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Formation and function of ceramide-enriched membrane platforms with CD38 during M₁-receptor stimulation in bovine coronary arterial myocytes

Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia

Submitted 11 June 2008 ; accepted in final form 20 August 2008

CD38 contains an ADP ribosylcyclase domain that mediates intracellular Ca²⁺ 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 (M₁) receptor stimulation in bovine coronary arterial myocytes (CAMs). By confocal microscopic analysis, oxotremorine (Oxo), an M₁ receptor agonist, was found to increase LR clustering on the membrane with the formation of a complex of CD38 and LR components such as GM₁, acid sphingomyelinase (ASMase), and ceramide, a typical ceramide-enriched macrodomain. At 80 µM, Oxo increased LR clustering by 78.8%, which was abolished by LR disruptors, methyl-β-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 M₁ 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 Ca²⁺ 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 M₁ receptor activation to produce second messenger cADPR.

lipid microdomains; sphingolipid; lipid mediator; smooth muscle; calcium signaling

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