Calcium channel activation in arterioles from genetically hypertensive rats.

Enhanced contractile responsiveness to the calcium channel agonist Bay K 8644 has been documented in large conduit arteries and small muscular arteries from hypertensive rats. The present study examined the effects of Bay K 8644 on the intracellular calcium concentration ([Ca2+]i) in microvessels from stroke-prone spontaneously hypertensive rats and normotensive Wistar-Kyoto rats. Using microspectrofluorometry of fura-2, [Ca2+]i was measured in smooth muscle cells localized on arteriolar fragments (15-35 microns external diameter) isolated after collagenase digestion of the pancreas. Resting [Ca2+]i in hypertensive arterioles (94 +/- 6 nM, n = 29) did not differ from that in normotensive vessels (81 +/- 4 nM, n = 40). KCl (50 mM), applied alone and in the presence of Bay K 8644 (30 nM), stimulated increases in [Ca2+]i that were reversed in calcium-free solution and with nifedipine (10 microM), consistent with activation of potential-operated calcium channels. Potassium-induced calcium transients were consistently potentiated by Bay K 8644. The change in [Ca2+]i evoked by KCl alone or in combination with Bay K 8644 did not differ between arterioles from hypertensive and normotensive rats. In 24% of the vessels from hypertensive rats and in 29% of those from normotensive rats, Bay K 8644 evoked an increase in [Ca2+]i that did not differ significantly between the two strains. The findings indicate that, in contrast to observations made in larger arteries, there is no evidence of a functional abnormality in potential-operated calcium channels in very small arterioles from genetically hypertensive rats.     

Ionic currents in single smooth muscle cells of the canine renal artery.

Membrane currents from single smooth muscle cells enzymatically isolated from canine renal artery were recorded using the patch-clamp technique in the whole-cell and cell-attached configurations. These cells exhibited a mean resting potential, input resistance, membrane time constant, and cell capacitance of -51.8 +/- 2.1 mV, 5.2 +/- 0.98 G omega, 116.2 +/- 16.4 msec, and 29.1 +/- 2.0 pF, respectively. Inward current, when elicited from a holding potential of -80 mV, activated near -50 mV, reached a maximum near 0 mV and was sensitive to the dihydropyridine agonist Bay K 8644 and dihydropyridine antagonist nisoldipine. Two components of macroscopic outward current were identified from voltage-step and ramp depolarizations. The predominant charge carrier of the net outward current was identified as K+ by tail-current experiments (reversal potential, -61.0 +/- 0.8 mV in 10.8 mM [K+]o 0 mM [K+]i). The first component was a small, low-noise, voltage- and time-dependent current that activated between -40 and -30 mV (IK(dr)), and the second component was a larger, noisier, voltage- and time-dependent current that activated at potentials positive to +10 mV (IK(Ca)). Both IK(dr) and IK(Ca) displayed little inactivation during long (4-second) voltage steps. IK(Ca) and IK(dr) could be pharmacologically separated by using various Ca2+ and K+ channel blockers. IK(Ca) was substantially inhibited by external NiCl2 (500 microM), CdCl2 (300 microM), EGTA (5 mM), tetraethylammonium (Ki at +60 mV, 307 microM), and charybdotoxin (100 nM) but was insensitive to 4-aminopyridine (0.1-10 mM). IK(dr) was inhibited by 4-aminopyridine (Ki at +10 mV, 723 microM) and tetraethylammonium (Ki at +10 mV, 908 microM) but was insensitive to external NiCl2 (500 microM), CdCl2 (300 microM), EGTA (5 mM), and charybdotoxin (100 nM). Two types of single K+ channels were identified in cell-attached patches. The most abundant K+ channel that was recorded exhibited voltage-dependent activation, was blocked by external tetraethylammonium (250 microM), and had a large single-channel conductance (232 +/- 12 pS with 150 mM K+ in the patch pipette, 130 +/- 17 pS with 5.4 mM K+ in the patch pipette). The second channel was also voltage dependent, was blocked by 4-aminopyridine (5 mM), and exhibited a smaller single-channel conductance (104 +/- 8 pS with 150 mM K+ in the patch pipette, 57 +/- 6 pS with 5.4 mM K+ in the patch pipette). These results suggest that depolarization of canine renal artery cells opens dihydropyridine-sensitive Ca2+ channels and at least two K+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of adenosine-induced inhibition of calcium current in guinea pig ventricular cells

