hCG receptors by membrane lipid fluidity

Molecular and Cellular Endocrinology, 44 (1986) 69-76 Elsevier Scientific Publishers Ireland, Ltd.

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Modulation of rat testicular L H / h C G receptors by membrane lipid fluidity J. Kolena, P. BlaZi~ek,S. Horkovics-Kovhts, K. Ondria~ and E. Seb6kovg Institute of Experimental Endocrinology and Institute of Experimental Pharmacology, Centre of Physiological Sciences, SIovak A cademy of Sciences, VIg~rska3, Bratislava (Czechoslovakia) (Received 17 June 1985; accepted 14 October 1985)

Key words: L H / h C G receptors; spin label; fluorescence polarization; membrane fluidity; cAMP production.

Summary 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), 1(12,3) and CAT 16 incorporated into the membrane. Incubation of membrane with cholesteryl-hemisuccinate 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 L H / h C G 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 L H / h C G 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 L H / h C G receptors are not coupled with the adenylate cyclase system.

Polypeptide hormone receptors belong to the most specific regulators of biological function. The majority of peptide hormones has been shown to cause activation of adenylate cyclase. An understanding of the principles involved in the interaction of hormone-receptor complex with adenylate cyclase has led to the framing of the mobile receptor hypothesis (Cuatrecasas, 1974). Receptors are a heterogenous population of molecules which interact with membrane components, e.g. phospholipids and protein (Catt and Dufau, 1977). Lateral movement of these components is modulated by membrane composition, physiological and physical conditions (Shinitzky and Henkart, 1979). It is possible to insert specific lipids into cell membrane and to study the resulting changes in membrane properties. Thus the functions of serotonin, insulin and prolactin receptors were found to be

affected by changes in membrane microviscosity (Heron et al., 1980; Ginsberg et al., 1981; Dave and Witorsch, 1983). These studies have suggested that modifying the physical state of the surrounding lipid matrix in membranes might affect the accessibility and thereby the functioning of receptors. Our previous results suggested that the L H / hCG receptor in the rat undergoes variations which depend on the functional state of the gonads (Kolena et al., 1978, 1980). In addition, significant positive correlations between testicular membrane fluidity and L H / h C G receptors during development of the rat have been observed (Kolena and Ondriag, 1984). The present study demonstrates that incorporation of lipids into testicular membranes can directly influence gonadotropin binding.

0303-7207/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland, Ltd.

70 Materials and methods

Materials Purified hCG (CR 123, 12780 IU mg -1) and ovine LH (NIH-LH-S 13, 0.093 x NIH-LH-S 1 IU mg 1) was generously supplied by NIAMDD, NIH, Bethesda, MD. Na12SI was purchased from the Radiochemical Centre, Amersham. Percoll was obtained from Pharmacia. Cholesteryl-hemisuccinate, fatty acids, polyvinylpyrrolidone (PVP) and 1,6-diphenyl-l,3,5-hexatriene (DPH) were products of Sigma. Stearic acid spin probes with a dimethyloxazolidinyl group at the 5th (1(12,3)), or 16th carbon positions (I(1,14)) were purchased from Syva (Palo Alto, CA). Spin probe 1-oxyl2,2,6,6-tetramethyl-4-dimethylaminopiperidine, cetyl bromide (CAT 16) was obtained from the Institute of Organic Chemistry, Sofia, Bulgaria. Other reagents used were of analytical reagent grade. Methods Crude membrane preparation Male Wistar rats aged 55-65 days were killed by decapitation. The homogenate of decapsulated testes in ice-cold 50 mM Tris-HC1 (pH 7.4) was filtered through 6 layers of surgical gauze, centrifuged at 1000 x g for 15 min and the supernatant was further centrifuged at 20000 x g for 30 min (Kolena, 1976). The final pellet was resuspended in the same buffer (200 mg of tissue per ml).

