Cholesterol depletion perturbs calcium handling by rat submandibular glands.
The sensitivity to cholesterol depletion of calcium handling by rat submandibular glands was investigated. The glands were digested with collagenase. After homogenization, the lysate was extracted at 4 degrees C with 0.5% Triton X-100 and the extract was submitted to an ultracentrifugation in a sucrose discontinuous gradient. A population of detergent-resistant membranes (DRM) was collected at the 5%-35% interface. The DRM had a higher content of cholesterol, saturated and long-chain fatty acids. Caveolin-1 and alpha(q/11) were located in these membranes. They were more ordered than vesicles from total cellular lysate as determined by anisotropy measurement. They disappeared after cholesterol extraction with methyl-beta-cyclodextrin (MCD). Exposure of the cellular suspension with MCD nearly abolished the response to carbachol, epinephrine, and substance P and inhibited the activation of phospholipase C (PLC) by these agonists and by sodium fluoride. MCD did not affect the mobilization of intracellular pools of calcium by thapsigargin. It increased the uptake of extracellular calcium or barium and did not inhibit the uptake of calcium after depletion of the intracellular stores of this ion. From these results, it is concluded that Triton X-100 can extract a fraction of membrane resistant to detergents. Treatment of the cells with MCD disrupts these membranes. The coupling between the heterotrimeric GTP-binding protein G(q/11) and poly-phosphoinositide-specific PLC is affected by disruption of these membrane fractions. At the opposite, the store-operated calcium channel (SOCC) is not affected by DRM-disruption.
Garcia-Marcos
M
M
Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias, Universidad del Pais Vasco, Bilbao, Spain.
Tandel
S
S
Pochet
S
S
Genin
J
J
De Lorenzi
M
M
Gomez
F
F
Kumps
A
A
Marino
A
A
Dehaye
J P
JP
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D051242:Caveolin 1; D022461:Caveolins / Q000187:drug effects / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D045604:Extracellular Fluid / Q000187:drug effects / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000187:drug effects / Q000378:metabolism; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D008297:Male; D017830:Octoxynol / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D013363:Submandibular Gland / Q000187:drug effects / Q000378:metabolism*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000494:pharmacology
D015220:Calcium Channels; C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D017830:Octoxynol; D002784:Cholesterol; D010738:Type C Phospholipases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium