Comparison of Na+-Ca2+ exchange current elicited from isolated rabbit ventricular myocytes by voltage ramp and step protocols

 In this study, the effects of three different voltage protocols on the Na⁺-Ca²⁺ exchange current (I _Na-Ca) of rabbit right ventricular myocytes were studied. Whole-cell patch-clamp recordings were made using a Cs⁺-based internal dialysis solution and external solutions designed to block major interfering currents. I _Na-Ca was measured at 35–37°C as (5 mM) Ni-sensitive current elicited by: a 2 s descending ramp (DR: +80 to –120 mV); a 2 s ascending ramp (AR: –120 to +80 mV) and 500 ms voltage steps (VS) between –120 and +80 mV. DR and AR were applied from –40 mV and elicited I _Na-Ca with reversal potentials (E _rev) of –17.6±2.5 mV (mean±SEM; n=16) and –46.2±4.1 mV (n=10; P=0.0001) respectively. This difference was maintained when the holding potential was –80 mV (–44.0±2.1 mV, n=24 and –86.3±4.8 mV, n=10; P=0.0001), when the internal Ca chelator (EGTA) was replaced with BAPTA (–19.5±1.8 mV and –46.3±1.6 mV, n=6; P=0.0003) and when DR and AR were applied alternately to the same cell. Experiments using modified ramp waveforms suggested a possible mechanism for these differences. Increases in subsarcolemmal Ca caused by Ca entry (coupled to Na extrusion) during the initial positive potential phase of the DR might have induced I _Na-Ca reversal at less negative potentials than observed with AR, during the initial phase of which subsarcolemmal Ca would not have accumulated. These data suggest that I _Na-Ca during voltage-clamp experiments can be significantly influenced by the type of voltage protocol chosen, as the protocol appears to induce subsarcolemmal changes in Ca and Na concentration that are independent of Ca buffering in the bulk cytosol and can occur on a pulse-to-pulse basis. Received: 23 October 1998 / Received after revision: 8 January 1999 / Accepted: 11 January 1999     

Estimation of outward currents in isolated human atrial myocytes using inactivation time course analysis

 The aim was to investigate outward currents in single, isolated, human, atrial myocytes and to determine the relative contribution of individual current components to the total outward current. Currents were recorded using the whole-cell patch-clamp technique at 36–37°C. Individual outward current components were estimated from recordings of total outward current using a mathematical procedure based on the inactivation time course of the respective currents. This method allows estimation of outward currents without the use of drugs or conditioning voltage-clamp protocols to suppress individual current components. A rapidly activating and partially inactivating total outward current was recorded when myocytes were voltage clamped at potentials positive to –20 mV (peak current density 24.0±0.97 pA/pF at +40 mV; n=107 cells, 33 patients). This total outward current comprised three overlapping currents: a rapidly inactivating, transient, outward current (I _to1) a slowly and partially inactivating current (ultrarapid delayed rectifier, I _Kur) and a third current component which most probably reflects a non selective cation current (not characterized). The average current densities at +40 mV were 8.92±0.44 pA/pF for I _to1 and 15.1±0.72 pA/pF for I _Kur (n=107 cells). Recovery from inactivation was bi-exponential for both currents and was faster for I _to1. A slowly activating delayed rectifier current (I _K) was not found. The current densities of peak I _to1 and I _Kur varied strongly between individual myocytes, even in those from the same patient. The ratio I _Kur/I _to1 was 0.5–6.9 with a mean of 1.98±0.11 (n=107 cells), suggesting that I _Kur is the main repolarizing current. The amplitudes of the total outward current, I _to1 and I _Kur, and the ratio of the latter two were independent of patient age (16–87 years). Received: 2 February 1998 / Received after revision: 12 March 1998 / Accepted: 31 March 1998     

Calcium-activated chloride conductance in a pancreatic adenocarcinoma cell line of ductal origin (HPAF) and in freshly isolated human pancreatic duct cells

