In SANC constituitive AC generates high basal cAMP, inducing PKA-dependent phosphorylation that regulates Ca2+ cycling, that is essential for normal pacemaker function. Our goals were to identify, in rabbit SANC, the types of AC expressed, and their Ca2+ sensitivity and location. Radioimmunoassay (with total phosphodiasterase inhibition) showed a high Ca2+ activated basal AC activity. AC activity increased 5-fold from Ca2+ free (EGTA) to 1 uM free Ca2+. RT PCR (using specifically designed rabbit primers) showed that AC types II and V, and Ca2+ activated types, I and VIII, are expressed in SANC. The organization of these distinct AC types within calveolar or non-calveolar membrane microdomains was determined in pooled SANC isolated from 5 hearts, using triton x100, and sucrose gradient ultracentrifugation. Lipid domains segregated into caveolin containing and non-caveolin containing membrane microdomains, where AC activity was concentrated (fig
, AC activity). Immunoblots demonstrated localization of different AC types between these two membrane domains, with AC I, II, V/VI localizing to caveolin containing lipid rafts, and AC VIII present in both caveolin and GM1 lipid domains, and also in the soluble fraction (fig
). In summary, multiple ACs, both Ca2+ activated and non-CA2+ activated types, are expressed in SANC, and these reside in distinct calveolar and non-calveolar lipid domains. We conclude that constituitive basal AC activity is, generated, in part, at least, by a Ca2+ activated AC. type.