The mechanism of adenosine-induced inhibition of Ca2+ currents was studied by recording single-channel Ca2+ currents from cell-attached patches on isolated guinea pig ventricular cells with pipettes containing 50 or 100 mM Ba2+. Numerous 100-msec depolarization steps were applied repetitively at 2 Hz from the resting potential of approximately -70 mV. The addition of 0.1 mM adenosine to the superfusate in the presence of 100 nM isoproterenol depressed the isoproterenol-induced increase in ensemble-averaged current: in peak amplitude, from 258 +/- 105% (mean +/- SD) (p less than 0.01) that of control in the presence of isoproterenol to 153 +/- 41% (n = 7) (p less than 0.05) by the addition of adenosine. In these patches, adenosine did not much affect the open probability in nonblank sweeps (control, 0.10 +/- 0.05; isoproterenol, 0.11 +/- 0.08; adenosine and isoproterenol, 0.08 +/- 0.05), but significantly decreased channel availability, defined as the rate of current-containing sweeps in total sweeps (control, 0.36 +/- 0.17; isoproterenol, 0.69 +/- 0.16 [p less than 0.01]; adenosine and isoproterenol, 0.45 +/- 0.20 [p less than 0.01]). The decrease of channel availability by adenosine was due to the shortening of the duration of the available state and the prolongation of that of the unavailable state. The depressive action of adenosine was suppressed by 0.1 mM theophylline. The single-channel conductance in the presence of 100 mM Ba2+ and Bay K 8644 was 26 pS and was not affected by isoproterenol or adenosine. We conclude that adenosine decreases Ca2+ current under beta-adrenergic stimulation mainly by reducing channel availability.     

Inotropic and calcium kinetic effects of calcium channel agonist and antagonist in isolated cardiac myocytes from cardiomyopathic hamsters.

The mechanism by which heart cells of cardiomyopathic (CM) hamsters become calcium overloaded is not known. We examined the number of slow calcium channels, calcium uptake via slow calcium channels, calcium pool sizes, and the contractile response to Bay K 8644, verapamil, and nifedipine using isolated cardiac myocytes from 8-9-month-old CM hamsters (BIO 14.6) and age-matched normal controls. The number of dihydropyridine binding sites as assessed by specific binding of [3H]PN200-110 was similar in the two groups (control hearts: Bmax = 333 +/- 89 [mean +/- SD] fmol/mg; CM hearts: Bmax = 357 +/- 75 fmol/mg; n = 5 experiments, p = 0.6). Current density through L-type calcium channels was determined using the whole-cell clamp technique (at -50 mV holding potential and -10 mV test potential) and was the same in CM myocytes (17.8 +/- 1.5 [mean +/- SD] pA/pF) and control myocytes (18.6 +/- 2.1 pA/pF) (n = 5 experiments, p = 0.5). The current-voltage relation (test potentials varied from -40 to +50 mV) was also the same in CM and control cells, as was apparent threshold, peak current, and reversal potential. However, the initial rate of 45Ca influx as well as the size of the rapidly exchangeable calcium pool was significantly greater in myocytes obtained from CM than from normal hamsters. In both myocyte preparations, Bay K 8644 increased the rate of 45Ca uptake by 25% at 60 seconds; verapamil decreased 45Ca uptake at 60 seconds by 16% and 17% in normal and CM hamsters, respectively. A similar inhibitory effect was observed with nifedipine. The amplitude of cell motion in cells driven at 1.5 Hz as assessed by an optical-video system increased progressively with increasing concentrations of extracellular calcium or Bay K 8644 in cardiac myocytes from normal or CM hamsters. However, the concentration-effect curves for the two effectors were shifted to the left in CM cells compared with cells from normal hamsters. Both preparations demonstrated similar contractile responses to verapamil and nifedipine. These findings demonstrate that single enzymatically dissociated cardiac myocytes from CM hamsters have impaired contractile properties analogous to those seen in the intact heart and thus provide a useful experimental system in which to study underlying cellular mechanisms operative in this model of heart failure. Our results further indicate that calcium overload in CM hamster cardiac myocytes may not be due to increased calcium influx via dihydropyridine-sensitive calcium channels, as suggested previously, but rather to abnormalities of intracellular calcium homeostasis.     

Hypoxia inhibits contraction but not calcium channel currents or changes in intracellular calcium in arteriolar muscle cells

OBJECTIVE: We tested the hypothesis that hypoxia inhibits currents through L-type Ca(2+) channels and inhibits norepinephrine-induced rises in intracellular Ca(2+) in cremasteric arteriolar muscle cells, thus accounting for the inhibitory effect of hypoxia on norepinephrine-induced contraction of these cells. METHODS: Single smooth muscle cells were enzymatically isolated from second-order and third-order arterioles from hamster cremaster muscles. The effects of hypoxia (partial pressure of oxygen: 10-15 mm Hg) were examined on Ba(2+) (10 mM) currents through L-type Ca(2+) channels by use of the perforated patch clamp technique. Also, the effect of hypoxia on norepinephrine-induced calcium changes was studied using Fura 2 microfluorimetry. RESULTS: Hypoxia inhibited the norepinephrine-induced (10 microM) contraction of single arteriolar muscle cells by 32.9 +/- 5.6% (mean +/- SE, n = 4). However, hypoxia had no significant effect on whole-cell currents through L-type Ca(2+) channels: the peak current densities measured at +20 mV were -3.83 +/- 0.40 pA/pF before hypoxia and -3.97 +/- 0.36 pA/pF during hypoxia (n = 15; p > 0.05). In addition, hypoxia did not inhibit Ca(2+) transients in arteriolar muscle cells elicited by 10 microM norepinephrine. Instead, hypoxia increased basal Ca(2+) (13.8 +/- 3.2%) and augmented peak Ca(2+) levels (29.4 +/- 7.3%) and steady-state Ca(2+) levels (15.2 +/- 5.4%) elicited by 10 microM norepinephrine (n = 21; p < 0.05). CONCLUSIONS: These data indicate that hypoxia inhibits norepinephrine-induced contraction of single cremasteric arteriolar muscle cells by a mechanism that involves neither L-type Ca(2+) channels nor norepinephrine-induced Ca(2+) mobilization. Instead, our findings suggest that hypoxia must inhibit norepinephrine-induced contraction by affecting a component of the signaling pathway that lies downstream from the increases in Ca(2+) produced by this neurotransmitter.     

