Membrane fluidization increases low-affinity muscarinic receptor binding in brain: Changes with aging

ExperimentalGerontology, Vol.21, pp. 37-44, 1986 Printedin the U.S.A. Allrightsreserved.

0531-5565/86$3.00 + .00 Copyright~ 1986PergamonJournalsLtd

MEMBRANE FLUIDIZATION INCREASES LOW-AFFINITY MUSCARINIC RECEPTOR BINDING IN BRAIN: CHANGES WITH AGING

GERHARD FREUND 1, THOMAS R. BROPHY III, a n d JAMES D. SCOTT Veterans Administration Medical Center and Departments of Medicine and Neuroscience, Center for Alcohol Research, University of Florida, Gainesville, Florida Abstract-Specific cholinergic muscarinic receptor binding was determined with L-[~H] quinuclidinyl benzilate ([~H]QNB) in homogenates from crude synaptosomal pellets prepared from mouse whole-brain homogenates. Specific total (high- and low-affinity) binding was determined in the absence of the agonist carbachol and low-affinity binding in its presence. These membrane preparations were fluidized by adding in vitro aliphatic alcohols ranging from ethanol to hexanol and by increasing the incubation temperatures. At 23 °C hexanol (14.7 mM) nearly doubled the low-affinity binding in the presence of carbachol (0.32 mM) and decreased high-affinity binding by the same amount. This suggested a change of muscarinic receptors from high- to lowaffinity conformation. Increase of incubation temperature from 24 °C to 37 °C nearly tripled lowaffinity binding. Brain homogenates from female C57BL/6J mice, ages 6, 12, 18, and 30 months, showed a progressively lower stimulation by hexanol of low-affinity [3H]QNB binding in the presence of carbachol. We postulate that this diminished change with age of [3H]QNB-receptor binding in response to alcohols may be a result of increasing membrane rigidity with advancing age. Rigidity of membranes may link aging at the membrane level, synaptic receptors, and impaired learning behavior. Key Words: muscarinic receptor binding, membrane fluidization, mouse brain, aging of brain receptors INTRODUCTION AGING AND c h r o n i c alcohol c o n s u m p t i o n i m p a i r l e a r n i n g a n d m e m o r y in h u m a n s a n d a n i m a l s ( F r e u n d , 1980, 1982a,b; K u b a n i s et al., 1982). The causes o f these deficits could r a n g e o n a c o n t i n u u m f r o m loss o f entire cells, their processes, a n d synapses to purely m o l e c u l a r changes in synapses. T h e c o m m o n t e r m i n a l p a t h w a y is i m p a i r e d or lost synaptic f u n c t i o n (Bartus et al., 1982; S a m o r a j s k i , 1981). A g i n g a n d alcohol could affect transmitter p r o d u c t i o n , receptor n u m b e r s a n d affinity, m e m b r a n e e n v i r o n m e n t o f receptors, p o s t s y n a p t i c c o u p l i n g , or effector events, or a c o m b i n a t i o n o f these. O n e of the factors that is t h o u g h t to i n f l u e n c e a n d restrict receptor b i n d i n g is the lipid m e m b r a n e e n v i r o n m e n t o f the receptors (Birdsall et al., 1983). 'Correspondence and requests for reprints should be addressed to Gerhard Freund, M.D., Medical Service (111), Veterans Administration Medical Center, GainesviUe,FL 32602. (Received 24 October 1985, Revised 4 December 1985, Accepted 3 January 1986) 37

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G. FREUND, T.R. BROPHY 111, AND J.D. SCOTT

