Alternative splicing of the Na(+)-Ca2+ exchanger gene, NCX1.

We describe an analysis of the NCX1 gene and show that various tissues express different alternatively spliced forms of the gene. Alternative splicing has been confirmed by the genomic analysis of the Na(+)-Ca2+ exchanger gene. We also describe the Drosophila Na(+)-Ca2+ exchanger as having many of the same structural characteristics of the mammalian exchangers and this locus as possibly undergoing alternative splicing in the same region that has been described in the NCX1 gene. The general structure of the exchangers is similar to that of the alpha-subunit of the (Na(+)+ K+)-A Pase. Finally, sequence comparison of the various molecules demonstrates that structural characteristics of these molecules are more strongly conserved than the primary sequence of these products.

Ultra-slow voltage-dependent inactivation of the calcium current in guinea-pig and ferret ventricular myocytes.

L-type Ca2+ current, iCa, has been recorded in guinea-pig ventricular myocytes at 36 degrees C using the whole cell patch clamp technique. Intracellular Ca2+ was buffered with ethylenebis(oxonitrilo)tetraacetate (EGTA). An increase in the rate of stimulation from 0.5 to 3 Hz resulted in an abrupt decrease in iCa in the first beat at the high rate, followed by a progressive decrease (tau approx. 7 s) over the next 30 s. The changes were not the result of Ca(2+)-dependent inactivation, because similar changes occurred with either Ba2+ or Na+ as the charge carrier. During 20-s voltage clamp pulses there was an ultra-slow phase of inactivation of Ba2+ or Na+ current through the Ca2+ channel (tau approx. 6 s at 0 mV). This was confirmed by applying test pulses after conditioning pulses of different duration: the Ba2+ current during the test pulse decreased progressively when the duration of the conditioning pulse was increased progressively to 20 s. Ultra-slow inactivation of Ba2+ current was voltage dependent and increased monotonically at more positive potentials. Recovery of Ba2+ current from ultra-slow inactivation occurred with a time constant of 3.7 s at -40 mV and 0.7 s at -80 mV. The gradual decrease in iCa on increasing the rate to 3 Hz may have been the result of the development of ultra-slow voltage-dependent inactivation.

Measurement of intracellular Ca2+ concentration using Indo-1 during simultaneous flash photolysis to release Ca2+ from DM-nitrophen.

We have constructed a modular instrument to measure intracellular [Ca2+] ([Ca2+]i) in single isolated cells while simultaneously imposing step changes in [Ca2+]i using "caged Ca2+". By combining the outputs of a xenon arc lamp with a frequency-tripled (Nd:YAG) laser, the instrument can operate with low maintained illumination to measure [Ca2+]i using a ratiometric Ca(2+)-sensitive fluorophore and also activate the release of Ca2+ from a caged-Ca2+ compound with a high energy pulse of ultraviolet light. This instrument is simple to assemble, introduces little electrical noise, provides a wide range of illumination power, produces only moderate photobleaching of the Ca2+ indicator and can be readily adapted to diverse cellular preparations. We demonstrate the use of this system to measure step changes in [Ca2+]i in adult rat ventricular myocytes and a human embryonic kidney cell line (293 cells) in culture.

A confocal laser scanning microscope designed for indicators with ultraviolet excitation wavelengths.

In this paper we describe the modifications necessary to upgrade, at affordable cost, a commercially available confocal laser scanning microscope for use with ultraviolet (UV) excitation. The optical problems associated with these modifications are described in detail, and easy solutions to solve them are suggested. The optical resolution of the instrument was tested with fluorescent beads and was found to be close to diffraction limited. The light losses due to lateral chromatic aberration were assessed in a thick fluorescent specimen and were found to be comparable to those usually observed with visible light. For a more visual example of the resolution of this instrument, isolated ventricular heart muscle cells were loaded with the fluorescent Ca2+ indicator indo 1. This allowed us to visualize subcellular structural detail and to illustrate the optical sectioning capability of the UV confocal microscope when recording indo 1 emission. Dual-emission line scans were used to perform ratiometric time-resolved detection of Ca2+ transients in voltage-clamped heart muscle cells loaded with the salt form of indo 1. The system presented in this paper should significantly broaden the range of fluorescent indicators that can be used in confocal microscopy.

