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Cardiac alternans refers to a condition in which there is a periodic beat-to-beat oscillation in electrical activity and the strength of cardiac muscle contraction at a constant heart rate. Clinically, cardiac alternans occurs in settings that are typical for cardiac arrhythmias and has been causally linked to these conditions. At the cellular level, alternans is defined as beat-to-beat alternations in contraction amplitude (mechanical alternans), action potential duration (APD; electrical or APD alternans) and Ca(2+) transient amplitude (Ca(2+) alternans). The cause of alternans is multifactorial; however, alternans always originate from disturbances of the bidirectional coupling between membrane voltage (Vm ) and intracellular calcium ([Ca(2+) ]i ). Bidirectional coupling refers to the fact that, in cardiac cells, Vm depolarization and the generation of action potentials cause the elevation of [Ca(2+) ]i that is required for contraction (a process referred to as excitation-contraction coupling); conversely, changes of [Ca(2+) ]i control Vm because important membrane currents are Ca(2+) dependent. Evidence is mounting that alternans is ultimately caused by disturbances of cellular Ca(2+) signalling. Herein we review how two key factors of cardiac cellular Ca(2+) cycling, namely the release of Ca(2+) from internal stores and the capability of clearing the cytosol from Ca(2+) after each beat, determine the conditions under which alternans occurs. The contributions from key Ca(2+) -handling proteins (i.e. surface membrane channels, ion pumps and transporters and internal Ca(2+) release channels) are discussed., (© 2014 Wiley Publishing Asia Pty Ltd.)

Rationale: Intra-sarcoplasmic reticulum (SR) free [Ca] ([Ca](SR)) provides the driving force for SR Ca release and is a key regulator of SR Ca release channel gating during normal SR Ca release or arrhythmogenic spontaneous Ca release events. However, little is known about [Ca](SR) spatiotemporal dynamics., Objective: To directly measure local [Ca](SR) with subsarcomeric spatiotemporal resolution during both normal global SR Ca release and spontaneous Ca sparks and to evaluate the quantitative implications of spatial [Ca](SR) gradients., Methods and Results: Intact and permeabilized rabbit ventricular myocytes were subjected to direct simultaneous measurement of cytosolic [Ca] and [Ca](SR) and FRAP (fluorescence recovery after photobleach). We found no detectable [Ca](SR) gradients between SR release sites (junctional SR) and Ca uptake sites (free SR) during normal global Ca release, clear spatiotemporal [Ca](SR) gradients during isolated Ca blinks, faster intra-SR diffusion in the longitudinal versus transverse direction, 3- to 4-fold slower diffusion of fluorophores in the SR than in cytosol, and that intra-SR Ca diffusion varies locally, dependent on local SR connectivity. A computational model clarified why spatiotemporal gradients are more detectable in isolated local releases versus global releases and provides a quantitative framework for understanding intra-SR Ca diffusion., Conclusions: Intra-SR Ca diffusion is rapid, limiting spatial [Ca](SR) gradients during excitation-contraction coupling. Spatiotemporal [Ca](SR) gradients are apparent during Ca sparks, and these observations constrain models of dynamic Ca movement inside the SR. This has important implications for myocyte SR Ca handling, synchrony, and potentially arrhythmogenic spontaneous contraction.

