Multicellular Preparations Research Articles

Carbon monoxide inhibits depolarization- induced Ca rise and increases cyclic GMP in visceral smooth muscle cells

Monocytes were isolated from the urinary bladder of the guinea-pig. By means of the voltage clamp technique, whole cells were depolarized from −65 to + 10 mV in order to increase the intracellular calcium concentration [Ca 2+] i and to monitor this increase by means of the calcium activated potassium current I K.Ca. Superfusion of the cells with carbon monoxide-containing solutions for 2 min inhibited the signal to about 50% of the control suggesting depression of the depolarization-induced increase in [Ca 2+] i. The CO-mediated inhibition of I K.Ca was partially reversed by wash-off of CO; flashes of high light intensity accelerated the rate of recovery. Sodium nitroprusside (0.01–1 mM) depressed the depolarization-induced increase in [Ca 2+] i similar to CO. In multicellular preparations of the urinary bladder, CO-containing media were shown to increase the cGMP concentration by a factor of 2 in the absence and by a factor of 3 in the presence of 1 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). According to our previous work, CO binds to and activates soluble guanylate cyclase [Brüne B and Ullrich V, Mol Pharmacol 32: 497–504, 1987; Utz J and Ullrich V, Naunyn Schmiedebergs Arch Pharmacol 337 (Suppl): 299, 1988] and the rise in cGMP could thus effect [Ca 2+] i by still unknown mechanisms.

Electrophysiologic and Antiarrhythmic Actions of Pirmenol on Rabbit and Guinea Pig Cardiac Preparations

The electrophysiological and antiarrhythmic effects of pirmenol HCl were examined using the microelectrode technique applied to multicellular preparations and the suction-pipette whole-cell clamp method applied to ventricular myocytes from rabbit and guinea pig hearts. Pirmenol at 5 microM and higher doses suppressed the sinus node automaticity by depressing the slow diastolic depolarization without changing the maximum diastolic potential. Pirmenol at 1 microM and higher doses depressed the maximum upstroke velocity (Vmax) of action potentials and prolonged the action potential duration at 90% repolarization in atrial muscles and Purkinje fibers without affecting resting membrane potentials. Pirmenol at 5 microM depressed the early part of the plateau and lengthened the final repolarization of the action potentials in ventricular myocytes, of which effects were attributed to the depression of the calcium current and the delayed outward K+ current. Triggered tachyarrhythmias arising from delayed afterdepolarizations in papillary muscles and ventricular myocytes were markedly inhibited by 1-5 microM pirmenol. The drug changed the amplitude and appearance of the transient inward current in ventricular myocytes. These results suggest that pirmenol has electrophysiologic properties that could provide an antiarrhythmic action on various types of arrhythmias.

A mathematical model of a bullfrog cardiac pacemaker cell

Previous models of cardiac cellular electrophysiology have been based largely on voltage-clamp measurements obtained from multicellular preparations and often combined data from different regions of the heart and a variety of species. We have developed a model of cardiac pacemaking based on a comprehensive set of voltage-clamp measurements obtained from single cells isolated from one specific tissue type, the bullfrog sinus venosus (SV). Consequently, sarcolemmal current densities and kinetics are not influenced by secondary phenomena associated with multicellular preparations, allowing us to realistically simulate processes thought to be important in pacemaking, including the Na(+)-K+ pump and Na(+)-Ca2+ exchanger. The membrane is surrounded extracellularly by a diffusion-limited space and intracellularly by a limited myoplasmic volume containing Ca2(+)-binding proteins (calmodulin, troponin). The model makes several predictions regarding mechanisms involved in pacing. 1) Primary pacemaking cannot be attributed to any single current but arises from both the lack of a background K+ current and a complex interaction between Ca2+, delayed-rectifier K+, and background leak currents. 2) Ca2+ current displays complex behavior and is important during repolarization. 3) Because of Ca2+ buffering by myoplasmic proteins, the Na(+)-Ca2+ exchanger current is small and has little influence on action potential repolarization but may modulate the maximum diastolic potential. 4) The Na(+)-K+ pump current does not play an active role in repolarization but is of sufficient size to modulate the rate of diastolic depolarization. 5) K+ accumulation and Ca2+ depletion may occur in the extracellular spaces but play no role in either the diastolic depolarization or repolarization of a single action potential. This model illustrates the importance of basing simulations on quantitative measurements of ionic currents in myocytes and of including both electrogenic transporter mechanisms and Ca2+ buffering by myoplasmic Ca2(+)-binding proteins.

