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. A hyposmotic challenge applied by removal of sucrose from the standard bathing solution reduced exchanger current, measured as the Ni(2+)-sensitive component of whole-cell transsarcolemmal current. These changes were fully reversible. 3. No response of whole-cell current to hyposmosis was observed when Ca2+ was removed from the bathing solution by chelation with 1 mM EGTA. 4. Inclusion of 25 microM exchanger inhibitory peptide (XIP) in the pipette solution caused a marked reduction in the Ni(2+)-sensitive component of membrane current, but the percentage change in Ni(2+)-sensitive membrane slope conductance evoked by hyposmosis was the same as when XIP was omitted from the pipette solution. 5. Exposure of cells to hyperosmotic solutions produced variable responses. In a majority of cells, solutions 30% hyperosmotic compared with control evoked a persistent increase in exchanger current, whereas for solutions 50% hyperosmotic, a larger but transient increase in current was observed. 6. Over a wide range of osmolalities (50-130% of isosmotic) the changes in Ni(2+)-sensitive membrane slope conductance were linearly related to the changes in extracellular osmotic pressure. 7. We propose that one consequence of exposing ventricular myocytes to anisosmotic solutions is modulation of Na(+)-Ca2+ exchange current.

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. GTP gamma S prevented enhancement of ICa by isoprenaline with an IC50 of approximately 10 microM and considerably reduced the ability of forskolin to increase ICa. In addition GTP gamma S also reduced the time-to-peak response for potentiation of ICa by forskolin. Responses to forskolin were abolished by co-dialysis of cells with the cyclic AMP antagonist, Rp-adenosine-3'-5'-mono-thionophosphate (Rp-cAMPS). 3. Photoreleased GTP gamma S (PR-GTP gamma S; approximately 23 microM) generally induced a biphasic increase in ICa. This response was also inhibited by chronic intracellular dialysis with GTP gamma S with an IC50 of approximately 1 microM. 4. Pretreatment of cells with pertussis toxin (PTX) reversed the inhibitory effect of 100 microM GTP gamma S on isoprenaline-induced stimulation of ICa. However, PTX pretreatment did not restore the activating action of PR-GTP gamma S inhibited by chronic application of GTP gamma S. 5. Photoreleased cyclic AMP (approximately 5 microM; PR-cyclic AMP) increased peak ICa. This effect was inhibited by dialysis of cells with Rp-cAMPS and by stimulation of ICa by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Co-dialysis of cells with uncaged GTP gamma S reduced the time-to-peak for PR-cyclic AMP mediated activation of ICa but did not affect the magnitude of the response. 6. It is concluded that chronically applied GTP gamma S can (i) inhibit activation of ICa by isoprenaline by interacting with a PTX-sensitive guanosine nucleotide binding (G-) protein located upstream of adenylate cyclase (possibly Gi) and (ii) accelerate the response to cyclic AMP dependent phosphorylation possibly by interacting with a G-protein coupled directly to the channel. 7. In view of this diverse range of effects, care should be taken when using GTP gamma S to characterize G-protein-mediated events, since the resulting physiological response may be due to activation of several G-protein containing pathways.

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 0.42-1.23 mmol/l). Increasing [Mg2+]o from 0.5 mmol/l to either 10.5 or 20 mmol/l caused small increases in [Mg2+]i. Decreasing [Na+]o by 50% had no effect on the [Mg2+]i and there was no change in [Na+]i on increasing [Mg2+]o from 0.5 to 10.5 mmol/l. Varying pHo or changing pHi with NH4Cl did not influence the [Mg2+]i. In vitro calibration of mag-fura-2 and -5 using the ratio method gave values for K'd (experimentally determined dissociation constant) of 22.2 +/- 2.7 (mean +/- S.D., n = 7) and 25.7 +/- 1.3 (n = 4) mmol/l respectively. Mag-fura-2 reacted to physiological concentrations of Ca2+ and mag-fura-5 to changes in pH. In isolated myocytes, Na+ removal gave an apparent increase of [Mg2+]i with mag-fura-2 but not with mag-fura-5. However, when the pHi was altered with NH4Cl mag-fura-5 showed an apparent decrease in [Mg2+]i on application and an apparent increase on removal, with a time course similar to the pHi changes. It is concluded that Mg2+ extrusion in guinea-pig myocardium is not via a Na(+)-Mg2+ exchanger. The use of mag-fura-2 and -5 are limited in their application because of Ca2+ and H+ sensitivity respectively.

