tion, since AA and its acyclic derivative hepoxilin A3 elicit both direct reactions of the pyramidal neurons of the CA1 field of the rat hippocampus, i.e., ...
ARACHIDONIC ACID AND ITS ACYCLIC DERIVATIVES REGULATE SHORT-TERM PLASITICITY OF THE CHOLINORECEPTORS OF NEURONS OF THE EDIBLE SNAIL A. S. Pivovarov, E. I. Drozdova, and B. I. Kotlyar*
UDC 612.822.1+615.78+612.822.3
The influence of eicosanoids on the dynamics of the exlinction of the inward current, induced by repeated iontophoretie appplications of acetylcholine to the soma has been demonstrated in identified RPa3 and LPa3 neurons of the edible snail by means of bielectrode recording of the membrane potential. The extracellular action of arachidonic acid (50-100 lunole/l) intensifies the extinction. Quinacrine (100-600 #mole/l), an inhibitor of phosphopholipase A2, which decreases the content of arachidonic acid in the cells, acts ambiguously. Nordihydroguaiaretic acid (3-10 I.tmole/l), which is an inhibitor of the lipoxygenase oxidation of arachidonic acid, attenuates the extinction. Indomethacin (10-50 tanole/l), a blocker of cyclooxygenase oxidation of arachidonic acid, does not influence extinction. All of the compounds investigated decrease the amplitude of the inward current induced by acetylcholine. The results obtained make it possible to hypothesize that arachidonic acid and its acyclic metabolites, which are formed as a result of lipoxygenase oxidation, regulate short-term plasticity of the cholinoreceptors of neurons of the edible snail. The cyclic eicosanoids which are formed during the cyclooxygenase oxidation of arachidonic acid do not exert a regulatory effect on the plasticity of the cholinoreceptors.
We have found by studying the role of messengers in the regulation of the short-term plasticity of the cholinoreceptors of the identified neurons of the edible snail, that cGMP intensifies the extinction of the inward current induced by repeated administrations of acetylcholine (AC) to the soma (the AC current) [1]. The enzyme guanylate cyclase, which catalyzes the formation of cGMP from GTP, is localized primarily in the cytoplasm, and is activated via a more complex pathway than adenylate cyclase. It has been demonstrated that the formation of cGMP is mediated by acyclic metabolites of arachidonic acid (AA) which are formed during its lipoxygenase oxidation [18]. This information explains our interest in AA as a probable participant in the regulatory cascade of the plasticity of the cholinoreceptors. Arachidonic acid (5,8,11,14-eicosatetraenoic acid), a natural polyunsaturated fatty acid with four double bonds, is formed from unsaturated fatty acids of the cells membranes: linoleic and linolenic. It is found in bound form (in the second position) in lipids, namely, phospholipids (phosphatidylinositol, phosphatidylethanolamine, phosphatidylamine), glycerol lipids (1,2diacylgtycerol), and phosphatidic acid. AA is liberated under the influence of phospholipases (primarily phospholipase A2), and is then subjected to further enzymatic oxidation to a series of low molecular weight bioregulators, collectively termed the "eicosanoids" [10, 11, 19]. Two principal pathways of the metabolism of AA have been identiifed: the lipoxygenase and the cyclooxygenase. The acyclic eicosanoids, hydroperoxyeicosatetraenoic acids (HPETE), the mono- and dieicosatetraenoic acids (diHETE, HETE), the leukotrienes, the trioxytetraenes (Iipoxins), the hepoxilins, and the trioxilins, are formed under the influence of lipoxygenases. Cyclooxygenase oxidizes AA to cyclic derivatives, the prostaglandins, prostacyclins, and thromboxanes [10, 11, 19]. It is assumed that the eicosanoids, along with the cyclic nucleotides, Ca2+ ions, diacylglycerol, the phosphatidylinositides, and oligo(A), belong to the universal regulators of cell metabolism [3]. The eicosanoids freely penetrate the membrane of cells and can therefore fulfill the role of both intra- and extracellular signal molecules and participate in the transsynaptic regulation of the activity of neurons. The regulatory functions of the eicosanoids have been demonstrated in a number of investigations. AA inhibits the inward ionic current in response to carbachol in the superior cervical ganglion of the rat in vitro [14], stimulates the muscarine-sensitive K + channels of isolated cells of the guinea pig atrium through a receptor-inde*Deceased. Department of Higher Nervous Activity of the Biological Faculty, M. V. Lomonosov Moscow State University. Translated from Zhurnal Vysshei Nervnoi Deyatel'nosti imeni I. P. Pavlova, Vol. 41, No. 4, pp. 796-805, July-August, 1991. Original article submitted December 3, 1990; revision submitted January 1, 1991. 0097-0549/92/2205-0393512.50
