Extracellular osmotic pressure modulates sodium-calcium exchange in isolated guinea-pig ventricular myocytes.

1. The sensitivity of the cardiac Na(+)-Ca2+ exchange current to changes in osmotic pressure was investigated in guinea-pig ventricular myocytes, using the whole-cell patch-clamp technique. 2.

Modulation of cardiac L-type Ca2+ channels by GTP gamma S in response to isoprenaline, forskolin and photoreleased nucleotides.

1. Using the patch-clamp recording technique, we have investigated the effects of chronic intracellular application of guanosine thiotriphosphate (GTP gamma S) by cell dialysis, on the potentiation of L-type Ca2+ currents (ICa) by isoprenaline and forskolin and also by GTP gamma S and cyclic AMP released intracellularly by flash-photolysis of their caged derivatives. 2.

The regulation of intracellular Mg2+ in guinea-pig heart, studied with Mg(2+)-selective microelectrodes and fluorochromes.

Because of the reported presence of a Na(+)-Mg2+ exchanger in guinea-pig but not in ferret myocardium, the Mg2+ extrusion mechanism in guinea-pig myocardium has been reinvestigated using Mg(2+)- and Na(+)- selective microelectrodes and the fluorochromes mag-fura-2 and -5. The mean [Mg2+]i measured with microelectrodes in trabeculae or papillary muscles was 0.72 mmol/l (n = 22, thirteen experiments; range 0.

Activation of L-type Ca2+ currents in cardiac myocytes by photoreleased GTP.

L-type calcium currents (ICa) were recorded from isolated ventricular myocytes by using standard patch-clamp methods. In the absence of agonist, photorelease of GTP by flash photolysis of intracellularly applied caged-GTP rapidly increased the amplitude of ICa over a wide range of membrane potentials. Control experiments clearly demonstrated that this effect was not due to either the release of photolytic by-products or to the light flash itself.

Temperature dependence of electrophysiological properties of guinea pig and ground squirrel myocytes.

The effects of changing temperature on the electrophysiology of isolated cardiac myocytes of the guinea pig and Richardson's ground squirrel were studied by patch-clamp techniques. In cells from both species, the resting membrane potential declined on cooling from 36 to 12 degrees C by approximately 6 mV. The duration of the plateau of the action potential in guinea pig cells increased monotonically on cooling.

Flash photolysis of intracellular caged GTP gamma S increases L-type Ca2+ currents in cardiac myocytes.

L-type calcium currents were recorded from isolated ventricular myocytes using standard patch-clamp methods. Rapid release of photolabile guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), tetralithium salt, by flash photolysis of intracellular caged-GTP gamma S increased the magnitude of the L-type calcium current: an effect independent of the ultraviolet flash per se or the production of photolytic by-products. This increase in calcium current was markedly reduced by intracellularly applied guanosine 5-O-(thiodiphosphate), trilithium salt or by an excess of GTP gamma S.

Cytosolic free Ca2+ during operation of sodium-calcium exchange in guinea-pig heart cells.

1. Membrane current generated by the Na(+)-Ca2+ exchange mechanism was recorded in single guinea-pig ventricular myocytes using the whole-cell voltage-clamp technique and the intracellular free calcium concentration ([Ca2+]i) was monitored using the fluorescent probe Indo-1, applied intracellularly through a perfused patch pipette. The reversal potential of the exchanger (ENa, Ca) was measured from records of the 2 mM-Ni(2+)-sensitive current and used in an attempt to clamp [Ca2+]i at a level determined by the ionic compositions of the external and pipette solutions.

Acute effects of adriamycin on the macromolecular organization of the cardiac muscle cell plasma membrane.

Adriamycin is a potent chemotherapeutic agent used in the treatment of human neoplastic disease. A major side effect limiting the use of this drug is its toxic effect on the heart, and congestive heart failure becomes an increasingly common complication as the cumulative dose of the drug rises. To learn more about the mechanism of adriamycin cardiotoxicity and, in particular, to investigate its initial effects on the cardiac muscle cell plasma membrane, isolated guinea pig myocytes were exposed to the drug in vitro.

