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Effect of vitamin E on acetylcholine-induced current in molluscan neurons: Role of cytoplasmic free calcium and arachidonic acid
03064522/92 55.00 + 0.00 Pergamon Press Ltd 0 1992 IBRO
Neuroscience Vol. 48, No. 3, pp. 145-152, 1992 Printed in Great Britain
EFFECT OF VITAMIN E ON ACETYLCHOLINE-INDUCED CURRENT IN MOLLUSCAN NEURONS: ROLE OF CYTOPLASMIC FREE CALCIUM AND ARACHIDONIC ACID V. A. DYATLOV Laboratory of Neurobiology, A. A. Bogomoletz Institute of Physiology, Ukrainian Academy of Sciences, Kiev 24, 252601 GSP, Ukraine of cytoplasmic concentration of free Ca2+ ([Cal,) and arachidonic acid in potentiating the effect of vitamin E CDL-CL -tocopherol) on acetylchoiine receptor activity in H&x acre neurons was studied using a two-microe~~tr~e intracellular recording, voltage clamp and fluorescent calcium probe fura- technique. Elevation of [Cali, by intracellular injection from a microelectrode or by depolarizing pulses and application of 0.1 pM-O.1 mM vitamin E enhanced the acetylcholine-induced chloride current both in LPI1 and RBc4 neurons. Application of 10 PM arachidonic acid to the same neurons decreased this current. The use of fluorescent probe showed that vitamin E did not essentially change [Cal,, but an increase of [Cal. intensified both the enharming effect of vitamin E and the depressing effect of a~~hidonic acid. The enhancing effect of calcium influx was considerably decreased after vitamin E
Abstract-Role
application. The antioxidant action of vitamin E was probably not involved in the mechanism of its enhancing effect on acetylcholine-induced current, since synthetic antioxidant, ionol, depressed acetylcholine responses. A spectrum analysis has shown the interaction between vitamin E and arachidonic acid in solution. This interaction may be considered as the molecular mechanism responsible for the prevention by vitamin E of steady arachidonic acid production from membrane phospholipids and its down-regulatory effect on acetylcholine receptor activity. Our rest&s support this suggestion, since an inhibitor of phosphoiipa~ A,, 4-bromoph~acyl bromide, mimicked the enhancing effect of vitamin E.
In previous papers,8*‘3we described the vitamin E-dependent regulation of the properties of acetylcholine (ACh) receptors in identified neurons of the snail Helix pomatia. This may be related to the fact that lipid-soluble tocopherols (different forms of vitamin E) act as antioxidants and slow down the rate of metabolic processes. It has aiso been suggested that some of the effects of vitamin E may be connected with calcium accumulation in cells that is enhanced under vitamin E deficiency.” The structural stability of biological membranes may be supported by vitamin E via its reaction with lipid peroxide radicals, protection against destructive action of arachidonic acid and quenching of singlet mokcular oxygen.3*‘5In the present paper, we have extended the analysis of the possible roles of cytoplasmic Ca*+, arachidonic acid and antioxidant properties of vitamin E in the enhancing effect of vitamin E on the ACh-induced chloride current in identified neurons of the mollusc Helix pomatia. Abbreviations:ACh, acetylcholine; [Cal,, cytoplasmic concentration of free Ca2+; DMSO. dimethvl sulohoxide: EGTA, ethyleneglycol-bis(aminoethylether)te&acetic acid; HEPES, ~-2-hydroxyethylpi~~ne-~-2-eth~~ &phonic acid.
EXPERIMENTAL PROCEDURES
Subjects The perioesophageal ganglionic ring together with the buccal ganglia was isolated from Helix pomatia snails and fixed inside a plastic chamber exposing the ventral surface of the ganglia. The gli~onn~tive envelope of the ganglia was removed under microscopic visualization. Neurons Dl, D4, E4, LPl 1 and RBc4 were identified in different preparations according to their position and their responses to drugs and stimulation of the main nerves.18*33For the right buccal and the left pleural ganglia the abbre~ations RBc and LPI were applied, respectively. The preparation was bathed in continuously circulating saline solution with the following composition (in mM): NaCl 100, KC1 4, CaCl, 10, MgCl, 4, HEPES-NaOH 10, pH 7.6. Electroph~~~oiog~cal recordings The electrophysiological experiments were carried out under voltage-clamp conditions, using a twomicroelectrode set-up. In some experiments intracellular recordings were made from non-clamped neurons. We used glass micr~lectrodes filled with 745
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2.5 M KC1 or 0.6 M KSO, of 8-12 MR.
and having a resistance
Drug applications Acetylcholine was applied locally to the neuronal somata by iontophoresis or by pressure ejection from a pipette with a 556-pm tip. To avoid desensitization, the cell was washed between two subsequent responses with saline solution for 3 min. Ca’+ and EGTA were injected intracellularly by iontophoresis or by depolarizing pulses as described earlier.7~‘o Vitamin E (0.1 p M-O. 1 mM; DL-x-tocopherol), 0.1 mM ionol, 10 PM arachidonic acid and 20 PM 4-bromophenacyl bromide (all dissolved in saline solution) were applied to individual cells by pressure from a pipette with a 5-6-pm tip positioned 15-20 pm from the cell. DL-a-Tocopherol and arachidonic acid were first dissolved in absolute ethanol (lo-’ M) and then diluted in the saline solution. Arachidonic acid was dissolved in ethanol under a gentle stream of nitrogen immediately before use. Arachidonic acid and fura-Z/AM were purchased from Calbiochem (Switzerland); EGTA, HEPES, ACh chloride and 4-bromophenacyl bromide from Sigma (U.S.A.); DL-a-tocopherol from Serva (F.R.G.) and ionol from Reachem (Russia). Measurement
qf’ cytoplasmic ,free Ca
Intracellular free Ca was monitored with the fluorescent probe fura- as described by Kostyuk et 01.~’ Briefly, isolated non-identified neurons” were loaded with fura- for 3040 min at 20-21 C by adding 0. I % stock solution of fura-2/AM in dimethyl sulphoxide (DMSO) (final concentration 5jlM) to cell suspen-
Measurement of absorption spectra The UV absorption spectra of DL-CX-tocopherol in ethanol were measured before and after addition of arachidonic acid on a Beckman 35 spectrophotometer in l-cm cuvettes. All experiments were performed at room temperature (20-21 C). RESULTS The effects of calcium influx, vitamin E, ionol, arachidonic acid and 4-bromophenacyl bromide on the amplitude of ACh-induced current were studied in Dl, D4, E4, LPI 1 and RBc4 neurons, where a similar ionic mechanism of ACh responses associated with an initial increase in conductance to choloride ions was found earlier.%” The elevations of cytoplasmic concentration of free Ca’+ ([Cal,,) in Dl, D4 and E4 neurons induced by intracellular injection of Ca’+ or by depolarizing pulses” led to a faint decrease of ACh-induced chloride current (< 18%, n = 12) in these neurons; applications of 0.1 pM-O.1 mM vitamin E, 0.1 mM ionol, IOpM arachidonic acid and 20pM 4-bromophenacyl bromide were ineffective. Another situation was observed in LPI1 and RBc4 neurons, where calcium influx into these cells enhanced the ACh-induced chloride current (Fig. I).
