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Local anesthetics: Stimulation of incorporation of inositol into phosphoinositides in guinea pig cerebral cortical synaptoneurosomes
Biochimica et Biophysica Acta, 1054 (1990) 213-218
213
Elsevier BBAMCR 12746
Local anesthetics" stimulation of incorporation of inositol into phosphoinositides in guinea pig cerebral cortical synaptoneurosomes Yukio Nishizawa *, Fabian Gusovsky and John W. Daly Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestioe and Kidney Diseases, National Institutes of Health, Bethesda, MD (U.S.A.)
(Received 8 November 1989) (Revised manuscript received12 April 1990)
Key words: Phosphoinositidemetabolism; Anesthetic; Brain; lnositol lipid metabolism; (Guinea pig)
Various local anesthetics enhanced the incorporation of [3Hlinositol into phosphoinositides in guinea pig cerebral cortical synaptoneurosomes. Dibucaine, QX-572 and dimethisoquin showed maximum stimulation at 100 pM, tetracaine and diphenhydramine at 300 #M, and QX-314 at 1 mM, while quinacrine, lidocaine and cocaine showed no or only slight stimulation. There was no correlation between local anesthetic activity, estimated by inhibition of the 22Na+ flux elicited by the sodium channel activator batrachotoxin, and the potency for stimulation of inositol incorporation. A quaternary, relatively weak, local anesthetic, QX-572, was the most potent agent in stimulation of inositol incorporation, while the next most potent agent was dibucaine, a tertiary, very potent, local anesthetic. Dihucaine did not affect the uptake of [3H]inositol by synaptoneurosomes. The incorporation of [3H]inositol into phosphoinositides was increased in calcium-free buffer. The presence of dibucaine resulted in further stimulation of [3H]inositol incorporation in calcium-free buffer. Although dibucaine and QX-572 markedly stimulated incorporation of [3H]inositol into phosphoinositides, only QX-572 significantly enhanced the incorporation of 32po43- into phosphoinositides. The results suggest that certain local anesthetics enhance a pathway involving an exchange reaction between inositol and the phosphoinositol ester bond of phosphatidylinositol, but do not markedly affect the de novo pathway of phosphoinositide synthesis.
Introduction Local anesthetics are known to affect the functions of a variety of membrane proteins including channel proteins [1-4], ATPases [5], adenylate and guanylate cyclases [6,7] and phospholipases [8]. The effects of such agents as blockers of voltage-sensitive sodium channels appear to be fundamental to the local anesthetic activity on neurons [1], while other effects are ancillary in nature. Recently activation of sodium channels in synaptoneurosomes was shown to elicit breakdown of phosphoinositides [9-12]. Local anesthetics blocked such responses, primarily through blockade of sodium channels [13]. However, some local anesthetics also
blocked receptor-mediated phosphoinositide breakdown. The breakdown of phosphoinositides by phospholipases, either through receptor or ion channel activation, results in the formation of two classes of intracellular second messengers, the inositol phosphates and diacylglycerides [14]. Cellular responses are mediated by both classes of phosphoinositide metabolites; inositol phosphates can activate the mobilization of calcium ions and diacylglycerides can activate protein kinase C. The present paper concerns the stimulatory effects of a variety of local anesthetics on the incorporation of inositol into phosphoinositides.
Materials and Methods Materials
* Present address: Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-26, Japan. Correspondence: J.W. Daly, Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, U.S.A.
Batrachotoxin was isolated as reported previously [15]. Local anesthetics were obtained from the following sources; dibucaine, Ciba-Geigy Corp. (Ardsley, NY); tetracaine, quinoacrine, and diphenhydramine (Sigma, St. Louis, MO); dimethisoquin, Smith-Kline French Laboratories (Philadelphia, PA); cocaine, Merck, Sharp
0167-4889/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (Biomedical Division)
214 and Dohme (West Point, PA); QX-572, QX-314, and lidocaine were provided by Dr. L.-Y.M. Huang, formerly of the National Institute of Mental Health, Bethesda, MD. [3H]Inositol (14-17 Ci/mmol) was from New England Nuclear (Boston, MA), [32p]orthophosphate from Amersham (Arlington Heights, IL). Anion exchange resin, AG1-X8 (formate form) was from Bio-Rad (Richmond, CA), and Hydrofluor and Betafluor from National Diagnostics (Sommerville, N J).
with 4 #Ci [32p]orthophosphate at 3 7 ° C for 90 min. Total lipids were extracted as described above from the trichloroacetic acid precipitates and the chloroform layer of 3 tubes were pooled (1.4 ml). An aliquot of the chloroform layer (0.2 ml) was then transferred to a scintillation vial and chloroform was evaporated. Radioactivity was then measured after addition of 4 ml of Betafluor. The remainder was subjected to thin-layer chromatography (see below).
Synaptoneurosomes
Uptake of [3H]inositol by synaptoneurosomes
Cerebral cortical synaptoneurosomes were prepared by the method of Hollingsworth et al. [16]. Brain cortex of Hartley guinea pigs was homogenized in 7-10 volumes of Krebs-Henseleit buffer (pH 7.4), using a glass homogenizer. The homogenate was centrifuged at 100 × g for 15 min. The resulting pellet was resuspended in an appropriate volume of buffer and used for assay.
The synaptoneurosome suspension (300 ~tl) was distributed in 5-ml tubes and incubated with 5.7 /~Ci [3H]inositol at 37 ° C for 90 min. Inositol concentrations were from 1 /~M to 10 raM. After the incubation, the suspension was centrifuged and the resulting pellet was washed twice by adding 1 ml of Krebs-Henseleit buffer followed by centrifugation. The resulting pellet was mixed with 1 ml of 6% trichloroacetic acid and then centrifuged. The supernatant fraction was removed and an aliquot (100 /xl) of the fraction was used for measurement of radioactivity after adding 4 ml of Hydrofluor. The radioactivity consists of mainly free [3H]inositol (90.4 + 1.0%, n = 3) as shown by elution with water from anion exchange AG1-X8 column chromatography, performed as described [11]. The precipitates after treatment with trichloroacetic acid were used as described above for determination of incorporation of [3H]inositol into lipids.
