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W J Lederer

Publications and source records attributed to W J Lederer.

At least 127 records · Page 7Linked to original sources
PubMed1990

Modulation of ATP-sensitive K+ channel activity and contractile behavior in mammalian ventricle by the potassium channel openers cromakalim and RP49356.

We have investigated the effects of potassium channel opening drugs on the ATP-dependence of ATP-sensitive K+ channel activity and on contractile activity in rat and guinea pig ventricular myocytes. The results show that cromakalim (BRL34915), and RP49356, agents reported to open ATP-sensitive K+ channels, do so by shifting the intracellular [ATP] required to cause half-maximal inhibition of channel activity (ki) to higher [ATP]. In guinea pig ventricular myocytes at 37 degrees C, the ki was shifted from 79 to 152 microM by 40 microM cromakalim and, in rat myocytes at room temperature, the ki was also shifted to higher [ATP] by 50 microM RP49356. The effect of externally applied RP49356 on the contractile activity of intact rat ventricular myocytes was investigated. At 100 microM the drug was without effect in the presence of normal bathing solution containing 10 mM glucose. When glucose in the bathing medium had been replaced by 2-deoxyglucose for 84 +/- 2 min, 100 microM RP49356 decreased the twitch amplitude to 23 +/- 4% of control. The negative inotropic effect of 100 microM RP49356 increased with time after perfusion with 2-deoxyglucose, and the negative inotropic effect diminished on reperfusing with glucose; 83 +/- 3 min after reperfusing with glucose, twitch amplitude was decreased by only 52 +/- 6% on exposure to 100 microM RP49356. These results suggest that the effect of the potassium channel opening drugs on contractility and electrical behavior will depend critically on the intracellular [ATP]. The results provide an explanation of how potassium channel openers may become clinically useful as cardioprotective agents without interfering with normal function.

Adenosine Triphosphate↗
PubMed1989

Excitation-contraction coupling in heart muscle.

We have investigated the links between electrical excitation and contraction in mammalian heart muscle. Using isolated single cells from adult rat ventricle, a whole-cell voltage-clamp technique and quantitative fluorescence microscopy, we have measured simultaneously calcium current (ICa) and [Ca2+]i (with fura-2). We find that the voltage-dependence of ICa and the [Ca2+]i-transient and the dependence of [Ca2+]i-transient on depolarization-duration cannot both be readily explained by a simple calcium-induced Ca-release ('CICR') mechanism. Additionally, we find that when [Ca2+]i and [Na+]i are at their diastolic levels, activation of the Na-Ca exchange mechanism by depolarization does not measurably trigger the release of Ca2+i. Finally, measuring ICa in adult and neonatal rat heart cells and using the alkaloid ryanodine, we have carried out complementary experiments. These experiments show that there may be an action of ryanodine on ICa that is independent of [Ca2+]i and independent of a direct action of the alkaloid on the calcium channel itself. Along with experiments of others showing that ryanodine binds to the sarcoplasmic reticulum calcium-release channel/spanning protein complex, our data suggests a model to explain our findings. The model links the calcium channels responsible for ICa to the sarcoplasmic reticulum by means of one or more of the spanning protein(s). Information from the calcium channel can be communitated to the sarcoplasmic reticulum by this route and, presumably, information can move in the opposite direction from the sarcoplasmic reticulum to the calcium channel.

Animals↗
PubMed1989

The mechanism of early contractile failure of isolated rat ventricular myocytes subjected to complete metabolic inhibition.

