General anesthetics can competitively interfere with sensitive ...

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anesthetics with proteins was shown for bacterial and firefly luciferase, which are soluble proteins free of lipid (2, 3), For the bacterial luciferase the anesthetics ...

Proc. Nati. Acad. S(i. USA Vol. 84, pp. 5972-5975, August 1987 Neurobiology

General anesthetics can competitively interfere with sensitive membrane proteins (glioma C6 cells/NaI/KI/CL- cotransporter/halothane/l-alkanols)

PIET W. L. TAS*, HANS G. KRESSt, AND KLAUS KOSCHEL* *Institute for Virology and Immunology, University of Wbrzburg, Versbacher Strasse 7, D-8700 Wirzburg, Federal Republic of Germany; and Anaesthesiology, University of Wuirzburg, Josef-Schneider-Strasse 2, D-8700 Wurzburg, Federal Republic of Germany

tInstitute for

Communicated by W. K. Joklik, April 27, 1987

ABSTRACT It is not known whether proteins or lipids are the primary target of anesthetic action. The resolution of this problem is hampered by the fact that it is not possible to investigate the biological activity of integral membrane pro-

isoflurane, and methoxyflurane were from Abbott (Wiesbaden, Federal Republic of Germany). The 1-alkanols and furosemide were from Sigma. All other chemicals were from Merck (Darmstadt, Federal Republic of Germany). Cell Culture. C6 rat glioma cells (ATTC CCL107) (5) were cultured in plastic tissue culture flasks (Nunc, Roskilde, Denmark) in Dulbecco's modified Eagle's medium (DMEM) (GIBCO) with 10%o (vol/vol) fetal calf serum (GIBCO) in a 10% C02/90% humidified air atmosphere and were passaged by trypsinization (0.25% trypsin). 86Rb+-Uptake Experiments. Permanent cultures of rat glioma C6 cells on 3-cm Petri dishes were preincubated for 15 min at 360C in 150 mM NaCl/5 m KCI/2 mM CaCl2/0.4 mM MgSO4/25 mM glucose/25 mM Hepes Tris, pH 7.3, followed by a 10-min incubation in hyperosmolar medium (same medium with 250 mM sorbitol) containing 1 ,uCi/ml 86Rb+. The reaction was stopped by rinsing the dishes three times with 2 ml of ice-cold PBS (140 mM NaCl/2.68 mM KCl/8.33 mM Na2HPO4/1.15 mM KH2PO4/1.14 mM CaC12/0.49 mM MgCl2, pH 7.2). The cells were then denatured with 1 ml of cold 5% (wt/vol) trichloroacetic acid and centrifuged, and the amount of radioactivity in the supernatant was determined by Cerenkov counting. The protein content of each dish was determined using the method of Lowry et al. (6). Na+/K+/Cl- cotransporter activity was determined as the loop diuretic (e.g., 1 mM furosemide or 10 ,uM bumetanide)sensitive part of the 86Rb+ uptake (7). For measurements in the presence of volatile anesthetics, the petri dishes were incubated in an air-tight-plastic gas flow chamber on a rocker platform under a flow of air supplemented with the desired anesthetic concentration using vaporizers specific for that anesthetic (Dragerwerk, Lubeck, Federal Republic of Germany). At the start of the measurement, hyperosmolar medium with 86Rb+, preequilibrated for 15-30 min with anesthetic by vigorously bubbling the medium with an air stream containing the anesthetic, was added to the dishes. The time needed to saturate the medium with anesthetic was determined for each anesthetic using gas chromatographic detection (8). The concentration of the anesthetic in the air was checked with an EMMA analyzer (Engstr0m multigas monitor for anesthetics, Gambro Engstr0m AB, Bromma, Sweden). For concentrations exceeding the range of the vaporizer, the anesthetics were added as solution to the incubation medium and dissolved by stirring. The 1-alkanols and the anesthetics diethyl ether, chloroform, and trichloroethylene were also added as solution to the incubation medium and dissolved by stirring in a closed glass flask. For very volatile substances such as diethyl ether and chloroform a calculated amount of these substances was added in a glass Petri dish to the air-tight chamber to obtain the corresponding anesthetic concentration in the air.

teins in the absence of lipids. However, certain characteristics of membrane protein function inhibition by anesthetics cannot be explained on the basis of an indirect inhibition by disturbance of the lipid bilayer and, therefore, most likely are the result of a direct anesthetic-protein interaction. This is the case (i) when the anesthetics competitively interfere with the binding of an endogenous ligand to the membrane protein and (ii) when the size of the anesthetic molecule is Qf importance for the potency and/or mechanism of inhibition. The present study shows that this is true for a membrane transport system, the Na+/K+/Cl- cotransport in glial-type cells.