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. The timecourse for activation of ICa by photolysis of caged-GTP was markedly altered by intracellular application of either GDP beta S or GTP gamma S. Upon maximal stimulation of ICa by intracellular dialysis with cAMP, photoreleased GTP induced a small, rapid increase in ICa followed by a gradual inhibition. The presence of Rp-cAMPS intracellularly reduced both the magnitude of the response to photoreleased GTP and its time to peak. Similar effects were observed when protein kinase inhibitor dialysed the cell interior, suggesting that both cAMP-dependent and independent processes were involved in this effect. We conclude that rapid release of GTP within ventricular myocytes, in the absence of agonist, causes rapid activation of L-type Ca2+ current. Mechanisms underlying this effect include stimulation of adenylate cyclase, together with other, as yet uncharacterized, GTP-dependent pathways for increasing ICa in the heart.

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. In contrast, the action potential of ground squirrel cells showed a biphasic response, increasing in duration from 36 to 24 degrees C and then decreasing on cooling from 24 to 12 degrees C. From voltage-clamp studies, the properties of L-type calcium currents (ICa) on cooling were compared in the two species and were found to be similar: In both cases, ICa decreased in amplitude from approximately 2 nA peak current at 36 degrees C to less than 400 pA at 12 degrees C. The Q10 of both the maximum amplitude and time to peak for ICa in both species was approximately 1.8. The time for half inactivation had a greater Q10 of 2.5-3. It is concluded that, surprisingly, factors affecting the resting membrane potential and properties of L-type calcium channels are not major contributors to cardiac dysfunction on cooling. Rather, it is sarcoplasmic reticulum calcium release and reuptake that are likely to be the most important cold-sensitive processes.

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. It is therefore likely that rapid release of GTP gamma S intracellularly in the absence of an agonist can, via a G protein-mediated mechanism, cause L-type calcium current activation. In the presence of Rp-adenosine 3,5-mono-thionophosphate (Rp-cAMP), photoreleased GTP gamma S results in a transient and much reduced increase in the amplitude of the L-type calcium current. We conclude that activation of Gs coupled to adenylate cyclase and ultimately cAMP-dependent phosphorylation may be primarily responsible for L-type channel activation, although a fast membrane-delimited (direct) pathway, not involving cytoplasmic second messengers, may also contribute to this effect.

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. 2. Measurements of ENa, Ca indicated that [Ca2+]i was close to that in the pipette solution when the holding potential was set at the ENa, Ca expected for a 3Na+:1Ca2+ exchanger. The measured value of ENa, Ca was more positive than the theoretical value when the membrane potential was held positive to ENa, Ca and the opposite was true when the holding potential was more negative than the expected ENa, Ca. 3. As Indo-1 diffused into the cell from the whole-cell clamp electrode, the intensities of the fluorescent signals measured at 405 and 480 nm increased with time, with no obvious saturation over a 10-45 min recording period. However, the ratio of these two signals reached a steady level within 5 min after rupture of the patch membrane, when the holding potential was set at the expected ENa, Ca of the exchanger. The intensity ratios measured using pipette solutions containing 600 and 803 nM [Ca2+] were almost equal to the ratios obtained extracellularly from internal solutions of identical compositions, but in experiments using pipette solutions having lower [Ca2+] the intensity ratios measured in myocytes were higher than those obtained extracellularly. 4. If the membrane was depolarized or hyperpolarized, the fluorescence ratio either increased or decreased, respectively. These changes in the fluorescence ratio were virtually blocked by the extracellular application of 2 mM-Ni2+. 5. When the concentration of bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the recording pipette was reduced from 30 to 1 mM, an increase in [Ca2+]i was observed during a depolarizing ramp pulse. The Ca2+ influx estimated by integrating the 2 mM-Ni(2+)-sensitive current during the pulse correlated with the increase in [Ca2+]i estimated from Indo-1 using the extracellular calibration curve, but the values of the influx determined directly from Indo-1 fluorescence were always larger than those calculated from the exchanger current.(ABSTRACT TRUNCATED AT 400 WORDS)

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. Plasma membrane macromolecular structure was examined by freeze-fracture electron microscopy of myocytes exposed to 0.1, 1 and 2 mM adriamycin for periods up to 105 minutes. The principal effect of adriamycin was rapid induction of smooth (protein-poor) domains in the membrane, with displacement and clustering of intramembrane particles (the structures representing integral membrane proteins). These effects, which are time-dependent and dose-dependent, culminate in the formation of saucer-shaped lesions in the membrane, which broadly resemble deformations of the membrane induced by polymyxin B. Both adriamycin and polymyxin are known to have the ability to interact selectively with anionic phospholipids. It is concluded that an important initial effect of adriamycin on cardiac muscle cells is alteration of the macromolecular architecture of the plasma membrane, probably through interaction with anionic phospholipids, and that this may represent the underlying cause of a range of sarcolemmal dysfunctions associated with exposure to the drug.