1. Twitch shortening of isolated rat ventricular myocytes was measured on exposure to complete metabolic blockade (2 mM-cyanide in the presence of 10 mM-2-deoxyglucose). Under these conditions twitch shortening declines to undetectable levels over 1-15 min. This 'early' contractile failure is followed by the development of a maintained contracture. 2. Contractures induced by caffeine (20 mM) were similar in amplitude before and after 'early' contractile failure. This result suggests that 'early' contractile failure is not due to depletion of Ca2+ from the sarcoplasmic reticulum. 3. The action potential shortened as the twitch magnitude declined during 'early' contractile failure, raising the possibility of a causal link. Voltage-clamp experiments show that an enormous increase in K+ conductance (greater than 20-fold) occurs during the period of 'early' contractile failure, and presumably underlies the action potential shortening. 4. If the K+ conductance changes are inhibited by replacement of intracellular K+ with N-methyl glucosamine and inclusion of 2 mM-tolbutamide in intra- and extracellular solutions, good voltage control can be achieved. Under these conditions, 'early' contractile failure did not occur on exposure to complete metabolic blockade and neither Ca2+ current nor the twitch were completely abolished until a maintained contracture had begun to occur. 5. Injection of ATP following 'early' contractile failure could partially restore the twitch and prolong the foreshortened action potential. 6. These results are consistent with the hypothesis that 'early' contractile failure occurring under non-voltage-clamped conditions is due principally to failure of activation of the Ca2+ current because of the shortening of the action potential. Although a decline in the availability of Ca2+ current also occurs, action potential shortening results mainly from increased conductance through ATP-sensitive K+ channels which are activated by a fall of intracellular [ATP]. Contractile failure arises not because of a primary alteration, or defect, in the coupling of excitation to contraction, but because the cell membrane is effectively clamped at a potential close to the K+ equilibrium potential.

Action Potentials↗
PubMed1989

Intramembrane charge movement in guinea-pig and rat ventricular myocytes.

1. Non-linear capacitative current (charge movement) was studied in isolated guinea-pig and rat ventricular myocytes. Linear capacitance was subtracted using standard procedures. Most of the experiments were done with guinea-pig myocytes, while rat myocytes were used for comparison. 2. When a myocyte was held at -100 mV, depolarizing clamp steps produced a rapid outward current transient, which was followed by an inward current transient upon repolarization. This current was identified as the movement of charged particles in the cell membrane, rather than ionic movement across the membrane, for the following reasons: (1) the current saturated at membrane potentials positive to +20 mV; (2) the current was capacitative in nature, having no reversal potential; (3) in general, the charge moved during depolarization (Qon) approximated the charge moved during repolarization (Qoff). 3. Qoff was significantly less than Qon for a depolarization from -100 mV to 0 mV. However, the Qoff/Qon ratio approached unity if the cell was instead repolarized to -140 mV. This was interpreted as being due to the immobilization of a fraction of the charge during the depolarization, which recovered rapidly enough to be measured at -140 mV, but recovered too slowly at -100 mV. 4. Charge movement in these cells had a sigmoidal dependence on the membrane potential, which could be empirically described by the two-state Boltzmann equation Q = Qmax/(1 + exp[-(V-V*)/kappa]), where Q is the charge movement at potential V, Qmax is the maximum charge, V* is the membrane potential at Q = Qmax/2, and kappa is a slope factor. Qmax was 11.7 nC/microF, V* was -18 mV and kappa was 16 mV in guinea-pig myocytes held at -100 mV, while the values in rat myocytes were 10.9 nC/microF, -32 mV and 13 mV. 5. The charge movement could be partially immobilized by a prior depolarization. This effect developed over a broad voltage range, from -120 to +20 mV. The fraction of charge that could be immobilized by a 10 s pre-pulse to +20 mV was 59%. 6. The time course of decay of both Qon and Qoff could basically be described as a single-exponential process. The time constant was largest at -40 mV and decreased at both more positive and negative test potentials. A second, slower Qoff time constant, possibly representing remobilization of immobilized charge, could be seen under some conditions. 7. The temperature dependence of charge movement was studied between 11 and 35 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1989

Nucleotide modulation of the activity of rat heart ATP-sensitive K+ channels in isolated membrane patches.

