Properties of membrane currents in isolated smooth muscle cells from guinea-pig trachea

Tracheal smooth muscle cells were enzymatically isolated from guinea-pig trachea. These cells contracted in response to acetylcholine (0.01–10 M) in a concentration-dependent fashion. Under current-clamp conditions with 140 mM K⁺ in the pipette solution, the membrane potential oscillated spontaneously at around –30 mV. Under voltage-clamp conditions, there appeared spontaneous but steady oscillations of outward current (I _o). On depolarization from a holding potential at –40 mV, three components of outward current were elicited: transient outward current (I _T), steady-state outward current (I _s) and I _o. These three components of outward current reversed around the K⁺ equilibrium potential and were abolished by Cs⁺ in the pipette, indicating that K⁺ was the major charge carrier of these outward currents. All these three components were completely suppressed by extracellular tetraethylammonium (10 mM). Both I _T and I _o were depressed by quinidine (1 mM), 4-aminopyridine (10 mM) and nifedipine (100 nM), but I _s was not affected. I _T and I _o were suppressed by a Ca²⁺-free perfusate with less than 1 nM Ca²⁺ in the pipette, while with 10 nM Ca²⁺ in the pipette, only I _o was suppressed. In both conditions, I _s was not affected by the Ca²⁺-free perfusate. Therefore, it is suggested that I _o, I _T and I _s are separate types of K⁺ current. With Cs⁺ in the pipette, K⁺ currents were almost completely suppressed and a transient inward current was observed during depolarizing pulses. The inward current was not affected by tetrodotoxin and increased when the concentration of extracellular Ca²⁺ was raised, indicating that the current is a Ca²⁺ channel current. Even with a holding potential of –80 mV, the low-threshold inward current could not be observed. The high-threshold Ca²⁺ current was abolished by nifedipine (100 nM) and was enhanced by Bay K 8644 (100 nM). The order of permeation of divalent cations through the Ca²⁺ channel was Ba²⁺ >Sr²⁺ Ca²⁺. Cd²⁺ blocked the Ca²⁺ current more effectively than Ni²⁺. These results may indicate that the Ca²⁺ current of tracheal smooth muscle cells is mainly composed of the current through an L-type Ca²⁺ channel.     

Ultra-slow voltage-dependent inactivation of the calcium current in guinea-pig and ferret ventricular myocytes

L-type Ca²⁺ current, i _Ca, has been recorded in guinea-pig ventricular myocytes at 36° C using the whole cell patch clamp technique. Intracellular Ca²⁺ was buffered with ethylenebis(oxonitrilo)tetraacetate (EGTA). An increase in the rate of stimulation from 0.5 to 3 Hz resulted in an abrupt decrease in i _Ca in the first beat at the high rate, followed by a progressive decrease ( approx. 7 s) over the next 30 s. The changes were not the result of Ca²⁺-dependent inactivation, because similar changes occurred with either Ba²⁺ or Na⁺ as the charge carrier. During 20-s voltage clamp pulses there was an ultra-slow phase of inactivation of Ba²⁺ or Na⁺ current through the Ca²⁺ channel ( approx. 6 s at 0 mV). This was confirmed by applying test pulses after conditioning pulses of different duration: the Ba²⁺ current during the test pulse decreased progressively when the duration of the conditioning pulse was increased progressively to 20 s. Ultra-slow inactivation of Ba²⁺ current was voltage dependent and increased monotonically at more positive potentials. Recovery of Ba²⁺ current from ultra-slow inactivation occurred with a time constant of 3.7 s at –40 mV and 0.7 s at –80 mV. The gradual decrease in i _Ca on increasing the rate to 3 Hz may have been the result of the development of ultra-slow voltage-dependent inactivation.     