Richards et al. (1) have proposed that direct interactions of general anesthetics with hydrophobic areas of target proteins are responsible for the induction of the anesthetic state. They further suggested that small molecules may interact with one set of hydrophobic sites and larger molecules with a different set of hydrophobic sites. A direct interaction of general anesthetics with proteins was shown for bacterial and firefly luciferase, which are soluble proteins free of lipid (2, 3), For the bacterial luciferase the anesthetics competed with alkyl aldehyde binding and for the firefly luciferase the anesthetics competed with luciferin binding to the enzyme. On the basis of these data Franks and Lieb (2) have speculated that general anesthetics interfere competitively with the function of sensitive membrane proteins by binding to hydrophobic pockets on these molecules. The only evidence so far that fits this speculation is the allosteric inhibition of the acetylcholine receptor by barbiturates; however, other general anesthetics inhibit this receptor by different mechanisms (4). It is necessary, therefore, to continue the search for membrane functions sensitive to clinical concentrations of general anesthetics, as well as to determine whether these functions are inhibited in a competitive or noncompetitive manner. Due to the presence of lipid it is not always possible to decide whether certain anesthetics interfere directly with protein or not. A detailed study of the effect of anesthetics on the Na+/K'CVcotransporter of rat glioma C6 cells shows that besides the competitive interference other characteristics can be found that suggest that anesthetics can directly interact with membrane proteins and, thereby, inhibit their function.

MATERIALS AND METHODS Materials. Rubidium-86 (specific activity, 2 mCi/mg; 1 Ci = 37 GBq) was obtained from New England Nuclear; halothane was from Hoechst (Frankfurt); enflurane,

RESULTS AND DISCUSSION Inhibition of the Na+/K+/Cl- Cotransporter by Volatile Anesthetics. We have studied the influence of general anes-

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thetics on the Na+/K+/Cl- cotransporter of rat glioma cells (7). This membrane protein simultaneously transports 1 Na+, 1 K+, and 2 Cl- ions into the cell. The energy for this transport is provided by the sodium gradient, which is maintained by the Na+/K+ ATPase (9). Na+/K+/Cl- cotransport has also been demonstrated in primary rat (unpublished data) and human (10) astrocyte cultures. Evidence suggests that this cotransporter is involved in the control of cell volume and in the regulation of potassium homeostasis in the extracellular space surrounding neuronal cells (8, 11). Cotransport activity was measured as the furosemide-sensitive part of the 86Rb+ [as substitute for potassium (12)] uptake into the rat glioma C6 cells. To rule out possible nonspecific effects of furosemide, we have performed some of the experiments also with bumetanide, which is considered to be a more specific inhibitor of the Na+/K+/Cl- cotransporter. Similar results were obtained with this inhibitor. To increase the furosemide sensitive part of the total 86Rb+ uptake, hyperosmolar (550 mosM) medium was used, which increases the activity of the cotransporter 5-fold compared with normal (300 mosM) medium (Fig. 1). Fig. 2 shows the effect of 1, 2, or 4% (vol/vol) halothane on the activity of the Na+/K+/Clcotransporter. The cotransporter was significantly inhibited at clinical concentrations of the anesthetic (33% inhibition at 1% halothane; corresponding with 0.40 mM halothane in the uptake buffer). Removal of the halothane atmosphere and replacement of the halothane-containing incubation medium by a halothanefree medium led to a rapid reversal of cotransport activity (Fig. 2). In contrast with halothane, other commonly, used volatile, halogenated anesthetics such as enflurane and methoxyflurane only inhibited cotransporter function far above clinically relevant concentrations (Table 1). Similar results were obtained with primary rat astrocytes (data not shown). Inhibition of Na+/K+/Cl- Cotransport by 1-Alkanols. Since the halothane molecule is rather small compared to the above mentioned volatile anesthetics, we hypothesized that the molecular size of the anesthetic might be an important parameter in cotransporter inhibition. To study this in more detail we tested the inhibitory action of a homologous series of 1-alkanols on cotransporter function in rat glioma cells. Interestingly, the smaller anesthetics (methanol, ethanol, 1-propanol, 1-butanol and 1-pentanol) inhibited cotransporter functions at concentrations that correlate very well with the concentrations that anesthetize 50% of the tadpoles in an established assay system

