic BB/W-rat

It has been suggested that L-type Ca 2+ channel antagonists exert a beneficial effect on nerve conduction velocity (NCV) slowing in short-term experimental diabetic neuropathy. We examined the effects of long-term treatment with the L-channel blocker, nimodipine, on two aspects of neuronal function previously documented to be abnormal in the spontaneously diabetic BB/W-rat: nerve conduction velocity and calcium influx in dorsal root ganglion (DRG) neurons. Treatment with 20 mg/kg nimodipine i.p. every 48 h from onset of diabetes for 6 months led to a transient, non-significant (30%) improvement in NCV. Intervention with the same regimen from 3 to 6 months of diabetes had no corrective effect on the already established NCV defect. Voltage activated calcium currents were recorded in isolated DRG neurons from nimodipine-treated and untreated diabetic and non-diabetic age-matched BB/W control rats. The peak high-threshold calcium current density ( I DCa, pA pF ) was significantly larger in non-treated diabetic rats compared with control rats (157 ± 12 vs. 66 ± 5.5 ( P < 0.05)). Long-term treatment with nimodipine was associated with a non-significant (28%) decrease (112 ± 29) in the I DCa compared with non-treated diabetic rats. We conclude that L-channel mediated perturbations of cytosolic Ca 2+ levels are only of minor pathophysiologic significance in the development of chronic diabetic neuropathy.

A TTX‐sensitive inward sodium current contributes to spontaneous activity in newborn rabbit sino‐atrial node cells.

1. Single cells were isolated from the sinus node region of rabbits (2 days old to adult) to study the age-dependent contribution of the sodium current (iNa) to pacemaker activity. 2. Experiments were conducted in 50 mM Na(+)-Ca(2+)-free solution. All newborn cells (2-19 days) exhibited a TTX-sensitive, Mn(2+)-insensitive fast inward Na+ current (peak current density 115.5 +/- 11.9 pA pF-1 at 0 mV). Fifty per cent of young cells (20-40 days) possessed the current, but only one in ten adult cells. Current density decreased with development independently of cell capacitance. 3. Newborn cells exhibited a noticeable window current. With development, the position of the activation curve was shifted in the positive direction, while the inactivation was unaltered, resulting in reduced overlap of the two curves and hence less window current. 4. In newborn cells, 3 microM TTX significantly reduced all measured parameters of spontaneous action potentials, slowing rate by 63%. In contrast, there was no significant effect of TTX on rate or most of the same parameters in adult cells. 5. These results indicate that cells of the sinus node region exhibit a substantial TTX-sensitive current at birth. With development, both the density and frequency of occurrence of this current within the sinus node decrease, as does its contribution to automaticity.

Differential oxygen sensitivity of calcium channels in rabbit smooth muscle cells of conduit and resistance pulmonary arteries.

1. Calcium currents were recorded from smooth muscle cells dispersed from conduit and resistance rabbit pulmonary arteries. We tested the hypothesis that Ca2+ channel activity was regulated by environmental O2 tension. 2. Conduit (proximal) and resistance (distal) myocytes differ in their Ca2+ channel density and responses to low PO2. Ca2+ current density in distal myocytes (20.7 +/- 7.4 pA pF-1, n = 10) is almost twice the value in proximal myocytes (12.6 +/- 5.5 pA pF-1, n = 39). In proximal myocytes, the predominant response to reductions in PO2 is inhibition of the calcium current (n = 12) at membrane potentials below 0 mV, whereas potentiation of current amplitude is observed in distal myocytes (n = 24). 3. Hypoxia also produces opposite shifts in the conductance-voltage relationships along the voltage axis. The average displacements induced by low PO2 are +5.05 +/- 2.98 mV (n = 5) in proximal myocytes and -6.06 +/- 2.45 (n = 10) in distal myocytes. 4. These findings demonstrate longitudinal differences in Ca2+ channel density and O2 sensitivity in myocytes along the pulmonary arterial tree. These results may help to understand the differential reactivity to hypoxia of the pulmonary vasculature: vasodilatation in conduit arteries and vasoconstriction in resistance vessels.

Opiate-mediated inhibition of calcium signaling is decreased in dorsal root ganglion neurons from the diabetic BB/W rat.