1. We have measured the ATP dependence of KATP channel activity, and the effect of various metabolites on this relationship, in inside-out membrane patches isolated from rat ventricular myocytes. 2. The inhibition of KATP channel activity by ATP could be described as a sigmoid function of [ATP] with a Hill coefficient (HATP) of 2 and a half-maximal inhibition at an ATP concentration (Ki, ATP) of 25 microM, in the presence of 0 mM, or 0.5 mM, total [Mg2+]. The non-hydrolysable ATP analogue, AMP-PNP, also inhibited the channel with Ki, AMP-PNP = 60 microM and HAMP-PNP = 2. 3. Acidosis caused a small, but significant, increase in Ki, ATP from 25 microM at pH 7.25 to 50 microM at pH 6.25, but phosphate and lactate were without effect (at 20 mM) on channel activity. 4. In the absence of ATP or Mg2+, ADP3- inhibited channel activity with Ki, ADP = 275 microM, and HADP = 1.2. Other purine and pyrimidine triphosphates, diphosphates and monophosphates also inhibited the channel with apparent order of inhibitory effectiveness ATP greater than AMP-PNP greater than ADP greater than CTP greater than GDP = AMP = ITP. 5. In the absence of Mg2+, but in the presence of 40 microM-ATP, channel inhibition by GTP, ITP, CTP, GDP, ADP or AMP was additive with inhibition by ATP. 6. In the presence of 0.5 mM-Mg2+ and 40 microM-ATP, inhibition by GTP, GMP and AMP was still additive with inhibition by ATP. The diphosphates ADP and GDP, however, paradoxically increased channel activity in the presence of ATP. This increase in channel activity appeared to result from a competitive increase in Ki, ATP, MgADP did not appear to cause any inhibition of channel activity. 7. We conclude that, in cardiac tissue, KATP channels are regulated by [ATP], and that this regulation is sensitive to other intracellular nucleotides, Mg2+, and pH, but not to phosphate or lactate. A simple, interactive two binding-site model is consistent with the nucleotide-dependent regulation that we observe.

Adenosine Triphosphate↗
PubMed1989

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.

Animals↗
PubMed1988

Anoxic contractile failure in rat heart myocytes is caused by failure of intracellular calcium release due to alteration of the action potential.

Anoxia of the heart causes failure of contraction before any irreversible injury occurs; the mechanism by which anoxia blocks cardiac excitation-contraction coupling is unknown. Studies in whole muscle are confounded by heterogeneity; however, achieving the low oxygen tensions required to study anoxia in a single myocyte during electrophysiological recording has been a barrier in experimental design. Guided by calculations of oxygen transport, we developed a system to insulate myocytes in an open dish from oxygen by a laminar counterflowing argon column, permitting free access to the cell by microelectrodes while maintaining a PO2 less than 0.02 torr (1 torr = 133 Pa). In the absence of glucose, the amplitude of stimulated contraction of anoxic ventricular myocytes fell to zero over 2 min after a lag period attributable to the consumption of endogenous glycogen. The cytosolic calcium concentration transient, measured by indo-1 fluorescence, fell to zero simultaneously with contraction. After the twitch had failed, microinjection of caffeine around the cell still caused a large calcium release and contraction, indicating that sarcoplasmic reticular calcium stores were not depleted. Twitch failure was accompanied by shortening and then failure of the action potential; under voltage clamp, large outward currents, reversing at the resting potential, developed during contractile failure. After failure of action potential-mediated contraction, voltage-clamp depolarization, with a large command voltage to compensate for the series-resistance error due to outward currents, restored normal twitch contraction. We conclude that anoxic contractile failure in the rat myocyte is due to alteration of the action potential and the distal pathways of excitation-contraction coupling remain essentially intact.

Action Potentials↗
PubMed1988

Changes in the calcium current of rat heart ventricular myocytes during development.

1. Calcium current (ICa) was recorded in single rat heart cells at two periods during development: (1) at 2-7 days post-partum (neonatal), and (2) at 6-8 weeks (adult). 2. We measured both transient and steady-state components of ICa and could describe ICa in terms of the steady-state activation (d infinity) and inactivation (f infinity) parameters, the channel reversal potential (Echannel) and a relative conductance parameter, gr. 3. In adult single cells, the application of ryanodine (10 microM), an agent known to alter the function of the sarcoplasmic reticulum (SR), abolished contraction rapidly and increased ICa. Ryanodine also produced a 13 mV shift in f infinity towards more positive potentials and altered its slope, while producing a small increase in gr but no effect on d infinity. In neonatal single cells, ryanodine (10 microM) had no significant effect on contraction, ICa, d infinity, f infinity, or gr. Caffeine (10 mM), a less specific agent widely used to investigate sarcoplasmic reticulum function, had actions similar to those of ryanodine. 4. In adult myocytes, when EGTA (10 or 20 mM) or bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, 10 mM) were included in the pipette solution, contractions were rapidly abolished, while a small (4 mV) shift of f infinity to more positive potentials was seen. A large additional shift of f infinity was observed when ryanodine (10 microM) was added to the superfusion solution in the continued presence of EGTA or BAPTA. The alterations of ICa in EGTA (or BAPTA) plus ryanodine were the same as those seen in ryanodine alone. In neonatal cells, in contrast, when EGTA or BAPTA were included in the pipette solution we observed only a small effect on f infinity and the application of ryanodine had no effect. 5. Electron micrographs of our preparations show that the dissociated adult cells have sharp sarcolemmal borders, fully developed sarcomeres with T-tubules and sarcoplasmic reticulum membranes. In contrast, the neonatal cells that we use have few of these intracellular structures. Our observations in these preparations are consistent with the work of others (e.g. Penefsky, 1974; Hirakow & Gotoh, 1975; Ishikawa & Yamada, 1975; Legato, 1975; Hoerter, Mazet & Vassort, 1981). 6. Our data suggest that fully developed sarcoplasmic reticulum in rat heart cells can affect ICa.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1988

