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P Lipp

Publications and source records attributed to P Lipp.

63 records · Page 4Linked to original sources
PubMed1991

Measurement of Ca2(+)-release-dependent inward current reveals two distinct components of Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes.

Ca2+ current (L-type) and inward current caused by Ca2+ release from the sarcoplasmic reticulum and carried by electrogenic Na+/Ca2+ exchange have been measured in cultured atrial myocytes from hearts of adult guinea-pigs using whole-cell voltage clamp techniques. The pipette solution, used for internal dialysis of the cells, contained a high concentration, 60 mM or 25 mM, of citrate as a non-saturable low-affinity Ca2(+)-chelating compound. It has been shown previously that Ca2(+)-release-dependent inward current under these conditions is carried by electrogenic Na+/Ca2+ exchange. Furthermore, Ca2(+)-release-dependent inward current (the release signal) can be completely separated from triggering Ca2+ current if brief depolarizations for activating ICa are used. In the majority of cells that did not produce spontaneous Ca2+ release, conditions could be found that caused the release signal to be split into two components: an early component of variable amplitude and a late component of rather constant amplitude. The delay of the late component with regard to triggering ICa was inversely related to the amplitude of the first one. Below a certain amplitude of the first component, the second one failed to be elicited. This suggests the second component to be triggered by the first one. Weakly Ca2(+)-buffered cells produced spontaneous Ca2+ release, resulting in irregular "transient inward currents" at constant membrane-holding potential. Synchronization by trains of step depolarizations unmasked two components also in the spontaneous release signals. In none of the cells studied was any indication of more than two components of the release signal detected. The results are discussed in terms of two distinct compartments of sarcoplasmic reticulum with different properties of Ca2+ release.

Animals↗
PubMed1991

High-resolution measurement and calibration of Ca(2+)-transients using Indo-1 in guinea-pig atrial myocytes under voltage clamp.

Spherical atrial myocytes obtained by enzymatic dispersion of hearts from adult guinea-pigs were loaded with the fluorescent Ca(2+)-indicator Indo-1 via patch-clamp pipettes. The dialysing solution additionally contained citrate (60 mM) as low-affinity ('linear') Ca(2+)-chelating compound in order to slow intracellular Ca(2+)-transients. Changes in Indo-1 fluorescence under voltage-clamp due to Ca(2+)-entry and/or release from the SR were calibrated using an in vivo procedure to determine the limiting fluorescence ratios. Sample recordings will be presented to demonstrate that components of a [Ca2+]i-transient due to entry via L-type Ca(2+)-channels and due to Ca(2+)-release from the SR can be directly visualized.

Animals↗
PubMed1990

Simultaneous recording of Indo-1 fluorescence and Na+/Ca2+ exchange current reveals two components of Ca2(+)-release from sarcoplasmic reticulum of cardiac atrial myocytes.

Simultaneous measurements of intracellular Ca2(+)-concentration ([Ca2+]i) using Indo-1 and the current generated by electrogenic Na+/Ca2(+)-exchange (INaCa) have been performed on atrial myocytes from hearts of adult guinea-pigs. Whereas the fluorescence-measurements provide information on global [Ca2+]i, InaCa which is a linear function of Ca2(+)-concentration, indicates subsarcolemmal [Ca2+]. Under conditions in which intracellular Ca2(+)-transients due to Ca2(+)-release from the sarcoplasmic reticulum (SR) have been artificially slowed, a deviation between the two different Ca2(+)-signals can be found. During onset of release signals Ca2(+)-concentration seen by the membrane is higher than global [Ca2+]i. Our results provide evidence that in atrial myocytes, lacking a T-system, Ca2(+)-induced Ca2(+)-release occurs first from a subsarcolemmal compartment of the SR. The resulting Ca2(+)-transient serves to trigger Ca2(+)-release from deeper SR-compartments.

Animals↗
PubMed1988

Transient inward current in guinea-pig atrial myocytes reflects a change of sodium-calcium exchange current.