Incorporation of [3H]inositol into lipids The incorporation of [3H]inositol into lipids was measured as reported previously [11]. The synaptoneurosomes (about 50 mg of protein) were suspended in 14 ml of fresh Krebs-Henseleit buffer containing 200 #Ci [3H]innositol (1 /~M). Aliquots of the synaptoneurosome suspension (300 #1) were distributed in 5-ml tubes and test agent or buffer was added to a final volume of 400/xl. The synaptoneurosome suspension was then incubated at 37 ° C for 90 min. After the incubation, the suspension was centrifuged to remove free [3H]inositol, and the pellet was resuspended in 1 ml of Krebs-Henseleit buffer, followed by centrifugation. The resulting pellet was mixed with 1 ml of 6% trichloroacetic acid and centrifuged. The precipitate was pelleted by centrifugation for the measurement of incorporation of [3H]inositol into phosphoinositides. The pellet was suspended in 0.5 ml of the mixture of 1 M KC1/10 mM inositol and methanol (1 : 1). Chloroform (0.5 ml) was added to the suspension and lipids were extracted by shaking for 5 min. An aliquot of the chloroform layer (0.2 ml) was transferred to a scintillation vial and evaporated to dryness. Radioactivity in lipid fraction was measured after 4 ml of Betafluor was added. For the determination of radioactivity incorporated into each class of phosphoinositides, chloroform layers of 3 tubes were combined (1.4 ml), and radioactivity in 0.2 ml of the extract was measured after addition of 4 ml of Betafluor. The remainder was subjected to thin-layer chromatography (see below).
Thin-layer chromatography of phosphoinositides Phosphoinositides were separated by thin-layer chromatography essentially as described [17]. Thin-layer plates (Whatman LK5DF, 0.25 mm thickness) were developed in 1% sodium oxalacetate, dried at room temperature and activated at 110 ° C for 20 min prior to use. An aliquot of lipid extract (0.3 ml) was evaporated to dryness under nitrogen. Lipids were dissolved in 50 /~1 of a mixture of chloroform/ m e t h a n o l / conc. HCI (200 : 100 : 0.75) and 20/~1 of the solution was applied to the thin-layer plate. The plate was developed in chlorof o r m / a c e t o n e / m e t h a n o l / acetic a c i d / w a t e r (40 : 15 : 13 : 12 : 8). Localization of phosphoinositides was performed by iodine vapor detection of standard phosphoinositides. The silica gel for each phosphoinositide was scraped into scintillation vials and 1 ml of methanol was added. The methanol suspension was sonicated for 15 rain in a bath-type sonicator and then radioactivity was measured after adding 10 ml of Betafluor.
Incorporation of [ 32p]orthophosphate into phosphoinositides
Results
The synaptoneurosome suspension (300 #1) in Krebs-Henseleit buffer was distributed in 5-ml tubes as above. The synaptoneurosomes were then incubated
A variety of local anesthetics inhibit the breakdown of phosphoinositides elicited by sodium channel activator, batrachotoxin [13]. A representative potent local
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anesthetic, dibucaine, inhibited inositol phosphate formation elicited by batrachotoxin in a dose-dependent manner with an IC50 at about 1/~M (Fig. 1). Dibucaine also enhanced the incorporation of [3H]inositol into lipids both in the absence and presence of batrachotoxin. The stimulatory effect of dibucaine on [3H]inositol incorporation occurred at 10-100 ~tM in the absence of
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Inhibition of Sodium Flux Fig. 3. Relationship between IC50 values of local anesthetics versus sodium flux elicited by 1 ~M batrachotoxin and the incorporation of inositol into lipids in guinea pig cerebral cortical synaptoneurosomes. 1, dibucaine; 2, tetracaine; 5, dimethisoquin; 6, quinacrine; 8, QX-572; 9, QX-314; 10, diphenhydramine; 11, lidocaine; 15, cocaine. IC50 values for sodium flux are Ref. 13. The incorporations of inositol are the maximum mean values of three determinations.
batrachotoxin. Batrachotoxin itself reduced incorporation of [3H]inositol, and effect that was reversed by dibucaine. A series of local anesthetics were examined with respect to the effects on inositol incorporation into lipids. QX-572, dibucaine, and dimethisoquin were most potent stimulators of inositol incorporation; maximum stimulations were achieved at about 100/~M (Fig. 2A). Tetracaine and diphenhydramine were nearly as potent, showing maximum stimulation at 300 /xM. All these local anesthetics inhibited the incorporation of inositol into lipids at 1 mM. QX-314 at higher than 1 mM caused a slight stimulation of inositol incorporation,
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Fig. 4. Effects of dibucaine on the uptake of [3H]inositoi by guinea pig cerebral cortical synaptoneurosomes and on the incorporation of [3H]inositol into lipids. Synaptoneurosomes were incubated with [3H]inositol for 90 min at 37°C in the presence (O) or absence (o) of 100 /zM dibucaine and radioactivity present in tricMoroacetic acid soluble fraction (uptake, A) and in lipid fraction (B) was measured. Each value represents the mean of 3 determinations. The error (S.E.M.) was for all points smaller than the symbol.
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Fig. 5. Effects of dibucaine, calcium-free buffer, and calcium-free buffer containing EGTA on the incorporation of [3H]inositol into lipids in guinea pig cerebral cortical synaptoneurosomes. Synaptoneurosomes were incubated with [3H]inositol in regular KrebsHenseleit buffer containing 2.5 mM CaC12, in calcium-free buffer, or in calcium-free buffer containing EGTA in the presence (•) or absence (0) of 100/tM dibucaine for 90 min at 37 o C. Radioactivity in the lipid fraction was measured. Each value represents the mean of three determinations. The error (S.E.M.) was for all points except one smaller than the symbol.
while c o c a i n e a n d l i d o c a i n e h a d m i n i m a l effects (Fig. 2B). Q u i n a c r i n e h a d a m i n i m a l s t i m u l a t o r y effect a n d m a r k e d l y i n h i b i t e d the inositol i n c o r p o r a t i o n at 3 0 0 / ~ M and 1 mM. T h e p o t e n c i e s o f local anesthetics as i n h i b i t o r s o f b a t r a c h o t o x i n - a c t i v a t e d s o d i u m flux a n d efficacy as s t i m u l a t o r s of inositol i n c o r p o r a t i o n i n t o lipids s h o w e d n o c o r r e l a t i o n (Fig. 3). T h e m o s t efficacious s t i m u l a t o r s of inositol i n c o r p o r a t i o n i n c l u d e d d i b u c a i n e , a very p o t e n t local anesthetic, a n d QX-572, a relatively w e a k local anesthetic.