Inositol-1,4,5-trisphosphate (IP(3))-dependent Ca(2+) release represents the major Ca(2+) mobilizing pathway responsible for diverse functions in non-excitable cells. In the heart, however, its role is largely unknown or controversial. In intact cat atrial myocytes, endothelin (ET-1) increased basal [Ca(2+)](i) levels, enhanced action potential-evoked [Ca(2+)](i) transients, caused [Ca(2+)](i) transients with alternating amplitudes (Ca(2+) alternans), and facilitated spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR) in the form of Ca(2+) sparks and arrhythmogenic Ca(2+) waves. These effects were prevented by the IP(3) receptor (IP(3)R) blocker aminoethoxydiphenyl borate (2-APB), suggesting the involvement of IP(3)-dependent SR Ca(2+) release. In saponin-permeabilized myocytes IP(3) and the more potent IP(3)R agonist adenophostin increased basal [Ca(2+)](i) and the frequency of spontaneous Ca(2+) sparks. In the presence of tetracaine to eliminate Ca(2+) release from ryanodine receptor (RyR) SR Ca(2+) release channels, IP(3) and adenophostin triggered unique elementary, non-propagating IP(3)R-dependent Ca(2+) release events with amplitudes and kinetics that were distinctly different from classical RyR-dependent Ca(2+) sparks. The effects of IP(3) and adenophostin were prevented by heparin and 2-APB. The data suggest that IP(3)-dependent Ca(2+) release increases [Ca(2+)](i) in the vicinity of RyRs and thus facilitates Ca(2+)-induced Ca(2+) release during excitation-contraction coupling. It is concluded that in the adult mammalian atrium IP(3)-dependent Ca(2+) release enhances atrial Ca(2+) signalling and exerts a positive inotropic effect. In addition, by facilitating Ca(2+) release, IP(3) may also be an important component in the development of Ca(2+)-mediated atrial arrhythmias.

The glycolytic product pyruvate has beneficial effects on cardiac contractile function. The postulated cellular mechanisms underlying the positive inotropic effect of pyruvate, however, are contradictory or have remained elusive. Therefore, we studied the effects of pyruvate on cardiac Ca2+ regulation, intracellular pH (pHi) and flavoprotein oxidation using fluorescence confocal microscopy in intact and permeabilized rat ventricular myocytes and single channel recordings from rat cardiac ryanodine receptors (RyRs) incorporated into planar lipid bilayers. In intact cells extracellular pyruvate (10 mM) elevated diastolic [Ca2+]i, which was due, at least in part, to a concomitant acidification of the cytosol. Furthermore, pyruvate increased the amplitude and slowed the kinetics of the electrically evoked [Ca2+]i transient, and augmented sarcoplasmic reticulum (SR) Ca2+ content. Recording of flavoprotein (FAD) fluorescence indicated that pyruvate caused a reduction of mitochondrial redox potential, which is proportional to an increase of the rate of ATP synthesis. Inhibitors of mitochondrial monocarboxylate transport (alpha-cyano-4-hydroxycinnamate, 0.5 mM), adenine nucleotide translocation (atractyloside, 0.3 mM) and the electron transport chain (cyanide, 4 mM) abolished or attenuated the pyruvate-mediated increase of the amplitude of the [Ca2+]i transient, but did not change the effect of pyruvate on diastolic [Ca2+]i. Results from experiments with permeabilized myocytes indicated a direct correlation between ATP/ADP ratio and SR Ca2+ content. Furthermore, pyruvate (4 mM) reduced the frequency of spontaneous Ca2+ sparks by approximately 50%. Single RyR channel recordings revealed a approximately 60% reduction of the open probability of the channel by pyruvate (1 mM), but no change in conductance. This effect of pyruvate on RyR channel activity was neither Ca2+ nor ATP dependent. Taken together, these findings suggest that, in cardiac tissue, pyruvate has a dual effect on SR Ca2+ release consisting of a direct inhibition of RyR channel activity and elevation of SR Ca2+ content. The latter effect was most probably mediated by an enhanced SR Ca2+ uptake due to an augmentation of mitochondria-dependent ATP synthesis.