 Using the whole-cell patch-clamp technique, a calcium-activated chloride conductance (CACC) could be elicited in HPAF cells by addition of 1 μM ionomycin to the bath solution (66 ± 22 pA/pF;V _m + 60 mV) or by addition of 1 μM calcium to the pipette solution (136 ± 17 pA/pF; V _m + 60 mV). Both conductances had similar biophysical characteristics, including time-dependent inactivation at hyperpolarising potentials and a linear/slightly outwardly rectifying current/voltage (I/V) curve with a reversal potential (E _rev) close to the calculated cloride equilibrium potential. The anion permeability sequence obtained from shifts in E _rev was I > Br ≥ Cl. 4,4′-Diisothiocyanatostilbene disulphonic acid (DIDS, 500 μM) caused a 13% inhibition of the current (V _m + 60 mV) while 100 μM glibenclamide, 30 nM TS-TM-calix[4]arene and 10 μM tamoxifen, all chloride channel blockers, had no marked effects (8%, –6% and –2% inhibition respectively). Niflumic acid (100 μM) caused a voltage-dependent inhibition of the current of 48% and 17% (V _m ± 60 mV, respectively). In freshly isolated human pancreatic duct cells (PDCs) a CACC was elicited with 1 μM calcium in the pipette solution (260 ± 62 pA/pF; V _m + 60 mV). The presence of this CACC in human PDCs could provide a possible therapeutic pathway for treatment of pancreatic insufficiency of the human pancreas in cystic fibrosis. Received: 2 October 1997 / Received after revision: 28 November 1997 / Accepted: 1 December 1997     

Modulation by mexiletine of action potentials, L-type Ca current and delayed rectifier K current recorded from isolated rabbit atrioventricular nodal myocytes

 Using whole-cell patch clamp recordings at 37°C, we have examined the effects of externally applied mexiletine (a class 1b antiarrhythmic agent) on action potentials, L-type Ca current (I_Ca,L) and delayed rectifier K current (I_K) in single isolated rabbit atrioventricular nodal (AVN) myocytes. In spontaneously active AVN cells, 30–100 μM mexiletine depolarised the maximum diastolic potential and slowed both action potential upstroke and repolarisation. Under selective recording conditions for I_Ca,L, mexiletine reduced peak I_Ca,L (at +10 mV) amplitude in a dose-dependent fashion (41.8 ± 3.0% inhibition by 100 μM and 16.4 ± 1.8% at 30 μM). The voltage dependence of I_Ca,L activation was unaffected by both concentrations of the drug. Under selective recording conditions, I_K amplitude was measured as the peak of the deactivating tail current following a depolarising voltage pulse to +20 mV. 30 μM mexiletine inhibited I_K by 34.3 ± 5.8%, whilst 100 μM mexiletine reduced the current by 52.7 ± 6.1%. The effects of mexiletine on I_Ca,L and I_K are likely to contribute significantly to the changes in action potentials observed in spontaneously active cells. These findings are also suggestive of key roles for I_Ca,L and I_K in determining the shape and rate of action potentials in this region of the heart. Received: 21 May 1997 / Received after revision: 25 July 1997 / Accepted: 19 August 1997     

Voltage dependence of the Fura-2 transient in rabbit left atrial myocytes at 37°C

 We used the whole-cell patch-clamp technique and monitoring of Fura-2 fluorescence to investigate the voltage dependence of the L-type Ca current (I _Ca,L) and intracellular Ca (Ca_i) transient in rabbit atrial myocytes at 37°C. Imaging the atrial cell membrane with Di-4-ANNEPS showed (in contrast to ventricular cells) that atrial cells had very few transverse tubules. We measured I _Ca,L using a Cs-based internal dialysis solution to eliminate interfering K currents. The voltage dependence of peak I _Ca,L amplitude was bell-shaped: I _Ca,L was maximal at +10 mV and declined at more negative and positive potentials. For measuring the Fura-2 (Ca_i) transient, we used a K-based internal dialysis solution to preserve normal excitation–contraction coupling. Ryanodine (20 μM) plus thapsigargin (2 μM) (blockers of the sarcoplasmic reticulum, SR) abolished the phasic component of the Fura-2 transient (n = 5), demonstrating that the phasic Fura-2 transient provided an index of the magnitude of SR release. The Fura-2 transient also showed bell-shaped voltage dependence, but this was different from that for I _Ca,L. The Fura-2 transient peaked at +30 mV and partially declined at more positive potentials; but at potentials where inward I _Ca,L was small (if not absent), the phasic Fura-2 transient still attained a significant amplitude. We used a rapid application of nifedipine (32 μM), and of nifedipine plus 5 mM Ni, to assess the ability of I _Ca,L and reverse-mode Na-Ca exchange to trigger SR Ca release. With test pulses to +10 mV and +60 mV, a rapid switch to nifedipine (which blocked I _Ca,L) produced no significant reduction in phasic Fura-2 transient amplitude. This suggests that in the absence of I _Ca,L, another mechanism was able to trigger SR release. With pulses to +10 and +60 mV, a single beat switch to nifedipine plus 5 mM Ni almost completely abolished the phasic transient. Since 5 mM Ni inhibits Na-Ca exchange, this suggests that, in the absence of I _Ca,L, trigger Ca entry via reverse Na-Ca exchange was able to activate SR Ca release in atrial cells at 37°C. The mechanisms underlying the Fura-2 transient in atrial cells, and differences with pre-existing data from rabbit ventricular cells, are discussed. Received: 24 September 1996 / Received after revision and accepted: 19 December 1996     