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sion in the normal saline solution. The internal calcium concentration was calculated from the ratio between the values of fluorescence excited both at 387 nm and at 362 nm according to the procedure given by Grynkiewicz ev ul.“’
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Fig. 1. Effects of calcium influx and vitamin E apptieation on ACh-induced current in LPI1 neuron. Holdine. ootential - 60 mV. ACh application (250 nA, 2 s duration) is indicated by arrows. (A) Control. (B, C) xfter intracellular injection b‘f CaZ+ (26 nA, 1min duration) and vitamin E application (50phl, 3 min exposure) 10 min after this injection. (D) After 150 min washout. (E, F) After vitamin E application (50 PM, 3 min exposure) and intracellular injection of Cazf (20 nA, 1 min duration) after this application.
Effect of vitamin E on ACh-induced current The enhancement of ACh~ind~c~ current after vitamin E appheation was always greater after preliminary injection of Ca*+ (Fig. IC) than in control (Fig- IE). The enh~n~ment induced by vitamin E application at holding potential -60 mV reached 98&26% (n = 4) in 1Omin after Ca2+ injection (20 nA, I min duration) and only 57 + 12% (n = 4) in control. Preliminary injection of EGTA (10 nA, 1 mm duration) into the LPI1 and RBc4 neurons at hoiding potential - 60 mV decreased both the AChinduced current in these neurons by 32 li: 12% (n = 4) and the enhancing effect of vitamin E by 21 + 11% (n = 4). The preliminary application of vitamin E aboiished the enhancing effect of calcium infiux into the LPll and RBc4 neurons on ACh-induced current (Fig. IF). After 3 min exposure to 10 p M vitamin E the ~ntra~jIular injection of GaZ+ @OnA, 1 min duration), which evoked in control the enhancement of ACh-induced current by 87 It 25% (n = 8), either did not alter the ACh-induced current (n = 3) or increased it only by 28 f 10% {n = 5). The calcium infiux into the LPll and RBc4 neurons and vitamin E appii~ation augments the ACh-induced current without shifting its reversal potential (data not shown). The elevation of the ACh-induced current appeared in 60 f 10 s (n = 12) after the canning of calcium injection and 6 + 3 min (n = 12) after the onset of 10 .uM vitamin E application, which became maximal in 15 f 3 min (n = 12) and 3&60 min (n = IZ), respectively, after the beginning. This enhancement of the ACh-induced current was reversible at low concentrations of vitamin E (0.1-5 PM) and small Ca2+ injections (G 10 nA), but was only partially reversible at greater concentrations of vitamin E ~lO~M~.~ mM) and stronger Ca2+ injections (> IO nA) even during prolonged (22 h) washout, TO define a possible involvement of cytoplasmic free Ca*+ in the enhancing effect of vitamin E on ACh-induced current, we estimated the changes of [Calin in tested neurons after vitamin E application. Vitamin E (0.1-10 PM) did not change this concentration (n = 4) and 0.1 mM vitamin E evoked its faint monotonous increase (Fig. 2). The possibility of antioxidant action of vitamin E as a mechanism underlying the vitamin E-induced
enhancement of ACh-indu~d current in Lpi1 and RBc4 neurons was tested by using a synthetic antioxidant, ionol. Ionol (0.i mM) did not mimic the enhancing effect of vitamin E but, on the contrary, evoked a depression of the ACh-induced current in LPll and RBc4 neurons. In other investigated neurons, ion01 was ineffective. Applications of 10 p M arachidonic acid to LPI1 and RBc4 neurons evoked a deerease of the ACh-induced current (Fig. 3). This depression appeared 3 + 2 min (n = 8) after the beginning of arachidonic acid application and became maximal in 30 & 10 min (n = 6). The decrease of the ACh-induced current after application of arachidonic acid was only partially reversible (74 rt_18% from control values, n = 6) even after prolonged ( > 2 h) washout. No shift in the reversal potential of this current was observed in LPll and RBc4 neurons after such application. The depressing effect of arachidonic acid was dependent on holding potential. At holding potential -25 mV the depressing effect of arachidonic acid on the ACh-induced current in LPll and RBc4 neurons (Fig. 4) was greater by 45 f 14% (n = 4) than at holding potential -60 mV (Fig. 3). Application of arachidonic acid to spontaneously active non-c~arn~d RBc4 neuron at resting potential -47 mV (Fig. 5) evoked a decrease of the ACh hyperpolarizing response and an appearance of spontaneous activity with irregular firing rate. It is known that vitamin E can stabilize lipid bilayers via the Van der Waals interaction of tocopherols with unsaturated fatty acids of the phospholipids and in this way decrease damages of biomembrnnes caused by free fatty acids.15 We measured the UV spectra of ~~-~-t~o~herol in ethanol solution before and after addition of increasing concentrations of arachidonic acid. The addition of arachidonic acid to DL-c(-tocopherol solution evoked a decrease of the absorption maxima at 214-215 nm, but did not affect the absorbance at 293 nm (Fig. 6). Besides, a decrease of the absorbantes at 200-230 nm was observed. These changes induced immediateiy after the addition of arachidonic acid were not eliminated by subsequent incubation for 1.5-30 min.