Ca(2+) alternans is a potentially arrhythmogenic beat-to-beat alternation of the amplitude of the action potential-induced [Ca(2+)](i) transient in cardiac myocytes. Despite its pathophysiological significance the cellular mechanisms underlying Ca(2+) alternans are poorly understood. Recent evidence, however, points to the modulation of Ca(2+)-induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) by localized alterations in energy metabolism as an important determinant of Ca(2+) alternans. We therefore studied the subcellular properties of Ca(2+) alternans in field-stimulated cat atrial myocytes employing fast two-dimensional fluorescence confocal microscopy. Ca(2+) alternans was elicited by an increase in stimulation frequency or by metabolic interventions targeting glycolysis. Marked subcellular variations in the time of onset, the magnitude, and the phase of alternans were observed. Longitudinal and transverse gradients of Ca(2+) alternans were found as well as neighbouring subcellular regions alternating out-of-phase. Moreover, focal inhibition of glycolysis resulted in spatially restricted Ca(2+) alternans. When two adjacent regions within a myocyte alternated out-of-phase, steep [Ca(2+)](i) gradients developed at their border giving rise to delayed propagating Ca(2+) waves. The results demonstrate that Ca(2+) alternans is a subcellular phenomenon caused by modulation of SR Ca(2+) release, which is mediated, at least in part, by local inhibition of energy metabolism. The generation of arrhythmogenic Ca(2+) waves by subcellular variations in the phase of Ca(2+) alternans represents a novel mechanism for the development of atrial disrhythmias.

In atrial myocytes, an initial exposure to isoproterenol (ISO) acts via cAMP to mediate a subsequent acetylcholine (ACh)-induced activation of ATP-sensitive K(+) current (I(K,ATP)). In addition, beta-adrenergic receptor (beta-AR) stimulation activates nitric oxide (NO) release. The present study determined whether the conditioning effect of beta-AR stimulation acts via beta(1)- and/or beta(2)-ARs and whether it is mediated via NO signaling. 0.1 microM ISO plus ICI 118,551 (ISO-beta(1)-AR stimulation) or ISO plus atenolol (ISO-beta(2)-AR stimulation) both increased L-type Ca(2+) current (I(Ca,L)) markedly, but only ISO-beta(2)-AR stimulation mediated ACh-induced activation of I(K,ATP). 1 microM zinterol (beta(2)-AR agonist) also increased I(Ca,L) and mediated ACh-activated I(K,ATP). Inhibition of NO synthase (10 microM L-NIO), guanylate cyclase (10 microM ODQ), or cAMP-PKA (50 microM Rp-cAMPs) attenuated zinterol-induced stimulation of I(Ca,L) and abolished ACh-activated I(K,ATP). Spermine-NO (100 microM; an NO donor) mimicked beta(2)-AR stimulation, and its effects were abolished by Rp-cAMPs. Intracellular dialysis of 20 microM protein kinase inhibitory peptide (PKI) abolished zinterol-induced stimulation of I(Ca,L). Measurements of intracellular NO ([NO](i)) using the fluorescent indicator DAF-2 showed that ISO-beta(2)-AR stimulation or zinterol increased [NO](i). L-NIO (10 microM) blocked ISO- and zinterol-induced increases in [NO](i). ISO-beta(1)-AR stimulation failed to increase [NO](i). Inhibition of G(i)-protein by pertussis toxin significantly inhibited zinterol-mediated increases in [NO](i). Wortmannin (0.2 microM) or LY294002 (10 microM), inhibitors of phosphatidylinositol 3'-kinase (PI-3K), abolished the effects of zinterol to both mediate ACh-activated I(K,ATP) and stimulate [NO](i). We conclude that both beta(1)- and beta(2)-ARs stimulate cAMP. beta(2)-ARs act via two signaling pathways to stimulate cAMP, one of which is mediated via G(i)-protein and PI-3K coupled to NO-cGMP signaling. Only beta(2)-ARs acting exclusively via NO signaling mediate ACh-induced activation of I(K,ATP). NO signaling also contributes to beta(2)-AR stimulation of I(Ca,L). The differential effects of beta(1)- and beta(2)-ARs can be explained by the coupling of these two beta-ARs to different effector signaling pathways.