There is evidence that increased m e m b r a n e lipid microviscosity in older mice is associated with a decrease of t r a n s m i t t e r - r e c e p t o r b i n d i n g (Samuel et al., 1982). However, m a n y changes associated with aging have n o significant effect o n f u n c t i o n . W e therefore wanted to test two hypotheses. First, the c h a n g i n g of m e m b r a n e fluidity with alcohols a n d incub a t i o n t e m p e r a t u r e s will alter receptor b i n d i n g if the lipid m e m b r a n e m i c r o e n v i r o n m e n t is relevant to receptor b i n d i n g . Second, if the age-associated increased m e m b r a n e rigidity has a potential effect o n receptor b i n d i n g , then the older, rigid m e m b r a n e s should be more resistant to f l u i d i z a t i o n - i n d u c e d receptor b i n d i n g changes by the same c o n c e n t r a t i o n of a n alcohol. The p u r p o s e o f this investigation was to d e t e r m i n e how increasing m e m b r a n e fluidity with aliphatic alcohols a n d higher i n c u b a t i o n t e m p e r a t u r e s affect m u s c a r i n i c cholinergic receptor b i n d i n g in b r a i n h o m o g e n a t e s o f old a n d y o u n g mice. Brain postsynaptic m u s c a r i n i c receptors are t h o u g h t to exist in at least two i n t e r c o n v e r t i b l e f u n c t i o n a l a n d c o n f o r m a t i o n a l states (Birdsall et al., 1983). Briefly, the receptor that is n o t coupled to an effector (enzyme or ion channel) is in a " h i g h - a f f i n i t y " state that converts to a "lowaffinity" state with coupling. A p p r o p r i a t e c o n c e n t r a t i o n s o f m u s c a r i n i c a n t a g o n i s t s such as q u i n u c l i d i n y l benzylate (QNB), because of a very high a f f i n i t y for the receptor, occupy almost all o f the available receptor b i n d i n g sites irrespective o f their degree o f affinity. In contrast, agonist agents, like carbachol, have generally lower affinities for the receptor a n d therefore in high c o n c e n t r a t i o n s compete for a n d occupy mostly h i g h - a f f i n i t y receptor sites. The l o w - a f f i n i t y agonist c a r b a c h o l in a p p r o p r i a t e l y high c o n c e n t r a t i o n s will block most h i g h - a f f i n i t y sites b u t will leave the l o w - a f f i n i t y ( p r e s u m a b l y effector-coupled) receptor sites available for b i n d i n g with low c o n c e n t r a t i o n s o f h i g h - a f f i n i t y QNB. The Q N B b i n d i n g in the presence of c a r b a c h o l reported here is therefore a m e a s u r e of the n u m b e r of " l o w - a f f i n i t y " m u s c a r i n i c receptor b i n d i n g sites. MATERIALS AND METHODS Female C57BL/6J mice (age 2 months) were purchased and housed in animal facilities until they were 6, 12, 18, or 30 months old. The mice were fed Purina Laboratory Chow (Ralston Purina Company, St. Louis, MO) ad libitum. At the specified ages the mice were decapitated, the brains were removed, and the cerebellum and olfactory bulbs were separated. The brains were immediately homogenized with a Brinkman Polytron homogenizer at power setting 5 for 15 seconds in ice-cold 0.05-M sodium potassium-phosphate buffer (pH 7.4). The resulting concentration was approximately 50 mg tissue/ml buffer (5% w/v, 4.5 mg protein/ml buffer). Crude synaptosomal fractions were prepared by centrifugation at 50,000 × g for 10 minutes. The pellets were resuspended in the original volume of fresh Na-K buffer by the homogenizer at setting 5 for 5 seconds. This resulted in concentration of approximately 2.1 mg of 50,000 g resuspended pellet protein per milliliter of buffer. Individual incubates contained resuspended 50,000 × g homogenate, 50 #l/ml buffer. Muscarinic cholinergic receptors were determined by the method of Yamamura and Snyder (1974) adapted as described previously (Freund, 1980). 3lH]L-quinuclidinyl benzylate ([3H]QNB), 40 Ci/mmol, was purchased from New England Nuclear Corporation, Boston, MA. [3H]QNB was added in the concentration of 5.4 × 10-~° M to all tubes. To assay specific binding, we incubated 100 #1 of the homogenates for 1 hour at various temperatures with 2 ml of 0.05 M sodium-potassium (Na-K) phosphate buffer, pH 7.4, containing the [3H]QNB. Nonspecific binding was determined in the presence of unlabeled 2.5 tzM QNB (Yamamura and Snyder, 1974), a gift from Hoffman La Roche, Nutley, NJ. Every determination was performed in triplicate. The incubation was terminated by vacuum filtration through glass-fiber filters (GF/B, Whatman). The filters were washed three times with 3 ml ice-cold buffer. The filters were transferred to liquid scintillation vials. Glacial acetic acid, 50 ~1, and 0.5 ml Protosol (New England Nuclear) were added, and the mixture was heated in a water bath at 57 °C for 30 min. After the mixture was cool, 10 ml of Econofluor (New England Nuclear) were added; the vials were stirred and counted after 36 hours of adaptation to dark. Carbamylcholine chloride (carbachol; Sigma, St. Louis, MO) was added to the incubated samples in concentration 0.32 raM, which suppressed specific QNB binding to approximately 30°7o of baseline (McKinney and Coyle, 1982). Hexylalcohol (hexanol; Eastman Kodak, Rochester, NY) was added in the concentration of 14.7

39

B R A I N M U S C A R I N I C R E C E P T O R S IN A G I N G

mM. The other alcohols were obtained from Fisher Scientific Co. Protein concentrations were determined in 50-#1 aliquots from the resuspended pellet homogenates by the method of Lowry et al. (1951), using bovine albumen as standard. Specific binding was calculated as total minus nonspecific. The results were expressed as the meant + SEM of the number of experiments indicated. Statistical comparisons were made using Student's t test (Dixon and Massey, 1969).

