Angiotensin II Increases Neuronal Delayed Rectifier K+ Current: Role of 12-Lipoxygenase Metabolites of Arachidonic Acid

Mingyan Zhu,1 Rama Natarajan,2 Jerry L. Nadler,3 Jennifer M. Moore,1 Craig H. Gelband,1 and Colin Sumners1

 1Department of Physiology, College of Medicine and University of Florida Brain Institute, University of Florida, Gainesville, Florida 32610;  2Gonda Diabetes Center, City of Hope Medical Center, Duarte, California 91010; and  3Division of Endocrinology, University of Virginia Health Science Center, Charlottesville, Virginia 22908


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Zhu, Mingyan, Rama Natarajan, Jerry L. Nadler, Jennifer M. Moore, Craig H. Gelband, and Colin Sumners. Angiotensin II Increases Neuronal Delayed Rectifier K+ Current: Role of 12-Lipoxygenase Metabolites of Arachidonic Acid. J. Neurophysiol. 84: 2494-2501, 2000. Angiotensin II (Ang II) elicits an Ang II type 2 (AT2) receptor-mediated increase in voltage-dependent delayed rectifier K+ current (IKV) in neurons cultured from newborn rat hypothalamus and brain stem. In previous studies, we have determined that this effect of Ang II is mediated via a Gi protein, activation of phospholipase A2 (PLA2), and generation of arachidonic acid (AA). AA is rapidly metabolized within cells via lipoxygenases (LO), cyclooxygenase (COX) or p450 monooxygenase enzymes, and the metabolic products are known regulators of K+ currents and channels. Thus in the present study, we have investigated whether the AT2 receptor-mediated effects of Ang II on neuronal IKV require AA metabolism and if so, which metabolic pathways are involved. The data presented here indicate that the stimulatory actions of Ang II and AA on neuronal IKV are attenuated by selective blockade of 12-LO enzymes. However, the effects of Ang II are not altered by blockade of 5-LO or p450 monooxygenase enzymes. Furthermore, the actions of Ang II are mimicked by a 12-LO metabolite of AA, but 5-LO metabolites such as leukotriene B4 and C4 do not alter neuronal IKV. These data indicate that the AT2 receptor-mediated stimulation of neuronal IKV is partially mediated through 12-LO metabolites of AA.


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It is well established that mammalian brain contains angiotensin II (Ang II) type 2 (AT2) receptors, and that these sites are more prevalent in neonates (Lenkei et al. 1996; Millan et al. 1991; Nuyt et al. 1999; Tsutsumi and Saavedra 1991). A number of studies have suggested that these brain AT2 receptors are involved in a variety of different physiological and pathological processes (Gallinat et al. 2000). For example, the fact that they are present in neonates has led to the suggestion that AT2 receptors are involved in development and differentiation (Cook et al. 1991; Millan et al. 1991). Support for this has come from studies that have determined that stimulation of AT2 receptors causes neurite outgrowth in undifferentiated neuroblastoma × glioma cells (Laflamme et al. 1996) and plays a role in apoptosis of pheochromocytoma PC12W cells and neurons cultured from newborn rat brain (Shenoy et al. 1999; Yamada et al. 1996). Studies from mutant mice that lack the AT2 receptor gene have revealed that the knockout animals exhibit decreased exploratory behavior, spontaneous movements, an impaired drinking response to water deprivation, and lower basal body temperature (Hein et al. 1995; Ichiki et al. 1995). In fact, more recent studies in these mutant mice suggest an important role for brain AT2 receptors in stress-induced hyperthermia (Watanabe et al. 1999). A pathological role for CNS AT2 receptors has been indicated by studies that show that global ischemia elicits a transient increase in AT2 receptor mRNA in rat brain (Makino et al. 1996) and that glutamate-induced toxicity of cultured cortical neurons is associated with increased expression of AT2 receptor mRNA and increased AT2 receptor binding (Shibata et al. 1998). Thus AT2 receptors may be involved in the central control of certain behaviors and physiological responses, and in the pathological processes that result from ischemia in the brain. It is also clear that stimulation of brain AT2 receptors elicits physiological effects at the fundamental level of changes in neuronal firing rate and membrane ionic currents. For example, in situ electrophysiological recordings have indicated that Ang II elicits an AT2 receptor-mediated excitation of inferior olivary neurons (Ambuhl et al. 1992) and depresses glutamate-induced depolarization in locus coeruleus (Xiong and Marshall 1994). Our studies using neurons cultured from neonatal rat brain stem and hypothalamus have attempted to define the ionic basis of such AT2 receptor-mediated changes in neuronal activity. We have determined that Ang II elicits an AT2 receptor-mediated increase in neuronal delayed rectifier K+ current (IKV) (Kang et al. 1994), and that this effect is mediated mostly through activation of phospholipase A2 (PLA2) and generation of arachidonic acid (AA) (Zhu et al. 1998). AA itself is a well-known modulator of neuronal K+ current and channels (Premkumar et al. 1990; Schweitzer et al. 1990; Zona et al. 1993). However, AA is rapidly metabolized via three major groups of enzymes within cells, namely the lipoxygenases (LO), cyclooxygenases (COX), and cytochrome p450 monooxygenases, and it is clear that AA metabolites produced from each of these pathways have the ability to modulate K+ channels in various tissues (Arima et al. 1997; Duerson et al. 1996; Harder et al. 1997; Roman and Alonso-Galicia 1999; Twitchell et al. 1997; Yu 1995). In particular, one study has indicated that a p450 metabolite of AA mediates the actions of Ang II, via AT2 receptors, in vascular tissue (Arima et al. 1997). Thus it was possible that the actions of Ang II on IKV involved AA itself or AA metabolites, or a combination of these factors. Our preliminary studies have suggested that the stimulation of IKV by Ang II involves generation of LO, but not COX metabolites of AA (Zhu et al. 1998). Thus in the present study, we have investigated the role of AA metabolism in the Ang II-induced increase in neuronal IKV, in particular the role of LO metabolites. The results indicate that the AT2 receptor-mediated stimulation of neuronal IKV by Ang II is partially mediated through 12-LO metabolite of AA, but does not involve 5-LO metabolites nor p450 monooxygenase metabolites of AA. However, the data leave open the possibility of a further signaling mechanism that is responsible for the residual action of Ang II on IKV.


