Biomedical subjects

J C Hancox

Publications and source records attributed to J C Hancox.

At least 55 records · Page 3Linked to original sources

PubMed1997

Cultured adult rabbit myocytes: effect of adding supplements to the medium, and response to isoprenaline.

INTRODUCTION: The aims of this study were to investigate: (1) the effect of supplementing the culture medium on preservation of L-type calcium channel current (1Ca,L) in adult rabbit ventricular myocytes cultured for 4 days; and (2) preservation of the ICa,L response in cultured myocytes to the beta-adrenergic agonist isoprenaline. METHODS AND RESULTS: Adult rabbit myocytes were cultured on laminin-pretreated glass coverslips. The basic, serum-free culture medium was supplemented with 2 mM L-carnitine, 5 mM creatine, and 5 mM taurine. Myocytes were whole cell patch-clamped, and the L-type Ca channel current was recorded selectively as Ba flux (IBa,L) via the channels. IBa,L density (i.e., IBa,L amplitude normalized to membrane capacitance) in myocytes maintained in supplemented medium did not change significantly during culture (P > 0.1). By comparison, IBa,L density in myocytes cultured in nonsupplemented medium declined by 36% after 24 hours in culture (day 1) and then recovered by the fourth day (day 4). There was no significant difference in the response to isoprenaline of acutely isolated myocytes and 4-day cultured myocytes. Isoprenaline 100 nM increased peak IBa,L by 149% +/- 32% (mean +/- SEM) in acutely isolated myocytes (n = 4 cells), and by 224% +/- 60% (n = 6 cells) and 159% +/- 24% (n = 8 cells) in day 1 and 4 cultured myocytes, respectively. CONCLUSIONS: Supplemented medium improved IBa,L density in cultured myocytes. beta-Adrenergic receptors and intracellular messenger pathways appear to remain intact in adult rabbit myocytes cultured for up to 4 days.

Adrenergic beta-Agonists↗

PubMed1997

Role of intracellular sodium overload in the genesis of cardiac arrhythmias.

A number of clinical cardiac disorders may be associated with a rise of the intracellular Na concentration (Na(i)) in heart muscle. A clear example is digitalis toxicity, in which excessive inhibition of the Na/K pump causes the Na(i) concentration to become raised above the normal level. Especially in digitalis toxicity, but also in many other situations, the rise of Na(i) may be an important (or contributory) cause of increased cardiac arrhythmias. In this review, we consider the mechanisms by which a raised Na(i) may cause cardiac arrhythmias. First, we describe the factors that regulate Na(i), and we demonstrate that the equilibrium level of Na(i) is determined by a balance between Na entry into the cell, and Na extrusion from the cell. A number of mechanisms are responsible for Na entry into the cell, whereas the Na/K pump appears to be the main mechanism for Na extrusion. We then consider the processes by which an increased level of Nai might contribute to cardiac arrhythmias. A rise of Na(i) is well known to result in an increase of intracellular Ca, via the important and influential Na/Ca exchange mechanism in the cell membrane of cardiac muscle cells. A rise of intracellular Ca modulates the activity of a number of sarcolemmal ion channels and affects release of intracellular Ca from the sarcoplasmic reticulum, all of which might be involved in causing arrhythmia. It is possible that the increase in contractile force that results from the rise of intracellular Ca may initiate or exacerbate arrhythmia, since this will increase wall stress and energy demands in the ventricle, and an increase in wall stress may be arrhythmogenic. In addition, the rise of Na(i) is anticipated to modulate directly a number of ion channels and to affect the regulation of intracellular pH, which also may be involved in causing arrhythmia. We also present experiments in this review, carried out on the working rat heart preparation, which suggest that a rise of Na(i) causes an increase of wall stress-induced arrhythmia in this model. In addition, we have investigated the effect on wall stress-induced arrhythmia of maneuvers that might be anticipated to change intracellular Ca, and this has allowed identification of some of the factors involved in causing arrhythmia in the working rat heart.

Action Potentials↗

PubMed1997

Ion channel and exchange currents in single myocytes isolated from the rabbit atrioventricular node.