Fluorescence lifetime imaging of intracellular calcium in COS cells using Quin-2.

We describe the first fluorescence lifetime images of cells. To demonstrate this new capability we measured intracellular images of Ca2+ in COS cells based on the Ca(2+)-dependent fluorescence lifetime of Quin-2. Apparent fluorescence lifetimes were measured by the phase-modulation method using a gain-modulated image intensifier and a slow-scan CCD camera. We describe methods to correct the images for photobleaching during acquisition of the data, and to correct for the position-dependent response of the image intensifier. The phase angle Quin-2 images were found to yield lower than expected Ca2+ concentrations, which appears to be the result of the formation of fluorescent photoproducts by Quin-2. Fluorescence lifetime imaging (FLIM) does not require wavelength-radiometric probes and appears to provide new opportunities for chemical imaging of cells.

On establishing primary cultures of neonatal rat ventricular myocytes for analysis over long periods.

INTRODUCTION: Primary cultures of neonatal rat ventricular myocytes include a population of rapidly dividing nonmyocardial cells that can alter the properties of myocytes and complicate experimental interpretations. Without any intervention, nonmyocyte proliferation restricts the utility of primary cultures in biochemical and electrophysiologic studies to 4-5 days. However, with the recent interest in regulation of cardiac gene expression and the effects of growth factors on cardiac function, long-term studies with stable heart cultures are warranted. METHODS AND RESULTS: In the present study an immunohistochemical staining strategy was developed that allowed for reliable quantitation of myocytes and nonmyocytes in cultures maintained for extended periods under different culture conditions. Density gradient purification of myocytes was found valuable in limiting nonmyocyte levels to < 20% at early times. Further treatment of cultures with a mitotic inhibitor, 0.1 mM bromodeoxyuridine, or 3500 rads of gamma-irradiation effectively blocked the proliferation of nonmyocardial cells, while it had no effect on cardiocyte levels. However, bromodeoxyuridine displayed side effects on the myocytes; the spontaneous beating rate and intracellular glycogen content were markedly depressed. In contrast, a systematic investigation of the properties of the irradiated myocytes, including spontaneous beating rates, dihydropyridine receptors, glycogen content, sarcoplasmic reticulum function, and phosphoinositide signaling, revealed that irradiation did not alter cardiac cell function. Although ionizing radiation can stimulate gene expression in some cell types, gamma-irradiation did not evoke c-fos expression or cause sarcomere formation, responses seen in cardiac cells to several trophic factors. CONCLUSION: This study establishes a system of stable, functional, primary cultured cardiac cells that can be used in long-term molecular and electrophysiologic studies of at least 2 weeks.

Two-photon-excitation fluorescence imaging of three-dimensional calcium-ion activity.

Two-photon excitation of the ultraviolet-absorbing fluorescent calcium indicator Indo-1 in laser scanning microscopy makes possible a quantitative, three-dimensional recording of intracellular free calcium activity ([Ca(2equation)](i)) distributions and dynamics with low background and minimal photobleaching. We have constructed a simple optical system that facilitates collection of the 400-500-nm Indo-1 fluorescence without the use of a confocal spatial filter. Instead of the fluorescence being descanned as is normally required in confocal microscopy, the fluorescence is deflected by a dichroic mirror into a separate detection pathway. Images of [Ca(2+)](i) distributions with three-dimensional submicrometer resolution and 10% precision are obtained at100-µM Indo-1 concentration and 3-s recording time for 384 × 512 pixels. Data on [Ca(2+)](i) in tumor mast cells and cardiac myocytes illustrate the capabilities of this technique.

The role of Na(+)-Ca2+ exchange in paired pulse potentiation of ferret ventricular muscle.