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Materials

Newborn Sprague-Dawley rats were obtained from our breeding colony, which originated from Charles River Farms (Wilmington, MA). Dulbecco's modified Eagle's medium (DMEM) and TRIzol reagent were obtained from GIBCO-BRL (Gaithersburg, MD). Plasma-derived horse serum (PDHS) was from Central Biomedia (Irwin, MO). Renaissance enhanced chemiluminescence (ECL) kits were purchased from Dupont-NEN (Boston, MA). Losartan potassium (Los) was generously provided by W. Henckler, Merck (Rahway, NJ). PD123,319 (PD), and 5,6,7-trihydroxyflavone (baicalein) were purchased from Research Biochemicals International (Natick, MA). Tetrodotoxin (TTX) was purchased from Calbiochem (La Jolla, CA). Gene-Amp RT-PCR kits and all reagents for RT-PCR were purchased from Perkin Elmer Biotechnologies (Norwalk, CT). MK 886 and cinnamyl-3,4-dihydroxy-alpha -cyanocinnamate (CDC) were from Biomol (Plymouth Meeting, PA). Leukocyte 12-LO standard was from Oxford Biomedical (Oxford, MI). Ang II, sodium GTP, cadmium chloride (CdCl2), HEPES, ethylene glycol-bis (beta -aminoethyl ether)-N,N,N',N'-tetracetic acid (EGTA), dipotassium ATP, peroxidase-conjugated affinity purified goat anti-rabbit IgG, 5, 8, 11, 14-eicosatetraynoic acid (ETYA), 17-octadecynoic acid (17-ODYA), Leukotriene B4, Leukotriene C4, and 12-(S)-hydroxy-(5Z, 8Z, 10E, 14Z)-eicosatetraenoic acid [12-(S)-HETE] were purchased from Sigma Chemical (St. Louis, MO). All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA). Oligonucleotide primers for the rat AT2 receptor gene (Mukoyama et al. 1993), the rat brain cytosolic PLA2 gene (Owada et al. 1994) and the 12-LO gene (Watanabe et al. 1993) were synthesized by Gemini Biotech (Alachua, FL). The sequences of these primers are as follows: 1) AT2 receptor gene: sense, 5'-TGGTGTATGGCTTGTCTGTC-3'; antisense, 5'-CACACTACGGAGCTTCTGTT-3', 2) PLA2 gene: sense, 5'-GCTCCACATGGTACATGTCA-3'; antisense, 5'-CCTCAAGCTACTCAAGGTCG-3', 3) 12-LO: sense, 5'-ACCTGATCTCAGAGAGAGGCT-3'; antisense, 5'-TCAGTGATCTCTCGACACCAG-3'.

