AND DONALD. M. BERS. Division of Biomedical Sciences, ...... BERS, D. M., J. H. B. BRIDGE, AND K. T. MACLEOD. The mecha- nism of ryanodine action in ...
Rat vs. rabbit ventricle: Ca flux and intracellular Na assessed by ion-selective microelectrodes MICHAEL J. SHATTOCK AND DONALD M. BERS Division of Biomedical Sciences, University of California, Riverside,
SHATTOCK, MICHAEL J., AND DONALD M. BERS. Rat VS. rabbit ventricle: Ca flux and intracellular Na assessed by ionselective microelectrodes. Am. J. Physiol. 256 (Cell Physiol. 25): C813-C822, 1989.-Trans sarcolemmal Ca movements in rabbit and rat ventricular muscle were compared using extracellular double-barreled Ca-selective microelectrodes. In rabbit ventricle, steady-state twitches were associated with transient extracellular Ca (Ca,) depletions, indicative of Ca uptake during the twitch. In contrast, steady-state twitches in rat ventricle were associated with net cellular Ca extrusion. Rest periods in rabbit ventricle lead to a net loss of cell Ca and resumption of stimulation induces a net uptake of Ca by the cells. Conversely, in rat ventricle rest periods lead to cellular Ca gain and resumption of stimulation induces a net Ca loss from the cells. Thus stimulation is associated with net Ca gain in rabbit ventricle and net Ca loss in rat ventricle. These observations provide an explanation for some of the functional differences between rat and rabbit ventricle (e.g., negative force-frequency staircase and rest potentiation in rat vs. positive staircase and rest decay in rabbit). Resting intracellular Na activity (ah,) was 12.7 t 0.6 mM in rat and 7.2 * 0.5 mM in rabbit ventricle. This ak, in rat ventricle is sufficiently high that Ca entry via Na-Ca exchange is thermodynamically favored at the resting membrane potential. This may explain why rest potentiation is observed in rat ventricle. In contrast, the lower ah, in rabbit ventricle would favor Ca extrusion via Na-Ca exchange at rest (and consequent rest decay). In rat ventricle, the increase of intracellular [ Ca] ( [Ca] i) associated with contraction, coupled with the short action potential duration, strongly favor Ca extrusion via Na-Ca exchange and explain the observed Ca, accumulation observed during twitches in rat. The high plateau of the rabbit ventricular action potential tends to prevent Ca extrusion via Na-Ca exchange during the contraction and explains the Ca, depletions observed in rabbit. It is concluded that the higher ak, and shorter action potential duration in rat vs. rabbit ventricle can explain many of the functional differences observed in these tissues. excitation-contraction frequency relationship;
coupling; extracellular calcium; sodium-calcium exchange
force-
RAT VENTRICLE demonstrates a negative forcefrequency staircase (27, 41), a strong rested-state contraction, rest potentiation, and a pattern of postrest recovery that is different from that of many other ventricular muscle preparations (3,5). The rest potentiation exhibited by rat ventricle is one of many characteristics that distinguish this tissue from other mammalian tissues, such as rabbit ventricle (3). Rabbit ventricle shows rest decay (i.e., gradual decline in the size of the first contraction after rest). This is believed to reflect the ADULT
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gradual emptying of the sarcoplasmic reticulum (SR) Ca stores during rest (1). SR Ca content can be assessed using rapid cooling contractures (5, 12). These appear to be independent of Ca influx, and rapid cooling may induce release of all the SR Ca, as opposed to the graded release which occurs during the twitch (5, 26). Recent experiments using rapid cooling contractures have suggested that the rest decay of contractions is indeed closely associated with a decline in the SR Ca content (5, 12). In rat ventricle, an increase in SR Ca content (as assessed by rapid cooling contractures) appears to be involved in rest potentiation (5), and the rest potentiation can be abolished by SR inhibitors such as ryanodine or caffeine (3,62). Thus, as concluded by Bers (5), it seemsthat rest decay in rabbit ventricle is associated with a loss of SR Ca during the period of rest while conversely in rat ventricle, rest potentiation appears to be associated with a gain in SR Ca. One aim of the present study was to examine the rest and activation-dependent Ca fluxes in rat and rabbit ventricular muscle using extracellular double-barreled Ca microelectrodes. Specifically, we aim to compare directly extracellular Ca depletions and repletions in rat and rabbit ventricular muscle and to determine whether differences in trans sarcolemmal Ca fluxes explain some of the functional differences between these two preparations. In rabbit ventricle, activation-dependent increases in Ca influx and net Ca uptake after a rest period have been reported from experiments using extracellular Ca ([Cal,) microelectrodes (2-4, 9, 47), [Cal,-sensitive dyes (36) and radioactive Ca (53). The cumulative Ca depletions observed on resumption of stimulation were interpreted to be due to net cellular Ca uptake and refilling of SR Ca stores which had become depleted as a function of rest duration (9,47). In all respects this interpretation agrees with conclusions from experiments using rapid cooling contractures (5, 7, 12). Thus, in rabbit ventricle, it seemsclear that rest decay is due to the loss of SR Ca to the cytoplasm and subsequent extrusion of that Ca into the extracellular space. Furthermore, rest decay of rabbit ventricular twitches, rapid cooling contractures, and [Cal, depletions are all inhibited by a reduction of the trans sarcolemmal [Na] gradient (6,9,12,61). These studies suggest that Ca extrusion via Na-Ca exchange is largely responsible for the rest-induced loss of cellular (and SR) Ca.
