- Email: [email protected]
12-Lipoxygenase increases neuronal excitability by inhibiting M-type potassium channels
International Congress Series 1233 (2002) 365 – 369
12-Lipoxygenase increases neuronal excitability by inhibiting M-type potassium channels Yoshitaka Takahashi a, Tanihiro Yoshimoto a,*, Naoto Hoshi b, Haruhiro Higashida b a
Department of Molecular Pharmacology, Kanazawa University Graduate School of Medicine, Kanazawa 920-8640, Japan b Department of Biophysical Genetics, Kanazawa University Graduate School of Medicine, Kanazawa 920-8640, Japan
Abstract To elucidate a role of 12-lipoxygenase in neuronal functions, cDNA for human 12-lipoxygenase of platelet type was permanently introduced to mouse neuroblastoma rat glioma hybrid NG108-15 cells which expressed many neural properties. The number of action potentials evoked by depolarizing current steps at a current-clamp mode was strikingly increased in the 12-lipoxygenase-expressing NG108-15 cells as compared with the wild-type cells which hardly had enzyme activity. The voltagedependent M-type potassium current (M-currents) which was one of the critical regulators of neuronal excitability was significantly reduced in the transformed cells. The other outward and inward currents were not affected by the 12-lipoxygenase expression so far as examined. Treatment of the transformed cells with a lipoxygenase inhibitor reversed the membrane excitability and the M-current amplitude to the control level. These results indicate that 12-lipoxygenase and/or its metabolites target potassium channels and up-regulate the membrane excitability. D 2002 Elsevier Science B.V. All rights reserved. Keywords: 12-Lipoxygenase; M-current; Potassium channel; NG108-15
1. Introduction Arachidonate 12-lipoxygenase is a dioxygenase which incorporates one molecule of oxygen regiospecifically and stereospecifically into unsaturated fatty acid such as arachidonic acid. Although a number of papers have reported various biological activities of 12-lipoxygenase metabolites, a definitive function of the enzyme applicable to many Abbreviations: DMEM, Dulbecco’s modified Eagle medium; NDGA, nordihydroguaiaretic acid. * Corresponding author. Tel.: +81-76-265-2186; fax: +81-76-234-4227. E-mail address: [email protected] (T. Yoshimoto). 0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 5 5 1 - 4
366
Y. Takahashi et al. / International Congress Series 1233 (2002) 365–369
animal species has not been established [1]. To determine the role of the enzyme in the nervous system, we permanently transfected 12-lipoxygenase cDNA into NG108-15 cells exhibiting many of the neural properties that are observed in intact neurons [2]. This paper clearly demonstrates the increased membrane excitability of the cells which was attributed to the specific inhibition of a voltage-dependent K+ channel generating Mcurrent (IK(M)), a critical regulator of neuronal excitability [3].
2. Material and methods 2.1. Plasmids An expression vector pEF-BOS [4] was ligated to neomycin-resistant gene to yield pBOSNeo that allowed for selection of transfected cells by geneticin. The plasmid was then ligated to human platelet 12-lipoxygenase cDNA [5] attached by the XbaI sites at both ends. NG108-15 cells were transfected with the expression vector using lipofectamine and selected in the medium containing geneticin. 12-Lipoxygenase-expressing cells were identified by slot blot analysis of RNA. Mock-transfected cells were established by the transfection of parental pBOSNeo. 2.2. Enzyme assay 12-Lipoxygenase activity was determined using [1-14C] arachidonic acid as a substrate as described previously [6]. The radioactive products extracted by diethyl ether were separated by thin layer chromatography, and quantified by a Fujix BAS1000 imaging analyzer (Tokyo, Japan). 2.3. Electrophysiology Cells were plated onto 35-mm plastic dishes coated with 0.01% polyornithine and differentiated for 7 – 14 days in DMEM supplemented with 1% fetal calf serum, 100 AM hypoxanthine, 16 AM thymidine and 0.25 mM dibutyryl cAMP [7]. Electrophysiological recordings were performed as previously described [8]. IK(M) deactivation tails were evoked by a hyperpolarizing voltage step for 1 s to 40 mV from a holding potential of 20 mV. For recording outward currents, the membrane potential was stepped from a holding potential of 80 to + 20 mV for 1 s. The inward peak current was measured at a command potential at 30 mV for a holding potential at 80 mV.