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Fig. 6. Effects of receptor agonists and a local anesthetic, dibucaine, on incorporation of [3H]inositoi into lipids of guinea pig cerebral cortical synaptoneurosomes. Synaptoneurosomes were incubated with [3H]inositol in Kxebs-Henseleit buffer for 60 rain at 37 ° C, followed by addition of carbamylchoIine (2 raM), norepinephrine (100 /zM), and/or dibucaine (100 btM) or tetrodotoxin (1 /~M) and further incubation for 90 rain at 37 ° C. Radioactivity in lipid fraction was measured. Each value represents the mean of three determinations.
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Fig. 7. Effects of dibucaine and QX-572 on the incorporation of [32p]orthophosphate or [3H]inositol into phosphoinositides in guinea pig cerebral cortical synaptoneurosomes. Synaptoneurosomes were incubated with [32p]orthophosphate or [3H]inositol for 90 min at 37 o C in the presence or absence of (A) dibucaine or (B) QX-572 (100 btM). Open bars: incorporation of [32p]orthophosphate. Hatched bars: incorporation of [3H]inositoi. Lipids were extracted and separated by thin-layer chromatography. Radioactivity present in each inositol phospholipid (PI, PIP, PIP2) and phosphatidic acid (PA) was measured. Each value represents the mean+ S.E. (n = 3). Control values were as follows: [3H]inositol incorporation (cpm): PI 1295 + 128; PIP 211 + 11; PIP2 46 + 3. [32P]Phosphate incorporation (cpm): PI 3046 + 116; PIP 1602 + 94; PIP2 1443 + 42; PA 4758 + 93.
T h e effect of d i b u c a i n e on the u p t a k e of [3H]inositol b y s y n a p t o n e u r o s o m e s a n d o n [3H]inositol i n c o r p o r a tion into p h o s p h o i n o s i t i d e s were c o m p a r e d (Fig. 4). T h e i n c o r p o r a t i o n of inositol i n t o lipids was e n h a n c e d b y 100 # M d i b u c a i n e at the c o n c e n t r a t i o n s of inositol b e t w e e n 1 /~M a n d 10 m M , while u p t a k e , w h i c h was m e a s u r e d as r a d i o a c t i v i t y in t r i c h l o r o a c e t i c acid s o l u b l e fraction, was u n a f f e c t e d b y d i b u c a i n e . I n o s i t o l i n c o r p o r a t i o n i n t o lipids was e n h a n c e d in c a l c i u m - f r e e K r e b s - H e n s e l e i t b u f f e r c o m p a r e d with the b u f f e r c o n t a i n i n g 2.5 m M CaC12 (Fig. 5). T h e a d d i t i o n of E G T A to c a l c i u m - f r e e b u f f e r f u r t h e r e n h a n c e d the inositol i n c o r p o r a t i o n . T h e s t i m u l a t i o n of inositol incorp o r a t i o n into lipids b y d i b u c a i n e (100 ~tM) o c c u r r e d b o t h in c a l c i u m - f r e e a n d c a l c i u m - c o n t a i n i n g buffers. I n d e e d , the effect of d i b u c a i n e a p p e a r e d greater t h a n a d d i t i v e with t h a t d u e to l a c k of calcium. A f t e r l a b e l i n g with [3H]inositol in the p r e s e n c e of QX-572, s u b s e q u e n t s t i m u l a t i o n of p h o s p h o i n o s i t i d e b r e a k d o w n b y b a t r a c h o t o x i n o r r e c e p t o r agonists was greatly r e d u c e d ( d a t a n o t shown), p e r h a p s d u e in p a r t to difficulties in r e m o v i n g the high c o n c e n t r a t i o n (300 /xM) o f QX-572 b y washing. R e c e p t o r agonists, such as c a r b a m y l c h o l i n e (2 m M ) a n d n o r e p i n e p h r i n e (100/~M), s t i m u l a t e d i n c o r p o r a t i o n of [3H]inositol b y 50% a n d 30%, respectively (Fig. 6). C o m b i n a t i o n s of c a r b a m y l c h o l i n e or n o r e p i n e p h r i n e with d i b u c a i n e a p p e a r e d to have less t h a n a d d i t i v e effects on [3H]inositol i n c o r p o r a t i o n . T e t r o d o t o x i n , a specific b l o c k e r of s o d i u m channels, p a r t i a l l y r e d u c e d the r e s p o n s e to c a r b a m y l c h o l i n e a n d to n o r e p i n e p h r i n e (Fig. 6).
