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


METHODS
We isolated fresh rat preglomerular VSMCs with a magnetized microsphere/sieving technique; cytosolic Ca2+ ([Ca2+]i) was measured with fura-2 ratiometric fluorescence.


RESULTS
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.


CONCLUSIONS
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.
Fellner
S K
SK
Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7545, USA. sfellner@med.unc.edu
Arendshorst
W J
WJ
D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D007678:Kidney Glomerulus / Q000098:blood supply* / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000201:enzymology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D051381:Rats; D011921:Rats, Inbred WKY; D012079:Renal Circulation / Q000502:physiology; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D007211:Indoles; D010726:Phosphodiesterase Inhibitors; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D002110:Caffeine; D004533:Egtazic Acid; D000252:Calcium-Transporting ATPases; D002118:Calcium; C000543:cyclopiazonic acid