Effects of quinidine on the sodium current of guinea pig ventricular myocytes. Evidence for a drug-associated rested state with altered kinetics

In guinea pig cardiac myocytes quinidine (20 microM) caused less than 10% tonic block reduction of the sodium current at -120 mV, but a fast pulse train reduced it more than 90%. Recovery from use-dependent block was time and voltage dependent, and was always slow (tau = 34 +/- 10 seconds at -160 mV; tau = 90 +/- 35 seconds at -120 mV; n = 15, mean +/- SD, p less than 0.001, paired t test). However, in association with repeated activation a fast component of recovery from block was observed: use-dependent unblocking. Availability of sodium channels for use-dependent unblocking was enhanced by hyperpolarization until a plateau was reached near -160 mV. Compared with the availability of drug-free sodium channels (h-curve), the voltage dependence of availability for use-dependent unblocking (h'-curve) was shifted by about 30 mV to more negative potentials, and its slope was reduced 2.5-fold. At -160 mV, the kinetics of development of availability of sodium channels for use-dependent unblocking were rapid (tau less than 10 msec). Depolarization to -120 mV reduced the availability of sodium channels for fast unblocking with a time constant of 191 +/- 46 msec (n = 14). Finally, block established by frequent brief depolarizations (activations) declined during prolonged inactivation. From these results we concluded that the time and voltage dependence of the availability of sodium channels for unblocking are considerably different from the availability for activation of drug-free channels, that rested drug-associated channels do exist, and that drug-associated channels do not conduct (or at least have a greatly reduced conductance) upon activation unless they first unblock. Furthermore, activated and inactivated channels have a different affinity for quinidine, and since quinidine can occupy the channel receptor even when "guarded," our results are incompatible with the guarded receptor hypothesis but can be explained within the framework of the modulated receptor hypothesis.     

Long-term and immediate effect of testosterone on single T-type calcium channel in neonatal rat cardiomyocytes

In the cardiovascular system, T-type calcium channels play an important role for the intracellular calcium homeostasis and spontaneous pacemaker activity and are involved in the progression of structural heart diseases. Androgens influence the cardiovascular physiology and pathophysiology. However, their effect on native T-type calcium currents (I(Ca,T)) remains unclear. To test the chronic effect of testosterone on the cardiac I(Ca,T), cultured neonatal rat ventricular cardiomyocytes were treated with testosterone (1 nM-10 microM) for 24-30 h. Current measurements were performed after testosterone washout to exclude any acute testosterone effects. Testosterone (100 nm) pretreatment significantly increased whole-cell I(Ca,T) density from 1.26 +/- 0.48 pA/pF (n = 8) to 5.06 +/- 1.75 pA/pF (n = 7; P < 0.05) and accelerated beating rate. This was attributed to both increased expression levels of the pore-forming subunits Ca(v)3.1 and Ca(v)3.2 and increased T-type single-channel activity. On single-channel level, the increase of the ensemble average current by testosterone vs. time-matched controls was due to an increased availability (58.1 +/- 4.2 vs. 21.5 +/- 4.0%, P < 0.01) and open probability (2.78 +/- 0.29 vs. 0.85 +/- 0.23%, P < 0.01). Cotreatment with the selective testosterone receptor antagonist flutamide (10 mum) prevented these chronic testosterone-induced effects. Conversely, acute application of testosterone (10 microM) decreased T-type single-channel activity in testosterone pretreated cells by reducing the open probability (0.78 +/- 0.13 vs. 2.91 +/- 0.38%, P < 0.01), availability (23.6 +/- 3.3 vs. 57.6 +/- 4.5%, P < 0.01), and peak current (-20 +/- 4 vs. -58 +/- 4 fA, P < 0.01). Flutamide (10 microM) did not abolish the testosterone-induced acute block of T-type calcium channels. Our results indicate that long-term testosterone treatment increases, whereas acute testosterone decreases neonatal rat T-type calcium currents. These effects seem to be mediated by a genomic chronic stimulation and a nongenomic acute inhibitory action.     