Electromechanical alternans was characterized in single cat atrial and ventricular myocytes by simultaneous measurements of action potentials, membrane current, cell shortening and changes in intracellular Ca2+ concentration ([Ca2+]i). Using laser scanning confocal fluorescence microscopy, alternans of electrically evoked [Ca2+]i transients revealed marked differences between atrial and ventricular myocytes. In ventricular myocytes, electrically evoked [Ca2+]i transients during alternans were spatially homogeneous. In atrial cells Ca2+ release started at subsarcolemmal peripheral regions and subsequently spread toward the centre of the myocyte. In contrast to ventricular myocytes, in atrial cells propagation of Ca2+ release from the sarcoplasmic reticulum (SR) during the small-amplitude [Ca2+]i transient was incomplete, leading to failures of excitation-contraction (EC) coupling in central regions of the cell. The mechanism underlying alternans was explored by evaluating the trigger signal for SR Ca2+ release (voltage-gated L-type Ca2+ current, ICa,L) and SR Ca2+ load during alternans. Voltage-clamp experiments revealed that peak ICa,L was not affected during alternans when measured simultaneously with changes of cell shortening. The SR Ca2+ content, evaluated by application of caffeine pulses, was identical following the small-amplitude and the large-amplitude [Ca2+]i transient. These results suggest that the primary mechanism responsible for cardiac alternans does not reside in the trigger signal for Ca2+ release and SR Ca2+ load. beta-Adrenergic stimulation with isoproterenol (isoprenaline) reversed electromechanical alternans, suggesting that under conditions of positive cardiac inotropy and enhanced efficiency of EC coupling alternans is less likely to occur. The occurrence of electromechanical alternans could be elicited by impairment of glycolysis. Inhibition of glycolytic flux by application of pyruvate, iodoacetate or beta-hydroxybutyrate induced electromechanical and [Ca2+]i transient alternans in both atrial and ventricular myocytes. The data support the conclusion that in cardiac myocytes alternans is the result of periodic alterations in the gain of EC coupling, i. e. the efficacy of a given trigger signal to release Ca2+ from the SR. It is suggested that the efficiency of EC coupling is locally controlled in the microenvironment of the SR Ca2+ release sites by mechanisms utilizing ATP, produced by glycolytic enzymes closely associated with the release channel.

1. The cellular mechanisms governing cardiac atrial pacemaker activity are not clear. In the present study we used perforated patch voltage clamp and confocal fluorescence microscopy to study the contribution of intracellular Ca2+ release to automaticity of pacemaker cells isolated from cat right atrium. 2. In spontaneously beating pacemaker cells, an increase in subsarcolemmal intracellular Ca2+ concentration occurred concomitantly with the last third of diastolic depolarization due to local release of Ca2+ from the sarcoplasmic reticulum (SR), i.e. Ca2+ sparks. Nickel (Ni2+; 25-50 microM), a blocker of low voltage-activated T-type Ca2+ current ((ICa,T), decreased diastolic depolarization, prolonged pacemaker cycle length and suppressed diastolic Ca2+ release. 3. Voltage clamp analysis indicated that the diastolic Ca2+ release was voltage dependent and triggered at about -60 mV. Ni2+ suppressed low voltage-activated Ca2+ release. Moreover, low voltage-activated Ca2+ release was paralleled by a slow inward current presumably due to stimulation of Na+-Ca2+ exchange (INa-Ca). Low voltage-activated Ca2+ release was found in both sino-atrial node and latent atrial pacemaker cells but not in working atrial myocytes. 4. These findings suggest that low voltage-activated ICa,T triggers subsarcolemmal Ca2+ sparks, which in turn stimulate INa-Ca to depolarize the pacemaker potential to threshold. This novel mechanism indicates a pivotal role for ICa,T and subsarcolemmal intracellular Ca2+ release in normal atrial pacemaker activity and may contribute to the development of ectopic atrial arrhythmias.