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. To explain this apparent paradox, repair mechanisms, operating to seal the membrane lesions caused by gap junction tearing, have been hypothesized, but evidence for their existence has previously been lacking. Using freeze-fracture electron microscopy, the present study identifies repair sites as smooth membrane domains that are continuous with the neighbouring plasma membrane, thus forming intact seals. That these structures are not chemically-induced artefacts is demonstrated by their presence in myocytes that were frozen directly from the living state. Subsarcolemmal vesicle clusters, detected in thin sections and freeze-fracture replicas, are associated with the smooth sealing domains. These structures may represent either rounded-up fragments of mechanically disrupted membrane or structures concerned with the synthesis of new lipid. From their freeze-fracture morphology, the sealing domains appear to be lipid-rich and protein-poor. Cytochemical studies using Ruthenium Red, cationized ferritin and lectins show in addition that they have a lower content of negatively-charged membrane components than the neighbouring plasma membrane, and that the carbohydrate residues normally associated with plasma membrane glycolipids and glycoproteins are absent.

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. Serial sectioning was used to establish that at least part of the annular gap junction population in the freshly isolated myocyte represents truly discrete cytoplasmic vesicles; thus, some gap junctions are rapidly endocytosed after myocyte separation. Analysis of the surface-located-to-annular gap junction ratio suggested that no further endocytosis occurred in rabbit and cat myocytes maintained for 22 and 15 hours, respectively. Guinea pig myocytes, by contrast, did appear to continue endocytosis in culture. Analysis of the distance of gap junctional structures from the cell surface suggested that little if any inward migration of gap junction vesicles occurred. Hypoxia had no detectable effect on the internalization or inward movement of gap junctions. The quantity of ultrastructurally detectable gap junction membrane appeared to remain constant over time, as did the incidence of "complex structures" (i.e., annular gap junction profiles with features previously suggested to represent degradation). New gap junction formation was negligible, and a reappraisal of the nature of "complex structures" led to the conclusion that the origin of these structures need not be related to degradation. Taken together, the findings suggest that degradation and disappearance of gap junctional membrane after isolation of the mature myocyte constitute a much slower process than previously believed, and the possibility that the cardiac gap junction protein has a longer half-life than its counterpart in liver remains open.

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. This current usually has peak amplitude when repolarization is imposed during the first 50 ms after the action potential upstroke, but becomes negligible once the final phase of repolarization is reached. The envelope of peak current tail amplitudes strongly resembles that of the intracellular calcium transient recorded in other studies. 2. Repetitive stimulation producing normal action potentials at a frequency of 2 Hz progressively augments the tail current recorded immediately after the stimulus train. Conversely, if each action potential is prematurely terminated at 0.1 Hz, repetitive stimulation produces a tail current much smaller than the control value. The control amplitude of inward current is only maintained if interrupted action potentials are separated by at least one full 'repriming' action potential. These effects mimic those on cell contraction (Arlock & Wohlfart, 1986) and suggest that progressive changes in tail current are controlled by variations in the amplitude and time course of the intracellular calcium transient. 3. When intracellular calcium is buffered sufficiently to abolish contraction, the tail current is abolished. Substitution of calcium with strontium greatly reduces the tail current. 4. The inward tail current can also be recorded at more positive membrane potentials using standard voltage clamp pulse protocols. In this way it was found that temperature has a large effect on the tail current, which can change from net inward at 22 degrees C to net outward at 37 degrees C. The largest inward currents are usually recorded at about 30 degrees C. It is shown that this effect is attributable predominantly to the temperature sensitivity of activation of the delayed potassium current, iK, whose decay can then mask the slow tail current at high temperatures. 5. Studies of the relationship between the tail current and the membrane calcium current, iCa, have been performed using a method of drug application which is capable of perturbing iCa in a very rapid and highly reversible manner. Partial block of iCa with cadmium does not initially alter the size of the associated inward current tail. When iCa is increased by applying isoprenaline, the percentage augmentation of the associated tail current is much greater but occurs more slowly.(ABSTRACT TRUNCATED AT 400 WORDS)