高胆固醇血症对大鼠心室肌细胞离子电流的作用

目的：观察高胆固醇血症对大鼠心室肌细胞离子电流的作用。方法： 通过全细胞膜片钳技术记录用酶解法分离的正常和高胆固醇饮食的大鼠心室肌细胞离子电流。结果： 高胆固醇组（组Ⅱ）血清总胆固醇水平明显高于正常组（组Ⅰ）［（3.10±0.62)mmol·L-1 vs (1.18±0.37)mmol·L-1, P<0.01, n=20］。组Ⅱ血清甘油三酯也明显高于组Ⅰ［（1.51±0.30)mmol·L-1 vs (0.43±0.15)mmol·L-1, P<0.01, n=20］。组Ⅱ大鼠心室肌细胞动作电位时程（APD）与组I相比明显延长，APD50从(70.86±8.12)ms延长至(116.16±6.90)ms (n=10, P<0.01); APD90 从(95.10±7.27)ms延长至(144.04±7.39)ms (n=10, P<0.01)；在实验电压 -120 mV， Ik1从(-16.98±4.54) pA/pF（组I）增加到(-19.92±4.08) pA/pF（组Ⅱ）(n=12, P<0.05)；在实验电压 0 mV， ICa-L从(-8.56±1.29) pA/pF（组Ⅰ）减少到(-5.24±0.90) pA/pF（组Ⅱ）(n=10, P<0.01)；在实验电压 +60 mV，Ito从(13.20±1.97) pA/pF（组I）减少到(10.30±1.97) pA/pF（组Ⅱ）(n=8, P<0.05)。结论： 高胆固醇血症可显著改变心肌细胞离子电流密度的大小，对心脏具有毒性作用。     

Effects of calcitonin gene-related peptide on membrane currents in mammalian cardiac myocytes

We examined the effects of calcitonin gene-related peptide (CGRP) on the membrane currents of single atrial and ventricular cells of guinea pig heart. The tightseal whole-cell voltage-clamp technique was used. In atrial cells, like isoproterenol, CGRP increased the L-type Ca channel current (I _Ca.L) in a concentration-dependent manner. Human CGRP-(8-37), a putative CGRP receptor antagonist, completely abolished the CGRP-induced increase of I _Ca.L. Although the effects of CRGP were similar to those of isoproterenol, propranolol, a -adrenergic receptor antagonist, did not affect the CGRP-induced increase of I _Ca.L. After I _Ca.L had been maximally activated by isoproterenol (2 M) or intracellular cyclic adenosine 5-monophosphate (100 M), CGRP failed to increase I _Ca.L. Acetylcholine antagonized the effects of CGRP on I _Ca.L. Unlike the effects on atrial cells, CGRP had no significant effects on the membrane currents of ventricular myocytes. Thes results indicate that CGRP increases I _Ca.L via adenylate cyclase activation by binding to specific membrane receptors in cardiac atrial myocytes. Furthermore, CGRP receptors are expressed in atrial cells but probably not in ventricular cells.     

Effects of the protein kinase A inhibitor H-89 on Ca2+ regulation in isolated ferret ventricular myocytes

 We investigated the effects of a protein kinase A (PKA) inhibitor, H-89 {N-[2-(p-bromocinnamylamino)ethyl]-5-iso-quinolinesulphonamide}, on Ca²⁺ regulation in Fura-2-loaded ferret myocytes. H-89 (10 μmol/l) decreased the amplitude of the Fura-2 transient to 28.2±4.3% (P<0.001) of control and prolonged its duration, characterized by a decrease in the rate of decline of Ca²⁺ to diastolic levels: t _1/2 increased from 311±35 ms to 547±43 ms (P<0.001, n=7). Reduced Ca²⁺ uptake by the sarcoplasmic reticulum (SR) in the presence of H-89 was also indicated by a decrease in the SR Ca²⁺ content, as assessed with caffeine. The apparent slowing of the SR Ca²⁺-ATPase was not caused by changes in phosphorylation of phospholamban (PLB). However, Ca²⁺ uptake in microsomal vesicles prepared from canine hearts and fast-twitch rat skeletal muscle (which lacks PLB) was decreased by 34.1 and 46.8% (n=3), respectively, suggesting that H-89 has a direct inhibitory effect on the SR Ca²⁺-ATPase. In electrophysiological experiments, 5.0 μmol/l H-89 decreased the L-type Ca²⁺ current (I _Ca) by 39.5% (n=6) and slowed the upstroke of the action potential and, in some cases, caused loss of excitability without changes in the resting membrane potential. In summary, data show that [Ca²⁺ ]_i regulation, and hence contraction, is sustained by PKA-mediated phosphorylation, even in the absence of β-agonists. However, the use of H-89 as a tool to study the role of this signalling pathway is limited by the non-specific effects of H-89 on the SR Ca²⁺-ATPase. Received: 4 September 1998 / Received after revision: 19 October 1998 / Accepted: 20 October 1998     