Integrity of the dissociated adult cardiac myocyte: gap junction tearing and the mechanism of plasma membrane resealing.

Dissociation of adult cardiac myocytes by collagenase perfusion techniques requires separation of the junctional contacts that link the cells physically, electrically and metabolically in the intact heart. Gap junctions, one of three types of intercellular junction present at the cardiac intercalated disc, are not split into their component membranes when myocytes are dissociated; they are ripped from the plasma membrane of one cell, to be retained by its neighbour. Partitioning of junctions in this way might be expected to constitute a serious threat to the ionic integrity of dissociated myocytes, but in practice, high yields of functionally intact cells, suitable for experimental studies, are routinely obtained.

Fate of gap junctions in isolated adult mammalian cardiomyocytes.

The fate of gap junctions in dissociated adult myocytes, maintained for up to 22 hours in culture medium, was investigated by semiquantitative analysis of thin sections and by freeze-fracture electron microscopy. Gap junctions in the dissociated myocyte are intact bimembranous structures seen either as invaginated surface-located structures or as annular profiles in the cytoplasm. Surface-located junctions are sealed from the exterior by a sheet of nonjunctional membrane originating (together with the "outer" junctional membrane) from the formerly neighboring cell.

Sodium-calcium exchange during the action potential in guinea-pig ventricular cells.

1. Slow inward tail currents attributable to electrogenic sodium-calcium exchange can be recorded by imposing hyperpolarizing voltage clamp pulses during the normal action potential of isolated guinea-pig ventricular cells. The hyperpolarizations return the membrane to the resting potential (between -65 and -88 m V) allowing an inward current to be recorded.

On the mechanism of isoprenaline- and forskolin-induced depolarization of single guinea-pig ventricular myocytes.

1. Isoprenaline (10 nM to 1 microM) and forskolin (0.6-100 microM) depolarized single guinea-pig myocytes studied in vitro.

Calcium-activated inward current and contraction in rat and guinea-pig ventricular myocytes.

1. Single ventricular cells from rat and guinea-pig hearts were voltage clamped, and contraction was monitored with an optical method. 2.

Electrical activity and contraction in cells isolated from rat and guinea-pig ventricular muscle: a comparative study.

1. Contraction in single ventricular muscle cells from rat and guinea-pig heart was measured using an optical technique, while at the same time either action potentials were recorded or transmembrane currents were measured under voltage-clamp conditions. 2.

An isoprenaline activated sodium-dependent inward current in ventricular myocytes.

In the heart, catecholamines affect pacemaker activity by shifting the activation curve for the nonspecific inward current and increasing both the calcium current, and the delayed potassium current. We report here that in mammalian ventricle there is another mechanism that seems to involve a sodium-dependent inward current. This is elicited by agents that increase intracellular cyclic AMP concentration, such as the beta-adrenergic agonist isoprenaline, and is unaffected by agents which block the three currents listed above, but is absent when external sodium is replaced with tetramethylammonium.

Electrical properties and response to noradrenaline of individual heart cells isolated from human ventricular tissue.

The analysis of the electrical properties and response to catecholamines of cardiac tissue is greatly simplified by the use of single cell preparations. In this study individual cells isolated from human ventricular tissue were used to estimate cellular sarcolemmal resistance and capacitance and to record the time course of the response to ionophoretically applied noradrenaline. The mean input capacitance of the cells is consistent with a surface membrane area of approximately 15,000 micron2 if the specific membrane capacitance is 1 microF X cm-2.

Determination of intracellular potassium ion concentration in isolated rat ventricular myocytes.