Effects of lidocaine on single cardiac sodium channels

Lidocaine block of single cardiac sodium channels was studied in cell free inside-out patches of ventricular cells isolated from guinea-pig hearts. When applied to the inner surface of the membrane lidocaine depressed Na channel currents by decreasing the probability P of the channels to open measured from the peaks of the averaged currents. In parallel to the decrease in P the relative number of empty sweeps (nulls) was increased. Half maximum block of the activity of single Na channels was observed at 2.9 microM. Lidocaine affected the gating behaviour of Na channels by shortening of the mean open time tau 0 from 0.44 +/- 0.17 (control) to 0.19 +/- 0.13 (5 microM lidocaine, holding potential-120 mV, test potential -60 mV). Five micromolar lidocaine completely suppressed burst-like openings of Na channels and abolished the slow decaying phase of the averaged currents. A shift from - 120 towards - 160 mV exerted relief from the effect of both P and tau 0.     

Direct evidence for calcium conductance of hyperpolarization-activated cyclic nucleotide-gated channels and human native If at physiological calcium concentrations

AIMS: The hyperpolarization-activated cyclic nucleotide-gated (HCN) current I(f)/I(HCN) is generally thought to be carried by Na(+) and K(+) under physiological conditions. Recently, Ca(2+) influx through HCN channels has indirectly been postulated. However, direct functional evidence of Ca(2+) permeation through I(f)/I(HCN) is still lacking. METHODS AND RESULTS: To possibly provide direct evidence of Ca(2+) influx through I(HCN)/I(f), we performed inside-out and cell-attached single-channel recordings of heterologously expressed HCN channels and native rat and human I(f), since Ca(2+)-mediated I(f)/I(HCN) currents may not readily be recorded using the whole-cell technique. Original current traces demonstrated HCN2 Ca(2+) inward currents upon hyperpolarization with a single-channel amplitude of -0.87+/-0.06 pA, a low open probability of 3.02+/-0.48% (at -110 mV, n=6, Ca(2+) 2 mmol/L), and a Ca(2+) conductance of 8.9+/-1.2 pS. I(HCN2-Ca2+) was significantly activated by the addition of cAMP with an increase in the open probability and suppressed by the specific I(f) inhibitor ivabradine, clearly confirming that Ca(2+) influx indeed was conducted by HCN2 channels. Changing [Na(+)] (10 vs. 100 mmol/L) in the presence or absence of 2 mmol/L Ca(2+) caused a simple shift of the reversal potential along the voltage axis without significantly affecting Na(+)/Ca(2+) conductance, whereas the K(+) conductance of HCN2 increased significantly in the absence of external Ca(2+) with increasing K(+) concentrations. The mixed K(+)-Ca(2+) conductance, however, was unaffected by the external K(+) concentration. Notably, we could also record hyperpolarization-activated Ca(2+) permeation of single native I(f) channels in neonatal rat ventriculocytes and human atrial myocytes in the presence of blockers for all known cardiac calcium conduction pores (Ca(2+) conductance of human I(f), 9.19+/-0.34 pS; amplitude, -0.81+/-0.01 pA; open probability, 1.05+/-0.61% at -90 mV). CONCLUSION: We directly show Ca(2+) permeability of native rat and, more importantly, human I(f) at physiological extracellular Ca(2+) concentrations at the physiological resting membrane potential. This might have particular implications in diseased states with increased I(f) density and HCN expression.     

Incorporation of calcium channels from cardiac sarcolemmal membrane vesicles into planar lipid bilayers.

When purified porcine cardiac sarcolemmal membrane vesicles are incorporated into planar lipid bilayers formed at the tip of patch electrode pipettes, individual divalent cation channels can be monitored. Channel activity is increased in the presence of the Ca2+ channel agonist Bay K 8644, is voltage dependent, and selects for divalent cations over anions. The activity does not inactivate because it is maintained during prolonged depolarizations. Determination of divalent cation selectivity from the reversal potential of single-channel currents indicates a relative permeability ratio for Ba/Ca/Mg of 1:0.45:0.08. Mean channel conductance in 0.1 M Ba2+/0.01 M Mg2+ is 8 pS. Channels are reversibly blocked by the Ca2+ channel inhibitor nitrendipine, and inhibition can be competitively antagonized by Bay K 8644. Binding studies with 3H-labeled D-600 demonstrate the presence of high-affinity receptors for D-600 in sarcolemmal membranes (Kd = 6.4 X 10(-9) M; Bmax = 3 pmol per mg of protein). In addition, experiments with resolved D-600 stereoisomers indicate that (-)D-600 is at least 25-fold more potent than (+)D-600 in competing for this aralkyl amine receptor. Consistent with this, (-)D-600 is much more effective than the (+) isomer in inhibiting bilayer-incorporated channels. These results demonstrate that the divalent cation channel that has been reconstituted in planar lipid bilayers possesses many of the characteristics of voltage-regulated Ca2+ channels in heart and suggest that receptors for Ca2+ entry blockers are functionally associated with this channel.     

