Store-operated Ca2+ entry is exaggerated in fresh preglomerular vascular smooth muscle cells of SHR.
BACKGROUND
Regulation of preglomerular vasomotor tone vessels ultimately control glomerular filtration rate, sodium reabsorption and systemic blood pressure. To gain insight into the complex renal hemodynamic factors that may result in hypertension, we studied calcium signaling pathways.


METHODS
Fresh, single, preglomerular vascular smooth muscle cells (VSMC) were isolated from 5- to 6-week-old SHR and WKY utilizing a magnetized microsphere/sieving technique. Cytosolic Ca2+ ([Ca2+]i) was measured with fura-2 ratiometric fluorescence. To examine store-operated calcium entry (SOC), VSMC were activated in calcium-free buffer containing nifedipine. To deplete the sarcoplasmic reticulum (SR) of Ca2+, vasopressin-1 receptor agonist [V1R; inositol trisphosphate (IP3)-mediated mobilization], ryanodine (non-IP3 induced mobilization), and cyclopiazonic acid (CPA; Ca2+-ATPase inhibition) were utilized. Addition of external calcium followed by quenching of the fura/Ca2+ signal with Mn2+ permitted assessment of divalent cation entry via SOC.


RESULTS
V1R caused greater mobilization in SHR than WKY (P < 0.01) as well as greater calcium entry (P < 0.001). Ryanodine and CPA both caused SR calcium depletion that was not statistically different between strains, but absolute calcium entry through SOC was more than double in SHR following either maneuver (P < 0.001). 2-Amino-ethoxybiphenyl borane (2-APB), an inhibitor not only of IP3 receptors, but also of SOC, blocked calcium entry in the ryanodine and CPA experiments independent of IP3. As well, Gd3+, a selective inhibitor of SOC, inhibited the Ca2+ response. We also studied L-channel calcium entry stimulated by V1R. The total calcium response was greater in SHR as was the absolute inhibition by nifedipine. As a percent of the total response, participation of L-type channels sensitive to nifedipine was about 45% in both strains of rat.


CONCLUSIONS
Utilizing three separate mechanisms to deplete the SR of Ca2+ in order to activate SOC, we show for the first time, that SOC is exaggerated in preglomerular VSMC of young SHR.
Fellner
Susan K
SK
Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, 27599-7545, USA. sfellner@med.unc.edu
Arendshorst
William J
WJ
D000818:Animals; D000831:Animals, Newborn / Q000378:metabolism; D001127:Arginine Vasopressin / Q000494:pharmacology; D001160:Arterioles; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D007211:Indoles / Q000494:pharmacology; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007678:Kidney Glomerulus / Q000098:blood supply*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D051381:Rats; D011918:Rats, Inbred SHR / Q000378:metabolism*; D011921:Rats, Inbred WKY; D018160:Receptors, Cytoplasmic and Nuclear / Q000187:drug effects / Q000378:metabolism; D017483:Receptors, Vasopressin / Q000819:agonists; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D014667:Vasopressins / Q000378:metabolism
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D007211:Indoles; D053496:Inositol 1,4,5-Trisphosphate Receptors; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D017483:Receptors, Vasopressin; D019837:Ryanodine Receptor Calcium Release Channel; D014667:Vasopressins; D001127:Arginine Vasopressin; D012433:Ryanodine; D000252:Calcium-Transporting ATPases; D002118:Calcium; C000543:cyclopiazonic acid