Preparation of cultured neurons

Neuronal co-cultures were prepared from the hypothalamus and brain stem of newborn Sprague-Dawley rats exactly as described previously (Sumners et al. 1991). Cultures were grown on 35-mm plastic culture dishes in DMEM/10% PDHS for 10-14 days, at which time they consisted of 90% neurons and 10% astrocyte glia and microglia, as determined by immunofluorescent staining (Sumners et al. 1991).

Electrophysiological recordings

Macroscopic K+ current was recorded using the whole cell configuration of the patch-clamp technique as detailed previously (Hamill et al. 1981; Kang et al. 1994). Experiments were performed at room temperature (22-23°C) using an Axopatch 1D amplifier and Digidata 1200 B interface (Axon Instruments, Burlingame, CA). Neurons were bathed in a modified Tyrode's solution containing (in mM) 137 NaCl, 5.4 KCl, 2 MgSO4, 1.35 CaCl2, 0.3 NaH2PO4, 0.3 CdCl2, 10 dextrose, 10 HEPES, and 0.0015 TTX, pH 7.4 (NaOH). The patch electrodes had resistances of 3-4 MOmega when filled with an internal pipette solution containing (in mM) 130 KCl, 2 MgCl2, 0.25 CaCl2, 1.0 ATP, 8 dextrose, 0.1 GTP, 10 HEPES, and 5 EGTA, pH 7.2 (KOH). For whole cell recordings, cell capacitance was canceled electronically, and the series resistance (<10 MOmega ) was compensated for by 75-80%. Data acquisition and analysis were performed using pCLAMP 6.04. Whole cell currents were digitized at 3 kHz and filtered at 1 kHz (-3 dB frequency filter). Standard recording conditions for K+ current were achieved by stepping from a holding potential of -80 to +10 mV for 100 ms every 10 s. Under these recording conditions, both the IKV and A-type K+ current were obtained. Therefore the tracings shown here should reflect both Kv and A-type currents, except that not all of the neurons used here contain the latter current. As a result of this, the current measurements from which mean current densities were derived were made at the end of the test pulse, at which time they reflect only IKV (Kang et al. 1994). In some recordings IKV was measured directly by stepping from holding potential of -40 to +10 mV for 100 ms. Under these recording conditions IA is completely inactivated. Current density was derived by dividing transmembrane current (pA) by membrane capacitance (pF), which was calculated as detailed by us recently (Zhu et al. 1999). The average cell capacitance for neurons used in this study was 33.7 ± 15.5 pF (mean ± SE; n = 34 neurons).

Selective stimulation of neuronal AT2 receptors causes an increase in IKV (Zhu et al. 1998), whereas selective stimulation of neuronal AT1 receptors causes a decrease in neuronal IKV (Zhu et al. 1999). Some of the neurons in the cultures used here contain both AT1 and AT2 receptors (Gelband et al. 1997). Since the aim of the present studies was to measure AT2 receptor-mediated effects of Ang II on IKV, all electrophysiological recordings were performed in the presence of 1 µM Los to block AT1 receptors. Los did not alter basal IKV.

Extraction of total RNA and reverse transcriptase-polymerase chain reaction (RT-PCR)

