Rigidity Of Membrane Lipids Research Articles

Ligand-induced rapid desensitization causes structural alteration of rat ovarian LH/hCG receptor.

The role of the physical state of ovarian membranes was studied in the process of the early desensitization of the LH/hCG receptor. Thirty min after injection of a desensitizing dose of hCG to rats, the hCG-responsive adenylylcyclase activity was reduced, whereas hCG binding to ovarian membranes was still normal. Early desensitization decreased rigidity of membrane lipids, determined by fluorescence polarization of DPH. Possible structure-functional properties of the LH/hCG receptor were analyzed by thermal perturbation technique. Desensitization decreased thermal stability of the LH/hCG receptor in membranes and in proteoliposomes. Desensitization modified the quenching of protein fluorescence and intrinsic fluorescence spectral properties of membranes. The Stern-Volmer constants for control and desensitized membranes were found to be 4.3 and 5.5, respectively, indicating that desensitization elevated the accessibility of fluorophores for acrylamide. The changes of the physical properties of membranes resulting from desensitization was exhibited solely in the treatment with hCG in vivo, but not in vitro. The results suggest that the hCG-induced alteration of the physical state of luteal membranes may be a requirement for the induction of changes that lead to desensitization.

Effect of phospholipids on the reconstitution and thermal stability of delipidated rat ovarian luteinizing hormone/human chorionic gonadotropin receptors in proteoliposomes

The role of lipids and a possible structure-functional alteration of delipidated LH/hCG receptor reconstituted into proteoliposomes was analyzed by thermal perturbation techniques. Delipidated receptor lost to a great extent its binding activity and thermal stability. The LH/hCG receptor was almost fully reactivated by the reconstitution into proteoliposomes with phosphatidylcholine (PC) and sphingomyelin (SpM) and partly with phosphatidylethanolamine (PE) and phosphatidic acid (PA). The heat inactivation profile of delipidated LH/hCG binding sites was shifted to a lower temperature by about 4°C ( T 50 values). Thermal inactivation of the receptor by delipidation was entirely inverted by treatment with soybean PC, dioleoyl PC and dipalmitoyl PC and partially with SpM. The presence of negatively charged phospholipids, phosphatidylserine (PS), phosphatidylglycerol (PGl) and PA, did not change the heat-inactivation profile of the LH/hCG binding sites of delipidated proteoliposomes. On the other hand, the next decline of thermal stability of the receptor was found in proteoliposomes reconstituted in the presence of lyso-derivatives of PC, as well as of cholesteryl hemisuccinate. Delipidation of the LH/hCG receptor modified the differential scanning calorimetric profile and the quenching of protein fluorescence characteristic for control proteoliposomes. Delipidation increased membrane lipid rigidity. Reconstitution of delipidated proteoliposomes with soybean PC, dioleoyl PC, PGl, PS and PA decreased, and that of dipalmitoyl PC, lysoPC, SpM and cholesterol increased the degree of fluorescence polarization of DPH of proteoliposomes. The different action of phospholipids on the reconstitution, thermal inactivation of the receptor and membrane lipid fluidity in proteoliposomes suggests that lipid fluidity is not related to the stabilizing action of phospholipids on the LH/hCG receptor.

Effects of partial delipidation of rat ovarian membranes on thermal stability of LH/hCG receptors

The role of lipids and of possible structure-functional properties of the LH/hCG receptor were analyzed by thermal perturbation techniques in its native membrane environment. A method for the reversible removal of lipids from membranes with a mild detergent Tween 20 was developed. The receptor was reactivated with phosphatidylcholine (PC) by its reconstitution into proteoliposomes. The heat inactivation profile of LH/hCG binding sites in delipidated membranes was shifted to a temperature lower by approx. 8 C° ( T 50 values). Thermal inactivation of the delipidated LH/hCG receptor was found to be a quick process. Occupation of receptor binding sites by the agonist before thermal perturbation induced stabilization of the receptor. Thermal inactivation of the receptor by delipidation was fully reversed by treatment with soybean PC, diolcoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC), partly with phosphatidylethanolamine (PE) and sphingomyelin (SpM), but not with phosphatidylserine (PS), phosphatidylglycerol (PGI) or cholesterol. Delipidation modified the differential scanning calorimetric profile characteristic of control membranes. Delipidation of ovarian membranes also increased membrane lipid rigidity. The addition of PC, DOPC and PS to delipidated membranes decreased, that of DPPC and SpM increased, while PGI did not change the degree of fluorescence polarization of DPH, suggesting that membrane lipid fluidity was not involved in the stabilizing action of specific phospholipids against thermal inactivation of the ovarian LH/hCG receptor.

Early hCG-induced desensitization of rat ovarian LH/hCG receptors is associated with altered physical state of membranes

Injection of pseudopregnant rats with pharmacological doses of hCG produced desensitization of adenylylcyclase and steroidogenic systems in ovarian membranes and luteal cells. Membrane lipid rigidity, as determined by fluorescence polarization of DPH, decreased as early as 0.5 h after injection of hCG. Desensitization also modified the differential scanning calorimetric profile characteristic of control membranes. The accessibility of ovarian LH/hCG receptors was unchanged. The results indicate that the hCG-induced decrease of membrane lipid rigidity is preceded by the process of desensitization of the rat ovary.

