Calcium and chloride channel activation by angiotensin II-AT1receptors in preglomerular vascular smooth muscle cells

Abstract

The pathways responsible for the rapid and sustained increases in [Ca2+]ifollowing activation of ANG II receptors (AT1) in renal vascular smooth muscle cells were evaluated using fluorescence microscopy. Resting intracellular calcium concentration [Ca2+]iaveraged 75 ± 9 nM. The response to ANG II (100 nM) was characterized by a rapid initial increase of [Ca2+]iby 74 ± 6 nM ( n = 35) followed by a decrease to a sustained level of 12 ± 2 nM above baseline. The average time from peak to 50% reduction from the peak value (50% time point) was 32 ± 4 s. AT1receptor blockade with 1 μM candesartan ( n = 5) prevented the responses to ANG II. In nominally calcium-free conditions ( n = 8), the peak increase in [Ca2+]iaveraged 42 ± 7 nM but the sustained phase was absent and the 50% time point was reduced to 11 ± 4 s. L-type calcium channel blockade with diltiazem reduced the peak [Ca2+]ito 24 ± 8 nM and the sustained level to 4 ± 2 nM ( n = 10). In cells preincubated in low Cl−(3.0 mM), the peak response to ANG II was suppressed as was the sustained response. Blockade of chloride channels with DIDS eliminated both the peak and sustained responses ( n = 11); chloride channel blockade with DPC ( n = 17) suppressed the peak increase in [Ca2+]ito 18 ± 5 and also prevented the sustained response. IP3 receptor blockade by 10 μM TMB-8 ( n = 6) reduced the peak to 22 ± 8 and prevented the sustained response. Exposure to 10 μM TMB-8 in the presence of Ca2+-free medium prevented the ANG II response ( n = 9). In the presence of 100 μM DPC and 10 μM TMB-8 ( n = 7), the ANG II response was also prevented. Thus the rapid initial increase in [Ca2+]iis due not only to release from intracellular stores, but also to Ca2+influx from the extracellular fluid. Although Ca2+entry via L-type calcium channels is responsible for the major portion of the sustained response, other entry pathways participate. The finding that chloride channel blockers markedly attenuate both rapid and sustained responses indicates that chloride channel activation contributes to, rather than being the consequence of, the initial rapid response.