Modulation of Calcium Fluxes Across Synaptosomal Plasma Membrane by Local Anesthetics

Journal of Neurochemis1ry Raven Press, Ltd., New York 0 1990 International Society for Neurochemistry

Modulation of Calcium Fluxes Across Synaptosomal Plasma Membrane by Local Anesthetics

Elena Garcia-Martin, Soledad Gonzhlez-Cabanillas, and Carlos Gutien-ez-Merino

Departamento de Bioquimica y Biologia Molecular y Genktica, Laboratorio de Bioquimica. Facultad de Ciencias, Badajoz, Spain

Abstract: We have studied the effects of local anesthetics (dibucaine, tetracaine, lidocaine, and procaine) on calcium fluxes through the plasma membrane of synaptosomes. All these local anesthetics inhibit the ATP-dependent calcium uptake by inverted plasma membrane vesicles at concentrations close to those that promote an effective blockade of the action potential. The values obtained for the of inhibition of calcium uptake are the following: 23 pM(dibucaine), 0.44 &(lidocaine), 1.5 &(procaine), and 0.8 mM(tetracaine). There is a good correlation between these values and the concentrations of the local anesthetics that inhibit the Ca2'- dependent Mg2+-ATF'ase of these membranes. In addition, except for procaine, these local anesthetics stimulate sever-

alfold the Ca2+ outflow via the Na+/Ca2+ exchange in these membranes. This effect, however, is observed at concentrations slightly higher than those that effectively inhibit the ATP-dependent Ca2+ uptake, e.g., 80-700 pM dibucaine, 2- 10 mM lidocaine, and 1-3 mM tetracaine. The results suggest that the Ca2+ buffering of neuronal cytosol is altered by these anesthetics at pharmacological concentrations. Key Words: Local anesthetics-Synaptosomes-Calcium transport- Ca2+,Mg2+-ATPase-Na+/Ca2+ exchange-Anesthesia. Garcia-Martin E. et al. Modulation of calcium fluxes across synaptosomal plasma membrane by local anesthetics. J. Neurochem. 55, 370-378 (1990).

Despite the large effort devoted to the understanding of anesthesia, its molecular mechanism remains elu- sive. In this regard it is to be recalled that the change of membrane fluidity produced by these drugs is quite low at their pharmacological concentrations, and thus it is unlikely to account for the cellular functional perturbations leading to the phenomenon of anesthesia (Roth, 1979; Franks and Lieb, 1982; Dodson and Moss, 1984; Escudero and Gutikrrez-Merino, 1987; Garcia- Martin and Gutikrrez-Merino, 1990).

Besides the well-known blockade of the Na+ channels of axonal membranes by anesthetics (Strichartz, 1976), other functional perturbations of the nerve cells are likely to be involved in the phenomenon of anesthesia. It should be noted that local anesthetics have been reported to be antagonists of calmodulin action at pharmacological concentrations (Tanaka and Hi- daka, 198 1; Volpi et al., 198 l), thus altering the modulation of nerve cell metabolism by Ca2+. In addition, the Ca2+,Mg2+-ATPase of the isolated nerve terminals (synaptosomes) is also strongly inhibited at concentra-

tions of local anesthetics close to their pharmacological doses (Garcia-Martin and Gutikrrez-Merino, 1986, 1990). Because Ca2+ plays a major role in cellular excitability and in particular in nerve cell functions, we undertook a systematic study of the effects of local anesthetics on the Ca2' fluxes across the plasma membrane of synaptosomes.

There is now agreement that the two most important transport processes of Ca2+ through the nerve cell plasma membrane are the Ca2+ pump and the Na+/ Ca2+ exchange (Gill et al., 198 1, 1984; Akerman and Nicholls, 1983; Sanchez-Armass and Blaustein, 1987). Both operate normally by extruding Ca2+ from the neural cytosol toward the extracellular space to maintain low cytosolic levels of free Ca". It has been suggested that the Ca2+ pump, coupled to the Ca2',Mg2+- ATPase activity of the plasma membrane, and the Na+/ Ca2+ exchange are the main pathways for Ca" extrusion from the neural cytosol (Akerman and Nicholls, 1983; Sanchez-Amass and Blaustein, 1987). Subcel- lular organelles in nerve cell terminals, e.g., mitochon-

~

Received August 8, 1989; accepted December 7, 1989. Address correspondence and reprint requests to Dr. C. Gutitrrez-

Merino at Laboratorio de Bioquimica, Departamento de Bioquimica y BiolOgia Molecular y GenCtica, Facultad de Ciencias, 06080-Ba- dajoz, Spain.

Abbreviations wed: CTC, chlortetracycline; TES, 2-{ [I-hydroxy- 1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.

3 70

LOCAL ANESTHETICS AND Ca2+ FLUXES IN SYNAPTOSOMES 3 71

dria and endoplasmic reticulum, have been suggested to play only a secondary role in buffering Ca2+ in nerve terminals under normal resting physiological conditions (Akerman and Nicholls, 1983; Gill et al., 1984).