1. Stimulation of cardiac muscle with pairs of stimuli ('paired pulse stimulation') results in a large inotropic effect and experiments have been carried out on ferret ventricular muscle to investigate the underlying mechanism. 2. Aequorin was used to measure sarcoplasmic Ca2+ in papillary muscles. During paired pulse stimulation the first aequorin light transient (i.e. Ca2+ transient) and contraction of the pair increased in amplitude, whereas the second aequorin light transient and contraction were small. When the interval between the pair was decreased, the second aequorin light transient and contraction of the pair were smaller, but the increase in the first aequorin light transient and contraction was greater. 3. The relationship between contraction and the aequorin light transient was the same during paired pulse stimulation and on raising the bathing Ca2+ concentration. It is concluded that there was no change in the myofilament sensitivity to Ca2+ during paired pulse stimulation. 4. The increase in the aequorin light transient and contraction during paired pulse stimulation was prevented by ryanodine, an inhibitor of the sarcoplasmic reticulum (SR). 5. During paired pulse stimulation of ventricular myocytes there was little change in the first action potential of the pair, but the second action potential was shorter than control when the interval between the pair was short. During paired pulse stimulation of ventricular myocytes under voltage clamp control there was little change in the first Ca2+ current (iCa) of the pair, but the second iCa was smaller than control when the interval between the pair was short. Because paired pulse potentiation was greatest when the interval between the pair was short, it is concluded that paired pulse potentiation was not the result of a prolongation of the action potential or increase in iCa. 6. During paired pulse stimulation of ventricular myocytes under voltage clamp control the increase in contraction was greater, the more positive the membrane potential during the second pulse of the pair. This voltage dependence is consistent with a role for the Na(+)-Ca2+ exchanger in paired pulse potentiation. 7. During paired pulse stimulation of ventricular myocytes under voltage clamp control, changes in putative Na(+)-Ca2+ exchange current were observed consistent with a decrease of Ca2+ efflux (or increase of Ca2+ influx) via the exchanger during the second pulse of the pair. 8. A computer model of excitation-contraction coupling (Harrison, McCall & Boyett, 1992) has been used to simulate paired pulse stimulation and the results described above.(ABSTRACT TRUNCATED AT 400 WORDS)

Does the use of DM-nitrophen, nitr-5, or diazo-2 interfere with the measurement of indo-1 fluorescence?

Emission spectra of the photolabile Ca2+ chelators DM-nitrophen, nitr-5, and diazo-2 were studied alone, and in the presence of indo-1, to investigate potential interactions that would make the simultaneous manipulation and ratiometric measurement of the intracellular Ca2+ concentration difficult. Neither diazo-2 nor its photoproduct were found to be significantly fluorescent, and consequently concentrations of diazo-2 up to 20 times that of indo-1 did not distort the emission spectra of indo-1. DM-nitrophen was scarcely fluorescent, but its fluorescence did increase upon photolysis. In contrast to diazo-2 and DM-nitrophen, nitr-5 itself was found to be quite fluorescent, and this fluorescence was significantly increased upon photolysis. Thus, combined use of nitr-5 and indo-1 poses the most difficulty. The emission spectra of all the investigated compounds were used to define experimental conditions and calibration procedures that make possible simultaneous measurement and manipulation of the intracellular Ca2+ concentration.

Sodium/calcium exchanger in heart muscle: molecular biology, cellular function, and its special role in excitation-contraction coupling.

The Na/Ca exchanger has been examined with respect to its molecular biology, its cellular function, and its role in excitation-contraction coupling. The Na/Ca exchanger plays a central part in excitation-contraction coupling, setting the level of sarcoplasmic reticular calcium and contributing to the triggering of sarcoplasmic reticular calcium release. Functional biophysical studies with isolated single cells and caged calcium provide evidence that the Na/Ca exchanger works as a two step sequential transporter. In the heart there are about 250 exchangers.mu-2, operating at a turnover rate of up to about 2500.s-1, with the exchanger carrying -2.56 charges under normal conditions. The Na/Ca exchanger has been recently cloned from diverse mammalian species and several tissues and is largely conserved. It is clear, however, that the function of the Na/Ca exchanger is different in the different tissues. Thus work is in progress in several laboratories, including ours, to determine how the Na/Ca exchanger achieves its tissue specific function. Several modulatory motifs have been seen in studies of the exchanger that may explain some of the tissue specific differences. Interestingly the modulation of the Na/Ca exchanger (for example, by protons, sodium, calcium, ATP, calmodulin) seems to arise from interactions with the intracellular loop.