OBJECTIVE: To review findings from the authors' laboratory in studies of electrophysiological properties of single rod- and spindle-shaped myocytes from rabbit atrioventricular node (AVN). DESIGN: Single cells were isolated from the AVN of the rabbit heart with the use of enzymatic and mechanical dispersion. For recording, cells were superfused with a Tyrode's solution at 33 to 37 degrees C, and recordings were made with microelectrodes or patch pipettes under 'current' or voltage' clamp conditions. Results are expressed as mean +/- SEM. RESULTS: AVN cells had a mean membrane capacitance of 40 +/- 3.9 pF and membrane resistance of 565 +/- 167 M omega (n = 9). Spontaneously active cells exhibited pacemaker activity showing a clear diastolic depolarization and overshooting action potential (AP) with a relatively slow upstroke velocity (7.4 +/- 0.9 V/s, n = 6) and a maximum diastolic potential of -70.5 +/- 2.9 mV. Under voltage clamp conditions, depolarizing pulses from 40 mV elicited L-type calcium currents sensitive to inhibition by nifedipine and managanese or cadmium ions, which could also block spontaneous APs. Depolarizing pulses also activated delayed rectifier potassium current (IK). IK showed rapid activation, and IK 'tails' in AVN cells were blocked by 5 microM E4031, consistent with the rapidly activating subtype of IK (IKr). IK was similar in AVN and ventricular myocytes, except for the time-course of deactivation, which was faster in AVN cells. In 80% to 90% of cells, hyperpolarizing voltage steps activated a small time-independent current. Ten per cent to 20% of cells showed the hyperpolarization-activated current (I(f)), but I(f) amplitude was only significant at potentials more negative than the pacemaker potential. AVN cells showed an apparent absence of inwardly rectifying potassium current. CONCLUSIONS: The high membrane resistance of AVN cells suggests that only small changes in ionic currents could significantly affect membrane potential. L-type calcium current is important in generating the AP upstroke, and IKr may play a role in both AP repolarization and diastolic depolarization. The ionic basis underlying spontaneous activity is not yet clear, but in some cells I(f) is not required because cells without I(f) can generate spontaneous APs.

Animals↗

PubMed1996

The effect of internal sodium and caesium on phasic contraction of patch-clamped rabbit ventricular myocytes.

1. The voltage dependence of phasic contraction was assessed in rabbit ventricular myocytes. Phasic contraction at all potentials was abolished by exposure to ryanodine-thapsigargin, showing that it was due primarily to Ca2+ release from the sarcoplasmic reticulum (SR). Experiments were performed at 35 degrees C, cells were whole-cell patch clamped and contraction was measured optically as unloaded shortening. Cells were held at -40 mV to inactivate the Na+ current (INa) and T-type Ca2+ current. A standard cellular Ca2+ load was established by applying a train of conditioning pulses at 0.5 Hz before each test pulse. The effect of replacing K+ with Cs+ in the dialysing pipette solution, and the effect of altering dialysing [Na+] between 0 and 20 mM, was assessed on contraction. 2. Cells dialysed with a K(+)-based, Na(+)-free solution exhibited a 'bell-shaped' voltage dependence of the L-type Ca2+ channel current (ICa,L), with a maximum ICa,L at +10 mV. Replacing internal K+ with Cs+, or altering pipette [Na+], did not affect the voltage dependence of ICa,L. 3. The voltage dependence of phasic contraction in cells dialysed with a K(+)-based solution was modulated by pipette [Na+]. The voltage dependence of phasic contraction was bell-shaped with 0 Na+, became much loss bell-shaped with 10 mM Na+ and with 20 mM Na+ the phasic contraction elicited at +100 mV was 1.6-fold larger than that at +10 mV. 4. Replacing 80% of K+ with Cs+ in the pipette dialysis solution led to a significant reduction in contraction amplitude and a more rapid decline in contraction amplitude after beginning the dialysis of the cell. 5. Cells dialysed with a Cs(+)-based solution displayed a voltage dependence of phasic contraction which was more bell-shaped (i.e. more similar to that of ICa,L) than that obtained with the corresponding K(+)-based dialysis solution. The level of pipette [Na+] still modulated the voltage dependence of phasic contraction in cells dialysed with a Cs(+)-based solution. 6. Time-to-peak contraction (tpk) also displayed voltage dependence; it had a minimum value between 0 and +20 mV (the voltage range for maximum ICa,L), but increased at more negative and positive potentials. Alteration of tpk contraction is discussed in relation to the stochastic behaviour of L-type Ca2+ channels and SR Ca2+ release channels. 7. The shape of the voltage dependence of contraction in rabbit myocytes at 35 degrees C is modulated by dialysing [Na+] over the tested range, 0-20 mM. Modulation of voltage dependence of contraction by dialysing [Na+] is consistent with an influence of reverse Na(+)-Ca2+ exchange in triggering intracellular Ca2+ release, in addition to the trigger Ca2+ which enters via ICa,L. 8. The marked effect of dialysing Cs+ on contraction amplitude, and on the voltage dependence of phasic contraction, does not appear to have been reported previously. Internal dialysis with Cs+ is a commonly used technique for blocking interfering outward K+ currents, in order to measure ICa,L more selectively. The present study suggests that Cs+ might also interfere with processes involved in excitation-contraction coupling and indicates that it might be wise to exercise caution with the use of internal Cs+ in experiments investigating excitation-contraction coupling.