In this article we report the results obtained in a systematic study of the effects of the local anesthetics dibucaine, tetracaine, lidocaine, and procaine on the Ca2' transport processes referred to above. In addition, because it has been shown that high concentrations of some local anesthetics (e.g., tetracaine) produce mi- cellization of biological membranes and also because we have recently shown that some of the local anesthetics studied here (i.e., dibucaine and tetracaine) significantly alter the passive permeability of sarcoplasmic reticulum membranes to Ca2+ (Escudero and Gutiir- rez-Merino, 1987), we have also studied the effects of these drugs on the passive Ca2+ influx across the plasma membrane of synaptosomes. The reported asymmetry of the Na+/Ca2+ exchange system on both sides of the plasma membrane (Gill et al., 1984; Rahamimoff and Spanier, 1984; Rega and Garrahan, 1985) prompted us to examine also the effects of these anesthetics directly on Ca2+ influx rather than only on the outflow of Ca2+ from preloaded synaptosomes. This was achieved using the fluorescence of chlortetracycline (CTC), which is shown to monitor the Na+/Ca2+ exchange through the plasma membrane of synaptosomes (Garcia-Martin et al., 1989). In addition, this approach generates continuous data that can be more appropri- ately handled for mathematical analysis. The results have been checked using the classic approach based on the use of 45Ca2+ and Millipore filtration (Gill et al., 1981, 1984).

MATERIALS AND METHODS

Synaptosomes were prepared from Wistar rat brain as indicated elsewhere (Garcia-Martin and Gutiirrez-Merino, 1986), basically following the method of Michaelis et al. (1983). Synaptic vesicles were prepared as done by Gill et al. (1986). Briefly, synaptosomes were subjected to osmotic lysis with a hypotonic solution containing 5 mM 2-{ [2-hydroxy- 1,1 -bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid (TES), 1 mM EDTA, and 2 mM P-mercaptoethanol (pH 7.4). After 40 min at room temperature, a low-speed centrifugation run at -8,000 g for I0 min was done to remove mitochondria and remaining synaptosomes; then the supernatant was centrifuged at -40,000 g for 30 min to pellet the plasma membrane synaptic vesicles. The percentage of inverted vesicles was estimated to be -40% from measurements of the Na+,K+-ATPase and of the Ca2+-ATPase activities of these membranes, as in Gill et al. (1986). Protein concentration was measured by the method of Lowry et al. (195 1) using bovine serum albumin as standard. The total Ca2+,Mg2+-ATPase activity of these membrane preparations was measured using the coupled enzyme system pyruvate kinase/lactate dehydrogenase. The composition of the assay medium was as follows: 50 mM TES (pH 7.4), 0.1 A4 KCI, 2 mMMgCI2, 2 mMATP, 50 pMCaCI2 (free Ca" 18.6 p M ) , 2 mM 8-mercaptoethanol, 0.42 m M phosphoenolpyruvate, 0.22 mil4 NADH, 5 mM sodium azide, 10 IU/ml pyruvate

kinase, and 28 IU/ml lactate dehydrogenase. 45Ca2f uptake was measured by Millipore filtration through HAWP02500 filters. The reaction medium for Ca2+ uptake contained the following: 50 mM TES (pH 7.4), 0.1 M KCI, 2 m M MgClz, 1 mM ATP, 50 pM CaC12 (0.4 pCi/ml, free Ca2+ 42 pM), 2 mM P-mercaptoethanol, and 0.13 mg synaptosomal protein per milliliter. Aliquots of the reaction mixture were pooled at 2 and 5 min and at 10 min after addition of A23187 (19 p M ) . These aliquots were added to 3 ml of a solution of ice- cold Lac13 (0.4 mM) to stop the reaction, then vacuum fil- tered through Millipore filters. The filters were washed with 6-9 ml of ice-cold solution containing 50 mMTES (pH 7.4), 0.1 M KCI, 1 mM MgClz, and 1 mM LaCI3 ; they were then dissolved with methyl glycol and counted with a Beckman scintillation counter, using [2-(4'-butylphenyl)-5-(4'-biphen- yly1)- I ,3,4-oxadiazole/toluene ( 5 g/L) as the scintillation cocktail.

The intrasynaptosomal free Ca2+ concentration was measured using the fluorescence indicator quin-2 following the procedure described by Ashley et al. (1984). The tetracetox- ymethylester of quin-2 was dissolved in dimethyl sulfoxide at a concentration of 5 mM. The calibration of the method and calculation of the intrasynaptosomal free Ca2+ concentration were carried out as done by Ashley et al. ( I 984), except that, following Bryant ( 1 985), the apparent dissociation constant (KD,app) of the quin-2-Ca2+ complex was calculated from the expression

where K F = 2.9 k 0.2 nM and K F = 89 k 5 pM (Bryant, 1985).

The measurement of Ca2+ fluxes from CTC fluorescence is described in Appendix.