Isolated cells of the frog sinus venosus: properties of the inward current activated during hyperpolarization

Single sinus venosus cells from frog, Rana esculenta, were isolated using an enzymic dispersion procedure, involving applications of collagenase and protease. About 40%-60% of the cells showed spontaneous contractions. Isolated cells were studied in the whole-cell configuration. Regenerative action potentials were tetrodotoxin-insensitive and similar to those recorded in multicellular preparations. Hyperpolarizing pulses in the voltage range negative to -50 mV induced the activation of a time-dependent inward current, which was blocked by 4 mM caesium but less affected by barium ions. A lower concentration of caesium (1 mM) exerted a voltage-dependent reduction of the current and decreased the spontaneous pacing rate. The activation range of the hyperpolarization-activated current approximately extended from -50 mV to -110 mV, but varied from cell to cell. A high variability was observed in the behaviour of the activation kinetics. The current had a reversal potential near -20 mV that was shifted positively by increasing the external potassium concentration (from 3 mM to 30 mM) and negatively by reducing the external sodium concentration (from 115 mM to 30 mM). The hyperpolarization-activated inward current of the frog sinus venosus cell appears to be carried by both sodium and potassium ions. It shows electrophysiological properties similar to those of the If current of the mammalian heart. The role of the current in the spontaneous activity is discussed.

Electrophysiological properties and responses to simulated ischemia in cat ventricular myocytes of endocardial and epicardial origin.

In multicellular preparations, there are differences in action potential configuration between endocardium and epicardium, and electrophysiological alterations induced by ischemia are more drastic in epicardium than in endocardium. The present study was designed to examine electrophysiological properties of single cardiac myocytes enzymatically isolated from the endocardial and epicardial surfaces of the cat left ventricle and to determine whether the differential responses to ischemia of intact tissue occur in single cells. Action potentials recorded from the isolated single cells of epicardial surface had lower action potential amplitude and a prominent notch between phase 1 and phase 2, compared with those of the cells isolated from the endocardial surface; these findings are similar to those in intact endocardial and epicardial preparations. Resting membrane potentials recorded from both endocardial and epicardial single cells were sensitive to the change in extracellular K+ concentration and had properties of a K+ electrode. Action potential duration was frequency dependent in both cell types and was shorter in epicardial cells than in endocardial cells at a stimulation rate of 3 Hz. When the cells were superfused with Tyrode's solution that was altered to mimic an ischemic environment in vivo (PO2, 30-40 mm Hg; pH 6.8; [K+], 10 mM; and glucose free), resting membrane potential, action potential amplitude, and action potential duration were reduced, and the refractory period was shortened in both endocardial and epicardial single cells, but there were no differences in the degree of changes in action potentials and refractory periods induced between the two cell types. Action potential changes induced by L-alpha-lysophosphatidylcholine (5-40 mg/l) were also similar in endocardial and epicardial single cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium release from cardiac sarcoplasmic reticulum induced by photorelease of calcium or Ins(1,4,5)P3

The ability of Ca2+ or inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to release Ca2+ from cardiac sarcoplasmic reticulum (SR) was investigated using saponin-skinned ventricular trabeculae from rats. To overcome diffusion delays, rapid increases in the concentrations of Ca2+ and Ins(1,4,5)P3 were produced by laser photolysis of "caged Ca2+" (Nitr-5) and "caged Ins(1,4,5)P3". Photolysis of Nitr-5 to produce a small jump in [Ca2+] from pCa 6.8 to 6.4 induced a large and rapid force response (t1/2 = 0.89 s at 12 degrees C); the source of the Ca2+ that activated the myofibrils was judged to be the SR, since it was blocked by 0.1 mM ryanodine or 5 mM caffeine. A smaller, slower, and less consistent release of SR Ca2+ was produced by photorelease of Ins(1,4,5)P3. The results demonstrate that these caged compounds can be used to study excitation-contraction coupling in skinned multicellular preparations of cardiac muscle. The data are consistent with a major role for Ca2(+)-induced Ca2+ release in cardiac activation, whereas the role for Ins(1,4,5)P3 may be to modulate, rather than directly stimulate, SR Ca2+ release.

