Ca2+ is an essential messenger in numerous key biological processes. However, the cellular mechanisms of the control of intracellular free Ca2+ concentration (Ca2+ activity) are still incompletely understood. The present proposed studies will take the advantage of newly developed experimental techniques (e.g. ion-sensitive microelectrodes and fluorescent indicator quin 2) to explore the following questions: (1) What are the relative contributions of sarcolemmal Na-Ca exchange versus Ca-pump for the Ca2+ extrusion at physiologial resting state? Do Ca-pump, Na-Ca exchange and Na-K pump coordinate effectively to keep intracellular Ca2+ and Na+ low at unphysiological conditions (e.g. Na-K pump is inhibited completely)? (2) Is the Na-Ca exchange electrogenic? (3) What is the relative importance between sarcolemmal efflux systems and intracellular buffering systems for the Ca2+ homeostasis? (4) How does cAMP modulate ca2+ transport and metabolism? We will combine the use of ion-sensitive microelectrodes, voltage clamp and tension recording to measure simultaneously intracellular ion activities, membrane voltage, membrane current and muscle contraction in sheep cardiac Purkinje fibers and guinea pig ventricular papillary muscles. We will also measure cytosolic free Ca2+ concentration in isolated cardiac myocytes from rat and guinea pig ventricles. The Ca2+ transport systems in plasma membrane and intracellular organelles will be distinguished by chemical or pharmacological manipulations. For instance, Na-Ca exchange can be inhibited by removing all Na+ in external solution and sarcoplasmic reticulum can be blocked by caffeine. These proposed studies will allow us to analyze quantitatively the contribution of several Ca2+ controlling systems for the homeostasis of this ion. Furthermore, they will provide the information about the universal role of Ca2+ in many physiological and pathological states of the heart. Therefore, these studies will broaden our understanding on the fundamental principles of normal and abnormal cardiac excitation and contraction.
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