The sarcoplasmic potassium concentration of a suspension of rat ventricular myocytes, prepared by collagenase-induced disruption of the myocardial mass, was determined by a null-point technique. Addition of digitonin resulted in a release of potassium from the cells which was interpreted as a flux from the sarcoplasm. The intracellular potassium concentration was estimated to be 113 +/- 6mM.

Influence of a change in stimulation rate on action potentials, currents and contractions in rat ventricular cells.

The effects of a change in stimulation rate on electrical activity and accompanying contraction were investigated in ventricular cells isolated from rat heart; the cells were stimulated to contract either by brief depolarization pulses which evoked action potentials, or, under voltage-clamp conditions, by step depolarizations. An increase in stimulation rate from 0.3 to 3 Hz resulted in a gradual reduction in the amplitude of contraction and attenuation of the late phase of the action potential.

Ultrastructure of the sarcolemma and intercalated disc in isolated rat myocytes.

The ultrastructure of the sarcolemma in isolated calcium-tolerant myocytes has been compared with that of myocytes in the intact heart, using thin section and freeze-fracture electron microscopical techniques. Data on the density and distribution of intramembrane particles and on the topography of Z-folds in the general (i.e.

Morphometric analysis of the isolated calcium-tolerant cardiac myocyte. Organelle volumes, sarcomere length, plasma membrane surface folds, and intramembrane particle density and distribution.

Using morphometric analysis of thin sections and freeze-fracture replicas, the ultrastructure of isolated rat myocytes prepared by collagenase digestion (Powell et al. 1980) was compared with that of myocytes fixed by perfusion of intact myocardium. The volumes of myofibrils, mitochondria, nuclei, sarcoplasmic reticulum and lipid droplets in the isolated myocytes did not differ from those of their counterparts in the intact heart, but the volume occupied by transverse tubules was apparently reduced.

Morphometric analysis of calcium-tolerant myocytes isolated from the adult rat heart.

The ultrastructure of calcium-tolerant isolated myocytes has been compared with that of intact heart myocytes using morphometric techniques. Isolated myocytes were prepared by the method of Powell et al. [1] from four adult rat hearts, and multiple left ventricular tissue samples were obtained from a further four rat hearts after fixation by Langendorff perfusion.

The effects of sodium, hydrogen and magnesium ions on mitochondrial calcium sequestration in adult rat ventricular myocytes.

The effect of Na+, H+ and Mg2+ ions on net calcium exchange induced in digitonin-treated myocytes has been investigated. Raising the [Na] from 1.4 to 31.

Net calcium exchange in adult rat ventricular myocytes: an assessment of mitochondrial calcium accumulating capacity.

Net calcium exchange has been measured in a suspension of cardiac myocytes after treatment with digitonin. The exchange is believed to be across the mitochondrial membranes and can be stimulated or inhibited by agents augmenting or blocking mitochondrial electron transport. The uptake of calcium shows a strong dependence on suspension pCa but is not evident below 1 microM (pCa 6.

Cytoplasmic free calcium measured by quin2 fluorescence in isolated ventricular myocytes at rest and during potassium-depolarization.

We have measured intracellular free Ca2+ concentration in rat and guinea pig ventricular myocytes using the fluorescent Ca2+-indicator quin2. Our results indicate a resting concentration in heart cells that is considerably lower than previous estimates. The mean value of 137 nM for rat ventricle that we have recorded is consistent with the hypothesis that resting intracellular [Ca2+] is controlled by a voltage-dependent, sarcolemmal exchange mechanism.

Strontium, nifedipine and 4-aminopyridine modify the time course of the action potential in cells from rat ventricular muscle.

Action potentials, initiated by brief depolarizing pulses, were recorded from single cells isolated from rat ventricular muscle. These action potentials showed a rapid upstroke to about +30 mV, followed by two phases of repolarization referred to as the early and late phases of the action potential. Nifedipine (1 microM), which blocks the second inward current (Isi) carried by Ca in these cells, shortened the early phase.