3. Results 3.1. 12-Lipoxygenase overexpression in NG108-15 cells To explore a role of 12-lipoxygenase in neuronal functions, we transfected NG108-15 cells with an expression vector harboring human platelet 12-lipoxygenase cDNA. Positive
Y. Takahashi et al. / International Congress Series 1233 (2002) 365–369
367
clones were isolated by slot blot analysis using a 32P-labeled 12-lipoxygenase cDNA probe. Four clones expressing 12-lipoxygenase with the specific activities ranging from 1 to 5 nmol/10 min/mg of protein were selected and used for further experiments. In contrast, the mock-transfected as well as wild-type cells hardly metabolized arachidonic acid. 3.2. 12-Lipoxygenase increases membrane excitability We examined the effect of 12-lipoxygenase overexpression on the membrane excitability as an overall neuronal characteristic. We selected cells with resting membrane potentials of more negative than 40 mV by a patch-clamp method. By monitoring the number of action potentials evoked by a series of depolarizing current steps at a currentclamp mode, we found the striking increase in the excitability in NG108-15 cells overexpressing 12-lipoxygenase. Fig. 1A shows typical results obtained by a wild-type cell and a 12-lipoxygenase-expressing cell. In the wild-type cell, a 1-s depolarizing current
Fig. 1. Repetitive action potential discharges in NG108-15 cells overexpressing 12-lipoxygenase (12-LOX). (A) Upper, a typical voltage trace in a wild-type cell, a 12-lipoxygenase-expressing cell, and a 12-lipoxygenaseexpressing cell treated with 0.5 AM NDGA for 5 days is shown. Lower, depolarizing currents of 1 s, 0.32 nA were injected at a resting membrane potential which was more negative than 40 mV. (B) The relationship between the number of action potentials generated during 1-s depolarization and the magnitude of the depolarizing current up to 0.4 nA is shown. Data are representative of two to nine separate experiments.
368
Y. Takahashi et al. / International Congress Series 1233 (2002) 365–369
step of 0.32 nA evoked only four action potentials at the resting membrane potential of 47 mV. In sharp contrast, 11 action potentials were evoked by the identical stimulus in the 12-lipoxygenase-expressing cell at 43 mV. This elevation of excitability was observed over a wide range of currents injected (Fig. 1B). This effect did not depend on resting membrane potentials, because essentially the same results were obtained when the membrane potential was shifted to 50 or to 80 mV (data not shown). To confirm this increased excitability, we treated the 12-lipoxygenase-expressing cells for 5 days with 0.5 AM nordihydroguaiaretic acid (NDGA), a general lipoxygenase inhibitor. As expected, the NDGA treatment of the cells depressed the excitability. In a typical case shown in Fig. 1A, only four action potentials were elicited by the 0.32 nA current injection for 1 s at a resting membrane potential of 47 mV. The excitability observed over a range of injected currents was almost the same level as that in wild-type cells (Fig. 1B). 3.3. 12-Lipoxygenase suppresses the voltage-dependent M-type potassium channels To examine what type of ion channels is responsible for changing the membrane excitability in 12-lipoxygenase-expressing cells, we first inspected IK(M) under voltageclamp recording mode. The inward current relaxations evoked by stepping to 40 mV from a holding potential of 20 mV were reduced in the four clonal cells overexpressing 12-lipoxygenase. The amplitudes of IK(M) which were normalized to the surface area of each cell by the simultaneous measurement of the capacitance of the cell were significantly smaller in the 12-lipoxygenase-expressing cells than control cells. The average normalized amplitudes of IK(M) in 12-lipoxygenase-expressing cells were reduced by 45– 78% as compared with the wild-type and mock-transfected cells. 3.4. 12-Lipoxygenase expression does not affect other outward or inward currents We next examined whether the other types of channel were affected in 12-lipoxygenase-expressing cells. The outward currents observed on stepping for 1 s from a holding potential of 80 mV to a command potential of + 20 mV are known to be composed of both Ca2+ -dependent K+ currents and Ca2+ -independent, delayed rectifier K+ currents in differentiated NG108-15 cells [9]. The densities of these outward K+ currents in the four 12-lipoxygenase-expressing cells were not statistically different from those in wild-type cell and in mock-transfected cells. Furthermore, we examined the inward peak currents observed on stepping from a holding potential of 80 mV to a command potential of 30 mV which are mainly carried by Na+ and Ca2+ ions [10]. Again, the inward peak currents were not affected by 12-lipoxygenase overexpression. These results suggest that 12-lipoxygenase expression does not occlude or open ion channels non-specifically, but specifically inhibits the M-type K+ channels.