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%o Halothane FIG. 2. Effect of the general anesthetic halothane on the activity of the NaI/KI/CL- cotransporter in rat glioma C6 cells. Monolayers of rat glioma C6 cells were preincubated for 10 min at 360C in normal (300 mosM) medium followed by a 10-min incubation in hyperosmolar medium containing 86Rb+ at 1 ,tCi/ml in the presence or absence of different concentrations of halothane. Bar R shows the return of cotransport activity upon removal of halothane. For this experiment cells were preincubated in normal medium in the presence of 1% halothane followed by a 10-min incubation in hyperosmolar medium containing 16Rb+ at 1 uCi/ml in the absence of halothane. Cotransport activity was determined as the 1 mM furosemide-sensitive part of the total 86Rb+ uptake. The activity of the NaI/KI/Cl- cotransporter in the absence of halothane was taken as 100%, and activity in the presence of the anesthetic was calculated as percent of the uninhibited activity. Data are means ± SD of three experiments performed in triplicate.

(Fig. 3). For molecules larger than 1-pentanol, this correlation ceased to exist. This finding suggested that, besides lipid solubility, the size of the anesthetic molecule itself is an important parameter for inhibiting the Na+/K+/Clcotransporter. A similar cut-off phenomenon dependent on the size of anesthetics was also observed for the alkane binding site of P-lactoglobulin (16). The occurrence of a cut-off in the alkanol series for molecules larger than 1-pentanol cannot be explained on the basis of decreased lipid solubility (17) and, 0

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FIG. 1. Stimulation of the furosemide-sensitive component of the

"6Rb+ uptake by hyperosmolar medium. Monolayers of rat glioma C6 cells on 3-cm Petri dishes were allowed to take up 86Rb+ for 10 min at 360C in the presence or absence of 1 mM furosemide in normal medium (300 mosM) or hyperosmolar medium containing 300 mM sorbitol. Data are means ± SD of triplicate determinations. Open bar, total 86Rb+ uptake; hatched bar, 86Rb+ uptake sensitive to 1 mM furosemide; mosm, mosM.

FIG. 3. Relation between general anesthetic concentrations needed to anesthetize 50% of tadpoles (ED,,() and the concentrations that inhibit NaI/KI/Cl- cotransport by 50%. The data are plotted as potencies, defined as reciprocals of aqueous ED5j( concentrations (M-l). The line through the origin represents the line of identity for which the anesthetic concentration is identical to the concentration that inhibits NaI/K/Cl-' cotransport by 50% (IC5(). The ICs5s for cotransporter inhibition by 1-alkanols and acetone were determined by testing several concentrations of these compounds from which an ICs,, was extrapolated. The animal potency data of most compounds were from table 5 of ref. 14. The anesthetic potency of 1-pentanol was extrapolated from the same table using the membrane/buffer partition coefficient of table 1 of ref. 13. The anesthetic potency of halothane was from ref. 15. 1-Alkanols are indicated by numbers: 1, methanol; 2, ethanol; . 8, octanol. A, acetone; H, halothane.