The effect of diabetes mellitus on opiate-mediated inhibition of calcium current density (I(D Ca) [pA pF-1]) and cytosolic calcium response ([Ca2+]i nM) to depolarization with elevated KCl and capsaicin was assessed. Experiments were performed on isolated, acutely dissociated dorsal root ganglion (DRG) neurons from diabetic, BioBreeding/Worcester (BB/W) rats and age-matched control animals. Sciatic nerve conduction velocity was significantly decreased in diabetic animals compared to controls. Mean I(DCa) and [Ca2+]i responses to capsaicin and elevated KCl recorded in DRGs from diabetic animals were significantly larger than those recorded in DRG neurons from controls. In neurons from diabetic animals, the opiate agonist dynorphin A (Dyn A; 1, 3, and 5 microM) had significantly less inhibitory effect on I(D Ca) and KCl-induced [Ca2+]i responses compared to controls. Omega-conotoxin GVIA (omega-CgTX; 10 microM) and pertussis toxin (PTX; 250 ng ml-1) abolished Dyn A-mediated inhibition of I(DCa) and [Ca2+]i in control and diabetic neurons, suggesting that Dyn A modulated predominantly N-type calcium channels coupled to opiate receptors via PTX-sensitive (Gi/o) inhibitory G proteins. These results suggest that opiate-mediated regulation of PTX-sensitive, G protein-coupled calcium channels is diminished in diabetes and that this correlates with impaired regulation of cytosolic calcium.

Voltage-dependent currents and modulation of calcium channel expression in zona fasciculata cells from rat adrenal gland.

1. Whole-cell voltage-activated currents from single zona fasciculata (ZF) cells from rat adrenal glands were studied. T- and L-type Ca2+ currents and a slowly inactivating A-type K+ current were the three major currents observed. 2. In freshly isolated cells, the A-type K+ current and the T-type Ca2+ current were predominant. The A-type current was activated at -50 mV and inhibited by 4-amino-pyridine with a half-maximal block (IC50) at 130 microM while the T-type current was activated at -70 mV and blocked by Cd2+, Ni2+ and amiloride with IC50 values of 24.1, 132.4 and 518.9 microM, respectively. 3. Under current clamp, depolarizing current pulses produced a single Ca2+ action potential with Cs+ in the pipette internal solution. Upon replacement of Cs+ by K+, the half-amplitude width of the action potential was shortened and membrane potential oscillations were seen after the spike. 4. In freshly isolated cells and during the first 24 h after plating, the T-type current was observed in all cells, with L-type current being observed in < 2% of cells, even in the presence of (+)SDZ 202,791, a dihydropyridine Ca2+ channel agonist. With time in culture, the T-type current disappeared, and a high-voltage-activated L-type current became increasingly apparent. In cells tested after > 2 days in culture, (+)SDZ 202,791 potentiated L-type current by 407 +/- 12% and the antagonist (-)SDZ 202,791 blocked this increase. The L-type current was activated between -30 and -20 mV and was sensitive to nitrendipine and omega-conotoxin GVIA. 5. Pre-incubation of cultured ZF cells with adrenocorticotrophic hormone (ACTH) or vasoactive intestinal peptide (VIP) for 3 days resulted in a high, sustained level of expression of T-type current, with a mean amplitude of 34.2 +/- 5.5 pA pF-1 for ACTH-treated cells compared with 3.4 +/- 1.8 pA pF-1 for untreated cells. Cycloheximide strongly inhibited this effect. Neither treatment affected L-type current expression. 6. The expression of both Ca2+ current types was unaffected by pre-incubation with 8-bromo-cAMP or forskolin. The protein kinase A antagonist, H89, did not inhibit the ACTH-induced upregulation of T-type Ca2+ currents. 7. It is concluded that the main voltage-dependent currents involved in cell excitability and steroidogenesis in rat adrenal ZF cells are an A-type K+ current and a T-type Ca2+ current. The physiological role and control of expression of L-type Ca2+ channels in rat ZF cells remain less clear.

Volume regulation in a toad epithelial cell line: role of coactivation of K+ and Cl- channels.