Effects of extracellular ATP on hair cells isolated from the guinea-pig semicircular canals

Externally applied ATP (100 μM) induced membrane currents in type I and type II vestibular hair cells enzymatically isolated from guinea-pig semicircular canals. In whole-cell voltage-clamp and with 140 mM K⁺ in the pipette solution, ATP evoked an inwardly directed current in 58% of the cells when held at potentials below −40 mV. In the remainder, external ATP produced an outward current. After block of the K currents, an inward current activated by ATP was revealed at −50 mV. Intracellular Ca²⁺ levels were monitored using the Ca²⁺ indicator Fura-2 and were found to rise in both hair cell types in response to ATP. These results strongly suggest that ATP directly controls the entry of Ca²⁺ into crista hair cells which can then further modulate K⁺ currents.     

Voltage clamp of single freshly dissociated smooth muscle cells: Current-voltage relationship for three currents

Voltage-clamp experiments on single freshly dissociated (i.e. uncultured) vertebrate smooth muscle cells were carried out under conditions where the inital inward current, as well as various phases of outward current, could be studied. Current-voltage relationships were obtained for the initial current, the peak outward current, and a later, steady-state outward current, over a potential range of approximately –130 mV to +50 mV. Evidence is presented that the initial current is carried by Ca⁺⁺ ions and is responsible for the rising phase of the action potential and that the peak in the outward current is due to Ca⁺⁺ activation of K⁺ conductance.     

Photoelectric recording of mechanical responses of cardiac myocytes

A method to monitor contraction of isolated myocytes by transmicroscopic photometry is illustrated. Two photodiodes are mounted inside an inverse microscope used for visual control of a cell. Illumination of one diode varies in proportion to changes in cell length. The contraction signal is amplified in a comparator circuit. Spatial resolution of the device is in the order of 1 m which corresponds to about 5% of cell shortening in the fully activated state of contraction. The method was tested on isolated myocytes from guinea-pig ventricle. Optical records of contraction in response to action potentials or during voltage clamp compare well with the contractile behaviour of multicellular preparations.     

Action potential and membrane currents of single pacemaker cells of the rabbit heart

Single, viable pacemaker cells were isolated from sinoatrial (S-A) and atrioventricular (A-V) nodes by treating with collagenase. In normal Tyrode solution containing 1.8 mM Ca²⁺, these pacemaker cells had a round configuration and contracted rhythmically at a frequency of about 150–260/min. The amplitude, duration, and maximum rate of rise of the spontaneous action potentials recorded using patch clamp electrodes were similar to those obtained from multicellular preparations. Amplitudes of the recorded membrane current were normalized with reference to the surface area of the cell by assuming the cell shape as a plane oblate spheroid. The membrane resistance of the isolated nodal cells was 14.9±4.0 k·cm² (n=12) at about –35 mV and the membrane capacitance was 1.30±0/24 F/cm² (n=18). The inactivation time course of the slow inward current,i _si, was fitted with a sum of two exponentials with time constants of 6.7±0.6 ms and 46.6±15.3 ms (n=4) at +10 mV. The amplitude ofi _si peaked at 0+10 mV in the current-voltage relationship and was 18.2±8.4 A/cm². The potassium current,i _K was activated in the voltage range positive to –50 mV and was saturated at about +20 mV. The amplitude of the fully-activatedi _K at –40 mV was 3.3±1.4 A/cm² (n=10) and showed an inward-going rectification. The activation of the hyperpolarization-activated current was observed at potentials negative to –70 mV in seven of 14 experiments. The current density and membrane capacitance calculated could be overestimated and the membrane resistance underestimated, because of the presence of caveolae on the cell surface. However, these data give the nearest possible estimates of the electrical constants in the nodal cells, which cannot be measured accurately in the conventional multicellular preparations.     