217 The effects of local anesthetics on the incorporation of [32p]orthophosphate into phosphoinositides were compared with effects on the incorporation of [3H]inositol into phosphoinositides (Fig. 7). Dibucaine did not affect the incorporation of [32p]orthophosphate into phosphatidic acid, phosphatidylinositol, phosphatidylinositol 4-phosphate, or phosphatidylinositol 4,5-bisphosphate, while markedly enhancing the incorporation of [3H]inositol into these lipids. QX-572 only slightly stimulated the incorporation of [32p]orthophosphates into phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidic acid, while markedly enhancing the incorporation of [3H]inositol into phosphoinositides. There was no significant effect of dibucaine on incorporation of [32p]orthophosphate into phosphatidylcholine/phosphatidylethanolamine (data not shown). Discussion
A variety of local anesthetics inhibit both sodium flux induced by sodium channel activator, batrachotoxin, and phosphoinositide breakdown elicited by batrachotoxin [13]. Batrachotoxin, in addition to stimulating phosphoinositide breakdown, decreases incorporation of [3H]inositol into lipids (Fig. 1, see also Ref. 11). The decrease in incorporation of [3H]inositol into lipids elicited by batrachotoxin is reversed by dibucaine, over the same concentration range in which dibucaine inhibits batrachotoxin-elicited phosphoinositide breakdown (Fig. 1). Somewhat higher concentrations of dibucaine cause a further increase in inositol incorporation in the presence of batrachotoxin. A similar stimulatory effect of high concentrations of dibucaine on incorporation occurs in the absence of batrachotoxin, where phosphoinositide breakdown is not stimulated. Tetrodotoxin, a specific and potent blocker of sodium channels, which inhibits both batrachotoxin-elicited sodium flux and batrachotoxin-elicited phosphoinositide breakdown [12], reverses the inhibitory effect of batrachotoxin on [3H]inositol incorporation, but does not further stimulate the incorporation of [3H]inositol into lipids (data not shown). The local anesthetics dibucaine, QX-572, dimethisoquin, tetracaine, and diphenhydramine proved to be the most potent and efficacious in stimulating inositol incorporation into lipids (Fig. 2). QX-314, another quaternary local anesthetic similar in structure to QX572, and lidocaine, cocaine, and quinacrine show slight or minimal stimulation of inositol incorporation. All local anesthetics, except QX-314 and lidocaine, have inhibitory effects on inositol incorporation at high concentrations. There is no correlation between the efficacy of local anesthetics towards stimulation of inositol incorporation and the potency in inhibition of sodium flux elicited by batrachotoxin (Fig. 3). For example,
dibucaine and dimethisoquin are much more potent in blocking sodium flux elicited by batrachotoxin (ICs0 0.7-1 #M) than in stimulating [3H]inositol incorporation (ECs0 60-80 #M), while QX-572 shows similar potency for blockade of sodium flux (IC50 90/~M) and in stimulating [3H]inositol incorporation (ECs0 70/~M) (see Fig. 2 and Ref. 13). The stimulatory effects of these agents on inositol incorporation would appear to occur through mechanism(s) different in structural requirements than those for blockade of sodium channels. One possible mechanism to account for stimulatory effects of local anesthetics on inositol incorporation is that such effects result from enhancement of uptake of inositol into the synaptoneurosomes. However, dibucaine had no effect on uptake of inositol over a wide range of concentrations of inositol (Fig. 4). Batrachotoxin at 1/~M does reduce uptake of 1/~M [3H]inositol by about 30% (data not shown). Uptake of inositol by brain slices is partially 'sodium dependent' [18], and thus, an alteration of sodium gradient induced by batrachotoxin might be responsible for reduction in [aH]inositol uptake in the presence of batrachotoxin. Calcium ions in extracellular medium inhibit the labeling of phosphatidylinositol by [3H]inositol in brain preparations [19]. Calcium-free buffer did enhance the incorporation of inositol into lipids in synaptoneurosomes (Fig. 5). Calcium ions are proposed to affect the labeling of phosphatidylinositol by [3H]inositol through an inhibition of the CDP-diacylglycerol :inositol transferase pathway, while markedly stimulating the phosphatidylinositol:inositol exchange pathway [20-22]. The stimulatory effects of dibucaine on the inositol incorporation appear to be enhanced in both calciumfree buffer and in calcium-free buffer that contained EGTA; i.e., the effects of lack of extracellular calcium and of dibucaine on incorporation are greater than additive (Fig. 5). It is possible that calcium ions inhibit local anesthetic-stimulated inositol incorporation and indeed, local anesthetics and calcium ions do compete for membrane sites [23]. The local anesthetic, dibucaine, was reported to stimulate labeling of phosphoinositides by [32p]orthophosphate in rabbit vagus nerves, in 1972 [24]. Subsequent studies have demonstrated that a variety of local anesthetics and drugs with local anesthetic activity stimulate incorporation of both [ 32P]orthophosphate and [3H]inositol into phosphoinositides in rodent brain slices [25-27]. The mechanism(s) involved have been unclear. In the present study in synaptoneurosomes, it is apparent that dibucaine and QX-572 have no or only slight effects, respectively, on the labeling of phosphoinositides or phosphatidic acid by [32p]orthophosphate, while these local anesthetics show profound effects on the incorporation of [3H]inositol into phosphoinositides (Fig. 7). Thus, in synaptoneurosomes, local anesthetics have little effect on the de novo pathway, the only
218 p a t h w a y that results in labeling b y [32p]orthophosphate, and, therefore, m u s t stimulate [3H]inositol i n c o r p o r a tion p r i m a r i l y t h r o u g h effects o n the other pathway, n a m e l y p h o s p h a t i d y l i n o s i t o l - i n o s i t o l exchange. T w o m e t a b o l i c pools of phosphoinositides, o n e sensitive to s t i m u l a t i o n b y receptor agonists, the other insensitive, occur i n most cells (see Ref. 28). T h e n a t u r e of the pool(s) whose labeling is s t i m u l a t e d b y lcoal anesthetics could n o t be defined, because of difficulties i n r e m o v i n g local anesthetics, which c a n i n h i b i t agonist responses [23], b y washing. T h e local anesthetics all had first s t i m u l a t o r y a n d t h e n i n h i b i t o r y effects o n inositol inc o r p o r a t i o n a n d b o t h these effects occur at c o n c e n t r a tions significantly higher t h a n those required for blockade of s o d i u m channels.
References 1 Catterail, W.A. (1987) Trends Pharmacol. Sci. 8, 57-65. 2 Fishman, M.C. and Spector, I. (1981) Proc. Natl. Acad. Sci. USA 78, 5245-5249. 3 Frelin, C., Vigne, P. and Lazdunski, M. (1982) Biochem. Biophys. Res. Commun. 106, 967-973. 4 Aksentsev, S.L., Konev, S.V., Rakovich, A.A., Okun, I.M. and Kulikov, A.V. (1982) Gen. Physiol. Biophys. 1,403-412. 5 Roufogalis, B.D. (1973) Biochim. Biophys. Acta 318, 360-370. 6 Voeikov, V.V. and Lefkowitz, R.J. (1980) Biochim. Biophys. Acta 629, 266-281. 7 Richelson, E., Prendergast, F.G. and Divenets-Romero, S.D. (1978) Biochem. Pharmacol. 27, 2039-2048. 8 Waite, M. and Sisson, P. (1972) Biochemistry 11, 3098-3105.