Action Potentials↗

PubMed1996

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 (IK) 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 IK in morphologically normal cells from the atrioventricular node (AVN) and ventricle of the rabbit heart. Cells were held at -40 mV and 5 microM external nifedipine was used to block L-type calcium current (ICa,L). Significant IK 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 IK 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 IK 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 IK and this suggested that only the rapid component of IK, called IKr, was present in either cell type. Moreover, IK tails after pulses to all potentials were blocked completely by E-4031, a selective blocker of IKr. The reversal potential of IK 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 IK channel were highly selective for K. The time constants for deactivation of IK 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 IK in AVN cells was a distinguishing feature and suggests that there may be differences in the IKr channel protein between ventricular and AVN cells.

Action Potentials↗

PubMed1996

Action potentials, ion channel currents and transverse tubule density in adult rabbit ventricular myocytes maintained for 6 days in cell culture.

Adult rabbit ventricular myocytes were cultured in a basic medium (Medium 199) for up to 6 days to assess preservation of morphology and ion channel currents. In culture, cells remained rod shaped and striated but their ends became progressively rounded. Cell cross-sectional area declined slightly (by 14%) over the first 24 h, in contrast, whole-cell capacitance (which reflects external surface membrane plus membrane infoldings) decreased by 42% over the same time. Using whole-cell patch-clamp, we observed that the typical "N" shape steady-state current-voltage (I-V) relation became flattened after 24 h in culture. L-type Ca channel density was assessed as barium flux (IBa,L) via the channel. IBa,L (normalised to cell capacitance) declined by 50% after 24 h and recovered partially by days 4 and 6. The density of inward rectifier K current declined by 54% after 24 h and showed no recovery subsequently. In contrast, there was no significant decline in the density of transient outward K current after 24 h, but it declined subsequently by 65% after 6 days. We speculate that the time course of change in each ion channel density may reflect a change in pattern of ion channel expression, or differential membrane loss since the density of transverse tubules decreased by 57% after 6 days in culture. These results suggest that even by 24 h in culture, ion channel density in myocytes has changed substantially from the acutely isolated state.

Action Potentials↗

PubMed1996

The Fura-2 transient can show two types of voltage dependence at 36 degrees 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 (ICa,L) and intracellular Ca (Cai) transient in ventricular myocytes isolated from the rat heart. Intracellular Ca was monitored using Fura-2 and the experiments were carried out at 36 degrees C. We measured ICa,L by using a caesium-based internal dialysis solution to eliminate interfering K currents. The voltage dependence of peak ICa,L amplitude was bell-shaped: ICa,L was maximal at +10 mV and declined at more positive potentials. When ICa,L was integrated over the first 25 ms to estimate the magnitude of Ca entry, this had a very similar voltage dependence to peak ICa,L. In all cells, phasic Fura-2 transients were abolished by 5 microM 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 Cai 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 ICa,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 trigger Ca entry via ICa,L at this potential. This would suggest that, in these cells, another trigger for SR Ca release (in addition to ICa,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.

Animals↗

PubMed1996

A method for making rapid changes of superfusate whilst maintaining temperature at 37 degrees C.

