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     

A novel, rapid and reversible method to measure Ca buffering and time-course of total sarcoplasmic reticulum Ca content in cardiac ventricular myocytes

 This paper outlines a simple method of estimating both the Ca-buffering properties of the cytoplasm and the time-course of changes of sarcoplasmic reticulum (s.r.) Ca concentration during systole. The experiments were performed on voltage-clamped ferret single ventricular myocytes loaded with the free acid of fluo-3 through a patch pipette. The application of caffeine (10 mM) resulted in a Na-Ca exchange current and a transient increase of the free intracellular Ca concentration ([Ca²⁺]_i). The time-course of change of total Ca in the cell was obtained by integrating the current and this was compared with the measurements of [Ca²⁺]_i to obtain a buffering curve. This could be fit with a maximum capacity for the intrinsic buffers of 114±18 μmol l^–1 and K _d of 0.59±0.17 μM (n=8). During the systolic rise of [Ca²⁺]_i, the measured changes of [Ca²⁺]_i and the buffering curve were used to calculate the magnitude and time-course of the change of total cytoplasmic Ca and thence of both s.r. Ca content and Ca release flux. This method provides a simple and reversible mechanism to measure Ca buffering and the time-course of both total cytoplasmic and s.r. Ca. Received: 14 October 1998 / Accepted: 6 November 1998     

”Voltage-activated Ca release” in rabbit, rat and guinea-pig cardiac myocytes, and modulation by internal cAMP

 It is widely believed that Ca release from the sarcoplasmic reticulum (SR) in heart muscle is due to ”Ca-induced Ca-release” (CICR), triggered by transmembrane Ca entry. However, in intact guinea-pig cells or cells dialysed with cAMP there may be an additional mechanism – SR release may be activated directly by membrane depolarisation without Ca entry. The first objective of the present study was to investigate whether this ”voltage-activated Ca release” (VACR) mechanism is present across species such as rabbit, rat and guinea-pig. The second objective was to characterise the dependence of a VACR mechanism on internal [cAMP]. Membrane current was measured with the whole-cell patch-clamp technique, intracellular [Ca] was monitored with Fura-2 (or a combination of Fluo-3/SNARF-1). Rapid changes of superfusate (within 100 ms) were made using a system which maintained cell temperature at 37°C. We used a train of conditioning pulses to ensure a standard SR load before each test pulse. In rabbit myocytes dialysed with 100 μM cAMP, 89.6 ± 7.0% of the control intracellular Ca (Ca_i) transient was still elicited by depolarisation during a switch to 5 mM Ni, which blocked pathways for Ca entry. This suggested that rabbit myocytes possess a VACR mechanism. The percentage of control Ca_i transient elicited by depolarisation in the presence of 5 mM Ni (i.e. magnitude of VACR) increased in a graded fashion with the pipette [cAMP] between zero and 100 μM. In rat myocytes dialysed with 50 μM cAMP, 64.4 ± 6.2% of SR release was activated by depolarisation in the presence of 5 mM Ni, suggesting the presence of a VACR mechanism. The extent to which VACR triggered SR release increased with the pipette [cAMP] between zero and 50 μM. In guinea-pig myocytes dialysed with 100 μM cAMP, 74.6 ± 3.6% of the control Ca_i transient was elicited by depolarisation in the presence of 5 mM Ni. The degree to which VACR triggered SR release was also graded with the pipette [cAMP] between zero and 100 μM. It therefore appears that each of the three species might possess a VACR mechanism which can be modulated by the internal [cAMP]. This may reflect an effect of cAMP to phosphorylate key proteins involved in excitation–contraction coupling. Under normal physiological conditions with a basal [cAMP] between 2 and 20 μM, VACR may play a role in triggering SR release. The role of VACR may increase under conditions which increase internal [cAMP]. Received: 4 April 1997 / Received after revision: 2 July 1997 / Accepted: 7 July 1997     

Activation of Ca2+-sensitive Cl– current by reverse mode Na+/Ca2+ exchange in rabbit ventricular myocytes