糖尿病家兔心室肌细胞L型钙电流对模拟缺血-再灌注呈

方法: 采用四氧嘧啶静脉注射建立6周的糖尿病家兔模型,胶原酶分离家兔左心室肌细胞,以膜片钳全细胞模式记录糖尿病家兔和正常对照家兔心室肌细胞在基线状态,模拟缺血灌流5 min和再灌注5 min三个时相的I_Ca,L。 结果: 糖尿病组和对照组心室肌细胞最大I_Ca,L密度在基线状态无显著差异;对照组细胞(n=11)最大I_Ca,L密度在基线、缺血灌流后和再灌注后分别为(-8.36±1.63)pA/pF、(-5.90±1.75)pA/pF 和 (-4.22±1.02)pA/pF,缺血时I_Ca,L小于基线(P<0.01),而再灌注后I_Ca,L较之基线(P<0.01)和缺血时(P<0.05)均显著减小;糖尿病组细胞(n=9)最大I_Ca,L密度在基线、缺血灌流后和再灌注后分别为(-7.55±1.62)pA/pF、(-6.05±1.58)pA/pF和(-5.12±1.13)pA/pF,仅再灌注后I_Ca,L明显小于基线(P<0.01),而缺血时I_Ca,L分别与基线(P>0.05)和再灌注后(P>0.05)相比均无显著差异。 结论: 糖尿病状态下的心室肌细胞I_Ca,L对急性缺血损伤呈现"钝化"反应,随缺血进程的衰减较正常细胞缓慢,而缺血后再灌注则对于有无糖尿病的心肌均强力抑制I_Ca,L。本研究结果可能有助于提示糖尿病条件下的缺血-再灌注心肌损伤机制以及对合并缺血性心脏病的糖尿病患者的治疗方案。     

Effect of stretch on calcium channel currents recorded from the antral circular myocytes of guinea-pig stomach

The effect of membrane stretch on voltage-activated Ba²⁺ current (I _Ba) was studied in antral circular myocytes of guinea-pig using the whole- cell patch-clamp technique. The changes in cell volume were elicited by superfusing the myocytes with anisosmotic solutions. Hyposmotic superfusate (202 mosmol/l) induced cell swelling and increased peak values of I _Ba at 0 mV (from −406.6 ± 45.5 pA to −547.5 ± 65.6 pA, mean ± SEM, n = 8) and hyperosmotic superfusate (350 mosmol/l) induced cell shrinkage and decreased peak values of I _Ba at 0 mV (to −269.5 ± 39.1 pA, n = 8). Such changes were reversible and the extent of change was dependent on the osmolarity of superfusate. The values of normalized I _Ba at 0 mV were 1.43 ± 0.04, 1.30 ± 0.06, 1.23 ± 0.04, 1.19 ± 0.04, 1 and 0.68 ± 0.06 at 202, 220, 245, 267, 290 and 350 mosmol/l, respectively (n = 8). I _Ba was almost completely blocked by nicardipine (5 μM) under hyposmotic conditions. The values of steady-state half-inactivation voltage (−37.7 ± 3.3 and −36.5 ± 2.6 mV, under control and hyposmotic conditions, respectively) or the half-activation voltage (−13.6 ± 2.3 and −13.9 ± 1.9 mV) of I _Ba were not significantly changed (P > 0.05, n = 6). Cell membrane capacitance was slightly increased from 50.00 ± 2.86 pF to 50.22 ± 2.82 pF by a hyposmotic superfusate (P < 0.05, n = 6). It is suggested that cell swelling increases voltage-operated L-type calcium channel current and that such a property is related to the response of gastric smooth muscle to mechanical stimuli. Received: 14 November 1995/Received after revision and accepted: 8 January 1996     