1. We have measured changes in cell volume, membrane potential and ionic currents in distal nephron A6 cells following a challenge with hypotonic solutions (HTS). 2. The volume increase induced by HTS is compensated by a regulatory volume decrease (RVD), which is inhibited by both 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and quinine. Quinine (500 microM) completely blocked RVD, whereas 100 microM NPPB delayed and attenuated RVD. 3. The resting potential in A6 cells was -52.3 +/- 4.8 mV (n = 53), and shifted to -35.1 +/- 2.2 mV (n = 33) during HTS. 4. Resting membrane current in A6 cells was 0.35 +/- 0.12 pA pF-1 at -80 mV and 0.51 +/- 0.16 pA pF-1 at +80 mV (n = 5). During cell swelling these values increased to 11.5 +/- 1.1 and 29.3 +/- 2.8 pA pF-1 (n = 29), respectively. 5. Quinine (500 microM) completely blocked the HTS-activated current at -15 mV, the reversal potential for Cl- currents, but exerted only a small block at -100 mV (K+ equilibrium potential). NPPB (100 microM) inhibited the current at both potentials almost to the same extent. The HTS-induced net current reversed at -41 +/- 2.5 mV (n = 15), which is close to the measured resting potential during HTS. 6. The quinine-insensitive current reversed near the Cl- equilibrium potential. The quinine-sensitive current reversed near the K+ equilibrium potential. The respective conductances activated by HTS at the zero-current potential were 2.1 +/- 0.7 nS for K+ and 5.2 +/- 1.3 nS for Cl- (n = 15). 7. Single channel analysis unveiled activation of at least two different channels during HTS. A 36 pS channel reversing at the Cl- equilibrium potential showed increased open probability at depolarized potentials. HTS also activated a K+ channel with a 29 pS conductance in high-K+ extracellular solutions (130 mM) or 12 pS in 2.5 mM K+. 8. This coactivation of K+ and Cl- channels shifts the membrane potential towards a value between EK and ECl (the reversal potentials for K+ and Cl-), where a net efflux of Cl- (Cl- inward current) and K+ (K+ outward current) under zero-current conditions occurs. Block of either the K+ or the Cl- conductance will shift the zero-current potential towards the equilibrium potential of the unblocked channel, preventing net efflux of osmolytes and RVD. This coactivation of K+ and Cl- currents causes a shift of osmolytes out of the cells, which almost completely accounts for the observed RVD.

Non‐selective cation current of guinea‐pig endocardial endothelial cells.

1. Endocardial endothelial (EE) cells, isolated by the enzymatic treatment of guinea-pig heart, were used for whole-cell voltage clamp experiments. 2. The inward rectifier K+ current was observed in about half of the experiments. The contribution of Ca(2+)-dependent K+ current to the resting membrane conductance was also suggested. 3. After the K+ conductances were suppressed, removal of external Na+ revealed an inward cation current (1.2 pA pF-1, at -45 mV), whose slope conductance was a saturable function of external Na+ concentration. When Na+ was totally replaced by various monovalent cations, the order of the membrane conductances was K+ > Rb+ > Cs+ > Na+ > Li+. 4. This basal non-selective cation current was blocked by either Gd3+ or La3+, and showed slight outward rectification. 5. Addition of 20 mM Ca2+ or Ba2+, but not Mg2+ or Mn2+, to the Na(+)-free solution, induced an inward current, indicating that this current possesses a significant Ca2+ permeability. 6. In approximately 15% of the experiments, ATP and histamine induced another type of non-selective cation current, which showed different ion selectivity (Na+ > K+, Cs+) and rectification (inward). 7. The basal non-selective cation current is responsible for both the low resting potential and the leak Ca2+ influx of EE cells.

A highly calcium-selective cation current activated by intracellular calcium release in MDCK cells.