 We investigated how Ca²⁺-sensitive transient outward current, I _to(Ca), is activated in rabbit ventricular myocytes in the presence of intracellular Na⁺ (Na⁺ _i) using the whole-cell patch-clamp technique at 36°C. In cells dialysed with Na⁺-free solutions,the application of nicardipine (5 μM) to block L-type Ca²⁺ current (I _Ca) completely inhibited I _to(Ca). In cells dialysed with a [Na⁺]_i≥5 mM, however, I _to(Ca) could be observed after blockade of I _Ca, indicating the activity of an I _Ca-independent component. The amplitude of I _Ca-independent I _to(Ca) increased with voltage in a [Na⁺]_i-dependent manner. The block of Ca²⁺ release from the sarcoplasmic reticulum by caffeine, ryanodine or thapsigargin blocked I _Ca-independent I _to(Ca). In Ca²⁺-free bath solution I _to(Ca) was completely abolished. The application of 2 mM Ni²⁺ or the newly synthesized compound KBR7943, a selective blocker of the reverse mode of Na⁺/Ca²⁺ exchange, or perfusion with pipette solution containing XIP (10 μM), a selective blocker of the exchanger, blocked I _Ca-independent I _to(Ca). From these results we conclude that, in the presence of Na⁺ _i, I _to(Ca) can be activated via Ca²⁺-induced Ca²⁺ release triggered by Na⁺/Ca²⁺ exchange operating in the reverse mode after blockade of I _Ca. Received: 20 January 1998 / Received after revision: 6 July 1998 / Accepted: 25 July 1998     

Rapid effects of cytochalasin-D on contraction and intracellular calcium in single rat ventricular myocytes

 Contraction and intracellular calcium ([Ca²⁺]_i) transients were recorded using a video edge detector and fluorescence spectrophotometry, respectively, in rat ventricular myocytes at 22–24°C stimulated at a frequency of 1 Hz. Application of the F-actin disrupter cytochalasin-D (Cyt-D) caused a large reduction in the amplitude of contraction and a small increase in the [Ca²⁺]_i transient. These responses began within a few seconds of application and were complete after 2 min of exposure. Phase-plane relationships of contraction and [Ca²⁺]_i were consistent with cytochalasin-D causing a decrease in myofilament responsiveness to Ca²⁺. Received: 18 May 1998 / Received after revision: 8 July 1998 / Accepted: 13 July 1998     

A measurable reduction of s.r. Ca content follows spontaneous Ca release in rat ventricular myocytes

 The Ca content of the sarcoplasmic reticulum (s.r.) was measured in voltage-clamped rat ventricular myocytes from the integral of the Na-Ca exchange current evoked by applying caffeine to release the s.r. Ca content. Following spontaneous release of Ca from the s.r., the s.r. Ca content was decreased. The magnitude of this decrease was equal to that of the amount of calcium directly measured to have been pumped out of the cell during the spontaneous release. Following a spontaneous release, the s.r. Ca content recovered linearly. These results are shown to be consistent with the hypothesis that the frequency of spontaneous release is determined by the time taken for the cell and s.r. to reaccumulate the Ca²⁺ ions pumped out of the cell during spontaneous release. Received: 9 July 1997 / Accepted: 21 July 1997     

Sarcomere length during contraction of isolated guinea-pig ventricular myocytes

 An improved method was developed for measuring sarcomere length (SML) during twitch contractions of single cardiac ventricular myocytes, using a charge-coupled photodiode array self-scanning at a rate of 1.5 ms/element. The average resting SML of 111 cells was 1.88±0.04 μm (mean ±SD). When contractions were triggered by action potentials under perforated-patch conditions, the time course of SML shortening closely followed changes in cell length. A large variation was observed in contraction time course between myocytes, some cells having a phasic component with a duration at 50% shortening (full-width at half-maximum; FWHM) of approximately 40 ms, while others shortened more slowly (FWHM of phasic component @100 ms). FWHM was highly correlated with relaxation half-time, but with neither action potential duration nor resting SML. The kinetics of slowly contracting cells could not be converted to the rapid type by using conditioning trains or applying isoprenaline. The steady-state SML/pCa relation in ventricular myocytes was measured by applying solutions of various pCa immediately after localized punctures of the surface membrane using a focal laser beam. The Hill coefficient, n _H, was @4–5 and K _1/2@400–500 nM, but there was no evidence of two populations of cells with different Ca²⁺ sensitivities. Received: 28 September 1998 / Received after revision: 11 December 1998 / Accepted: 14 December 1998     

Effects of cytosolic Ca2+ on the Ca2+ content of the sarcoplasmic reticulum in saponin-permeabilized rat ventricular trabeculae

