Study On The Mechanisms Of Calcium's Effects On Spontaneous ACh Secretion From Isolated Myocytes

Exocytosis is the pathway by which cells selectively externalize compounds and complement plasma membrane proteins, as such, it is essential for a number of basic biological processes. Details of its molecular mechanism have been studied already. In specialized cell types, like neurons and endocrine cells, exocytosis of neurotransmitters and hormones depends on an elevation of intracellular free calcium. Calcium, as one of the most important second messengers in a cell, plays a serious part in the process of exocytosis.Calcium ions in cytoplasm have two sources: extracellular and intracellular. Calcium has been known to be related with spontaneous acetylcholine (ACh) release from isolated myocytes, but the respective role of extracellular Ca²⁺ and intracellular Ca²⁺ on ACh release has not yet been identified.In this research, we used cultured Xenopus myocytes as a model to research the detail mechanism of calcium's effect on ACh release by patch clamp technique. Myocytes loaded with exogenous ACh into the cytoplasm in the absence of extracellular Ca²⁺ undergo spontaneous quantal ACh secretion as detected by the appearance of pulsatile miniature endplate currents (MEPCs). Analysis of the frequencies, amplitudes and time courses of these currents suggests similar cellular mechanisms are involved in the secretions of ACh in normal and Ca²⁺-free medium. Various doses of ryanodine were used to regulate the intracellular Ca²⁺ to different levels. The spontaneous ACh secretion from myocytes in Ca²⁺-free medium was decreased by reducing intracellular Ca²⁺ levels and enhanced by increasing cytosolic Ca²⁺ levels. These observations demonstrate that the spontaneous secretion from isolated myocytes and the effect of ryanodine on ACh-loaded cells are both independent of extracellular Ca²⁺ while Ca²⁺ in the sarcoplasmic reticulum plays a crucial role in the secretions.