1. The whole-cell patch clamp technique and fluorescence microscopy with the Ca2+ indicators fura-2 and fluo-3 were used to measure the whole-cell current and the free intracellular Ca2+ concentration ([Ca2+]i) in Madin-Darby canine kidney (MDCK) cells. 2. In a Ca(2+)-free bath solution, thapsigargin (TG) caused a transient increase of [Ca2+]i. Subsequent addition of Ca2+ caused a long lasting elevation of [Ca2+]i. 3. In a Ca(2+)-free bath solution, extracellular application of TG, ATP or ionomycin, or intracellular application of inositol 1,4,5-trisphosphate (IP3), caused a small but significant inward current (Iin) and a transient outward Ca(2+)-dependent K+ current (IK(Ca)), consistent with intracellular Ca2+ release. Subsequent addition of Ca2+ induced a prominent Iin with a current density of -4.2 +/- 0.7 pA pF-1. This Iin was unaffected by inositol 1,3,4,5-tetrakisphosphate (IP4). 4. Na+ replacement by mannitol, N-methyl-D-glucamine+ (NMG+), aminomethylidin-trimethanol+ (Tris+) or choline+ reduced Iin by 54, 65, 52 and 56%, respectively. This indicates an apparent Ca2+ selectivity over Na+ of 26:1. Iin was, however, unaffected by replacing Cl- with gluconate- or by the K+ channel blocker charybdotoxin (CTX). 5. Iin was completely blocked by La3+ (IC50 = 0.77 microM). Consistently, La3+ completely reversed the TG-induced elevation of [Ca2+]i. SK&F 96365 (1-[3-(4-methoxyphenyl)-propoxyl]-1-(4-methoxy-phenyl)-ethyl-1H-im idazole) HCl did not inhibit the TG-induced Iin. It did, however, exhibit a biphasic effect on [Ca2+]i, consisting of an initial Ca2+ decay and a subsequent Ca2+ elevation. La3+ completely reversed the SK&F 96365-induced elevation of [Ca2+]i. 6. In the absence of Na+, Iin was dependent on the bath Ca2+ concentration (EC50 = 1.02 mM). Ca2+ replacement by Ba2+ or Mn2+ resulted in a reduction of Iin by 95 and 94%, respectively. 7. From these experiments we conclude that Ca2+ release from intracellular Ca2+ stores, induced by different independent methods, stimulates La(3+)-inhibitable Ca2+ entry in MDCK cells. Ca2+ entry is at least, in part, mediated by a cation current, which is highly, but not exclusively, selective for Ca2+ over Na+ and insensitive to SK&F 96365.

Sustained calcium influx activated by basic fibroblast growth factor in Balb‐c 3T3 fibroblasts.

1. We have investigated the ionic events elicited in Balb-c 3T3 fibroblasts by basic fibroblast growth factor (bFGF), a peptide that binds to membrane receptors with tyrosine kinase activity and has a mitogenic action on many cell types. The peptide (0.2-100 ng ml-1) caused the appearance of an inward current, as observed in whole-cell patch-clamp experiments at a holding potential of -50 mV, that could last for tens of minutes and had a peak density of 4.6 +/- 2.6 pA pF-1. The reversal potential was 18.8 +/- 16.7 mV. 2. The current was reversibly abolished by removal of bFGF from the external bath. Inhibition of low-affinity FGF receptors had no effect on the activation of the inward current; it was completely abolished when cells were pre-incubated with tyrphostin or 5'-methylthioadenosine (MTA), two inhibitors of the tyrosine kinase activity of the high-affinity FGF receptors. The inward current was not activated by the emptying of internal calcium stores, as tested with 200 nM thapsigargin. 3. Values of peak current density comparable to control ones were obtained when either all Na+ ions or all Ca2+ ions were removed from the external solution; when both ions were completely removed, no inward current could be observed. The inward current was not affected by 2 microM nifedipine, and was reversibly blocked by the imidazole derivative SK&F 96365-A. 4. Measurements of free intracellular calcium concentration ([Ca2+]i) with the dye fura-2 showed that bFGF elicited sustained increases in [Ca2+]i that were completely dependent on external calcium and on the presence of the agonist and could last more than 1 h. 5. Single channel currents (conductance 7.9 pS) in response to bFGF stimulation could be recorded in the cell-attached configuration with 100 mM CaCl2 in the pipette. When the resting potential was brought near to 0 mV by external perfusion in a high-K+ solution, Vrev was about 0 mV. 6. We conclude that in Balb-c 3T3 cells bFGF induces an inward current that is carried at least partially by Ca2+ ions; this current in turn causes a long-lasting increase in intracellular Ca2+ concentration. The amplitude and time course of these bFGF-activated ionic events are compatible with their involvement in the control of cell proliferation.

Functional availability of sodium channels modulated by cytosolic free Ca2+ in cultured mammalian neurons (N1E-115).

