| dc.description | Excitation-contraction (EC) coupling refers to a process whereby electrical stimulation of the sarcolemmal (SL) membrane of a myocyte is translated into mechanical contraction. A rapid increase in intracellular Ca2+ concentration ([Ca2+];) is required for contraction to occur. Most of this rise in [Ca2+]; occurs through release from the sarcoplasmic reticulum (SR). The following two fundamental mechanisms have been proposed to explain the process by which stimulation of the SL causes release of Ca2+ from the SR; (1) calcium-induced calcium release (CICR), whereby trans sarcolemmal Ca2+ influx stimulates opening of SR ryanodine receptors (RyRs) and release of stored Ca2+, and; (2) a voltage sensitive release mechanism (VSRM), whereby SR release of Ca2+ is stimulated by depolarization of the SL and is independent of transsarcolemmal Ca2+ influx. The objective of the present study was to evaluate the use of tetracaine and ryanodine, two pharmacological agents known to interact with SR RyRs, as tools to investigate the contribution of each mechanism to cardiac contraction. Single electrode voltage clamp and video edge detection were used to measure membrane currents and contractions respectively, in freshly dissociated guinea-pig ventricular myocytes at 37°C. Both high resistance (15--20 MO) and patch electrodes (1--3 MO) were used in the present study. Initial experiments showed that tetracaine preferentially inhibited VSRM contractions at concentrations which also inhibited Na+ current. However, inhibition of the VSRM by tetracaine persisted in the presence of prior Na+ channel blockade by lidocaine or lidocaine plus tetrodotoxin. Rapid application of moderate concentrations of tetracaine (≥300 muM) selectively inhibited VSRM contractions in undialyzed myocytes. However, longer-duration (∼3 rains.) continuous exposure was required for inhibition of VSRM contractions in dialyzed myocytes. Continuous exposure also was associated with an increase in SR Ca2+ stores, however this effect was independent of the effects of tetracaine on the VSRM. The tetracaine-insensitive component of contraction was inhibited by rapid application of Cd2+, disruption of SR release, or block of ICa-L which suggests that this component represents CICR. Thus, effects of tetracaine on cardiac EC-coupling are opposite to that which has been reported in skeletal muscle where this agent selectively inhibits CICR. The results of the present study show that inhibition of the VSRM by tetracaine is not due to a shift in the properties of activation or inactivation of this mechanism nor is it due to the local anesthetic actions of this agent. Higher concentrations (1 mM) became less selective and inhibited both contractions. The effects of ryanodine on contractions also were examined in the present study. The current view about ryanodine is that inhibition of contractions by low concentrations of this agent results from depletion of SR Ca2+ stores. However, the present study demonstrates that inhibition of VSRM contractions by nanomolar concentrations of ryanodine is independent of depletion of SR stores as assessed by rapid application of 10 mM caffeine. The effects of tetracaine on VSRM contractions occur within seconds, therefore rapid application of this agent will be a useful technique for evaluation of the contributions of the VSRM to total contraction. By comparison, ryanodine has a slow mechanism of action and therefore cannot be applied rapidly, however the high selectivity and affinity of this agent for SR RyRs makes it a valuable tool for investigation of the VSRM. In conclusion, the results of this study illustrate that both tetracaine and ryanodine can be used as tools to investigate the relative contributions of the VSRM to cardiac contraction. | en_US |