We have developed a technique for making a rapid solution change, whilst at the same time maintaining the temperature of the preparation at 37 degrees C. It is technically difficult to use rapid solution changes when experiments are performed at normal mammalian body temperature. As a solution is heated from room temperature to 37 degrees C, gas bubbles form in the rapid-flowing solution streams, and these disturb a cell or attached recording pipettes. We describe a system that has been developed to eliminate these problems. We show how to construct the different components of the system, and we have designed an electronic circuit to control solution changes. We have performed tests to characterise the function of this system. Solution flow out of the nozzle of the device (0.88 ml min-1, linear flow velocity 11.6 cm s-1) caused a fall in the steady-state temperature at the experimental preparation of only 0.3 degrees C. The device which takes between 0.5 and 1 s to completely change the superfusate of a single cell, was used routinely with five different experimental solutions. This system may be valuable in studies which require rapid solution changes to be performed at a normal mammalian body temperature.

Animals↗

PubMed1996

Actions of the digitalis analogue strophanthidin on action potentials and L-type calcium current in single cells isolated from the rabbit atrioventricular node.

1. The atrioventricular node (AVN) of the heart is vital to normal cardiac function and is a major site of antiarrhythmic drug action. This study describes the effects of the cardiac glycoside analogue strophanthidin on spontaneous action potentials and L-type calcium current recorded from single AVN cells isolated from the rabbit heart. 2. With a standard KCl-based internal dialysis solution, exposure to 50 microM strophanthidin produced a progressive depolarization of the maximum diastolic potential and a reduction in action potential amplitude and upstroke velocity. Sustained application resulted in the loss of action potentials and occurrence of spontaneous 'bell-shaped' depolarizations. 3. Cells were whole-cell voltage clamped at -40 mV and depolarizing voltage clamps applied. With a standard KCl-based internal dialysis solution, exposure to 50 microM strophanthidin caused a large reduction of ICa,L at all potentials between -30 and +40 mV (n = 4). At + 10 mV, the mean ICa,L amplitude was reduced from -232 +/- 65 pA to -48 +/- 26 pA (P < 0.05; 1 test; n = 5 cells). 4. To record ICa,L more selectively, cells were dialysed with a Cs-based pipette solution. A short strophanthidin exposure reduced ICa,L amplitude from -250 +/- 31 pA to -88 +/- 19 pA (P < 0.001; n = 8 cells). For both KCl and CsCl-based solutions it was observed that sustained exposure to strophanthidin for several minutes caused spontaneous inward fluctuations in the membrane current record similar to the 'ITI' (arrhythmogenic oscillatory transient inward) current shown for other cardiac cells. 5. When the calcium chelator BAPTA was added to the pipette solution (10 mM), the reduction in ICa,L by strophanthidin was largely eliminated (P > 0.1), and no spontaneous inward current fluctuations were observed after sustained exposure to strophanthidin (n = 8 cells). 6. When external Ca in the perfusate was replaced with Ba, strophanthidin did not significantly reduce the Ba current through L-type calcium channels (n = 5 cells). 7. We conclude that strophanthidin reduces ICa,L by an indirect action, mediated by the rise in intracellular calcium (Cai) which follows inhibition of the Na/K pump caused by cardiac glycosides. The appearance of spontaneous ITI with strophanthidin would also seem to be mediated by a rise in Cai, and may contribute to the spontaneous oscillations in membrane potential observed after prolonged strophanthidin exposure.

Action Potentials↗

PubMed1995

Calcium transients which accompany the activation of sodium current in rat ventricular myocytes at 37 degrees C: a trigger role for reverse Na-Ca exchange activated by membrane potential?