A model study of the contribution of active Na [sbnd]K transport to membrane repolarization in cardiac cells

A biochemical model of active Na-K transport in cardiac cells was studied in conjunction with a representation of the passive membrane currents and ion concentration changes. The active transport model is based on the thermodynamic and kinetic properties of a six-step reaction scheme for the Na,K-ATPase. It has a fixed Na:K stoechiometry of 3:2, and its activation is governed by three parameters: membrane potential intracellular Na+ concentration, and interstitial K+ concentration. The Na-K pump current is directly proportional to the density of Na,K-ATPase molecules. The passive membrane currents and ion concentration changes involve only Na+ and K+ ions, and no attempt was made to provide a precise representation of Ca2+ currents or Ca2+ concentration changes. The surface-to-volume ratio of the interstitial compartment is 55 times larger than that of the intracellular compartment. The flux balance conditions are such that the original equilibrium concentration values are re-established at each stimulation cycle. The underlying assumptions of the model were checked against experimental measurements on Na-K pump activity in a variety of preparations. In addition, the qualitative validation of the model was carried out by comparing its behavior following sudden frequency shifts to corresponding experimental observations. The overall behavior of the model is quite satisfactory and it is used to provide the following indications: (1) when the intracellular and interstitial volumes are relatively large, the ion concentration transients are small and the pumping rate depends essentially on average concentration levels. (2) An increase in internal Na+ concentration potentiates the response of the Na-K pump to rapid membrane depolarizations. (3) When the internal Na+ concentration is large enough, the Na-K pump current transient plays an important role in shaping the plateau and repolarization phase of the action potential. (4) A rapid increase in external K+ concentration during voltage clamp in multicellular preparations could saturate the Na-K pump response and lead to a fairly linear dependence of the pump activity on the internal Na+ concentration.

Limitations of approximate solutions for computing the extracellular potential of single fibers and bundle equivalents

The mathematical description of the extracellular field generated by activity in an excitable fiber in an unbounded volume conductor will depend on assumptions made about the sources and the source-field relationship. This paper examines and compares the rigorous and conventional approximate solutions of Laplace's equation used to evaluate the extracellular potential of a single, cylindrical fiber. The single fiber is considered as both a prototypical element (such as a nerve or muscle fiber) and an elementary model of an entire multicellular preparation (e.g., nerve bundle or Purkinje strand). The effects of the fiber radius, the intracellular and extracellular conductivities, and the shape and extent of the source function (either the transmembrane potential or the intracellular potential) on the solutions are discussed. The results show that, in general, the approximate solutions are unsatisfactory for computing the surface extracellular potential when the single fiber is used to represent a large bundle (greater than 300 microns).

Rapid cooling contractures as an index of sarcoplasmic reticulum calcium content in rabbit ventricular myocytes

Rapid cooling contractures (RCCs) were used to assess changes in sarcoplasmic reticulum (SR) Ca content in both isolated rabbit ventricular myocytes and multicellular preparations. The main difference observed between these preparations was the magnitude of RCCs relative to twitches, apparently due to differences in measured parameters, i.e., unloaded shortening vs. isometric tension. When multicellular preparations were unloaded, RCC shortening was similar to that observed in myocytes. RCC magnitude decreased as the time between the last electrical stimulation and the RCC was increased (rest decay). RCC rest decay closely paralleled that of postrest twitches, suggesting that SR Ca loss is responsible for this process. Paired RCC experiments were used to investigate RCC relaxation and rest decay. When a second RCC (RCC2) was induced immediately after the first (RCC1), a large contracture was still observed (RCC2/RCC1 x 100 = 77.8 +/- 7.3%, mean +/- SD), indicating that the SR resequestered the majority of Ca on rewarming. This fraction was increased (to 92.9 +/- 5.5%) if Na and Ca-free solution was used during RCCs and rewarming, indicating that Na-Ca exchange also contributes to RCC relaxation. Increasing the interval between paired RCCs led to a decrease in RCC2, analogous to rest decay. This rest decay was abolished by inhibiting Na-Ca exchange, indicating that SR Ca loss during rest is mediated primarily by this process. RCCs were abolished by 10 mM caffeine. Ryanodine (1 microM) greatly accelerated RCC rest decay but had less effect on RCCs generated immediately after a train of stimulation. This accelerated rest decay was also dependent on Na-Ca exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular origins of the transient inward current in cardiac myocytes. Role of fluctuations and waves of elevated intracellular calcium.