1. Whole-cell sodium currents (INa) were measured in mouse neuroblastoma cells (N1E-115) at different [Ca2+]i values using appropriate Ca-EGTA buffers in the pipettes. 2. INa was found to be larger at pCa 7 than at pCa 8 or 9 with a ratio of 1:0.65 or 0.55, respectively. The steady-state inactivation (h infinity curve) was independent of [Ca2+]i, thus excluding surface charge effects as a cause of the Ca2+ effect. 3. Recovery of INa from slow inactivation after changing from resting (-30 to -40 mV) to holding potential (-70 mV) occurred in a similar way at all pCa values. The Ca2+ effect appears to be independent of slow inactivation and to occur within the first 2 min of pipette buffer-cytoplasm equilibration. 4. The cell membrane capacitance (Cm) was independent of [Ca2+]i, thus excluding exo- or endocytosis of sodium channel-containing membrane as a cause of the Ca2+ effect. 5. Non-stationary fluctuation analysis was used to determine simultaneously the single channel current (iNa) and the size of INa. At pCa values of 7 and 9, iNa was identical, i.e. 0.59 and 0.58 pA, while INa/Cm differed, i.e. 41.1 and 22.2 pA pF-1, respectively. The peak open probability at 0 mV was about 0.5 for both pCa values indicating that [Ca2+]i controls the fraction of channels available for activation. 6. Since [Ca2+]i in other neurons varies between 30 and 100 nM in the resting and active state, respectively, the present data suggest a modulatory role for [Ca2+]i in neuronal excitability.

Skeletal muscle DHP receptor mutations alter calcium currents in human hypokalaemic periodic paralysis myotubes.

1. Mutations in the gene encoding the alpha 1-subunit of the skeletal muscle dihydropyridine (DHP) receptor are responsible for familial hypokalaemic periodic paralysis (HypoPP), an autosomal dominant muscle disease. We investigated myotubes cultured from muscle of patients with arginine-to-histidine substitutions in putative voltage sensors, IIS4 (R528H) and IVS4 (R1239H), of the DHP receptor alpha 1-subunit. 2. Analysis of the messenger ribonucleic acid (mRNA) in the myotubes from such patients indicated transcription from both the normal and mutant genes. 3. In control myotubes, the existence of the slow L-type current and of two rapidly activating and inactivating calcium current components (T-type with a maximum at about -20 mV and 'third type' with a maximum at +10 to +20 mV) was confirmed. In the myotubes from patients with either mutation, the third-type current component was seen more frequently and, on average, with larger amplitude. 4. In myotubes with the IVS4 mutation (R1239H) the maximum L-type current density was smaller than control (-0.53 +/- 0.31 vs. -1.41 +/- 0.71 pA pF-1). The voltage dependence of activation was normal, and hyperpolarizing prepulses to -120 mV for 20 s did not increase the reduced current amplitude during test pulses. 5. In myotubes with the IIS4 mutation (R528H) the L-type current-voltage relation, determined at a holding potential of -90 mV, was normal. However, the voltage dependence of inactivation was shifted by about 40 mV to more negative potentials (voltage at half-maximum inactivation, V1/2 = -41.5 +/- 8.2 vs. -4.9 +/- 4.3 mV in normal controls).(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophysiological properties of neonatal mouse cardiac myocytes in primary culture.

1. The increasing utility of transgenic mice in molecular studies of the cardiovascular system has motivated us to characterize the ionic currents in neonatal mouse ventricular myocytes. 2. Cell capacitance measurements (30 +/- 1 pF, n = 73) confirmed visual impressions that neonatal mouse ventricular myocytes in primary culture are considerably smaller than freshly isolated adult ventricular myocytes. With the use of electron microscopy, mitochondria and sarcoplasmic reticulum were found in close association with myofibrils, but transverse tubules were not observed. 3. Action potential durations, measured at 50 and 90% repolarization, were 23 +/- 1 and 42 +/- 2 ms respectively (n = 46). Application of 4-aminopyridine (4-AP; 5 mM) prolonged action potential duration at 50% repolarization by 26 +/- 5% (n = 3). The brevity of the action potential is explained by the rapid activation of a transient outward K+ current upon voltage-clamp depolarization to plateau potentials. 4. Potassium currents identified include an inward rectifier, a large 4-AP-sensitive transient outward, a slowly inactivating 4-AP-insensitive outward, a slowly activating delayed rectifier and a small rapidly activating E-4031 (10 microM)-sensitive delayed rectifier K+ current. 5. Sodium currents (-305 +/- 50 pA pF-1, n = 21) were recorded in 40 mM Na+ with Ni2+ (1 mM) to block Ca2+ currents and with K+ replaced by Cs+. The relative insensitivity of the Na+ current to block by tetrodotoxin (IC50 = 2.2 +/- 0.3 microM, n = 4) is distinctive of the cardiac Na+ channel isoform. 6. Nitrendipine-insensitive (10 microM) Ba2+ currents elicited during steps from -90 to -30 mV measured -25 +/- 5 pA pF-1 (n = 7, 30 mM Ba2+). Decay of these currents was complete during 180 ms depolarizations, even with Ba2+ as the charge carrier. These currents were not present when the holding potential was set at -50 mV. These data support the presence of a low threshold, T-type Ca2+ current. 7. The maximal nitrendipine-sensitive L-type Ca2+ current density was -10 +/- 2 pA pF-1 (n = 8) in 2 mM Ca2+ and -38 +/- 5 pA pF-1 (n = 9) in 30 mM Ba2+. Exposure to isoprenaline (1 microM) resulted in an 82% increase (n = 3) in the amplitude of the Ba2+ currents elicited at 0 mV. 8. Neonatal mouse cardiac myocytes in primary culture possess surprisingly large inward currents given the brevity of their action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)