 Rat ventricular trabeculae were mounted for isometric tension recording, and then permeabilized with saponin. The Ca²⁺ concentration ([Ca²⁺]) within the permeabilized preparation (cytosolic [Ca²⁺]) was monitored continuously using Indo-1 and the integrals of Ca²⁺ transients resulting from brief caffeine application used as an index of the sarcoplasmic reticulum (SR) Ca²⁺ content. The relationship between SR Ca²⁺ content and cytosolic [Ca²⁺] was studied within the reported physiological range (i.e. 50–250 nmol · l^–1 Ca²⁺). Increasing cytosolic [Ca²⁺] from 50 nmol · l^–1 to 250 nmol · l^–1 increased the steady-state SR Ca²⁺ content about threefold. However, increasing [Ca²⁺] above 250 nmol · l^–1 typically resulted in spontaneous SR Ca²⁺ release, with no further increase in SR Ca²⁺ content. The SR Ca²⁺ content increased only slowly when cytosolic [Ca²⁺] was increased; it was unchanged 20 s after a rapid increase in cytosolic [Ca²⁺], but increased progressively to a new steady-state level during the following 1–2 min. In a parallel series of experiments using intact papillary muscles, increasing extracellular [Ca²⁺] (from 0.5 to 5 mmol · l^–1) significantly increased twitch tension within 20 s of the solution change. These results support previous suggestions that the SR Ca²⁺ content may increase when diastolic cytosolic [Ca²⁺] rises during inotropic interventions such as increased stimulus rate or extracellular [Ca²⁺]. However, the rate at which SR Ca²⁺ responds to changes in cytoplasmic [Ca²⁺] within the diastolic range does not appear rapid enough to explain the early potentiation of twitch tension in intact preparations after an increase in extracellular [Ca²⁺]. Received: 26 August 1997 / Accepted: 28 October 1997     

Ca2+ entry through cardiac L-type Ca2+ channels modulates β-adrenergic stimulation in mouse ventricular myocytes

 β-adrenergic receptor (β-AR) stimulation increases cardiac L-type Ca²⁺ channel (CaCh) currents via cAMP-dependent phosphorylation. We report here that the affinity and maximum response of CaCh to isoproterenol (Iso), in mouse ventricular myocytes were significantly higher when Ba²⁺ was used as the charge carrier (I _Ba) instead of Ca²⁺ (I _Ca). The EC₅₀ and maximum increase of peak currents were 43.7 ± 7.9 nM and 1.8 ± 0.1-fold for I _Ca and 23.3 ± 4.7 nM and 2.4 ± 0.1-fold for I _Ba. When cells were dialyzed with the faster Ca²⁺ chelator, BAPTA, both sensitivity and maximum response of I _Ca to Iso were significantly augmented compared to cells with EGTA (EC₅₀ of 23.1 ± 5.2 nM and maximal increase of 2.2 ± 0.1-fold). Response of I _Ca to forskolin was also significantly increased when cells were dialyzed with BAPTA or when currents were measured in Ba²⁺. In contrast, depletion of the sarcoplasmic reticulum (SR) Ca²⁺ stores by ryanodine did not alter sensitivity of I _Ca to Iso or forskolin. These results suggest that the Ca²⁺ entering through CaCh regulates cAMP-dependent phosphorylation, and such negative feedback may play a significant role in cellular Ca²⁺ homeostasis and contraction in cardiac cells during β-AR stimulation. Received: 10 December 1997 / Received after revision: 19 January 1998 / Accepted: 21 January 1998     

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     

Strophanthidin-induced gain of Ca2+ occurs during diastole and not systole in guinea-pig ventricular myocytes

 We have investigated the effects of inhibiting the Na-K pump with strophanthidin on the intracellular Ca²⁺ concentration ([Ca²⁺]_i), sarcoplasmic reticulum (s.r.) Ca²⁺ content and membrane currents. s.r. Ca²⁺ content was measured by integrating the Na-Ca exchange current resulting from application of 10 mM caffeine. The application of strophanthidin increased both diastolic and systolic [Ca²⁺]_i. This was accompanied by an increase of s.r. Ca²⁺ content from a resting value of 17.9±1.5 μmol/l to 36.9±3.3 μmol/l (n=16) after 5 min. Systolic fluxes of Ca²⁺ into and out of the cell before and during strophanthidin application were also measured. Ca²⁺ efflux (measured as the integral of the Na-Ca exchange tail current) rose steadily in the presence of strophanthidin, while Ca²⁺ influx (the integral of the L-type Ca²⁺ current) was reduced. In spite of this, s.r. Ca²⁺ content rose substantially. In the presence of Cd²⁺ (100 μM), which inhibits the L-type Ca²⁺ current, strophanthidin had negligible effects on current suggesting that Ca²⁺ influx via Na-Ca exchange during depolarization does not account for the increase of s.r. Ca²⁺ content. This suggests that changes of Ca²⁺ flux during systole are not responsible for the strophanthidin-induced increase of s.r. Ca²⁺. We conclude that the primary mechanism by which the cardiac cell gains Ca²⁺ when the Na-K pump is inhibited is by a net influx during diastole. Received: 2 November 1998 / Received after revision: 8 December 1998 / Accepted: 9 December 1998     