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. Under voltage clamp both agents caused an inward current to flow. 2. These effects were abolished by propranolol (100 nM) and the beta1-antagonist metoprolol (100-200 nM), but not by the beta2-agonist [corrected] salbutamol (1 microM). 3. The interaction of isoprenaline with forskolin, caffeine or isobutylmethylxanthine (IBMX) on current amplitude was as expected if all of these drugs were causing inward current by increasing intracellular levels of cyclic adenosine monophosphate (cyclic AMP). Low concentrations of forskolin (less than 600 nM) or IBMX (less than 20 microM) potentiated the effect of isoprenaline, whereas isoprenaline caused no further inward current in cells in which high concentrations of forskolin (600 nM-100 microM) or IBMX (20 microM-1 mM) were already evoking maximum inward current. 4. Isoprenaline-induced inward current was reduced 30-50% by acetylcholine (10-30 microM). This action of acetylcholine was blocked by atropine (100 nM). 5. The effect of isoprenaline on holding current was critically dependent on temperature. The onset of the current was delayed and its amplitude reduced as the myocyte was cooled from 37 degrees C to ambient temperature (22-24 degrees C). 6. Isoprenaline-induced inward current was not affected by the potassium channel blockers barium (2 mM) or tetraethylammonium (TEA; 10-20 mM). The amplitude of the inward current did not vary as a function of [K+]o. 7. The inward current was not affected by the calcium channel blockers cadmium 1 mM, or nifedipine (10 microM), or when internal calcium was reduced by including EGTA in the recording electrode filling solution. 8. The amplitude of the current was also unaffected by caesium (5 mM), which blocks the hyperpolarization-activated, non-specific channel if, or by strophanthidin (10 microM) which blocks the Na+-K+ pump. It was unchanged by substitution of external chloride by isethionate. 9. The inward current was absent when external sodium was replaced by the impermeant ion tetramethylammonium (TMA). 10. Isoprenaline- and forskolin-induced inward currents were associated with an increase in both membrane chord conductance and noise. The increase in conductance was most readily measured at potentials where the inwardly rectifying potassium channel, iK1, was small, or when iK1 was blocked by the addition of barium (2 mM).(ABSTRACT TRUNCATED AT 400 WORDS)

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. When the membrane was depolarized to 0 mV, there was a phasic and a tonic component of the contraction in guinea-pig cells, whereas in rat cells only the phasic component was obvious. In both species the depolarizations evoked the second inward current (Isi). 3. In rat cells, when the membrane potential during a depolarization was varied over the range -40 to +60 mV, the amplitude of contraction first increased to a peak at a potential close to 0 mV, and then declined as the membrane potential became more positive. In contrast, contraction in guinea-pig cells measured under similar conditions continued to increase as the depolarization was increased, and the tonic component of contraction became more obvious at more positive potentials. Contraction amplitude in guinea-pig cells could also be increased by increasing pulse duration under conditions where the tonic component of contraction was prominent. 4. Contraction during depolarization was suppressed by ryanodine in rat cells, whereas in guinea-pig cells contraction persisted, but with a modified time course. Ryanodine did inhibit spontaneous contractions of guinea-pig cells during exposure to low extracellular sodium. 5. Nifedipine suppressed Isi and phasic contraction in both rat and guinea-pig cells. In guinea-pig cells these effects developed contemporaneously, but in rat cells substantial reduction of Isi occurred before marked suppression of contraction. 6. In rat cells exposed to strontium in place of external calcium, inactivation of Isi was slowed and contraction was prolonged, with a slower time-to-peak and relaxation. The time course of the action potential was modified and ryanodine no longer inhibited contraction of rat cells in the presence of strontium. 7. It is concluded that the amplitude of contraction in rat and guinea-pig ventricular cells is determined by calcium both entering through the surface membrane and released from internal stores, and that under normal conditions the balance is towards release from stores in rat cells, and towards entry through the surface in guinea-pig cells.

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. In rat cells, short (2-10 ms) depolarizing pulses to 0 mV from a holding potential of -40 mV evoked current carried by calcium, and on repolarization to -40 mV there was a slow 'tail' current which decayed much more slowly than the expected deactivation of calcium current at this potential. 3. When rat cells were loaded with EGTA diffusing into the cytosol from an intracellular electrode, contraction and the tail current were both abolished, whereas the peak calcium current was not reduced. 4. Exposure of rat cells to ryanodine (1-2 microM) suppressed both contraction and the tail current, but not peak calcium current. 5. The tail current was unaffected by tetrodotoxin (10 microM), but was reduced by lowering extracellular sodium to 10% by replacement with lithium or choline. 6. In rat cells, exposure to nifedipine (1-5 microM) initially caused a marked reduction of calcium current while substantial contraction and tail current remained; longer exposure to nifedipine suppressed both contraction and the tail current. Isoprenaline (50-100 nM) caused a marked increase in peak calcium current, while under these conditions there was little or no increase in either contraction or tail current. 7. The amplitude of the tail current in rat cells varied with the duration of the depolarization at 0 mV; the tail current evoked by repolarization to -40 mV reached a peak just as contraction was beginning to develop and was back to undetectable levels just as relaxation became significant, as might be expected if the tail current were determined by the cytosolic calcium transient which triggered contraction. 8. In guinea-pig cells, a tail current was also recorded on repolarization to a holding potential of -40 mV, and, as in rat cells, the tail was suppressed by cytosolic EGTA and reduced by exposure of the cells to low-sodium solution. 9. It is concluded that the tail currents recorded in both rat and guinea-pig cells represent current activated by a rise in cytosolic calcium; in rat cells this is markedly dependent on ryanodine-sensitive release of calcium from internal stores. The origin of this current, and its possible role during the plateaux of action potentials are discussed.

