inhibition of rat liver cholesterol esterase by local anaesthetics

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Page 1: Inhibition of rat liver cholesterol esterase by local anaesthetics

68

Biochimica et Biophysics Acta, 409 (1975) 68-74 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 56681

INHIBITION OF RAT LIVER CHOLESTEROL ESTERASE BY LOCAL ANAESTHETICS

JOHN R. TRAYNOR and HANS KUNZE

Department of Biochemical Pharmacology, Max-Planck-Institute for Experimental Medicine, Hermann-Rein-Strasse 3, 34 Gb: ttingen (G. F. R.)

(Received April 29th, 1975)

Summary

1. A number of local anaesthetics was shown to inhibit rat liver cholester- ol esterase activity towards radioactively labelled cholesterol oleate. The anaes- thetics inhibited in the order dibucaine > chlorpromazine > tetracaine > benzocaine > procaine > lidocaine > cocaine.

2. The mode of inhibition was seen to be non-competitive with respect to the substrate and is probably independent of any involvement of Ca”.

3. The inhibition by tetracaine is partially reversed by sodium deoxycho- late. However, all ionic and non-ionic detergents studied, sodium deoxycholate, sodium taurocholate, Triton X-100, and cetyltrimethylammonium bromide are capable of inhibiting the rat liver cholesterol esterase in a concentration depen- dent manner. Only sodium taurocholate stimulates enzymic activity.

Introduction

Local anaesthetics are capable of inhibiting various enzymes [ 11 . Among lipid metabolising enzymes both phospholipases A [ 2-41 and C [3] are in- hibited and dibucaine, a potent local anaesthetic, inhibits the hormone-stimu- lated lipolysis in fat cells of rat adipose tissue [5]. Also, we have recently shown that triglyceride lipase activity in homogenates of bovine seminal vesi- cles can be inhibited by certain local anaesthetics [2]. In order to further extend studies of the interaction of this class of drugs with lipolytic enzymes we have investigated their effects on the cholesterol esterase (EC 3.1.1.13) of rat liver, an enzyme occurring in the particle free cytoplasm of the cell [6] and being responsible for the immediate metabolic fate of almost all chylomicron cholesterol ester [ 71. Despite alffering from the disadvantage of being extreme- ly unstable, this enzyme requires no co-factors for optimal activity [6], which makes for clearer interpretation of results. Furthermore, local anaesthetics are

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known to be hydrolysed by microsomal esterases in the liver [8] but not by the particle-free cytoplasm (own observation).

Materials and Methods

Enzyme source Liver homogenates were prepared from fed 160 to 250 g female rats of

the Wistar strain essentially by the method of Deykin and Goodman [6]. For experiments reported in this paper the 100 000 X g supernatant fraction, freed from floating fat layer, was routinely employed. Protein was determined by the Biuret method [ 91 using bovine serum albumin (Armour Pharmaceutical Corp., Chicago, U.S.A.) as standard.

Substances All reagents and solvents used were of analytical grade. Cholesterol [l-

’ 4 C] oleate (20 mCi/mmol) purchased from Amersham Buchler (Braunschweig, G.F.R.) was diluted with unlabelled cholesterol oleate (Serva, Heidelberg, G.F.R.) to 485 dpm per nmol; [4-14 C] cholesterol oleate (55.5 mCi/mmol) purchased from New England Nuclear (Boston, Mass., U.S.A.) was diluted to 5309 dpm per nmol. Samples of the local anaesthetics benzocaine, procaine and tetracaine were gifts from Hoechst (Frankfurt, G.F.R.), chlorpromazine from Bayer (Leverkusen, G.F.R.), cocaine from E. Merck (Darmstadt, G.F.R,), lidocaine from Pharma-Stern (Wedel, G.F.R.), and dibucaine from Ciba-Geigy (Basel, Switzerland); see reference [2] for the structures of these compounds. Dimilume scintillation fluid was purchased from Packard Instruments Co. (Warrenville, U.S. A.).

