Ryanodine receptor and capacitative Ca2+ entry in fresh preglomerular vascular smooth muscle cells.

Abstract

A multiplicity of hormonal, neural, and paracrine factors regulates preglomerular arterial tone by stimulating calcium entry or mobilization. We have previously provided evidence for capacitative (store-operated) Ca2+ entry in fresh renal vascular smooth muscle cells (VSMCs). Ryanodine-sensitive receptors (RyRs) have recently been identified in a variety of nonrenal vascular beds. We isolated fresh rat preglomerular VSMCs with a magnetized microsphere/sieving technique; cytosolic Ca2+ ([Ca2+]i) was measured with fura-2 ratiometric fluorescence. Ryanodine (3 micromol/L) increased [Ca2+]i from 79 to 138 nmol/L (P = 0.01). Nifedipine (Nif), given before or after ryanodine, was without effect. The addition of calcium (1 mmol/L) to VSMCs in calcium-free buffer did not alter resting [Ca2+]i. In Ca-free buffer containing Nif, [Ca2+]i rose from 61 to 88 nmol/L after the addition of the Ca2+-ATPase inhibitor cyclopiazonic acid and to 159 nmol/L after the addition of Ca2+ (1 mmol/L). Mn2+ quenched the Ca/fura signal, confirming divalent cation entry. In Ca-free buffer with Nif, [Ca2+]i increased from 80 to 94 nmol/L with the addition of ryanodine and further to 166 nmol/L after the addition of Ca2+ (1 mmol/L). Mn2+ quenching was again shown. Thus, emptying of the sarcoplasmic reticulum (SR) with ryanodine stimulated capacitative Ca2+ entry. Preglomerular VSMCs have functional RyR, and a capacitative (store-operated) entry mechanism is activated by the depletion of SR Ca2+ with ryanodine, as is the case with inhibitors of SR Ca2+-ATPase.