Biomedical subjects
Publications and source records attributed to P Hess.
This article reviews the new insight into the function of cardiac calcium channels made possible by electrophysiological recordings from single cells and single channels. The unitary properties of the two different types of cardiac calcium channels are discussed and contrasted, and special emphasis is given to the mechanisms of channel permeation, gating, and modulation by exogenous and endogenous ligands.
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Freshly dispersed rabbit ear artery cells were studied using patch-clamp techniques to measure membrane currents in whole cells and single-channel currents in membrane patches. Whole-cell calcium currents recorded at physiologic extracellular calcium concentrations were small (approximately 10 pA). Recordings with 110 mM external barium gave much larger currents and revealed two current components with properties similar to those in other vascular smooth muscle preparations and to the calcium currents designated as T and L in heart cells and neurons. T current was activated with weak depolarizations and inactivated rapidly, while L current was activated with relatively strong depolarizations and inactivated more slowly. L current was increased by dihydropyridine agonists and decreased by dihydropyridine antagonists, while T current was unaffected. Recordings from cell-attached and outside-out membrane patches with 100 mM external barium showed unitary calcium channel currents with conductances of 8 and 25 pS. The small conductance channels had kinetic properties that accounted for T current in the whole-cell recordings, while the 25-pS channels showed the voltage dependence, the time dependence, and the dihydropyridine sensitivity expected for L-type channels. We conclude that vascular smooth muscle cells contain two types of calcium channels with properties very similar to those described for T- and L-type calcium channels in other cell types; the L current appears to be the predominant current component in whole-cell recordings.
Electrophysiological recordings were used to analyze single calcium channels in planar lipid bilayers after membranes from bovine cardiac sarcolemmal vesicles had been incorporated into the bilayer. In these cell-free conditions, channels in the bilayer showed unitary barium or calcium conductances, gating kinetics, and pharmacological responses that were similar to dihydropyridine-sensitive calcium channels in intact cells. The open channel current varied in a nonlinear manner with voltage under asymmetric (that is, physiological) ionic conditions. However, with identical solutions on both sides of the bilayer, the current-voltage relation was linear. In matched experiments, calcium channels from skeletal muscle T-tubules differed significantly from cardiac calcium channels in their conductance properties and gating kinetics.
Cardiac calcium channel activity is markedly increased by beta-adrenergic agents or calcium agonists such as Bay K 8644. The molecular mechanisms underlying these important modulatory effects have been studied with patch clamp techniques by several groups. This paper presents new experiments and reviews published evidence from fluctuation analysis of whole cell calcium current and unitary recordings of single calcium channel activity. Two different factors underlie the enhancement of calcium channel activity seen with beta-stimulation or cyclic AMP: (1) increased availability of calcium channels, expressed in whole cell recordings as an increase in the number of functional channels and in single channel recordings as an increase in the proportion of non-blank sweeps. (2) changes in opening probability, due to alteration of the fast kinetics of channel opening and closing. Both factors contribute to the beta-adrenergic enhancement in frog, rat, and guinea-pig ventricular cells although their quantitative importance is somewhat variable. Unlike beta-adrenergic agents, calcium agonists such as Bay K 8644 promote a mode of channel gating that is characterized by long openings and short closings, seen only rarely in control or with beta-stimulation.
Single channel and whole cell recordings were used to study ion permeation through Ca channels in isolated ventricular heart cells of guinea pigs. We evaluated the permeability to various divalent and monovalent cations in two ways, by measuring either unitary current amplitude or reversal potential (Erev). According to whole cell measurements of Erev, the relative permeability sequence is Ca2+ greater than Sr2+ greater than Ba2+ for divalent ions; Mg2+ is not measurably permeant. Monovalent ions follow the sequence Li+ greater than Na+ greater than K+ greater than Cs+, and are much less permeant than the divalents. These whole cell measurements were supported by single channel recordings, which showed clear outward currents through single Ca channels at strong depolarizations, similar values of Erev, and similar inflections in the current-voltage relation near Erev. Information from Erev measurements stands in contrast to estimates of open channel flux or single channel conductance, which give the sequence Na+ (85 pS) greater than Li+ (45 pS) greater than Ba2+ (20 pS) greater than Ca2+ (9 pS) near 0 mV with 110-150 mM charge carrier. Thus, ions with a higher permeability, judged by Erev, have lower ion transfer rates. In another comparison, whole cell Na currents through Ca channels are halved by less than 2 microM [Ca]o, but greater than 10 mM [Ca]o is required to produce half-maximal unitary Ca current. All of these observations seem consistent with a recent hypothesis for the mechanism of Ca channel permeation, which proposes that: ions pass through the pore in single file, interacting with multiple binding sites along the way; selectivity is largely determined by ion affinity to the binding sites rather than by exclusion by a selectivity filter; occupancy by only one Ca ion is sufficient to block the pore's high conductance for monovalent ions like Na+; rapid permeation by Ca ions depends upon double occupancy, which only becomes significant at millimolar [Ca]o, because of electrostatic repulsion or some other interaction between ions; and once double occupancy occurs, the ion-ion interaction helps promote a quick exit of Ca ions from the pore into the cell.