Phorbol ester increases calcium current and simulates the effects of angiotensin II on cultured neonatal rat heart myocytes.

The effects of increased protein kinase C activity were studied in neonatal rat myocytes grown in primary culture. The changes in mechanical and electrical behavior, as well as protein phosphorylation, that followed the apparent activation of protein kinase C by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined. As spontaneous beating frequency was increased minimally by 10 nM TPA and by 100% with 85 nM TPA, shortening amplitude, shortening velocity, and relaxation velocity decreased concomitantly. In contrast, 4-alpha-phorbol-12,13-didecanoate (alpha-PDD), which does not activate protein kinase C, had no effect on beating behavior at 800 nM. In voltage-clamped single myocytes, both steady-state and transient components of the cadmium-sensitive calcium current were increased by the addition of TPA (65 nM). Neither the time constant for the inactivation of the transient component of this calcium current nor the reversal potential was altered by TPA. The phosphorylation state of a discrete set of proteins, with apparent molecular weights of 32 and 83 kDa, was enhanced when TPA was added to intact myocytes. Angiotensin II enhances the phosphorylation state of the same set of proteins as observed with TPA. We conclude that activation of protein kinase C can modify mechanical behavior and increase L-type Ca2+ channel activity in cultured neonatal rat ventricular myocytes. The remarkable similarity in mechanical, electrical, and protein phosphorylation responses of cultured neonatal myocytes following TPA or angiotensin II application indicate that protein kinase C may mediate the action of angiotensin II.

Angiotensin II↗
PubMed1988

Angiotensin II increases spontaneous contractile frequency and stimulates calcium current in cultured neonatal rat heart myocytes: insights into the underlying biochemical mechanisms.

The effect of angiotensin II on cultured neonatal rat heart myocytes was studied by measuring changes in cell length, the magnitude and kinetics of the calcium current, and changes in cyclic adenosine 3',5'-monophosphate (cAMP) and phosphoinositide metabolism. Spontaneous beating frequency of multicellular networks was increased by angiotensin II with a maximal increase of 100% above control values at concentrations of 5 nM or greater. The half-maximal response occurred at 0.6 nM angiotensin II. Shortening amplitude, shortening velocity, and relaxation velocity decreased concomitantly with the increasing contractile rate. In voltage-clamped single myocytes, both steady-state and transient components of the calcium current were increased by the addition of angiotensin II. Angiotensin II had no effect on either control or isoproterenol-stimulated adenylate cyclase activity in myocyte membranes. Neither the basal levels nor the isoproterenol-stimulated cAMP accumulation in intact cells was affected by addition of hormone. In myocytes labeled with [3H]inositol, angiotensin II stimulated the formation of [3H]inositol phosphates. One minute after addition of 5 nM angiotensin II, inositol monophosphate and inositol bisphosphate levels were increased to 73% and 99%, respectively, above control values and remained elevated at 10 minutes. Inositol trisphosphate levels were not significantly different from control values at either time point. Nifedipine (10 microM) had no effect on angiotensin II-induced increases in [3H]inositol phosphates. We conclude that the increases in both spontaneous beating rate and calcium current in angiotensin II-stimulated cultured neonatal heart cells are not dependent on cAMP or inositol trisphosphate levels but may involve sustained phosphoinositide hydrolysis.