Multiple types of Ca2+ currents in single canine Purkinje cells

Whole-cell Ca2+ channel currents were recorded from isolated single canine Purkinje and ventricular cells to determine whether there were multiple types of Ca2+ channels in these two cell types, as in many other excitable tissues. The experimental conditions were such that currents other than Ca2+ channel currents were largely suppressed. The charge carrier was either Ca2+ or Ba2+ (5mM). In every canine Purkinje cell studied (n = 36), we saw T and L Ca2+ channel currents that are similar to their counterparts in other tissues. Neither current was affected by tetrodotoxin (30 microM), but both were reduced by Mn2+ (5mM). Ni2+ (50 microM) blocked T more than L current. Nisoldipine (1 microM) apparently abolished the L current but also decreased the T current by 50%. Substitution of Ba2+ for Ca2+ augmented and prolonged L current but did not affect T current significantly. At 36 degrees C and with 5 mM [Ca2+]o, T current inactivated over a voltage range from -70 to -30 mV whereas L current inactivated between -30 and +20 mV. T current was detectable in only some of the ventricular cells studied (8 out of 12). In these cells the ratio of maximal T current to maximal L current (0.2 +/- 0.1, n = 8) was lower than the T/L ratio in Purkinje cells (0.6 +/- 0.2, n = 6). The density of peak L current in ventricular cells (7.5 +/- 1.7 pA/pF, n = 8) was higher than that in Purkinje cells (4.4 +/- 3.4 pA/pF, n = 6). Therefore, in ventricular cells the L current is the main Ca2+ current whereas in Purkinje cells, the T current also contributes significantly to membrane electrical activity. In Purkinje cells, beta-adrenoceptor stimulation by isoproterenol (1 microM) increased L current but did not affect T current. On the other hand, in 70% (7 out of 10) of the Purkinje cells, alpha-adrenoceptor stimulation by 10 microM norepinephrine (in the presence of 2 microM propranolol) increased the T current. Our observations show that the distribution of the two types of Ca2+ channels in canine ventricle is heterogeneous and that the two types of Ca2+ channels are modulated by catecholamines by different receptors.     

Late sodium current and its contribution to action potential configuration in guinea pig ventricular myocytes

We used the patch clamp technique to study the nature of the late sodium current in guinea pig ventricular myocytes. In a cell attached mode of single channel recording at room temperature (22-24 degrees C) two kinds of late (100 msec or more after beginning of the depolarizing pulse) sodium channel activities were recognized. One is isolated brief openings appearing once for about 120 depolarizations per channel (background type), while the other type is sustained openings with rapid interruptions (burst type) that occurred only once for 2,700 depolarizations per channel. The time constant obtained from the open time histogram of the burst type (1.05 msec) was about five times longer than that of background type (0.18 msec, measured at the potential 10 mV above the threshold). Magnitude of the late sodium current flowing through the entire surface of a myocyte was estimated with tetrodotoxin (60 microM), a specific inhibitor of sodium channels, in whole-cell clamp experiments. The steady tetrodotoxin-sensitive current of 12 to 50 pA was registered at -40 mV (26 +/- 14 pA, mean +/- SD, n = 5), in good agreement with the late sodium current calculated from the single channel recording. Tetrodotoxin produced small (congruent to 10%) but significant decreases in the action potential duration. These results suggest the presence of a small but significant late sodium current with slow inactivation kinetics and that this current probably plays a significant role in maintaining the action potential plateau and the duration in guinea pig ventricular myocytes.     

High molecular weight proteins purified from cardiac junctional sarcoplasmic reticulum vesicles are ryanodine-sensitive calcium channels

The cardiac high molecular weight proteins/ryanodine receptors were purified to homogeneity from junctional sarcoplasmic reticulum membranes and shown to exhibit large conductance calcium channel activity. High molecular weight proteins were solubilized from junctional sarcoplasmic reticulum in zwitterionic detergent and purified by size-exclusion chromatography followed by sucrose density gradient centrifugation. The purified proteins exhibited an apparent Mr = 400,000-350,000, and bound [3H]ryanodine with a Kd of 4.6 nM and a Bmax of 140-280 pmol/mg protein. High molecular weight proteins demonstrated divalent cation channel activity after incorporation into planar lipid bilayers. Two channel types were identified. Large conductance channels had a slope conductance of 96 +/- 13 pS and a Erev of 42 +/- 9 mV (n = 5); small conductance channels had a slope conductance of 5.5 +/- 1 pS [1.0 microM cis CaCl2; 50 mM trans Ba(OH)2]. Reducing cis calcium from 1 microM to 1 nM reduced the large conductance channel open time from 7 +/- 1% to 0.1% (holding potential, -100 mV). Adding ATP (1 mM) to the cis chamber increased channel open time from 6 +/- 1% to 52 +/- 4% (holding potential, -100 mV); 10 nM ryanodine increased and 100 microM ryanodine decreased percent of open time of the 96 pS channel, without altering unitary channel conductance. The large conductance channel was similar to the calcium release channel detected in native canine cardiac junctional sarcoplasmic reticulum vesicles. Our data suggest that the ryanodine receptor, the calcium-release channel, and the high molecular weight proteins are all identical proteins containing allosteric regulatory sites for calcium, ATP, and ryanodine.     

