Nonanesthetic Alcohols Dissolve in Synaptic Membranes Without Perturbing Their Lipids (Anesthesia Cut-Off/Rana Pipiens/Torpedo/Lipid Order Parameter) K
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Proc. Natl. Acad. Sci. USA Vol. 86, pp. 1084-1087, February 1989 Neurobiology Nonanesthetic alcohols dissolve in synaptic membranes without perturbing their lipids (anesthesia cut-off/Rana pipiens/Torpedo/lipid order parameter) K. W. MILLER*tt, L. L. FIRESTONE*t§, J. K. ALIFIMOFFt, AND P. STREICHERt Departments of *Biological Chemistry and Molecular Pharmacology and of tAnesthesia, Harvard Medical School and Massachusetts General Hospital, Boston, MA 02114 Communicated by Frank Brink, Jr., November 16, 1988 (received for review July 25, 1988) ABSTRACT While many theories of general anesthesia and tetradecanol (4, 8, 9). However, it is possible that the postulate a lipid site of action, there has been no adequate cut-off in vivo may have been underestimated because of explanation for the lack of anesthetic potency of the highly experimental conditions. For most anesthetics, the free hydrophobic primary alkanols with more than 12 carbons (the aqueous concentration does not differ significantly from the cut-off). Some work suggests that these nonanesthetic alcohols measured total concentration of anesthetic. However, long- do not dissolve in membranes. Other work contradicts this and chain alcohols are so insoluble in water that significant suggests that an anesthetic site on a protein provides a better depletion of the alcohol may occur by adsorption to the explanation. Here we show that both the anesthetic dodecanol apparatus (10) or uptake into animals, leading to uncertainty and the nonanesthetic tetradecanol are taken up equally well in their final free aqueous concentrations. into the tissues of animals and into isolated postsynaptic To reassess the issues raised by these reports, we have membranes. When a group of Rana pipiens tadpoles were reexamined the cut-off both in vivo and in postsynaptic treated with dodecanol, half were anesthetized by 4.7 /AM (free membranes. First, we have redetermined the position of the aqueous concentration), and the corresponding concentration cut-off for normal alcohols in aquatic animals by using in the tissues was found to be 0.4 mmol per kg wet weight. radiolabeled agents to monitor both the free aqueous con- Prolonged exposure (92 hr) to tetradecanol produced even centration to which the animals were exposed and the uptake higher tissue concentrations (0.7 mmol per kg wet weight), yet of the anesthetic and nonanesthetic alcohols into their tis- no anesthetic effects were observed. Furthermore, general sues. Second, we reexamined the ability of long-chain alco- anesthetics are thought to act on postsynaptic membranes but hols to partition into and disorder postsynaptic membranes. both alkanols partitioned into postsynaptic membranes from For this work, we chose to use high specific activity acetyl- Torpedo electroplaques. The spin label, 12-doxyl stearate, was choline receptor-rich membranes purified from Torpedo incorporated into these membranes. The lipid order parameter electroplaques, whose lipid composition (11) is broadly it reported was decreased by the anesthetic alcohols (octanol, similar to that of the central nervous system (12), rather than decanol, and dodecanol), whereas the nonanesthetic alcohols which are of either did not change it significantly (tetradecanol) or actually postsynaptic membranes from brain, heteroge- increased it (hexadecanol and octadecanol). Thus, although neous origin. lipid solubility is unable to account for the pharmacology ofthe cut-off in potency of the long-chain alcohols, lipid perturba- EXPERIMENTAL METHODS tions provide an accurate description. Anesthetic Concentration (EC50) and Total Alcohol Concen- tration in Tadpoles. Rana pipiens tadpoles were exposed to The structural diversity of general anesthetics has led to the anesthetics in oxygenated aqueous solutions of '4C-labeled development of theories which assume that anesthetics must alkanols (ICN and New England Nuclear) and their ability to dissolve in and perturb the lipid bilayer of excitable mem- Animals were branes rather than bind to a site on a protein, and which right themselves was tested as described (13). predict that all lipophilic compounds will be anesthetics (for observed until a stable response level had been obtained and reviews see refs. 1-3). Thus, it has always been puzzling that were then killed, weighed, dissolved in BTS-450 (Beckman), highly lipophilic primary alcohols with more than 12 carbons and stored so as to minimize chemiluminescence, and their are not general anesthetics (4) (an effect referred to as the alkanol content was assayed by liquid scintillation spectros- cut-off in anesthetic potency). One possible explanation for copy. the cut-off is that the nonanesthetic alcohols are not taken up Alkanol Partitioning into Membranes. Postsynaptic mem- into membranes in sufficient concentration. However, recent branes from Torpedo electroplaques were prepared by dif- work shows that these 1-alkanols do partition into lipid ferential and sucrose density-gradient centrifugation