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. 1999 Dec 15;521(Pt 3):567–574. doi: 10.1111/j.1469-7793.1999.00567.x

12-Lipoxygenase overexpression in rodent NG108-15 cells enhances membrane excitability by inhibiting M-type K+ channels

Yoshitaka Takahashi *, Hiroo Kawajiri *, Tanihiro Yoshimoto *, Naoto Hoshi *, Haruhiro Higashida *
PMCID: PMC2269696  PMID: 10601489

Abstract

  1. 12-Lipoxygenase produces 12-hydroperoxy acid from arachidonic acid released from membrane phospholipids. To elucidate the role of the enzyme in neuronal functions, mouse neuroblastoma × rat glioma hybrid NG108-15 cells were permanently transfected with the cDNA for human 12-lipoxygenase.

  2. The number of action potentials evoked by depolarizing current steps in a current-clamp mode was strikingly increased in 12-lipoxygenase-expressing NG108-15 cells as compared with the wild-type cells which hardly had the enzyme activity.

  3. In the transformed cells, the M-type voltage-dependent K+ current was significantly reduced with little or no change in other ion channel currents.

  4. Treatment of the transformed cells with a 12-lipoxygenase inhibitor recovered the action potential frequency and the M-current amplitude to the control level.

  5. These results indicate that 12-lipoxygenase and/or its metabolites target K+ channels and upregulate the membrane excitability, and thereby modulate neuronal signalling.

Arachidonate 12-lipoxygenase is a dioxygenase which incorporates one molecule of oxygen regiospecifically and stereospecifically into unsaturated fatty acids such as arachidonic acid or linoleic acid. When arachidonic acid is released from membrane phospholipids by the reaction of phospholipases, 12-lipoxygenase converts it to 12-hydroperoxyeicosatetraenoic acid (12-HPETE) as a primary product. In the intact cell, 12-HPETE is readily reduced to 12-hydroxy acid (12-HETE) by glutathione peroxidase or similar enzymes. Alternatively, 12-HPETE is converted to hepoxilins or a 12-keto acid (12-KETE) depending on the cell type (Shimizu & Wolfe, 1990). Although a number of papers have reported various biological activities of these metabolites, none of them clearly demonstrated a definitive function of 12-lipoxygenase applicable to many animal species. Thus, the physiological significance of 12-lipoxygenase has not been established, and is still a subject of investigations (Yoshimoto & Yamamoto, 1995; Funk, 1996; Kuhn & Thiele, 1999).

In the nervous system, the function of 12-lipoxygenase has been studied by using enzyme inhibitors or metabolites. The 12-lipoxygenase metabolites applied in the micromolar range in the bath or by intracellular perfusion are reported to modulate ion channel conductances in both stimulatory and inhibitory directions in Aplysia, bullfrog and rat neuronal cells (Piomelli et al. 1987; Carlen et al. 1989; Buttner et al. 1989; Yu, 1995; Vaughan et al. 1997). In these studies, the effects were not always consistent probably due to the technical difficulty of handling these unstable and hydrophobic lipid metabolites in aqueous solution. Furthermore, it is usually impossible to determine whether such high concentrations of metabolites could be produced in vivo by 12-lipoxygenase localized in these cells (Shimizu & Wolfe, 1990).

To circumvent these problems in previous pharmacological studies, an approach using 12-lipoxygenase itself is necessary. We chose to overexpress 12-lipoxygenase cDNA rather than use topical application of enzyme metabolites. NG108-15 cells are dividing culture cells obtained by fusion of mouse neuroblastoma and rat glioma cells, but still express many of the neural properties that are observed in intact neurons (Nirenberg et al. 1983). In particular, the differentiated NG108-15 cells become electrically excitable with the expression of voltage- and Ca2+-gated ion channel molecules (Higashida & Brown, 1986). They also promote neurite extension, form synapses with cultured striated muscle cells and release acetylcholine (Nirenberg et al. 1983). We have established NG108-15 cells which overexpress 12-lipoxygenase. Wild-type NG108-15 cells do not have detectable enzyme activity. A striking elevation of membrane excitability is observed in the cells which are permanently transfected with 12-lipoxygenase cDNA. We clearly demonstrate here that the increased membrane excitability of the 12-lipoxygenase-expressing NG108-15 cells can be attributed to the specific inhibition of a voltage-dependent K+ channel generating M-current (IK(M)), a critical regulator of neuronal excitability (Brown & Adams, 1980; Brown, 1988).