Activation of the transient inward current (ITI) by a rise in intracellular calcium concentration ([Ca2+]i) is believed to be responsible for generating triggered cardiac arrhythmias. In this study, the cellular basis of the rise in [Ca2+]i that activates ITI and aftercontractions in single rat ventricular myocytes was examined. [Ca2+]i was measured both indirectly by cell contraction and directly with fura-2. Under conditions that caused steady-state [Ca2+]i to increase (i.e., calcium overload) membrane repolarization after a voltage-clamp depolarization resulted in the appearance of ITI that was similar in many respects to that observed in multicellular preparations. This ITI occurred at the same time that [Ca2+]i spontaneously increased and preceded the aftercontraction by 60-90 msec. However, ITI recorded from a single cell was variable in time course and amplitude (unlike that observed in multicellular preparations). Examination of cell contraction and digital imaging of fura-2 fluorescence showed that ITI was often associated with propagating regions of increased [Ca2+]i, which arose from discrete sites of origin within the cell. Apparently synchronous aftercontractions could also be associated with multiple propagating waves of [Ca2+]i. The variation in the time course and amplitude of ITI in single cells appeared to be due to changes in the location and number of sites of origin for the waves of [Ca2+]i. After the first aftercontraction and ITI, desynchronization of the sites of origin of increased [Ca2+]i occurred, and this resulted in a decrease in the amplitude of ITI and an increase in its duration. We conclude that the variability seen in single cells arises from changes in the pattern of spontaneous Ca2+ release. Such phenomena will seriously complicate interpretation of multicellular data, even when [Ca2+]i is measured directly.

Ionic currents in single smooth muscle cells from the ureter of the guinea‐pig.

1. Ionic currents underlying the action potential were recorded from enzymatically isolated smooth muscle cells of guinea-pig ureter. 2. The action potential recorded from a single cell was similar to that from a multicellular preparation. It showed repetitive spikes on a plateau potential which followed the first spike. Treatment with 10 mM-tetraethylammonium (TEA) increased the amplitude and duration of the plateau phase and abolished the repetitive spikes. 3. Under voltage clamp mode, at least two (maybe three) kinds of outward currents were activated during depolarizing pulses. The main outward current was Ca2+-dependent K+ current (IK(Ca], which was mostly blocked in Ca2+-free solution, or by application of 1 mM-cadmium (Cd2+) or 2 mM-tetraethylammonium (TEA). IK(Ca) was greatly decreased by treatment with 5 mM-caffeine or an addition of 10 mM-EGTA in a pipette solution. 4. In the presence of 1 mM-Cd2+ and 2 mM-TEA, a small transient outward current remained. 4-Aminopyridine (1 mM) suppressed the transient outward current by about 40%. Time- and voltage-dependent delayed rectifier outward currents were small in ureter cells. An inwardly rectifying K+ current was not detected. 5. An application of 1 mM-Cd2+, 5 mM-cobalt (Co2+), 1 mM-lanthanum (La3+) or 0.1 microM-nifedipine completely blocked the action potential. Replacement of 80-90% of extracellular Na+ with Li+ or Tris almost abolished the plateau potential and repetitive spikes but did not change significantly the first spike. 6. In the presence of 30 mM-TEA, the inward current elicited by depolarization was monophasic and lasted for more than 1 s. Application of 1 mM-Cd2+, 1 mM-La3+, 0.1 microM-nifedipine, or 5 mM-Co2+ completely blocked inward current. The replacement (87%) of extracellular Na+ ions with Li+, Tris, sucrose or TEA speeded up the decay of inward current; the inward current decreased by 10-60% at the end of a 500 ms pulse. 7. Even in low-Na+ solution (120 mM-TEA), the inactivation of ICa had a quite slow component (tau = 1 s), in addition to another faster component (tau = 100 ms) at 0 mV. When short depolarizing clamp pulses (50 ms) were repetitively applied at short intervals (50 ms) and with interpulse voltage of -10 or -20 mV to mimic the repetitive spikes on the plateau of the action potential, the decline of peak Ca2+ current during the train of pulses was smaller than the decay of Ca2+ current during a long pulse.(ABSTRACT TRUNCATED AT 400 WORDS)