1. Enzymatically isolated, cultured myocytes from hearts of adult guinea-pigs were voltage clamped with a whole-cell patch-clamp technique. The pipette-filling solution for internal dialysis contained 65 mM-citrate and 50 microM-EGTA as Ca2+-chelating agents and 20 mM-Na+. Potassium channel currents were blocked by replacing this ion on both sides of the membrane by Cs+. 2. In the above conditions myocytes develop spontaneous transient inward currents (Iti) at constant negative membrane holding potentials. At a given membrane potential Iti can be recorded with constant amplitude and frequency for periods of up to ca. 40 min. A membrane current with similar properties can be evoked by superfusion of the cell with caffeine-containing (5-10 mM) solution. 3. Depolarization results in a reduction of Iti amplitude and a prolongation of its duration. After a step change of the membrane potential to ca. -10 mV or a less-negative level only one inward current change is observed. Thereafter the membrane current remains inward with regard to the instantaneous current at this membrane potential. Complete relaxation of Iti then is only observed after repolarization to a more-negative membrane potential. 4. The current change caused by sarcoplasmic Ca2+ release is inward in a range of membrane potentials between -90 and +75 mV. A reversal of Iti was never detected. 5. Both the instantaneous current-voltage (I-V) relation and voltage dependence of peak Iti display distinct outward rectification. Both I-V relations can be described by a formalism suggested for a membrane current caused by electrogenic Na+-Ca2+ exchange (INa, Ca) assuming a 3:1 stoichiometry and a single energy barrier in the electric field of the membrane. 6. An increase of the time integral of Iti at the holding potential is observed after depolarizations to positive membrane potentials, where the outward-rectifying current component is prominent. This supports the view that the outward current represents INa, Ca in the 'reverse mode', carrying Ca2+ ions into the cell. 7. After prolonged cell dialysis a run-down of Iti is observed. Since strong depolarizations in this condition still can cause inward currents upon repolarization, run-down is likely to reflect an impairment of sarcoplasmic reticulum function rather than an effect of cell dialysis on the exchanger. 8. We conclude that under the present conditions a membrane current is measured, which to a large extent determines the 'passive' I-V curve of the myocytes. This current is modified by a rise in Ca2+(i) following sarcoplasmic Ca2+ release.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1988

Voltage dependence of sodium-calcium exchange current in guinea-pig atrial myocytes determined by means of an inhibitor.

1. Spontaneous transient inward currents (Iti) caused by cyclic release of Ca2+ ions from the sarcoplasmic reticulum were studied in cultured atrial myocytes from hearts of adult guinea-pigs. K+ channel currents were blocked by replacing K+ on both sides of the membrane by Cs+; the L-type Ca2+ current was inhibited by D600. 2. The voltage dependence of peak Iti and the background current displayed distinct outward-going rectification. The I-V curves for both currents approach each other at strongly positive membrane potentials but do not intersect. 3. 3'-4'Dichlorobenzamil (DCB) causes a concentration-dependent inhibition of peak Iti and a shift of the holding current (at -60 to -40 mV) in the inward direction. Inhibition of Iti is half-maximal at a concentration of 30 microM. 4. DCB reduces the outward-rectifying component of both peak Iti and the background current. The I-V curves of the control and DCB-inhibited currents intersect at ca. +10 mV (peak Iti) and negative to -75 mV (background current), suggesting the reversal potential of the DCB-inhibited current to be shifted by ca. 85 mV in the positive direction if Cai2+ rises following Ca2+ release. 5. The voltage dependence of the DCB-inhibited currents is highly compatible with the concept of Na+-Ca2+ exchange being the charge-carrying mechanism of the outward-rectifying background current. Ca2+ release from the SR alters the I-V curve of this current according to the shift of the thermodynamic driving force.

Action Potentials↗
PubMed1987

Effects of calcium release from sarcoplasmic reticulum on membrane currents in guinea pig atrial cardioballs.