Enzyme assays Cholesterol esterase activities were measured with [4-l 4 C] cholesterol

oleate or cholesterol [l- ’ 4C] oleate as substrate. The incubation medium, un- less otherwise stated, consisted of 35 mM potassium phosphate buffer (pH 7.45), 750 (~1 of the 100 000 X g supernatant with 23-27 mg protein, and 44-220 PM substrate, added in 50 ~1 of acetone, in a total volume of 2 ml. Incubations were carried out for 45 min at 37°C in a shaking water bath. Blanks without enzyme were included in all experiments. For kinetic assays a higher concentration of acetone (100 ~1) was employed, however, this had no effect on the activity.

Assays utilising [ 4- ’ 4 C] cholesterol oleate as substrate were stopped and extracted according to Goller and Sgoutas [ 111, and the extracts subjected to thin-layer chromatography on silica gel H with hexane/diethyl ether/acetic acid

(83 : 16 : 1, by vol.). Areas corresponding to cholesterol oleate and free cholesterol were scraped off and transferred directly into scintillation vials, and suspended in a mixture of 13 ml Dimilume and water (10 : 3, by vol.).

For most routine assays cholesterol [l-l 4C] oleate was used as substrate and enzyme activity determined by assaying the unchanged ester. In these cases reactions were terminated by addition of 10 ml isopropanol/heptane/ 1 M po- tassium hydroxide (40 : 10 : 1, by vol.) to the whole reaction mixture with a final pH of about 11. Unlabelled cholesterol oleate (1 mg) was added to each

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sample as carrier material. After standing at room temperature for 30 min 6 ml heptane and 6 ml water were added for phase separation. The heptane layers, containing unchanged cholesterol ester, were transferred directly to scintilla- tion vials and the aqueous phases washed twice with 3 ml heptane. The com- bined washings from each sample were added to the original extract and the solvent removed at 40°C under reduced pressure. The residues were dissolved in 10 ml Dimilume for counting. Control experiments showed the overall recovery of the labelled steroid ester to be greater than 98% whilst more than 99% of the released [l- ’ 4 C] oleic acid remained in the aqueous phase.

Radioactivity measurements were carried out using a Beckman LS 250 scintillation spectrometer and corrected for quenching by means of an external standard.

Enzyme activities were corrected for non-enzymatic hydrolysis, which did not exceed 3%, and expressed as initial velocities in pmol substrate hydrolysed per mg protein per min. The rate of reaction was linear within the time of incubation and protein concentrations employed. Groups of data for direct comparison were obtained on the same day with the same enzyme preparation since the specific activities of preparations varied, and storage at +4”C or -20°C resulted in a rapid loss of enzyme activity.

Results

At a concentration of 1 mM all seven local anaesthetics tested inhibited the enzymic hydrolysis of labelled cholesterol oleate by the rat liver cholesterol esterase (Table I). The inhibitory potency of the drugs decreased in the order dibucaine > chlorpromazine > tetracaine > benzocaine > procaine > lidocaine > cocaine. Parallel dose response curves were obtained for tetracaine, in which the aromatic lipophilic and tertiary amine moieties of the local anaesthetic molecule are joined by an ester grouping, and dibucaine, which has an amide link. None of the local anaesthetics stimulated hydrolysis at concentrations below inhibitory values.

TABLE I

RELATIVE INHIBITORY ACTIVITY OF LOCAL ANAESTHETICS ON THE HYDROLYSIS OF CHOLESTEROL [1-14Cl OLEATE BY RAT LIVER 100000 X B SUPERNATANT

The complete reaction mixture (2 ml) contained 35 mM potassium phosphate buffer (PH 7.45). 750 ~1 high-speed supernatant (with 24 mg protein), 11 or 50 PM cholesterol [l-14Cloleate (added in 50 !~l acetone), and 1 mM local anaesthetic. Incubations were carried out at 37’C for 45 min. The data represent means of duplicates.