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     

Na+-dependent regulation of the free Mg2+ concentration in neuropile glial cells and P neurones of the leech Hirudo medicinalis

 To investigate the Mg²⁺ regulation in neuropile glial (NG) cells and pressure (P) neurones of the leech Hirudo medicinalis the intracellular free Mg²⁺ ([Mg²⁺]_i) and Na⁺ ([Na⁺]_i) concentrations, as well as the membrane potential (E _m), were measured using Mg²⁺- and Na⁺-selective microelectrodes. The mean steady-state values of [Mg²⁺]_i were found to be 0.91 mM (mean E _m=–63.6 mV) in NG cells and 0.20 mM (mean E _m=–40.6 mV) in P neurones with a [Na⁺]_i of 6.92 mM (mean E _m=–61.6 mV) and 7.76 mM (mean E _m=–38.5 mV), respectively. When the extracellular Mg²⁺ concentration ([Mg²⁺]_o) was elevated, [Mg²⁺]_i in P neurones increased within 5–20 min whereas in NG cells a [Mg²⁺]_i increase occurred only after long-term exposure (6 h). After [Mg²⁺]_o was reduced back to 1 mM, a reduction of the extracellular Na⁺ concentration ([Na⁺]_o) decreased the inwardly directed Na⁺ gradient and reduced the rate of Mg²⁺ extrusion considerably in both NG cells and P neurones. In P neurones Mg²⁺ extrusion was reduced to 15.4% in Na⁺-free solutions and to 6.0% in the presence of 2 mM amiloride. Mg²⁺ extrusion from NG cells was reduced to 6.2% in Na⁺-free solutions. The results suggest that the major [Mg²⁺]_i-regulating mechanism in both cell types is Na⁺/ Mg²⁺ antiport. In P neurones a second, Na⁺-independent Mg²⁺ extrusion system may exist. Received: 11 August 1998 / Received after revision: 14 October 1998 / Accepted: 15 October 1998     

Actions of myristyl-FRCRCFa, a cell-permeant blocker of the cardiac sarcolemmal Na-Ca exchanger, tested in rabbit ventricular myocytes

 In cardiac muscle, the electrogenic Na-Ca exchanger plays important roles in determining action potential shape and in the beat-to-beat homeostasis of intracellular calcium. In this study we tested the actions of a putative cell-permeant blocker of the cardiac sarcolemmal Na-Ca exchange, ”Myristyl- (Myr-) FRCRCFa”. Experiments were performed using isolated rabbit right ventricular myocytes and whole-cell patch-clamp at 35–37°C. The Na-Ca exchange current (I _Na-Ca), L-type calcium current (I _Ca,L), inward rectifier potassium current (I _K1) and delayed rectifier potassium current (I _K) were compared in untreated cells and cells incubated in a solution containing N-myristylated FRCRCFa. With other major currents blocked, I _Na-Ca was measured as the Ni-sensitive component of current during a voltage ramp applied from the holding potential of –40 mV, between +80 and –120 mV (ramp velocity 0.1 V s^–1). In untreated cells, I _Na-Ca at +60 mV was 7.1±0.6 pA/pF and at –100 mV was –2.7±0.3 pA/pF (n=9). After a 15-min pre-incubation with 20 μM Myr-FRCRCFa, I _Na-Ca was reduced to 4.2±0.3 pA/pF at +60 mV and –1.5±0.2 pA/pF at –100 mV (P<0.02; n=7). After incubation with 20 μM Myr-FRCRCFa for 1 h, I _Na-Ca at both potentials was further reduced (2.3±0.8 pA/pF at +60 mV; –0.9±0.3 pA/pF at –100 mV; P<0.008 compared with control; n=4). Under selective recording conditions for I _Ca,L, there was little difference in I _Ca,L density between untreated and cells incubated with Myr-FRCRCFa. A Boltzmann fit to the I _Ca,L/V relation showed no significant alteration of half-maximal activation potential or slope factor of activation. I _K1 was also largely unaffected by pre-incubation of cells with Myr-FRCRCFa. I _K, measured as deactivating tail current following 1-s test depolarisations to a range of test potentials, was also not significantly altered by Myr-FRCRCFa. The suppression of I _Na-Ca in cells incubated in Myr-FRCRCFa suggests that addition of the myristyl group to FRCRCFa peptide conveys cell permeancy to the peptide and that Myr-FRCRCFa applied externally to rabbit ventricular myocytes is moderately effective as an I _Na-Ca blocker. I _Ca,L, I _K1 and I _K were largely unaffected by Myr-FRCRCFa. N-Myristylation of such conformationally constrained hexapeptides may, therefore, provide a means of producing cell-permeant inhibitors of the cardiac Na-Ca exchanger. Received: 6 February 1997 / Received after revision: 8 April 1998 / Accepted: 9 April 1998     