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Fig. 2. Changes in basal cytoplasmic micromolar concentration of free eakium (top) and ~~ernbr~e current {bottom) in a non-j~en~~~ neuron after application of 0.1 mM vitamin E,
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Fig. 3. Effect of vitamin E and arachidonic acid on the ACh-induced current in RBc4 neuron. Holding potential -60 mV. ACh application (500 nA, 2.5 s duration) is indicated by arrows. (A) Control. (B) After 5 min exposure to 10 FM vitamin E. (C) After 3 h washout. (D) After 5 min exposure to 10FM
arachidonic acid. (E) After 3 h washout.
These results together with the described data about the influences of vitamin E and arachidonic acid on the ACh responses in LPI1 and RBc4 neurons stimulated us to define the possibility of continuous production of arachidonic acid in investigated neurons. Since arachidonic acid can be produced directly by the action of phospholipase A,,’ we used a substance that can inhibit phospholipase A,, 4-bromophenacyl bromide.‘4,29 4-Bromophenacyl bromide (20 ,uM) evoked elevation of the ACh-induced chloride current in LPI1 and RBc4 neurons (four out of six cells), but was ineffective in Dl, D4 and E4 neurons (in all cells, n = 6). The time characteristics of 4-bromophenacyl bromide action in LPI1 and RBc4 neurons were similar to those of the vitamin E-induced effect. The enhancing effect of vitamin E on the ACh-induced current was always less (four out of four cells) after preliminary application of 4-bromophenacyl bromide (Fig. 7E) than in control (Fig. 7D), i.e. the effects of4-bromophenacyl bromide and vitamin E were non-additive.
To avoid possible synaptic influences on the LPI1 and RBc4 neurons from other cells in the ganglia. we cut different intraganglionic nerves. Trans-section of buccal and cerebral commissures or cerebral-buccal. cerebral-pedal and cerebral-pleural connectives had no essential effect on the ACh-induced current in LPI1 neuron or on modifications of this current after applications of vitamin E and arachidonic acid (six out of six cells). Cutting of the buccal and cerebral commissures or cerebral-pedal and cerebral-pleural connectives either had no effect on the ACh-induced current in RBc4 neurons and its modifications after applications of vitamin E or arachidonic ‘&id (six out of six cells), but cutting of the right cer;bral-buccal connective led to the increase of the ACh-induced current by 32 f 14% (n = 3). This increase appeared in 75-90 s after the trans-section and became maximal in 15-20 min. Vitamin E (0.1 pM-O.1 mM) did not affect the ACh-induced current in RBc4 neuron of the right cerebral-buccal after trans-section connective, but the depressing effect of exogenous
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Fig. 4. Effects of vitamin E and arachidonic acid on the ACh-induced current in RBc4 neuron. Holding potential -25 mV. ACh application (500 nA, 2.5 s duration) is indicated by arrows. (A) Control. (B) After 5 min exposure to 10pM vitamin E. (C) After 3 h washout. (D) After 5 min exposure to 10pM arachidonic acid. (E) After 2 h washout.
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Effect of vitamin E on ACh-induced current
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Fig. 5. Effect of arachidonic acid on the ACh-induced current in non-clamped RBc4 neuron. Resting potential -47 mV. Applications of ACh (600 nA) and arachidonic acid (AA) are indicated by lines. (A) Control. (B) After 3 min exposure to 10 pM arachidonic acid.
arachidonic acid on the ACh-induced current in this neuron remained (four out of four cells). Lonol and 4-bromophenacyl bromide became ineffective 20 min after the trans-section of the right cerebral-buccal connective (four out of five cells). DISCUSSION
The results of our experiments on identified Dl, D4, E4, LPI1 and RBc4 neurons, in which similar ionic m~hanism of ACh responses associated with an increase in membrane conductivity toward chloride ions was found earlier,%13 indicate that LPll and RBc4 neurons possess ACh-receptors of a special sub-type. Calcium influx into these neurons 1
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Fig. 6. UV absorption spectra of DL-a-tocopherol (0. I mM) in ethanol before (A) and after addition of 10 PM (B) and 5O@M (Cc) arachidonic acid.
selectively enhances the ACh-induced chloride current, whereas it leads to its decrease in the rest of the investigated neurons. Only ACh receptors on LPll and RBc4 neurons react to vitamin E by the increase of the ACh-induced chloride current. The fact that the reversal potential of this current does not change in the investigate neurons under the described influences proves the non-’ olvement of other ions in the ACh-induced clrvw rrent after drug applications. The similarity between the actions of calcium ions and vitamin E on the ACh-induced current (Fig. 1) does not prove the identity of their mechanisms of action. Calcium injection (20 nA, 1 min duration) induced approximately a two-fold increase of [Ca],;” vitamin E either did not change the basal level of [Cal, at 0.1 PM-10 mM concentration of a-tocopherol or slightly enhanced this level at 0.1 mM concentration (Fig. 2). Such elevation of [Cal, under the action of large doses of vitamin E may be of a nonspecific nature, e.g. the consequence of formation of hydrophilic pores in the cell membraneal The absence of [Cali, increase at physiological doses of vitamin E and the differences in the time characteristics (latency, time to the maximum increase) between the Ca2+- and vitamin E-induced effects suggest that the main action of vitamin E might not be related to the changes in the [Ca]in. The dependence of the vitamin E-induced effect on intracellular injections of CaZ+ and EGTA may be only a reflection of Ca-dependent secondary mechanisms, which control the ACh-indu~ current (e.g. it was reported’* that ACh receptors in the LPil and RBc4 neurons are Ca/calmodulin dependent). The antoxidant action of vitamin ES also does not seem to be related to the vitamin E-induced increase of ACh responses in LPll and RBc4 neurons, since
750
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Fig. 7. Effects of 4-bromophenacyl bromide and vitamin E on the ACh-induced current in RBc4 neuron. Holding potential -60 mV. ACh application (500 nA, I s duration) is indicated by arrows. (A) Control. (B) After 5 min exposure to 20 PM 4-bromophenacyl bromide. (C) After 90 min washout. (D) After 5 min exposure to IOpM vitamin E. (E) After 5 min exposure to 20pM 4-bromophenacyl bromide and following 5 min exposure to 10 PM vitamin E 5 min after the beginning of 4-bromophenacyl bromide application. (F) After 3 h washout.