Preparation of Leydig cells Crude interstitial cells were prepared by collagenase digestion of decapsulated testes (Sebrkovfi and Kolena, 1978). Approximately 108 cells in 2 ml of Medium-199 with 1 mg ml -~ BSA were layered on top of continuous Percoll gradient and centrifuged at 600 x g for 20 min (Browning et al., 1981). The purified Leydig cells were in the third of 4 bands generated on the gradient. Preparation of Percoll gradients was described previously (Kolena et al., 1983). Incubation of tissues with lipids 10 mg of lipid was dissolved in 0.2 ml of hot glacial acetic acid. On stirring the solution, it was diluted to 50 vols. with 50 mM Tris-HC1, pH

7.4 + 3.5% PVP. The pH was readjusted to 7.4 with solid Tris-base (Heron et al., 1980). 1.5 ml of crude testicular membrane fraction in Tris-HCl buffer or 1.5 ml washed Leydig cells in Medium199 + 1 mg ml 1 BSA were incubated 90 min with varying concentrations of lipid suspensions at 25°C. Controls were incubated with the same concentration of Tris-acetate buffer with PVP. The testicular membranes were then centrifuged at 20000 × g for 30 rain and pellets were washed once with Tris-HC1 buffer and once with 0.05 mol 1-1 phosphate buffer (pH 7.4) with 0.015 mol 1 sodium chloride (PBS). The final pellets were suspended in PBS buffer (200 mg ml 1). After incubation with lipids, Leydig cells were centrifuged at 150 x g for 10 rain. The cells were washed twice and distributed in Medium-199 + 1 mg ml- 1 BSA.

hCG binding assay 0.1 ml aliquots of testicular membrane fraction or Leydig cells were incubated 16 h at room temperature with 0.1 ml PBS + 1 mg ml- i BSA with or without 100-fold excess of unlabeled hCG and 0.1 ml [125I]hCG (1-1.5 ng, spec. act. about 2.3 TBq g - l ) . After incubation and centrifugation the pellets were washed twice with 2 ml of cold PBS (Kolena and Sebrkov~, 1983; Kolena et al., 1983). The results are expressed as specific binding per mg protein. Protein was determined by standard procedure (Lowry et al., 1951). Scatchard analysis (Scatchard, 1949) of data obtained from saturation curves was used to estimate the number of receptor sites and was carried out using a computer program. Production of cAMP Highly purified Leydig cells (0.1 ml, about 4 x 105 viable cells) were suspended in 0.7 ml Medium-199 containing 20 mmol 1-1 Hepes (pH 7.4), 0.2 mmol 1-1 1-methyl-3-isobutylxanthine (MIX) and 1 mg ml-1 BSA fraction V and incubated at 37°C with or without LH. The cells in 0.06% trypan blue were counted in a hemocytometer. The medium was then aspirated, centrifuged at 2000 x g for 15 min and stored at - 20°C prior to determination of cAMP by protein binding assay as described previously (Kolena and Channing, 1972; Sebrkovfi and Kolena, 1978).

71

Fluorescence polarization and electron spin resonance (ESR) measurements

Fluorescence polarization was measured with an Aminco Bowman SPF spectrofluorometer (Shinitzky and Inbar, 1976). Crude testicular membranes (100 #g protein) or Leydig cells (1.5 × 106 cells) were incubated with 2 ml of DPH (2 ~tmol 1-1 ) in PBS buffer for 1 h at 24°Co The fluorescence polarization was computed by the equation: p = I~v - Ivh ( I h v / I h h ) Ivv + ]vh ( I h , , / I h h ) where I vv and I vh a r e the fluorescence intensities detected through a polarizer oriented in parallel and perpendicularly to the direction of vertically polarized light. The Ihv/Ihh represents the ratio when the excitation is polarized horizontally and the emission observed through the analyzer oriented perpendicularly and in parallel, respectively. Lipid microviscosity was estimated by the empirical relation 2P/(0.46 - P) (Heron et al., 1980). Samples for ESR measurements were prepared as follows: 10 /~g of spin probe were mixed with 0.06 ml of crude testicular membrane (3.1 mg protein). ESR spectra were recorded using an ERS 230 spectrometer. The typical instrumental setting was: 5 mW microwave power, modulation amplitude 1.6 × 10 - 4 T. The inner A . hyperfine splitting parameters of the spin probes in testicular membrane fraction were determined as previously reported (Kolena and OndriaL 1984) from ESR spectra (Gaffney, 1976). The spin probes I(12,3) and I(1,14) reflect the mobility of the hydrophobic membrane part at the C 5 and C~6 carbon membrane depths (Griffith and Jost, 1976). The CAT 16 spin probe reflects the mobility of the polar part of the membrane (Lassmann and Herrmann, 1984). The ESR spin probe parameters A . were evaluated in order to estimate the effect of cholesterol on the testicular membrane fluidity at different membrane depths. The parameter A± is proportional to the order parameter S (Gaffney, 1976). Because of the complexity of the membrane system, a decrease in S and an increase in A l is interpreted as an increase in the membrane fluidity at the given depth.