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Table 1. Comparison of the concentrations of several general anesthetics that anesthetize 50% of the patients and that inhibit Na+/K+/Cl- cotransport by 50% MAC equivalent,t MAC value,* IC50 for Na+/K+/Clcotransport inhibition, mM mM % vol/vol Anesthetic 44 9.7 1.9 Diethyl ether 10l 0.52 1.7 Enflurane 10l 0.34 1.40 Isoflurane 9 0.50 0.79 Chloroform 10 0.28 0.16 Methoxyflurane 22 0.13 0.2 Trichloroethylene 0.70 0.24 0.75 Halothane *The anesthetic potency data of the compounds for humans, expressed as minimum alveolar concentration (MAC) values, were from table 6 of ref. 13. tThe MAC values were recalculated in aqueous concentrations using the water/gas distribution coefficients (from ref. 13) of the various compounds. tThe IC50 values for enflurane and isoflurane were estimated from data at lower concentrations, since high concentrations of these anesthetics are toxic for the cells.

therefore, strongly suggest that anesthetics interact with a hydrophobic region of circumscribed dimension of the cotransporter molecule. Not all anesthetics smaller than 1hexanol fit in such a region of the cotransporter. Acetone and halothane (Fig. 3) apparently fit, but not chloroform and trichloroethylene (Table 1). This fact can be explained by assuming that the molecular shape or sterical factors prevent these anesthetics from binding. How Do Anesthetics Inhibit Cotransport Activity? Because Na+/K+/Cl- cotransport is inhibited by loop diuretics that are assumed to interact competitively with Cl- for binding to the cotransporter molecule (18, 19), we investigated whether general anesthetics interfere in a similar way with cotransport function. With higher Cl- concentration, we consistently observed a progressive decrease in the Inhibitory action of 30 mM 1-propanol (Fig. 4A, Inset), which suggested a competitive type of interaction. However, the inhibitory action of 10 mM 1-hexanol on cotransporter activity was not affected by the Clconcentration (Fig. 4B, Inset), which suggested a noncompet-

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itive type of inhibition. To decide whether the inhibition was competitive or noncompetitive, modified Lineweaver-Burk plots (20, 21) for enzyme functions having two substrate binding sites were constructed. The involvement of two chloride ions in the cotransport process was strongly suggested by Hill plots for Na+, K+, and Cl- that showed a stoichiometry of 1:1:2 (unpublished result). A Lineweaver-Burk plot of 1/v vs. 1/[CIF gives linear curves (Fig. 4). The intercept with the ordinate gives the Vmax of the cotransport activity. At infinite Cl- concentration, 1-propanol did not affect the Vmax of the reaction, suggesting competitive inhibition (Fig. 4A); however, 1-hexanolas representative of the group of molecules that do not inhibit the cotransporter according to its anesthetic potency-contributed a noncompetitive type of inhibition. A similar competitive inhibition of cotransport activity was also found for the volatile anesthetic halothane (Fig. 5). Measurement of the cut-off in inhibitory potency in the 1-alkanol series provided interesting data. It was found that, at clinically relevant concentrations, only anesthetics that fit into a hydrophobic region can interfere

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FIG. 4. Competitive inhibition of propanol (A) and noncompetitive inhibition of hexanol (B) of the furosemide-sensitive 86Rb+ uptake into rat glioma C6 cells. (A, Inset) Furosemide-sensitive 86Rb+ uptake (nmol per mg of protein per min) in the absence (0) and presence (A) of 30 mM propanol. (B, Inset) Furosemide-sensitive IRbV uptake (nmol per mg of protein per min) in the absence (e) and presence (A) of 10 mM hexanol. All 86Rb+ uptake measurements in the presence and absence of anesthetic were performed with and without 1 mM furosemide to obtain the furosemide-sensitive part of the total IRbV uptake. Incubation was performed as described in the legend to Fig. 1. The chloride ion was replaced by gluconate to obtain incubation medium with various Cl- concentrations, keeping the concentration of the other ions constant. A and B are Lineweaver-Burk plots of the reciprocal of 86Rb+ transport activity 1/v against 1/[CI-]2 in the presence and absence of anesthetics [units for v are nmol per mg of protein per min]. Data are duplicate measurements. The experiment presented in A was performed five times, and the experiment presented in B was performed three times with basically the same results. Data are means ± SD of duplicate measurements.

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Pharmaceuticals (Ballerup, Denmark) for a kind gift of bumetanide. This work was supported by a grant from the Wilhelm Sander-Stiftung, Neustadt/Donau, Federal Republic of Germany (83.007.2).

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