Growth media were removed from neuronal cultures that were then washed once with ice-cold Tyrode's solution, pH 7.4. After this, neurons were lysed in TRIzol reagent (0.5 ml/dish), and total RNA was extracted as detailed previously (Huang et al. 1997). The overall procedures for RT-PCR of the AT2 receptor, PLA2, and 12-LO from single neurons were as detailed by us previously (Zhu et al. 1998). In brief, following recordings of IKV, the neuronal intracellular contents were drawn into the tip of the patch pipette using negative pressure, and the tip was broken off inside the RT-PCR tube. The volume of intracellular contents and patch pipette solution in the broken tip was adjusted to 8 µl for the RT-PCR, which was performed using Gene-Amp RT-PCR kits. The RT was performed at 25°C for 10 min, followed by 42°C for 60 min, 99°C for 5 min, and 4°C for 10 min. A first PCR was performed at 94°C for 4 min, followed by 38 cycles at 94°C for 45 s, 55°C for 1 min (for AT2 receptor), or 63°C for 1 min 40 s (for PLA2 and 12-LO), and 72°C for 2 min. Final extension was achieved at 72°C for 10 min and 4°C for 30 min. A second PCR was performed (on 20 µl of the 1st PCR products) at 94°C for 4 min, followed by 36 cycles (for AT2 receptor) or 30 cycles (for PLA2 or 12-LO) at 94°C for 45 s, 55°C for 1 min (for AT2 receptor) or 63°C for 1 min 40 s (for PLA2 or 12-LO), and 72°C for 2 min. After final extension at 72°C for 10 min and 4°C for 30 min, the PCR products were electrophoresed on a 2% agarose gel containing 1 µg/ml ethidium bromide. Using these conditions, we observed the production of a 495 bp AT2 receptor-specific DNA, a 263 bp PLA2-specific DNA and a 295 bp12-LO-specific DNA, which correspond to the AT2 receptor, PLA2, and 12-LO mRNAs, respectively. In all situations, exclusion of either RNA or MuLV reverse transcriptase resulted in no visible bands following gel electrophoresis.

Western blot analysis

Detection of 12-LO protein in control cultured neurons and in rat hypothalamus and brain stem was performed by Western blot analysis using a polyclonal peptide antibody to the leukocyte type of 12-LO (Natarajan et al. 1996). All procedures, including extraction of total cell protein, were as detailed previously (Gelband et al. 1999).

Drug and antibody applications

Ang II and drugs were dissolved in the appropriate solvent, followed by dilution in superfusate solution, patch pipette solution, or DMEM, depending on the route of administration. Intracellular application of anti-12-LO antibodies and 12-(S)-HETE were achieved by injection through the patch pipette as detailed by us previously (Zhu et al. 1999). In brief, a sidearm pipette holder is attached to the head stage of the Axopatch. One side arm is used to supply suction for seal formation, and the second side arm is used to advance a very fine polyethylene catheter (PE-50) down the inside of the patch pipette. Control measurements of K+ current are made 5 min after the whole cell configuration is established in a given neuron. After this, the peptide solution (5 µl) is injected into the tip of the recording electrode via the PE-50 tube. From the pipette tip, the anti-12-LO antibodies are allowed to diffuse into the neuron, and measurements of K+ current are made 4 min later, at which time a stable peak response is obtained. Care is take not to overperfuse the neuron, and this is monitored electrically via the Axopatch and on the TV monitor. Thus the concentrations of anti-12-LO antibodies that are given in the results refer to the amounts that are injected at the pipette tip, and so are likely higher than the amounts that reach the site of action.

Experimental groups and data analysis

Electrophysiological analyses were performed with the use of multiple 35-mm dishes of cultured neurons. Comparisons were made with the use of a one-way ANOVA followed by Newman-Keuls test to assess statistical significance.


