Spontaneous transient elevations of intracellular calcium (Ca2+i) have functional roles in the development of Xenopus spinal neurons. However, less is known about the roles of elevations of Ca2+i in the differentiation of other cell types. We have examined Xenopus myocytes as a first step in determining if Ca2+i transients are a more general feature of differentiation in excitable cells. We find that cultured myocytes, like neurons, exhibit spontaneous Ca2+i transients during an early developmental period. These transients average 1.4 min in duration and occur at an average frequency of 6/hr in cultures containing myocytes and neurons. Culture conditions can influence transient production; for example, myocyte-enriched cultures have a lower incidence of transient-producing cells. Transients persist in 0-Ca2+ medium, indicating that they arise from intracellular stores. Caffeine-sensitive Ca2+ stores are present in these cells, and depletion or block of these stores eliminates transient production. To determine if transients play a functional role during development, we blocked their production with intracellular BAPTA, a rapid Ca2+ chelator. Cellular differentiation is significantly inhibited only when BAPTA is applied early in development, during the period of transient production, while later BAPTA treatments have no effect. Blocking transient production severely perturbed myofibril organization and sarcomere assembly. However, other aspects of myocyte differentiation were not affected by transient blockade, indicating that not all myogenic differentiation programs are regulated in this manner. Our results suggest that spontaneous Cai2+ transients play a role in cytoskeletal organization during myofibrillogenesis.

Electric fish of the genus Sternopygus produce a sinusoidal electric organ discharge (EOD) of low frequencies in males, high frequencies in females, and overlapping and intermediate frequencies in juveniles. Correspondingly, the cells of the electric organ, the electrocytes, generate action potentials which are of long duration in mature males, short duration in females, and intermediate duration in immatures. The androgen dihydrotestosterone (DHT) lowers EOD frequency and increases electrocyte action potential duration. We examined the electrocytes under voltage clamp to determine whether variations in the kinetic properties of the Na+ current might underlie these phenomena. We found that the fast inactivation time constants of the peak Na+ current (0 mV) ranged from 0.5 to 4.7 msec and varied systematically with EOD frequency and action potential duration. Voltage dependence of steady- state inactivation also varied with EOD frequency with the midpoint of inactivation being more positive in fish with low EOD frequencies. There was no correlation between the voltage at which the Na+ current activates, voltage at peak current, reversal potential, rate of recovery from inactivation, or TTX sensitivity and EOD frequency. We tested whether DHT influenced Na+ current inactivation by recording from electrocytes before and after juvenile fish of both sexes were implanted with a DHT-containing or empty capsule. We found that inactivation time constants were significantly slower in DHT implanted, but not control, fish. This is the first observation of functionally relevant individual variation in the kinetics of a Na+ current and the first demonstration that the kinetics of a Na+ current may be modulated by an androgen.

Spontaneous calcium release from intracellular stores occurs during myofibrillogenesis, the process of sarcomeric protein assembly in striated muscle. Preventing these Ca2+ transients disrupts sarcomere formation, but the signal transduction cascade has not been identified. Here we report that specific blockade of Ca2+ release from the ryanodine receptor (RyR) activated Ca2+ store blocks transients and disrupts myosin thick filament (A band) assembly. Inhibition of an embryonic Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) by blocking the ATP-binding site, by allosteric phosphorylation, or by intracellular delivery of a pseudosubstrate peptide, also disrupts sarcomeric organization. The results indicate that both RyRs and MLCK, which have well-described calcium signaling roles in mature muscle contraction, have essential developmental roles during construction of the contractile apparatus.

Embryonic Xenopus myocytes generate spontaneous calcium (Ca(2+)) transients during differentiation in culture. Suppression of these transients disrupts myofibril organization and the formation of sarcomeres through an identified signal transduction cascade. Since transients often occur during myocyte polarization and migration in culture, we hypothesized they might play additional roles in vivo during tissue formation. We have tested this hypothesis by examining Ca(2+) dynamics in the intact Xenopus paraxial mesoderm as it differentiates into the mature myotome. We find that Ca(2+) transients occur in cells of the developing myotome with characteristics remarkably similar to those in cultured myocytes. Transients produced within the myotome are correlated with somitogenesis as well as myocyte maturation. Since transients arise from intracellular stores in cultured myocytes, we examined the functional distribution of both IP(3) and ryanodine receptors in the intact myotome by eliciting Ca(2+) elevations in response to photorelease of caged IP(3) and superfusion of caffeine, respectively. As in culture, transients in vivo depend on Ca(2+) release from ryanodine receptor (RyR) stores, and blocking RyR during development interferes with somite maturation.

In Sternopygus macrurus, electrocyte action potential duration determines the electric organ discharge pulse duration. Since the electric organ discharge is a sexually-dimorphic behavior under the control of steroid hormones, and because electrocyte action potential durations can range from 3-14 ms, the electrocytes provide a unique opportunity to study how sex steroids regulate membrane excitability. In this study, the voltage-sensitive ionic currents of electrocytes were identified under current- and voltage-clamp as a prelude to further studies on their regulation by sex steroid hormones. Bath application of TTX completely abolished the spike and eliminated an inward current under voltage clamp, indicating that the action potential is due primarily to a sodium current. Calcium-free saline had no effect on spike waveform or voltage-clamp currents, indicating that neither calcium nor calcium-dependent currents contribute to the action potential. Application of potassium channel blocking agents, such as tetraethylammonium and cesium ions, caused changes in the spike which, together with voltage-clamp results, indicate the presence of two potassium currents: an inward rectifier and a classical delayed rectifier. In addition, these cells have a large, presumably voltage-insensitive, chloride current. Differences in one or more of these currents could be responsible for the range of action potential durations found in these cells and for the steroid-mediated changes in spike duration.