Light-scattering measurements Measurements of the Caz+ permeabilities following the

light-scattering method have been camed out as described in detail elsewhere (Kometani and Kasai, 1978; Escudero and GutiCrrez-Merino, 1987). The operational procedure followed in the osmotic shock experiments is outlined next. One volume of synaptosomes or vesicles preequilibrated at 25°C dialyzed against 10 mM TES (pH 7.4) was mixed with I vol of 10 mM TES buffer containing the appropriate salt (i.e., NaCl or CaCI2) at a final concentration of 100 mM. Upon mixing with a hyperosmotic solution, vesicles and synaptosomes shrink as water flows out to compensate osmolanty between compartments, and then they swell as salts of the external medium diffuse inside accompanied by water (Ko- metani and Kasai, 1978). To avoid an extensive lysis of membranes, the sucrose and TES of preparation buffers were removed in three dialysis steps of 2-3 h each, so that synaptosomes were dialyzed first against 10 mM TES and 300 mM sucrose, then against 10 mM TES and 100 mM sucrose, and finally against 10 mM TES. Light-scattering intensity at 400-410 nm was measured at 25°C as a function of time after mixing using a Hitachi-Perkin Elmer spectrofluorometer (model 650-40). The analysis of the slow recovery phase of the light-scattering data was carried out as indicated in Es- cudero and Gutitrrez-Merino (1987). The data can be sat- isfactorily fitted to one exponential of the form

[ I , - = [ I , - I ~ ] ~ / ~ . e+' (2)

where fo, I, , and I , are the light-scattering intensities at the

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3 72 E. GARCfA-MARTiN ET AL.

peak after mixing, at time t , and at equilibrium after mixing, respectively, and D is the diffusion coefficient of the cation through channels in the membrane. Equation 2 is derived under the assumption that the anion, chloride in our exper- iment, is a much faster permeant species than the cation. This assumption is largely justified in the case of the plasma membrane of synaptosomes (Hille, 198 1). The results of osmotic shock experiments presented in this article represent the average of duplicate experiments camed out with at least three different preparations of synaptosomes or synaptic vesicles.

Chemicals Bovine serum albumin, LaC13, ATP, CTC, phosphoenol-

pyruvate, EDTA, EGTA, dibucaine, procaine, lidocaine, tetracaine, phenylmethylsulfonyl fluoride, P-mercaptoethanol, quin-2 acetoxymethyl ester, and TES were obtained from Sigma. A23 187, pyruvate kinase, and lactate dehydrogenase were purchased from Boehringer Mannheim. All the other chemicals used in this study were of analytical grade and obtained from Merck or from Carlo Erba. Tetracaine solu- tions were freshly prepared before use.

RESULTS

Inhibition of calcium pump by local anesthetics The effect of these local anesthetics on the steady-

state ATP-dependent Ca2' accumulation by plasma membrane vesicles, prepared from synaptosomes as indicated in Materials and Methods, was measured by Millipore filtration. Both the fraction of inverted vesicles (40%) estimated as indicated above and the amount of Ca2+ accumulated at 2 and 5 min in the absence of anesthetic (3.4 and 5.4 nmol Ca2+/mg membrane protein, respectively; results not shown) agree well with the values reported by other investigators in similar experimental conditions (Papazian et al., 1979; Gill et al., 198 1, 1984; Banilai and Rahami- moff, 1983). Figure 1 shows the effect of several concentrations of the local anesthetics on the ATP-dependent Ca2+ accumulation. From these results the following values can be derived: 23 pM (dibucaine), 0.44 mM(lidocaine), 1.5 mM(procaine), and 0.8 mM (tetracaine). The Ca2+,Mg2+-ATPase activity of these membranes is also inhibited by these concentrations of local anesthetics (Garcia-Martin and Gutitrrez- Merino, 1986, 1990). On the contrary, the concentrations of the local anesthetics studied herein did not significantly alter the total calcium binding capacity of synaptosomal membranes, i.e., the Ca2+ binding per milligram of protein in the presence of A23 187, which was found to be 2.7 +- 0.2 nmol Ca2'/mg protein.

Effect of local anesthetics on calcium permeability of plasma membrane of synaptosomes

The observed decrease in the level of Ca2+ uptake by synaptosomes (see above) could be due either to the inhibition of the Caz+ pump or to a large increase of the passive permeability of the plasma membrane of synaptosomes to Ca2+ or to both effects simultaneously. We thus measured the passive permeability of synap tosomes to Ca2+ in the presence of various concentra-

.-.. r - 4

L" 120 Y C 0 u + 0

x 80 - ol Y m Q 40 a 3

E 3 r i

c( u o m -5.5 - 4 . 5 - 3 . 5 -2 .5 -1.5 U

Log [Local a n e s t h e t i c ]

FIG. 1. Dependence of the ATP-dependent Ca2+ uptake upon the total local anesthetic concentration in the assay medium. Calcium uptake was determined 2 min after the addition of 1 mM ATP and is expressed as percentage of the control value measured in the absence of local anesthetic in the assay medium. The results shown have been averaged from those obtained in duplicate experiments carried out with three different synaptosome preparations. (0). dibucaine; (A), lidocaine; (4, tetracaine; (O), procaine. Other experimental conditions are indicated in the Materials and Methods.

tions of the local anesthetics. We used the osmotic shock approach as described in Materials and Methods. The possibility of changes in the aggregation state of synaptosomes under these experimental conditions was explicitly considered, as indicated in Escudero and Gutitrrez-Merino (1987), and this putative contribu- tion to the light-scattering change was found to be neg- ligible.

