Extraction of membrane cholesterol disrupts caveolae and impairs serotonergic (5-HT2A) and histaminergic (H1) responses in bovine airway smooth muscle: role of Rho-kinase.
Some receptors and signaling molecules, such as Rho-kinase (ROCK), localize in caveolae. We asked whether the function of histamine receptors (H(1)) and 5-hydroxytryptamine (serotonin) receptors (5-HT(2A)) in bovine tracheal smooth muscle are modified after caveolae disruption and if so, whether the altered ROCK activity plays a role in this modification. Methyl-beta-cyclodextrin (MbetaCD), used to deplete membrane cholesterol, was shown to disrupt caveolae and diminish sustained contractions to histamine (approximately 80%), 5-HT (100%), alpha-methyl-5-HT (100%), and KCl (approximately 30%). Cholesterol-loaded MbetaCD (CL-MbetaCD) restored the responses to KCl and partially restored the responses to agonists. ROCK inhibition by Y-27632 diminished contractions to histamine (approximately 85%) and 5-HT (approximately 59%). 5-HT or histamine stimulation augmented ROCK activity. These increases were reduced by MbetaCD and partially reestablished by CL-MbetaCD. The increase in intracellular Ca(2+) that was induced by both agonists was reduced by MbetaCD. The presence of caveolin-1 (Cav-1), H1, 5-HT(2A), and ROCK1 was corroborated by immunoblotting of membrane fractions from sucrose gradients and by confocal microscopy. H(1) receptors coimmunoprecipitated with Cav-1 in caveolar and noncaveolar membrane fractions, whereas 5-HT(2A) receptors appeared to be restricted to noncaveolar membrane fractions. We conclude that caveolar and cholesterol integrity are indispensable for the proper functionality of the H(1) and 5-HT(2A) receptors through their Rho/ROCK signaling.
Sommer
Bettina
B
Departamento de Investigacion en Hiperreactividad Bronquial, Instituto Nacional de Enfermedades Respiratorias, Mexico DF, Mexico.
Montaño
Luis M
LM
Carbajal
Verónica
V
Flores-Soto
Edgar
E
Ortega
Alicia
A
Ramírez-Oseguera
Ricardo
R
Irles
Claudine
C
El-Yazbi
Ahmed F
AF
Cho
Woo Jung
WJ
Daniel
Edwin E
EE
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D021941:Caveolae / Q000502:physiology*; D051242:Caveolin 1 / Q000502:physiology; D002784:Cholesterol / Q000502:physiology*; D008563:Membrane Lipids / Q000502:physiology*; D009130:Muscle, Smooth / Q000502:physiology*; D044402:Receptor, Serotonin, 5-HT2A / Q000502:physiology*; D011969:Receptors, Histamine H1 / Q000502:physiology*; D015398:Signal Transduction; D014132:Trachea / Q000502:physiology*; D047392:beta-Cyclodextrins / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000502:physiology*
D051242:Caveolin 1; D008563:Membrane Lipids; D044402:Receptor, Serotonin, 5-HT2A; D011969:Receptors, Histamine H1; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol; D054460:rho-Associated Kinases; D002118:Calcium