On the role of G protein activation and phosphorylation in desensitization to acetylcholine in guinea-pig atrial cells.

1. The ACh-activated K+ current (IK,ACh) has been investigated in guinea-pig atrial cells at 36 degrees C using the whole-cell patch-clamp technique. 2. During an exposure to ACh, IK,ACh faded as a result of desensitization. Throughout the fade of the current, the current reversed at EK and showed inward-going rectification. The fade was, therefore, the result of a genuine decrease in IK,ACh. 3. The onset of desensitization (as judged by the fade of IK,ACh) was biphasic and the time constants of the fast and slow phases of desensitization were 1.58 +/- 0.14 (n = 16) and 148.2 +/- 12.8 s (n = 18) respectively. Recovery from the fast and slow phases of desensitization (after 30 s and 5 min exposures to ACh respectively) occurred with time constants of 52 and 222 s respectively. This suggests that two processes are involved in desensitization. 4. The Q10 of the rate constant of the fast phase of desensitization was 2.2 +/- 0.3 (n = 6). 5. Intracellular perfusion with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or extracellular perfusion with AlF4- were used to bypass the muscarinic receptor and trigger IK,ACh by directly activating the G protein, GK, that links the muscarinic receptor to the K+ channel. Both GTP gamma S and AlF4- activated a current with the same reversal potential and the same degree of inward-going rectification as the ACh-activated current. 6. Desensitization still occurred when the muscarinic receptor was bypassed and IK,ACh was triggered by direct activation of GK with either GTP gamma S or AlF4-. This suggests that desensitization is, in part, the result of a modification of either GK or the K+ channel. 7. Activation of the muscarinic receptor by ACh resulted in greater desensitization than direct activation of GK; at the end of a 5 min exposure to ACh, current was only 22 +/- 1% (n = 19) of its peak value, whereas, after direct activation of GK by GTP gamma S for 5 min, current was 42 +/- 6% (n = 5) of its peak value. This suggests that desensitization also involves the muscarinic receptor. 8. When cells were perfused with GTP gamma S, the fast phase of desensitization could still occur, but the slow phase was reduced. This suggests that the fast phase involves GK or the K+ channel, whereas the slow phase involves the muscarinic receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Scorpion toxins targeted against the sarcoplasmic reticulum Ca(2+)-release channel of skeletal and cardiac muscle.

We report the purification of two peptides, called "imperatoxin inhibitor" and "imperatoxin activator," from the venom of the scorpion Pandinus imperator targeted against ryanodine receptor Ca(2+)-release channels. Imperatoxin inhibitor has a M(r) of approximately 10,500, inhibits [3H]ryanodine binding to skeletal and cardiac sarcoplasmic reticulum with an ED50 of approximately 10 nM, and blocks openings of skeletal and cardiac Ca(2+)-release channels incorporated into planar bilayers. In whole-cell recordings of cardiac myocytes, imperatoxin inhibitor decreased twitch amplitude and intracellular Ca2+ transients, suggesting a selective blockade of Ca2+ release from the sarcoplasmic reticulum. Imperatoxin activator has a M(r) of approximately 8700, stimulates [3H]ryanodine binding in skeletal but not cardiac sarcoplasmic reticulum with an ED50 of approximately 6 nM, and activates skeletal but not cardiac Ca(2+)-release channels. These ligands may serve to selectively "turn on" or "turn off" ryanodine receptors in fragmented systems and whole cells.

Thapsigargin inhibits contraction and Ca2+ transient in cardiac cells by specific inhibition of the sarcoplasmic reticulum Ca2+ pump.