4. Discussion We have demonstrated that the overexpression of 12-lipoxygenase dramatically increases the membrane excitability in NG108-15 neuronal cells. This phenotypic change is
Y. Takahashi et al. / International Congress Series 1233 (2002) 365–369
369
associated with the inhibition of the M-type K+ current, one of known regulators of their subthreshold electrical excitability [3]. Other outward or inward currents, including the voltage-gated Na+ channel which is another determinant of the neuronal excitability, do not seem to be affected by 12-lipoxygenase overexpression, suggesting that 12-lip- oxygenase specifically inhibits the M-type K+ current and thereby increases membrane excitability. There has been little consistent evidence that 12-lipoxygenase products or other arachidonic metabolites are involved in the modulation of M-currents under physiological conditions [11]. Therefore, this is the first conclusive and definitive report which has established the physiological significance of 12-lipoxygenase in general neuronal functions.
References [1] T. Yoshimoto, S. Yamamoto, Arachidonate 12-lipoxygenase, J. Lipid Mediators Cell Signal. 12 (1995) 195 – 212. [2] M. Nirenberg, S. Wilson, H. Higashida, A. Rotter, K. Krueger, N. Busis, R. Ray, J.G. Kenimer, M. Adler, Modulation of synapse formation by cyclic adenosine monophosphate, Science 222 (1983) 794 – 799. [3] D.A. Brown, M-currents: an update, Trends Neurosci. 11 (1988) 294 – 299. [4] S. Mizushima, S. Nagata, pEF-BOS, a powerful mammalian expression vector, Nucleic Acid Res. 18 (1990) 5322. [5] T. Yoshimoto, Y. Yamamoto, T. Arakawa, H. Suzuki, S. Yamamoto, C. Yokoyama, T. Tanabe, H. Toh, Molecular cloning and expression of human arachidonate 12-lipoxygenase, Biochem. Biophys. Res. Commun. 172 (1990) 1230 – 1235. [6] T. Hada, H. Hagiya, H. Suzuki, T. Arakawa, M. Nakamura, S. Matsuda, T. Yoshimoto, S. Yamamoto, T. Azekawa, Y. Morita, Arachidonate 12-lipoxygenase of rat pineal glands: catalytic properties and primary structure deduced from its cDNA, Biochim. Biophys. Acta 1211 (1994) 221 – 228. [7] H. Higashida, J. Robbins, A. Egorova, M. Noda, M. Taketo, N. Ishizaka, S. Takasawa, H. Okamoto, D.A. Brown, Nicotinamide-adenine dinucleotide regulates muscarinic receptor-coupled K + (M) channels in rodent NG108-15 cells, J. Physiol. (London) 482 (1995) 317 – 323. [8] J. Robbins, J. Trouslard, S.J. Marsh, D.A. Brown, Kinetic and pharmacological properties of the M-current in rodent neuroblastoma glioma hybrid cells, J. Physiol. (London) 451 (1992) 159 – 185. [9] D.A. Brown, H. Higashida, Voltage- and calcium-activated potassium currents in mouse neuroblastoma rat glioma hybrid cells, J. Physiol. (London) 397 (1988) 149 – 165. [10] R. Bodewei, S. Hering, B. Schubert, A. Wollenberger, Sodium and calcium currents in neuroblastoma glioma hybrid cells before and after morphological differentiation by dibutyryl cyclic AMP, Gen. Physiol. Biophys. 4 (1985) 113 – 127. [11] N.V. Marrion, Control of M-current, Annu. Rev. Physiol. 59 (1997) 483 – 504.
