Calcium signaling mechanisms were examined in vessel segments and dispersed single smooth muscle cells (SMC) of interlobular arteries and afferent arterioles (<50 μm diameter) from the rat kidney. These resistance vessels were isolated from rat kidneys, using an iron oxide-sieving technique with subsequent collagenase digestion. Individual cells were identified by their characteristic oval appearance and positive staining for smooth muscle-specific α-actin and heavy chain myosin SM-1 and SM-2. Cytosolic calcium concentration ([Ca2+]i) was measured using fura 2 ratiometric fluorescence at 340 and 380 nm wavelength with a microscope-based photometer. Angiotensin II (ANG II) and arginine vasopressin (AVP), at concentrations of 10−10–10−6M, produced dose-dependent increases in [Ca2+]i; maximum increases were 221 ± 49 nM for ANG II and 237 ± 49 nM for AVP. The temporal response patterns for both agonists were characterized by a square-shaped, immediate step increase in [Ca2+]ito a near maximum level that was maintained through the recording period of 150–200 s. Responses of individual dispersed SMC and short vessel segments were similar. Losartan antagonized the action of ANG II, indicating mediation by AT1 receptors on preglomerular arteriolar SMC. The V1-selective antagonist [d(CH2)5Tyr(Me)2Tyr(NH2)9]AVP completely inhibited AVP-induced [Ca2+]ichanges. The importance of calcium entry in hormone-induced changes in [Ca2+]iwas demonstrated by the finding that neither ANG II nor AVP elicited a [Ca2+]iresponse in media rendered nominally calcium free by addition of ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid. Calcium entry occurred primarily through L-type, voltage-gated calcium channels as the dihydropyridine, nifedipine, completely prevented or reversed [Ca2+]ichanges normally elicited by either hormone. Our results provide new information about the similarity of calcium signaling in single SMC and short segments freshly isolated from renal interlobular arteries and afferent arterioles. The observations indicate that AT1 and V1 receptors are coupled to signal transduction pathways leading to rapid changes in [Ca2+]i. Calcium mobilization appears to play a minor to nonexistent role under the experimental conditions. The predominant mechanism involves calcium entry through dihydropyridine-sensitive, voltage-gated calcium channels in single SMC from these resistance vessels.
Antagonism of T-type calcium channels inhibits high-fat diet–induced weight gain in mice