The passive Ca2+ influx through the plasma membrane of synaptosomes can be described as a simple exponential process, as illustrated in the Fig. 2. The rate constant of this process can be estimated as indicated in Materials and Methods, and we obtained a value of 0.013 f 0.002 min-' (half-time of 54 f 9 min; mean of > 10 experiments with three different preparations of synaptosomes). This value agrees well with the one that can be approximately derived from data published by Gill et al. (1984), which were obtained using the Millipore filtration technique to monitor the efflux of 45Ca2+ from synaptosomes preloaded with this cation, and it is similar to the rate of passive permeability to Ca2+ of membranes from other mammalian cells, such as the sarcoplasmic reticulum (Escudero and Gutitrrez-Merino, 1987). We have found that the permeability to Ca2+ is not significantly enhanced by procaine (up to 15 mM), lidocaine (up to 5 a), dibucaine (up to 0.8 mM), or tetracaine (up to 0.5 mM). Ethanol (up to 2%) does not significantly enhance the permeability to Ca2+ of these membranes either (E. Garcia-Martin, S. Gonzhlez-Cabanillas, and C. Gu- titrrez-Merino, unpublished results). This observation has been particularly useful for studying the effect of higher concentrations of some of these local anesthetics, such as lidocaine and dibucaine, which were added in ethanolic solution because of their low solubility in

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3 73 LOCAL ANESTHETICS AND Ca2+ FLUXES IN SYNAPTOSOMES

7 . 5 , I

D l I 6 . 5

3.5.1 0 9 18 27 36 45

T i m e ( mln )

FIG. 2. Dependence of the light-scattering intensity (I?) of vesicles prepared from synaptosomes after an osmotic shock with 100 mM CaCI,. The experimental conditions are indicated in Materials and Methods. The symbols are representative points taken at different times from the continuous recorder trace and the error bars are indicative of the noise of the signal. The data shown are representative of those obtained in >10 measurements carried out with three different membrane preparations. The experimental data are plotted as ($) * In(/, - I , ) to allow their analysis as indicated in Materials and Methods. I, is the light-scattering intensity at osmotic equilibrium after the osmotic shock.

water. It is to be recalled that at these local anesthetic concentrations, both the ATP-dependent Ca2+ uptake and the Ca2+,Mg2+-ATPase activity are strongly inhibited. In addition, these results indicate that the membrane is not disrupted by concentrations of the local anesthetics studied herein. This point was further checked by studying the effect of these local anesthetics on the permeability of synaptosomes to sucrose. None of them at the concentration referred to above significantly increases the rate of sucrose inflow through these membranes.

Effects of local anesthetics on Nat/Ca2+ exchange The effects of local anesthetics on Ca2+ outflow

through the Na+/Ca2+ exchange were studied by monitoring with CTC the release of Ca2+ from synaptosomes preloaded with this cation in the presence of 100-150 mM NaCl in the external medium (see Ap- pendix). Synaptosomes were preloaded with CaC12 by preincubation with 5-20 mA4 CaC12 for 2-3 h at 4°C or alternatively by freezing and thawing and 30-min incubation at 37°C. Both experimental approaches gave us identical results within experimental errors. This short preincubation time was used to avoid the flocculation of membranes that occurs upon overnight incubation with these concentrations of CaC12 ; in addition, using 45Ca2f we have confirmed that these treatments produce a large load of Ca2+ sufficient to almost equilibrate outer and inner Ca2+ concentrations, both in synaptosomes and in plasma membrane vesicles. On the other hand, 1 mMCaC12 in the incubation medium gave us a low signal to accurately monitor the Ca2+ outflow. The time dependence of the decay of CTC fluorescence in the presence of Na+ in the external medium closely followed the release of 45Ca2+ from

preloaded synaptosomes (results not shown). To further ensure that we were monitoring the decay of CTC fluorescence, which paralleled the release of Ca2+ coupled to Na' inflow, we camed out experimental series such as that shown in the Fig. 3, using both synaptosomes and plasma membrane vesicles prepared from them. It is readily evident from the data presented in this figure that the enhancement of the decay rate of CTC fluorescence is dependent upon the presence of Na+ in the external medium and that K+ does not produce any significant increase in this slow decay. Further- more, millimolar Li+ concentrations, which are known to decrease the rate of the Na+/Ca2+ exchange process (Gill et al., 198 I ) , clearly slow down the rate of change of the CTC fluorescence in the presence of NaCl. Figure 4 shows the effects of several concentrations of lidocaine on the time dependence of the decay of the intensity of CTC fluorescence after dilution of synaptosomes preloaded with 20 mM Ca2+ into a medium depleted of calcium by addition of 1 mA4 EGTA and containing 150 mMNaC1. It can be observed that millimolar concentrations of lidocaine stimulate severalfold the rate of Ca2+ efflux from synaptosomes. Similar results have been obtained using 45Ca2+ to monitor calcium efflux from preloaded synaptosomes in the presence of 150 mM NaCl in the external medium. It is to be noted, however, that the extent of the observed stimulation is only two- to threefold. However, by use of 45Ca2+, the decrease of total 45Ca2+ within all synaptosomal compartments (e.g., mitochondrial, vesicular, and cytosolic) is the monitored parameter. This likely decreases the apparent effect on Ca2+ efflux through the plasma membrane, mostly owing to the high capacity of mitochondria to accumulate Ca2+. The rate constant of Ca2+ outflow determined using the fluorescence of CTC (0.15 f 0.02 min-') is nearly identical to that measured using 45Ca2+ (results not shown). A similar stimulation has been obtained with tetracaine and dibucaine, whereas procaine (up to 10 mM) was found to have no effect. The rate constant of Ca2+ outflow has been estimated as indicated in Appendix, and the stimulation of the rate of Ca2+ outflow by these local anesthetics is quantitatively presented in Table 1.