We investigated the role of the fast sodium current (INa) in triggering Ca release from the sarcoplasmic reticulum (SR), using adult rat left ventricular myocytes, loaded with Fura-2 to measure intracellular Ca (Cai), which were whole-cell patch-clamped at 35-37 degrees C. Before each test pulse, a series of 400-ms conditioning pulses to +10 mV were applied to establish a constant level of SR Ca load. Pulses were applied every 15 s. A test pulse from -80 mV to -50 mV elicited a rapid INa and a phasic Cai transient. When the solution perfusing a myocyte was rapidly switched for 15 s before a test pulse to one containing the L-type Ca channel blocker nifedipine (20 microM), the test pulse still activated INa and a phasic Cai transient, the amplitude of which was not significantly different from control (P > 0.05; t-test). When a rapid switch to 20 microM nifedipine plus 30 microM tetrodotoxin (TTX) was made 15 s before a test pulse, both INa and the Cai transient were completely abolished (n = 6). When a switch was made to Na-free (Li) solution, which contained 20 microM nifedipine to block L-type Ca current, ICa,L, there was no significant difference in the Cai transient amplitude from that of control (P > 0.05; n = 6). Brief depolarising test pulses (-80 mV to +20 mV, 10 ms duration) to simulate membrane potential escape also elicited a Cai transient which attained 90.0% (+/-2.8%; n = 7) of the Cai transient activated by a conditioning pulse to +10 mV. The Cai transient with a brief pulse was not significantly affected by application of 20 microM nifedipine (P > 0.05), but adding TTX with nifedipine reduced the Cai transient amplitude to 76.9% (+/-6.8%; P < 0.02; n = 8). In four cells, the Cai transient remaining in the presence of nifedipine plus TTX was abolished by adding 5 mM Ni. These data are consistent with voltage escape during activation of INa leading to a trigger Ca entry via a mechanism other than L-type Ca channels or subsarcolemmal Na accumulation with reverse Na-Ca exchange. The block by Ni of the Cai transient suggests that a brief membrane potential escape might directly activate reverse mode Na-Ca exchange to trigger SR release, and this mechanism would seem to account largely for the Cai transient which accompanies INa in rat myocytes, under these experimental recording conditions.

Animals↗

PubMed1995

Spontaneous bursting induced by convulsant agents in an identified insect neurone.

1. Using an in vitro preparation, intracellular recordings were made from the cell body of the "fast" coxal depressor motoneurone (Df) from the cockroach, Periplaneta americana. 2. This cell was normally quiescent in the absence of injected current but could respond to applied depolarizing current with one or more plateau potentials: regenerative depolarizations which can drive production of axonal impulses. However, when the convulsant agents picrotoxin (PTX; 10(-5) M) or pentylenetetrazole (PeTZ; 25 mM) were applied Df exhibited spontaneous bursting behaviour. 3. The bursting activity induced by the two agents was qualitatively different. The bursting pattern induced by PTX was irregular and occurred only after a delay of 20-40 min; the transformation to bursting activity occurred without any consistent changes in membrane potential and effective membrane resistance (5.9 +/- 0.4 M omega; n = 25; mean +/- SEM) remained unchanged (P > 0.1). PTX did not induce spontaneous bursting or cause a significant change in plateau potential threshold or membrane potential in isolated somata. Therefore, it appeared to exert its actions or more distant regions of the neurone, probably by enhancing the effects of excitatory synaptic input. 4. In contrast, PeTZ rapidly (30 sec-2 min) induced regular bursting activity. This agent could also produce spontaneous bursting activity in isolated somata; thus it exerted its actions primarily upon intrinsic membrane properties, although it also enhanced the effects of electrically driven synaptic stimulation of the non-isolated Df somata. 5. Experiments with PTX also revealed that bursting could be recorded in cells in which it was not possible to evoke plateau potentials by electrically depolarizing the soma. This suggests that other regions of the cell possess the machinery necessary to produce burst-like behaviour.

Animals↗

PubMed1995

Barium enhances the excitability of a motoneurone from the cockroach (Periplaneta americana).

1. An isolated ganglion preparation was used to investigate the effects of barium ions (Ba2+) on excitability of the soma of the 'fast' coxal depressor motoneurone (Df) from the cockroach Periplaneta americana. 2. Under current-clamp, short (50 ms) depolarising pulses applied to the soma of Df elicited damped membrane oscillations in normal external solution. In the presence of 10 mM Ba2+, similar pulses produced all-or-none action potentials (n = 12). 3. Under voltage-clamp, addition of Ba2+ to the external solution suppressed the Ca-dependent K+ conductance (IC) in Df (n = 4). 4. Modulation of IC may offer a means of altering the excitability, and therefore output, of motoneurone Df.