Near-membrane [Ca2+] may differ significantly from bulk cytosolic [Ca2+], particularly during rapid Ca2+ signalling events related to cardiac muscle excitation-contraction coupling. We used the lipophilic membrane-associated Ca2+ indicator Ca(2+)-Green C-18 (C-18) and laser-scanning confocal microscopy to detect extracellular [Ca2+] and changes of t-tubular [Ca2+] in cultured neonatal rat myocytes and in freshly isolated adult guinea pig ventricular myocytes. Changes of extracellular [Ca2+] were readily detected by the C-18 located in the cell membrane. Control experiments were carried out with 100 mM extracellular nickel to rapidly quench the fluorescent indicator accessible form the extracellular space. After exposure to Ni2+, C-18 fluorescence was lower than measured in Ca(2+)-free conditions indicating that C-18 was located in the outer leaflet of the cell membrane. In contrast, the lipophilic derivative of Indo-1 (FIP-18) was significantly internalized, as visualized using two-photon excitation of FIP-18. Surprisingly, in low extracellular [Ca2+], C-18 located in the outer leaflet of the cell membrane also reported transient elevations of intracellular [Ca2+] during application of 10 mM caffeine. In the absence of extracellular Na+ to inhibit Ca2+ removal via Na/Ca exchange, the intracellular Ca2+ signals evoked by caffeine were prolonged, as recorded with Fura-Red. However, the near-membrane Ca2+ signal simultaneously detected by C-18 did not increase during caffeine stimulation in the absence of extracellular Na+. These results suggest that the C-18 signal reports extrusion of cytosolic Ca2+ from the subsarcolemmal space mediated by Na/Ca exchange. C-18 was also used to analyze the extracellular accessibility of the t-tubular lumen in isolated guinea pig ventricular myocytes. After stepwise increases of [Ca2+]o with a rapid superfusion device, a wave-like Ca2+ gradient travelled along the t-tubules at a velocity of 3.4-16.3 microns/s. The solution change within the t-tubules was delayed by 0.63-2.3 s and wash-out of Ca2+ from the t-tubules slowed from t1/2 = 0.9 s at the surface to 1.7 s in deeper regions of the t-tubular system. This slow exchange of the solution within the t-tubules, lasting several seconds, may give rise to spatially inhomogeneous accumulation and/or depletion resulting from ion fluxes across the t-tubular membrane during physiological activity.

In heart, spontaneous local increases in cytosolic Ca2+ concentration ([Ca2+]i) called "Ca2+ sparks" may be fundamental events underlying both excitation-contraction coupling and resting Ca2+ leak from the sarcoplasmic reticulum (SR). In this study, resting Ca2+ sparks were analyzed in rabbit and rat ventricular myocytes with laser scanning confocal microscopy and the fluorescent Ca2+ indicator fluo 3. During the first 20 s of rest after regular electrical stimulation, both the frequency of Ca2+ sparks and SR Ca2+ content gradually decreased in rabbit. When rabbit SR Ca2+ content was decreased by reduction of stimulation rate. the initial resting spark frequency was also decreased, even though resting [Ca2+]i was unchanged. The rest-dependent decrease in spark frequency in rabbit cells was prevented by inhibition of Na+/Ca2+ exchange (which also prevents SR Ca2+ depletion during rest). These results suggest that elevation of SR Ca2+ content can increase Ca2+ spark frequency. In contrast to rabbit cells, 20 s of rest produced a gradual increase in spark frequency in rat cells, although SR Ca2+ content was constant and Ca2+ influx was completely prevented. This indicates that there is a time-dependent increase in spark probability during rest that is independent of [Ca2+]i or SR Ca2+. This effect was also apparent in rabbit cells when SR Ca2+ depletion was prevented by blocking Na+/Ca2+ exchange. Stimulation of Ca2+ extrusion via Na+/Ca2+ exchange in the rat (by Ca2+-free superfusion, which slowly depletes SR Ca2+ content) converted the normal rest-dependent increase in spark frequency to a decrease. The amplitude of individual Ca2+ sparks increased with increasing SR Ca2+ content. In the Ca2+-overloaded state, fusion of sparks or long-lasting localized increases of [Ca2+]i were observed with increased spark frequency. We conclude that the resting frequency of Ca2+ sparks can be independently affected by changes in SR Ca2+ content, [Ca2+]i, or rest period. The latter may reflect recovery of the SR Ca2+ release channels from inactivation or adaptation.