a synthetic antioxidant, ionol, did not mimic this effect. The depressant effect of arachidonic acid on the ACh-induced current (Fig. 3) observed only in LPI1 and RBc4 neurons, where vitamin E evoked the enhancement of the same current, impelled us to study the possible role of arachidonic acid in this effect of vitamin E. The measurement of UV spectra of DL-a-tocopherol before and after addition of increasing concentrations of arachidonic acid revealed a decrease of the UV absorption maxima at 214-215 nm, but the absence of absorbance changes at 293 nm, thus indicating a-tocopherol interaction with arachidonic acid.15 Similar spectral changes caused by addition of synthetic fatty acid derivatives to a-tocopherol solution in ethanol have been reported earlier. ** The measurements of the cr-tocopherol fluorescence spectra suggest that tr-tocopherol forms complexes with free fatty acids including arachidonic acid not only in solutions but also in membrane systems (e.g. liposomes and sarcoplasmic reticulum membrane suspensions).15 The data on 4-bromophenacyl bromide action show that arachidonic acid might be continuously produced in LPll and RBc4 neurons by the activity of phospholipase A,. Phospholipase A, is activated in vitro at high concentrations of Ca*+ but, nevertheless, it has been suggested that calcium is the major regulator of this enzyme in oivo.2.6Phospholipase A, can also be activated by somatostatin29 and FMRFamide.*’ We could not establish the cause of permanent activation of phospholipase A, in the LPll neuron, since the trans-section of connections when isolating this neuron from possible synaptic influences from other neurons was ineffective. But in the RBc4 neuron we found that trans-section of the right
cerebral-buccal connective led to the enhancement of the ACh-induced current, probably by abolishing a depressing influence of an unknown agent, the secretion of which was stimulated through this connective. Such an agent might be of peptide nature.*’ The enhancing effects of 4-bromophenacyl bromide and a-tocopherol on the ACh-induced current in LPI1 and RBc4 neurons as well as the depressing effect of arachidonic acid on the same current may indicate that the steady down-regulation of ACh receptor function may be due to the synthesis in the neuron of such biologically active lipoxygenase or cyclooxygenase metabolites or arachidonic acid as leukotrienes and prostaglandins. Calcium ions are activators of both phospholipase A, and its metabolite synthesis. 2,6That is why the effect of arachidonic acid might be dependent on membrane potential (Figs 3-5) or preliminary injection of Ca’+. The enhancing effect of vitamin E on the ACh-induced current may be associated with the intensity of arachidonic acid production from membrane phospholipids; thereby it can depend on preliminary injection of Ca*+. The injection of Ca ions into the LPI1 and RBc4 neurons shortly before application of vitamin E poorly influenced the effect of vitamin E* but preliminary injection of Ca*+ long before application of vitamin E led to the increase of the enhancing effect of vitamin E on the ACh-induced current (Fig. 1). It may be a result of long-lasting metabolic processes of arachidonic acid (e.g. it takes about 5-10 min for the synthesis of leukotrienes and prostaglandins in cells).27~35 Vitamins play an essential role in the metabolism of an organism, being almost always an initial material for the synthesis of coenzymes and enzymes3~*’ The participation of vitamins A and K, or their
Effect of vitamin E on ACh-induced current
751
derivatives in the processes of photoreception30,36 calcium exchange processes,22324inhibition of protein allows one to assume that not only hormoness~p~“~2’~32kinase C34 or inhibition of the interaction of arachibut vitamins can also infiuenct: the processes of donic acid and its metabolites with various enzymes,4,6.23,25,26,3L The primary importance of our reception, transmission and analysis of information in isolated nervous system and in isolated nerve cells. results is that they demonstrate for the first time that vitamin E and endogenous arachidonic acid exert The present results support this assumption for vitaantagonistic modulatory effect on the activity of ACh min E. receptors in nerve cells. The data obtained in the present study do not exclude other possible mechanisms of vitamin E Acknowledgements-I am indebted to Dr P. V. Fklan for action. These might be: changes in the structure and advice and help in the use of fluorescent probe fura- and fluidity of the lipid bilayer,‘s3 formation of complexes Dr T. V. Belyaeva for assistance in measuring the UV of vitamin E with linolenic acid and other saturated absorption spectra. It is a pleasure to thank Acad. P. G. and unsaturated fatty acids in the membrane,ls Kostyuk for his support of these experiments aid helpful discussion of the results. changes in the metabolism of vitamin D as related to REFERENCES 1. Arvanov V. L., Takenaka T. and Ayrapetian S. N. (1986) The effects of short-chain fatty acids on the neuronal membrane cholinor~ptive properties. Cell. molec. Neurobio~. 6, 165-177. 2. Berridge M. J. (1984) Inositol triphosphate and diacylglycerol as second messengers. Eiochem. J. 22% 345-360. 3. Bicknell F. and Prescott F. (1953) The Vitamins in Medicine William Heinemann Medical Books, London. leukocytes. Structural 4. Borgeat P. and Samuelsson B. (1979) Metabolism of arachidonic acid in pol~orphonuclear analysis of novel hydroxylated compounds. J. biof. Chem. 254, 2643-2646. 5. Boynd P. J., Osborne N. N. and Walker R. J. (1987) Localization of arg-vasopressin-like material in central neurones and mechanism of action of arg-vasotocin on identified neurones of the snail Helix aspersa. Neuropharmacology 26, 1633-1647. 6. Burgoyne R., Cheek T. R. and O’Sullivan A. J. (1987) Receptor-activation of phospholipase AZ in cellular s&nailing. Trends biochem. Sci. 12, 332-333. I. Dyatlov V. A. (1988) Role of calcium ions in the processes of serotonin-induced modulation of neuronal response to a~tyl~holine application in Helix pomatia. Ne~rophysjo~ogy, Kim 20, 489-492. 8. Dyatlov V. A. (1990) Regulation of acetylcholine receptor surface topography by vitamin E in molluscan neurons. Dokl. Akad. Nauk SSSR. 314, 748-752 (in Russian). 9. Dyatlov V. A. (1990) Modulatory effects of oxytocin on functional properties of three types of cholinoreceptors in molluscan neurons. ~europhysioiogy, Kiev 22, 72-77.