Pseudopregnancy-dependent changes in rat ovarian LH/hCG receptors in relation to membrane lipid fluidity

The specific binding of [125I] hCG to ovarian membrane preparations as well as membrane fluidity have been investigated in immature rats during hormonally-induced pseudopregnancy. Membrane fluidity was monitored either by fluorescence polarization analysis of 1,6-diphenyl-1,3,5-hexatriene or by electron spin resonance of 16-, 12-, 5-doxyl stearic acid and CAT 16. A significant positive correlation was found between membrane lipid rigidity and the number of LH/hCG receptors. Luteinization of the ovary induced mobility of molecules in the hydrophobic membrane part at about the C16 carbon level. The changes in rigidity of membrane lipid were the apparent result of alterations in the cholesterol to phospholipids ratio. The results suggest that the increased rigidity of membrane lipid during pseudopregnancy may maximally expose ovarian LH/hCG receptors maintained in a cryptic form.

Modulation of rat testicular LH/hCG receptors by membrane lipid fluidity

The specific binding of [ 125I]hCG to rat testicular membrane preparations was investigated as a function of membrane fluidity changed by lipids. Membrane fluidity was measured either by fluorescence depolarization of diphenylhexatriene or ESR spectra of I(1,14), I(12,3) and CAT 16 incorporated into the membrane. Incubation of membrane with cholesteryl-hemisucinnate increased both the rigidity of membrane lipids and the specific binding of [ 125I]hCG. A similar rigidifying action of saturated fatty acids was, however, not accompanied by increased accessibility of LH/hCG receptors. Treatment of testicular membranes with unsaturated fatty acids enhanced membrane fluidity but specific binding of the gonadotropin disappeared. In spite of the increase of LH/hCG receptors in cell membranes treated with cholesteryl-hemisuccinate, Leydig cells showed decreased sensitivity to cAMP response to LH stimulation. The results indicate that newly exposed LH/hCG receptors are not coupled with the adenylate cyclase system.

Fluorescence studies of the blood platelet membranes associated with fibrinogen

To follow microviscosity changes in membranes associated with fibrinogen binding to human platelets, specific fluorescent probes were used and their fluorescence anisotropy was analysed. The degree of fluorescence anisotropy of diphenylhexatriene, anilinonaphthalene sulfonate (ANS) and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Fluorescence polarization analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase in the membrane lipid rigidity. On the other hand, changes in the fluorescence anisotropy of membrane tryptophans and N-(3- pyrene) maleimide suggest augmented mobility of the membrane proteins. The binding of fibrinogen to the membrane receptors is not accompanied by any change in the fluorescence intensity of ANS attached to the membranes. This may suggest that covering of platelets with fibrinogen molecules does not influence the surface membrane charge.

Microenvironment changes of human blood platelet membranes associated with fibrinogen binding.

Alterations in the membrane organization caused by fibrinogen binding to human blood platelets and their isolated membranes were analyzed by fluorescence and electron spin resonance measurements. The degree of fluorescent anisotropy of DPH, ANS and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Both fluorescence and ESR analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase of the membrane lipid rigidity. This effect seems to be indirect in nature and is mediated by altered membrane protein interactions. As it has been shown that an increased membrane lipid rigidity leads to a greater exposure of membrane proteins, including fibrinogen receptors, this might facilitate a formation of molecular linkages between neighboring platelets. On the other hand, changes of fluorescence anisotropy of membrane tryptophans and N-(3-pyrene) maleimide suggest the augmented mobility of the membrane proteins. Evidence is presented which indicated that the binding of fibrinogen to the membrane receptors is not accompanied by any changes in the fluorescence intensity of ANS attached to the membranes. It may suggest that the covering of platelets with fibrinogen does not influence the surface membrane charge. In contrast to fibrinogen, calcium ions caused an increase of the fluorescence intensity resulting from the more efficient binding of ANS to the platelet membranes.

Elevation of apparent membrane viscosity in ovarian granulosa cells by follicle-stimulating hormone

Continued exposure of cultured granulosa cells to follicle-stimulating hormone (FSH) induced: (i) a rise in apparent membrane microviscosity, as reflected by an increase in fluorescence polarization of the lipid-soluble probe, 1,6-diphenyl-1,3,5,-hexatriene; and (ii) a progressive decline in the cyclic AMP response to renewed challenge with the same hormone. Both changes were reduced or prevented by pretreatment of the cells with oleic or linoleic acid, agents which reduce membrane viscosity, but not by elaidic or palmitic acid which increase the rigidity of membrane lipids. Other agents that inhibited FSH-induced changes in membrane fluidity (gonadotropin-releasing hormone, actinomycin D and cycloheximide) also prevented desensitization to FSH. Cyclic AMP and cyclic GMP derivatives did not mimic the effects of FSH on apparent membrane viscosity or desensitization. Changes in membrane fluidity are unlikely to be the sole cause of desensitization since (i) pretreatment of the cells with fatty acids that increase lipid viscosity did not induce desensitization to FSH, and (ii) desensitization of granulosa cells to lutropin and prostaglandin E 2 by exposure to the homologous hormone was not attended by increased membrane viscosity. The experiments described provide the first example of a hormonally induced increase in the target cell apparent membrane viscosity.