At concentrations between 1 to 10 raM, caffeine reduced the Ca-accumulating capacity of fragmented reticulum obtained from frog and rabbit muscle. With 8 mM caffeine enough Ca was released from frog reticulum to account for the force of the contracture. Caffeine did not affect all reticulum membranes equally. The fraction which was spun down at 2000 g was more sensitive than the lighter fractions. The percentage of the total accumulated Ca released by caffeine decreased with decreasing Ca content of the reticulum. In parallel with their known effects on the caffeine contracture, a drop in temperature increased the caffeine-induced Ca release while procaine inhibited it. Caffeine also inhibited the rate of Ca uptake, which may in part account for the prolongation of the active state caused by caffeine.
The vesicles of fragmented sarcoplasmic reticulum, i.e. the physiological relaxing factor, remove most of the exchangeable Ca bound to actomyosin and myofibrils. The extent to which they remove Ca and the extent to which they inhibit myofibrillar activity are closely correlated. Previous work has shown that those in vitro reactions of actomyosin with ATP which are equivalent to its contraction, i.e. superprecipitation and a high ATPase activity, require the formation of a Ca-actomyosin complex, and that actomyosin after the removal of most of its bound Ca is inhibited by physiological concentrations of ATP. The evidence now suggests that the factor achieves its relaxing effect through the dissociation of the Ca-actomyosin complex and that it has no other direct influence on the biological activity of actomyosin. Experiments, showing that under similar conditions the vesicles of the factor are capable of reducing the concentration of ionized Ca in the surrounding medium to about 0.02 #M and less, suggest that the factor competes successfully with actomyosin for the available Ca through its mechanism of Ca accumulation.
Bovine adrenal chromaffin cells were isolated by removal of the cortex and sequential collagenase digestion of the medulla. The catecholamine secretory function of these cells was characterized with respect to acetylcholine stimulation, cation requirements, and cytoskeletal elements. The dose-response curve for stimulated release had its half-maximum value at 10-5 M acetylcholine, and maximum secretion was on the average 7 times that of control basal secretion. The differential release of epinephrine versus norepinephrine after stimulation with 0.1 mM acetylcholine occurred in proportion to their distribution in the cell suspension. The cholinergic receptors were found to be predominantly nicotinic. The kinetics of catecholamine release were rapid, with significant secretion occurring in less than 60 sec and 85% of maximum secretion within 5 min. A critical requirement for calcium in the extracellular medium was demonstrated, and 80% of maximum secretion was achieved at physiologic calcium concentrations. Stimulation by excess potassium (65 mM KCI) also induced catecholamine secretion which differed from acetylcholine stimulation in being less potent, in having a different dependence on calcium concentration, and in its response to the local anesthetic tetracaine. Tetracaine, which is thought to inhibit membrane cation permeability, was able to block acetylcholine-stimulated but not KCI-stimulated secretion. The microtubule disrupting agent vinblastine was able to block catecholamine release whereas the microfilament disrupter cytochalasin B had little effect. The results show the isolated bovine chromaffin cells to be viable, functioning, and available in large quantity. These cells now provide an excellent system for studying cell surface regulation of hormone and neurotransmitter release.The term "stimulus-secretion coupling" was originally coined by Douglas and Rubin (1, 2) more than a decade ago to describe the sequence of events initiated by acetylcholine (ACh) stimulation of adrenal chromaffin cells and leading to secretion of catecholamines by exocytosis. They had in mind the close similarity to the phenomenon of "excitation-contraction coupling" in muscle (namely, the key role of calcium in mediating both secretion and contraction and a parallel set of electrical and ionic events at the plasma membrane in response to ACh). Much progress has been made during the past 2 decades in elucidating the various steps in the mechanism of secretion, with a substantial portion of the data deriving from studies on the perfused adrenal gland (2-4). There remain, however, many important aspects of cell surface receptor regulation of secretion that are still poorly understood, such as (i) the molecular nature of the calcium permeability sites that are altered by activated cholinergic receptors and potassium depolarization, (ii) the mechanism by which calcium entry into the cell induces membrane fusion and exocytosis, (iii) the possible role of cyclic nucleotides coupled with ion translocation in mediating horm...
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