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1991)
Neuroscience Vol. 48, No. 3, pp. 145-152, 1992 Printed in Great Britain
EFFECT OF VITAMIN E ON ACETYLCHOLINE-INDUCED CURRENT IN MOLLUSCAN NEURONS: ROLE OF CYTOPLASMIC FREE CALCIUM AND ARACHIDONIC ACID V. A. DYATLOV Laboratory of Neurobiology, A. A. Bogomoletz Institute of Physiology, Ukrainian Academy of Sciences, Kiev 24, 252601 GSP, Ukraine of cytoplasmic concentration of free Ca2+ ([Cal,) and arachidonic acid in potentiating the effect of vitamin E CDL-CL -tocopherol) on acetylchoiine receptor activity in H&x acre neurons was studied using a two-microe~~tr~e intracellular recording, voltage clamp and fluorescent calcium probe fura- technique. Elevation of [Cali, by intracellular injection from a microelectrode or by depolarizing pulses and application of 0.1 pM-O.1 mM vitamin E enhanced the acetylcholine-induced chloride current both in LPI1 and RBc4 neurons. Application of 10 PM arachidonic acid to the same neurons decreased this current. The use of fluorescent probe showed that vitamin E did not essentially change [Cal,, but an increase of [Cal. intensified both the enharming effect of vitamin E and the depressing effect of a~~hidonic acid. The enhancing effect of calcium influx was considerably decreased after vitamin E
Abstract-Role
application. The antioxidant action of vitamin E was probably not involved in the mechanism of its enhancing effect on acetylcholine-induced current, since synthetic antioxidant, ionol, depressed acetylcholine responses. A spectrum analysis has shown the interaction between vitamin E and arachidonic acid in solution. This interaction may be considered as the molecular mechanism responsible for the prevention by vitamin E of steady arachidonic acid production from membrane phospholipids and its down-regulatory effect on acetylcholine receptor activity. Our rest&s support this suggestion, since an inhibitor of phosphoiipa~ A,, 4-bromoph~acyl bromide, mimicked the enhancing effect of vitamin E.
In previous papers,8*‘3we described the vitamin E-dependent regulation of the properties of acetylcholine (ACh) receptors in identified neurons of the snail Helix pomatia. This may be related to the fact that lipid-soluble tocopherols (different forms of vitamin E) act as antioxidants and slow down the rate of metabolic processes. It has aiso been suggested that some of the effects of vitamin E may be connected with calcium accumulation in cells that is enhanced under vitamin E deficiency.” The structural stability of biological membranes may be supported by vitamin E via its reaction with lipid peroxide radicals, protection against destructive action of arachidonic acid and quenching of singlet mokcular oxygen.3*‘5In the present paper, we have extended the analysis of the possible roles of cytoplasmic Ca*+, arachidonic acid and antioxidant properties of vitamin E in the enhancing effect of vitamin E on the ACh-induced chloride current in identified neurons of the mollusc Helix pomatia. Abbreviations:ACh, acetylcholine; [Cal,, cytoplasmic concentration of free Ca2+; DMSO. dimethvl sulohoxide: EGTA, ethyleneglycol-bis(aminoethylether)te&acetic acid; HEPES, ~-2-hydroxyethylpi~~ne-~-2-eth~~ &phonic acid.
EXPERIMENTAL PROCEDURES
Subjects The perioesophageal ganglionic ring together with the buccal ganglia was isolated from Helix pomatia snails and fixed inside a plastic chamber exposing the ventral surface of the ganglia. The gli~onn~tive envelope of the ganglia was removed under microscopic visualization. Neurons Dl, D4, E4, LPl 1 and RBc4 were identified in different preparations according to their position and their responses to drugs and stimulation of the main nerves.18*33For the right buccal and the left pleural ganglia the abbre~ations RBc and LPI were applied, respectively. The preparation was bathed in continuously circulating saline solution with the following composition (in mM): NaCl 100, KC1 4, CaCl, 10, MgCl, 4, HEPES-NaOH 10, pH 7.6. Electroph~~~oiog~cal recordings The electrophysiological experiments were carried out under voltage-clamp conditions, using a twomicroelectrode set-up. In some experiments intracellular recordings were made from non-clamped neurons. We used glass micr~lectrodes filled with 745
V. A I)YAII.O\
746
2.5 M KC1 or 0.6 M KSO, of 8-12 MR.
and having a resistance
Drug applications Acetylcholine was applied locally to the neuronal somata by iontophoresis or by pressure ejection from a pipette with a 556-pm tip. To avoid desensitization, the cell was washed between two subsequent responses with saline solution for 3 min. Ca’+ and EGTA were injected intracellularly by iontophoresis or by depolarizing pulses as described earlier.7~‘o Vitamin E (0.1 p M-O. 1 mM; DL-x-tocopherol), 0.1 mM ionol, 10 PM arachidonic acid and 20 PM 4-bromophenacyl bromide (all dissolved in saline solution) were applied to individual cells by pressure from a pipette with a 5-6-pm tip positioned 15-20 pm from the cell. DL-a-Tocopherol and arachidonic acid were first dissolved in absolute ethanol (lo-’ M) and then diluted in the saline solution. Arachidonic acid was dissolved in ethanol under a gentle stream of nitrogen immediately before use. Arachidonic acid and fura-Z/AM were purchased from Calbiochem (Switzerland); EGTA, HEPES, ACh chloride and 4-bromophenacyl bromide from Sigma (U.S.A.); DL-a-tocopherol from Serva (F.R.G.) and ionol from Reachem (Russia). Measurement
qf’ cytoplasmic ,free Ca
Intracellular free Ca was monitored with the fluorescent probe fura- as described by Kostyuk et 01.~’ Briefly, isolated non-identified neurons” were loaded with fura- for 3040 min at 20-21 C by adding 0. I % stock solution of fura-2/AM in dimethyl sulphoxide (DMSO) (final concentration 5jlM) to cell suspen-
Measurement of absorption spectra The UV absorption spectra of DL-CX-tocopherol in ethanol were measured before and after addition of arachidonic acid on a Beckman 35 spectrophotometer in l-cm cuvettes. All experiments were performed at room temperature (20-21 C). RESULTS The effects of calcium influx, vitamin E, ionol, arachidonic acid and 4-bromophenacyl bromide on the amplitude of ACh-induced current were studied in Dl, D4, E4, LPI 1 and RBc4 neurons, where a similar ionic mechanism of ACh responses associated with an initial increase in conductance to choloride ions was found earlier.%” The elevations of cytoplasmic concentration of free Ca’+ ([Cal,,) in Dl, D4 and E4 neurons induced by intracellular injection of Ca’+ or by depolarizing pulses” led to a faint decrease of ACh-induced chloride current (< 18%, n = 12) in these neurons; applications of 0.1 pM-O.1 mM vitamin E, 0.1 mM ionol, IOpM arachidonic acid and 20pM 4-bromophenacyl bromide were ineffective. Another situation was observed in LPI1 and RBc4 neurons, where calcium influx into these cells enhanced the ACh-induced chloride current (Fig. I).