For statistical evaluation Student's t-test was used. The results are expressed as mean + SE. Results

The effect of changing the microviscosity of crude testicular membranes on the specific binding of [I:SI]hCG is shown in Fig. 1. An important point is that in parallel with increasing microviscosity of membrane lipids by treatment with rising concentration of cholesteryl-hemisuccinate, increased specific binding of [I25I]hCG was observed. On the other hand, incubation of crude liver membrane with cholesterol had no effect on [125I]hCG specific binding in this non-target tissue. Scatchard plots derived from the binding data of 4 experiments showed no appreciable effect of 0.75

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Fig. 1. Effect of cholesterol on microviscosity and [125I]hCG specific binding to membrane preparations. Aliquots of crude testicular (full line) or hepatic (dashed line) membranes were incubated with increasing amounts of cholesteryl-hemisuccinate in Tris-acetate buffer (pH 7.4) containing 3.5% PVP for 90 min at 25°C. After washing, membranes were assayed for membrane lipid microviscosity, using DPH as a probe and for [~25I]hCG specific binding. Each point is the m e a n + S E of 5 estimations. The experiment was repeated 4 times with similar results.

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mg ml-I cholesteryl-hemisuccinate on the affinity of testicular,binding sites for hCG (9.3 + 2.5 and 6.28 + 1.3 nmo1-1 for control and cholesteroltreated, respectively). However, exposure of testicular membrane to cholesterol resulted in a 2.2-fold increase in the maximum binding capacity of the membrane from 76.5 + 13.3 to 168 + 28.4 fmol per mg protein ( P < 0.001). Incubation of testicular membrane preparations with cholesterol at temperatures below 37°C did not change specific binding of [125I]hCG (Fig. 2). An even more detailed evaluation of the fluidity of the testicular membrane preparation was carried out by electron spin resonance. Cholesterol decreased the fluidity of the testicular membrane at both the hydrophobic and the polar membrane parts. As shown in Fig. 3, the parameters A ± of spin probes CAT 16, I(12,3) and 1(1,14) increase with temperature both with and without cholesterol treatment. Furthermore, membrane with added

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cholesterol exhibits lower A _~ values. On comparing the temperature and cholesterol effects on membrane fluidity (Ondria~ et al., 1983) spin probes I(1,14), I(12,3) and CAT 16 cholesterol were found to have similar effects on parameter A ± as chilling control membranes by about 30, 17 and 15°C. The linear temperature dependences of the A ± parameters of the samples with cholesterol exhibit breaks at about 20-25°C, possibly indicating some structural membrane changes. To determine whether other lipids with a rigidifying effect on plasmatic membrane can increase L H / h C G receptors in a manner similar to that exerted by cholesterol, we investigated the effect of saturated fatty acids on [125I]hCG specific binding to testicular membrane. The results are presented in Fig. 4. The increase of microviscosity of membrane lipids was not accompanied by increased specific [12SI]hCG binding. Both arachidic and stearic acids had effects on membrane fluidity similar to those seen after treatment with cholesterol. Yet specific binding of [IESI]hCG to testicular membrane was either not changed (ara-

chidic acid) or was even decreased (stearic acid). Maximum decrease of the accessibility of gonadotropin receptors was observed with palmitic acid, although this fatty acid had an unequivocal rigidifying effect on the membrane. It was possible to affect the accessibility of L H / h C G receptors also by insertion of unsaturated fatty acids into the membrane. Incubation of i

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Fig. 7. Responsiveness of Leydig cells to LH in cAMP formation. Percoll purified Leydig cells (about 4 x 105 viable cells) were preincubated with cholesteryi-hemisuccinate 0.8 (I), 0.08 (II) or 0.008 (III) m g m l - i . Washed cells were incubated at 37°C in Medium-199 with 20 mmol l - ] Hepes (pH 7.4), 0.2 mmol 1-1 MIX, and 1 mg m1-1 BSA fraction V without (control) or with 5 p g m1-1 LH. Each point is the m e a n + S E of 4 estimations. These results were confirmed in 2 independent experiments.