as de- bilayers (5), questioning the previous measurements in which scribed (14). In some experiments, 1 ml of the membrane biomembranes were used (reviewed in ref. 1). suspension was mixed with 4 ml of a saturated aqueous Other explanations for the cut-off are possible. Nonanes- solution of 14C-labeled alkanol, and in other experiments 10 thetic alcohols may dissolve in membrane lipids but not Al of an ethanolic stock solution of radiolabeled alcohol was significantly perturb them (6), or the cut-off in anesthetic added to 1 ml of the membrane suspension (8.8 mg of potency may be due to the specificity of anesthetic-protein protein). These mixtures were layered on top of continuous interactions. Indeed, luciferase, a pure protein sensitive to gradients, made from two cycles of freezing and thawing 1.2 anesthetics, was not inhibited by those alcohols with more M sucrose, and centrifuged to equilibrium (64,000 X g, 4 hr, than 16 carbons (7). In comparison, previous work places the cut-offfor general anesthesia in tadpoles between dodecanol tTo whom reprint requests should be addressed at: Department of Anesthesia, Massachusetts General Hospital, Fruit Street, Boston, MA 02114. The publication costs of this article were defrayed in part by page charge §Present address: Department of Anesthesiology and Critical Care payment. This article must therefore be hereby marked "advertisement" Medicine, University of Pittsburgh School of Medicine, Pittsburgh, in accordance with 18 U.S.C. §1734 solely to indicate this fact. PA 15261. 1084 Downloaded by guest on September 25, 2021 Neurobiology: Miller et al. Proc. Natl. Acad. Sci. USA 86 (1989) 1085 40C), and fractions were collected and assayed for protein aqueous-phase radioactivity (5). To avoid such problems, we and alcohol content. The purified electroplaque membranes did not aim to determine partition coefficients but only to contained 1.5 nmol of acetylcholine sites per mg of protein, determine whether nonanesthetic alkanols could exist in and 400-425 phospholipid and 200-250 cholesterol molecules biomembranes in higher concentrations than those predicted per acetylcholine binding site. by the lipid solubility hypothesis of general anesthesia. After Order Parameter Measurements in Membranes. 12-Doxyl a period ofequilibration, mixtures containing the membranes stearate [12-(2,2-dimethyl-N-oxyloxazolidine) stearate] was and 14C-labeled alkanols were centrifuged down sucrose deposited from a methanolic stock solution by evaporation density gradients (Fig. 1). A peak ofradioactivity, coincident for 6 hr under vacuum. Torpedo electroplaque membranes with the narrow membrane band, was found for both anes- were spin labeled by gently shaking them for 6 hr at 40C with thetic and nonanesthetic alkanols. the spin label. Final probe concentration was <1% of Higher alkanol concentrations were obtained by adding membrane lipids (mol/mol). Membranes were then washed alkanols mixed with ethanol to the same concentration of by three cycles of sedimentation (6000 x g, 10 min, 40C) and membranes as described above and then removing the resuspended in 3 vol of ice-cold Torpedo Ringer's solution ethanol. Similar results were obtained. In one experiment, (14) to minimize signal from unbound spin label. Small ali- [14C]dodecanol was 0.5-0.75 ,LM in the aqueous phase and 70 quots (<25 Al) ofethanolic stock solutions ofeach 14C-labeled mM in membrane lipid (phospholipid plus cholesterol). For alcohol were added to 250 A1 ofthe spin-labeled membranes (4 [14C]tetradecanol, the corresponding figures were 0.1-0.2 mg per ml of protein) and incubated at room temperature for 30 min. Membranes were then washed free of ethanol, as 2.5 indicated by gas chromatography, using three cycles of sedi- mentation (6000 x g, 10 min, 40C), resuspended in equal A volumes of fresh buffer, and loaded into glass capillary tubes (o.d. 2 mm), which were then flame-sealed on ice. Electron spin resonance spectra were obtained at 20.0 ± 0.10C with a 1.5 - Bruker ER200 operating at 9.5 GHz, 3250 G, modulation amplitude of 0.32 G, frequency of 100 kHz and 10 mW of microwave power. Order parameters (S) were calculated by the method ofHubbell and McConnell (15) from the hyperfine splittings, which were corrected for solvent polarity (16). 0.5 Following spectroscopy, the samples were sedimented (6000 X g, 15 min) and the supernatants were discarded. The pellets were dispersed in scintillation cocktail and the 14C-labeled alcohol content was determined. 25r RESULTS B Tetradecanol Is Not an Anesthetic, Although It Is Taken up by Tadpoles. When we exposed groups of tadpoles to [14C]- 15 dodecanol, the aqueous concentration fell by almost 20% x over 2 hr, and the animals reached a steady level ofanesthesia with an EC50 of4.7 ,uM. Exposure to supersaturated solutions of [14C]tetradecanol for up to 92 hr did not cause anesthesia. CU The concentration of dodecanol in tadpoles was found to be 5 - 0.4 mmol per kg wet weight at the anesthetic concentration. The concentration of ['4C]tetradecanol was 0.7 mmol per kg wet weight after 92 hr of exposure, demonstrating that the cut-off cannot be explained by lack of uptake of the alcohols into the tadpoles (Table 1).