The Fura-2 transient can show two types of voltage dependence at 36°C in ventricular myocytes isolated from the rat heart

We used the whole-cell patch-clamp method to investigate the voltage dependence of the L-type Ca current (I _Ca,L) and intracellular Ca (Ca_i) transient in ventricular myocytes isolated from the rat heart. Intracellular Ca was monitored using Fura-2 and the experiments were carried out at 36° C. We measured I _Ca,L by using a caesium-based internal dialysis solution to eliminate interfering K currents. The voltage dependence of peak I _Ca,L amplitude was bell-shaped: I _Ca,L was maximal at +10 mV and declined at more positive potentials. When I _Ca,L was integrated over the first 25 ms to estimate the magnitude of Ca entry, this had a very similar voltage dependence to peak I _Ca,L. In all cells, phasic Fura-2 transients were abolished by 5 μM ryanodine (a blocker of the sarcoplasmic reticulum, SR) showing that the Fura-2 transient provided an index of the magnitude of SR Ca release. For experiments measuring the Ca_i transient, we used a K-based internal dialysis solution to preserve normal excitation-contraction coupling. In 30–40% of cells, we found that the Fura-2 transient had a bell-shaped voltage dependence. This suggests that, in these cells, the primary trigger mechanism for Ca-induced Ca-release might have been Ca entry via I _Ca,L. In the remaining 60–70% of cells, the voltage dependence of the Fura-2 transient was not bell-shaped. The Fura-2 transient reached a maximum with a pulse to +10 mV, and the amplitude of the transient did not decline significantly at more positive potentials to this. In cells with a non-bell-shaped voltage dependence of the Fura-2 transient, pulses to potentials as far positive as +140 mV elicited phasic Fura-2 transients. Since this potential exceeded the Nernst potential for Ca, it was unlikely there was any tigger Ca entry via I _Ca,L at this potential. This would suggest that, in these cells, another trigger for SR Ca release (in addition to I _Ca,L) might be present. We conclude that rat ventricular myocytes, produced using a standard isolation technique and under standard recording conditions, can show either a bell-shaped or a sigmoidal voltage dependence of the Fura-2 transient. Received: 13 October 1995/Received after revision and accepted: 10 January 1996     

Mechanism of hyperthyroidism-induced modulation of the L-type Ca2+ current in guinea pig ventricular myocytes

The positive inotropic effects of thyroid hormone in the heart, increased force and velocity of contraction have been mostly attributed to modulation of myosin ATPase isoenzymes (V₁, V₂ and V₃), and sarcoplasmic reticulum Ca²⁺ pumping activity. In addition, we have suggested that the effects on ventricular contraction result from a thyroid hormone-induced increase in L-type Ca²⁺ current (I _{Ca, L}). Due to the central role of I _{Ca, L} in excitation-contraction coupling, we studied mechanisms whereby thyroid hormone augments this current. Since thyroid hormone modulates adenylate cyclase activity in various tissues, we tested the hypothesis that the hormone activates adenylate cyclase, leading to increased cyclic adenosine monophosphate (cAMP) levels, protein kinase A activation, Ca²⁺ channel phosphorylation and increased I _{Ca, L}. We therefore stimulated or inhibited different sites along the adenylate cyclase cascade, and measured I _{Ca, L} and isometric twitch in ventricular myocytes and papillary muscles from euthyroid and hyperthyroid guinea pigs. Our major findings were as follows. In euthyroid myocytes, 0.1 M isoproterenol (Iso) increased I _{Ca, L} (at V _M=0 mV) from –7.04±0.72 to –22.26±1.88 pA/pF, P<0.05, while in hyperthyroid myocytes (I _{Ca, L}=-21.48±2.94 pA/pF), Iso was ineffective. In euthyroid myocytes, intracellular application of cAMP (50 M) was as potent as Iso, but ineffective in hyperthyroid myocytes. In hyperthyroid myocytes, a protein kinase A inhibitor (2 M) lowered I _{Ca, L} from –26.82±1.54 to -10.17±1.70 pApF (P<0.05), but had no effect in euthyroid myocytes. In hyperthyroid myocytes, acetylcholine (ACh) (1 M) decreased I _{Ca, L} from –26.86±1.49 to –18.33±1.25 pA/pF (P<0.05), while in euthyroid myocytes ACh decreased I_{Ca, L} from –6.80±0.61 to –6.00±0.39 pA/pF (NS). Accordingly, in hyperthyroid papillary muscles, ACh decreased twitch tension by 36.4±2.8%, but in euthyroid preparations only by 9.4±5.1% (P<0.05). These findings suggest that thyroid-hormone-induced increase in I _{Ca, L} contributing to positive inotropy, is mediated by activation of the adenylate cyclase cascade.     