Regulation of the level of ionized calcium, [Ca2+]i, is critical for its use as an important intracellular signal. In cardiac and skeletal muscle the control of fluctuations of [Ca2+]i depend on sarcolemmal and sarcoplasmic reticulum ion channels and transporters. We have investigated the sesquiterpine lactone, thapsigargin (TG), because of its reported action to alter cellular calcium regulation in diverse cell types, including striated muscle cells. We have combined biochemical and physiological methods at the cellular level to determine the site of action of this agent, its specificity, and its cellular effects. Using a patch-clamp method in whole cell configuration while measuring [Ca2+]i with Indo-1 salt, we find that TG (100 nM) largely blocks the contraction and the [Ca2+]i transient in rat ventricular myocytes. Analysis of these data indicate that no sarcolemmal current or transport system is directly altered by TG, although indirect [Ca2+]i-dependent processes are affected. In permeabilized myocytes, TG blocked oxalate-stimulated calcium uptake (half-maximal effect at 10 nM) into the SR. However, TG (100 microM) had no effect on Ca(2+)-induced Ca(2+)-release in purified muscle (ryanodine-receptor enriched) vesicles while clearly blocking Ca(2+)-ATPase activity in purified (longitudinal SR) vesicles. We conclude that in striated muscle TG markedly alters calcium metabolism and thus alters contractile function only by its direct action on the Ca(2+)-ATPase.

The length, width and volume of isolated rat and ferret ventricular myocytes during twitch contractions and changes in osmotic strength.

The length and width of rat and ferret ventricular myocytes have been measured using a linear photodiode array; the volume of the myocytes has been calculated based on the assumption that the cells were elliptical cylinders. During a twitch contraction, there was a decrease in cell length, but no significant change in the calculated cell volume, because the cells increased in width. Inotropic interventions not only resulted in a greater shortening of the cell during each contraction, but also a greater increase in cell width. Changes in cell length, width and volume on changing the osmotic strength of the bathing solution have also been investigated. The increase in volume in hypotonic solution, and the decrease in hypertonic solution, were the result of changes in the cell width; there were no significant changes in the cell length. It is concluded from the latter experiment that the lateral compliance of a cell is greater than its longitudinal compliance, and, therefore, during a twitch contraction, when the cell shortens, the displacement of the cell contents from the two ends of the cell and the expansion of the cell laterally will not act as a large force to oppose shortening.

Mechanical alternans during acidosis in ferret heart muscle.

Acidosis leads to mechanical alternans (i.e., alternation of large and small contractions) in ferret papillary muscles. This alternation in the size of the contraction is paralleled by alternation in the size of the intracellular Ca2+ transient (monitored using the photoprotein aequorin). In isolated myocytes, the large contraction is accompanied by a prolonged action potential. Mechanical alternans also can be induced by acidosis in isolated myocytes during a train of voltage-clamp pulses. Thus, it appears unlikely that the mechanical alternans is secondary to changes in action potential duration; it is more likely that the observed changes in action potential duration are secondary to changes in the size of the Ca2+ transient. The observation that a Ca2(+)-activated inward current also shows alternation during mechanical alternans provides a possible mechanism for the link between Ca2+ and action potential duration. The alternation in the size of the Ca2+ transient may be secondary to the slowed mechanical restitution observed in papillary muscles during acidosis. This also could explain the observation that decreasing stimulation rate can abolish the alternans.

Adriamycin cardiomyopathy in the rabbit: alterations in contractile proteins and myocyte function.