We studied the blocking actions of external Ca2+, Mg2+, Ca2+, and other multivalent ions on single Ca channel currents in cell-attached patch recordings from guinea pig ventricular cells. External Cd or Mg ions chopped long-lasting unitary Ba currents promoted by the Ca agonist Bay K 8644 into bursts of brief openings. The bursts appear to arise from discrete blocking and unblocking transitions. A simple reaction between a blocking ion and an open channel was suggested by the kinetics of the bursts: open and closed times within a burst were exponentially distributed, the blocking rate varied linearly with the concentration of blocking ion, and the unblocking rate was more or less independent of the blocker concentration. Other kinetic features suggested that both Cd2+ and Mg2+ lodge within the pore. The unblocking rate was speeded by membrane hyperpolarization or by raising the Ba concentration, as if blocking ions were swept into the myoplasm by the applied electric field or by repulsive interaction with Ba2+. Ca ions reduced the amplitude of unitary Ba currents (50% inhibition at approximately 10 mM [Ca]o with 50 mM [Ba]o) without detectable flicker, presumably because Ca ions exit the pore very rapidly following Ba entry. However, Ca2+ entry and exit rates could be resolved when micromolar Ca blocked unitary Li+ fluxes through the Ca channel. The blocking rate was essentially voltage independent, but varied linearly with Ca concentration (rate coefficient, 4.5 X 10(8) M-1s-1); evidently, the initial Ca2+-pore interaction is outside the membrane field and much faster than the overall process of Ca ion transfer. The unblocking rate did not vary with [Ca]o, but increased steeply with membrane hyperpolarization, as if blocking Ca ions were driven into the cell. We suggest that Ca is both an effective permeator and a potent blocker because it dehydrates rapidly (unlike Mg2+) and binds to the pore with appropriate affinity (unlike Cd2+). There appears to be no sharp dichotomy between "permeators" and "blockers," only quantitative differences in how quickly ions enter and leave the pore.
A case of transposition of the sartorius muscle is presented. This method was successfully used for treatment of an exposed knee joint with subtotal loss of the extensor apparatus of the knee. A proximally based muscle flap proved very efficient for coverage of the knee joint and restoration of function.
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We used the patch clamp technique to record unitary calcium (Ca2+) channel activity in freshly dissociated ventricular myocytes from adult guinea pigs. We found two types of Ca2+ channels with distinct permeation and gating properties and different sensitivity to pharmacological agents. One channel (T-type) requires negative membrane potentials to remove inactivation. It gives rise to a transient mean current and is not affected by dihydropyridines or isoproterenol. The other Ca2+ channel (L-type) has a larger unitary barium-conductance, activates at more positive potentials and its averaged current decays much more slowly. It shows a distinct gating pattern with different gating modes, the proportion of which is drastically altered by dihydropyridine Ca2+-channel agonists and antagonists. L-type channel activity is modulated by beta-adrenergic stimulation by a mechanism of action which differs from that of dihydropyridine Ca2+-channel agonists.
Microelectrodes filled with neutral carrier selective to Ca2+ were used to measure the free intracellular Ca2+ concentration [( Ca2+]i) in sheep cardiac tissue and frog skeletal muscle. Calibration of the electrodes was performed in the presence of a solution resembling the cationic composition of the cytoplasm. [Ca2+]i at rest in normal physiological saline (20-22 degrees C) was 240 nM in Purkinje fibres, 270 nM in ventricular muscle, and 52 nM in skeletal muscle. In Purkinje fibres, elevation of [Ca2+]o from 1.8 mM to 5.4 mM produced a 1.7-fold increase in [Ca2+]i. Elevation of [Ca2+]o from 1.8 mM to 18 mM induced a 2.6-fold increase in [Ca2+]i. Exposure to Na+-free solution (Li+-substituted) gave rise to elevation of [Ca2+]i by factors of 5.8 and 14 in ventricular muscle and Purkinje fibres, respectively. These latter changes in [Ca2+]i were associated with the development of contractures which reached 34% and 172% of the corresponding twitch tension.
Two-dimensional echocardiography can provide better information than conventional radiological volume measurements concerning the amount of hypertrophy and dilatation, which contribute to the cardiac enlargement found in athletes. Linear cardiac measurements in standardised planes were obtained by two-dimensional echocardiography; the findings in 21 women athletes obtained before and after a 100-mile race were compared with a number of normals. All cardiac chambers were found to be equally enlarged and the aortic root and the ascending aorta were dilated. The generalised dilatation of the heart must be due to increased ejection volume in these athletes.