I
I
I -
J A
sion in the normal saline solution. The internal calcium concentration was calculated from the ratio between the values of fluorescence excited both at 387 nm and at 362 nm according to the procedure given by Grynkiewicz ev ul.“’
1 1 C
B
-I
-e
E
D
F
::
1
0.1nA
-J 10s
Fig. 1. Effects of calcium influx and vitamin E apptieation on ACh-induced current in LPI1 neuron. Holdine. ootential - 60 mV. ACh application (250 nA, 2 s duration) is indicated by arrows. (A) Control. (B, C) xfter intracellular injection b‘f CaZ+ (26 nA, 1min duration) and vitamin E application (50phl, 3 min exposure) 10 min after this injection. (D) After 150 min washout. (E, F) After vitamin E application (50 PM, 3 min exposure) and intracellular injection of Cazf (20 nA, 1 min duration) after this application.
Effect of vitamin E on ACh-induced current The enhancement of ACh~ind~c~ current after vitamin E appheation was always greater after preliminary injection of Ca*+ (Fig. IC) than in control (Fig- IE). The enh~n~ment induced by vitamin E application at holding potential -60 mV reached 98&26% (n = 4) in 1Omin after Ca2+ injection (20 nA, I min duration) and only 57 + 12% (n = 4) in control. Preliminary injection of EGTA (10 nA, 1 mm duration) into the LPI1 and RBc4 neurons at hoiding potential - 60 mV decreased both the AChinduced current in these neurons by 32 li: 12% (n = 4) and the enhancing effect of vitamin E by 21 + 11% (n = 4). The preliminary application of vitamin E aboiished the enhancing effect of calcium infiux into the LPll and RBc4 neurons on ACh-induced current (Fig. IF). After 3 min exposure to 10 p M vitamin E the ~ntra~jIular injection of GaZ+ @OnA, 1 min duration), which evoked in control the enhancement of ACh-induced current by 87 It 25% (n = 8), either did not alter the ACh-induced current (n = 3) or increased it only by 28 f 10% {n = 5). The calcium infiux into the LPll and RBc4 neurons and vitamin E appii~ation augments the ACh-induced current without shifting its reversal potential (data not shown). The elevation of the ACh-induced current appeared in 60 f 10 s (n = 12) after the canning of calcium injection and 6 + 3 min (n = 12) after the onset of 10 .uM vitamin E application, which became maximal in 15 f 3 min (n = 12) and 3&60 min (n = IZ), respectively, after the beginning. This enhancement of the ACh-induced current was reversible at low concentrations of vitamin E (0.1-5 PM) and small Ca2+ injections (G 10 nA), but was only partially reversible at greater concentrations of vitamin E ~lO~M~.~ mM) and stronger Ca2+ injections (> IO nA) even during prolonged (22 h) washout, TO define a possible involvement of cytoplasmic free Ca*+ in the enhancing effect of vitamin E on ACh-induced current, we estimated the changes of [Calin in tested neurons after vitamin E application. Vitamin E (0.1-10 PM) did not change this concentration (n = 4) and 0.1 mM vitamin E evoked its faint monotonous increase (Fig. 2). The possibility of antioxidant action of vitamin E as a mechanism underlying the vitamin E-induced
enhancement of ACh-indu~d current in Lpi1 and RBc4 neurons was tested by using a synthetic antioxidant, ionol. Ionol (0.i mM) did not mimic the enhancing effect of vitamin E but, on the contrary, evoked a depression of the ACh-induced current in LPll and RBc4 neurons. In other investigated neurons, ion01 was ineffective. Applications of 10 p M arachidonic acid to LPI1 and RBc4 neurons evoked a deerease of the ACh-induced current (Fig. 3). This depression appeared 3 + 2 min (n = 8) after the beginning of arachidonic acid application and became maximal in 30 & 10 min (n = 6). The decrease of the ACh-induced current after application of arachidonic acid was only partially reversible (74 rt_18% from control values, n = 6) even after prolonged ( > 2 h) washout. No shift in the reversal potential of this current was observed in LPll and RBc4 neurons after such application. The depressing effect of arachidonic acid was dependent on holding potential. At holding potential -25 mV the depressing effect of arachidonic acid on the ACh-induced current in LPll and RBc4 neurons (Fig. 4) was greater by 45 f 14% (n = 4) than at holding potential -60 mV (Fig. 3). Application of arachidonic acid to spontaneously active non-c~arn~d RBc4 neuron at resting potential -47 mV (Fig. 5) evoked a decrease of the ACh hyperpolarizing response and an appearance of spontaneous activity with irregular firing rate. It is known that vitamin E can stabilize lipid bilayers via the Van der Waals interaction of tocopherols with unsaturated fatty acids of the phospholipids and in this way decrease damages of biomembrnnes caused by free fatty acids.15 We measured the UV spectra of ~~-~-t~o~herol in ethanol solution before and after addition of increasing concentrations of arachidonic acid. The addition of arachidonic acid to DL-c(-tocopherol solution evoked a decrease of the absorption maxima at 214-215 nm, but did not affect the absorbance at 293 nm (Fig. 6). Besides, a decrease of the absorbantes at 200-230 nm was observed. These changes induced immediateiy after the addition of arachidonic acid were not eliminated by subsequent incubation for 1.5-30 min.