9 Gusovsky, F., Hollingsworth, E.B. and Daly, J.W. (1986) Proc. Natl. Acad. Sci. USA 83, 3003-3007. 10 Hollingsworth, E.B., Sears, E.B., de la Cruz, R.A., Gusovsky, F. and Daly, J.W. (1986) Biochim. Biophys. Acta 883, 15-25. 11 Gusovsky, F. and Daly, J.W. (1988) Neuropharmacology 27, 95105. 12 Gusovsky, F., McNeal, E.T. and Daly, J.W. (1987) Mol. Pharmacol. 32, 479-487. 13 Nishizawa, Y., Gusovsky, F. and Daly, J.W. (1988) Mol. Pharmacol. 34, 707-713. 14 Berridge, M.J. (1987) Ann. Rev. Biochem. 56, 159-193. 15 Tokuyama, T. and Daly, J.W. (1983) Tetrahedron 39, 41-57. 16 Hollingsworth, E.B., McNeal, E.T., Burton, J.L., Williams, R.J., Daly, J.W. and Creveling, C.R. (1985) J. Neurosci. 5, 2240-2253. 17 Jolles, J., Schrama, L.H. and Gispen, W.H .(1981) Biochim. Biophys. Acta 666, 90-98. 18 Howerton, T.C. and Rutledge, C.O. (1988) Biochem. Pharmacol. 37, 911-915. 19 Kendall, D.A. and Nahorski, S.R. (1984) J. Neurochem. 42, 13881394. 20 Egawa, K., Takenawa, T. and Sachtor, B. (1981) Mol. Cell. Endocrinol. 21, 29-35. 21 Tolbert, E.M., White, A.C., Aspry, K., Cutts, J. and Fain, J.N. (1980) J. Biol. Chem. 255, 1938-1944. 22 Pripi6, V., Blackmore, P.F. and Exton, J.H. (1982) J. Biol. Chem. 257, 11315-11322. 23 Low, P.S., Lloyd, D.H., Stein, T.M. and Royers, III, J.A. (1979) J. Biol. Chem. 254, 4119-4125. 24 Salway, J.G. and Hughes, I.E. (1972) J. Neurochem. 19, 1233-1240. 25 Pappu, A.S. and Hauser, G. (1981) J. Neurochem. 37, 1006-1014. 26 Pappu, A.S. and Hauser, G. (1981) Biochem. Pharmacol. 30, 3243-3246. 27 Hokin-Neaverson, M. (1980) Biochem. Pharmacol. 29, 2697-2700. 28 Monaco, M.E. (1987) J. Biol. Chem. 262, 13001-13006.
213
Elsevier BBAMCR 12746
Local anesthetics" stimulation of incorporation of inositol into phosphoinositides in guinea pig cerebral cortical synaptoneurosomes Yukio Nishizawa *, Fabian Gusovsky and John W. Daly Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestioe and Kidney Diseases, National Institutes of Health, Bethesda, MD (U.S.A.)
(Received 8 November 1989) (Revised manuscript received12 April 1990)
Key words: Phosphoinositidemetabolism; Anesthetic; Brain; lnositol lipid metabolism; (Guinea pig)
Various local anesthetics enhanced the incorporation of [3Hlinositol into phosphoinositides in guinea pig cerebral cortical synaptoneurosomes. Dibucaine, QX-572 and dimethisoquin showed maximum stimulation at 100 pM, tetracaine and diphenhydramine at 300 #M, and QX-314 at 1 mM, while quinacrine, lidocaine and cocaine showed no or only slight stimulation. There was no correlation between local anesthetic activity, estimated by inhibition of the 22Na+ flux elicited by the sodium channel activator batrachotoxin, and the potency for stimulation of inositol incorporation. A quaternary, relatively weak, local anesthetic, QX-572, was the most potent agent in stimulation of inositol incorporation, while the next most potent agent was dibucaine, a tertiary, very potent, local anesthetic. Dihucaine did not affect the uptake of [3H]inositol by synaptoneurosomes. The incorporation of [3H]inositol into phosphoinositides was increased in calcium-free buffer. The presence of dibucaine resulted in further stimulation of [3H]inositol incorporation in calcium-free buffer. Although dibucaine and QX-572 markedly stimulated incorporation of [3H]inositol into phosphoinositides, only QX-572 significantly enhanced the incorporation of 32po43- into phosphoinositides. The results suggest that certain local anesthetics enhance a pathway involving an exchange reaction between inositol and the phosphoinositol ester bond of phosphatidylinositol, but do not markedly affect the de novo pathway of phosphoinositide synthesis.
Introduction Local anesthetics are known to affect the functions of a variety of membrane proteins including channel proteins [1-4], ATPases [5], adenylate and guanylate cyclases [6,7] and phospholipases [8]. The effects of such agents as blockers of voltage-sensitive sodium channels appear to be fundamental to the local anesthetic activity on neurons [1], while other effects are ancillary in nature. Recently activation of sodium channels in synaptoneurosomes was shown to elicit breakdown of phosphoinositides [9-12]. Local anesthetics blocked such responses, primarily through blockade of sodium channels [13]. However, some local anesthetics also
blocked receptor-mediated phosphoinositide breakdown. The breakdown of phosphoinositides by phospholipases, either through receptor or ion channel activation, results in the formation of two classes of intracellular second messengers, the inositol phosphates and diacylglycerides [14]. Cellular responses are mediated by both classes of phosphoinositide metabolites; inositol phosphates can activate the mobilization of calcium ions and diacylglycerides can activate protein kinase C. The present paper concerns the stimulatory effects of a variety of local anesthetics on the incorporation of inositol into phosphoinositides.
Materials and Methods Materials
* Present address: Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-26, Japan. Correspondence: J.W. Daly, Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, U.S.A.
Batrachotoxin was isolated as reported previously [15]. Local anesthetics were obtained from the following sources; dibucaine, Ciba-Geigy Corp. (Ardsley, NY); tetracaine, quinoacrine, and diphenhydramine (Sigma, St. Louis, MO); dimethisoquin, Smith-Kline French Laboratories (Philadelphia, PA); cocaine, Merck, Sharp
0167-4889/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (Biomedical Division)
214 and Dohme (West Point, PA); QX-572, QX-314, and lidocaine were provided by Dr. L.-Y.M. Huang, formerly of the National Institute of Mental Health, Bethesda, MD. [3H]Inositol (14-17 Ci/mmol) was from New England Nuclear (Boston, MA), [32p]orthophosphate from Amersham (Arlington Heights, IL). Anion exchange resin, AG1-X8 (formate form) was from Bio-Rad (Richmond, CA), and Hydrofluor and Betafluor from National Diagnostics (Sommerville, N J).
with 4 #Ci [32p]orthophosphate at 3 7 ° C for 90 min. Total lipids were extracted as described above from the trichloroacetic acid precipitates and the chloroform layer of 3 tubes were pooled (1.4 ml). An aliquot of the chloroform layer (0.2 ml) was then transferred to a scintillation vial and chloroform was evaporated. Radioactivity was then measured after addition of 4 ml of Betafluor. The remainder was subjected to thin-layer chromatography (see below).