0 10 20 30 40 50 60

Time l m i n l

FIG. 3. Dependence of the fluorescence of CTC (normalized to the maximum value, F-) upon time after addition of synaptosomes preloaded with 20 mM CaCI2 to a medium containing 10 pM CTC, 1 mM EGTA, and 50 mM KCI. The arrows indicate the addition of 0.1 M KCI (a) or 0.1 M NaCl (b) to otherwise duplicateexperiments.

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3 74 E. GARCIA-MARTIN ET AL.

- 2 1001 I

L L 1 6 11 16 21 26

T i m e (min) FIG. 4. Effect of lidocaine on the time dependence of CTC fluorescence after dilution of synaptosomes, preincubated at 4°C with 20 mM CaCI2 for 2-3 h, in a Ca2+-free medium plus 150 mM NaCl (a& arbitrary units). The total concentrations of lidocaine in the medium were 0 (a), 2 (b), 4 (c), 6 (d), and 8.1 (e) mM. The results shown are a typical experimental series obtained with one preparation of synaptosomes and are representative of the data obtained with three different preparations. Other experimental conditions are indicated in Materials and Methods.

Adsorption of local anesthetics on synaptosomal membranes

values listed above are given as the total local anesthetic concentrations in the cuvette. Calcium uptake, osmotic shock, and CTC measurements were carried out at membrane concentrations of -0.1-0.2 mg protein/ml (see above). The extent of local anesthetic bound to synaptosomal membranes was estimated by measuring the concentration of the local anesthetic in the supernatant after pelleting synaptosomes preincubated with a given anesthetic concentration for 30 min by centrifugation at 20,000 g for 20 min. The concentration of the anesthetics before addition of synaptosomes and in the supernatant after pelleting them was measured from their respective absorbance peaks, i.e., 328 nm for dibucaine, 3 10 nm for tetracaine, 295 nm for procaine, and 273 nm for lidocaine.

In the presence of a synaptosomal concentration of 0.6 mg protein/ml, the following values were obtained for the partition coefficient Kp (expressed as the ratio between the total local anesthetic bound to the membrane and the anesthetic in the aqueous solution): 1.2 f 0.2 (dibucaine), 0.76 & 0.07 (tetracaine), 0.28 k 0.02 (procaine), and to. 1 (lidocaine). These Kp values are similar to those obtained for sarcoplasmic reticulum membranes (GutiCrrez-Merino et al., 1989). The value of Kp has been determined from measurements at several total local anesthetic Concentrations. Because it has been shown that interactions of local anesthetics with lipid bilayers exhibit saturation characteristics (Ohki, 1984; Eftink et al., 1985), the data presented correspond to the values obtained with nonsaturating local anesthetic concentrations, i.e., those obtained at total anesthetic concentrations lower than 25% of total lipid concentration in the medium. Consistently, at

The

higher anesthetic concentrations the apparent Kp values tend to be smaller (results not shown). In addition, these values of Kp appear to be linearly dependent upon the concentration of membranes, being about three times lower at a protein concentration of 0.2 mg protein/ml (results not shown). It follows that a maximum value of 10-20% of total local anesthetic concentration should be bound to synaptosomes in the experiments reported in this article. Furthermore, considering that the lipid/protein ratio (wt/wt) of the plasma membrane of synaptosomes is 1: 1 (Hitzemann et al., 1984), at 0.1 mg/ml the estimated total concentration of lipid is - 130 pM. Therefore, taking into account that the saturation of the lipid bilayer by these local anesthetics occurs at molar ratios of around five lipids per anesthetic molecule (Eftink et al., 1985), it follows that the maximum concentration of local anesthetic bound to synaptosomes (expressed per unit of solution volume) in CTC or in osmotic shock experiments is -26 pM. Thus, only the lower concentrations of dibucaine used in this study require a significant correction for binding to synaptosomes.