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The present study is focused on which particular LO isozyme mediates the stimulation of IKV by Ang II, and whether other AA metabolic pathways (e.g., cytochrome p450 monooxygenase) are involved in this response. Thus the first series of experiments were performed to confirm that the Ang II-induced stimulation of IKV involved AA metabolism. The data presented in Fig. 1 indicate that the stimulation of IKV by Ang II was attenuated by superfusion of 10 µM ETYA, which is an analogue of AA and a general inhibitor of AA metabolism (Collins and Davies 1998; Muller et al. 1998; Schmitt and Meves 1995). Full reversal of this Ang II effect was obtained with subsequent superfusion of 1 µM PD123,319, a selective AT2 receptor blocker. PD123,319 (1 µM) alone did not alter baseline IKV (data not shown), in concert with our previous findings (Kang et al. 1993). Our data also indicate that superfusion of ETYA (10 µM) alone did not alter neuronal IKV (Fig. 1). Thus the data from the first set of experiments confirm our previous findings that the Ang II stimulation of neuronal IKV involves AA metabolism. In the next series of experiments, we examined the roles of LO isozymes (5-LO and 12-LO) in this Ang II response, with the use of selective inhibitors of each isozyme. Treatment of cultured neurons with 10 µM MK 886, a selective inhibitor of 5-LO activating protein (FLAP) (Lammers et al. 1996), had no significant effects on the stimulation of IKV by Ang II (Fig. 2). In contrast, pretreatment of cultures with selective 12-LO inhibitors significantly attenuated the Ang II-induced increase in IKV. For example, inclusion of specific anti-12-LO antibodies (Kim et al. 1995) in the pipette solution reduced the effects of Ang II on IKV (Fig. 3A). Inclusion of normal rabbit IgG in the pipette solution did not alter the stimulation of IKV produced by Ang II (not shown). Similar inhibitory effects were observed following treatment of cultured neurons with the selective 12-LO inhibitors CDC (200 nM; Fig. 3A) (Wen et al. 1996) or baicalein (1-10 µM) (Nadler et al. 1987). In the latter case, baicalein at 1 and 10 µM reduced the stimulatory effects of Ang II on neuronal IKV by 28.2 ± 2.4% and 60 ± 5.2%, respectively (n = 5 neurons in each case). Analysis of IKV under recording conditions in which IA was inactivated produced similar results, i.e., Ang II produced a significant increase in IKV that was attenuated 40-80% by 200 nM CDC (Fig. 3B). The role of 12-LO was further demonstrated by experiments in which the stimulatory action of Ang II on neuronal IKV was partially reversed by acute administration of anti-12-LO antibodies via intracellular application (Fig. 4). Full reversal of the Ang II effect was obtained with subsequent superfusion of 1 µM PD123,319 (Fig. 4). Control recordings of neuronal IKV in the presence of the 5-LO and 12-LO inhibitors (including 12-LO antibodies) were not significantly different from the recordings of neuronal IKV made in untreated neurons (Figs. 2 and 3). In addition, higher concentrations of the 12-LO inhibitors produced no further attenuation of Ang II-modulated IKV (data not shown). These data therefore indicate that 12-LO is involved in the AT2 receptor-mediated increase in neuronal IKV by Ang II. This idea is supported by experiments that indicate that intracellular application of the 12-LO metabolite 12-(S)-HETE (1 µM), the receptors for which are primarily intracellular (Herbertsson et al. 1999), elicits a significant increase in neuronal IKV (Fig. 5). By contrast, superfusion of either of the 5-LO metabolites leukotriene B4 (LTB4; 10 nM) or leukotriene C4 (LTC4; 1 µM), the receptors for which are G-protein coupled sites located on the plasma membrane (Gaudreau et al. 1998; Geirsson et al. 1998), produced no significant changes in neuronal IKV (Fig. 5). To further establish a role for 12-LO metabolites of AA in the Ang II-induced increase in neuronal IKV, cultured neurons were superfused with AA (50 µM) in the absence or presence of 5-LO or 12-LO inhibitors. The data presented in Fig. 6 indicate that superfusion of AA elicits a significant increase in neuronal IKV, an effect that is attenuated by pretreatment of cells with the 12-LO inhibitor baicalein (1 µM), but that is not altered by 5-LO activating protein inhibitor MK 886 (10 µM). Collectively, the present data and our previous studies (Zhu et al. 1998) indicate that the AT2 receptor-mediated stimulation of neuronal IKV involves a pathway that includes activation of PLA2 and of 12-LO. To further establish these signaling molecules as mediators of the Ang II-induced increase in neuronal IKV, it is necessary to demonstrate their presence within the Ang II-responsive neurons. The Western immunoblot data presented in Fig. 7 (top) demonstrate the presence of a 72-kDa 12-LO in cultured neurons, similar to that found in rat hypothalamus and brain stem and reported previously (Watanabe et al. 1993). Single-cell RT-PCR studies indicate that AT2 receptor, PLA2, and 12-LO mRNAs are present within a single neuron that had responded to Ang II with an increase in IKV (Fig. 7, bottom). Thus the immunoblot and RT-PCR data provide strong support for a role of PLA2 and 12-LO in the Ang II-induced stimulation of neuronal IKV.



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Fig. 1. Stimulation of neuronal voltage-dependent delayed rectifier K+ current (IKV) by angiotensin II (Ang II) is attenuated by 5, 8, 11, 14-eicosatetraynoic acid (ETYA). K+ was recorded during 100-ms voltage steps from a holding potential of -80 to +10 mV. A: representative time course showing the effects of superfusion of 10 µM ETYA on the stimulation of IKV produced by superfusion of 100 nM Ang II. Once the effects of ETYA had stabilized, 1 µM PD123,319 was added to the superfusate. B: bar graphs are means ± SE of IKV current densities recorded during the application of Ang II, ETYA, and PD123,319. Data are from 5 neurons. * P < 0.05 compared with control; ** P < 0.05 compared with Ang II alone. C: effects of superfused ETYA (10 µM) on K+ current in untreated neurons. Control (CON) recordings were made before the application of ETYA. Bar graphs show means ± SE of IKV current densities obtained in each treatment situation. Data are from 5 neurons.