STUDY OBJECTIVE: The aim was to determine the mechanism of the cardiotoxic effect of adriamycin, particularly at the level of the function of the cardiac myocyte. DESIGN: After chronic exposure to adriamycin, the contractile responses of single isolated cardiac myocytes to increasing calcium and isoprenaline were measured, as well as oxygen consumption of myocyte suspensions. Creatine kinase and myosin isoforms were investigated in whole ventricle. The degree of fibrosis of the ventricle was quantified using histological methods. SUBJECTS: 24 white male New Zealand rabbits were treated with adriamycin (1 mg.kg-1) twice a week for eight weeks, and allowed to recover for two weeks. There were 29 untreated controls. Six further rabbits were implanted with mini osmotic pumps delivering a constant infusion of isoprenaline for one week; five controls had pumps containing saline. MEASUREMENTS AND MAIN RESULTS: Cardiac myocytes were enzymatically isolated, and their contraction amplitude and velocity monitored. Cells isolated from adriamycin treated rabbits had a lower contraction amplitude than those from controls when maximally activated with calcium, at 11.1(0.9)% shortening (n = 11) v 13.6(0.5)% (n = 14), p less than 0.02; or with isoprenaline, at 11.6(0.6)% shortening v 13.1(0.4)%, p less than 0.05. Contraction, but not relaxation, velocity in maximum calcium or isoprenaline was also significantly lower in cells from adriamycin treated animals. Oxygen consumption per 10(6) cells was lower in preparations from treated animals (p less than 0.05), but the relative effects of glucose, acetate, 2,4-DNP, and cyanide were unaffected. There was no significant change in creatine kinase or myosin isozyme composition or in amounts of fibrosis following adriamycin treatment. However, the quantity of myosin per g wet weight of tissue was significantly reduced from 8.04(0.45) mg.g-1 wet tissue, n = 4, in controls to 5.76(1.55) mg.g-1, n = 6, in adriamycin treated animals (p less than 0.001). The EC50 for isoprenaline was unchanged in cells from treated animals. Together with the unaltered maximum isoprenaline/calcium ratio, this implies that there is no change in beta adrenoceptor sensitivity following adriamycin treatment. To confirm that it was possible to desensitise rabbit cardiac beta adrenoceptors, and to detect changes in sensitivity on single cells, rabbits were treated with isoprenaline for one week. Such treatment decreased the maximum isoprenaline/calcium contraction amplitude ratio from 0.97(0.15), n = 5, to 0.47(0.12), n = 6 (p less than 0.05), and increased the EC50 from 7.9 to 224 nM (p less than 0.05). CONCLUSIONS: Single cardiac myocytes isolated from the hearts of adriamycin treated rabbits show a decrease in contraction amplitude, velocity, and oxygen consumption compared to controls. The decreased contractility of individual myocytes may relate to their low myosin content, and could contribute to the reduced cardiac output produced by adriamycin treatment. Heart failure induced by adriamycin in the rabbit is not accompanied by beta adrenoceptor desensitisation.

The control of calcium influx by cytoplasmic calcium in mammalian heart muscle.

The role of Ca2+ in the initiation and maintenance of contraction has been extensively studies. Many of these studies have focused on how Ca2+ influx and efflux affect cytoplasmic Ca2+ (Cai) and, therefore, contraction in cardiac muscle. However, it has recently become apparent that Cai itself may play a major role in the control of Ca2+ influx and efflux from cardiac muscle. Here we review current ideas on the mechanisms underlying Ca2+ homeostasis in cardiac muscle, with specific attention to how Cai may control Ca2+ influx, both under normal and pathological conditions.

An improved apparatus for the optical recording of contraction of single heart cells.

An optical system for measuring changes in cell length during unloaded contractions of cardiac myocytes is described. A one-dimensional video "image" of a cell is obtained every 4 ms with a linear photodiode array, which is aligned with the longitudinal axis of the cell. The circuit used to process the image from the photodiode array has a variety of features to aid in the accurate determination of the distance between the ends of the cell, i.e. the cell length. First, the video image of the cell is divided into two "windows", one encompassing the "front" edge of the cell, the other encompassing the "rear" edge. Other cells or debris beyond the cell edges are excluded. Changes in the general light level, for example as a result of debris floating above the cell, have little effect because within the windows the "background light level" is subtracted from the signals before they are processed further. To detect the cell edges, the system determines when the signals within the windows exceed (front edge) or drop below (rear edge) chosen thresholds, which are different for the front and rear edges. The system has "memory" and it identifies the rear edge of the cell as the last time the signal falls below the threshold; because of this "bright spots" within the cell are not mistaken for the end of the cell. The system has "hysteresis", which enables it to ignore small fluctuations in brightness around the threshold. The system is easy to use, accurate, readily calibrated, and it has good spatial and time resolution (about 0.25 micron and 4 ms respectively).

Rapid regulation of the 'second inward current' by intracellular calcium in isolated rat and ferret ventricular myocytes.