Concomitant activation of Cl- and K+ currents by secretory stimulation in human epithelial cells.

1. Whole-cell currents were investigated in the model salt-secreting epithelium, human T84 cell line, by means of the perforated patch-clamp technique. In the control extracellular medium containing Cl-, depolarizing voltage ramps evoked current responses which peaked at 5.43 +/- 0.81 pA pF-1 at +60 mV and had a reversal potential (Erev) of -38.4 +/- 2.5 mV (n = 23). 2. Activation of the cAMP pathway with forskolin increased the current at +60 mV from 3.81 +/- 0.61 to 20.79 +/- 5.08 pA pF-1 (n = 18). In thirteen cells, Erev was initially shifted towards positive potentials (Erev of the cAMP-activated initial current was -18.2 +/- 1.2 mV) and subsequently shifted towards more negative potentials, consistent with the activation of both Cl- and K+ currents during cAMP stimulation. 3. Increasing the intracellular Ca2+ concentration, [Ca2+]i, with ionomycin (1 microM) or with acetylcholine (1 microM), increased the current at +60 mV from 7.79 +/- 1.57 to 57.50 +/- 12.10 pA pF-1 (n = 6) and from 6.36 to 34.13 pA pF-1 (n = 4), respectively. With both agonists, Erev was shifted either towards the reversal potential for potassium, EK, or towards the reversal potential for chloride, ECl, depending on the cell. 4. In the absence of chloride ions (gluconate substituted), stimulation of the Ca2+ pathway activated a time-independent outward current of large amplitude. This current exhibited inward rectification at positive voltages, reverted at -89.5 +/- 0.2 mV and was markedly reduced by charybdotoxin (10 nM), a specific blocker of Ca(2+)-activated K+ channels. When a voltage step protocol was used, increased [Ca2+]i also activated an outward current at potentials more positive than -40 mV which slowly relaxed during depolarizing steps. 5. The activation of both (i) a time-dependent inwardly rectifying charybdotoxin-sensitive K+ current, and (ii) a time-dependent slowly inactivating current was also produced by cAMP stimulation. 6. We concluded that (i) in the T84 epithelial cells, both Cl- and K+ currents are concomitantly increased by secretagogue stimulation, and (ii) two different types of K+ conductances are activated by either the cAMP or the intracellular Ca2+ secreting pathways.

Stimulation of chloride secretion by P1 purinoceptor agonists in cystic fibrosis phenotype airway epithelial cell line CFPEo-.