Ca2+]i in single isolated cardiac cells: a review of recent results obtained with digital imaging microscopy and fura-2.

The use of fluorescent Ca2+ indicators to observe [Ca2+]i transients in voltage-clamped single cells has many advantages over previous methods, such as the use of aequorin in multicellular preparations, for studying excitation-contraction coupling. In the studies reviewed in this article, [Ca2+]i in single isolated mammalian ventricular myocytes was observed through the use of the fluorescent Ca2+ indicator, fura-2. Individual cells, loaded with fura-2 either by internal perfusion or by exposure to fura-2/AM, were generally studied with the use of inverted microscopes equipped with ultraviolet epifluorescence illumination, intensified silicon intensifier target cameras (ISIT), and (or) a photomultiplier tube. Analysis of subcellular patterns of fura-2 fluorescence was performed by digital analysis of the images obtained with the ISIT camera. Variation of membrane voltage and exposure of cells to ryanodine (which was assumed to selectively block the release of Ca2+ from the sarcoplasmic reticulum) were used to investigate the cellular processes that determine the [Ca2+]i transient. The main results of these studies are the following. (1) In any population of enzymatically isolated heart cells, there are (i) mechanically quiescent cells in which [Ca2+]i is spatially uniform, constant over time, and relatively low; (ii) spontaneously contracting cells, which have a relatively elevated [Ca2+]i, but in which the spatial uniformity of [Ca2+]i is interrupted periodically by spontaneous, propagating waves of high [Ca2+]i; and (iii) cells that are hypercontracted (rounded up) and that have higher levels of [Ca2+]i than the other two types.(ABSTRACT TRUNCATED AT 250 WORDS)

An ATP‐sensitive conductance in single smooth muscle cells from the rat vas deferens.

1. The whole-cell voltage-clamp technique was used to study the effects of extracellular ATP on smooth muscle cells isolated from the rat vas deferens. 2. ATP (1-200 microM) elicited an inward-rectifying current that was rapid in onset (less than or equal to 100 ms), reached a peak value that depended on [ATP], and desensitized in the continued presence of ATP (half-time approximately 2 s). 3. Cells recovered from desensitization when incubated in the absence of ATP (resensitization half-time approximately 2 min). 4. A comparison was made of the ability of ATP and several of its structural analogues to stimulate inward current at a negative holding potential. ATP was by far the most effective compound among the series ATP, ADP, AMP, adenosine, GTP, UTP and ITP. ADP elicited a current that was 20-25% as large as that produced by ATP, while the other compounds were ineffective at a concentration which produced a maximal ATP response. 5. AMP-CPP (alpha, beta-methylene ATP), AMP-PCP (beta, gamma-methylene ATP), and AMP-PNP (beta, gamma-imido ATP), which are relatively resistant to hydrolysis, were similarly compared to ATP. While none of these analogues elicited a current resembling the ATP-induced current, AMP-CPP and AMP-PNP each produced a small, relatively sustained inward current; AMP-PCP had little or no effect. 6. The ATP-sensitive conductance is cation selective, but does not appear to discriminate strongly between Na+, K+ and Mg2+. 7. Analysis of the fluctuations which accompany the ATP-induced current suggests that ATP controls a population of channels with a unitary current greater than 0.5 pA at -130 mV. 8. The ATP-evoked current discussed in this report may be responsible for the depolarizing effect of ATP previously described in multicellular preparations of the vas deferens.

Relationship between force and Ca2+ concentration in smooth muscle as revealed by measurements on single cells.