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. Most interestingly, the intracellular pathway(s) linking the beta-receptor(s) to activation of the Ca current and the Na-dependent current, which in both cases presumably involves the intracellular concentration of cAMP, differ, as isoprenaline causes a persistent augmentation of the calcium current whereas the Na-dependent current often fades. These effects of isoprenaline are antagonized by acetylcholine. In unclamped cells, the Na-dependent current depolarizes the membrane to the potential range at which repetitive firing occurs. It may therefore be involved in the generation of ventricular arrhythmias.

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. This is larger than might be expected from the measured external dimensions of the cell and is compatible with the presence of surface membrane infoldings and caveolae. At membrane potentials close to -75 mV the mean cell input resistance was approximately 40 M omega, giving a specific membrane resistance of 6 omega X cm2 if mean membrane area is 15,000 micron2 and consistent with the assumption that the isolated cells have sealed intercalated discs under the experimental conditions used. Ionophoretically applied noradrenaline produced a pronounced prolongation of the plateau phase of the action potential, but this effect developed over many seconds. The slow onset of action is not compatible with the kinetics of free extracellular diffusion of catecholamine but may reflect molecular events that occur between noradrenaline binding to membrane receptors and the final cellular response. Under voltage-clamp conditions, the cells showed a time dependent inward current consistent with the rapid activation and decay of a sarcolemmal calcium conductance.

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. These changes were less marked at more depolarized potentials. The ventricular cells were voltage clamped at -40 mV and initially stimulated at 0.3 Hz by step depolarizations to 0 mV for 10 or 100 ms, which activated the second inward current (Isi) and an accompanying contraction. The amplitude and time course of contraction were similar with the two pulse durations. When the duration of the depolarization was 100 ms, an increase in stimulation rate to 3 Hz caused a gradual decline in the amplitude of Isi and of the evoked contraction; at the same time extra contractions and small, transient inward currents appeared in addition to the evoked contractions and Isis. There was a reduction in the early component of decay of Isi at 3 Hz. With a depolarizing pulse duration of 10 ms, an increase in stimulation rate to 3 or to 4.2 Hz did not change the amplitude of the evoked Isi or contraction and no extra contractions or currents appeared. Intracellular EGTA abolished all contractions in the cells and an increase in the rate of stimulation with 100 ms pulses did not then induce transient inward currents. There was some decrease in the Isi amplitude but this was not as marked as in the absence of EGTA and the time course of current decay was similar at the two rates. Ryanodine prevented the appearance of extra contractions and currents when the stimulation rate was increased to 3 Hz and, as in the presence of intracellular EGTA, there was a small decrease in Isi amplitude while the time course of decay was similar at the two stimulation rates. The time course of recovery of Isi from inactivation, as shown by a double-pulse procedure, was altered when the duration of the first pulse was reduced from 100 to 10 ms, an extra inactivation of Isi being seen at pulse intervals of 20-100 ms. This extra component of inactivation was not seen with intracellular EGTA or in the presence of ryanodine.(ABSTRACT TRUNCATED AT 400 WORDS)

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. non-disc) sarcolemma are summarised. The fate of gap junctions on separation of the intercalated disc membranes has been studied i) at intervals after isolation, ii) using cationized ferritin as an extracellular marker and iii) by serial sectioning. The results of these studies help explain how ionic integrity of the individual isolated cells may be maintained.

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. The isolated cells had significantly shorter sarcomeres than did cells in the intact tissue, and this was associated with an altered topography of plasma membrane surface folds at the level of the Z-lines. Plasma membrane intramembrane particles were randomly distributed and showed the same numerical density on the E-faces of both isolated and intact-heart myocytes. However, P-face particle density was slightly reduced in the isolated cells. It is concluded that the few differences detected in the isolated cells do not reflect any fundamental derangement of their properties.