Characterization of dihydropyridine-sensitive calcium channels in rat brain synaptosomes.

We examined the effects of dihydropyridine Ca2+-channel agonists on synaptosomal voltage-dependent Ca2+ entry and endogenous dopamine release. The (-) isomer of Bay K 8644 and the (+) isomer of Sandoz compound 202-791 were 100-1000 times more potent than their respective opposite enantiomers in enhancing Ca2+ uptake and dopamine release from striatal synaptosomes. The active isomer of each of these compounds increased Ca2+ entry and dopamine release to the same extent at a concentration of 1 nM. Fast-phase Ca2+ entry into synaptosomes isolated from cerebellum, cortex, and hippocampus was sensitive to nanomolar concentrations of Bay K 8644. No effect of Bay K 8644 was observed in synaptosomes isolated from brainstem. Bay K 8644 increased synaptosomal Ca2+ uptake and endogenous dopamine release from striatal synaptosomes only during the initial seconds of KCl-induced depolarization. The greatest increase was observed during the first second of depolarization. No effect was observed after greater than or equal to 5 sec of depolarization. Bay K 8644 did not alter Ca2+ uptake or dopamine release under resting conditions (5 mM KCl) or in response to KCl at greater than 15 mM. The activity of Bay K 8644 was also attenuated by lowering the concentrations of divalent cations in the incubation medium. Agonist activity was observed at Mg2+ concentrations greater than 500 microM (Ca2+ held at 100 microM) and Ca2+ concentrations greater than 100 microM (Mg2+ held at 1000 microM). These results suggest that the Ca2+ channels present in synaptosomes are sensitive to nanomolar concentrations of dihydropyridine agonists under a narrow range of experimental conditions.     

A role for a glibenclamide-sensitive, relatively ATP-insensitive K+ current in regulating membrane potential and current in rat aorta

OBJECTIVE: ATP-sensitive K+ channels have been classified based on their inhibition by cytoplasmic ATP. Recent evidence in vascular smooth muscle has suggested that these channels show weak sensitivity to intracellular ATP. However, it is not known whether these channels regulate the resting K+ conductance in vascular smooth muscles. Therefore, the aim of the present investigation was to characterize this current in rat aorta myocytes and to examine whether it contributes to setting the membrane potential. METHODS: The conventional and nystatin-permeablised whole cell patch clamp techniques were used to characterize the effect of glibenclamide on membrane potential and K+ current in enzymatically dispersed rat aorta myocytes. RESULTS: The mean resting potential measured in current clamp mode using the permeabilized patch approach was -54 +/- 5 mV (n = 8). Glibenclamide (10 microM) caused a reversible 24-mV depolarization in these cells. In symmetrical K+ (135 mM) solution an inward glibenclamide-sensitive (10 microM) current (-4.1 +/- 0.7 pA/pF; n = 5), hereafter termed Iglib, was observed at a membrane potential of -80 mV when cells held at -60 mV were ramped from -80 to +80 mV. In the absence of any nucleotide in the pipette solution, Iglib measured by the conventional whole-cell method was -23.69 +/- 4.65 pA/pF (n = 9). With 1 and 3 mM ATP in the pipette, the average current density was -25 +/- 6.3 pA/pF (n = 8), and -9.4 +/- 2.7 pA/pF (n = 9), respectively. In the absence of ATP, 1 mM GDP significantly (P < 0.01) increased Iglib (-44.8 +/- 8.4 pA/pF; n = 13). Inclusion of 1 mM ATP in the GDP-containing pipette solution had no significant effect on the current amplitude (-56.4 +/- 10.7 pA/pF; n = 7). Iglib fell to -11.0 +/- 2.9 pA/pF (n = 10) if 1 mM GDP and 3 mM ATP were present. In symmetrical K+, the Iglib observed in the presence of 1 mM ATP in the pipette was increased by more than two-fold in the presence of 10 microM levcromakalim. In PSS containing 5 mM K+, a significant glibenclamide-sensitive current was observed at -45 mV membrane potential when cells dialyzed with 1 mM ATP were ramped between -80 to 30 mV. CONCLUSION: These results demonstrate that Iglib channels in rat aorta myocytes differ from classical KATP channels, being relatively insensitive to intracellular ATP. Iglib therefore appears to have an important role in contributing to the maintenance of the resting potential in rat aortic smooth muscle.     