Local anaesthetic

Dibucaine Chlorpromazine Tetracaine Benzocaine Procaine Lidocaine Cocaine

% Inhibition at:

11 PM cholesterol oleate

100 91 45 24 15 14 10

50 PM cholesterol oleate

100 98 45 21 20 18 11

Page 4: Inhibition of rat liver cholesterol esterase by local anaesthetics

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Tetracame

0’ 1 I

10-d 10-3 Local anaesthetic CM)

I

lCJ2

Fig. 1. Dose-response curves for the effect of tetracaine and dibucaine on the hydrolysis of cholesterol [l-14Cloleate by rat liver 100 000 X g supernatant. In a total volume of 2 ml each reaction mixture contained 35 mM potassium phosphate buffer (PH ‘7.46). 26.7 mg protein, 50.7 PM cholesterol [l- 14Cl oleate (added in 50 ~1 acetone) and the local anaesthetics as indicated. Incubations were carried out for 45 min at 3VC.

The mode of inhibition was studied at varying substrate concentrations using tetracaine 1 mM as inhibitor. The results, presented as a double reciprocal plot [ll] , show the inhibition to be non-competitive with respect to the sub- strate (Fig. 2). An apparent Km of 83 PM and V of 30 pmol of cholesterol oleate hydrolysed per mg of protein per min were calculated for the enzyme preparation and a Ki for tetracaine of 1.49 mM [ 121. However, since the actual concentration of cholesterol oleate in true solution remained an undetermined factor the values of Km and Ki were calculated with no consideration of particle size. All of the remaining local anaesthetics inhibit in a similar manner to tetracaine, per cent inhibition being independent of the substrate concentra- tion (Table I). Ca”, 5 mM, had no effect on the inhibition caused by tetra- Caine.

Since, at the pH of the incubation mixture the local anaesthetics exist mainly in the positively charged form [l] it was of interest to see if the inhibition could be reversed by negatively charged surface active agents. Thus,

‘/[S] (/.i M-‘)

Fig. 2. Effect of tetracaine on the hydrolysis of [4-14C1 cholesterol oleate at various substrate concentra- tions. The complete reaction mixture (2 ml) contained 35 mM potassium phosphate buffer. 26.6 mg protein and 22 to 111 MM [4- 14C1 cholesterol oleate (added in 100 1.t1 acetone) m without, or -. with. 1 mM tetracaine. Incubations were carried out for 45 min at 37’C. Points represent means of duplicates. The lines are calculated for best fit.

Page 5: Inhibition of rat liver cholesterol esterase by local anaesthetics

-20’ 4 I ,

0 10-4 10-3 I

5x10-j

Bde sait (MI

Fig. 3. Effects of the bile salts sodium deoxycholate and sodium taurocholate on the hydrolysis of cholesterol [1-14Cl oleate by rat liver 100 000 X g supematant in the presence and absence of tetracaine. The complete reaction mixture (2 ml) contained 35 mM potassium phosphate buffer (PH 7.45). 750 ~1 supematsnt (with 25.5 mg protein). 58.6 PM cholesterol [1-‘4Cl oleate (added in 50 ~1 acetone), and the indicated amounts of sodium deoxycholate O-.--O. and sodium taurocholate A- A. in the absence, or in the presence (-0 and A- A. respectively) of 1 mM tetracaine.

although both tetracaine and sodium deoxycholate separately inhibit the en- zymic hydrolysis of cholesterol oleate together they are antagonistic, until above 1 mM bile salt concentration a rapid increase in inhibition occurs up to maximum. By comparison sodium taurocholate shows stimulation of enzyme activity at low concentrations but above 1 mM exhibits a reversal to an inhibi- tory effect both in the presence and absence of tetracaine, with apparently no antagonism between the bile salt and the local anaesthetic (Fig. 3).