METHODS

Cell culture

NG108-15 hybrid cells were used as an untransfected control (wild-type) and transfected with human platelet 12-lipoxygenase cDNA (clones NGHP59, NGHP60, NGHP110 and NGHP115). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5 % fetal calf serum, 100 μm hypoxanthine, 0.4 μm aminopterin and 16 μm thymidine at 37°C in a humidified atmosphere of 90 % air-10 % CO2 (Higashida & Brown, 1986).

Transfection of expression vectors

An expression vector, pEF-BOS, having a powerful elongation factor-1 α promoter (Mizushima & Nagata, 1990) was kindly provided by Dr S. Nagata of Osaka University. The pEF-BOS was digested by SapI, blunted by Klenow fragment, and ligated to a neomycin-resistance gene to yield pBOSNeo, which allowed for selection of transfected cells by geneticin. The plasmid was digested with XbaI, and ligated to human platelet 12-lipoxygenase cDNA (Yoshimoto et al. 1990) attached by the XbaI sites at both ends. NG108-15 cells were transfected with the expression vector (pBOSNeoHP) using lipofectamine (Gibco BRL) under the manufacturer's instructions. After 5 days the transfected cells were split at a ratio of 1:20 in culture medium containing 1.5 mg ml−1 geneticin. After 2 weeks we picked 120 clones, and identified cells by slot blot analysis of RNA using 32P-labelled cDNA probes. Mock-transfected cells were established by the transfection of parental pBOSNeo.

12-Lipoxygenase assay

12-Lipoxygenase activity was determined as described previously (Hada et al. 1994). Briefly, the cell homogenate was incubated in a 200 μl mixture of 50 mm Tris-HCl buffer at pH 7.4 and 25 μm[1-14C]arachidonic acid (1.85 kBq, Amersham). The reaction was performed at 30°C for 10 min with constant mixing, and quenched by the addition of an ice-cold mixture of diethyl ether:methanol:1 M citric acid (30:4:1, by volume). The ether layer was spotted onto a silica gel thin layer plate which was developed at 4°C for 60 min with a solvent system of diethyl ether:petroleum ether:acetic acid (85:15:0.1, by volume). The radioactive products (12-HPETE and 12-HETE) on the plate were detected and quantified by a Fujix BAS1000 imaging analyser (Tokyo, Japan). Protein concentration was determined with bovine serum albumin as a standard (Lowry et al. 1951).

Electrophysiology

Cells were plated onto 35 mm diameter plastic dishes coated with 0.01 % polyornithine and differentiated for 7–14 days in DMEM supplemented with 1 % fetal calf serum, 100 μm hypoxanthine, 16 μm thymidine and 0.25 mm dibutyryl cAMP (Higashida et al. 1995). For electrophysiological recordings, the culture medium was replaced with 10 mm Hepes-buffered DMEM. The whole-cell variant of the patch-clamp technique was used in the discontinuous voltage-clamp mode (Axoclamp 2, Axon Instruments), as previously described (Robbins et al. 1992). Electrodes were filled with a solution containing (mm): potassium citrate, 90; KCl, 20; Hepes, 40; MgCl2, 3; EGTA, 3; and CaCl2, 1, pH 7.4. The electrode resistance was about 5 MΩ, and the seal resistance was greater than 2–3 GΩ. 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 mV to +20 mV for 1 s. The inward peak current was measured at a command potential of −30 mV for a holding potential of −80 mV. Voltage and current were recorded on a thermal-array recorder (Nihonkoden, Model RTA-1100, Tokyo, Japan). IK(M) was processed by pCLAMP6 (Axon Instruments). The same software was used to generate stepped voltage commands. Results are expressed as means ±s.e.m. unless otherwise stated.

