Organization of Ca2+ signalling pathways in A7r5 vascular smooth muscle cells

I Pino de la Huerga, E J A Taylor & C W Taylor, Department of Pharmacology, University of Cambridge, CB2 1PD, UK.

In A7r5 vascular smooth muscle cells, vasopressin (AVP) causes a long-lasting inhibition of adenylyl cyclase (AC) mediated by IP3-evoked Ca2+ release and consequent activation of Ca2+-calmodulin-dependent kinase II (CaMKII) (Dyer et al., 2005). AVP can also reciprocally regulate capacitative and non-capacitative Ca2+ entry pathways via its ability to stimulate nitric oxide synthase-III (NOS-III) (Moneer et al., 2003). However, 5-HT, which shares with AVP an ability to stimulate phospholipase C, appears not to activate NOS-III and stimulates only capacitative Ca2+ entry. These observations suggest rather precise associations between receptors, Ca2+ signalling pathways, and proteins that are regulated by Ca2+. Here we combine subcellular fractionation with immunoprecipitation assays to examine possible associations between these signalling proteins.

Discontinuous sucrose gradients were used to separate light from heavy membrane fractions (Ostrom et al., 2002). Immunoprecipitation of solubilized membranes was performed using appropriate primary antibodies and Protein A/G Plus-agarose beads. Proteins in immunoprecipitates or sucrose gradient fractions were then identified by immunoblotting. Proteins were separated by SDS-PAGE on 4-12% Bis-Tris gradient gels, transferred to PVDF membranes, blocked and incubated with primary and secondary antibodies using standard methods. All experiments were performed at least 3 times.

Immunoblotting established that AC3, AC5/6, CaMKII, type 3 IP3 receptors and NOS-III were expressed in A7r5 cells. Both V1A receptors for AVP and 5-HT2A receptors were expressed predominantly in non-caveolar membranes, whereas NOS-III and type 3 IP3 receptors were more abundant in fractions that included caveolae. Immunoprecipitation established an association between NOS-III and caveolin 3, and between type 3 IP3 receptors and CaMKII. Our results suggest that the selective ability of AVP, but not 5-HT, to activate NOS-III is unlikely to result from colocalization of V1A receptors with NOS-III in caveolae. An association of type 3 IP3 receptors with CaMKII may underlie the selective ability of IP3-evoked Ca2+ signals to inhibit AC activity via CaMKII (Dyer et al., 2005).

Dyer, JL et al. (2005) J. Biol. Chem. 280, 8936-8944.
Moneer, Z et al. (2003) J. Biochem. 370, 439-448.
Ostrom, RS et al. (2002) Mol. Pharm. 62, 983-992.

Supported by the Wellcome Trust.