DISCUSSION

The Ca2+ pump of the plasma membrane of nerve cells operates by extruding Ca2+ from the cytosol to the extracellular space and is one of the more important transport systems of the neuron involved in maintain- ing submicromolar free Ca2+ levels in the cytosol (Gill

TABLE 1. Effects of local anesthetics on Na+lCa2+ exchange across plasma membrane of synaptosomes

Aqueous local anesthetic concentration Relative rate of Ca2+ efflux"

No anesthetic

Lidocaine (mM) -

2 4 6 8.1

0.5 1 3

Tetracaine (mM)

Dibucaine ( p M ) (60 f 4) 134 214 454

1 .o

1.5 2.6 3.7 6.0

1.5 4.2 6.5

1.2 1.9 3.2 6.5

Data are the average of results obtained from duplicate experiments carried out at 37°C with at least three different synaptosome prep arations. Estimated error of these data: f 10%. The rate constant of Ca2+ efflux coupled to Na+ influx in the absence of local anesthetics has been calculated to be 0.15 f 0.02 m i d , using synaptosomes preloaded with 20 mM Ca" and with 150 mM NaCl in the external medium.

Estimated from the decay of CTC fluorescence.

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3 75 LOCAL ANESTHETICS AND Ca2+ FLUXES IN SYNAPTOSOMES

TABLE 2. Concentrations of local anesthetics producing an effecctive inhibition of ATP-dependent Caz+ uptake and of Ca2+, Mg"-A TPase of synaptosomal plasma membrane

and effective blockade of action Dotential

Ko.sa K0.2' G o ' Local anesthetic (M) (M)

Dibucaine 0.023 S O . 1 0.008 Tetracaine 0.8 0.4 + 0.1 0.02 Lidocaine 0.44 0.75 f 0.25 0.8 Procaine 1.5 2 + 0.5 1.6

a &.s is the local anesthetic concentration needed to produce -50% inhibition of Ca2+ uptake. Values are the &., values after correction for local anesthetic adsorption to the membrane (see the text).

&.2 is the local anesthetic concentration needed to produce -20% inhibition of total Ca2+, Mg2+-ATPase activity, which can be taken as the && of the Ca2+, Mg2+-ATPase activity as indicated in Garcia- Martin and GutiCrrez-Merino (1986).

Concentration of local anesthetic that reduces the amplitude of the Aa action potential in sciatic nerve by 50%, taken from Greenberg and Tsong (1982).

et al., 198 1; Akerman and Nicholls, 1983). The results presented in this article show that at local anesthetic concentrations close to their respective pharmacological concentrations, these drugs inhibit the active Ca2+ transport across the plasma membrane of synaptosomes (see Table 2). Moreover, Table 2 also shows that, except for the case of tetracaine, the Ca2+ pump of the synaptosomal plasma membrane is inhibited by local anesthetics in the same concentration range that effectively blocks the Aa action potential of the sciatic nerve (Greenberg and Tsong, 1982). It is to be recalled that in vivo the large membrane potential of the plasma membrane favors a stronger adsorption of the charged local anesthetic species to the membrane, as has been shown for the case of dibucaine adsorption on dimyristoylphosphatidylcholine vesicles (Mayer et al., 1985), where the amount of this anesthetic bound to the lipid bilayer increases approximately eightfold upon a change in the membrane potential from 0 to about -70 mV, the resting potential of the nerve cell plasma membrane. Thus, this indicates that in vivo the apparent values for Ca2+ pump inhibition by these local anesthetics should be even lower than those obtained in this study. The inhibition of the Ca2' pump cannot be attributed to membrane disruption or to an important structural alteration of the lipid bilayer, as is shown by the maintenance of the membrane permeability barrier toward Ca2+ and sucrose and by the lack of a significant change in the polarization of 1,6- diphenyl- 1,3,5-hexatriene at local anesthetic concentrations that inhibit Ca2+,Mg2+-ATPase (Garcia-Martin and Gutitrrez-Merino, 1990). Thus, the possibility that local anesthetics could raise the cytosolic Ca2+ level of the neuron deserves consideration and is discussed quantitatively below.

Other Ca2+ transport processes, such as Na+/Ca2+ exchange and passive Ca2+ flux, are involved in main- taining physiological Ca2+ gradients across the plasma

membrane of the neuron (Gill et al., 1981, 1984; Ak- erman and Nicholls, 1983; Mullins et al., 1985). Therefore, we have also studied and quantified the effects of local anesthetics on these Ca2+ transport processes.

The results obtained in our study gave a value for the rate of passive Ca2+ influx across the plasma membrane of synaptosomes of 0.013 f 0.002 min-' (tlI2 = 54 * 9 min). This value agrees well with experimental results of passive Ca2+ efflux from synaptosomes preloaded with 45Ca2f as reported by Gill et al. (198 1). Taking for the internal volume of vesicles of synaptosomal plasma membrane a value of 3-7 pl/mg protein (Rahamimoff and Abramovitz, 1978; Rahamimoff and Spanier, 1984), for a physiological gradient of Ca2+, i.e., 0.1 pA4 and 1 mM free Ca2+ inside and outside the vesicles, respectively, we obtain a maximum rate of passive Ca2+ flux of lower than 0.1 nmol/min/mg protein. This result is in agreement with reported data using 45Ca2+ and Millipore filtration (Gill et al., 198 1). As shown above, the local anesthetics studied herein do not significantly alter this rate of passive Ca2+ flux in the concentration range that inhibits the Ca2+ pump of these membranes.

