Angiotensin II-stimulated Ca2+ entry mechanisms in afferent arterioles: role of transient receptor potential canonical channels and reverse Na+/Ca2+ exchange

Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina Submitted 25 May 2007 ; accepted in final form 26 October 2007 In afferent arterioles, the signaling events that lead to an increase in cytosolic Ca 2+ concentration ([Ca 2+ ] i ) and initiation of vascular contraction are increasingly being delineated. We have recently studied angiotensin II (ANG II)-mediated effects on sarcoplasmic reticulum (SR) mobilization of Ca 2+ and the role of superoxide and cyclic adenosine diphosphoribose in these processes. In the current study we investigated the participation of transient receptor potential canonical channels (TRPC) and a Na + /Ca 2+ exchanger (NCX) in Ca 2+ entry mechanisms. Afferent arterioles, isolated with the magnetized polystyrene bead method, were loaded with fura-2 to measure [Ca 2+ ] i ratiometrically. We observed that the Ca 2+ -dependent chloride channel blocker niflumic acid (10 and 50 µ M) affects neither the peak nor plateau [Ca 2+ ] i response to ANG II. Arterioles were pretreated with ryanodine (100 µM) and TMB-8 to block SR mobilization via the ryanodine receptor and inositol trisphosphate receptor, respectively. The peak [Ca 2+ ] i response to ANG II was reduced by 40%. Addition of 2-aminoethoxydiphenyl borane to block TRPC-mediated Ca 2+ entry inhibited the peak [Ca 2+ ] i ANG II response by 80% and the plateau by 74%. Flufenamic acid (FFA; 50 µM), which stimulates TRPC6, caused a sustained increase of [Ca 2+ ] i of 146 nM. This response was unaffected by diltiazem or nifedipine. KB-R7943 (at the low concentration of 10 µM) inhibits reverse (but not forward) mode NCX. KB-R7943 decreased the peak [Ca 2+ ] i response to ANG II by 48% and to FFA by 38%. We conclude that TRPC6 and reverse-mode NCX may be important Ca 2+ entry pathways in afferent arterioles. renal microcirculation; voltage-gated calcium entry; vascular smooth muscle cell Address for reprint requests and other correspondence: S. K. Fellner, Dept. of Cell and Molecular Physiology, Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (e-mail: sfellner{at}med.unc.edu )