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Fig. 2. Stimulation of neuronal IKV by Ang II is not altered by MK 886. K+ current was recorded as described in Fig. 1. Top: from left to right, representative current tracings showing the effects of superfused Ang II on K+ current in untreated neurons, or in neurons pretreated with the selective 5-lipoxygenase (5-LO) inhibitor MK 886 (10 µM, added to the DMEM and to the superfusate). Control (CON) recordings were made before the application of Ang II, and all recordings were made in the presence of 1 µM Los to block AT1 receptors. Bottom: bar graphs showing the means ± SE of IKV current densities obtained in both treatment situations. Sample sizes were 13 and 4 neurons for the untreated and MK 886 groups, respectively. * P < 0.05 compared with the respective control.



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Fig. 3. Ang II-induced stimulation of neuronal IKV: attenuation by selective inhibition of 12-LO. A: treatment of neurons with the selective 12-LO inhibitors attenuates the stimulation of IKV produced by Ang II. K+ current was recorded as described in Fig. 1. Top: from left to right are representative current tracings showing the effects of superfused Ang II (100 nM) on K+ current in untreated neurons or in neurons treated with the 12-LO inhibitor cinnamyl-3,4-dihydroxy-alpha -cyanocinnamate (CDC; 200 nM for 30 min and added to superfusate) or with anti-12-LO antibodies (1:1,000 dilution into pipette solution). Control (CON) recordings were made before the application of Ang II, and all recordings were made in the presence of 1 µM Los to block AT1 receptors. Bottom: bar graphs showing means ± SE of IKV current densities obtained in the above treatment situations and in untreated neurons. Sample sizes were 17, 4, and 7 neurons for the untreated CDC and anti-12-LO groups, respectively. * P < 0.05 compared with respective control treatment. ** P < 0.05 compared with Ang II in untreated group. B: treatment of neurons with the selective 12-LO inhibitor CDC reduces the increase of the IKV elicited by Ang II. K+ current was recorded during 100-ms voltage steps from a holding potential of -40 to +10 mV. Top: from left to right are representative current tracings showing the effects of superfused Ang II (100 nM) or K+ current in untreated neurons or in neurons treated with CDC as in A. Control (CON) recordings were made before the application of Ang II, and all recordings were made in the presence of Los. Bottom: bar graph showing means ± SE of IKV current densities obtained under these treatment and recording conditions. Sample sizes were 3 and 4 neurons for the untreated and CDC groups, respectively. * P < 0.05 compared with the respective control treatment.



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Fig. 4. Effects of acute treatment with anti-12-LO antibodies on Ang II-stimulated increases in IKV. K+ current was recorded as described in Fig. 1. Top: representative time course showing the effects of intracellular application of anti-12-LO antibodies (1:1,000) on the stimulation of neuronal IKV produced by superfusion of 100 nM Ang II. Once the effects of a 12-LO had stabilized, 1 µM PD123,319 was added to the superfusate. Bottom: bar graphs are means ± SE of IKV current densities recorded during the application of Ang II, 12-LO, and PD123,319. Data are from 3 neurons. * P < 0.05 compared with control.



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Fig. 5. Effects of LO metabolites of arachidonic acid (AA) on neuronal IKV. K+ current was recorded as in Fig. 1. Bar graphs show the means ± SE of IKV current densities recorded as a percent of control IKV. Control (CON) recordings were made before the application of LO metabolites following superfusion of LTB4 (10 nM), LTC4 (1 µM), or intracellular application of 12-(S)-HETE (1 µM). Sample sizes were 7, 3, and 8 neurons for the LTB4, LTC4, and 12 (S) HETE groups, respectively. * P < 0.05 compared with respective control.