(1) Ca current (ICa) and membrane currents related to Ca-entry during activation of ICa have been studied in cultured atrial myocytes from hearts of adult guinea pigs by means of patch clamp pipettes. The pipettes were filled with solutions containing citrate (65 mM) as major Ca-chelating compound and Cs ions in order to block K currents. (2) In myocytes dialysed with such solutions a monophasic time course of inactivation of ICa is observed, which is 1-2 orders of magnitude slower as compared to studies on intact cardiac cells or cells dialysed with EGTA as only Ca-chelating compound. (3) During long-lasting or repetitive depolarization a second component of ICa inactivation, apart from the slow decay observed in cells dialysed with such solutions, can be seen. This component of inactivation is not related to the depolarization as such but to loading of the cells with Ca2+. Whenever the rapid component of inactivation occurs, a transient inward current (Iti) after repolarization to the holding potential (-40 to -50 mV) is recorded. Both, ICa inactivation and Iti can be mimicked by extracellular application of caffeine (5-10 mM), suggesting both current changes to be caused by a rise in Cai due to Ca release from sarcoplasmic reticulum. In the presence of caffeine the rapid component of ICa-inactivation and Iti are abolished. (4) In addition to ICa inactivation and activation of Iti sarcoplasmic Ca release causes openings of a novel ion channel with large conductance (greater than 200 pS), the function of which is unknown. (5) The results are consistent with the concept of Cai-dependent inactivation of Ca current, which can be caused either by Ca-entry or by Ca-release from the SR. The transient inward current is likely to reflect a process of Ca-removal from the cell, namely Na-Ca exchange.

Animals↗
PubMedDate not supplied

Confocal microscopic detection of potential-sensitive dyes used to reveal loss of voltage control during patch-clamp experiments.

We used a fast, fluorescent, potential-sensitive indicator (Di-8-ANEPPS) in combination with laser-scanning confocal microscopy in the line-scan mode (temporal resolution 500 Hz) to independently determine the transmembrane potential in voltage-clamped cells. While a linear relation between command voltage and Di-8-ANEPPS fluorescence was found in unexcitable Sf9 cells, pronounced nonlinearities were observed in cardiac myocytes. Comparison of the fluorescence records and current traces indicated that most of the observed nonlinearities could be attributed to voltage-escape during flow of membrane current. Voltage-escape during large membrane currents may lead to various experimental difficulties during voltage-clamp experiments. The voltage recording technique based on fluorescence was then used to compare the voltage-escape during flow of Na+ and Li+ ions via voltage-dependent (TTX sensitive) Na+ channels in cardiac myocytes. In these experiments, no significant differences in the degree of voltage-escape was found, suggesting that the two currents were similar in amplitude. In addition to the application presented in this paper, confocal microscopic detection of transmembrane potential with fluorescent dyes may be a useful technique for experiments in preparations that are difficult to impale with microelectrodes because of their small size.

Animals↗
PubMedDate not supplied

Functional InsP3 receptors that may modulate excitation-contraction coupling in the heart.

The roles of the Ca2+-mobilising messenger inositol 1,4,5-trisphosphate (InsP3) in heart are unclear, although many hormones activate InsP3 production in cardiomyocytes and some of their inotropic, chronotropic and arrhythmogenic effects may be due to Ca2+ release mediated by InsP3 receptors (InsP3Rs) [1-3]. In the present study, we examined the expression and subcellular localisation of InsP3R isoforms, and investigated their potential role in modulating excitation-contraction coupling (EC coupling). Western, PCR and InsP3-binding analysis indicated that both atrial and ventricular myocytes expressed mainly type II InsP3Rs, with approximately sixfold higher levels of InsP3Rs in atrial cells. Co-immunostaining of atrial myocytes with antibodies against type II ryanodine receptors (RyRs) and type II InsP3Rs revealed that the latter were arranged in the subsarcolemmal space where they largely co-localised with the junctional RyRs. Stimulation of quiescent or electrically paced atrial myocytes with a membrane-permeant InsP3 ester, which enters cells and directly activates InsP3Rs, caused the appearance of spontaneous Ca2+-release events. In addition, in paced cells, the InsP3 ester evoked an increase in the amplitudes of action potential-evoked Ca2+ transients. These data indicate that atrial cardiomyocytes express functional InsP3Rs, and that these channels could modulate EC coupling.

Animals↗