605
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-
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8
Fig. 2. Changes in basal cytoplasmic micromolar concentration of free eakium (top) and ~~ernbr~e current {bottom) in a non-j~en~~~ neuron after application of 0.1 mM vitamin E,
74x
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ov
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Fig. 3. Effect of vitamin E and arachidonic acid on the ACh-induced current in RBc4 neuron. Holding potential -60 mV. ACh application (500 nA, 2.5 s duration) is indicated by arrows. (A) Control. (B) After 5 min exposure to 10 FM vitamin E. (C) After 3 h washout. (D) After 5 min exposure to 10FM
arachidonic acid. (E) After 3 h washout.
These results together with the described data about the influences of vitamin E and arachidonic acid on the ACh responses in LPI1 and RBc4 neurons stimulated us to define the possibility of continuous production of arachidonic acid in investigated neurons. Since arachidonic acid can be produced directly by the action of phospholipase A,,’ we used a substance that can inhibit phospholipase A,, 4-bromophenacyl bromide.‘4,29 4-Bromophenacyl bromide (20 ,uM) evoked elevation of the ACh-induced chloride current in LPI1 and RBc4 neurons (four out of six cells), but was ineffective in Dl, D4 and E4 neurons (in all cells, n = 6). The time characteristics of 4-bromophenacyl bromide action in LPI1 and RBc4 neurons were similar to those of the vitamin E-induced effect. The enhancing effect of vitamin E on the ACh-induced current was always less (four out of four cells) after preliminary application of 4-bromophenacyl bromide (Fig. 7E) than in control (Fig. 7D), i.e. the effects of4-bromophenacyl bromide and vitamin E were non-additive.
To avoid possible synaptic influences on the LPI1 and RBc4 neurons from other cells in the ganglia. we cut different intraganglionic nerves. Trans-section of buccal and cerebral commissures or cerebral-buccal. cerebral-pedal and cerebral-pleural connectives had no essential effect on the ACh-induced current in LPI1 neuron or on modifications of this current after applications of vitamin E and arachidonic acid (six out of six cells). Cutting of the buccal and cerebral commissures or cerebral-pedal and cerebral-pleural connectives either had no effect on the ACh-induced current in RBc4 neurons and its modifications after applications of vitamin E or arachidonic ‘&id (six out of six cells), but cutting of the right cer;bral-buccal connective led to the increase of the ACh-induced current by 32 f 14% (n = 3). This increase appeared in 75-90 s after the trans-section and became maximal in 15-20 min. Vitamin E (0.1 pM-O.1 mM) did not affect the ACh-induced current in RBc4 neuron of the right cerebral-buccal after trans-section connective, but the depressing effect of exogenous
IO.lnA
Fig. 4. Effects of vitamin E and arachidonic acid on the ACh-induced current in RBc4 neuron. Holding potential -25 mV. ACh application (500 nA, 2.5 s duration) is indicated by arrows. (A) Control. (B) After 5 min exposure to 10pM vitamin E. (C) After 3 h washout. (D) After 5 min exposure to 10pM arachidonic acid. (E) After 2 h washout.
749
Effect of vitamin E on ACh-induced current
ACh
AA
1
50mV
10 5
Fig. 5. Effect of arachidonic acid on the ACh-induced current in non-clamped RBc4 neuron. Resting potential -47 mV. Applications of ACh (600 nA) and arachidonic acid (AA) are indicated by lines. (A) Control. (B) After 3 min exposure to 10 pM arachidonic acid.
arachidonic acid on the ACh-induced current in this neuron remained (four out of four cells). Lonol and 4-bromophenacyl bromide became ineffective 20 min after the trans-section of the right cerebral-buccal connective (four out of five cells). DISCUSSION
The results of our experiments on identified Dl, D4, E4, LPI1 and RBc4 neurons, in which similar ionic m~hanism of ACh responses associated with an increase in membrane conductivity toward chloride ions was found earlier,%13 indicate that LPll and RBc4 neurons possess ACh-receptors of a special sub-type. Calcium influx into these neurons 1
0.8
PI
,”
0.6
s
‘0
d Q
0.4
0.2
0
200
250 Wavelength
300
350
tnml
Fig. 6. UV absorption spectra of DL-a-tocopherol (0. I mM) in ethanol before (A) and after addition of 10 PM (B) and 5O@M (Cc) arachidonic acid.
selectively enhances the ACh-induced chloride current, whereas it leads to its decrease in the rest of the investigated neurons. Only ACh receptors on LPll and RBc4 neurons react to vitamin E by the increase of the ACh-induced chloride current. The fact that the reversal potential of this current does not change in the investigate neurons under the described influences proves the non-’ olvement of other ions in the ACh-induced clrvw rrent after drug applications. The similarity between the actions of calcium ions and vitamin E on the ACh-induced current (Fig. 1) does not prove the identity of their mechanisms of action. Calcium injection (20 nA, 1 min duration) induced approximately a two-fold increase of [Ca],;” vitamin E either did not change the basal level of [Cal, at 0.1 PM-10 mM concentration of a-tocopherol or slightly enhanced this level at 0.1 mM concentration (Fig. 2). Such elevation of [Cal, under the action of large doses of vitamin E may be of a nonspecific nature, e.g. the consequence of formation of hydrophilic pores in the cell membraneal The absence of [Cali, increase at physiological doses of vitamin E and the differences in the time characteristics (latency, time to the maximum increase) between the Ca2+- and vitamin E-induced effects suggest that the main action of vitamin E might not be related to the changes in the [Ca]in. The dependence of the vitamin E-induced effect on intracellular injections of CaZ+ and EGTA may be only a reflection of Ca-dependent secondary mechanisms, which control the ACh-indu~ current (e.g. it was reported’* that ACh receptors in the LPil and RBc4 neurons are Ca/calmodulin dependent). The antoxidant action of vitamin ES also does not seem to be related to the vitamin E-induced increase of ACh responses in LPll and RBc4 neurons, since
750
I A,-$i /
0.2 n A
Fig. 7. Effects of 4-bromophenacyl bromide and vitamin E on the ACh-induced current in RBc4 neuron. Holding potential -60 mV. ACh application (500 nA, I s duration) is indicated by arrows. (A) Control. (B) After 5 min exposure to 20 PM 4-bromophenacyl bromide. (C) After 90 min washout. (D) After 5 min exposure to IOpM vitamin E. (E) After 5 min exposure to 20pM 4-bromophenacyl bromide and following 5 min exposure to 10 PM vitamin E 5 min after the beginning of 4-bromophenacyl bromide application. (F) After 3 h washout.