12-Lipoxygenase increases neuronal excitability by inhibiting M-type potassium channels Yoshitaka Takahashi a, Tanihiro Yoshimoto a,*, Naoto Hoshi b, Haruhiro Higashida b a
Department of Molecular Pharmacology, Kanazawa University Graduate School of Medicine, Kanazawa 920-8640, Japan b Department of Biophysical Genetics, Kanazawa University Graduate School of Medicine, Kanazawa 920-8640, Japan
Abstract To elucidate a role of 12-lipoxygenase in neuronal functions, cDNA for human 12-lipoxygenase of platelet type was permanently introduced to mouse neuroblastoma rat glioma hybrid NG108-15 cells which expressed many neural properties. The number of action potentials evoked by depolarizing current steps at a current-clamp mode was strikingly increased in the 12-lipoxygenase-expressing NG108-15 cells as compared with the wild-type cells which hardly had enzyme activity. The voltagedependent M-type potassium current (M-currents) which was one of the critical regulators of neuronal excitability was significantly reduced in the transformed cells. The other outward and inward currents were not affected by the 12-lipoxygenase expression so far as examined. Treatment of the transformed cells with a lipoxygenase inhibitor reversed the membrane excitability and the M-current amplitude to the control level. These results indicate that 12-lipoxygenase and/or its metabolites target potassium channels and up-regulate the membrane excitability. D 2002 Elsevier Science B.V. All rights reserved. Keywords: 12-Lipoxygenase; M-current; Potassium channel; NG108-15
1. Introduction Arachidonate 12-lipoxygenase is a dioxygenase which incorporates one molecule of oxygen regiospecifically and stereospecifically into unsaturated fatty acid such as arachidonic acid. Although a number of papers have reported various biological activities of 12-lipoxygenase metabolites, a definitive function of the enzyme applicable to many Abbreviations: DMEM, Dulbecco’s modified Eagle medium; NDGA, nordihydroguaiaretic acid. * Corresponding author. Tel.: +81-76-265-2186; fax: +81-76-234-4227. E-mail address: [email protected] (T. Yoshimoto). 0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 5 5 1 - 4
366
Y. Takahashi et al. / International Congress Series 1233 (2002) 365–369
animal species has not been established [1]. To determine the role of the enzyme in the nervous system, we permanently transfected 12-lipoxygenase cDNA into NG108-15 cells exhibiting many of the neural properties that are observed in intact neurons [2]. This paper clearly demonstrates the increased membrane excitability of the cells which was attributed to the specific inhibition of a voltage-dependent K+ channel generating Mcurrent (IK(M)), a critical regulator of neuronal excitability [3].
2. Material and methods 2.1. Plasmids An expression vector pEF-BOS [4] was ligated to neomycin-resistant gene to yield pBOSNeo that allowed for selection of transfected cells by geneticin. The plasmid was then ligated to human platelet 12-lipoxygenase cDNA [5] attached by the XbaI sites at both ends. NG108-15 cells were transfected with the expression vector using lipofectamine and selected in the medium containing geneticin. 12-Lipoxygenase-expressing cells were identified by slot blot analysis of RNA. Mock-transfected cells were established by the transfection of parental pBOSNeo. 2.2. Enzyme assay 12-Lipoxygenase activity was determined using [1-14C] arachidonic acid as a substrate as described previously [6]. The radioactive products extracted by diethyl ether were separated by thin layer chromatography, and quantified by a Fujix BAS1000 imaging analyzer (Tokyo, Japan). 2.3. Electrophysiology Cells were plated onto 35-mm plastic dishes coated with 0.01% polyornithine and differentiated for 7 – 14 days in DMEM supplemented with 1% fetal calf serum, 100 AM hypoxanthine, 16 AM thymidine and 0.25 mM dibutyryl cAMP [7]. Electrophysiological recordings were performed as previously described [8]. IK(M) deactivation tails were evoked by a hyperpolarizing voltage step for 1 s to 40 mV from a holding potential of 20 mV. For recording outward currents, the membrane potential was stepped from a holding potential of 80 to + 20 mV for 1 s. The inward peak current was measured at a command potential at 30 mV for a holding potential at 80 mV.