cAMP-dependent inward rectifier current in neurons of the rat suprachiasmatic nucleus

Electrophysiological properties of the inward rectification of neurons in the rat suprachiasmatic nucleus (SCN) were examined by using the single-electrode voltage-clamp method, in vitro. Inward rectifier current (I _H) was produced by hyperpolarizing step command potentials to membrane potentials negative to approximately −60 mV in nominally zero-Ca²⁺ Krebs solution containing tetrodotoxin (1 μM), tetraethylammonium (40 mM), Cd²⁺ (500 μM) and 4-aminopyridine (1 mM).I _H developed during the hyperpolarizing step command potential with a duration of up to 5 s showing no inactivation with time.I _H was selectively blocked by extracellular Cs⁺ (1 mM). The activation of the H-channel conductance (G _H) ranged between −55 and −120 mV. TheG _H was 80–150 pS (n=4) at the half-activation voltage of −84±7 mV (n=4). The reversal potential ofI _H obtained by instantaneous current voltage (I/V) relations was −41±6mV (n=4); it shifted to −51±8mV (n=3) in low-Na⁺ (20 mM) solution and to −24±4 mV (n=4) in high-K⁺ (20 mM) solution. Forskolin (1–10 μM) produced an inward current and increased the amplitude ofI _H. Forskolin did not change the half-activation voltage ofG _H. 8-Bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP, 0.1–1 mM) and dibutyryl-cAMP (0.1–1 mM) enhancedI _H. 3-Isobutyl-1-methylxanthine (IBMX, 1 mM) also enhancedI _H. The results suggest that the inward rectifier cation current is regulated by the basal activity of adenylate cyclase in neurons of the rat SCN.     

Heterogeneity of hair cells in the bullfrog sacculus

 Morphometric and electrophysiological features of hair cells in the sacculus of the bullfrog (Rana catesbeiana) were studied. Confocal microscopy observations of hair cells in situ revealed that three classes of hair cells can be distinguished by their somata shape and macular location. Two of these, termed central cylindrical (CCHCs) and central flask-shaped (CFHCs) hair cells, were found in the central part of the macula. The third class, termed peripheral elongated hair cells (PEHC), was only found around the perimeter of the sacculus. Using the whole-cell patch-clamp technique CCHCs and CFHCs were distinguished by the amplitude of their voltage-activated calcium currents (I _Ca). The mean amplitudes of steady-state I _Ca at –20 mV were –900 ± 500 pA (n = 18) for CCHCs and –160 ± 70 pA (n = 10) for CFHCs. The two hair-cell types also differed in the possession of a Cs⁺-resistant, apamin-sensitive, calcium-sensitive potassium current, found only the CFHCs. This study indicates that several populations of hair cells with distinct morphological features exist in the bullfrog sacculus, and that at least two of these differ in their complement of membrane conductances. Received: 18 February 1997 / Received after revision: 24 June 1997 / Accepted: 23 July 1997     

Influence of postnatal changes in action potential duration on Na-Ca exchange in rabbit ventricular myocytes

 Cardiac Na-Ca exchanger (NCX) expression and current density are significantly greater in newborn rabbit hearts compared with adults. However, the relatively short action potential (AP) at birth may limit the impact of increased NCX expression by diminishing Ca²⁺ entry via Na-Ca exchange current (I _NaCa). To address the interdependence of AP duration and NCX activity, we voltage-clamped newborn (NB, 1–5 day), juvenile (JV, 10–14 day) and adult (AD) rabbit myocytes with a series of APs of progressively increasing duration (APD₉₀: 108–378 ms) under nominally chloride-free conditions. In each age group we quantified an increase in outward (Q _Exout) and inward (Q _Exin) Ni²⁺-sensitive charge movement in response to AP prolongation. Q _Exout and Q _Exin measured during age-appropriate APs declined postnatally [Q _EXout: NB (2 day) 0.19 ± 0.02, JV (10 day) 0.10 ± 0.01, AD 0.04 ± 0.002; Q _EXin: NB –0.2 ± 0.01, JV –0.11 ± 0.02; AD –0.04 ± 0.003 pC/pF] despite the significantly shorter APD₉₀ of newborn myocytes (NB 122 ± 10; AD 268 ± 22 ms). When Ca²⁺ fluxes by other transport pathways were blocked with nifedipine, ryanodine and thapsigargin, age-appropriate APs elicited contractions in NB and JV but not AD myocytes (NB 4.8 ± 0.5, JV 1.2 ± 0.3% resting length). These data demonstrate that a shorter AP does not negate the impact of increased NCX expression at birth. Received: 23 September 1997 / Received after revision: 2 January 1998 / Accepted: 5 January 1998     