Action Potentials↗

PubMed1995

The actions of nickel on membrane currents activated by hyperpolarisation in single cells from the rabbit atrioventricular node.

1. The atrioventricular node (AVN) is vital for cardiac function as it normally provides the only conduction route for the cardiac impulse from atria to ventricles and can act as a pacemaker for the ventricles if the sinoatrial node (SAN) fails. We have shown previously that whilst 80-90% of AVN myocytes do not possess If (we have termed these type 1 cells), a small proportion (10-20%) of AVN cells (type 2) do exhibit If. 2. The present study describes the effects of the divalent cation nickel (Ni) on membrane currents activated by hyperpolarising voltage clamps from -40/mV in type 1 and type 2 cells at 35 degrees C, using the whole cell patch clamp technique. In type 2 cells 5 mM Ni enhanced the amplitude of If. At -120 mV the mean Ni-activated If was -1.85 +/- 0.28 pA/pF (mean +/- SEM; n = 5). Ni significantly enhanced If at -70 mV and at all potentials negative to this (p < 0.05 at -70, -80, -90 and -110 mV; 0.05 < p < 0.1 at -100 mV; p < 0.005 at -120 mV). 3. In type 1 cells, which exhibit a small time-independent inward current on hyperpolarisation there was no activation of If by Ni (p > 0.1 at all potentials between -40 mV and -120 mV). 4. In type 1 cells 5 mM Ni significantly reduced the time-independent inward current activated by a hyperpolarising pulse to -120 mV (p < 0.02) and had a smaller effect at -110 and -100 mV (0.05 < p < 0.1 at these potentials). With pulses to less negative potentials there was no significant alteration of the time-independent current. 5. An additional observation was that the fast sodium current activated on repolarisation of the membrane potential to -40 mV after a hyperpolarising voltage clamp appeared to be blocked by Ni. However, this apparent blockade reflected a positive shift in the activation threshold for INa, since a repolarising step to -30 mV could still elicit INa. 6. Ni is known to block sarcolemmal Na/Ca exchange in cardiac cells, and one possible mechanism for the enhancement of If by Ni in type 2 cells is increased intracellular Ca via Na/Ca exchange blockade increasing If. The reduction in end pulse current in type 1 cells is also consistent with Na/Ca exchange current blockade. A second possibility of the enhanced If in type 2 cells with Ni is a positive shift of the activation curve for If in the presence of an increased concentration of external divalent cations.

Animals↗

PubMed1995

Na-Ca exchange tail current indicates voltage dependence of the Cai transient in rabbit ventricular myocytes.

INTRODUCTION: In mammalian cardiac myocytes, a rise of intracellular calcium (Cai) is well known to activate Ca extrusion via forward Na-Ca exchange, which generates an inward membrane current. This can be observed as an inward "tail" current (INa-Ca) when the membrane is repolarized after a depolarization-activated rise of Cai. If, during a voltage step, the membrane is repolarized at the time of the peak of the Cai transient, the size of the INa-Ca tail might be expected to reflect the magnitude of the Cai transient. Therefore, it might be possible to estimate the amplitude and voltage dependence of the Cai transient without, for instance, using fluorescent indicators that can interfere with Cai regulation. The first aim of this study was to use INa-Ca tails to investigate the voltage dependence of the Cai transient in whole cell patch clamped rabbit ventricular myocytes dialyzed with a "normal" level of internal Na. The second aim was to investigate how the voltage dependence of the INa-Ca tails varied with changes to the dialyzing Na concentration. The third aim was to test the correlation of voltage dependence of INa-Ca tails with the voltage dependence of the Cai transient obtained using a fluorescent Ca indicator. METHODS AND RESULTS: Experiments were performed at 35 degrees to 37 degrees C using whole cell patch clamp, and the holding potential was set at -40 mV. Depolarization elicited a Cai transient that peaked in 40 to 50 msec. We reasoned, therefore, that membrane repolarization after 50 msec would cause the raised level of Cai to activate an inward current on forward Na-Ca exchange. The amplitude of INa-Ca measured shortly (10 msec) after repolarization should reflect the peak amplitude of the Cai transient elicited by the depolarization. In cells dialyzed with 10 mM Na-containing solution and depolarized for 50 msec to differing test potentials, the INa-Ca tail on repolarization increased progressively after pulses to between -40 and +20 mV. The INa-Ca tail was maximal after a +20-mV pulse and showed no decline after depolarizations to more positive potentials, up to +100 mV (P > 0.1; n = 8). This implies that the Cai transient has a similar amplitude for depolarizing pulses between +20 and +100 mV. When Na-free solution dialyzed the cell, the voltage dependence of the INa-Ca tail became bell-shaped, with a maximum at +20 mV (n = 4). Voltage dependence of the INa-Ca tail was little affected by raising dialyzing Na from 10 to 20 mM (n = 4); but the amplitude of the INa-Ca tail increased. Inhibition of the Na-K pump with strophanthidin in cells dialyzed with 10 mM Na had qualitatively similar effects to increasing dialyzing Na. In Fura-2 loaded cells dialyzed with 10 mM Na, the Cai transient exhibited a similar voltage dependence to the INa-Ca tail (n = 6). CONCLUSION: The results of this study suggest that in cells dialyzed with 10 mM Na, the voltage dependence of the Cai transient is different from the L-type Ca current, since this current declines at potentials > +20 mV. The results obtained using Fura-2 suggest that the INa-Ca tail current measurement tracked the Cai sufficiently well to reflect the voltage dependence of the Cai transient. The data also confirm that the voltage dependence of the Cai transient in rabbit cells can be modulated by altering dialyzing Na concentration.

