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Mechanism of extracellular ATP‐induced increase of cytosolic Ca2+ concentration in isolated rat ventricular myocytes.
Author(s) -
Christie A,
Sharma V K,
Sheu S S
Publication year - 1992
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.1992.sp018929
Subject(s) - extracellular , cytosol , myocyte , mechanism (biology) , chemistry , microbiology and biotechnology , biophysics , medicine , biochemistry , biology , enzyme , philosophy , epistemology
1. Changes in the cytosolic Ca2+ concentration ([Ca2+]i) of isolated rat ventricular myocytes in suspension were measured in response to extracellular ATP using the fluorescent Ca2+ indicators Quin‐2 and Fura‐2. 2. ATP produced a concentration‐, time‐ and Mg(2+)‐dependent, biphasic increase of [Ca2+]i whereas slowly hydrolysable ATP analogues produced a slow, monophasic increase of [Ca2+]i and the non‐hydrolysable ATP analogues were without effect. 3. Extracellular Ca2+ was required for the ATP‐induced increase of [Ca2+]i and pre‐treatment of the cells with caffeine, ryanodine, verapamil or nimodipine partially inhibited the [Ca2+]i increase. 4. Whole‐cell patch‐clamp experiments revealed that ATP activated an ionic current that had a linear current‐voltage relationship with a reversal potential near O mV. Quinidine, a putative P2 purinergic receptor blocker, abolished the ATP‐activated current. The ATP‐activated current was Mg2+ dependent. 5. Associated with the ATP‐activated current was cellular depolarization. In a physiological solution, ATP depolarized cells to the threshold for the firing of action potentials. In the presence of the voltage‐activated ion channel blockers tetrodotoxin, 4‐aminopyridine, caesium and nitrendipine, ATP depolarized cells to ‐44 +/‐ 6 mV from a resting potential of ‐66 +/‐ 4 mV (n = 11). 6. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and autoradiography demonstrated that extracellular ATP stimulated the phosphorylation of several extracellular membrane‐bound proteins. The phosphorylation of these proteins was concentration, time and Mg2+ dependent. Pre‐treatment of cells with the slowly hydrolysable ATP analogues inhibited the ATP‐induced phosphorylation. Adenosine 5'‐O‐3‐thiotriphosphate (ATP gamma S) thiophosphorylated proteins with the same apparent molecular weight as the proteins phosphorylated by ATP. 7. These results suggest that the ATP‐induced increase of [Ca2+]i is a result of the activation, possibly by protein phosphorylation, of a novel ion channel carrying inward current. The ATP‐activated channel may be permeable to Na+ and Ca2+ and causes [Ca2+]i to rise. More importantly, this inward current depolarizes the cell to the threshold of inducing spontaneous firing of action potentials. The firing of action potentials results in the influx of Ca2+ through L‐type Ca2+ channels which would trigger Ca2+ release from the sarcoplasmic reticulum and lead to the increase in [Ca2+]i.