From the measurements of CTC fluorescence, using the rate constants given in Table 1, the maximum rate of Ca2+ efflux via the Na+/Ca2+ exchange in synaptosomes can be estimated to be 5- 10 nmol/min/mg protein for a physiological gradient of Na+ and an internal Ca2+ concentration far above the physiological range. In addition, the rate of Na'/Ca2' exchange obtained from our results agrees well with data published by other investigators (Gill et al., 198 1, 1984; Barzilai and Rahamimoff, 1983; Rahamimoff and Spanier, 1984). On the other hand, it has been demonstrated that the rate of this efflux is strongly dependent on free Ca2+ concentration within the synaptosomes (Gill et al., 1984). If preloading of synaptosomes with Ca2+ is omitted, the concentration of free Ca2+ within our preparation of synaptosomes is - 100- 150 nM in the presence of an energy donor substrate such as 10 mM glucose, as estimated from measurements of the fluorescence of quin-2 (see Materials and Methods). This result is in good agreement with that reported by Ashley et al. (1984) for synaptosomes prepared following a protocol slightly different from ours and it is close to the free Ca2+ level of resting nerve terminals (Akerman and Nicholls, 1983). Attempts to directly measure the level of free Ca2+ within the synaptosomes in the presence of local anesthetics have been unfruitful so far, because we have found that quin-2 and other fluorescent indicators of Ca2' interact with local anesthetics, resulting in large changes of their fluorescence intensity (Gutitrrez-Merino and Mach , 1989). The rate of Ca2+ efflux through the Na+/Ca2+ exchanger at a free Ca2+ concentration of 0.1 pM is - 15-fold lower than its value at a free Ca2+ concentration of 2 mM(Gil1 et al., 1984). Therefore, the maximum rate of Ca2+ efflux from the nerve terminals in resting physiological con-

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3 76 E. GARCfA-MARTIN ET AL.

ditions can be estimated from our data to be -0.3- 0.7 nmol Ca2+/min/mg protein. The results presented here show that local anesthetics stimulate by about twofold this rate at concentrations that inhibit the Ca2+ Pump-

On the other hand, the maximum rate of Ca2+ uptake by the plasma membrane of synaptosomes has been reported to be -2.2 nmol Ca2+/min/mg protein (Papazian et al., 1979; Gill et al., 1981; Barzilai and Rahamimoff, 1983) and in this study was -4.0 nmol/ min/mg protein (considering that only 40% are inverted vesicles). It follows from this analysis that stimulation of the Na+/Ca2+ exchange from the neural cytosol by local anesthetics cannot fully compensate for the inhibition of the Ca2+ pump and thus these drugs are likely to raise the level of free Ca2+ within the nerve terminals. It might be worthy to recall here that the role of the Na+/Ca2+ exchange in the extrusion of Ca2+ from the cytosol has been claimed to be secondary to that of the Ca2+ pump in resting conditions (Akerman and Nicholls, 1983; Gill et al., 1984; Sanchez-Armass and Blaustein, 1987). Our data and calculations fully support this hypothesis. Nevertheless, upon repetitive stimulation of the nerve terminal, it has been suggested that the Na+/Ca2+ exchange process can operate even more efficiently than the Ca2+ pump to lower the cytosolic Ca2+ level (Sanchez-Armass and Blaustein, 1987). For this case, the stimulation of this exchange by local anesthetics will certainly tend to avoid an ex- cessive rise of the cytosolic Ca2+ level, thus preventing it from reaching the neurotoxic range. In this regard, it is to be noted that relatively minor changes of free cytosolic Ca2+ in the nerve terminal, e.g., from 100 to 300-500 nM, have been associated with enhanced neurotransmitter release (Milani et al., 1989; Verhage et al., 1989).

A priori, it seems unlikely that drugs that typically decrease the cellular excitability can act via an increase of cytosolic free Ca2+ levels. Nevertheless, it might be worthy to note that the increase of cytosolic free Ca2+ by local anesthetics allows us to easily rationalize two basic observations: first, the cellular hyperexcitability caused by low doses of anesthetics (Siesjo, 1978), and second, the “anesthetic action” of high Mg2+ levels in the extracellular fluid, probably as a result of antago- nistic Ca2+ biochemical actions (Metzler, 198 1). These observations thus make it tempting to speculate that anesthesia could result from synaptic transmission failure due to depletion of neurotransmitter-loaded vesicles, a side-effect of sustained moderately high free Ca2+ levels in the neuronal cytosol. Depletion of the concentration of presynaptic vesicles loaded with neurotransmitter could also be, at least partially, induced by alternative mechanisms of action, such as inhibition of the H+-ATPase in catecholaminergic neurons (Barchfeld and Deamer, 1985; Akeson and Deamer, 1989). This hypothesis could account for the wide va- riety, and lack of chemical structural homology, of compounds inducing anesthesia and, in our opinion,

deserves further studies in vivo. In addition, local anesthetic concentrations higher than those promoting anesthesia should produce an even larger increase of cytosolic free Ca2+ as they completely inhibit the plasma membrane Ca2+ pump. Sustained high levels of free intracellular Ca2+ have been suggested to be an index of potential neurotoxicity (Komulainen and Bondy, 1988), and it is well known that high doses of anesthetics are neurotoxic (Siesjo, 1978).