RESULTS Aliphatic alcohols in vitro under appropriate conditions only diminished but did not increase specific high-affinity [3H]QNB binding in resuspended 50,000 g pellets from mouse whole-brain homogenates (data not shown). However, low-affinity binding in the presence of carbachol was enhanced (Figure 1), most marked by hexanol, and least by ethanol in descending order with decreasing chain length. This enhancement increased with increasing concentrations of alcohol up to a certain concentration. Beyond an optimal concentration of the alcohols, low-affinity binding was diminished to well below baseline control levels. All values were determined in triplicate. Figure 1 is one representative of three individual curves that varied less than 6°7o. Effect of incubation temperatures on specific, total (high- and low-affinity) QNB bind-

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40

G. FREUND, T.R. BROPHY 111, AND J.D. SCOTT

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FzG. 2. The effect of incubation temperatures on specific QNB ([~H]QNB 5.45 × 10-'°M) binding: (a) total (high- and low-affinity) without in vitro additions (CONTROL); (b) HEXANOL only 14.7 mM; (c) low-affinity binding in the presence of carbachol, 0.32 mM; and (d) low-affinity binding in the presence of both HEXANOL and CARBACHOL. Resuspended 50,000 × g pellets of mouse whole-brain homogenate (5o70w/v).

ing is shown in Figure 2. (a) The C O N T R O L curve shows the effect of increasing the incubation temperature f r o m 24 °C (room temperature) to 43 °C on [3H]QNB binding. The changes are small (7%). Binding is maximal at 37 °C and declines slightly at higher and lower temperatures. (b) When hexanol is added to the incubate, the total (high- and lowaffinity) binding at r o o m temperature decreases. At higher temperatures binding decreases even further. (c) When carbachol is added to the homogenate, only low-affinity binding is determined. It is evident from Figure 2 that low-affinity binding at room temperature decreases. At higher temperatures binding decreases even further. (c) When carbachol is added to the homogenate, only low-affinity binding is determined. It is evident from Figure 2 that low-affinity binding at r o o m temperature almost triples at body temperature and then slightly declines with even higher incubation temperatures. (d) Lowaffinity binding in the presence of carbachol is enhanced also by hexanol at r o o m temperature. Increasing the temperature to 2 9 ° C further increases low-affinity binding. However, with a further increase of temperature in the presence of hexanol, low-affinity binding markedly declines, probably as a result of over-fluidization by the combined fluidization effects of hexanol and raised temperature. Generally, at room temperature,

BRAIN MUSCARINICRECEPTORSIN AGING

41

hexanol increases the low-affinity binding (carbachol plus hexanol) to the same extent as it decreases the total (low- plus high-affinity) binding. This fact suggests that hexanol fluidization favors a change from a high- to a low-affinity conformation. This reciprocal relationship becomes even more pronounced at higher temperatures, up to approximately 30 °C. In contrast, hexanol combined with temperatures elevated beyond 30 °C, results in a decrease of all binding, probably because of overfluidization. Interestingly, with carbachol in the medium, raising the incubation temperature to 37 °C increased [3H]QNB binding. However, when the membranes were fluidized even more by the further addition of hexanol, [3H]QNB binding decreased. There was no significant effect on nonspecific binding (data not shown). The effect of increasing age on specific [3H]QNB binding is shown in Figure 3. As has been shown previously under different in vitro conditions, total (high- plus low-affinity)

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F]c. 3. Effect o f age on specific [3H]QNB binding. Total binding is high-affinity plus low-affinity binding. Lowaffinity binding is determined in the presence of carbachol. Low-affinity binding is stimulated by hexanol. Mouse whole-brain homogenate, [3H] QNB 5.45 x 10-'°M, carbachol 0.32 mM, hexanol 14.7 mM, incubation temperature, 23 °C, N = 8 per age group. The differences between hexanol-induced changes are significant ( p > 0.05) by Student's t test.

