Modification of smooth muscle Ca2+-sparks by tetracaine: Evidence for sequential RyR activation

Spontaneous Ca2+-sparks were imaged using confocal line scans of fluo-4 loaded myocytes in retinal arterioles. Tetracaine produced concentration-dependent decreases in spark frequency, and modified the spatiotemporal characteristics of residual sparks. Tetracaine (10 μM) reduced the rate of rise but prolonged the average rise time so that average spark amplitude was unaltered. The mean half-time of spark decay was also unaffected, suggesting that spark termination, although delayed, remained well synchronized. Sparks spread transversely across the myocytes in these vessels, and the speed of spread within individual sparks was slowed by approximately 60% in 10 μM tetracaine, as expected if the spark was propagated across the cell but the average Po for RyRs was reduced. Staining of isolated vessels with BODIPY-ryanodine and di-4-ANEPPS showed that RyRs were located both peripherally, adjacent to the plasma membrane, and in transverse extensions of the SR from one side of the cell to the other. Immuno-labelling of retinal flat mounts demonstrated the presence RyR2 in arteriole smooth muscle but not RyR1. We conclude that Ca2+-sparks in smooth muscle can result from sequential activation of RyRs distributed over an area of several μm2, rather than from tightly clustered channels as in striated muscle.