Effect of ipratropium bromide on calcium activated potassium channel in tracheal smooth muscle cells from chronically hypoxic rats]

OBJECTIVE: To study the effects of ipratropium bromide on large conductance calcium activated potassium (BK(Ca)) channel in tracheal smooth muscle cells (TSMC) from chronically hypoxic rat. METHODS: The chronically hypoxic rat model was established. Single TSMC was acutely isolated. Single channel currents were recorded by using the patch clamp technique in inside-out configuration. RESULTS: (1) Chronic hypoxia decreased BK(Ca) channel activity significantly in inside-out recording. Channel open probability (P(0)) of BK(Ca) channel in the chronically hypoxic rats reduced significantly compared with that in the control rats (0.17 +/- 0.07 for the hypoxia, 0.35 +/- 0.10 for the control, n = 30, t = 8.22, P < 0.01). Channel open time constants were significantly shortened in the chronically hypoxic group. The fast and slow open time constants (tau(O1), tau(O2))were significantly different from those in the control [tau(O1) (1.49 +/- 0.41) ms, (0.53 +/- 0.23) ms, respectively, n = 30, t = 12.07, P < 0.01; tau(O2) (11.9 +/- 3.2) ms, (3.8 +/- 1.4) ms, respectively, n = 30, t = 12.60, P < 0.01]. Closing time prolonged significantly. The fast and slow closing time constants (tau(c1), tau(c2)) were significantly different from those in the control [tau(c1) (2.7 +/- 0.9) ms, (5.7 +/- 1.5) ms, respectively, n = 30, t = 8.71, P < 0.01; tau(c2) (12.1 +/- 2.3) ms, (19.4 +/- 2.9) ms, respectively, n = 30, t = 14.05, P < 0.01]. Ipratropium bromide and salbutamol reversed the effect of chronic hypoxia on BK(Ca) channel. P(0) increased, tau(O1) and tau(O2) were prolonged significantly. tau(c1) and tau(c2) were shortened [P(0), 0.15 +/- 0.04, 0.28 +/- 0.09, 0.30 +/- 0.08, respectively, n = 25, F = 39.90, P < 0.01; tau(O1) (0.55 +/- 0.24) ms, (0.89 +/- 0.25) ms, (1.03 +/- 0.33) ms, respectively, n = 25, F = 15.32, P < 0.01; tau(O2) (3.6 +/- 1.4) ms, (6.3 +/- 1.9) ms, (6.9 +/- 2.0) ms, respectively, n = 25, F = 40.10, P < 0.01; tau(c1) (6.1 +/- 1.6) ms, (3.3 +/- 1.2) ms, (3.0 +/- 0.8) ms, respectively, n = 25, F = 57.14, P < 0.01; tau(c2) (20.1 +/- 2.5) ms, (12.4 +/- 2.6) ms, (13.0 +/- 2.0) ms, respectively, n = 25, F = 24.60, P < 0.01]. CONCLUSION: Chronic hypoxia decreased BK(Ca) channel activity.Ipratropium bromide and salbutamol reversed the effect of chronic hypoxia on BK(Ca) channel. The relaxing effect of ipratropium bromide and salbutamol on TSMC may be partly mediated via activation of BK(Ca) channel.     

Developmental changes in the beta-adrenergic modulation of calcium currents in rabbit ventricular cells.

We studied the developmental changes in the beta-adrenergic modulation of L-type calcium current (ICa) in enzymatically isolated adult (AD) and newborn (NB, 1-4-day-old) rabbit ventricular cells using the whole-cell patch-clamp method. ICa was measured as the peak inward current at a test potential of +15 mV by applying a 180-450-msec pulse from a holding potential of -40 mV with Cs(+)-rich pipettes and a K(+)-free bath solution at room temperature. In control, ICa density (obtained by normalizing ICa to the cell capacitance) was significantly higher in AD cells (5.5 +/- 0.2 [mean +/- SEM] pA/pF, n = 65) than in NB cells (2.6 +/- 0.1 pA/pF, n = 60). Isoproterenol (ISO, 1 nM-30 microM) increased ICa in a dose-dependent manner for both groups. The maximal effect (Emax) of ISO, expressed as percent increase in ICa over control levels, and the concentration for one half of the maximal effect (EC50) were 203% and 51 nM, respectively, for AD cells and 111% and 81 nM, respectively, for NB cells. The effect of ISO (1 microM) on ICa was decreased as the test potential was increased from -10 to +40 mV. However, the ratio of the percent increase in ICa for AD versus NB cells was almost constant (2.09-2.45) at each test potential. Dose-response curves of forskolin (FOR, 0.3-50 microM) gave Emax and EC50 of 268% and 0.74 microM, respectively, for AD cells and 380% and 1.15 microM, respectively, for NB cells. After stimulating ICa by 10 microM ISO, the addition of 10 microM FOR produced a further increase in ICa of only 12 +/- 2% in AD cells (n = 4) but a further increase of 140 +/- 41% in NB cells (n = 6). FOR (10 microM) did not produce any increase in ICa for AD and NB cells after stimulating ICa by intracellular application of 200 microM cAMP. ICa density stimulated by 10 microM ISO (17.8 +/- 1.1 pA/pF, n = 7), 10 microM FOR (21.0 +/- 1.3 pA/pF, n = 8), or 200 microM cAMP (18.0 +/- 1.3 pA/pF, n = 5) was equivalent in AD cells, whereas ICa density stimulated by 10 microM ISO (5.8 +/- 0.6 pA/pF, n = 9) was significantly lower than that stimulated by either 10 microM FOR (13.8 +/- 1.5 pA/pF, n = 7) or 200 microM cAMP (13.4 +/- 0.7 pA/pF, n = 7) in NB cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dihydropyridine receptors are primarily functional L-type calcium channels in rabbit ventricular myocytes.