RESULTS

Overexpression of 12-lipoxygenase in NG108-15 cells

As the first step to explore the role of 12-lipoxygenase in neuronal functions, we transfected NG108-15 cells with an expression vector harbouring human platelet 12-lipoxygenase cDNA, the transcription of which was driven by a powerful elongation factor-1 α promoter. The positive clones were selected by slot blot analysis of 12-lipoxygenase mRNA using a 32P-labelled cDNA probe, and their 12-lipoxygenase activities were examined. Figure 1 shows a silica gel thin layer chromatogram following incubation of [1-14C]arachidonic acid with the cell homogenates of four clones designated as NGHP cells. All four clones converted arachidonic acid to its 12-hydroperoxy and 12-hydroxy derivatives, indicating that these cells expressed the catalytically active 12-lipoxygenase. In contrast, the mock-transfected as well as wild-type cells hardly metabolized arachidonic acid (Fig. 1). The enzyme activities of the selected clones are shown in Table 1. The specific activities ranged between 1 and 5 nmol (10 min)−1 (mg of protein)−1.

Figure 1. Arachidonic acid metabolism by NG108-15 cells overexpressing 12-lipoxygenase.

Figure 1

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The wild-type, mock-transfected, and four clonal cells transfected with human platelet 12-lipoxygenase cDNA (NGHP59, 60, 110 and 115) were grown to confluence. The cells were harvested and sonicated. The cell homogenates (500 μg of protein) were incubated with 25 μm[1−14C]arachidonic acid at 30 °C for 10 min. The products were extracted and separated by thin layer chromatography. The migration positions of arachidonic acid (AA), 12-HPETE and 12-HETE are indicated.

Table 1.

12-Lipoxygenase activities in wild-type and transformed NG108-15 cells

Cell line Products formed (nmol (10 min)1 (mg of protein)−1)
NG108-15 < 0.10 (2)
Mock < 0.10 (2)
NGHP59 3.26 (2)
NGHP60 4.59 (2)
NGHP110 1.43 (3)
NGHP115 2.79 (3)
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Data are given as means (number of experiments in parentheses).

12-Lipoxygenase expression increases neuronal excitability

The morphology was not significantly different between wild-type and 12-lipoxygenase-expressing cells, either before or after differentiation. The growth rates of the transformed cells were essentially the same as that of wild-type cells.

We examined the effect of 12-lipoxygenase overexpression on the membrane excitability as an overall neuronal characteristic. We selected cells with resting membrane potentials 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 in a current-clamp mode, we found a striking increase in the excitability in NG108-15 cells overexpressing 12-lipoxygenase. Figure 2 shows typical results obtained in a wild-type cell and a 12-lipoxygenase-expressing NGHP59 cell. In the wild-type NG108-15 cell, a 1 s depolarizing current step of 0.32 nA evoked only four action potentials at the resting membrane potential of −47 mV. This number was of the same order of magnitude as that reported previously (Robbins et al. 1992). In sharp contrast, eleven action potentials were evoked by the identical stimulus in the NGHP59 cell at −43 mV (Fig. 2A). This elevation of excitability was observed over a wide range of currents injected (Fig. 2B). This effect did not depend on resting membrane potentials, because essentially the same results were obtained in the experiments where the membrane potential was shifted to −50 mV or to −80 mV.

Figure 2. Repetitive action potential discharges in NG108-15 cells overexpressing 12-lipoxygenase.

Figure 2

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A, upper, a typical voltage trace in a wild-type cell, an NGHP59 cell, and an NGHP59 cell treated with 0.5 μm NDGA for 5 days are 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 2–9 separate experiments.

To confirm that the increased excitability was attributable to 12-lipoxygenase expression, we treated the NGHP59 cells for 5 days with nordihydroguaiaretic acid (NDGA), a general lipoxygenase inhibitor. To avoid any toxic effect of NDGA, the concentration was lowered to 0.5 μm. As expected, the NDGA treatment of NGHP59 cells depressed the excitability. In a typical case shown in Fig. 2A, 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 at almost the same level as that in wild-type cells (n= 2, Fig. 2B). On average, the resting membrane potentials were not significantly different between 12-lipoxygenase-expressing NGHP59 cells and wild-type cells (-43.3 ± 0.63 mV (n= 4) vs.−46.7 ± 3.51 mV (n= 9), P= 0.53).

