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Angiotensin II Type 2 Receptor Stimulation of Neuronal Delayed- Rectifier Potassium Current Involves Phospholipase A2 and Arachidonic Acid

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Angiotensin II Type 2 Receptor Stimulation of Neuronal Delayed- Rectifier Potassium Current Involves Phospholipase A2 and Arachidonic Acid The Journal of Neuroscience, January 15, 1998, 18(2):679–686 Angiotensin II Type 2 Receptor Stimulation of Neuronal Delayed- Rectifier Potassium Current Involves Phospholipase A2 and Arachidonic Acid Mingyan Zhu, Craig H. Gelband, Jennifer M. Moore, Philip Posner, and Colin Sumners Department of Physiology, University of Florida, College of Medicine, Gainesville, Florida 32610 Angiotensin II (Ang II) elicits an Ang II type 2 (AT2 ) receptor- PLA2 and was mimicked by application of AA to neurons. 1 mediated increase in delayed-rectifier K current (IK ) in neu- Inhibition of lipoxygenase (LO) enzymes significantly reduced rons cultured from newborn rat hypothalamus and brain- both Ang II- and AA-stimulated IK , and the 12-LO metabolite stem. This effect involves a pertussis toxin (PTX)-sensitive Gi of AA 12S-hydroxyeicosatetraenoic acid (12S-HETE) stimu- protein and is abolished by inhibition of serine and threonine lated IK. These data indicate the involvement of a PLA2 , AA, phosphatase 2A (PP-2A). Here, we determined that Ang II and LO metabolite intracellular pathway in the AT2 receptor- 3 stimulates [ H]arachidonic acid (AA) release from cultured mediated stimulation of neuronal IK by Ang II. Furthermore, neurons via AT2 receptors. This effect of Ang II was blocked the demonstration that inhibition of PP-2A abolished the by inhibition of phospholipase A2 (PLA2 ) and by PTX. Be- stimulatory effects of Ang II, AA, and 12S-HETE on neuronal 3 cause AA and its metabolites are powerful modulators of IK but did not alter Ang II-stimulated [ H]-AA release sug- neuronal K 1 currents, we investigated the involvement of gests that PP-2A is a distal event in this pathway. PLA2 and AA in the AT2 receptor-mediated stimulation of IK 1 by Ang II. Single-cell reverse transcriptase (RT)-PCR analy- Key words: angiotensin II; AT2 receptor; delayed-rectifier K ses revealed the presence of PLA2 mRNA in neurons that current; phospholipase A2 ; arachidonic acid; lipoxygenase; responded to Ang II with an increase in IK. The stimulation of serine and threonine phosphatase 2A; hypothalamus and brain- neuronal IK by Ang II was attenuated by selective inhibitors of stem neurons Mammalian brain contains angiotensin II (Ang II) type 2 Similar to the in vivo situation, neurons cultured from the (AT2 ) receptors (Tsutsumi and Saavedra, 1991; Millan et al., hypothalamus and brainstem of newborn rats contain high levels 1992; Song et al., 1992), the functions of which are not well of AT2 receptors (Sumners et al., 1991), and we have used these established. The fact that these sites are expressed in high cultures to determine the AT2 receptor-mediated effects of Ang II 1 levels in neonatal tissues has led to the idea that they have a on membrane K currents. The rationale for this approach was role in development (Cook et al., 1991; Tsutsumi and Saavedra, that changes in these currents form the basis of changes in 1991; Millan et al., 1992). Support for this has come from neuronal activity and ultimately of behavioral and physiological studies that determined that stimulation of AT2 receptors effects. We determined that Ang II, via AT2 receptors, stimulates 1 1 causes neurite outgrowth in undifferentiated NG108–15 neu- neuronal delayed-rectifier K current (IK ) and transient K 3 roblastoma glioma cells (Laflamme et al., 1996) and causes current (IA ) (Kang et al., 1993), effects mediated by an inhibitory apoptosis of pheochromocytoma PC-12W cells and R3T3 fi- G-protein (Gi ) and abolished by inhibition of serine and threo- broblasts (Yamada et al., 1996; Horiuchi et al., 1997). Within nine phosphatase 2A (PP-2A) (Kang et al., 1994). Our present the brain, blockade of periventricular AT2 receptors potenti- investigations have centered around the possibility that phospho- ates the Ang II type 1 (AT1 ) receptor-mediated stimulation of lipase A2 (PLA2 ) is involved, because Ang II, via AT2 receptors, drinking and vasopressin secretion (Hohle et al., 1995), sug- causes stimulation of PLA2 activity in certain cell types (Lokuta gesting that AT1 and AT2 receptors may be antagonistic. In et al., 1994; Jacobs and Douglas, 1996). PLA2 catalyzes the addition, mutant mice lacking the gene encoding the AT2 generation of arachidonic acid (AA) from membrane phospho- receptor displayed decreased exploratory behavior and spon- lipids, and AA and its metabolites such as hydroxyeicosatetra- taneous movements compared with wild-type mice (Hein et al., enoic acid (HETE), leukotrienes, and prostaglandins are known 1 1995; Ichiki et al., 1995). Thus, AT2 receptors may be involved modulators of neuronal K channels (Premkumar et al., 1990; in the central control of certain behaviors and hormone secre- Schweitzer et al., 1993; Zona et al., 1993; Meves, 1994; Gubitosi- tion, as well as having putative roles in apoptosis and Klug et