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

In afferent arterioles, the signaling events that lead to an increase in cytosolic Ca2+ concentration ([Ca2+](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 Ca2+ 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+/Ca2+ exchanger (NCX) in Ca2+ entry mechanisms. Afferent arterioles, isolated with the magnetized polystyrene bead method, were loaded with fura-2 to measure [Ca2+](i) ratiometrically. We observed that the Ca2+-dependent chloride channel blocker niflumic acid (10 and 50 mu M) affects neither the peak nor plateau [Ca2+](i) response to ANG II. Arterioles were pretreated with ryanodine (100 mu M) and TMB-8 to block SR mobilization via the ryanodine receptor and inositol trisphosphate receptor, respectively. The peak [Ca2+](i) response to ANG II was reduced by 40%.Addition of 2-aminoethoxydiphenyl borane to block TRPC-mediated Ca2+ entry inhibited the peak [Ca2+](i) ANG II response by 80% and the plateau by 74%. Flufenamic acid (FFA; 50 mu M), which stimulates TRPC6, caused a sustained increase of [Ca2+](i) of 146 nM. This response was unaffected by diltiazem or nifedipine. KB-R7943 (at the low concentration of 10 mu M) inhibits reverse (but not forward) mode NCX. KB- R7943 decreased the peak [Ca2+](i) response to ANG II by 48% and to FFA by 38%. We conclude that TRPC6 and reverse-mode NCX may be important Ca2+ entry pathways in afferent arterioles.