Synaptoneurosomes
Uptake of [3H]inositol by synaptoneurosomes
Cerebral cortical synaptoneurosomes were prepared by the method of Hollingsworth et al. [16]. Brain cortex of Hartley guinea pigs was homogenized in 7-10 volumes of Krebs-Henseleit buffer (pH 7.4), using a glass homogenizer. The homogenate was centrifuged at 100 × g for 15 min. The resulting pellet was resuspended in an appropriate volume of buffer and used for assay.
The synaptoneurosome suspension (300 ~tl) was distributed in 5-ml tubes and incubated with 5.7 /~Ci [3H]inositol at 37 ° C for 90 min. Inositol concentrations were from 1 /~M to 10 raM. After the incubation, the suspension was centrifuged and the resulting pellet was washed twice by adding 1 ml of Krebs-Henseleit buffer followed by centrifugation. The resulting pellet was mixed with 1 ml of 6% trichloroacetic acid and then centrifuged. The supernatant fraction was removed and an aliquot (100 /xl) of the fraction was used for measurement of radioactivity after adding 4 ml of Hydrofluor. The radioactivity consists of mainly free [3H]inositol (90.4 + 1.0%, n = 3) as shown by elution with water from anion exchange AG1-X8 column chromatography, performed as described [11]. The precipitates after treatment with trichloroacetic acid were used as described above for determination of incorporation of [3H]inositol into lipids.
Incorporation of [3H]inositol into lipids The incorporation of [3H]inositol into lipids was measured as reported previously [11]. The synaptoneurosomes (about 50 mg of protein) were suspended in 14 ml of fresh Krebs-Henseleit buffer containing 200 #Ci [3H]innositol (1 /~M). Aliquots of the synaptoneurosome suspension (300 #1) were distributed in 5-ml tubes and test agent or buffer was added to a final volume of 400/xl. The synaptoneurosome suspension was then incubated at 37 ° C for 90 min. After the incubation, the suspension was centrifuged to remove free [3H]inositol, and the pellet was resuspended in 1 ml of Krebs-Henseleit buffer, followed by centrifugation. The resulting pellet was mixed with 1 ml of 6% trichloroacetic acid and centrifuged. The precipitate was pelleted by centrifugation for the measurement of incorporation of [3H]inositol into phosphoinositides. The pellet was suspended in 0.5 ml of the mixture of 1 M KC1/10 mM inositol and methanol (1 : 1). Chloroform (0.5 ml) was added to the suspension and lipids were extracted by shaking for 5 min. An aliquot of the chloroform layer (0.2 ml) was transferred to a scintillation vial and evaporated to dryness. Radioactivity in lipid fraction was measured after 4 ml of Betafluor was added. For the determination of radioactivity incorporated into each class of phosphoinositides, chloroform layers of 3 tubes were combined (1.4 ml), and radioactivity in 0.2 ml of the extract was measured after addition of 4 ml of Betafluor. The remainder was subjected to thin-layer chromatography (see below).
Thin-layer chromatography of phosphoinositides Phosphoinositides were separated by thin-layer chromatography essentially as described [17]. Thin-layer plates (Whatman LK5DF, 0.25 mm thickness) were developed in 1% sodium oxalacetate, dried at room temperature and activated at 110 ° C for 20 min prior to use. An aliquot of lipid extract (0.3 ml) was evaporated to dryness under nitrogen. Lipids were dissolved in 50 /~1 of a mixture of chloroform/ m e t h a n o l / conc. HCI (200 : 100 : 0.75) and 20/~1 of the solution was applied to the thin-layer plate. The plate was developed in chlorof o r m / a c e t o n e / m e t h a n o l / acetic a c i d / w a t e r (40 : 15 : 13 : 12 : 8). Localization of phosphoinositides was performed by iodine vapor detection of standard phosphoinositides. The silica gel for each phosphoinositide was scraped into scintillation vials and 1 ml of methanol was added. The methanol suspension was sonicated for 15 rain in a bath-type sonicator and then radioactivity was measured after adding 10 ml of Betafluor.
Incorporation of [ 32p]orthophosphate into phosphoinositides
Results
The synaptoneurosome suspension (300 #1) in Krebs-Henseleit buffer was distributed in 5-ml tubes as above. The synaptoneurosomes were then incubated
A variety of local anesthetics inhibit the breakdown of phosphoinositides elicited by sodium channel activator, batrachotoxin [13]. A representative potent local
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anesthetic, dibucaine, inhibited inositol phosphate formation elicited by batrachotoxin in a dose-dependent manner with an IC50 at about 1/~M (Fig. 1). Dibucaine also enhanced the incorporation of [3H]inositol into lipids both in the absence and presence of batrachotoxin. The stimulatory effect of dibucaine on [3H]inositol incorporation occurred at 10-100 ~tM in the absence of
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batrachotoxin. Batrachotoxin itself reduced incorporation of [3H]inositol, and effect that was reversed by dibucaine. A series of local anesthetics were examined with respect to the effects on inositol incorporation into lipids. QX-572, dibucaine, and dimethisoquin were most potent stimulators of inositol incorporation; maximum stimulations were achieved at about 100/~M (Fig. 2A). Tetracaine and diphenhydramine were nearly as potent, showing maximum stimulation at 300 /xM. All these local anesthetics inhibited the incorporation of inositol into lipids at 1 mM. QX-314 at higher than 1 mM caused a slight stimulation of inositol incorporation,
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Fig. 4. Effects of dibucaine on the uptake of [3H]inositoi by guinea pig cerebral cortical synaptoneurosomes and on the incorporation of [3H]inositol into lipids. Synaptoneurosomes were incubated with [3H]inositol for 90 min at 37°C in the presence (O) or absence (o) of 100 /zM dibucaine and radioactivity present in tricMoroacetic acid soluble fraction (uptake, A) and in lipid fraction (B) was measured. Each value represents the mean of 3 determinations. The error (S.E.M.) was for all points smaller than the symbol.