0 1989 the American
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The Na-Ca exchange mechanism, and hence the intracellular sodium ion activity (ah,), is an important determinant of the cellular Ca load and the inotropic state of the muscle. Indeed, the relationship between ah, and tension is very steep (23, 45, 46). This suggeststhat &, and Na-Ca exchange may be extremely important in the regulation of contractile force. It has been reported that ah, in rat ventricular muscle is unusually high (i.e., 28 30 mM) (63, 66). However, another report (30) suggests that & in rat ventricle (8.5 t 2.6 mM) is not very different fro.m that reported for other cardiac tissues. A second goal of the present study was to compare c& in rat and rabbit ventricle to assessthe potential role of the Na-Ca exchange in explaining the physiological differences between these species. METHODS
Hearts were excised from Sprague-Dawley rats (200250 g body wt) or New Zealand White rabbits after an injection of pentobarbital sodium (100 mg ip in rats and 150 mg iv in rabbits) and heparin (1,000 IU). Right ventricular papillary muscles or trabeculae were then tied with fine suture, excised, and mounted in a superfusion chamber (0.15 ml in vol). One end of the muscle was attached to a static hook fixed in the base of the chamber while the other was attached to a peizo-resistive force transducer (AE801, SensoNor, Norway). All experiments were performed at 30°C. After dissection, muscles were equilibrated for at least 1 h in normal Tyrode solution containing 2 mM CaClz (see below) while being stimulated at 0.5 Hz. Solutions The normal Tyrode solution contained (in mM) 140 NaCl, 6 KCl, 2 CaC12, 1 MgC1,, 10 glucose, 5 N-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) (pH 7.4). During dissection and experimental protocols using extracellular Ca-selective microelectrodes, the CaClz concentration of the Tyrode was reduced to 0.5 mM. All solutions were equilibrated with 100% oxygen. Caffeine (Sigma) was added as a solid to the Tyrode solution. A citrate-buffered Tyrode solution was formulated to provide an initial free [Ca] of 0.5 mM and a free [Mg] of 1 mM (as in the unbuffered solutions). The total added Ca and Mg were calculated using the binding constants from Martell and Smith (48) adjusted for pH, ionic strength, and temperature (31). The citrate-buffered Tyrode solution initially contained (in mM) 65 NaCl, 25 Nas citrate, 10.4 MgC12, 6.5 CaC12,6 KCl, 10 glucose, 10 HEPES (pH 7.4). However, buffering Ca, has been shown to result in an -20% reduction in developed tension of isolated muscles (58). The object of using the citrate-buffered solutions was to maintain a constant degree of contractility while buffering the [Cal,. Therefore, the total Ca concentration was titrated to maintain a relatively constant contractile response (usually requiring -1 mM additional Ca). Thus changing from the normal Tyrode solution to the citrate-buffered Tyrode resulted in little or no change in developed tension (although transient changes were sometimes observed).