12-Lipoxygenase expression suppresses the voltage-dependent potassium M-current

To identify what type of ion channels is responsible for changing the membrane excitability in 12-lipoxygenase-expressing cells, we first inspected IK(M) under voltage-clamp recording mode. Figure 3A shows typical current traces of each clone. 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 (NGHP59, 60, 110 and 115 cells). The amplitudes of IK(M) were normalized to the surface area of each cell by the simultaneous measurement of the capacitance of the cell (Cm), and the data were expressed as means of current densities (IK(M)Cm−1). As shown in Fig. 3B, the normalized amount of inward current relaxations was significantly smaller in the 12-lipoxygenase-expressing cells than the two types of control cells. While the averaged IK(M)Cm−1 was 0.35 ± 0.04 nA nF−1 (n= 26) in wild-type cells and 0.36 ± 0.05 nA nF−1 (n= 13) in mock-transfected cells, those of the 12-lipoxygenase-expressing cells were 0.17 ± 0.05 nA nF−1 (n= 9) in NGHP59, 0.06 ± 0.02 nA nF−1 (n= 8) in NGHP60, 0.21 ± 0.04 nA nF−1 (n= 21) in NGHP110 and 0.16 ± 0.03 nA nF−1 (n= 19) in NGHP115 cells (Fig. 3B).

Figure 3. M-currents in NG108-15 cells overexpressing 12-lipoxygenase.

Figure 3

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A, typical current traces in a wild-type NG108-15 cell, a mock-transfected cell, and four clonal cells transformed with 12-lipoxygenase cDNA. The cells were held at −20 mV to activate IK(M) and stepped to −40 mV for 1 s to deactivate IK(M). B, IK(M) was measured as shown in the inset, and normalized in each measurement to capacitance (Cm) in order to obtain the current density per cell surface area (IK(M)Cm−1). Data represent the means of 8–26 determinations and bars denote standard errors. * P < 0.05 and ** P < 0.005 from wild-type cells using one-way analysis of variance (ANOVA).

Figure 4 shows two examples of IK(M) in a series of current traces evoked by stepping to various voltages in a wild-type cell and a 12-lipoxygenase-expressing NGHP115 cell and the plots of their I–V curves. The outward rectification in the I–V curve associated with development of IK(M) between −80 and 0 mV observed in the wild-type cell was markedly reduced in the NGHP115 cell.

Figure 4. The I–V relationship of IK(M) in wild-type and NGHP115 cells.

Figure 4

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A, typical M-current traces in a wild-type NG108-15 cell (upper) and an NGHP115 cell overexpressing 12-lipoxygenase (middle). The cells were held at −20 mV and voltage was commanded for 1 s from 0 to −100 mV in 10 mV increments (bottom). B, plots of currents attained at the end of the voltage steps in response to 0 to −100 mV steps against command voltage from a holding potential of −20 mV. I–V curves were obtained by the measurement of traces shown in A.

We tested whether the control phenotype in IK(M) could be rescued from 12-lipoxygenase-expressing cells by enzyme inhibition with 0.5 μm NDGA. As shown in Fig. 5A, treatment of a 12-lipoxygenase-expressing cell by NDGA resulted in the dramatic recovery of the amplitude of IK(M), whereas the inhibitor had essentially no effect on the IK(M) in a wild-type cell. The upper panel of Fig. 5B shows the means of normalized IK(M) (IK(M)Cm−1) recorded in NDGA-treated cells. Although the normalized IK(M) in two clones did not recover to the level of wild-type cells (Fig. 5B, upper panel), a larger recovery in current (1.43-1.93) was observed in all four clonal cells treated with NDGA than in wild-type cells (1.08) (Fig. 5B, lower panel).

Figure 5. Effect of NDGA on IK(M) in control and 12-lipoxygenase-expressing cells.

