Membrane potentials of medullary chromaffin cells of the adrenal gland of the mouse were measured in situ. Resting potential (-54.3 +/- 8.8 mV) depended on extracellular [K+] as predicted by the constant-field equation with a permeability ratio, PNa/PK, of 0.09. Current-voltage (I-V) relationships showed that the current is rectified across the chromaffin cell membrane. A rectification ratio of 0.4 was calculated from the slopes of the I-V curves for positive (41 +/- 26 M omega) and negative (103 +/- M omega) currents. Because input resistance for a resting chromaffin cell in isolation is approximately 5 G omega, the chromaffin cells in situ behave as if they were electrically coupled. Most cells responded to depolarizing current pulses with repetitive action potentials, but only 50% of them showed spontaneous electrical activity. Spontaneous activity was often seen in the presence of tetrodotoxin (3 microM). Although the application of the K+-channel blockers tetraethylammonium and Ba2+ greatly increased the amplitude of the action potentials, only Ba2+ induced continuous electrical activity. Application of acetylcholine (ACh) always depolarized the cell membrane. This effect was blocked by atropine but not by D-tubocurarine, suggesting that ACh stimulation of chromaffin cells in the mouse involves activation of muscarinic receptors.

A B S T R A C T Fatigue and recovery from fatigue were related to metabolism in single fibers of the frog semitendinosus muscle. The fibers were held at a sarcomere length of 2.3 /.tin in oxygenated Ringer solution at 15~ and were stimulated for up to 150 s by a schedule of 10-s, 20-Hz tetanic trains that were interrupted by 1-s rest periods, after which they were rapidly frozen for biochemical analysis. Two kinds of fatigue were produced in relation to stimulus duration. A rapidly reversed fatigue occurred with stimulation for under 40 s and was evidenced by a decline in tetanic tension that could be overcome by 1 s of rest. A prolonged fatigue was caused by stimulation for 100-150 s. It was evidenced during stimulation by a fall in tetanic tension that could not be overcome by 1 s of rest, and after stimulation by a reduction, lasting for up to 82 rain, in the peak tension of a 200-ms test tetanus. Fiber phosphocreatine (PCr) fell logarithmically in relation to stimulus duration, from a mean of 121 + 8 nmol/mg protein (SEM, n = 12) to 10% of this value after 150 s of stimulation. PCr returned to normal levels after 90-120 min of rest. Stimulation for 150 s did not significantly affect fiber glycogen and reduced fiber ATP by at most 15%. It is suggested that the prolonged fatigue caused by 100-150 s of tetanic stimulation was caused by long-lasting failure of excitation-contraction coupling, as it was not accompanied by depletion of energy stores in the form of ATP. One possibility is that H + accumulated in fatigued fibers so as to interfere with the action of Ca 2+ in the coupling process.

At 15 degrees C, direct stimulation of frog single muscle fibers at a frequency of 20 Hz produced a tetanic tension that remained constant for 20 s and then declined. The decline was reversed during 1-s interruptions of the stimulus train in the first 50 s of stimulation, but not with longer stimulation. Posttetanic potentiation (PTP), characterized by prolonged twitch relaxation and contraction times and elevation of twitch height, remained for 10-40 min after a 10-s tetanus and for at least 90 min after a 50- to 150-s tetanus. Posttetanic fatigue appeared only after at least 50s of tetanic stimulation. Fatigue was manifested invariably by a reduction in the height of a 200-ms tetanic contraction and usually by a reduction in twitch height after PTP. Fatigued fibers recovered normal contractile responses in 40-160 min. Hypertonic solutions, which blocked contraction in response to tetanic stimulation, prevented posttetanic fatigue but not PTP. The observations suggest that fatigue is caused by a failure in excitation-contraction coupling, probably in relation to consumption of metabolic substrates. Even 10-s tetani which do not produce fatigue can affect muscle contractile function for up to 40 min.

Tension and metabolite concentrations were measured in single frog muscle fibers at 15 degrees C in vitro, in response to electrical stimulation or to immersion in caffeine- or potassium chloride-Ringer. Sarcomere length equaled 2.3 micrometers. Interrupted stimulation for 150 s at 20 Hz or stimulation for 7.5 min at 1 Hz was followed by at least 20 min of fatigue, evidenced by a reduced 200-ms test contraction. Fatigued fibers contracted maximally in potassium chloride- or caffeine-Ringer. They had high lactate and glucose 6-phosphate concentrations and a reduced phosphocreatine (PCr) concentration. Adenosine 5'-triphosphate (ATP) concentration was approximately normal but was markedly reduced by a caffeine contracture. A plot of PCr consumption against the tension-time integral at different stimulation frequencies (25, 35, or 50 Hz) and durations had an intercept of 25.5 nmol PCr/mg protein at time zero and a corrected slope of 0.65 nmol approximately P/mg protein per kg . s . cm-2. Prolonged fatigue is not due to energy exhaustion or to the inability of muscle fibers to consume residual ATP but probably arises from long-lasting interference in excitation-contraction coupling, which can be reversed by KCl- or caffeine-induced release of Ca2+ from intracellular stores.

Intracellular applications of a fixed amount (0.2 to 8 nmol) of inositol 1,4,5-trisphosphate (InsP3) over a brief period (2 s) into barnacle muscle fibers induced vigorous contractures. Peak tension attained during the first application depended on [InsP3]: the maximum tension evoked by the injection of 8 nmol was 1.6 kg/cm2. Peak tension during a second application of a high dose of InsP3 (greater than 10 microM) was always smaller than that during the first application. Extracellular Ca2+ could be omitted with no measurable effects on either the amplitude or time course of the contractures evoked by InsP3. Aequorin was used to measure InsP3-evoked Ca2+ release from intracellular stores in minced muscle fibers from lobster and in skinned muscle fibers from barnacle. Provided the sarcoplasmic reticulum was preloaded with Ca2+, application of InsP3 induced a transient Ca2+ release that was [InsP3] dependent. During each transient, [Ca2+] rose rapidly to a peak value (t1/2 less than 5 s) and then slowly returned (t1/2 less than 100 s) to a basal level. Maximum Ca2+ release was obtained at [InsP3] less than 100 microM and amounted to 4 nmol Ca2+/g of muscle, enough to increase [Ca2+]i from 0.1 to 8 microM had the Ca2+ release occurred in the intact fiber. Successive applications of a fixed amount of InsP3 elicited successive transient increases in Ca2+. The effects of [Ca2+] on the incorporation of [3H]inositol into the pools of phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate pools were measured.(ABSTRACT TRUNCATED AT 250 WORDS)