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pendent mechanism by acting directly on the G protein [9], and regulates the metabolism of phosphoinositides in the acinar ceils of the pancreas through a feedback mechanism, inhibiting the accumulation of inositol diphosphate and of inositol 1,4,5-triphosphate, and thus preventing an increase in the mobilization of deposited Ca2+ by cerulen [7]. AA effectively induces the mobilzation of of intracellular Ca 2§ in human thrombocytes [5, 8], and in rat mitochondria, microsomes, and permeable hepatocytes [16]. BW755C, a blocker of lipoxygenase oxidation, enhances the voltage-regulated Ca2+ current of neurons of the edible snail [2]. Endogenous metabolites of AA, 5-HETE, 12-HETE, and prostaglandins PGE2 and PGE2a, have been found in the synaptosomes and bodies of identified neurons of Aplysia [12, 17] during intracellular stimulation of histaminergic neurons. The inhibitory synaptic action of the neuroactive peptide, FMRF-amide, which induces the presynaptic inhibition of the release of the mediator by sensory neurons of Aplysia, was governed by AA and its lipoxygenase metabolite 12-HETE [13, 17, 20], which intensifies the S type of K + current of the sensory neurons of Aplysia through the increased probability of the opening of K+ channels [20]. The Ca2+-induced prolonged potentiation in hippocampal sections is accompanied by an increase in the liberation of AA, which is regarded as a possible transsynaptic mediator necessary for the development of prolonged potentiation [4]. The function of modulators of synaptic transmission in the hippocampus is ascribed to AA and the products of its lipoxygenase oxidation, since AA and its acyclic derivative hepoxilin A3 elicit both direct reactions of the pyramidal neurons of the CA1 field of the rat hippocampus, i.e., hyperpolarization, replaced by depolarization in some neurons, as well as modulator reactions, i.e., an increase in post-spike trace hyperpolarization, as well as in the early C1--dependent and the late K+-dependant phases of the orthodromic IPSP [6]. The study of the influences of AA, the inhibition of phospholipase A2, and of the two pathways of AA oxidation, the lipoxygenase and the cyclooxygenase, on the short-term plasticity of cholinoreceptors of the identified neurons of the edible snail formed the objective of the present investigation. METHODS The experiments were carried out in the RPa3 and LPa3 identified neurons of the edible snail (rlelix lucorum taurica Kryn) in a preparation of isolated ganglia. The circumesophageal nerve ring was fixed by means of steel micro-needles in a flow-through chamber, volume 1 ml. Following the treatment of the preparation with a 2% solution of collagenase (type IA, Sigma, USA) in a Ringer's solution for 30 min at room temperature, the connective tissue membranes covering the ganglia were removed. A Ringer's solution of the following composition flowed through the chamber containing the preparation (mmole/l): NaC1, 100; KC1, 4; CaCI2, 10; MgC12, 4; tris-HC1, 10; pH 7.5. The transmembrane ionic currents were recorded by means of the bieleclxode recording of the potential at the membrane. The intracellular electrodes were drawn from pyrex glass and filled with KC1 (2.5 mole/l; the resistance of the electrodes was 5-46 Mf~ (18.8 + 1.6 M~; M + m). The potentials were amplified by an MEZ-8201 microelectrode amplifier (Nihon Kohden, Japan). The recording currents were measured by means of an amplifier for the measurement of feedback current by the voltage drop at a resistance of 1.5 M.Q, and were recorded on a KSP-4 automatic recorder. A double-barreled micropipette was applied to the external surface of the soma. One channel served for the iontophoretic injection of AC and was filled with acetylcholine chloride (Serva, FRG) in distilled water (4 mole/l; pH 7.0; alkalinized with NaOH). Cationic currents (560-780 hA; 1.5-4 sec [2.4 _+0.1]) from t~SL-2 and 302-T (WPI, USA) electrostimulators were used for the the iontophoresis of the AC. In order to prevent the spontaneous diffusion of the AC from the phoretic pipette, a negative gate turn-off current (12-13 hA) was constantly passed through it. The other channel served for the balancing of the phoretic currents, and was filled with NaC1 (2 mole/l). A balancing current was passed through it which was opposite in direction to the phoretic current, but of the same strength and duration. The resistance of the pipettes was 20--30 M.Q. A series included 11-13 sequential iontophoretic applications of a AC current of constant direction, strength, and duration. The first 10 stimuli were delivered at an interval of 50-150 sec (77.4 + 2.4 sec) for the extinction of the AC current. The following stimuli were applied at an interval of 10 rain in order to assess the degree and rate of recovery of the extinguished reaction. The experiment consisted of several extinction series: control (before the pharmacological effect), experimental, and recovery. All the series were carried out without the flow-through of Ringer's solution. The experimental series were presented 20--60 min following the introduction of the compounds by means of microsyringe in volumes of 1-10 ~tl: AA (5,8,11,14-eicosatetraenoic acid; Serva, FRG); quinaerine, a phospholipase A2 inhibitor [21]; nordihydroguaiaretic acid, an inhibitor of lipoxygenases [15]; indomethacin, an inhibitor of cyclooxygenase [15] (all Sigma, USA). The AA and the
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Fig. 1. Influence of AC (A, 50 ~tmole/1), quinacrine (B, 300 ~tmole/1), nordihydroguaiaretic acid (C, 5 ~tmole/1), and indomethacin (D, 10 l.tmole/1) on the extinction curves of the AC current of identified neurons. A, D) Extinction curves of the AC current of the LPa3 neurons; B, C) of the RPa3 neurons: 1) Extinction of the AC current prior to the pharmacological action; 2) against the background of the action of the compound; 3) following the flushing of the preparation with Ringer's solution. Along the abscissa: the ordinal number of the application of AC in series; T, test for the spontaneous recovery of the extinguished response (application of AC to the soma from the same phoretic pipette 10 min after the end of the series of rhythmical stimulations); along the ordinate: the maximal amplitude of the AC current relative to its magnitude in response to the first stimulus in the series; %.
nordihydroguaiaretic acid were dissolved in ethanol, while the quinacrine and the indomethacin were dissolved in dimethylsulfoxide (Sigma, USA); the concentration of the solvents in the flow-through chamber did not exceed 0.5%. The significance of the influence of the compounds on the extinction process was assessed on the basis of nonparametric tests, the Wilcoxon paired T test (in individual neurons) and the signs test (in the entire population of the cells under investigation). The significance of the influence of the substances on the AC response was assessed on the basis of the Wilcoxon U test (Mann-Whitney). The statistical analysis of the results was carried out on an IBM XT personal computer by means of the specialized "DIASTA" program. The results were obtained in 46 neurons (24 RPa3 and 22 LPa3). The action of the AC was tested in 14 neurons; of the quinacrine in 11; of the nordihydroguaiaretic acid in 13; and of the indomethacin in 8. The membrane potential of the cells was (-42)-(-90) mV (-66.7 + 1.5 mV). Each cell was studied 3--7 h. 395
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l Fig. 2. Attenuation of the extinction of the AC current of a LPa3 neuron by arachidonic acid. Recordings of the entering current (deviation downwards) to the 1st, 3rd, 6th, and 10th repeated iontophoretic applications of AC (681 nA, 2 sec) with an interval of 50 sec are presented, as well as the test ejections of AC from the same phoretic pipette 10 min following the end of the rhythmical stimulation (T): A) in Ringer's solution of normal composition; B) against the background of the action of AA (50 ~rnole/1); C) following the flushing of the preparation with Ringer's solution for 75 min. The maintenance potential, 75 mV. Calibration: here and in Fig. 4 : 3 nA, 60 sec. TABLE 1. Influence of Arachidonic Acid, Inhibitors of its Oxidation, and Quinacrine on the Amplitude of the AC Current of the RPa3 and LPa3 Neurons Number of neurons investigated (degree i~th~ampli~ud~ o ~ t h e AC 9urrent~ %, e pnarmacologlcal acnlon) influence on the amplitude n~ t h o AC ellrrAnt - total increase decrease
I
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