FK-506 binding protein (FKBP) has been reported to be closely associated with the ryanodine receptor in skeletal and cardiac muscle and to modulate sarcoplasmic reticulum (SR) Ca2+ release channel gating in isolated channels. FK-506 can inhibit the activity of FKBP, thereby reversing its effects on SR Ca2+ release. We investigated the function of FKBP during normal contractions and Ca2+ transients in intact rat ventricular myocytes loaded with fluorescent Ca2+ indicators. FK-506 significantly increased steady state twitch Ca2+ transients and contraction amplitudes even under conditions in which the SR Ca2+ load and Ca2+ current were unaltered, suggesting that FK-506 increases the fraction of SR Ca2+ released during excitation-contraction (E-C) coupling. Action potentials were somewhat prolonged, consistent with the larger Ca2+ transients causing greater inward Na(+)-Ca2+ exchange current. FK-506 did not affect SR Ca2+ uptake but modestly decreased Ca2+ extrusion via Na(+)-Ca2+ exchange in intact cells (although no effect on Na(+)-Ca2+ exchange was seen in sarcolemmal vesicles). In most cells, FK-506 caused an increase in SR Ca2+ content during steady state stimulation, as assessed by caffeine-induced contractures. This was probably due to the inhibition of Ca2+ efflux via Na(+)-Ca2+ exchange. FK-506 also accelerated the rest decay of SR Ca2+ content and increased the frequency of resting Ca2+ sparks about fourfold. The increase in frequency of these basic Ca2+ release events was not associated with changes in the amplitude or duration of the Ca2+ sparks. We conclude that FK-506 increases the fraction of SR Ca2+ released during normal twitches and enhances the rate of SR Ca2+ release during rest. FK-506 also inhibits Na(+)-Ca2+ exchange, although this effect may be indirect. These effects are consistent with an important SR-stabilizing effect of FKBP in intact rat ventricular myocytes.

The quantitative analysis of the contribution of ion fluxes through membrane channels to changes of intracellular ion concentrations would benefit from the exact knowledge of the cell volume. It would allow direct correlation of ionic current measurements with simultaneous measurements of ion concentrations in individual cells. Because of various limitations of conventional light microscopy a simple method for accurate cell volume determination is lacking. We have combined the optical sectioning capabilities of fluorescence laser scanning confocal microscopy and the whole-cell patch-clamp technique to study the correlation between cell volume and membrane capacitance. Single cardiac myocytes loaded with the fluorescent dye calcein were optically sectioned to produce a series of confocal images. The volume of cardiac myocytes of three different mammalian species was determined by three-dimensional volume rendering of the confocal images. The calculated cell volumes were 30.4 +/- 7.3 pl (mean +/- SD) in rabbits (n = 28), 30.9 +/- 9.0 pl in ferrets (n = 23), and 34.4 +/- 7.0 pl in rats (n = 21), respectively. There was a positive linear correlation between membrane capacitance and cell volume in each animal species. The capacitance-volume ratios were significantly different among species (4.58 +/- 0.45 pF/pl in rabbit, 5.39 +/- 0.57 pF/pl in ferret, and 8.44 +/- 1.35 pF/pl in rat). Furthermore, the capacitance-volume ratio was dependent on the developmental stage (8.88 +/- 1.14 pF/pl in 6-month-old rats versus 6.76 +/- 0.62 pF/pl in 3-month-old rats). The data suggest that the ratio of surface area:volume of cardiac myocytes undergoes significant developmental changes and differs among mammalian species. We further established that the easily measurable parameters of cell membrane capacitance or the product of cell length and width provide reliable but species-dependent estimates for the volume of individual cells.