3. Results 3.1. 12-Lipoxygenase overexpression in NG108-15 cells To explore a role of 12-lipoxygenase in neuronal functions, we transfected NG108-15 cells with an expression vector harboring human platelet 12-lipoxygenase cDNA. Positive
Y. Takahashi et al. / International Congress Series 1233 (2002) 365–369
367
clones were isolated by slot blot analysis using a 32P-labeled 12-lipoxygenase cDNA probe. Four clones expressing 12-lipoxygenase with the specific activities ranging from 1 to 5 nmol/10 min/mg of protein were selected and used for further experiments. In contrast, the mock-transfected as well as wild-type cells hardly metabolized arachidonic acid. 3.2. 12-Lipoxygenase increases membrane excitability We examined the effect of 12-lipoxygenase overexpression on the membrane excitability as an overall neuronal characteristic. We selected cells with resting membrane potentials of more negative than 40 mV by a patch-clamp method. By monitoring the number of action potentials evoked by a series of depolarizing current steps at a currentclamp mode, we found the striking increase in the excitability in NG108-15 cells overexpressing 12-lipoxygenase. Fig. 1A shows typical results obtained by a wild-type cell and a 12-lipoxygenase-expressing cell. In the wild-type cell, a 1-s depolarizing current
Fig. 1. Repetitive action potential discharges in NG108-15 cells overexpressing 12-lipoxygenase (12-LOX). (A) Upper, a typical voltage trace in a wild-type cell, a 12-lipoxygenase-expressing cell, and a 12-lipoxygenaseexpressing cell treated with 0.5 AM NDGA for 5 days is shown. Lower, depolarizing currents of 1 s, 0.32 nA were injected at a resting membrane potential which was more negative than 40 mV. (B) The relationship between the number of action potentials generated during 1-s depolarization and the magnitude of the depolarizing current up to 0.4 nA is shown. Data are representative of two to nine separate experiments.
368
Y. Takahashi et al. / International Congress Series 1233 (2002) 365–369
step of 0.32 nA evoked only four action potentials at the resting membrane potential of 47 mV. In sharp contrast, 11 action potentials were evoked by the identical stimulus in the 12-lipoxygenase-expressing cell at 43 mV. This elevation of excitability was observed over a wide range of currents injected (Fig. 1B). This effect did not depend on resting membrane potentials, because essentially the same results were obtained when the membrane potential was shifted to 50 or to 80 mV (data not shown). To confirm this increased excitability, we treated the 12-lipoxygenase-expressing cells for 5 days with 0.5 AM nordihydroguaiaretic acid (NDGA), a general lipoxygenase inhibitor. As expected, the NDGA treatment of the cells depressed the excitability. In a typical case shown in Fig. 1A, only four action potentials were elicited by the 0.32 nA current injection for 1 s at a resting membrane potential of 47 mV. The excitability observed over a range of injected currents was almost the same level as that in wild-type cells (Fig. 1B). 3.3. 12-Lipoxygenase suppresses the voltage-dependent M-type potassium channels To examine what type of ion channels is responsible for changing the membrane excitability in 12-lipoxygenase-expressing cells, we first inspected IK(M) under voltageclamp recording mode. The inward current relaxations evoked by stepping to 40 mV from a holding potential of 20 mV were reduced in the four clonal cells overexpressing 12-lipoxygenase. The amplitudes of IK(M) which were normalized to the surface area of each cell by the simultaneous measurement of the capacitance of the cell were significantly smaller in the 12-lipoxygenase-expressing cells than control cells. The average normalized amplitudes of IK(M) in 12-lipoxygenase-expressing cells were reduced by 45– 78% as compared with the wild-type and mock-transfected cells. 3.4. 12-Lipoxygenase expression does not affect other outward or inward currents We next examined whether the other types of channel were affected in 12-lipoxygenase-expressing cells. The outward currents observed on stepping for 1 s from a holding potential of 80 mV to a command potential of + 20 mV are known to be composed of both Ca2+ -dependent K+ currents and Ca2+ -independent, delayed rectifier K+ currents in differentiated NG108-15 cells [9]. The densities of these outward K+ currents in the four 12-lipoxygenase-expressing cells were not statistically different from those in wild-type cell and in mock-transfected cells. Furthermore, we examined the inward peak currents observed on stepping from a holding potential of 80 mV to a command potential of 30 mV which are mainly carried by Na+ and Ca2+ ions [10]. Again, the inward peak currents were not affected by 12-lipoxygenase overexpression. These results suggest that 12-lipoxygenase expression does not occlude or open ion channels non-specifically, but specifically inhibits the M-type K+ channels.