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Fig. 5. Effects of dibucaine, calcium-free buffer, and calcium-free buffer containing EGTA on the incorporation of [3H]inositol into lipids in guinea pig cerebral cortical synaptoneurosomes. Synaptoneurosomes were incubated with [3H]inositol in regular KrebsHenseleit buffer containing 2.5 mM CaC12, in calcium-free buffer, or in calcium-free buffer containing EGTA in the presence (•) or absence (0) of 100/tM dibucaine for 90 min at 37 o C. Radioactivity in the lipid fraction was measured. Each value represents the mean of three determinations. The error (S.E.M.) was for all points except one smaller than the symbol.
while c o c a i n e a n d l i d o c a i n e h a d m i n i m a l effects (Fig. 2B). Q u i n a c r i n e h a d a m i n i m a l s t i m u l a t o r y effect a n d m a r k e d l y i n h i b i t e d the inositol i n c o r p o r a t i o n at 3 0 0 / ~ M and 1 mM. T h e p o t e n c i e s o f local anesthetics as i n h i b i t o r s o f b a t r a c h o t o x i n - a c t i v a t e d s o d i u m flux a n d efficacy as s t i m u l a t o r s of inositol i n c o r p o r a t i o n i n t o lipids s h o w e d n o c o r r e l a t i o n (Fig. 3). T h e m o s t efficacious s t i m u l a t o r s of inositol i n c o r p o r a t i o n i n c l u d e d d i b u c a i n e , a very p o t e n t local anesthetic, a n d QX-572, a relatively w e a k local anesthetic.
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Fig. 6. Effects of receptor agonists and a local anesthetic, dibucaine, on incorporation of [3H]inositoi into lipids of guinea pig cerebral cortical synaptoneurosomes. Synaptoneurosomes were incubated with [3H]inositol in Kxebs-Henseleit buffer for 60 rain at 37 ° C, followed by addition of carbamylchoIine (2 raM), norepinephrine (100 /zM), and/or dibucaine (100 btM) or tetrodotoxin (1 /~M) and further incubation for 90 rain at 37 ° C. Radioactivity in lipid fraction was measured. Each value represents the mean of three determinations.
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Fig. 7. Effects of dibucaine and QX-572 on the incorporation of [32p]orthophosphate or [3H]inositol into phosphoinositides in guinea pig cerebral cortical synaptoneurosomes. Synaptoneurosomes were incubated with [32p]orthophosphate or [3H]inositol for 90 min at 37 o C in the presence or absence of (A) dibucaine or (B) QX-572 (100 btM). Open bars: incorporation of [32p]orthophosphate. Hatched bars: incorporation of [3H]inositoi. Lipids were extracted and separated by thin-layer chromatography. Radioactivity present in each inositol phospholipid (PI, PIP, PIP2) and phosphatidic acid (PA) was measured. Each value represents the mean+ S.E. (n = 3). Control values were as follows: [3H]inositol incorporation (cpm): PI 1295 + 128; PIP 211 + 11; PIP2 46 + 3. [32P]Phosphate incorporation (cpm): PI 3046 + 116; PIP 1602 + 94; PIP2 1443 + 42; PA 4758 + 93.
T h e effect of d i b u c a i n e on the u p t a k e of [3H]inositol b y s y n a p t o n e u r o s o m e s a n d o n [3H]inositol i n c o r p o r a tion into p h o s p h o i n o s i t i d e s were c o m p a r e d (Fig. 4). T h e i n c o r p o r a t i o n of inositol i n t o lipids was e n h a n c e d b y 100 # M d i b u c a i n e at the c o n c e n t r a t i o n s of inositol b e t w e e n 1 /~M a n d 10 m M , while u p t a k e , w h i c h was m e a s u r e d as r a d i o a c t i v i t y in t r i c h l o r o a c e t i c acid s o l u b l e fraction, was u n a f f e c t e d b y d i b u c a i n e . I n o s i t o l i n c o r p o r a t i o n i n t o lipids was e n h a n c e d in c a l c i u m - f r e e K r e b s - H e n s e l e i t b u f f e r c o m p a r e d with the b u f f e r c o n t a i n i n g 2.5 m M CaC12 (Fig. 5). T h e a d d i t i o n of E G T A to c a l c i u m - f r e e b u f f e r f u r t h e r e n h a n c e d the inositol i n c o r p o r a t i o n . T h e s t i m u l a t i o n of inositol incorp o r a t i o n into lipids b y d i b u c a i n e (100 ~tM) o c c u r r e d b o t h in c a l c i u m - f r e e a n d c a l c i u m - c o n t a i n i n g buffers. I n d e e d , the effect of d i b u c a i n e a p p e a r e d greater t h a n a d d i t i v e with t h a t d u e to l a c k of calcium. A f t e r l a b e l i n g with [3H]inositol in the p r e s e n c e of QX-572, s u b s e q u e n t s t i m u l a t i o n of p h o s p h o i n o s i t i d e b r e a k d o w n b y b a t r a c h o t o x i n o r r e c e p t o r agonists was greatly r e d u c e d ( d a t a n o t shown), p e r h a p s d u e in p a r t to difficulties in r e m o v i n g the high c o n c e n t r a t i o n (300 /xM) o f QX-572 b y washing. R e c e p t o r agonists, such as c a r b a m y l c h o l i n e (2 m M ) a n d n o r e p i n e p h r i n e (100/~M), s t i m u l a t e d i n c o r p o r a t i o n of [3H]inositol b y 50% a n d 30%, respectively (Fig. 6). C o m b i n a t i o n s of c a r b a m y l c h o l i n e or n o r e p i n e p h r i n e with d i b u c a i n e a p p e a r e d to have less t h a n a d d i t i v e effects on [3H]inositol i n c o r p o r a t i o n . T e t r o d o t o x i n , a specific b l o c k e r of s o d i u m channels, p a r t i a l l y r e d u c e d the r e s p o n s e to c a r b a m y l c h o l i n e a n d to n o r e p i n e p h r i n e (Fig. 6).
