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The electrophysiological effects of the benzothiazepine 7-chloro-3,5-dihydro-5-phenyl-1H-4,1-benzothiazepine-2-one (CGP-37157) (CGP) were investigated on the canine (NCX1.1) and Drosophila (CALX1.1) plasmalemmal Na+-Ca2+ exchangers. These exchangers were selected for study because they show opposite responses to cytoplasmic regulatory Ca2+, thereby allowing us to examine the role of this regulatory mechanism in the inhibitory effects of CGP. CGP blocked Na+-Ca2+ exchange current mediated by both transporters with moderate potency (IC50 values = approximately 3-17 microM) compared with other recently reported blockers of Na+-Ca2+ exchange [e.g., 2-[4-[2,5-difluorophenyl) methoxy]phenoxy]phenoxy]-5-ethoxyaniline (KB-R7943) and 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (SEA0400)]. Experiments using alpha-chymotrypsin to remove autoregulation of Na+-Ca2+ exchange showed that block by CGP was reduced, suggesting that part of the effects of this drug may require intact ionic regulatory mechanisms. For NCX1.1, the inhibition produced by CGP was greater for outward Na+-Ca2+ exchange currents compared with inward currents. When CALX1.1 was examined, the extent of inhibition was similar for both inward and outward exchange currents. Although the extent and potency of CGP-mediated inhibition of Na+-Ca2+ exchange are less than those observed with SEA0400 and KB-R7943, our data demonstrate that CGP constitutes a novel class of plasmalemmal Na+-Ca2+ exchange inhibitors. Moreover, the widespread use of CGP as a selective mitochondrial Na+-Ca2+ exchange inhibitor should be reconsidered in light of these additional inhibitory effects.

The aim of this work was to determine the relationship between peak twitch amplitude and sarcoplasmic reticulum (SR) Ca2+ content during changes of stimulation frequency in isolated canine ventricle, and to estimate the extent to which these changes were dependent upon sarcolemmal Na(+)-Ca2+ exchange. In physiological [Na+]o, increased stimulation frequency in the 0.2-2-Hz range resulted in a positive inotropic effect characterized by an increase in peak twitch amplitude and a decrease in the duration of contraction, measured as changes in isometric force development or unloaded cell shortening in intact muscle and isolated single cells, respectively. Action potentials recorded from single cells indicated that the inotropic effect was associated with a progressive decrease of action potential duration and a marked reduction in average time spent by the cell near the resting potential during the stimulus train. The frequency-dependent increase of peak twitch force was correlated with an increase of Ca2+ uptake into and release from the SR. This was estimated indirectly using the phasic contractile response to rapid (less than 1 s) lowering of perfusate temperature from 37 degrees C to 0-2 degrees C and changes of twitch amplitude resulting from perturbations in the pattern of electrical stimulation. Lowering [Na+]o from 140 to 70 mM resulted in an increase of contractile strength, which was accompanied by a similar increase of apparent SR Ca2+ content, both of which could be abolished by exposure to ryanodine (1 x 10(-8) M), caffeine (3 x 10(-3) M), or nifedipine (2 x 10(-6) M). Increased stimulation frequency in 70 mM [Na+]o resulted in a negative contractile staircase, characterized by a graded decrease of peak isometric force development or unloaded cell shortening. SR Ca2+ content estimated under identical conditions remained unaltered. Rate constants derived from mechanical restitution studies implied that the depressant effect of increased stimulation frequency in 70 mM [Na+]o was not a consequence of a decreased rate of refilling of a releasable pool of Ca2+ within the cell. These results demonstrate that frequency-dependent changes of contractile strength and intracellular Ca2+ loading in 140 mM [Na+]o require the presence of a functional sarcolemmal Na(+)-Ca2+ exchange process. The possibility that the negative staircase in 70 mM [Na+]o is related to inhibition of Ca(2+)-induced release of Ca2+ from the SR by various cellular mechanisms is discussed.