Figure 5

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A, typical currents which were recorded in wild-type NG108-15 cells and NGHP59 cells, untreated or treated with 0.5 μm NDGA for 5 days are shown. The cells were held at −20 mV to activate IK(M) and stepped to −40 mV for 1 s to deactivate IK(M). B, upper histogram, IK(M) was recorded in the cells treated with NDGA. Data represent the means of IK(M) normalized to capacitance (Cm) (n= 4–13), and bars denote standard errors. Lower histogram, the relative ratios of the mean of normalized IK(M) recorded in the cells treated with NDGA to that in the non-treated cells

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. Figure 6A shows the means of the outward currents observed on stepping for 1 s from a holding potential of −80 mV to a command potential of +20 mV. These currents are known to be composed of both Ca2+-dependent K+ currents and Ca2+-independent, delayed rectifier K+ currents in differentiated NG108-15 cells (Brown & Higashida, 1988a). The densities of these outward K+ currents in the four 12-lipoxygenase-expressing clones were not statistically different from those in wild-type cells (P > 0.1) and in mock-transfected cells (P > 0.1). Furthermore, we examined the inward peak currents observed on stepping from a holding potential of −80 mV to a command potential of −30 mV. As shown in Fig. 6B, the means of the density of inward peak currents, which are mainly carried by Na+ and Ca2+ ions (Bodewei et al. 1985), were not affected by 12-lipoxygenase overexpression (P > 0.05). These results suggest that 12-lipoxygenase expression does not occlude or open ion channels non-specifically, but specifically inhibits the M-type K+ channels.

Figure 6. Outward and inward peak currents in NG108-15 cells overexpressing 12-lipoxygenase.

Figure 6

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A, the membrane potential was stepped from a holding potential of −80 mV to +20 mV for 1 s to measure outward peak current (x in inset). The leakage current was measured by hyperpolarizing a voltage step to −100 mV (y in inset), and the × 5 value of y was subtracted from x. The current was normalized in each measurement to capacitance in order to obtain the current density (nA nF−1). B, the inward peak current was measured at a command potential of −30 mV for a holding potential of −80 mV, and normalized to capacitance. The arrow in the inset shows the measured current. Data represent the means of 7–23 determinations and bars denote standard errors. Note, no statistical difference between wild-type and transformed cells by one-way ANOVA. 12-Lipoxygenase overexpression in rodent NG108-15 cells enhances the excitability by inhibiting M-type K+ channels.

DISCUSSION

In the present study, we have demonstrated that the overexpression of 12-lipoxygenase dramatically increases the membrane excitability in NG108-15 neuronal cells. This phenotypic change is associated with the inhibition of the M-type K+ current, one of the known regulators of their subthreshold electrical excitability (Brown & Adams, 1980; Brown, 1988). Other outward or inward currents, including the voltage-gated Na+ channel which is another determinant of the neuronal excitability (Catterall, 1993), do not seem to be affected by 12-lipoxygenase overexpression, suggesting that 12-lipoxygenase specifically inhibits the M-type K+ current and thereby increases membrane excitability. Our present data obtained by the genetic engineering technique are consistent with the results in previous studies which have shown the effects of 12-lipoxygenase metabolites on a K+ conductance by pharmacological methods (reviewed in Yoshimoto & Yamamoto, 1995). 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 (Marrion, 1997). Therefore, this is the first conclusive and definitive report which has established the physiological significance of 12-lipoxygenase in general neuronal functions.

Several potential mechanisms for the inhibition of M-type K+ current by 12-lipoxygenase can be considered. A direct interaction between the enzyme protein and the protein(s) which might affect M-currents would be possible, as well as the K+ channel inhibition by 12-lipoxygenase products. However, our results suggest that 12-lipoxygenase-generating metabolites play a more critical role than the 12-lipoxygenase itself, since the lipoxygenase inhibitor rescued not only the suppressive effect on M-currents but also the elevated membrane excitability which was brought about by the enzyme overexpression. Bath application of 12-HPETE was reported to increase a presynaptic K+ conductance in Aplysia sensory cells (Piomelli et al. 1987). It was also reported later that 12-HPETE could be replaced by 12-KETE and hepoxilin A3, both of which are metabolites of 12-HPETE (Piomelli et al. 1988, 1989). The modulation of small conductance K+ (SK) channels by application of 12-HPETE onto the Aplysia neuron was also reported (Buttner et al. 1989). Recently 12-HETE was found to modulate a voltage-dependent K+ conductance in rat periaqueductal grey (Vaughan et al. 1997) and the M-current in bullfrog neurons (Yu, 1995). Since 12-HPETE and 12-HETE are major products in 12-lipoxygenase-expressing NG108-15 cells (Fig. 1), these metabolites may play certain roles in inhibiting M-currents by mechanisms which remain to be elucidated. A previous report suggests that topical application of 12-HETE enhances M-currents in bullfrog sympathetic neurons (Yu, 1995). However, the results may need further investigation, since in the same report it is described that 12-HPETE which is very unstable and readily converted to 12-HETE in intact cells shows almost no effect on M-current amplitude.