APPENDIX

Measurement of Ca2+ emux using cI% fluorescence It has been shown that the fluorescence of CTC is notably

enhanced when its complex with Ca2+ is bound to a biological membrane (see, e.g., Millman et al., 1980). Thus, if CTC diffuses across the membrane with a rate faster than that of Ca2+ transport, it is possible to accurately estimate the rate of Ca2+ transport from the time dependence of CTC fluorescence (Dixon et al., 1984). Because the quantum yield of CTC fluorescence in solution, either free or complexed with Ca” (Ca-CTC), is much lower than that of the Ca-CTC bound to the membrane (Millman et al., 1980), the fluorescence of CTC in the presence of biological membranes directly mon- itors the Ca2+ bound to them, and thus reflects the free concentration of Ca” within the vesicles. Addition of vesicles preloaded with Ca2+ to a medium containing CTC and depleted of Ca2+ by EGTA initially produces a large enhancement of CTC fluorescence followed by a slow decay of its intensity (Fig. 5). It is likely that the rise of the fluorescence intensity directly reflects the diffusion of CTC within the vesicles, while the slow decay reflects the outflow of Ca” through the membrane. This interpretation rests on the assumption that the rate of formation of the Ca-CTC complex is much faster than the rate of diffusion of CTC across the membrane. Because the half-times of CTC diffusion across some mam-

0 . a - 10 E CL \ LL 0 . 4 -

0 . 0 4 0 5 10 15 20

T i m e ( m i n i FIG. 5. Dependence of the fluorescence of CTC (normalized to the maximum value, F-) upon time after addition of synaptosomes preloaded with 20 mM CaCI2 to a medium containing 10 &l CTC and 1 mM EGTA. Protein concentration, 0.1 mg/ml. The solid line corresponds to the best fit of the experimental data (broken line) to Eq. A1 . This fit was obtained using values for the rate constants K, and K. of 1.038 and 0.126 min-’, respectively, and a value for KcacTc of 384.6 M-’ (Millman et al., 1980). The data shown are representative of those currently obtained in our experimental conditions, averaged from >I 0 assays carried out with three different preparations of synaptosomes. See Appendix for further experimental details.

J. Neurochem., Vol. 55. No. 2, 1990

LOCAL ANESTHETICS AND Caz’ FLUXES IN SYNAPTOSOMES 3 77

malian membranes have been estimated to be -20-30 s in different experimental conditions (Millman et al., 1980; Dixon et al., 1984), this assumption appears to be largely justified.

Therefore, it can be written that

[CTCIT,, = [CTC]i + [Ca-CTC],

[Ca-CTCIi [Ca2+Ii [CTC], KCa-CTC =

[CTC]i = [CTC], ‘ ( I - e - K c . f ) (A41

where Fca.crc.Mb and F(t) are the intensity of the fluorescence of the “Ca-CTC-membrane” complex and the intensity of the fluorescence at a time ( after the addition of membranes to the reaction medium, respectively; subscripts i and o stand for concentrations inside and outside the vesicles, respectively; K, and K, are the rate constants of CTC diffusion inside the vesicles and of Ca2+ outflow, respectively; and [Ca”], = 0 is the concentration of Ca2+ within the vesicles at the moment of dilution in a Ca2+-free medium.

We have found that. as expected, the peak of the curve of the time dependence of the CTC fluorescence is directly pro- portional to the concentration of CaClz in the medium in which synaptosomes were preincubated (results not shown). The intensity of the CTC fluorescence reaches a limiting steady-state value after equilibration ofthe Ca2+ concentration across the membrane. This fluorescence intensity can be attributed to the complex of C‘TC with the Ca2+ that remains bound to the membranes and to that of free CTC. Thus, this component needs to be subtracted from the fluorescence intensity measurements to estimate K, and K,. The continuous line shown in Fig. 5 is the best fit of the corrected experimental data to Eq. A l , using the parameters indicated in the legend to that figure. It can be seen that the fit of the experimental data to the theoretical prediction is excellent. Synaptosomes are complex cellular fragments containing mitochondria and other subcellular organelles (Bradford, 1986), each with different Ca2+ accumulation capacity. However, the fit of the experimental data to the theoretical approach outlined above shows that under these experimental conditions CTC reflects only the concentration of Caz+ in a single internal compartment. This compartment is likely to be that in contact with the plasma membrane because the total concentration ofCTC is much lower than that of total lipids in the cuvette and CTC is in the external medium at the time of mixing. This hypothesis is supported by the fact that preincubation ofsyn- aptosomes with higher CTC concentration (above 0.1 mM) renders complex patterns of fluorescence decay, not ade- quately fitted by only a single exponential process (results not shown). In addition, because K, >> K,, from the fit of the decay of the intensity of CTC fluorescence to a simple exponential process, K, can be readily obtained. The values of K, referred to or presented in this article have been obtained by linear regression of In[F(co) - F(t)] versus t in the decay region of the time dependence of CTC fluorescence.

Acknowledgment: This work has been supported by grant no. 28 13/83 of the Spanish CAICYT.

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Modulation of Calcium Fluxes Across Synaptosomal Plasma Membrane by Local Anesthetics

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