Effects of various calmodulin antagonists on Na/Ca exchange current of single ventricular cells of guinea-pig

The effects of various calmodulin inhibitors were examined on the Na/Ca exchange current in single cardiac ventricular cells of the guinea-pig using the whole-cell patch-clamp technique. External application of W-7 and trifluoperazine inhibited Na/Ca exchange current in a dose-dependent manner with IC₅₀ values of 13 and 7 M, respectively. W-5 inhibited the exchange current but less potently than W-7. More specific calmodulin inhibitors such as CGS 9343B and calmidazolium did not, however, decrease the current as significantly as expected. All these drugs inhibited the Na current more strongly than the Na/Ca exchange current. Ruthenium red (RR), another type of calmodulin inhibitor, did not decrease the exchange current by internal application. Neither mastoparan or melittin (calmodulin-binding peptides) inhibited the exchange current appreciably. RR and the peptides did not affect the Na current either. These results indicate that calmodulin may not be involved in the activation of cardiac Na/Ca exchange or the Na current. Internal application of chymotrypsin inhibited the blocking effect of W-7 on the Na/Ca exchange current but not that on the Na current. These results indicate that W-7 blocks the Na/Ca exchange current not by binding to calmodulin but possibly by directly affecting an internal site of the exchanger itself and that the inhibitory action of W-7 is different on the Na/Ca exchange current and the Na current.     

Effect of acetylcholine on time-independent currents in sheep cardiac Purkinje fibers

Voltage-clamp experiments were carried out in sheep Purkinje fibers in order to find an explanation for the prolongation of the action potential, the positive shift of the plateau, the hyperpolarization of the maximum diastolic potential and the increase in rate of diastolic depolarization, occurring in the presence of acetylcholine (Ach).In the presence of Ach the instantaneous current-voltage relation is shifted in the inward direction for potentials positive to –75 mV, while the opposite shift is obtained for more negative potentials; the results suggest a decrease in background conductance.The contribution of K, Cl, Na and Ca to the Ach sensitive current was studied by varying K₀ concentration or adding 20 mmol·l^–1 Cs, by omitting Cl or Na, and by changing the Ca concentration.In 20 mmol·l^–1 Cs the apparent reversal potential of the Ach sensitive current is –50 mV, as compared to –75 mV in normal Tyrode. The component of the Ach sensitive current, which is suppressed by Cs, shows inward going rectification. In different K₀ concentrations the reversal potential of the Ach sensitive current is changed; the shift obeys the theoretical change in equilibrium potential of K. The results are consistent with a decrease in K background current by Ach (inward and outward rectifier).In Cl free media the Ach sensitive current is not decreased excluding a major contribution of Cl ions. The Ach effect also persists in Na free media; the reversal potential of the Ach sensitive current is slightly shifted in the hyperpolarizing direction. These results indicate that active electrogenic pumps (Na or Na–Ca) do not play an important role; they are in accord with a reduction in inward Na background current by Ach. The shift of the current-voltage relation by Ach was greater the lower the Ca_o concentration; the mechanism is not clear.The inward shift of the current at –40 mV was dependent on the Ach concentration. Half-maximum effect was obtained at 3·10^–7 mol·l^–1 Ach; the Hill coefficient was 1.12.It is concluded that Ach interacts in a one to one reaction with a muscarinic receptor and reduces the background current mainly carried by K (inward and outward rectifier), and less by Na (and probably Ca).Supported by F.G.W.O. Belgium 3.0087.74     

琥珀酸在海马CA1区对突触前GABA释放的影响

为了观察琥珀酸在大鼠海马CA1区对突触前GABA释放的影响,我们采用红外可视全细胞膜片钳技术记录了琥珀酸对γ-氨基丁酸(GABA)能自发性微小抑制性突触后电流(miniature inhibitory postsynaptic currents,mIPSCs)的作用。结果显示不同浓度的琥珀酸(10-6mol/L、10-5mol/L、10-4mol/L和10-3mol/L)在海马CA1区均能以浓度依赖的方式增强GABA能mIPSCs的频率,而对其电流幅度没有影响。10-4mol/L琥珀酸组GABA能mIPSCs的频率为2.25±0.99Hz,与正常对照组相比有显著性差异(n=8,P<0.01),而其电流幅度为31.63±6.16pA,与正常对照组相比没有差异(n=8,P>0.05)。以上实验结果表明琥珀酸能通过增强突触前GABA的自发性释放,对海马CA1区神经元产生超极化作用,此作用可能是琥珀酸抑制癫痫形成的主要方式之一。     