Characteristics of the delayed rectifier K current compared in myocytes isolated from the atrioventricular node and ventricle of the rabbit heart

The delayed rectifier potassium current (I _K) is known to be important in action potential repolarisation and may contribute to the diastolic pacemaker depolarisation in pacemaker cells from the heart. In this study, using whole-cell patch clamp, we investigated the characteristics of I _K in morphologically normal cells from the atrioventricular node (AVN) and ventricle of the rabbit heart. Cells were held at −40 mV and 5 μM external nifedipine was used to block L-type calcium current (I _Ca,L). Significant I _K was observed with pulses to potentials more positive than −30 mV. The steady-state activation curve in both cell types showed maximal activation at between + 10 and + 20 mV. Half-maximal activation of I _K occurred at −4.9 and −4.1 mV with slope factors of 8.3 and 12.4 mV in ventricular and AVN cells, respectively. Using pulses of increasing duration, significant I _K tails after repolarisation from + 40 mV were observed with pulses of 20 ms and increased with pulses up to 100–120 ms in both cell types. Pulses of longer duration did not activate further I _K and this suggested that only the rapid component of I _K, called I _Kr, was present in either cell type. Moreover, I _K tails after pulses to all potentials were blocked completely by E-4031, a selective blocker of I _Kr. The reversal potential of I _K varied with the concentration of external K. Superfusion of AVN cells with medium containing 4, 15 and 40 mM [K⁺]_o resulted in reversal potentials of −81, −56 and −32 mV, respectively, which are close to values predicted if the I _K channel were highly selective for K. The time constants for deactivation of I _K in ventricle and AVN on return to −40 mV after a 500-ms activating pulse to + 60 mV were 480 ms and 230 ms, respectively. The faster deactivation of I _K in AVN cells was a distinguishing feature and suggests that there may be differences in the I _Kr channel protein between ventricular and AVN cells. Received: 24 July 1995 /Received after revision: 20 October 1995 /Accepted: 23 October 1995     

A hyperpolarization-activated cation current (I h) contributes to resting membrane potential in rat superior cervical sympathetic neurones

 Using perforated-patch voltage-clamp recording, a prominent hyperpolarization-activated inward cation current (I _h) has been identified in dissociated, cultured and replated, superior cervical sympathetic (SCG) neurones from 17-day-old rats. I _h was identified as a slowly activated inward current on hyperpolarizing from –60 mV, with an extrapolated null potential (in 3 mM [K⁺]_out) of –42 mV. The activation range for I _h was –40 to –100 mV, with a half-activation voltage (V _0.5) of –63 mV. The current was suppressed by 1 mM Cs⁺ but not by 1 mM Ba²⁺. The reversal potential for the current change induced by Cs⁺ agreed with the null potential for I _h. I _h conferred strong inward rectification to the current-voltage curve negative to –55 mV in both voltage-clamp and current-clamp recording. This inward rectification was reduced by 1 mM Cs⁺. In a sample of eight cells with initial resting membrane potentials between –51 and –64 mV, Cs⁺ increased the resting potential of all cells by between 2.5 and 21 mV. These results indicate that I _h contributes a tonic inward (depolarizing) component to the maintenance of the resting membrane potential in SCG neurones. Received: 16 January 1998 / Received after revision and accepted: 1 April 1998     

Modulation of manganese currents by 1, 4-dihydropyridines,isoproterenol and forskolin in rabbit ventricular cells