74 testicular membrane preparations with unsaturated fatty acids reduced membrane microviscosity (data not shown), with simultaneous disappearance of [125I]hCG specific binding (Fig. 5). Exposure of membranes to cis-vaccenic (18 : 1), petroselinic (18 : 1), arachidonic (20:4), oleic (18 : 1) and linoleic acids (18:2) decreased total [125I]hCG binding while increasing non-specific binding. However, for this disappearance of L H / h C G receptors the presence of the acids in their free form was necessary, because esterification of the fatty acid (oleic acid palmityl ester) abolished such changes in receptors. A further series of experiments was performed to determine whether increased accessibility of L H / h C G receptors by cholesteryl-hemisuccinate is connected with increased function of Leydig cells. As can be seen in Fig. 6, cholesterol acted both on microviscosity and [125I]hCG specific binding to Leydig cells in a similar manner as in membrane preparations. Although cholesterol increased accessibility of L H / h C G receptors in Leydig cells, this was not related to enhanced responsiveness of the cells to LH in cAMP and testosterone secretion. Preincubation of Leydig cells with lower concentration of cholesterol (0.08 and 0.008 mg ml-1) did not change formation of cAMP, the higher concentration (0.8 mg ml 1) inhibited the adenylate cyclase enzymatic system (Fig. 7). Similar results were found with respect to testosterone secretion. Also fluidization of testicular membranes with unsaturated fatty acids did not cause activation of adenylate cyclase. The rise of non-specific binding of [125I]hCG up to reaching the level of total binding was due to the abolishment of stimulatory effects of LH on cAMP and testosterone formation by rat Leydig cells (data not shown). Discussion

The presented data indicate that modification of fluidity of crude testicular membrane by dispersion of various lipids has direct effects on L H / h C G receptors. After decreasing fluidity of membranes from rat testes by treatment with cholesterylhemisuccinate an increase in specific hCG binding sites was observed. Scatchard analysis of the presented results as well as data from other reports

(Cammeron and Stouffer, 1982; Dave and Witorsch, 1983) indicate that changes in hormone binding to membranes are associated with alterations in receptor number rather than with apparent receptor affinity. This finding, along with previous demonstrations showing that the binding of hormone to membrane can be increased by changes of lipid fluidity, suggests that the increase in binding is due to the exposure of a presynthetized pool of receptors maintained in a cryptic form. Membrane receptors can be differently affected by changes of lipid fluidity in different cells and after different treatments. When lipid microviscosity is decreased, prolactin receptors from the membranes of the ventral prostate (Dave and Witorsch, 1983) and beta-adrenergic receptors in liver cell membranes (Bakardjieva et al., 1979) become more exposed. On the other hand, increase of microviscosity enhances exposition in the case of beta-adrenergic receptors in rabbit reticulocyte (Strittmatter et al., 1979) and of serotonin receptors in mouse brain membranes (Heron et al., 1980). The increased accessibility of L H / h C G receptors in testicular membranes treated with cholesterol may be in consent with the concept of vertical displacement of membrane proteins (Borochov and Shinitzky, 1976). Along with the increase in lipid microviscosity the bulk membrane protein becomes more exposed to the aqueous medium. However, changes in membrane fluidity are unlikely to be the sole cause of this effect, because a comparable rigidifying action of saturated fatty acids did not increase the accessibility of L H / h C G receptors. The influence of cholesterol on testicular L H / h C G receptors may be connected with specific structural and chemical properties of cholesterol in biological membranes. Cholesterol is a major component of plasma membranes. It has a dual effect on membrane fluidity, increasing the mobility of hydrocarbon chains of bilayer lipids below the phase transition temperature and decreasing it above this temperature (Hsia and Boggs, 1972). The lipids of functional cell membranes at physiological temperature are in a fluid rather than gel state. Cholesterol enrichment decreases lipid fluidity of the outer membrane leaflet (Chabanel et al., 1983). The location of cholesterol within the bilayer was determined by

