Steroid hormone induced effects on membrane fluidity.

Abstract

The classical mechanism of steroid hormone action involves the niolecule binding to high affinity intracellular receptors. This steroid-receptor complex then travels to the nucleus where it modulates gene transcription. Some steroid hormone actions, however, are not due to genoniic changes, e.g., modification of behaviour by progesterone [ l ] and altered neuronal firing activity after gonadal and adrenal steroid hormone action 121. These actions are almost immediate or of R short latency and are insensitive to inhibitors of RNA and protein synthesis. Steroid horniones in such cases may act directly on plasma membranes. Indeed, receptors for oestradiol, testosterone and progesterone have been identified in synaptic membranes [ 3 ] . Steroid hormones may also alter the dynamic properties of membrane bilayers. The structural requirements for maxiniising the interaction of sterols with the bilayer appear to be R 3-p-hydroxy group, an isocaproyl side chain and a flat cholestene skeleton 14). Given the structural nnalogy between cholesterol and steroid hormones it is possible that such hormones both alter membrane fluidity and influence membrane protein organisation and function. Deliconstantinos [5] has observed a change in synaptic membrane ordering after exposure to steroid hormones. The influences of steroid hormones on model membrane bilayer fluidity and on natural membrane ordering was therefore investigated. Measurements of membrane fluidity were performed using the steady-state fluorescence polarisation of 1,6-diphenyl 1,3,5hexatriene (DPH) according to the method described by Shinitzky and Barenholz 161. The data obtained are expressed in terms of the emission anisotropy (r) where r = (Iw I@I + 244 . and IvH being the emission intensities observed parall8 and perpendicular to the polarisation plane of the exciting light. Lipid bilayer membranes were prepared by dispersing egg yolk phosphatidylcholine in 0.2M Tris-C1 buffer pH 7.4. Cholesterol was added to some liposomes to give a molar ratio of cholesterol to phospholipid of 0.8, similar to the ratio in synaptic membranes. Liposomal membranes containing steroid hormones were prepared by co-dissolving the hormone together with the requisite amount of phosphatidylcholine in chloroformmethanol (2: 1 v/v). The solvent was then removed under a stream of nitrogen. 0.2M Tris-CI buffer pH 7.4 was added to the dry hormone-lipid mixture and unilamellar liposomes formed by sonication. The final concentration of hormone varied between 2 and 1OpM. This method could not be used with natural membranes. In this case, the steroid hormones were incorporated into dipalmitoyl phosphatidylcholine (DPPC) liposomes. The hormone-associated DPPC WRS added in a 1:l molar ratio to the lipid in the membranes, resulting in transference of hormone from the DPPC bilayers to the membrane lipid. The suspension was then centrifuged on a 20% (w/v) sucrose layer on a 60% sucrose (w/v) cushion for 2 hours at 30,000 xg to separate the biomembrane from excess DPPC and any unassociated hormone. Progesterone was found to decrease membrane fluidity. Testosterone, however, caused no change in the ordering of the membrane whereas its metabolite 17-P-oestradiol increased membrane fluidity as did 17-a-oestradiol and oestriol. In both liposomes and natural membranes containing cholesterol the steroid hormone effect on membrane fluidity was less pronounced. It has been proposed [7] that cholesterol limits steroid hormones intercalating between lipid bilayers. They consequently bind more to the surface of the membrane, explaining the lower change in anisotropy. Thus in natural membranes the steroid hormones have more influence on sarcoplasmic reticulum membranes from rabbit white muscle with a neglible cholesterol content than in synaptic membranes. Steroid hormones have been shown to alter the dynamic properties of bilayers. Further work will show if this influences the activity of membrane-bound enzymes. Rorlesenme Oeslradiol