Cetrimide (cetyltrimethylammonium bromide) a cationic detergent com- pletely inhibits the enzymic hydrolysis over a range of 10d4 M-5 * lo-” M, whilst Triton X-100, a non-ionic detergent shows increasing inhibition with increasing concentrations. Addition of varying concentrations of Triton X-100 in the presence of 1 mM tetracaine results in increased inhibition (Fig. 4).

r--f6 I

0 0.001 0.01 0.1 % Trlton X-100 (w/vOI)

Fig. 4. The influence of Triton X-100 or7 the hydrolysis of cholesterol [1-“C] oleate by rat liver 100 000 X g supematant, in the absence and presetice of tetracaine. The complete reaction mixture (2 ml) con- tained 35 mM potassium phosphate buffer (PH 7.45). 23 mg protein. 50.2 MM cholesterol [1-14C1 oleate (added in 50 ~1 acetone), and the indicated amounts of Triton X-100 in the absence si----‘>, or in the presence o----.-o of 1 mM tetracaine.

Page 6: Inhibition of rat liver cholesterol esterase by local anaesthetics

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Discussion

Various local anaesthetics have been shown to inhibit the activity of rat liver cholesterol esterase in concentrations similar to those required for local anaesthesia. A close parallelism exists between the anaesthetic potency of these compounds and their ability to inhibit cholesterol esterase [ 1,131.

The drugs, regardless of structure, inhibit the enzymic reaction by a non- competitive mechanism. Although results of inhibition studies for enzymes acting at interfaces should be treated with caution [14] our findings suggest that interaction of the enzyme with the local anaesthetics is independent of the substrate and that the drugs do not alter the physical binding of the substrate to the enzyme. The inhibition appears to be independent of any involvement of Ca”. In contrast the inhibition of various phospholipases by local anaesthetics is competitive with Ca2’ [3,4] but not with substrate [ 41 whilst Ca” has also been implimented in the inhibition of hormone-stimulated lipolysis in rat fat cells by the anaesthetic dibucaine [ 51. However, whilst Ca” is involved in the mechanism of action of phospholipase AZ, phospholipase C, and the hormone- stimulated lipase it has, in accordance with earlier observations [6], no effect on rat liver cholesterol esterase activity.

Under the conditions employed cationic (Cetrimide), anionic (bile salts) and non-ionic (Triton X-100) detergents are capable of inhibiting the enzyme in a concentration dependent manner. Cetrimide, which like the charged form of the local anaesthetics has a quaternary nitrogen head group, is the most potent. Although it is difficult to clearly define the mechanisms by which the various detergents exert their effects some features can be described. Thus the rat liver cholesterol esterase with an alkaline pH optimum appears to differ from the acid cholesterol esterases of human and calf liver which are stimulated by Triton X-100 [ 151. The negatively charged detergents sodium deoxycholate and sodium taurocholate, above 1 mM, show common effects on the enzymic hydrolysis of cholesterol oleate both in the presence and absence of tetracaine. Such effects may be explained by the formation of bile salt micelles which are known to exert strong inhibitory effects on various lipase enzymes [ 161. The different effects shown by the two bile salts below their critical micellar con- centrations could be caused by dissimilar effects on the substrate or on the oil-water interface [14], or possibly by a differential binding of the 3a,l2cu- dihydroxy (deoxycholate) and the 3a,7a,l2a-trihydroxy (taurocholate) bile salts to the enzyme protein, as has been suggested for the pancreatic enzyme [ 17,181.

In the light of conflicting reports on the effects of bile salts on the cholesterol esterase of rat liver [ 191 and other cholesterol esterase [ 141, fur- ther work on standardised systems is necessary to explain more precisely the role played by bile salts in the hydrolytic activity of these enzymes and wheth- er this role is related to the antagonism of local anaesthetic-mediated inhibition of the rat liver enzyme.

References

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