Rest potentiation, believed to be due to increased utilization of sarcoplasmic reticular calcium, was converted to rest depression by BAY K 8644 (1 microM). Plateau height and duration of the postrest beat were enhanced by BAY K 8644, suggesting an enhancement of extracellular calcium entry. Caffeine (3 mM) also produced depression at all rest intervals, although to a lesser extent than BAY K 8644. Compared with BAY K 8644, treatment with caffeine resulted in an elevation of plateau amplitude and a shortening of action potential duration. Action potential configuration changes induced by rest were unaltered by caffeine despite reduction in rest potentiation. Caffeine-induced rest depression was associated with an increase in the time to peak tension. This was not observed with BAY K 8644. Treatment with both caffeine (3 mM) and BAY K 8644 (1 microM) greatly prolonged time to peak tension. Action potential duration and plateau height were either maintained or increased. Less rest depression was observed with the combination than with either agent alone. These results suggest that 1) BAY K 8644 and caffeine inhibit rest potentiation by different mechanisms, and 2) caffeine-induced inhibition of calcium uptake by the sarcoplasmic reticulum may enhance the effect of BAY K 8644-induced increase in calcium influx on the contractile apparatus.

1 Post-rest inotropy in canine ventricular myocardium has proved a useful indicator of sarcoplasmic reticular calcium release. This phenomenon is converted to rest depression by the calcium channel activator (agonist), Bay K 8644 as well as other chemically diverse agents such as caffeine and ryanodine. 2 Rapid cooling contractures and post-rest contraction amplitude were used as independent measures of sarcoplasmic reticular calcium content and release. Simultaneous recordings of transmembrane action potentials and their accompanying contractions were obtained to determine the association between electrophysiological and mechanical events. The present study was designed to elucidate the mechanism by which Bay K 8644, caffeine and ryanodine alter force production after variable periods of rest. 3 Bay K 8644 (1 μm) increased steady state contraction in response to a constant train of stimulation, caused rest-depression after 2 and 8 min rest, prolonged action potential duration and increased action potential plateau amplitude. Augmented steady state tension was not accompanied by any change in time to peak tension or rapid cooling contracture amplitude. However, the post-rest rapid cooling contracture was greatly diminished compared to that observed prior to Bay K 8644 treatment. 4 Caffeine (3 and 5 mm) caused rest-depression with an increase in steady state contraction amplitude. Along with this there was a slight decrease in action potential duration and plateau amplitude and an increase in time to peak tension. The rapid cooling contractures were virtually abolished at all conditioning intervals. The effect of caffeine on twitch tension and cooling contracture is consistent with the ability of this compound to inhibit calcium sequestration by the sarcoplasmic reticulum. 5 A combination of Bay K 8644 and caffeine caused significantly less rest-depression than that seen with Bay K 8644 alone. The augmented twitch tension was accompanied by a long time to peak tension and action potential duration. However, there was no increase in the amplitude of the rapid cooling contracture, either after a regular train of stimulation or after rest, compared to that seen after Bay K 8644. 6 Ryanodine (10 nm), produced rest-depression, reduced steady state twitch tension and augmented the rest-depression produced by Bay K 8644. The steady state rapid cooling contracture remained unchanged when both agents were present simultaneously, while the post-rest rapid cooling contracture was significantly depressed compared to that observed with Bay K 8644 alone. 7 Bay K 8644 and ryanodine appear to have similar actions with respect to promoting diastolic loss of calcium from the sarcoplasmic reticulum. Although caffeine also decreases post-rest potentiation, it antagonizes rest-depression caused by Bay K 8644. The data from these experiments suggest that this reversal is a result of depressed intracellular calcium buffering and enhanced myofilament sensitivity produced by caffeine in the presence of increased transmembrane calcium influx promoted by Bay K 8644.

The mechanism of the negative force staircase in thin rat ventricular trabeculae was investigated and compared with the positive force staircase in dog ventricular muscles of comparable diameter. Increasing stimulus frequency from 0.2 to 0.5 and 1 Hz resulted in a stepwise reduction of twitch amplitude that was demonstrated in both 1.25 and 2.5 mM external calcium concentration ([Ca]o). The negative staircase was associated with no change in the amplitude of postrest contraction or rapid-cooling contracture at either [Ca]o investigated. Hence, neither decreased loading nor overloading of the rat sarcoplasmic reticulum with calcium during the steady state can be invoked as a likely explanation for the negative staircase. The results are consistent with a frequency-dependent increase in the refractoriness of the sarcoplasmic reticular calcium release process or a decrease in the amount of trigger for calcium release, assuming that the amount of calcium present in the release pool is constant from one frequency to the next. In contrast to the rat, canine ventricular muscle exhibited a positive force staircase, the slope of which depended on [Ca]o, as well as a frequency-dependent increase in postrest contraction and rapid-cooling contracture. Data obtained from this series of experiments suggests that increased filling of the release pool within the sarcoplasmic reticulum with calcium underlies the inotropic effect of high-frequency stimulation in canine ventricular muscle.