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     

Ca-activated chloride current and Na-Ca exchange have different timecourses during sarcoplasmic reticulum Ca release in ferret ventricular myocytes

 The aim of this work was to measure membrane currents activated by Ca release from the cardiac sarcoplasmic reticulum (s.r.). Intracellular Ca concentration ([Ca²⁺]_i) was measured using fluo-3 in patch clamped cells. Calcium release from the s.r. (whether occurring spontaneously or evoked by caffeine) produced changes of membrane current which could be separated into a Ca-activated Cl current which was inhibited by DIDS or Cl removal and a Na-Ca exchange current. Both these currents had different time courses from the measured [Ca²⁺]_i. Furthermore the Ca-activated Cl current decayed more quickly than did Na-Ca exchange. Possible explanations for the different kinetics of these two Ca-sensitive currents are discussed. Received: 21 November 1997 / Received after revision: 12 December 1997 / Accepted: 15 December 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     

Estimation of myofibrillar responsiveness to Ca2+ in isolated rat ventricular myocytes

 To estimate myofibrillar responsiveness to Ca²⁺, we used the relation between cell length and intracellular [Ca²⁺] ([Ca²⁺]_i) during tetanic contractions of isolated ventricular myocytes. Enzymatically isolated rat ventricular myocytes were loaded with fura-2 AM (4 μM for 10 min) and excited alternately at 340 nm and 380 nm. The ratio (R) of fura-2 fluorescence at these wavelengths [F(340)/F(380), an index of [Ca²⁺]_i] and cell length (L) were measured simultaneously. Following treatment with thapsigargin (0.2 μM), myocytes were stimulated at 10 Hz for 10 s to produce a tetanic contraction every min and an instantaneous plot of R vs L (R-L trajectory) was constructed. The R-L trajectory followed the same path during cell shortening and re-lengthening, suggesting that dynamic equilibrium between R and L was achieved during tetanus. Increasing the extracellular [Ca²⁺] from 1 to 8 mM extended the R-L trajectory without a substantial shift of the relation. The Ca²⁺-sensitizing thiadiazinone derivative, EMD57033 (1 μM), shifted the R-L trajectory to the left (sensitization of the myofibrils to Ca²⁺), whereas the non-selective phosphodiesterase inhibitor, 3-isobutyl-1-methylxantine (200 μM), shifted the R-L trajectory to the right (desensitization of the myofibrils to Ca²⁺), in agreement with previous results obtained using skinned preparations. We conclude that the R-L trajectory is useful for estimating the myofibrillar responsiveness to Ca²⁺ in isolated myocytes and may be beneficial for the evaluation of inotropic agents. Received: 13 March 1998 / Received after revision: 4 May 1998 / Accepted: 2 June 1998     

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.     

Intracellular free magnesium in frog skeletal muscle studied with a new type of magnesium-selective microelectrode: interactions between magnesium and sodium in the regulation of [Mg]i

The application of a new type of intracellular magnesium-selective microelectrode based on the neutral carrier ETH5214 to measure intracellular free magnesium ([Mg]_i) in frog skeletal muscle fibers is reported. At room temperature (18–20° C) the average values for [Mg]_i was 0.93 mmol/l (pMg_i=3.03±0.42, SD; n = 38 experiments). The regulation of [Mg]_i was studied by measuring [Mg]_i and [Na]_i with ion-selective microelectrodes during alterations of the membrane potential and the transmembrane sodium and magnesium gradients. Depolarization by increasing external [K] from 2.5 mmol/l to 12.5 mmol/l did not significantly influence [Mg]_i. Increasing extracellular [Mg] from 1 mmol/l to 10 and 20 mmol/l caused a concentration-dependent rise in [Mg]_i and a decrease in [Na]_i, whereas removal of external magnesium did not affect [Mg]_i. Removal of external [Na] caused an increase in [Mg]_i and a decrease of [Na]_i. The results show that [Mg]_i in frog skeletal muscle is not in thermodynamic equilibrium and suggest that a Na/Mg exchange mechanism may be involved in maintaining low levels of [Mg]_i.