Although often used as a Ca²⁺ channel blocker, Mn²⁺, in fact, permeates through Ca²⁺ channels. Under Na⁺-free conditions, depolarizing pulses evoked slowly-decaying Mn²⁺ currents (I_Mn). Maximal I_Mn densities in the presence of 5 and 20 mM Mn²⁺ were 0.42±0.12 pA/pF (mean±SEM, n=17) and 1.23±0.10 pA/pF (n=40), respectively. At 5 mM, the ratio of maximal amplitude of I_Mn to that of the Ca²⁺ current (I_Ca) was 0.079±0.009 (n=8). I_Mn elicited from a holding potential of –50 mV was depressed by nitrendipine (1 µM) by ~70%. Nitrendipine (0.3 µM) shifted the steady-state inactivation curve to more negative potentials and shifted the potential for half-maximal inactivation (E_0.5) from 1.3 to –8.8 mV and also decreased the time constant of decay of I_Mn at 20 mV from 986.2 to 167.9 ms. BAY K 8644 (1 µM), isoproterenol (10 µM) and forskolin (10 µM) all increased I_Mn and shifted the current/voltage (I/V) relationship to more negative potentials. The small, slowly-inactivating I_Mn is thus modulated by dihydropyridine Ca²⁺ channel modulators and cyclic AMP-mediated phosphorylation in a manner similar to other L-type Ca²⁺ channel currents. L-type Ca²⁺ channels are involved in the regulation of intracellular [Mn] in ventricular myocytes.     

The peptide ”FRCRCFa”, dialysed intracellularly, inhibits the Na/Ca exchange in rabbit ventricular myocytes with high affinity

 We investigated the effect in rabbit ventricular myocytes of ”FRCRCFa”, a newly developed peptide inhibitor of the Na/Ca exchange. Myocytes were whole-cell patch clamped and experiments were carried out at 36°C. The Na/Ca exchange was measured selectively, by blocking interfering ion channel currents and the Na/K pump, as the membrane current which could be inhibited by 5 mM nickel (Ni; a known blocker of the Na/Ca exchange). Increasing concentrations of FRCRCFa dialysed into the cell from the patch-pipette inhibited the Na/Ca exchange current. The dose/response curve could be fitted by a function for co-operative ligand binding, which predicted a K_D for FRCRCFa-mediated inhibition of 22.7 ± 3.7 nM, with a Hill coefficient of 0.61 ± 0.06. Pipette FRCRCFa concentrations of 1 μM and above were sufficient to cause complete inhibition of Na/Ca exchange current. The inhibitory effect of FRCRCFa was independent of membrane potential and relatively selective: 10 μM FRCRCFa dialysed into the cell had no effect on the L-type Ca current and delayed rectifier and inward rectifier K currents. Thus FRCRCFa appears to be a potent and relatively selective inhibitor of the Na/Ca exchange in intact cardiac myocytes, and may be of value for studies of the Na/Ca exchange. Received: 9 July 1996 / Received after revision: 18 September 1996 / Accepted: 20 September 1996     

Enrichment of cardiac pacemaker-like cells: neuregulin-1 and cyclic AMP increase <Emphasis Type="Italic">I</Emphasis><Subscript>f</Subscript>-current density and connexin 40 mRNA levels in fetal cardiomyocytes

Generation of a large number of cells belonging to the cardiac pacemaker system would constitute an important step towards their utilization as a biological cardiac pacemaker system. The aim of the present study was to identify factors, which might induce transformation of a heterogenous population of fetal cardiomyocytes into cells with a pacemaker-like phenotype. Neuregulin-1 (α- and β-isoform) or the cAMP was added to fresh cell cultures of murine embryonic cardiomyocytes. Quantitative northern blot analysis and flowcytometry were performed to detect the expression of connexins 40, 43 and 45. Patch clamp recordings in the whole cell configuration were performed to determine current density of I _f, a characteristic ion current of pacemaker cells. Fetal cardiomyocytes without supplement of neuregulin or cAMP served as control group. Neuregulin and cAMP significantly increased mRNA levels of connexin 40 (Cx-40), a marker of the early differentiating conduction system in mice. On the protein level, flowcytometry revealed no significant differences between treated and untreated groups with regard to the expression of connexins 40, 43 and 45. Treatment with cAMP (11.2 ± 2.24 pA/pF; P < 0.001) and neuregulin-1-β (6.23 ± 1.07 pA/pF; P < 0.001) significantly increased the pacemaker current density compared to control cardiomyocytes (1.76 ± 0.49 pA/pF). Our results indicate that neuregulin-1 and cAMP possess the capacity to cause significant transformation of a mixed population of fetal cardiomyocytes into cardiac pacemaker-like cells as shown by electrophysiology and increase of Cx-40 mRNA. This method may allow the development of a biological cardiac pacemaker system when applied to adult or embryonic stem cells.     