217 The effects of local anesthetics on the incorporation of [32p]orthophosphate into phosphoinositides were compared with effects on the incorporation of [3H]inositol into phosphoinositides (Fig. 7). Dibucaine did not affect the incorporation of [32p]orthophosphate into phosphatidic acid, phosphatidylinositol, phosphatidylinositol 4-phosphate, or phosphatidylinositol 4,5-bisphosphate, while markedly enhancing the incorporation of [3H]inositol into these lipids. QX-572 only slightly stimulated the incorporation of [32p]orthophosphates into phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidic acid, while markedly enhancing the incorporation of [3H]inositol into phosphoinositides. There was no significant effect of dibucaine on incorporation of [32p]orthophosphate into phosphatidylcholine/phosphatidylethanolamine (data not shown). Discussion
A variety of local anesthetics inhibit both sodium flux induced by sodium channel activator, batrachotoxin, and phosphoinositide breakdown elicited by batrachotoxin [13]. Batrachotoxin, in addition to stimulating phosphoinositide breakdown, decreases incorporation of [3H]inositol into lipids (Fig. 1, see also Ref. 11). The decrease in incorporation of [3H]inositol into lipids elicited by batrachotoxin is reversed by dibucaine, over the same concentration range in which dibucaine inhibits batrachotoxin-elicited phosphoinositide breakdown (Fig. 1). Somewhat higher concentrations of dibucaine cause a further increase in inositol incorporation in the presence of batrachotoxin. A similar stimulatory effect of high concentrations of dibucaine on incorporation occurs in the absence of batrachotoxin, where phosphoinositide breakdown is not stimulated. Tetrodotoxin, a specific and potent blocker of sodium channels, which inhibits both batrachotoxin-elicited sodium flux and batrachotoxin-elicited phosphoinositide breakdown [12], reverses the inhibitory effect of batrachotoxin on [3H]inositol incorporation, but does not further stimulate the incorporation of [3H]inositol into lipids (data not shown). The local anesthetics dibucaine, QX-572, dimethisoquin, tetracaine, and diphenhydramine proved to be the most potent and efficacious in stimulating inositol incorporation into lipids (Fig. 2). QX-314, another quaternary local anesthetic similar in structure to QX572, and lidocaine, cocaine, and quinacrine show slight or minimal stimulation of inositol incorporation. All local anesthetics, except QX-314 and lidocaine, have inhibitory effects on inositol incorporation at high concentrations. There is no correlation between the efficacy of local anesthetics towards stimulation of inositol incorporation and the potency in inhibition of sodium flux elicited by batrachotoxin (Fig. 3). For example,
dibucaine and dimethisoquin are much more potent in blocking sodium flux elicited by batrachotoxin (ICs0 0.7-1 #M) than in stimulating [3H]inositol incorporation (ECs0 60-80 #M), while QX-572 shows similar potency for blockade of sodium flux (IC50 90/~M) and in stimulating [3H]inositol incorporation (ECs0 70/~M) (see Fig. 2 and Ref. 13). The stimulatory effects of these agents on inositol incorporation would appear to occur through mechanism(s) different in structural requirements than those for blockade of sodium channels. One possible mechanism to account for stimulatory effects of local anesthetics on inositol incorporation is that such effects result from enhancement of uptake of inositol into the synaptoneurosomes. However, dibucaine had no effect on uptake of inositol over a wide range of concentrations of inositol (Fig. 4). Batrachotoxin at 1/~M does reduce uptake of 1/~M [3H]inositol by about 30% (data not shown). Uptake of inositol by brain slices is partially 'sodium dependent' [18], and thus, an alteration of sodium gradient induced by batrachotoxin might be responsible for reduction in [aH]inositol uptake in the presence of batrachotoxin. Calcium ions in extracellular medium inhibit the labeling of phosphatidylinositol by [3H]inositol in brain preparations [19]. Calcium-free buffer did enhance the incorporation of inositol into lipids in synaptoneurosomes (Fig. 5). Calcium ions are proposed to affect the labeling of phosphatidylinositol by [3H]inositol through an inhibition of the CDP-diacylglycerol :inositol transferase pathway, while markedly stimulating the phosphatidylinositol:inositol exchange pathway [20-22]. The stimulatory effects of dibucaine on the inositol incorporation appear to be enhanced in both calciumfree buffer and in calcium-free buffer that contained EGTA; i.e., the effects of lack of extracellular calcium and of dibucaine on incorporation are greater than additive (Fig. 5). It is possible that calcium ions inhibit local anesthetic-stimulated inositol incorporation and indeed, local anesthetics and calcium ions do compete for membrane sites [23]. The local anesthetic, dibucaine, was reported to stimulate labeling of phosphoinositides by [32p]orthophosphate in rabbit vagus nerves, in 1972 [24]. Subsequent studies have demonstrated that a variety of local anesthetics and drugs with local anesthetic activity stimulate incorporation of both [ 32P]orthophosphate and [3H]inositol into phosphoinositides in rodent brain slices [25-27]. The mechanism(s) involved have been unclear. In the present study in synaptoneurosomes, it is apparent that dibucaine and QX-572 have no or only slight effects, respectively, on the labeling of phosphoinositides or phosphatidic acid by [32p]orthophosphate, while these local anesthetics show profound effects on the incorporation of [3H]inositol into phosphoinositides (Fig. 7). Thus, in synaptoneurosomes, local anesthetics have little effect on the de novo pathway, the only
218 p a t h w a y that results in labeling b y [32p]orthophosphate, and, therefore, m u s t stimulate [3H]inositol i n c o r p o r a tion p r i m a r i l y t h r o u g h effects o n the other pathway, n a m e l y p h o s p h a t i d y l i n o s i t o l - i n o s i t o l exchange. T w o m e t a b o l i c pools of phosphoinositides, o n e sensitive to s t i m u l a t i o n b y receptor agonists, the other insensitive, occur i n most cells (see Ref. 28). T h e n a t u r e of the pool(s) whose labeling is s t i m u l a t e d b y lcoal anesthetics could n o t be defined, because of difficulties i n r e m o v i n g local anesthetics, which c a n i n h i b i t agonist responses [23], b y washing. T h e local anesthetics all had first s t i m u l a t o r y a n d t h e n i n h i b i t o r y effects o n inositol inc o r p o r a t i o n a n d b o t h these effects occur at c o n c e n t r a tions significantly higher t h a n those required for blockade of s o d i u m channels.
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