The role of Ca2+ in regulating smooth muscle contraction was investigated by measuring isometric force and [Ca2+] simultaneously in individual single smooth-muscle cells. [Ca2+] was measured with fura-2 and a high time-resolution dual-wavelength digital microfluorimeter, and force was measured with an ultrasensitive force transducer attached to a probe around which was tied one end of the cell. Both [Ca2+] and force increase after maximal electrical stimulus, with [Ca2+] increasing considerably before the first detectable increase in force. Force development exhibited maximal sensitivity to [Ca2+] between 150 and 500 nM Ca2+. This Ca2+ sensitivity can account for the fact that many physiological stimuli produce full contraction even though such stimuli only increase Ca2+ to 600-800 nM. When Ca2+ was induced to increase rapidly, the relation between [Ca2+] and force exhibited hysteresis. During the onset of contraction, force at a given [Ca2+] was lower than during the muscle's return to rest, thus suggesting the existence of a slow step(s) linking Ca2+ and force development in smooth muscle. The direction of this hysteresis reversed during contractions in which Ca2+ increased slowly, suggesting that the contractile process becomes desensitized to [Ca2+] with time. These relations between calcium and force in intact single smooth-muscle cells differ in many respects from the relation found previously in chemically permeabilized multicellular preparations of smooth muscle.

Potential and current distributions in a cylindrical bundle of cardiac tissue

The intracellular and interstitial potentials associated with each cell or fiber in multicellular preparations carrying a uniformly propagating wave are important for characterizing the electrophysiological behavior of the preparation and in particular, for evaluating the source contributed by each fiber. The aforementioned potentials depend on a number of factors including the conductivities characterizing the intracellular, interstitial, and extracellular domains, the thickness of the tissue, and the distance (depth) of the field point from the surface of the tissue. A model study is presented describing the extracellular and interstitial potential distribution and current flow in a cylindrical bundle of cardiac muscle arising from a planar wavefront. For simplicity, the bundle is considered as a bidomain. Using typical values of conductivity, the results show that the intracellular and interstitial potential of fibers near the center of a very large bundle (greater than 10 mm) may be approximated by the potentials of a single fiber surrounded by a limited extracellular space (a fiber in oil), hence justifying a core-conductor model. For smaller bundles, the peak interstitial potential is less than that predicted by the core-conductor model but still large enough to affect the overall source strength. The magnitude of the source strength is greatest for fibers lying near the center of the bundle and diminishes sharply for fibers within 50 microns of the surface.

Sodium-pump activity and its inhibition by extracellular calcium in cardiac myocytes of guinea pigs

Myocardial sodium-pump activity was examined from ouabain-sensitive 86Rb + uptake using myocytes isolated from guinea-pig heart. Either sodium loading or the sodium ionophore, monensin, increased 86Rb + uptake by over 400%, indicating that the amount of Na + available to the pump is the primary determinant of its activity, and that the sodium pump has a substantial reserve capacity in quiescent myocytes. Moreover, the degree of the above stimulation is markedly higher than corresponding values reported with multicellular preparations, suggesting that diffusion barriers make it impossible to observe the capacity of the sodium pump in the latter preparations. Removal of extracellular Ca 2+ increased ouabain-sensitive 86Rb + uptake, probably by enhancing turnover of the sodium pump rather than increasing availability of Na + to the pump.

Characterization of the inward-rectifying potassium current in cat ventricular myocytes.

Whole-cell membrane currents were measured in isolated cat ventricular myocytes using a suction-electrode voltage-clamp technique. An inward-rectifying current was identified that exhibited a time-dependent activation. The peak current appeared to have a linear voltage dependence at membrane potentials negative to the reversal potential. Inward current was sensitive to K channel blockers. In addition, varying the extracellular K+ concentration caused changes in the reversal potential and slope conductance expected for a K+ current. The voltage dependence of the chord conductance exhibited a sigmoidal relationship, increasing at more negative membrane potentials. Increasing the extracellular K+ concentration increased the maximal level of conductance and caused a shift in the relationship that was directly proportional to the change in reversal potential. Activation of the current followed a monoexponential time course, and the time constant of activation exhibited a monoexponential dependence on membrane potential. Increasing the extracellular K+ concentration caused a shift of this relationship that was directly proportional to the change in reversal potential. Inactivation of inward current became evident at more negative potentials, resulting in a negative slope region of the steady state current-voltage relationship between -140 and -180 mV. Steady state inactivation exhibited a sigmoidal voltage dependence, and recovery from inactivation followed a monoexponential time course. Removing extracellular Na+ caused a decrease in the slope of the steady state current-voltage relationship at potentials negative to -140 mV, as well as a decrease of the conductance of inward current. It was concluded that this current was IK1, the inward-rectifying K+ current found in multicellular cardiac preparations. The K+ and voltage sensitivity of IK1 activation resembled that found for the inward-rectifying K+ currents in frog skeletal muscle and various egg cell preparations. Inactivation of IK1 in isolated ventricular myocytes was viewed as being the result of two processes: the first involves a voltage-dependent change in conductance; the second involves depletion of K+ from extracellular spaces. The voltage-dependent component of inactivation was associated with the presence of extracellular Na+.