1. Single cells were isolated from the ventricles of ferret and rat hearts. Cells were voltage clamped using a single conventional microelectrode. Membrane voltage, membrane currents and cell length were monitored. 2. The current elicited by decreasing the membrane potential from a holding potential of -40 or -45 mV to potentials more positive than -20 mV was abolished by D600, by Cd2+ and by removal of Ca2+ from the cell superfusate. This current activated within 20 ms and inactivated over several hundred milliseconds; it had a bell-shaped current-voltage relation, and was maximal at about +10 mV. It is concluded that this is the fast Ca2+ current ICa. 3. Increasing bathing [Ca2+] (Ca2+o) led to the appearance of transient inward currents (Iti). If ICa was triggered during Iti, it was reduced in magnitude, and inactivated more slowly. 4. The sarcoplasmic reticulum inhibitor ryanodine (1 mumol/l) abolished Iti, and reduced twitch contraction, but had no direct effect on the magnitude of ICa, although its rate of inactivation was slowed. 5. Iti produced by depolarization of the holding potential, or by lowering bathing [K+] or [Na+], led to similar changes to those described in paragraph 3. 6. Gradually increasing diastolic cytoplasmic [Ca2+] (Ca2+i) by rapid stimulation in the presence of ryanodine, by lowering bathing [K+], or lowering bathing [Na+], led to a parallel decrease of ICa. 7. The effects of lowering bathing [Na+] could be abolished by using an electrode-filling solution containing EGTA. 8. In some ferret cells a slow component of the second inward current was observed. The size of this current was directly related to the size of the twitch: changes in the size of the twitch produced by changing the pattern of stimulation or application of ryanodine were paralleled by changes in the size of this current, but had no effect on the size of ICa. 9. It is concluded that the magnitude of ICa can be decreased by an increase of either resting Ca2+i, or the spontaneous increase of Ca2+ which underlies Iti, but it is not affected by the size of the stimulated calcium transient (although the time course of inactivation is dependent on the calcium transient). The size of the slow component of the second inward current, however, is directly related to the size of the twitch and may, therefore, be activated by Ca2+.

The negative inotropic effect of acetylcholine on ferret ventricular myocardium.

1. The effects of acetylcholine (ACh) on developed tension and intracellular Ca2+ concentration (as measured with aequorin) were studied in ferret papillary muscles, and on twitch shortening, the action potential and membrane currents in ferret ventricular myocytes. 2. Addition of ACh to ferret papillary muscles resulted in decreases in developed tension and the intracellular Ca2+ transient, both of which then partially recovered in the continued presence of ACh ('fade' of the response). On wash-off of ACh both developed tension and the intracellular Ca2+ transient increased above control ('rebound') before returning to control values. 3. Addition of ACh to ferret ventricular myocytes resulted in a membrane hyperpolarization of 2 +/- 0.5 mV (mean +/- S.E.M.; n = 9), a decrease in action potential duration to 23 +/- 6% of control and a decrease in twitch shortening to 31 +/- 5% of control. In the continued presence of ACh these responses to ACh faded. Thirty seconds after the maximal effect of ACh, action potential duration had partially recovered to 34 +/- 6% of control and twitch shortening to 46 +/- 7% of control. 4. The effects of ACh on twitch shortening could be mimicked under voltage clamp by varying voltage clamp pulse duration to simulate the ACh-induced changes in action potential duration. 5. When ACh was applied during a train of voltage clamp pulses of constant duration, 81% of the cells showed less than a 20% decrease in Ca2+ current and twitch shortening. However in 19% of the cells twitch shortening and the apparent Ca2+ current decreased by more than 30%. 6. In the 81% of cells, the normal decrease in twitch shortening was wholly the result of the shortening of the action potential. This in turn was the result of an increase in an outward background current which increased the rate of repolarization during the action potential. The ACh-induced background current reversed at -89 +/- 2 mV and showed inward-going rectification; these properties suggest that it was carried by K+. 7. In the 19% of cells, the normal decrease in twitch shortening was only partly the result of the shortening of the action potential (due to both the increase in outward background current as well as the apparent decrease in Ca2+ current). In these cells the decrease in twitch shortening may also have been partly the direct result of the apparent decrease of Ca2+ current.