Animals↗

PubMed1994

Intracellular calcium transients recorded with Fura-2 in spontaneously active myocytes isolated from the atrioventricular node of the rabbit heart.

We have used the fluorescent Ca indicator Fura-2 to assess the changes in intracellular calcium (Cai) in single spontaneously active myocytes isolated from the rabbit atrioventricular node (AVN). Simultaneous recordings of membrane potential and the Fura-2 ratio signal (which reflects Cai) showed that a transient rise of Cai occurred with each spontaneous action potential (AP). The AP upstroke preceded the rise in Cai and repolarization of the AP occurred faster than the decline of Cai. The level of Cai remained raised and progressively declined towards a baseline diastolic level during the subsequent pacemaker depolarization. The Fura-2 (Cai) transient in spontaneously active AVN cells had a time-to-peak of 49.2 +/- 5.4 ms (mean +/- s.e.m.; n = 7) and declined with a single exponential time course (time constant = 139.8 +/- 23.9 ms; n = 7). Application of 10 microM ryanodine completely and irreversibly abolished the Cai transient, identifying the sarcoplasmic reticulum (SR) as the major source of releasable Ca. Both removal of external Ca and block of L-type Ca channels (with 2 microM nifedipine) also abolished Cai transients, suggesting that Ca entry via L-type Ca-channels is involved in triggering the SR Ca release underlying the Cai transient. Removal of external Na (in the presence of 20 microM nifedipine to block L-type Ca channels) caused a reversible increase in Cai, showing that Na/Ca exchange is present in AVN cells and that it is involved in Cai regulation. Spontaneous Cai transients were abolished by 1 microM acetylcholine, and this was associated with a hyperpolarization of membrane potential and cessation of action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗

PubMed1994

L-type calcium current in rod- and spindle-shaped myocytes isolated from rabbit atrioventricular node.

The atrioventricular node (AVN) is vital to normal cardiac function. The present report describes the properties of L-type calcium current (ICa) in rod- and spindle-shaped myocytes isolated from the rabbit AVN. With depolarizing voltage clamps from a holding potential of -40 mV, a rapidly activating ICa was observed, which peaked at +10 mV in most cells and exhibited a "bell-shaped" current-voltage relation. ICa was abolished by nifedipine (2-20 microM) and cadmium (100-200 microM) and was greatly reduced by manganese (1 mM). At +10 mV, time to peak ICa was 3.3 +/- 0.15 (SE) ms (n = 12) and ICa current density was 9.3 +/- 1.2 pA/pF (n = 9). Steady-state activation and inactivation curves for ICa showed half-maximal activation at -3.6 mV [slope factor (k) = 6.6 mV] and half-maximal inactivation at -25.8 mV (k = 6.5 mV). The time course of decay of ICa during a depolarizing pulse was voltage dependent and biexponential. The time course of recovery of ICa from inactivation was also biexponential (with two time constants tau 1 = 194.7 and tau 2 = 907.4 ms). Under current clamp, spontaneous action potentials from AVN cells were blocked by nifedipine as well as by cadmium, suggesting that L-type ICa was largely responsible for the action potential upstroke.