Effects of acetylcholine on time-dependent currents in sheep cardiac Purkinje fibers

Voltage clamp experiments were carried out on sheep Purkinje fibers to determine the effect of Ach on the time-dependent currents.On the pacemaker current (i _{K 2}) Ach 10^–6 mol·l^–1 had the following effects: shift of the activation curve by a few mV in the depolarizing direction, without change in the rectifier ratio. The potential dependence of the time constants for activation and deactivation was influenced in a similar way as the activation curve.Ach had no effect on the positive dynamic current (i _qr ) or the late plateau outward current (i _x ).The slow inward current (i _si ) as well as the transient inward current (T.I.) were reduced in amplitude and slowed in time course by Ach.The changes in pacemaker current are important in explaining the increased rate of diastolic depolarization in the presence of Ach. The decrease of slow inward current by Ach cannot be made responsible for the plateau shift or the prolongation of the action potential.Supported by F.G.W.O. Belgium 3.0087.74     

白藜芦醇经蛋白激酶G影响豚鼠心室肌细胞L-型钙通道

目的： 研究白藜芦醇经蛋白激酶G对正常豚鼠心室肌细胞L-型钙电流（I_Ca-L）的影响。 方法： 采用全细胞膜片钳技术,记录给予白藜芦醇前后I_Ca-L的变化，并分别记录给予蛋白激酶G（PKG）特异性激动剂8Br-cGMP(100 μmol·L^-1)、PKG特异性拮抗剂H8（5 μmol·L^-1）后白藜芦醇对I_Ca-L的影响。 结果： （1）白藜芦醇呈浓度依赖性抑制I_Ca-L，1、50、100 μmol·L^-1可使ICa-L峰值分别低18.31%±0.68％，56.20%±2.19％，84.51%±2.52％(n=5,P<0.05),但对I_Ca-L激活电位、峰电位、反转电位均没有影响；（2） 100 μmol·L^-1 8Br-cGMP轻度抑制I_Ca-L，电流密度低10.50%±1.11％，100 μmol·L^-1 8Br-cGMP+50 μmol·L^-1白藜芦醇使I_Ca-L电流密度降低87.58%±3.49％(n=6,P<0.05)；（3）5 μmol·L^-1 H8对I_Ca-L无影响，5 μmol·L^-1 H8+50 μmol·L^-1白藜芦醇对I_Ca-L无抑制作用。 结论： 白藜芦醇浓度依赖性抑制豚鼠心室肌细胞I_Ca-L，此作用机制可能与PKG激活有关。     

Inhibitory effect of phorbol 12, 13 dibutyrate on carbachol-activated nonselective cationic current in guinea-pig gastric myocytes

 The effect of protein kinase C (PKC) on carbachol (CCh)-activated nonselective cationic current (I _CCh) was investigated in guinea-pig gastric myocytes using a PKC activator, phorbol 12, 13 dibutyrate (PDBu). Pretreatment with 1 μ M PDBu suppressed I _CCh by 96.5 ± 2.9% (n = 14) in a reversible manner in nystatin-perforated mode. In the presence of 1 μM chelerythrine , a PKC inhibitor, inhibition of I _CChby PDBu was not seen. In whole-cell mode, the inhibition of I _CCh by PDBu was dependent on intracellular MgATP. In the presence of MgATP in the pipette, PDBu decreased I _CCh by 98.8 ± 1.2% (n = 5) as was observed in nystatin-perforated mode. However, PDBu had little effect on I _CCh in the absence of MgATP in the pipette; the extent of inhibition was 12.7 ± 4.3% (n = 8). PDBu also suppressed the generation of cationic current induced by intracellularly perfused GTP[γS]. In the PDBu-pretreated group (n = 9) and PDBu-untreated control group (n = 6), GTP[γS]-induced currents were 6.7 ± 2.4 pA and 236 ± 23 pA, respectively. These results suggest that PKC modulates I _CCh at postreceptor sites via protein phosphorylation. Received: 4 April 1997 / Received after revision: 27 June 1997 / Accepted: 3 June 1997     