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Fig. 6. Stimulation of neuronal IKV by AA is attenuated by baicalein. K+ current was recorded as in Fig. 1. Neurons were superfused with control solution (CON; superfusate) or AA (50 µM) in the absence (untreated) or presence of MK 886 (10 µM added to the DMEM for 30 min, and to the superfusate) or baicalein (1 µM added to the DMEM for 20 h, and to the superfusate). Bar graphs are means ± SE of IKV current densities obtained in each treatment situation. Sample sizes were 5, 5, and 6 neurons for the untreated, MK886, and baicalein groups, respectively. * P < 0.05 compared with respective controls. ** P < 0.05 compared with AA alone (no baicalein).



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Fig. 7. Localization of 12-LO, PLA2, and AT2 receptors within cultured neurons. Top: representative Western immunoblot showing the presence of 12-LO within cultured neurons. Data were obtained using a polyclonal anti-12-LO antibody as detailed in METHODS. Lane 1, 12-LO standard (5 µg); lane 2, blank; lane 3, cultured neurons; lane 4, cultured astroglia; lane 5, rat hypothalamus and brain stem. Bottom: presence of 12-LO, PLA2, and AT2 receptor mRNAs within the Ang II-responsive neurons. Left: current tracing from a representative neuron showing the increase in K+ current (AT2 receptor mediated) produced by Ang II (100 nM) in the presence of 1 µM Los. K+ current was recorded as in Fig. 1. After this recording the neuron was prepared for single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) as detailed in METHODS. Right: ethidium bromide-stained gel showing RT-PCR DNA products that correspond to the 12-LO, PLA2, and AT2 receptor mRNAs. Leftmost lane is 100 bp ladder. Lanes 1, 2, and 3 are the AT2 receptor, PLA2, and 12-LO mRNAs, respectively, from the responsive neuron shown at left.

Since activation of 12-LO is only partially responsible for the stimulation of IKV by Ang II, and COX is not involved in this Ang II action (Zhu et al. 1998), in further experiments we investigated the involvement of p450 monooxygenases. Treatment of cultured neurons with the selective cytochrome p450 monooxygenase inhibitor 17-ODYA (10 µM) did not alter the increase in neuronal IKV produced by either Ang II or AA (Fig. 8).



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Fig. 8. Stimulation of neuronal IKV by Ang II or AA is not altered by 17-ODYA. K+ current was recorded as described in Fig. 1. Neurons were superfused with control solution (superfusate; CON), Ang II (100 nM), or AA (50 µM) in the absence (untreated) or presence of the selective cytochrome p450 monooxygenase inhibitor 17-ODYA (10 µM, added to DMEM and to the superfusate). Top: representative current tracings showing the effects of Ang II and AA in each treatment situation. CON recordings were made before the application of Ang II or AA, and Ang II experiments were performed in the presence of 1 µM Los to block AT1 receptors. Bottom: bar graphs showing means ± SE IKV of current densities obtained in each treatment situation. Sample sizes were 14 and 5 neurons for the untreated and 17-ODYA groups (Ang II superfusions), respectively. Sample sizes were 5 and 5 neurons for the untreated and 17-ODYA groups (AA superfusions), respectively. * P < 0.05 compared with the respective control.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The physiological and behavioral effects that are stimulated by Ang II binding to its neuronal AT2 receptors in brain involve modulation of membrane ionic currents and firing rate. We have utilized neurons cultured from newborn rat brain stem and hypothalamus to investigate the AT2 receptor-mediated actions of Ang II on neuronal activity and on the underlying changes in membrane ionic currents. Our studies indicate that stimulation of neuronal AT2 receptors elicits an increase in neuronal firing rate (Zhu et al. 2000) via shortening of action potential duration, and this is consistent with our previous studies that indicate that Ang II increases both neuronal Kv and A-type K+ current (Kang et al. 1993). We have examined the intracellular mechanisms through which Ang II stimulates an increase in neuronal IKV. Our data indicate that Ang II elicits an AT2 receptor-mediated increase in PLA2 activity and generation of AA, and that this mechanism is mostly responsible for the observed increase in neuronal IKV (Zhu et al. 1998). Since AA and AA metabolites are able to modulate the activity of K+ channels (Arima et al. 1997; Colbert and Pan 1999; Duerson et al. 1996; Harder et al. 1997; Premkumar et al. 1990; Schweitzer et al. 1990; Twitchell et al. 1997; Zona et al. 1993), IKV, the stimulation of neuronal IKV by Ang II potentially involved these intracellular factors. Previous studies from our group gave an indication that this effect of Ang II involved LO, but not COX, metabolites of AA (Zhu et al. 1998). Results from the present study have confirmed this and have shown that the increase in neuronal IKV produced by Ang II is partially mediated through generation of 12-LO metabolites of AA. Furthermore, it appears that 5-LO metabolites and p450 monooxygenase metabolites of AA are not involved in this response. However, it should be emphasized that the 12-LO metabolic pathway is, according to our data, only partially responsible for this Ang II action. This leaves open the possibility that other pathways are involved in the Ang II effect on IKV. An obvious candidate is AA itself, since it has been shown to directly modulate K+ channels. It is also possible that other intracellular mechanisms separate from the PLA2/AA pathway are involved. For example, we cannot exclude the possibility that the Ang II-induced stimulation of neuronal IKV also involves direct membrane-delimited coupling of the AT2 receptor/Gi complex to the specific delayed rectifier K+ channel that is involved. A further possibility is that an alternate intracellular signaling pathway may be involved. For example, it has been determined that stimulation of AT2 receptors in PC12W pheochromocytoma cells increases the synthesis of the sphingolipid ceramide (Gallinat et al. 1999; Lehtonen et al. 1999). Considering that ceramide increases IKV in cortical cultured neurons (Yu et al. 1999), it is possible that the Ang II-induced increase in IKV seen in the present study also involves this sphingolipid. However, our preliminary studies indicate that short term (1-30 min) incubation of cultured neurons with Ang II does not alter cellular ceramide levels, nor does ceramide application alter neuronal IKV (E. M. Richards, M. Zhu, and C. Sumners, unpublished data). The identity of the other signaling component(s) that is involved in the stimulation of IKV by Ang II, in particular the possible direct action of AA at the Kv channel, will be the subject of further investigation once the identity of the channel that is involved has been ascertained.