There are several isoforms of 12-lipoxygenase with different substrate specificities and tissue distributions. For example, the leukocyte 12-lipoxygenase exhibits much broader substrate specificity than the platelet enzyme (Yoshimoto & Yamamoto, 1995). Our preliminary experiments show that NG108-15 cells transfected permanently with porcine leukocyte 12-lipoxygenase cDNA exhibit a similar phenotype with regard to the M-current inhibition. These data on the different 12-lipoxygenase isoforms also support our conclusion that 12-lipoxygenase metabolites play a role in the regulation of neuronal excitability. It is unlikely that 12-lipoxygenase directly oxygenates esterified fatty acids in membrane phospholipids to form reactive hydroperoxides which may cause ion channel oxidation and inactivation, since human platelet 12-lipoxygenase expressed in our system hardly reacted with esterified fatty acids (Takahashi et al. 1993). The specific inhibition of the M-type K+ channel without affecting other ion channels as far as investigated also disagrees with this hypothesis.

Recently the molecular correlation of the M-channel to KCNQ2 and KCNQ3 heteromultimeric potassium channels was reported (Wang et al. 1998). The expression pattern of KCNQ 2 and KCNQ 3 genes agreed with M-current expression observed in the rat cortex and hippocampus. On the other hand, there are only a limited number of reports which have examined the precise localization of 12-lipoxygenase in mammalian brain. It was reported that 12-lipoxygenase activity was detected in canine cerebrum, cerebellum, basal ganglia, hippocampus and olfactory bulb (Nishiyama et al. 1992). In rat brain, 12-lipoxygenase mRNA was detected most abundantly in the pineal gland, whereas faint bands were also seen in cortex, hypothalamus, cerebellum and pons upon Northern blot analysis (Hada et al. 1994). Neurons expressing both the M-current and 12-lipoxygenase have not yet been reported. In fact NG108-15 cells which express M-currents do not have the detectable 12-lipoxygenase activity. However, further experiments might reveal the colocalization of these K+ channel gene products and the enzyme in mammalian nervous systems to establish the physiological significance of the regulation of the M-current by 12-lipoxygenase.

The M-channel is inhibited by the application of bradykinin in NG108-15 cells (Higashida & Brown, 1986) or by acetylcholine in cells transformed to express muscarinic acetylcholine receptors (Fukuda et al. 1988). In response to bradykinin, the membrane phosphatidylinositol is hydrolysed to produce inositol 1,4,5-trisphosphate and diacylglycerol. The produced diacylglycerol activates protein kinase C which has been shown to inhibit M-currents, as originally reported in NG108-15 cells by one of the authors of the present work (Brown & Higashida, 1988b). On the other hand, inositol 1,4,5-trisphosphate raises the intracellular Ca2+ which is known to activate Ca2+-dependent phospholipase A2 and release arachidonic acid (Xu et al. 1998). In 12-lipoxygenase-expressing NG108-15 cells, the released arachidonic acid would be readily converted to 12-PETE, which may also contribute to the inhibition of M-current and increase of the cell excitability. Our findings suggested the effect of 12-lipoxygenase or its metabolites on the modulation of M-currents by G protein-coupled receptors such as bradykinin or m1 muscarinic receptor. Although it was demonstrated that bradykinin-induced acetylcholine release from differentiated NG108-15 cells was not a voltage-dependent process (Ogura et al. 1990), 12-lipoxygenase expression may change the sensitivity of the cells to bradykinin and modulate the bradykinin-induced release of acetylcholine. This possibility is now under investigation.

Acknowledgments

This work was supported by grants from the Ministry of Education, Science and Culture of Japan, Ono Pharmaceutical Company, the Mochida Memorial Foundation for Medical and Pharmaceutical Research, the Ichiro Kanehara Foundation and the Kisshokai Foundation.

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