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Microelectrodes Ca electrodes. Double-barreled Ca-selective microelectrodes were constructed from 2-mm diameter theta-glass (R and D Optical Systems, Spencerville, MD) as has been previously described in detail (2, 3, 9). Briefly, micropipettes were pulled and broken back to 5- to lopm diameter and one barrel was silanized bY exposure to a stream of N,N-dimethyltrimethylsilylamine (TMSDMA) vapor. To prevent silanization of both barrels, a stream of air was simultaneously passed through the second barrel. The silanized barrel was backfilled with a reference solution containing 10 mM CaClz and 100 mM KC1 and the nonsilanized barrel with a solution containing 140 mM NaCl. A column of the neutral Ca ion-exchange cocktail ETH 1001 (Fluka Chemical, Ronkonkoma, NY) 50-250 pm long was drawn into the silanized barrel. Ca electrodes with these tip diameters exhibit Nernstian behavior over the range 10 PM to 10 mM Ca (8). The resistance of these microelectrodes were typically l-5 GQ for the Ca-sensitive barrel and l-4 MQ for the reference barrel. The electrical response time of the reference and Ca-sensitive barrels were matched using a passive R-C filter (2). Electrodes showing poor matching of the two barrels were discarded. Potentials from both barrels were recorded via a high- impedence electrometer. The signals and their difference were continuously recorded, but only the difference signal (indicative of the [Cal,) is shown in Figs. 2-7. Small voltage offsets were observed in caffeine-containing and citratebuffered Tyrode solutions (7, 19), but measurements were made only after exposure to these solutions had reached steady state. These offsets did not change the Ca sensitivity and were accounted for. Therefore, they do not limit the interpretation of the data. Na electrodes. Single-barreled Na-selective microelectrodes were manufactured using the Na-selective neutral ion-exchange cocktail ETH 227 (Fluka). Micropipettes were pulled from filamented 1.5mm diameter borosilicate glass and were silanized by the method of Tsien and Rink (65) using TMSDMA. Micropipettes were then backfilled with a solution containing (in mM) 8 NaCl, 142 KCl, 1 MgC12, 5 HEPES, 2 ethylene glycol-bis(Paminoethyl ether)-N,N, N’, N’-tetraacetic acid (EGTA) (pH 7.1) that was forced down to the tip of the micropipette by applying positive pressure using a syringe. A small column of the ion exchange cocktail (40-200 pm long) was then pulled into the tip of the micropipette. Na electrodes manufactured in this way typically had resistances of -100 GQ. The signal from the Na electrodes (E& was passed to an electrometer (World Precision Instruments, FD 223) and was then displayed on a chart recorder. Membrane potentials were measured using conventional 3M KCl-filled microelectrodes, and all potential measurements were made with respect to a grounded Ag-AgCl wire placed in the experimental chamber. The membrane potential (E,) was displayed on the chart recorder along with the ENa, and the subtracted (E&&J or difference signal that is indicative of ah, (Ediff)
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Na electrodes were calibrated in solutions containing varying proportions of NaCl and KC1 (NaCl + KC1 =
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150 mM) plus (in mM) 1 MgClz, 5 HEPES, 2 EGTA (pH 7.1). Figure 1 shows the average of 13 calibration curves.
In Fig. 1, the solid line shows the theoretical electrode response as predicted by the Nikolsky equation and the dashed line is the linear regression of the points from 8 to 140 mM Na. The limit of detection of 2.7 mM and the selectivity for K vs. Na (or KNakpot) was 0.0171. In the ah, = 7 mM calibration buffer increasing free [Cal from 5 1 to 300 nM produced no change in electrode potential. Thus Ca at resting levels is not expected to interfere with the measured ah,. Sodium concentrations in the calibrating solutions were converted into activities assuming a Na activity coefficient of 0.76. Satisfactory Na-electrode impalements were confirmed by brief depolarizations in a Tyrode solution containing 30 mM KCl. This procedure depolarizes the muscle by -40 mV, and if the Na electrode and membrane potential electrode do not sensethe same E,, then an offset will rapidly appear on the Ediff trace on switching to 30 mM KC1 (or back to NT). Electrode impalements showing rapid changes in the Ediff signal of more than 2 mV were rejected. RESULTS
Measurements of [Cal0 in Rabbit Ventricle During Contraction and Rest Figure 2 shows the changes in [Cal, that accompany single contractions recorded (with signal averaging) during steady-state stimulation at 0.5 Hz of rabbit ventricle. Under control conditions, a net depletion of [Cal, (to -10 PM below that of the bulk solution) was observed early in the time course of activation. This depletion was considerably attenuated (to