75 examining its effects upon individual structural parameters. Cholesterol consists of a rigid plane with specific groups (3/3-OH, aliphatic chain) which establish an alignment with phospholipid molecules. Several authors using NMR studies on liposomes have observed interactions between the hydroxyl group of cholesterol and the phospholipid carbonyl group (Huang, 1976), whereas others found interactions of the hydroxyl group with the phospholipid phosphate group (Phillips and Finer, 1974). Dielectric measurements on egg phosphatidylcholine bilayers, however, showed the cholesterol hydroxyl group to be located between the phosphate group and the glycerol oxygens of the phospholipid molecules (Ashcroft et al., 1983). The cell surface receptor for L H / h C G , similarly to other membrane receptors, is thought to be an integral membrane protein associated with the membrane by strong hydrophobic bonds. These hydrophobic bonds can play an important role in the function of gonadotropin'hormone that may be influenced by cholesterol at this level by altering the lipid-receptor interaction. Diphenylhexatriene is an almost ideal probe for measuring fluidity of lipids, since it is hydrophobic and located with its symmetry axis normally to the plane of the membrane (Andrich and Vanderkooi, 1976) and it occupies the region near the centre of the bilayer (Engel and Prendergast, 1981). However, ESR spin probes can yield even more information on the order of membrane lipids. Our experiments with spin probes indicate that cholesterol greatly affects motion in both the hydrophobic and the polar membrane parts. The results are in agreement with the previously reported ordering action of cholesterol on the hydrophobic part of lipid (Oldfield et al., 1978) or biological membranes (Davies et al., 1980). Oldfield et al. (1978) using deuterium NMR have shown that cholesterol produces an almost uniform ordering of the acyl lipid chain when absolute changes are measured, but the greatest effect of cholesterol may be found near the middle of the acyl chain if percentage changes are determined. We have also found that the highest ordering effect of cholesterol, expressed on a temperature scale, was at the C~6 carbon depth. In the case of the polar part, studies with lipid membranes using deuterium NMR have shown

that cholesterol has little effect on the mobility of head lipid groups (Brown and Seelig, 1978), and that addition of cholesterol to choline-labeled lecithin results in complex behaviour of the head group deuterium quadrupole splitting as a function of temperature and cholesterol (Oldfield et al., 1978). In our study cholesterol decreased the mobility of the CAT 16 spin probe in the polar part of the testicular membrane in the temperature range from 10 to 40°C. On realizing the importance of temperature in all biological functions and of changes in cholesterol ordering effects at 15, 17 or 30°C (expressed in the temperature scale), biological responses to cholesterol added into membranes may possibly be a result of complex membrane events. But these spin labels of membrane lipids may be incorporated in environments different from that of the L H / h C G receptor whose activity was studied biochemically. Some experiments suggested heterogenous distributing of membrane lipids and Jain and White (1977) offered a model in which membrane lipids were segregated into a series of immiscible domains. In addition, the distribution of cholesterol may not be uniform in the plane of the bilayer, depending upon the chemical composition and structural organization of the testicular membrane, which confirms the possibility of changing membrane function at different locations of receptors. In spite of increased accessibility of L H / h C G receptors in cell membranes treated with cholesterol, Leydig cells showed a decreased sensitivity with respect to cAMP and testosterone responses to LH stimulation. These results are in agreement with experiments on Leydig cells in plated culture and in suspensions which indicates that newly exposed L H / h C G receptors would not be coupled with adenylate cyclase (Barafiao and Dufau, 1983). According to the collision-coupling model (Cuatrecasas, 1974) activation of adenylate cyclase becomes increased by higher membrane fluidity, which increases the chance of receptor interaction with the enzyme. Orly and Schramm (1975) and Hanski et al. (1979) reported that fluidization of turkey erythrocyte by unsaturated fatty acids activated adenylate cyclase. Cholesterol inhibition of Leydig cell responsiveness to LH can be interpreted as a result from an increase in membrane rigidity which, in turn, results in lower

76 interaction between adenylate cyclase and the h o r m o n e - r e c e p t o r c o m p l e x . I n d e e d , S i n h a et al. (1977) have shown that cholesterol enrichment of the membrane resulted in reduced fluidity and responsiveness of platelets to prostaglandin E 1 stimulation of adenylate cyclase.

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