We measured [3H]PN200-110 binding and patch-clamp currents in rabbit ventricular myocytes to determine if there is a disparity between the density of dihydropyridine-specific receptors and functional L-type calcium channels, as has been reported for skeletal muscle. The dihydropyridine receptor density was 74.7 +/- 4.2 fmol/mg protein (mean +/- SEM, Kd = 1.73 +/- 0.29 nM, n = 6) in ventricular homogenates and 147 +/- 6 fmol/mg protein (Kd = 1.15 +/- 0.16 nM, n = 4) in myocytes. Ventricular homogenates contained 121 +/- 9 mg protein/g wet wt (n = 7). These values were used to calculate a dihydropyridine receptor density of 12.9 dihydropyridine sites/micron2 for ventricular homogenates and 14.8 dihydropyridine sites/micron2 for myocytes. The number of functional L-type calcium channels (N) was calculated from measurements of whole-cell current (I), single-channel current (i), and open probability (po), where N = I/(i x po). We measured sodium current through calcium channels (I(ns)) to avoid calcium-induced inactivation. Whole-cell (I(ns)) and single-channel (i(ns) and po) measurements were obtained under similar ionic conditions at a test potential of -20 mV. In six cells, the peak I(ns) was approximately 105 pA/pF. The single-channel conductance was 40.8 +/- 2.6 pS (n = 12), and i(ns) at -20 mV was 1.96 pA. The mean po at -20 mV was 0.030 +/- 0.002 in 16 patches in which only a single channel was evident. The calculated density of functional L-type calcium channels was approximately 18 channels/micron2.(ABSTRACT TRUNCATED AT 250 WORDS)     

L- and T-type Ca2+ channels in canine cardiac Purkinje cells. Single-channel demonstration of L-type Ca2+ window current.

Canine cardiac Purkinje cells contain both L- and T-type calcium currents, yet the single Ca2+ channels have not been characterized from these cells. Additionally, previous studies have shown an overlap between the steady-state inactivation and activations curves for L-type Ca2+ currents, suggesting the presence of L-type Ca2+ "window" current. We used the on-cell, patch-clamp technique to study Ca2+ channels from isolated cardiac Purkinje cells. Patches contained one or more Ca2+ channels 75% of the time. L-type channels were seen in 69% and T-type channels in 73% of these patches. With 110 mM Ba2+ as the charge carrier, the conductances of the L- and T-type Ca2+ channels were 24.2 +/- 0.8 pS (n = 9) and 9.0 +/- 0.5 pS (n = 8), respectively (mean +/- SEM). With 110 mM Ca2+ as the charge carrier, the conductance of the L-type Ca2+ channel decreased to 9.7 +/- 1.2 pS (n = 4), whereas the T-type Ca2+ channel conductance was unchanged. Voltage-dependent inactivation was shown for both L- and T-type Ca2+ channels, although for L-type Ca2+ channel with Ba2+ as the charge carrier, inactivation took at least 30 seconds at a potential of +40 mV. After channel inactivation was complete, L-type Ca2+ channel reopenings were observed following repolarizing steps into the window voltage range. Thus, our data identify both L- and T-type Ca2+ channels in cardiac Purkinje cells and demonstrate, at the single-channel level, L-type channel transitions expected for a window current. Window current may play an important role in shaping the action potential and in arrhythmogenesis.     

L- and T-type voltage-gated Ca2+ channels in human granulosa cells: functional characterization and cholinergic regulation

Using the whole-cell configuration of the patch-clamp technique, we have characterized two types of ionic currents through voltage-dependent Ca2+ channels in human granulosa cells. One is long-lasting, activates at approximately -20 mV, reaches the peak at approximately +20 mV, has an inactivation time constant of 132.5 +/- 5.6 msec at 20 mV, and is sensitive to dihydropyridines. The other is transient, activates at approximately -40 mV, peaks at approximately -10 mV, has an inactivation time constant of 38.8 +/- 1.8 msec at -10 mV, displays a voltage-dependent inactivation, and is sensitive to 100 microm Ni2+, but not to dihydropyridines. Biophysical and pharmacological properties of these currents indicate that they are gated through L- and T-type calcium channels, respectively. The cholinergic receptor agonist carbachol (50 microm) reduces the amplitude of the currents through both L-type (-34.7 +/- 6.4%; n = 10) and T-type (-52.6 +/- 7.4%; n = 8) channels, suggesting a possible role of these channels in the cholinergic regulation of human ovarian functions.