Adenylyl Cyclases↗
PubMed1987

Effect of membrane potential changes on the calcium transient in single rat cardiac muscle cells.

The mechanism that links membrane potential changes to the release of calcium from internal stores to cause contraction of cardiac cells is unclear. By using the calcium indicator fura-2 under voltage-clamp conditions, changes in intracellular calcium could be monitored in single rat ventricular cells while controlling membrane potential. The voltage dependence of the depolarization-induced increase in intracellular calcium was not the same as that of the calcium current (Isi), which suggests that only a small fraction of Isi is required to trigger calcium release from the sarcoplasmic reticulum. In addition, sarcoplasmic reticulum calcium release may be partly regulated by membrane potential, since repolarization could terminate the rise in intracellular calcium. Thus, changes in the action potential will have immediate effects on the time course of the calcium transient beyond those associated with its effects on Isi.

Animals↗
PubMed1987

Cellular and subcellular heterogeneity of [Ca2+]i in single heart cells revealed by fura-2.

Digital imaging of calcium indicator signals (fura-2 fluorescence) from single cardiac cells has revealed different subcellular patterns of cytoplasmic calcium ion concentration ([Ca2+]i) that are associated with different types of cellular appearance and behavior. In any population of enzymatically isolated rat heart cells, there are mechanically quiescent cells in which [Ca2+]i is spatially uniform, constant over time, and relatively low; spontaneously contracting cells, which have an increased [Ca2+]i, but in which the spatial uniformity of [Ca2+]i is interrupted periodically by spontaneous propagating waves of high [Ca2+]i; and cells that are hypercontracted (rounded up) and that have higher levels of [Ca2+]i than the other two types. The observed cellular and subcellular heterogeneity of [Ca2+]i in isolated cells indicates that experiments performed on suspensions of cells should be interpreted with caution. The spontaneous [Ca2+]i fluctuations previously observed without spatial resolution in multicellular preparations may actually be inhomogeneous at the subcellular level.

Animals↗
PubMed1987

Effects of changes of intracellular pH on contraction in sheep cardiac Purkinje fibers.

Intracellular pH (pHi) was measured with a pH-sensitive microelectrode in voltage-clamped sheep cardiac Purkinje fibers while tension was simultaneously measured. All solutions were nominally CO2/HCO3 free and were buffered with Tris. The addition of NH4Cl (5-20 mM) produced an initial intracellular alkalosis that was associated with an increase of twitch tension. At the same time, a component of voltage-dependent tonic tension developed. Prolonged exposure (greater than 5 min) to NH4Cl resulted in a slow recovery of pHi accompanied by a decrease of tension. Removal of NH4Cl produced a transient acidosis that was accompanied by a fall of force. In some experiments, there was then a transient recovery of force. If extracellular pH (pHo) was decreased, then pHi decreased slowly. Tension also fell slowly. An increase of pHo produced a corresponding increase of both force and pHi. The application of strophanthidin (10 microM) increased force and produced an intracellular acidosis. The addition of NH4Cl, to remove this acidosis partially, produced a significant increase of force. The above results show that contraction is sensitive to changes of intracellular but not extracellular pH. This pH dependence will therefore modify the contractile response to inotropic maneuvers that also affect pHi.

Acid-Base Equilibrium↗
PubMed1987

Calcium current in isolated neonatal rat ventricular myocytes.

1. Calcium currents (ICa) from neonatal rat ventricular heart muscle cells grown in primary culture were examined using the 'whole-cell' voltage-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Examination of ICa was limited to one calcium channel type, 'L' type (Nilius, Hess, Lansman & Tsien, 1985), by appropriate voltage protocols. 2. We measured transient and steady-state components of ICa, and could generally describe ICa in terms of the steady-state activation (d infinity) and inactivation (f infinity) parameters. 3. We observed that the reduction of ICa by the calcium channel antagonist D600 can be explained by both a shift of d infinity to more positive potentials as well as a slight reduction of ICa conductance. D600 did not significantly alter either the rate of inactivation of ICa or the voltage dependence of f infinity. 4. The calcium channel modulator BAY K8644 shifted both d infinity and f infinity to more negative potentials. Additionally, BAY K8644 increased the rate of inactivation at potentials between +5 and +55 mV. Furthermore, BAY K8644 also increased ICa conductance, a change consistent with a promotion of 'mode 2' calcium channel activity (Hess, Lansman & Tsien, 1984). 5. We conclude that, as predicted by d infinity and f infinity, there is a significant steady-state component of ICa ('window current') at plateau potentials in neonatal rat heart cells. Modulation of the steady-state and transient components of ICa by various agents can be attributed both to specific alterations in d infinity and f infinity and to more complicated alterations in the mode of calcium channel activity.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1987

