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

Background A multiplicity of hormonal, neural, and paracrine factors regula tes preglomerular arterial tone by stimulating calcium entry or mobilizatio n. We have previously provided evidence for capacitative (store-operated) C a2+ entry in fresh renal vascular smooth muscle cells (VSMCs). Ryanodine-se nsitive receptors (RyRs) have recently been identified in a variety of nonr enal vascular beds. Methods. We isolated fresh rat preglomerular VSMCs with a magnetized micros phere/sieving technique; cytosolic Ca2+ ([Ca2+](i)) was measured with fura- 2 ratiometric fluorescence. Results. Ryanodine (3 mu mol/L) increased [Ca2+](i) from 79 to 138 nmol/L ( P = 0.01). Nifedipine (Nif), given before or after ryanodine, was without e ffect. The addition of calcium (1 mmol/L) to VSMCs in calcium-free buffer d id 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) in creased from 80 to 94 mmol/L with the ad dition 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 sarcoplasmi c reticulum (SR) with ryanodine stimulated capacitative Ca2+ entry. Conclusion. Preglomerular VSMCs have functional RyR, and a capacitative (st ore-operated) entry mechanism is activated by the depletion of SR Ca2+ with ryanodine, as is the case with inhibitors of SR Ca2+-ATPase.