Role of PKC in the effects of α1-adrenergic stimulation on Ca2+ transients, contraction and Ca2+ current in guinea-pig ventricular myocytes

 The effects of α₁-adrenoceptor stimulation on intracellular Ca²⁺ transients, contractility and L-type Ca²⁺ current (I _Ca,L) were studied in single cells isolated from ventricles of guinea-pig hearts. The aim of our study was to elucidate the mechanisms of the positive inotropic effect of α₁-adrenergic stimulation by focussing on the role of protein kinase C (PKC). Phenylephrine, an α₁-adrenergic agonist, at concentrations of 50–100 μM elicited a biphasic inotropic response: a transient negative inotropic response (22.9±6.0% of control) followed by a sustained positive inotropic response (61.0±8.4%, mean±SE, n=12). The Ca²⁺ transient decreased by 10.2±3.9% during the negative inotropic phase, while it increased by 67.7±10% (n=12) during the positive inotropic phase. These effects were inhibited by prazosin (1 μM), a α₁-adrenergic antagonist. Phenylephrine increased the I _Ca,L by 60.8±21% (n=5) during the positive inotropic phase. To determine whether activation of PKC is responsible for the increases in Ca²⁺ transients, contractile amplitude and I _Ca,L during α₁-adrenoceptor stimulation, we tested the effects of 4β-phorbol 12-myristate 13-acetate (PMA), a PKC activator, and of bisindolylmaleimide I (GF109203X) and staurosporine, both of which are PKC inhibitors. PMA mimicked phenylephrine’s effects on Ca²⁺ transients, contractile amplitude and I _Ca,L. PMA (100 nM) increased the Ca²⁺ transient, contractile amplitude and I _Ca,L by 131±17%, 137±25% (n=8), and 81.1±26% (n=5), respectively. Prior exposure to GF109203X (1 μM) or staurosporine (10 nM) prevented the phenylephrine-induced increases in Ca²⁺ transients, contractile amplitude and I _Ca,L. Our study suggests that during α₁-adrenoceptor stimulation increase in I _Ca,L via PKC causes an increase in Ca²⁺ transients and thereby in the contractile force of the ventricular myocytes. Received: 16 July 1998 / Received after revision and accepted: 20 October 1998     

心脏复极储备电流IKs在糖尿病QT间期延长性别差异中的作用

目的: 观察不同性别糖尿病家兔QT间期延长病理条件下的缓慢延迟整流钾电流(I_Ks)以及蛋白变化,为探讨糖尿病性长QT综合征性别差异的离子机制做基础。方法: 取体重2-2.5 kg家兔,一次性注射预热(37 ℃)的四氧嘧啶(140 mg/kg),8周后造成1型糖尿病模型,测定血糖,记录标准II导联心电图,采用酶解法分离家兔单个心室肌细胞,应用全细胞膜片钳技术记录动作电位时程(APD)和I_Ks,并且运用Western blotting法检测KvLQT1和mink蛋白表达变化。结果: 雌雄糖尿病组QT间期和APD均较对照组延长,雄性延长明显,且延长百分比差异显著(P<0.05)。在+40 mV到+70 mV测试电压范围内,雄性糖尿病组I_Ks step电流密度均低于对照组(P<0.05),在+70 mV时,由对照组(3.08±0.67)pA/pF(n=17)降低到(1.27±0.20)pA/pF(n=16),在0 mV~+70 mV测试电压范围内,雌性糖尿病组I_Ks step电流密度均高于对照组(P<0.05),在+70 mV时,由对照组的(1.56±0.20)pA/pF(n=13)增加到(3.65±0.50)pA/pF(n=14)。Western blotting结果显示雄性糖尿病组KvLQT1和mink蛋白表达水平分别下 调21.6%和18.5%;雌性糖尿病组KvLQT1和mink蛋白表达水平分别上调42.3%和20.5%(P<0.05)。结论: I_Ks参与了糖尿病QT间期延长的发生,并且存在性别差异。在雌性家兔早期糖尿病模型中,作为一个复极储备,代偿性上调,限制了QT间期的过度延长。