a synthetic antioxidant, ionol, did not mimic this effect. The depressant effect of arachidonic acid on the ACh-induced current (Fig. 3) observed only in LPI1 and RBc4 neurons, where vitamin E evoked the enhancement of the same current, impelled us to study the possible role of arachidonic acid in this effect of vitamin E. The measurement of UV spectra of DL-a-tocopherol before and after addition of increasing concentrations of arachidonic acid revealed a decrease of the UV absorption maxima at 214-215 nm, but the absence of absorbance changes at 293 nm, thus indicating a-tocopherol interaction with arachidonic acid.15 Similar spectral changes caused by addition of synthetic fatty acid derivatives to a-tocopherol solution in ethanol have been reported earlier. ** The measurements of the cr-tocopherol fluorescence spectra suggest that tr-tocopherol forms complexes with free fatty acids including arachidonic acid not only in solutions but also in membrane systems (e.g. liposomes and sarcoplasmic reticulum membrane suspensions).15 The data on 4-bromophenacyl bromide action show that arachidonic acid might be continuously produced in LPll and RBc4 neurons by the activity of phospholipase A,. Phospholipase A, is activated in vitro at high concentrations of Ca*+ but, nevertheless, it has been suggested that calcium is the major regulator of this enzyme in oivo.2.6Phospholipase A, can also be activated by somatostatin29 and FMRFamide.*’ We could not establish the cause of permanent activation of phospholipase A, in the LPll neuron, since the trans-section of connections when isolating this neuron from possible synaptic influences from other neurons was ineffective. But in the RBc4 neuron we found that trans-section of the right
cerebral-buccal connective led to the enhancement of the ACh-induced current, probably by abolishing a depressing influence of an unknown agent, the secretion of which was stimulated through this connective. Such an agent might be of peptide nature.*’ The enhancing effects of 4-bromophenacyl bromide and a-tocopherol on the ACh-induced current in LPI1 and RBc4 neurons as well as the depressing effect of arachidonic acid on the same current may indicate that the steady down-regulation of ACh receptor function may be due to the synthesis in the neuron of such biologically active lipoxygenase or cyclooxygenase metabolites or arachidonic acid as leukotrienes and prostaglandins. Calcium ions are activators of both phospholipase A, and its metabolite synthesis. 2,6That is why the effect of arachidonic acid might be dependent on membrane potential (Figs 3-5) or preliminary injection of Ca’+. The enhancing effect of vitamin E on the ACh-induced current may be associated with the intensity of arachidonic acid production from membrane phospholipids; thereby it can depend on preliminary injection of Ca*+. The injection of Ca ions into the LPI1 and RBc4 neurons shortly before application of vitamin E poorly influenced the effect of vitamin E* but preliminary injection of Ca*+ long before application of vitamin E led to the increase of the enhancing effect of vitamin E on the ACh-induced current (Fig. 1). It may be a result of long-lasting metabolic processes of arachidonic acid (e.g. it takes about 5-10 min for the synthesis of leukotrienes and prostaglandins in cells).27~35 Vitamins play an essential role in the metabolism of an organism, being almost always an initial material for the synthesis of coenzymes and enzymes3~*’ The participation of vitamins A and K, or their
Effect of vitamin E on ACh-induced current
751
derivatives in the processes of photoreception30,36 calcium exchange processes,22324inhibition of protein allows one to assume that not only hormoness~p~“~2’~32kinase C34 or inhibition of the interaction of arachibut vitamins can also infiuenct: the processes of donic acid and its metabolites with various enzymes,4,6.23,25,26,3L The primary importance of our reception, transmission and analysis of information in isolated nervous system and in isolated nerve cells. results is that they demonstrate for the first time that vitamin E and endogenous arachidonic acid exert The present results support this assumption for vitaantagonistic modulatory effect on the activity of ACh min E. receptors in nerve cells. The data obtained in the present study do not exclude other possible mechanisms of vitamin E Acknowledgements-I am indebted to Dr P. V. Fklan for action. These might be: changes in the structure and advice and help in the use of fluorescent probe fura- and fluidity of the lipid bilayer,‘s3 formation of complexes Dr T. V. Belyaeva for assistance in measuring the UV of vitamin E with linolenic acid and other saturated absorption spectra. It is a pleasure to thank Acad. P. G. and unsaturated fatty acids in the membrane,ls Kostyuk for his support of these experiments aid helpful discussion of the results. changes in the metabolism of vitamin D as related to REFERENCES 1. Arvanov V. L., Takenaka T. and Ayrapetian S. N. (1986) The effects of short-chain fatty acids on the neuronal membrane cholinor~ptive properties. Cell. molec. Neurobio~. 6, 165-177. 2. Berridge M. J. (1984) Inositol triphosphate and diacylglycerol as second messengers. Eiochem. J. 22% 345-360. 3. Bicknell F. and Prescott F. (1953) The Vitamins in Medicine William Heinemann Medical Books, London. leukocytes. Structural 4. Borgeat P. and Samuelsson B. (1979) Metabolism of arachidonic acid in pol~orphonuclear analysis of novel hydroxylated compounds. J. biof. Chem. 254, 2643-2646. 5. Boynd P. J., Osborne N. N. and Walker R. J. (1987) Localization of arg-vasopressin-like material in central neurones and mechanism of action of arg-vasotocin on identified neurones of the snail Helix aspersa. Neuropharmacology 26, 1633-1647. 6. Burgoyne R., Cheek T. R. and O’Sullivan A. J. (1987) Receptor-activation of phospholipase AZ in cellular s&nailing. Trends biochem. Sci. 12, 332-333. I. Dyatlov V. A. (1988) Role of calcium ions in the processes of serotonin-induced modulation of neuronal response to a~tyl~holine application in Helix pomatia. Ne~rophysjo~ogy, Kim 20, 489-492. 8. Dyatlov V. A. (1990) Regulation of acetylcholine receptor surface topography by vitamin E in molluscan neurons. Dokl. Akad. Nauk SSSR. 314, 748-752 (in Russian). 9. Dyatlov V. A. (1990) Modulatory effects of oxytocin on functional properties of three types of cholinoreceptors in molluscan neurons. ~europhysioiogy, Kiev 22, 72-77.
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