CD38 contains an ADP ribosylcyclase domain that mediates intracellular Ca(2+) signaling by the production of cyclic ADP-ribose (cADPR), but the mechanisms by which the agonists activate this enzyme remain unclear. The present study tested a hypothesis that a special lipid-raft (LR) form, ceramide-enriched lipid platform, contributes to CD38 activation to produce cADPR in response to muscarinic type 1 (M(1)) receptor stimulation in bovine coronary arterial myocytes (CAMs). By confocal microscopic analysis, oxotremorine (Oxo), an M(1) receptor agonist, was found to increase LR clustering on the membrane with the formation of a complex of CD38 and LR components such as GM(1), acid sphingomyelinase (ASMase), and ceramide, a typical ceramide-enriched macrodomain. At 80 microM, Oxo increased LR clustering by 78.8%, which was abolished by LR disruptors, methyl-beta-cyclodextrin (MCD), or filipin. With the use of a fluorescence resonance energy transfer (FRET) technique, 15.5+/-1.9% energy transfer rate (vs. 5.3+/-0.9% of control) between CD38 and LR component, ganglioside M(1) was detected, further confirming the proximity of both molecules. In the presence of MCD or filipin, there were no FRET signals detected. In floated detergent-resistant membrane fractions, CD38 significantly increased in LR fractions of CAMs treated by Oxo. Moreover, MCD or filipin attenuated Oxo-induced production of cADPR via CD38. Functionally, Oxo-induced intracellular Ca(2+) release and coronary artery constriction via cADPR were also blocked by LR disruption or ASMase inhibition. These results provide the first evidence that the formation of ceramide-enriched lipid macrodomains is crucial for Oxo-induced activation of CD38 to produce cADPR in CAMs, and these lipid macrodomains mediate transmembrane signaling of M(1) receptor activation to produce second messenger cADPR.