4. Discussion We have demonstrated that the overexpression of 12-lipoxygenase dramatically increases the membrane excitability in NG108-15 neuronal cells. This phenotypic change is
Y. Takahashi et al. / International Congress Series 1233 (2002) 365–369
369
associated with the inhibition of the M-type K+ current, one of known regulators of their subthreshold electrical excitability [3]. Other outward or inward currents, including the voltage-gated Na+ channel which is another determinant of the neuronal excitability, do not seem to be affected by 12-lipoxygenase overexpression, suggesting that 12-lip- oxygenase specifically inhibits the M-type K+ current and thereby increases membrane excitability. There has been little consistent evidence that 12-lipoxygenase products or other arachidonic metabolites are involved in the modulation of M-currents under physiological conditions [11]. Therefore, this is the first conclusive and definitive report which has established the physiological significance of 12-lipoxygenase in general neuronal functions.
References [1] T. Yoshimoto, S. Yamamoto, Arachidonate 12-lipoxygenase, J. Lipid Mediators Cell Signal. 12 (1995) 195 – 212. [2] M. Nirenberg, S. Wilson, H. Higashida, A. Rotter, K. Krueger, N. Busis, R. Ray, J.G. Kenimer, M. Adler, Modulation of synapse formation by cyclic adenosine monophosphate, Science 222 (1983) 794 – 799. [3] D.A. Brown, M-currents: an update, Trends Neurosci. 11 (1988) 294 – 299. [4] S. Mizushima, S. Nagata, pEF-BOS, a powerful mammalian expression vector, Nucleic Acid Res. 18 (1990) 5322. [5] T. Yoshimoto, Y. Yamamoto, T. Arakawa, H. Suzuki, S. Yamamoto, C. Yokoyama, T. Tanabe, H. Toh, Molecular cloning and expression of human arachidonate 12-lipoxygenase, Biochem. Biophys. Res. Commun. 172 (1990) 1230 – 1235. [6] T. Hada, H. Hagiya, H. Suzuki, T. Arakawa, M. Nakamura, S. Matsuda, T. Yoshimoto, S. Yamamoto, T. Azekawa, Y. Morita, Arachidonate 12-lipoxygenase of rat pineal glands: catalytic properties and primary structure deduced from its cDNA, Biochim. Biophys. Acta 1211 (1994) 221 – 228. [7] H. Higashida, J. Robbins, A. Egorova, M. Noda, M. Taketo, N. Ishizaka, S. Takasawa, H. Okamoto, D.A. Brown, Nicotinamide-adenine dinucleotide regulates muscarinic receptor-coupled K + (M) channels in rodent NG108-15 cells, J. Physiol. (London) 482 (1995) 317 – 323. [8] J. Robbins, J. Trouslard, S.J. Marsh, D.A. Brown, Kinetic and pharmacological properties of the M-current in rodent neuroblastoma glioma hybrid cells, J. Physiol. (London) 451 (1992) 159 – 185. [9] D.A. Brown, H. Higashida, Voltage- and calcium-activated potassium currents in mouse neuroblastoma rat glioma hybrid cells, J. Physiol. (London) 397 (1988) 149 – 165. [10] R. Bodewei, S. Hering, B. Schubert, A. Wollenberger, Sodium and calcium currents in neuroblastoma glioma hybrid cells before and after morphological differentiation by dibutyryl cyclic AMP, Gen. Physiol. Biophys. 4 (1985) 113 – 127. [11] N.V. Marrion, Control of M-current, Annu. Rev. Physiol. 59 (1997) 483 – 504.