The effect of a chemical phosphatase on single calcium channels and the inactivation of whole-cell calcium current from isolated guinea-pig ventricular myocytes

A chemical phosphatase, butanedione monoxime (BDM, at 12–20 mM), reduced open probability (P ₀) of single cardiac L-type Ca²⁺ channels in cellattached patches from guinea-pig ventricular myocytes, without effect on the amplitude of single-channel current, the mean open time or the mean shorter closed time, but it increased mean longer closed time and caused a fall in channel availability. A decrease in the mean time between first channel opening and last closing within a trace was principally due to an inhibition of the longer periods of activity. As a result, the time course of the mean currents, which resolved into an exponentially declining and a sustained component, was changed by an increase in the rate of the exponential phase and a profound reduction of the sustained current. Essentially similar results were obtained when studying whole-cell Ba²⁺ currents. The inactivation of the whole-cell Ca²⁺ currents was composed of two exponentially declining components with the slower showing a significantly greater sensitivity to BDM, an effect that was much more pronounced in myocytes exposed to isoprenaline with adenosine 5-O-(3-thiotriphosphate) (ATP[S]) in the pipette solution. The actions of BDM, which are the opposite of those produced by isoprenaline, suggest that the level of phosphorylation affects processes involved in the slow regulation of channel activity under basal conditions and that several sites (and probably several kinases) are involved. Channels with an inherently slow inactivation would seem to be converted into channels with a rapid inactivation by a dephosphorylation process.     

红藻氨酸对大鼠海马锥体细胞钙电流的影响

目的：应用膜片钳技术,观察红藻氨酸（KA）对大鼠海马锥体细胞ca2＋电流的影响,以研究癫痫的发病机制。方法：采用酶加机械分离法制备出生10～12d的大鼠海马锥体神经元标本,用全细胞膜片钳技术测定其生理学特性及观察KA对ca2＋电流的影响。结果：分离出的海马锥体细胞形态正常,有较长突起;用膜片钳技术证实,其保存了主要的离子通道活性。KA20μmol／L和100μmol／L的浓度均可使海马锥体细胞ca2＋电流峰值增大（n=8,P〈0．01）。结论：①大鼠海马锥体神经细胞具有明显的突起,细胞膜表面光洁、晕光好,适用于膜片钳实验研究;②KA使ca2＋内流增加,引起“Ca”超载”导致细胞毒性等一系列反应;③KA通过激活α-氨基羟甲基恶唑丙酸（AMPA）受体,诱发快速的兴奋性突触后电位（EPSP）,参与兴奋性突触传递。AMPA受体的激活可能是癫痫的发病机制之一。     

Effects of Angiotensin II on sustained outward currents in rat ventricular myocytes

We investigated the effects of angiotensin II (Ang II) on the sustained outward current (I _sus) and action potential of rat ventricular myocytes using the whole-cell patch-clamp technique. Ang II at 30 nM~3 µM inhibited I _sus with an IC₅₀ of 240 nM, a Hill coefficient of 1.0 and maximum inhibition of 19.4%. Ang II-mediated inhibition of I _sus was voltage independent, was due to a decrease in the K⁺ current and was abolished by the Ang II type-I (AT₁) receptor blocker, valsartan. The protein kinase C (PKC) inhibitors PKC19–36 or calphostin C, abolished Ang II-mediated inhibition of I _sus. In contrast, pretreatment with the protein kinase A (PKA) inhibitor PKA6–22 (100 µM) significantly enhanced the suppression of I _sus by 1 µM Ang II: (33.7±5.1% vs. control 17.1±2.3%). These results indicate that Ang II inhibits I _sus via the AT₁ receptor and activation of PKC. Ang II significantly prolonged action potential duration (APD) when the control APD was lengthened by a Ca²⁺ channel activator, BAY K8644. In myocytes with a relatively long APD, Ang II may prolong APD by inhibiting I _sus.