Animals↗

PubMed1993

One hump or two? The triggering of calcium release from the sarcoplasmic reticulum and the voltage dependence of contraction in mammalian cardiac muscle.

We have examined the hypothesis that the sarcolemmal Na/Ca exchanger is able to trigger calcium release from the sarcoplasmic reticulum in a direct fashion. We propose that when the cardiac muscle membrane is depolarised, for instance during the upstroke of the action potential or a square voltage clamp pulse, the voltage dependent Na/Ca exchanger generates an initial "spike" of calcium entry which is sufficient to trigger a fraction of the normal sarcoplasmic reticular calcium release, via calcium induced calcium release. For the last 20 years, it has been widely considered that calcium entry through L-type calcium channels is the only trigger for calcium release from sarcoplasmic reticulum in cardiac muscle. In the first section of this review, we examined some of the earlier studies of excitation-contraction coupling which used multicellular preparations of cardiac muscle. We suggested that these earlier studies do not support the idea that calcium entry via the calcium current (ICa) is the only trigger for sarcoplasmic reticular release. In contrast, more recent studies using isolated myocytes have supported ICa as the only trigger. However, these were performed mostly with a low or absent sodium inside the cell, or with an increased intracellular calcium buffering, or with other altered internal ions (eg, high magnesium or caesium in the pipette) or at a relatively low temperature. All these factors may have reduced or abolished the initial spike of calcium entry which the Na/Ca exchanger is expected to generate at the start of depolarisation. New studies on myocytes are presented, using conditions where cells are dialysed minimally, or where a normal level of internal sodium is preserved deliberately.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗

PubMed1993

A role for depolarisation induced calcium entry on the Na-Ca exchange in triggering intracellular calcium release and contraction in rat ventricular myocytes.

OBJECTIVE: The aim was to test whether depolarisation-induced calcium entry on the Na-Ca exchange is able to trigger calcium release from the sarcoplasmic reticulum in rat ventricular myocytes. METHODS: Myocytes were isolated enzymatically from the left ventricle of the rat heart. Cells were impaled with narrow tipped microelectrodes to minimise intracellular dialysis and maintain normal internal ionic conditions. Cells were voltage clamped, contraction was measured optically, and in some experiments intracellular calcium was measured with Fura-2. RESULTS: When the fast Na current was inactivated by using a holding potential of -40 mV, Ca entry via L-type Ca channels was expected to be the only mechanism capable of triggering sarcoplasmic reticular Ca release. In this situation, blocking L-type Ca channels should have abolished sarcoplasmic reticular release and the phasic twitch. However, after 2 min exposure to 20 microM nifedipine, which abolished the Ca current (ICa) completely, voltage clamp depolarisation from -40 mV to 0 mV still elicited 41(SEM 8.9)% of the control phasic twitch (n = 22 cells). This shows that there must be another mechanism, besides Ca entry via Ca channels, by which membrane depolarisation can trigger sarcoplasmic reticular release and the phasic twitch. The phasic twitch that remained in the presence of nifedipine increased progressively with the magnitude of step depolarisation, required a functional sarcoplasmic reticulum, was abolished by 5 mM external nickel, and was sensitive to both the Na and Ca transmembrane gradients. CONCLUSIONS: The voltage dependent sarcolemmal Na-Ca exchange is predicted theoretically to generate a transient Ca entry at the start of a step membrane depolarisation, when membrane potential suddenly becomes more positive than the reversal potential of the Na-Ca exchange. The results of this study indicate that in rat myocytes with normal internal ions, physiological levels of membrane depolarisation generate a sufficient Ca entry on the exchange to trigger sarcoplasmic reticular calcium release and contraction. In the absence of ICa, this mechanism is capable of triggering a calcium release which leads to about 40% of the phasic contraction in cells depolarised from -40 mV to 0 mV. The existence of this sarcoplasmic triggering mechanism may have significance for the normal control of cardiac muscle contraction.

Animals↗