Effect of reduced Na gradient on electrical activity in isolated bovine and feline ventricular myocytes

We have studied changes in electrical activity resulting from abrupt alterations of the Na gradient, using ventricular myocytes isolated from feline and bovine hearts. Attempting to investigate the ionic current possibly generated by Na-Ca exchange, we studied the effects of the changes in [Na]o in the presence of 20 mM CsCl to inhibit K currents. To facilitate the effect of Cs, we also used a K-free solution and a patch electrode filled with 150 mM cesium glutamate. The application of 20 mM Nao resulted in hyperpolarization and the action potential duration was reduced. Under voltage clamp, 20 of 45 mM Nao generated an outward current at all membrane potentials investigated. The initial part (100-200 ms) of this current was only partially inhibited by 5 mM NiCl2 which is known to fully block the Ca inward current. However, the outward current generated by the reduced [Na]o was fully inhibited by 20 mM MnCl2 (which presumably inhibits Na-Ca exchange). Our observations extend the work on multicellular cardiac preparations indicating that the outward current elicited by a sudden decrease in Na gradient could be generated by Na-Ca exchange. Although the characteristics of this outward current support certain concepts of the Na-Ca exchange in cardiac muscle, we cannot at present exclude a contribution of other membrane current(s).

Contractile properties of single isolated feline ventriculocytes.

Isolated single cardiac myocytes are becoming an increasingly popular experimental preparation. To date, however, the mechanical properties of these isolated adult mammalian cardiac myocyte preparations have not been thoroughly evaluated. The aims of this research were to 1) define the basic contractile properties of externally unloaded single feline ventriculocytes, 2) determine their response to standard inotropic stimuli, and 3) determine whether contractile properties vary as a function of cell size. Cell contractions were elicited by field stimulation (1 Hz) and recorded with the use of a photodiode array technique. In the 121 myocytes studied (2 mM Ca2+; 37 degrees C) the maximum extent of shortening averaged 7.5 +/- 0.2% resting cell length (L). The maximum rates of shortening and relengthening were 92 +/- 2.7 and 103 +/- 2.0% L/s, respectively. Elevating the extracellular Ca2+ and paired pulse stimulation increased the magnitude of twitch contractions as well as the rates of shortening and relengthening. The relationship between cell size and contractile performance was assessed in this research either by comparing the contractile properties of large and small myocytes or by plotting specific contractile parameters as a function of myocyte surface area. The results of this research support the idea that single feline ventricular myocytes retain normal contractile capabilities following isolation and respond to inotropic maneuvers in a similar fashion to that observed in multicellular preparations. In addition the present experiments showed that the contractile properties of the myocytes isolated from normal feline hearts are not cell size dependent.

Membrane currents in cardiac pacemaker tissue

The present work is a brief survey of the mechanism of the cardiac pacemaker in sinoatrial node cells. Information on the pacemaker mechanism in cardiac tissue has been greatly enhanced by the development of the single cell isolation technique and the patch clamp technique. These methods circumvent to a large extent the difficulties involved in voltage clamping multicellular preparations. The calcium current (ICa), delayed rectifier potassium current (IK), transient outward current (Ito;IA), and the hyperpolarization activated inward current (Ih or If) were found both in whole cell preparations and in single channel analysis. The physiological significance of these currents, together with the exchange current systems for the pacemaker depolarization are discussed.