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G. FREUND, T.R. BROPHY llI, AND J.D. SCOTT

binding is decreased in mouse whole brain with advancing age (Freund, 1980; Kubanis et al., 1982). In this experiment there is a decrease of 8.5% (p < 0.01) between the ages of 12 and 18 months. Low-affinity binding in the presence of carbachol is not significantly decreased with aging, but the magnitude of hexanol-induced increase of low-affinity binding diminishes progressively with age, from 431 + 20 to 334 ± 7 fmol/mg protein. The results obtained with pentanol (57 mM) and butanol (162 mM) were very similar (data not shown). DISCUSSION The impairment of cognitive function associated with aging and prolonged alcohol consumption may be the result of morphologically recognizable loss of neurons or their component parts, or both. In addition, only chemically detectable losses, such as decreased synaptic receptor densities and affinities (Bartus, 1982; Freund, 1984), may impair learning and memory. Finally, aging and chronic alcohol consumption may affect synaptic membrane structure, which, in turn, may affect the binding of synaptic transmitters to their respective receptors. The presence of ethanol in neuronal membranes increases their fluidity and compensatorily changes their chemical structural composition (Chin and Goldstein, 1977; Ingram, 1982a,b; Seeman, 1972). These changes in membrane structure and fluidity could, in turn, influence receptor binding. The purpose of this investigation was to determine whether fluidization of membranes by aliphatic alcohols and elevated incubation temperatures could alter receptor binding. We also wished to learn whether such changes were affected by age. We incubated crude synaptomal fractions obtained from mouse whole-brain homogenates at room temperature with the muscarinic cholinergic antagonist [3H]-QNB. The addition in vitro of aliphatic alcohols decreased the total specific (high- plus low-affinity) binding and increased proportionately the low-affinity binding determined in the presence of the unlabeled agonist carbachol. This could be interpreted as a direct effect of alcohols on the receptor molecules by changing them from a high- to a low-affinity conformation (Birdsall et al., 1978, 1980; McKinney and Coyle, 1982). Alternatively, this change could be induced indirectly by alterations in the membrane microenvironment of the receptors. Increasing the incubation temperature greatly increased low-affinity binding in the presence of carbachol. When the effects of hexanol and increased temperature were combined, the increase in [3H]QNB binding was much more limited at lower temperatures and markedly diminished at higher temperatures. There appeared to be an optimal degree of membrane fluidization near the body temperature without hexanol and at lower room temperature when membranes were further fluidized with hexanol. When fluidization with hexanol and with temperature were combined, the temperature for optimal binding was lowered to 31 °C. Higher degrees of membrane disorder, whether induced by hexanol or temperature, or both, caused a rapid decrease of binding below the baseline values. The homogenates from older mouse brains appeared to be more resistant to the disordering effect of hexanol, perhaps because their membranes were inherently more rigid. The relationships between membrane structure, receptor binding, and aging could be conceptualized as a triangle, each factor affecting the other two. The effects of membrane fluidization with either alcohols or temperature on muscarinic receptor binding have been studied previously. Gurwitz and Sokolovsky (1980) used 50 °C for 5 minutes before assay incubation (25 °C) and found a 10- to 12-fold decrease in agonist, but not antagonist,

BRAINMUSCARINICRECEPTORSIN AGING

43

binding in mouse medulla-pons and cerebellum, but not in cortex. E1-Fakahany and Richelson (1980) found that muscarinic receptor activation depended on temperature. Wei and Sulakhe (1982), who used a technique similar to the one described here, reported that cardiac muscarinic receptor binding at 37 °C, compared with 10 °C incubation temperatures, promoted conversion from high to low agonist affinity state receptor sites. However, heart and brain receptors may be different (Gibson et al., 1983). Under appropriate conditions temperature and aliphatic alcohol's greater than ethanol increase the rate of binding to nicotinic receptors (E1-Fakahany et al., 1983). Aging is associated with a decrease of shorter fatty acids, an increase of longer fatty acids, and a decrease in unsaturated fatty acids in brain phospholipids (Armbrecht et al., 1983; Rouser and Yamamoto, 1969). This would tend to decrease membrane fluidity or increase viscosity. Direct measurements of microviscosity in mouse-brain membranes of several brain regions confirm that microviscosity increases as age advances (Samuel et al., 1982). This increased membrane rigidity with advancing age also results in lesser disordering of membranes by ethanol in vitro (Armbrecht et al., 1983). The fact that the alcohols and their concentrations in these experiments are unphysiological is not considered important, because some physiological substances might alter the membrane order only in the restricted vicinity of the receptors in the same way that hexanol indiscriminately fluidizes all membranes everywhere. Physiologically relevant concentrations of ethanol in vitro do not change receptor binding. This probably means that changes in receptor binding are not related to acute intoxication. It does not mean, however, that prolonged in vivo exposure to ethanol could not change the membrane microenvironment of receptors and their function. All evidence taken together, aging appears to be associated with increased baseline membrane viscosity and with increased resistance to fluidization of membranes by alcohols. An increase in binding of low-affinity muscarinic receptors was induced by membrane fluidization with alcohols and with increasing incubation temperatures. This alcohol-induced increase in binding diminished with advancing age. We believe that this may be a link between age-related changes in membranes, receptors, and learning behavior. Acknowledgment-This study was supported by the Medical Research Service of the Veterans Administration.

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