Formation and function of ceramide-enriched membrane platforms with CD38 during M1-receptor stimulation in bovine coronary arterial myocytes.
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.
Jia
Su-Jie
SJ
Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
Jin
Si
S
Zhang
Fan
F
Yi
Fan
F
Dewey
William L
WL
Li
Pin-Lan
PL
D051997:ADP-ribosyl Cyclase 1 / Q000378:metabolism*; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects; D002417:Cattle; D002518:Ceramides / Q000378:metabolism*; D003331:Coronary Vessels / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D036563:Cyclic ADP-Ribose / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005372:Filipin / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D021962:Membrane Microdomains / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D018613:Microscopy, Confocal; D018721:Muscarinic Agonists / Q000494:pharmacology*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D010095:Oxotremorine / Q000494:pharmacology*; D043643:Receptor, Muscarinic M1 / Q000819:agonists* / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D014661:Vasoconstriction / Q000187:drug effects; D047392:beta-Cyclodextrins / Q000494:pharmacology
D002518:Ceramides; D018721:Muscarinic Agonists; D043643:Receptor, Muscarinic M1; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D036563:Cyclic ADP-Ribose; D010095:Oxotremorine; D005372:Filipin; D013108:Sphingomyelin Phosphodiesterase; D051997:ADP-ribosyl Cyclase 1