Effects of temperature and benzyl alcohol on the structure and adenylate cyclase activity of plasma membranes from bovine adrenal cortex.
Adenylate cyclase activation by corticotropin (ACTH), fluoride and forskolin was studied as a function of membrane structure in plasma membranes from bovine adrenal cortex. The composition of these membranes was characterized by a very low cholesterol and sphingomyelin content and a high protein content. The fluorescent probes 1,6-diphenylhexa-1,3,5-triene (DPH) and a cationic analogue 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) were, respectively, used to probe the hydrophobic and polar head regions of the bilayer. When both probes were embedded either in the plasma membranes or in liposomes obtained from their lipid extracts, they exhibited lifetime heterogeneity, and in terms of the order parameter S, hindered motion. Under all the experimental conditions tested, S was higher for TMA-DPH than for DPH but both S values decreased linearly with temperature within the range of 10 to 40 degrees C, in the plasma membranes and the liposomes. This indicated the absence of lipid phase transition and phase separation. Addition to the membranes of up to 100 mM benzyl alcohol at 20 degrees C also resulted in a linear decrease in S values. Membrane perturbations by temperature changes or benzyl alcohol treatment made it possible to distinguish between the characteristics of adenylate cyclase activation with each of the three effectors used. Linear Arrhenius plots showed that when adenylate cyclase activity was stimulated by forskolin or NaF, the activation energy was similar (70 kJ.mol-1). Fluidification of the membrane with benzyl alcohol concentrations of up to 100 mM at 12 or 24 degrees C produced a linear decrease in the forskolin-stimulated activity, that led to its inhibition by 50%. By contrast, NaF stabilized adenylate cyclase activity against the perturbations induced by benzyl alcohol at both temperatures. In the presence of ACTH, biphasic Arrhenius plots were characterized by a well-defined break at 18 degrees C, which shifted at 12.5 degrees C in the presence of 40 mM benzyl alcohol. These plots suggested that ACTH-sensitive adenylate cyclase exists in two different states. This hypothesis was supported by the striking difference in the effects of benzyl alcohol perturbation when experiments were performed below and above the break temperature. The present results are consistent with the possibility that clusters of ACTH receptors form in the membrane as a function of temperature and/or lipid phase fluidity.(ABSTRACT TRUNCATED AT 400 WORDS)
de Foresta
B
B
Equipe de Recherche No. 64-01 du Centre National de la Recherche Scientifique, Unité d'Enseignement et de Recherche Biomédicale des Saints-Pères, Paris, France.
Rogard
M
M
le Maire
M
M
Gallay
J
J
D000262:Adenylyl Cyclases / Q000032:analysis*; D000302:Adrenal Cortex / Q000187:drug effects / Q000201:enzymology*; D000324:Adrenocorticotropic Hormone / Q000494:pharmacology; D000818:Animals; D019905:Benzyl Alcohol; D001592:Benzyl Alcohols / Q000494:pharmacology*; D001593:Benzyl Compounds / Q000494:pharmacology*; D002417:Cattle; D002462:Cell Membrane / Q000032:analysis / Q000187:drug effects*; D005576:Colforsin / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005456:Fluorescent Dyes / Q000032:analysis; D005459:Fluorides / Q000494:pharmacology; D008560:Membrane Fluidity / Q000187:drug effects; D013696:Temperature
D001592:Benzyl Alcohols; D001593:Benzyl Compounds; D005456:Fluorescent Dyes; D005576:Colforsin; D000324:Adrenocorticotropic Hormone; D000262:Adenylyl Cyclases; D019905:Benzyl Alcohol; D005459:Fluorides