1. P1 purinoceptor agonists like adenosine have been shown to stimulate Cl- transport in secretory epithelia. In the present study, we investigated whether P1 agonist-induced Cl- secretion is preserved in cystic fibrosis airway epithelium and which signalling mechanism is involved. The effects of purinoceptor agonists on Cl- secretion were examined in a transformed cystic fibrosis airway phenotype epithelial cell line, CFPEo-. 2. Addition of adenosine (ADO; 0.1-1 mM) markedly increased 125I efflux rate. The rank order of potency of purinoceptor agonists in stimulating 125I efflux was ADO > AMP > ADP approximately equal to ATP. A similar order of potency was seen in transformed cystic fibrosis nasal polyp cells, CFNPEo- (ADO > ATP > AMP > ADP). These results are consistent with the activation of Cl- secretion via a P1 purinoceptor. 3. The P1 agonists tested (at 0.01 and 0.1 mM) revealed a rank order of potency of 5'-N-ethylcarboxamine adenosine (NECA) > 2-chloro-adenosine (2-Cl-ADO) > R-phenylisopropyl adenosine (R-PIA). 4. The known potent A2 adenosine receptor (A2AR) agonist, 5'-(N-cyclopropyl) carboxamidoadenosine (CPCA, 2 microM) but not the A1 adenosine receptor agonist, N6-phenyl adenosine (N6-phenyl ADO, 10 microM) markedly increased 125I efflux rate (baseline, 5.9 +/- 2.0% min-1, + CPCA, 10.9 +/- 0.6% min-1; P < 0.01). The stimulant effect of CPCA (10 microM) was abolished by addition of the A2AR antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) (100 microM; reported K(i) = 11 +/- 3 microM). These results favour the involvement of A2AR. 5. ADO (0.1-mM) and CPCA (2 microM) both induced a marked increase in intracellular [Ca2+] ([Ca2+]i); the effect of the latter was again abolished by pretreatment of the cells with DMPX. By contrast, N6-phenyl ADO did not affect [Ca2+]i. 6. In patch-clamp experiments, ADO (1 mM) induced an outwardly-rectified whole-cell Cl- current (baseline, 2.5 +/- 0.8 pA pF-1, + ADO, 78.4 +/- 23.8 pA pF-1; P < 0.02), which was largely inhibited in cells internally perfused with a selective inhibitory peptide of the multifunctional Ca2+/calmodulin-dependent protein kinase, CaMK [273-302] (20 microM), as compared to a control peptide, CaMK [284-302]. Addition of BAPTA (10 mM), a Ca2+ chelator, to the perfusion pipette also abolished the ADO-elicited Cl- current. 7. In conclusion, our results suggest that A2AR participates in regulation of airway C1 secretion via aCa2+-dependent signalling pathway, which involves CaMK and appears to be at least partially conserved in cystic fibrosis airway epithelial cells.

Background conductance attributable to spontaneous opening of muscarinic K+ channels in rabbit sino-atrial node cells.

Single myocytes were dissociated from the rabbit sino-atrial node, and the membrane background conductance produced by spontaneous opening of the muscarinic K+ channels was investigated by recording whole-cell and single channel currents in both normal K+ (5.4 mM) and high-K+ (145 mM) external solutions. Increasing external K+ concentration ([K+]o) from 5.4 to 145 mM induced a large inward shift of the whole-cell current accompanied by considerable current fluctuations at -50 mV. The high-K(+)-induced current was both K+ selective and voltage dependent, which was examined by varying [K+]o. This current was almost completely suppressed by 1-5 mM Ba2+ or 2-10 mM Cs+ and it was partly blocked by 10 microM atropine. In high-K+ (145 mM) solution, 20 nM acetylcholine (ACh) further increased the K+ conductance as well as the current noise. The power density spectrum of the noise was fitted with a sum of two Lorentzian functions. The corner frequencies of both the slow (approximately 5 Hz) and fast (approximately 120 Hz) components were comparable between the noise before and during the ACh application. Internal dialysis with a non-hydrolysable derivative of ATP, 5'-adenylylimido-diphosphate (AMP-PNP) or Mg(2+)-free solution markedly decreased both the amplitude and fluctuations of the high-K(+)-induced current. The relation between the variance of the current fluctuations and the mean current amplitude was linear in every experiment using dialysis of AMP-PNP or Mg(2+)-free internal solution, or using superfusion of ACh. The slopes of these relations gave comparable single channel current amplitudes of -0.7 pA at -50 mV. These results indicate that the spontaneous opening of the muscarinic K+ channels is largely responsible for the high-K(+)-induced current. In the high-K+ solution, the variance-mean relation at -50 mV showed that the muscarinic K+ channel provides an inward current of 3.12 +/- 2.13 pA pF-1 (n = 23), which was about 60% of the total inward background current. In the normal K+ solution, the variance-mean relation at -50 mV indicated that an outward current of 6.0 +/- 2.0 pA (0.33 +/- 0.28 pA pF-1, n = 8) was provided by the K+ channel. The single channel current amplitude was estimated to be 0.06 +/- 0.02 pA (n = 9). Cell-attached recordings in the absence of ACh demonstrated sporadic and brief openings of channels identical to the ACh-induced channels. The power density spectra of the single channel currents exhibited kinetic properties comparable with those of the whole-cell currents.(ABSTRACT TRUNCATED AT 400 WORDS)