The [Ca2+]-activated photoprotein aequorin was used to measure [Ca2+] in canine cardiac Purkinje fibers during the positive inotropic and toxic effects of ouabain, strophanthidin, and acetylstrophanthidin. The positive inotropic effect of these substances was associated with increases in the two components of the aequorin signal, L1 and L2. On the average, strophanthidin at 10(-7) M produced steady, reversible increases in L1, L2, and peak twitch tension of 20, 91, and 240%, respectively. This corresponds to increases in the upper-limit spatial average [Ca2+] from 1.9 X 10(-6) M to 2.1 X 10(-6) M at L1 and from 1.4 X 10(-6) M to 1.8 X 10(-6) M at L2. Elevation of diastolic luminescence above the control level was not detected. At higher concentrations (5 X 10(-7) M), strophanthidin produced aftercontractions, diastolic depolarization, and transient depolarizations, all of which were associated with temporally similar changes in [Ca2+]. During these events, diastolic [Ca2+] rose from the normal level of approximately 3 X 10(-7) M up to 1-2 X 10(-6) M. The negative inotropic effect of 5 X 10(-7) M strophanthidin was not associated with a corresponding decrease in the [Ca2+] transient but was associated with a change in the relationship between [Ca2+] and tension. Assuming the Na+-lag mechanism of cardiotonic steroid action, we conclude the following: at low concentrations of drug, increased Ca2+ uptake by the sarcoplasmic reticulum prevents a detectable rise in cytoplasmic [Ca2+] during diastole, but this increased Ca2+ uptake results in increased release of Ca2+ during the action potential. At higher drug concentrations, observable [Ca2+] changes during diastole activate tension and membrane conductance changes.
The effects of caffeine on tension, membrane potential, membrane currents, and intracellular [Ca2+], measured as the light emitted by the Ca2+-activated photoprotein aequorin, were studied in canine cardiac Purkinje fibers. An initial, transient, positive inotropic effect of caffeine was accompanied by a transient increase in the second component of the aequorin signal (L2) but not the first (L1). In the steady state, 4 or 10 mM caffeine always decreased twitch tension and greatly reduced both L1 and L2. At a concentration of 2 mM, caffeine usually reduced but occasionally increased the steady state twitch tension. However, 2 mM caffeine always reduced both L1 and L2. Caffeine eliminated the diastolic oscillations of intracellular [Ca2+] induced by high extracellular [Ca2+]. In voltage-clamp experiments, 10 mM caffeine reduced the transient outward current and the peak tension elicited by step depolarization from a holding potential of -45 mV. In the presence of 20 mM Cs+, 10 mM caffeine reduced slow inward current. However, the time course of this reduction was far slower than that in tension and light observed in separate experiments. The simplest explanation of the results is that caffeine inhibits the sequestration of Ca2+ by the sarcoplasmic reticulum. The results also suggest that in Purkinje fibers caffeine increases the sensitivity of the myofilaments to Ca2+.
Although the effect of gossypol on the testis and epididymis has been well documented, its effect on the somatic tissues of animals made infertile with the drug has been less well studied. In this study rats were treated daily with gossypol at either 20 mg/kg or 30 mg/kg for 6 weeks and at 10 mg/kg for 7 months. Complete tissue sets from control and gossypol-treated rats, including 26 organs, were subjected to histological examination. No significant histopathological differences were noted at the light microscope level between the control and infertile groups.
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The Ca++-sensitive bioluminescent protein aequorin was used to detect cytoplasmic Ca++ transients in dog Purkinje strands and cat papillary muscles. Isometric tension development was recorded in both preparations, and transmembrane potential was monitored in the Purkinje strands. Voltage-clamp studies were carried out on some of the Purkinje strands. The aequorin signal of the Purkinje strand normally consists of two readily discernible components, which we have provisionally attributed to the entry of Ca++ through the surface membrane, and to the release of Ca++ from intracellular stores. D600 and verapamil reduced or eliminated both of these components, as they did the net inward current in fibers depolarized from a holding potential of -45 mV. In cat papillary muscles D600, verapamil, nifedipine, diltiazem, and perhexiline all had qualitatively similar effects on the aequorin signals and contractions. The effects of these agents differed from those of reduced [Ca++] in two important respects: the relation between light intensity and tension development was altered, and the mechanical contraction was abbreviated by the Ca++ channel blockers. These changes persist when the depressant effects of the Ca++ channel blockers are offset by elevations of [Ca++]o; they probably reflect a reduction in the Ca++ sensitivity of the myofilaments.