In this article, we review briefly the available theories and data on [Ca2+]i-waves and [Ca2+]i-oscillations in mammalian cardiac and vascular smooth muscles. In addition to our review, we also report: (i) the existence and characterization of rapid agonist-induced [Ca2+]i-waves in cultured vascular smooth muscle cells (A7r5 cells); and (ii a new method for studying rapid [Ca2+]i-waves in mammalian cardiac ventricular cells. In mammalian cardiac muscle several types of Ca(2+)-release from sarcoplasmic reticulum (SR) are known to occur and might be involved in Ca(2+)-waves and Ca(2+)-oscillations: (a) Ca(2+)-induced release of Ca2+, of the type thought to be important in normal excitation-contraction coupling; (b) spontaneous, cyclic release of Ca2+ related to a Ca(2+)-overload of the SR; and (c) Ins(1,4,5)P3-induced Ca(2+)-release. The available data support the idea that [Ca2+]i-waves in heart propagate by a mechanism somewhat different than that involved in normal excitation-contraction coupling (a, above), perhaps involving spontaneous release of Ca2+ from an overloaded SR (b, above). In mammalian vascular smooth muscle, our data support the idea that agonist-receptor interaction (vasopressin, in this case) initiates [Ca2+]i-waves that then propagate via some form of Ca(2+)-induced release of Ca2+, perhaps in a manner similar to that proposed by Berridge and Irvine [1].

Aspects of pH regulation in ferret ventricular cells have been investigated by using pH- and sodium-selective microelectrodes in bicarbonate-free Tyrode's solution. An acid load was produced by the transient application of NH4Cl (10 or 20 mmol/l). A complete recovery from an acid load was still observed after multiple applications of NH4Cl, but amiloride (0.75 or 1 mmol/l), a blocker of the Na-H exchanger, increased the acidification and inhibited the recovery. Measurements of intracellular sodium concentration showed a transient decrease during the application of NH4Cl and a transient increase above control values during recovery from acidification. This increase was inhibited by amiloride. Intracellular sodium loading (strophanthidin [low calcium-low potassium Tyrode's solution]) did not initially cause an intracellular pH (pHi) change, but the acidification induced by amiloride under those circumstances was larger. Reducing extracellular sodium concentration from 155 to 5 or to 1.5 mmol/l caused an acidification. Changing extracellular pH (pHo) from 6.4 to 8.4 caused an average linear change in pHi in the same direction of 0.085 pHi units/pHo units. The mean intracellular buffering capacity measured with the NH4Cl method and with the proton extrusion mechanism blocked by amiloride was 36 +/- 15 mmol pH-1.l-1 (mean +/- SD), approximately half that of previous estimations. Changing the metabolic substrate from glucose to pyruvate in the superfusing solution caused an acidification of 0.21 pH units. This could be partially blocked by alpha-cyano-4-hydroxycinnamate, a finding consistent with a pyruvate-H+ cotransport and/or a pyruvate-OH- countertransport system being present in ventricular cells. The results of the present study show that ventricular cells can effectively buffer hydrogen ions and that an Na-H exchange system plays a major role in the regulation of pHi.

