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The sources and sequestration of Ca²⁺ contributing to neuroeffector Ca²⁺ transients in the mouse vas deferens

Keith L. Brain, Alina M. Cuprian, Damian J. Williams and Thomas C. Cunnane

The detection of focal Ca²⁺ transients (called neuroeffector Ca²⁺ transients, or NCTs) in smooth muscle of the mouse isolated vas deferens has been used to detect the packeted release of ATP from nerve terminal varicosities acting at postjunctional P2X receptors. The present study investigates the sources and sequestration of Ca²⁺ in NCTs. Smooth muscle cells in whole mouse deferens were loaded with the Ca²⁺ indicator Oregon Green 488 BAPTA-1 AM and viewed with a confocal microscope. Ryanodine (10 µM) decreased the amplitude of NCTs by 45 ± 6 %. Cyclopiazonic acid slowed the recovery of NCTs (from a time course of 200 ± 10 ms to 800 ± 100 ms). Caffeine (3 mM) induced spontaneous focal smooth muscle Ca²⁺ transients (sparks). Neither of the T-type Ca²⁺ channel blockers NiCl₂ (50 µM) or mibefradil dihydrochloride (10 µM) affected the amplitude of excitatory junction potentials (2 ± 5 % and -3 ± 10 %) or NCTs (-20 ± 36 % and 3 ± 13 %). In about 20 % of cells, NCTs were associated with a local, subcellular twitch that remained in the presence of the ₁-adrenoceptor antagonist prazosin (100 nM), showing that NCTs can initiate local contractions. Slow (5.8 ± 0.4 µm s^-1), spontaneous smooth muscle Ca²⁺ waves were occasionally observed. Thus, Ca²⁺ stores initially amplify and then sequester the Ca²⁺ that enters through P2X receptors and there is no amplification by local voltage-gated Ca²⁺ channels.

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