Regulation of twitch tension in sheep cardiac Purkinje fibers during calcium overload.

The dependence of twitch tension on the interval (delta t) between depolarizations was examined in voltage-clamped sheep cardiac Purkinje fibers under conditions of calcium overload. During the development of calcium overload (produced by sodium-pump inhibition), twitch amplitude changes from a monotonic function of delta t to an oscillatory one. We investigated the cellular processes underlying this oscillatory relationship. Measurable calcium current was blocked by D 600 (25 microM), but neither the twitch nor the oscillatory dependence of twitch tension on delta t was abolished. Caffeine (2 mM), applied to modify sarcoplasmic reticulum function, decreased the oscillatory period of the twitch/interval relationship as it increased the frequency of spontaneous fluctuations of resting tension. Our results suggest that the oscillatory relationship between twitch amplitude and delta t is not caused by changes in the calcium current per se but rather by fluctuations in the amount of releasable calcium in the sarcoplasmic reticulum. Additionally, we conclude that the calcium current may not be a necessary prerequisite for depolarization to trigger calcium release from the sarcoplasmic reticulum under conditions of calcium overload.

Animals↗
PubMed1987

Intracellular calcium in cardiac myocytes: calcium transients measured using fluorescence imaging.

We have examined the distribution of Ca2+ in voltage-clamped cardiac myocytes under resting conditions and during the Ca2+ transient. We find that the resting Ca2+ level in a quiescent rat myocyte bathed in 1 mM extracellular Ca is relatively low (between 60 and 100 nM) and uniform. At the peak of the Ca2+ transient, Ca2+ can rise to a level as high as 600 nM to 1.0 microM. Furthermore, the magnitude of the Ca2+ transient is dependent on the size of the membrane depolarization. There is good agreement between measurements made using video imaging and those made using a photomultiplier tube for the value of intracellular Ca2+ at the peak of the Ca2+ transient and for the subsequent slow changes in intracellular Ca2+. On repolarization, intracellular Ca2+ falls with a half-time of approximately 100 ms. The uniform distribution of Ca2+ reported in the Ca2+ images of myocytes at rest and at the peak of the Ca2+ transient under normal conditions is in contrast to what is observed during "Ca2+ overload" when subcellular regions of elevated Ca2+ are observed to propagate along the cell. Thus, the measurement of [Ca2+]i in cardiac myocytes with fura-2 has already yielded important new information that was not available using other techniques to measure [Ca2+]i in cardiac ventricular muscle.

Animals↗
PubMed1986

A novel experimental chamber for single-cell voltage-clamp and patch-clamp applications with low electrical noise and excellent temperature and flow control.

We describe a simple and inexpensive experimental chamber that is designed to overcome several problems encountered when doing electrical and optical studies on single cells or on isolated membrane patches. The bath is small enough to fit on the stage of a standard inverted microscope. It includes a novel solution level-detector, the output of which is used to actively control the level of solution in the experimental chamber. A Peltier-effect device is located adjacent to the flow-chamber and heats or cools the inflowing solution. Solutions can be rapidly switched using two electrically actuated microvalves. The attraction of this system is that, with appropriately quiet power supplies, not only is the bath solution-level held at a fixed height, but the temperature of the bathing solution can also be set over a wide temperature range (minimum range is 15 to 45 degrees C), and solutions can be rapidly changed. All of the construction details are supplied as are appropriate electrical circuits. Without modification, the chamber can be used for applications as diverse as fluorescence microscopy of living cells, time-lapse photomicroscopy and single-cell motion detection as well as single cell voltage-clamp and isolated membrane patch-clamp. With simple modification the system can be adapted for use in experiments on multicellular preparations.

Electric Wiring↗