We studied intracellular binding and possible compartmentalization of the fluorescent Ca2+ indicators, indo-1 and fura-2, in single mammalian cardiac ventricular cells that had been loaded with indo-1 and fura-2 by exposure to the acetoxymethylester form of the indicators (indo-1/AM and fura-2/AM). Techniques similar to those used in experiments on fluorescence recovery after photobleaching (FRAP) were used. It was assumed that reversible binding in myoplasm would be evident as slowed recovery of fluorescence after photobleaching, and that irreversible binding of the indicators to immobile myoplasmic sites (or "compartmentalization" in organelles) would be evident as incomplete recovery. Through the use of a mask, one half of a cell was exposed to high-intensity ultraviolet (UV) light to bleach the indo-1 or fura-2 in only that part of the cell. Upon removal of the mask and termination of the high-intensity UV illumination, fluorescence recovered in the bleached half of the cell, indicating diffusion of indo-1 and fura-2. Mathematical modeling of the diffusional redistribution of the indicators indicated that in these cells the apparent diffusion coefficient for indo-1 is 1.57 x 10(-7) cm2 s-1 (SD 0.48 x 10(-7) cm2 s-1; n = 5 cells, 21 degrees C), and for fura-2 is 3.19 x 10(-7) cm2 s-1 (SD 1.85 x 10(-7) cm2 s-1; n = 6 cells, 21 degrees C). These values are approximately 6 and 3, respectively, times smaller than those expected for free diffusion in the myoplasm. In the bleached half of the cell the recovered level of fluorescence never reached the final level in the half not exposed to UV light. The extent of incomplete recovery was variable amongst the cells. Our analysis indicated that, under the conditions we used, approximately one-third of the intracellular dye is not diffusible in the myoplasm.

Ventricular cells possess two Ca extrusion mechanisms, a Na/Ca exchange system and a Ca pump. Reversing the exchanger by extracellular Na removal causes [Na]i to decrease, and the cells take up mmolar quantities of calcium. Since [Ca]i shows only a marginal increase the calcium load must be buffered. The capacity of the SR is limited so the mitochondria probably buffer a large part of this load. However, when Ca uptake into the mitochondria is blocked, the gain in Ca is still mmolar and the increase in [Ca]i still marginal, suggesting an additional buffering site. Measurements of the Na/Ca stoichiometry on sarcolemmal vesicles gave a value of 3, but in ventricle values of around 2.5 or 3 are found. Reasons for this are discussed, as are the differences amongst the different methods of Ca measurement. The interaction of the sarcolemmal Ca pump and the exchanger are considered and it is suggested they could interact via [Na]i. At rest both systems could remove Ca from the cell but on a large perturbation the Na/Ca exchange would be the more important of the two.

The free Mg concentration ([Mg]i) was measured in ferret ventricular muscle using single-barrelled Mg-sensitive micro-electrodes. The electrodes were calibrated in solutions containing the [K]i and [Na]i measured in this tissue. In thirteen measurements from seven experiments conforming to strict criteria for calibration and penetration the estimated mean [Mg]i was 0.4 mmol/l. This is to be regarded as an upper limit since it was estimated by linear extrapolation of the calibration curve.

Determination of intracellular free magnesium [( Mg]i) with ion-selective Mg microelectrodes filled with the neutral carrier ETH 1117 is critically dependent on the values taken for K and Na in the calibrating solutions, for intracellularly the Mg microelectrodes also respond to these ions. This study was designed to test the influence of K and Na on the measured [Mg]i in ferret ventricular muscle. To estimate the effect of these ions, two methods were used: calculation based on the Nicolsky-Eisenman equation and calibration of the electrodes in solutions containing various concentrations of K and Na. Selectivity coefficients were measured for K and Na by the fixed interference method. While the Nicolsky-Eisenman equation could describe the behavior of an electrode in a given calibrating solution, the effects of changes in either the concentration of K and/or Na could not be predicted by the equation. Comparison between calibrating solutions containing the mean values for K and Na and calibrating solutions containing the lowest measured values for K and Na showed that the mean [Mg]i in ferret ventricle would increase from 0.4 to 0.9 mmol/l. A critical appraisal of the literature values for [Mg]i in muscle tissue suggests that values over 2 mmol/l were either unphysiological or too high for methodological reasons.