The present studies have raised a number of interesting questions concerning the mechanisms by which 12-LO metabolites of AA might alter IKV. For example, in previous studies we determined that the Ang II- and AA-induced stimulations of neuronal IKV are completely abolished by selective inhibition of serine/threonine phosphatase type 2A (PP-2A) (Kang et al. 1995; Zhu et al. 1998). This data, along with the observation that the Ang II stimulation of PLA2 in cultured neurons is not altered by PP-2A inhibitors (Zhu et al. 1998), suggests that PP-2A is downstream of the PLA2/AA/12 HETE pathway. It is well known that K+ channels can be modulated by phosphorylation and dephosphorylation of amino acid residues within the subunit proteins (Breitwieser 1996; Levitan 1999). Thus one possibility is that the 12-LO metabolites of AA or AA itself influence IKV via activation of PP-2A, which in turn modulate K+ channel activity. There is certainly a precedent for this since AA has been shown to stimulate the activity of PP-5, a novel serine/threonine phosphatase from brain (Skinner et al. 1997). A further possibility is that the 12-LO metabolites of AA modulate the K+ channel subunits directly, and that PP-2A has a permissive role over channel activation/inactivation. The actual mechanism of AA/12-HETE/PP-2A modulation of IKV will only be deciphered once certain fundamental questions have been answered. For example, do 12-HETE and/or AA stimulate PP-2A activity in cultured neurons? How does PP-2A modulate IKV: directly via dephosphorylation of channel subunits, or indirectly via activation of a kinase?

A final point concerns the fact that similar intracellular signaling pathways have been proposed for the modulation of K+ currents by calcium and somatostatin in neurons and pituitary tumor cells, respectively (Duerson et al. 1996; Yu 1995). Thus it is possible that a common series of intracellular events (PLA2/AA/12-LO metabolites of AA/PP-2A) are responsible for the modulation of K+ currents by different agents in different cell types. This commonality of signaling pathways might help to explain the fact that the AT2 receptor knockout mice did not exhibit more dramatic changes in physiological or behavioral responses; i.e., perhaps other factors that utilize the same intracellular signaling mechanisms were able to compensate for the lack of Ang II stimulation of AT2 receptors in these animals.


    ACKNOWLEDGMENTS

This work was supported by National Institutes of Health Grants HL-49130 (C. Sumners and C. H. Gelband) and DK-39721 (J. L. Nadler and R. Natarajan).


    FOOTNOTES

Address for reprint requests: C. Sumners, Dept. of Physiology, College of Medicine, University of Florida, Box 100274, Gainesville, FL 32610 (E-mail: csumners{at}phys.med.ufl.edu).

Received 4 February 2000; accepted in final form 28 July 2000.


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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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