al., 1995; Kim et al., 1995). Furthermore, modulation of 1 differentiation. K currents in sympathetic neurons and pituitary tumor cells involves lipoxygenase (LO) metabolites of AA and serine and Received Aug. 21, 1997; revised Oct. 27, 1997; accepted Nov. 4, 1997. threonine phosphatases (Duerson et al., 1996; Yu, 1995). The This work was supported by National Institutes of Health Grants NS19441, data presented here indicate that the AT2 receptor-mediated HL49130, and HL52189. We thank Jennifer Brock for typing this manuscript. stimulation of neuronal IK by Ang II involves an intracellular Correspondence should be addressed to Dr. Colin Sumners, Department of Physiology, University of Florida, Box 100274, 1600 Southwest Archer Road, pathway that includes PLA2 , AA, and LO metabolites of AA. In Gainesville, FL 32610. addition, the results suggest that PP-2A may be important for the Copyright © 1998 Society for Neuroscience 0270-6474/98/180679-08$05.00/0 stimulation of neuronal IK produced by Ang II and AA. • 680 J. Neurosci., January 15, 1998, 18(2):679–686 Zhu et al. AT2 Receptors and Neuronal Potassium Currents MATERIALS AND METHODS chloroform and methanol extraction, followed by isolation of the [ 3H]- LPE using TLC (chloroform:methanol:glacial acetic acid:water; 50:30:8: Materials. Newborn Sprague Dawley rats were obtained from our 3 breeding colony, which originated from Charles River Laboratories 3). Spots corresponding to [ H]-LPE were removed, and the data were expressed as dpm [ 3H]-LPE/well. (Wilmington, MA). DMEM and TRIzol reagent were obtained from 1 GIBCO-BRL (Gaithersburg, MD). Plasma-derived horse serum Electrophysiological recordings. Macroscopic K current was recorded (PDHS) was from Central Biomedia (Irwin, MO). [5,6,8,9,11,12,14,15- using the whole-cell configuration of the patch-clamp technique as de- 3H]Arachidonic acid ([ 3H]-AA; specific activity of 203 Ci/mmol) and scribed previously (Hamill et al., 1981; Kang et al., 1994). Experiments [1-3H]ethan-1-ol-2-amine HCL ([ 3H]ethanolamine; specific activity were performed at room temperature (22–23°C) using an Axopatch-1D of 18.0 Ci/mmol) were purchased from Amersham (Arlington Heights, amplifier and Digidata 1200A interface (Axon Instruments, Burlingame, IL). Losartan potassium was generously provided by Merck (Darmstadt, CA). Neurons were bathed in modified Tyrode’s solution containing (in Germany). PD123,319, nodularin (NDL), and antiflammin-2 were pur- mM): 140 NaCl, 5.4 KCl, 2.0 CaCl2 , 2.0 MgCl2 , 0.3 NaH2PO4 ,10 chased from Research Biochemicals (Natick, MA). AA was from Cal- HEPES, 0.0001 TTX, 0.1 CdCl2 , and 10 dextrose, pH 7.4 (NaOH). The patch electrodes had resistances of 3–4 MV when filled with an internal biochem (La Jolla, CA). Polyclonal anti-phospholipase A2 (anti-PLA2 ) antibodies were purchased from Upstate Biotechnology (Lake pipette solution containing (in mM): 140 KCl, 2 MgCl2 , 5 EGTA, 4 ATP, Placid, NY). Activated silicic acid (200–325 mesh; Unisil) was from 0.1 GTP, 10 dextrose, and 10 HEPES, pH 7.2 (KOH). For whole-cell Clarkson Chemical (Williamsport, PA). Gene-Amp reverse transcriptase recordings, cell capacitance was canceled electronically, and the series (RT)-PCR kits and all reagents for RT-PCR were purchased from resistance (,10 MV) was compensated for by 75–80%. Data acquisition Perkin-Elmer (Norwalk, CT). Bovine serum albumin (BSA), Ang II, and analysis were performed using pCLAMP 6.03. Whole-cell currents tetrodotoxin (TTX), dipotassium ATP, sodium GTP, pertussis toxin were digitized at 3 kHz and filtered at 1 kHz (23 dB frequency filter). (PTX), cadmium chloride (CdCl2 ), HEPES, phosphatidylcholine (PC), The current measurements from which mean current densities were phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidyl- derived were made 50 msec after the initiation of the test pulse, at which inositol (PI), 4-bromophenacyl bromide (4-BPB), nordihydroguiaretic time the current measurements reflect only IK (Kang et al., 1994). acid (NDGA), indomethacin, and 12S-hydroxy-(5Z,8Z,10E,14Z)- Current density was reported as pA/pF. The average cell capacitance for eicosatetraenoic acid (12S-HETE) were purchased from Sigma (St. neurons used in this study was 33.9 6 13.7 pF. Louis, MO). All other chemicals were purchased from Fisher Scientific Extraction of total RNA and RT-PCR. Growth media were removed from (Houston, TX) and were of analytical grade or higher. Oligonucleotide cultured neurons that were then washed once with ice-cold Tyrode’s solu- primers for the rat brain cytosolic PLA2 gene (Owada et al., 1994) and tion, pH 7.4. After this, neurons were lysed in TRIzol reagent (0.5 ml/dish), the rat AT2 receptor gene (Mukoyama et al., 1993) were synthesized in and total RNA was extracted as detailed previously (Huang et al., 1997). the DNA core facility of the Interdisciplinary Center for Biotechnology For the experiments using neurons from the whole dish, RT-PCR of the Research, University of Florida. The sequences of these primers AT receptor and PLA was performed using Gene-Amp RT-PCR kits 9 2 2 are as follows: PLA2 gene, sense: 5 -GCTCCACATGGTACATGTCA- essentially as described by Huang et al.
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