ANG II-mediated inhibition of neuronal delayed rectifier K+ current: role of protein kinase C-alpha

Sheng-Jun Pan, Mingyan Zhu, Mohan K. Raizada, Colin Sumners, and Craig H. Gelband

Department of Physiology, College of Medicine, and McKnight Brain Institute, University of Florida, Gainesville, Florida 32610


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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It was previously determined that ANG II and phorbol esters inhibit Kv current in neurons cultured from newborn rat hypothalamus and brain stem in a protein kinase C (PKC)- and Ca2+-dependent manner. Here, we have further defined this signaling pathway by investigating the roles of "physiological" activators of PKC and different PKC isozymes. The cell-permeable PKC activators, diacylglycerol (DAG) analogs 1,2-dioctanoyl-sn-glycerol (1 µmol/l, n = 7) and 1-oleoyl-2-acetyl-sn-glycerol (1 µmol/l, n = 6), mimicked the effect of ANG II and inhibited Kv current. These effects were abolished by the PKC inhibitor chelerythrine (1 µmol/l, n = 5) or by chelation of internal Ca2+ (n = 8). PKC antisense (AS) oligodeoxynucleotides (2 µmol/l) against Ca2+-dependent PKC isoforms were applied to the neurons to manipulate the endogenous levels of PKC. PKC-alpha -AS (n = 4) treatment abolished the inhibitory effects of ANG II and 1-oleoyl-2-acetyl-sn-glycerol on Kv current, whereas PKC-beta -AS (n = 4) and PKC-gamma -AS (n = 4) did not. These results suggest that the angiotensin type 1 receptor-mediated effects of ANG II on neuronal Kv current involve activation of PKC-alpha .

antisense; calcium; angiotensin type 1 receptor


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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MAMMALIAN BRAIN CONTAINS SPECIFIC angiotensin type 1 (AT1) receptors that are localized mainly in specific areas within the hypothalamus and brain stem (12, 27). Some of these areas lie outside the blood-brain barrier and sense circulating ANG II. ANG II acts at these receptors located on neurons to stimulate increases in blood pressure, arginine vasopressin release, salt appetite, and drinking behavior, effects that participate in the modulatory role of this peptide on extracellular fluid volume and cardiovascular hemodynamics (26, 33). In accordance with these physiological studies, it has been determined that ANG II elicits AT1 receptor-mediated increases in neuronal firing rate in the paraventricular nucleus, the subfornical organ, the supraoptic nucleus, and the rostral ventrolateral medulla, brain areas that are involved in cardiovascular regulation (2, 19, 29, 38). Changes in neuronal activity are governed by alterations in membrane currents through direct receptor-ion channel interactions or through receptor-mediated changes in intracellular messengers. Despite these well-documented physiological actions of ANG II in the brain, the mechanisms through which this peptide alters neuronal activity are not well established. An understanding of these mechanisms is crucial, since changes in neuronal activity induced by ANG II will ultimately lead to the above alterations in cardiovascular hemodynamics induced by this peptide.

Our group has utilized cultured neurons prepared from the hypothalamus and brain stem of newborn rats to investigate the mechanisms of AT1 receptor-mediated changes in neuronal activity and the intracellular signaling molecules that are involved (10, 29, 33, 35, 38, 39, 42). We have determined that activation of AT1 receptors in cultured neurons increases neuronal firing rate and that this involves inhibition of neuronal delayed rectifier K+ (Kv) current and transient (A-type) K+ currents. The inhibitory effects of ANG II on Kv current involve a signaling cascade revolving around Galpha q/11 protein, stimulation of phosphoinositide hydrolysis, an increase in intracellular free Ca2+ concentration, activation of protein kinase C (PKC), and modulation of Kv2.2. The major goals of the present study were to investigate the role of diacylglycerol (DAG) and determine which PKC isozyme is involved in this signaling pathway. This is important, because ANG II exerts a variety of AT1 receptor-mediated effects in neurons, many of which involve activation of PKC (29). For example, aside from the inhibition of Kv current, ANG II increases the phosphorylation of myristilated alanine-rich C kinase substrate protein via PKC-beta (20) and stimulates Fos-regulating kinase (13). Thus it is possible that these actions of ANG II involved different PKC isozymes, allowing this peptide to have differential effects on specific cellular responses. Furthermore, it is well known that the activity of K+ channels can be modulated by phosphorylation or dephosphorylation (17). One of our future goals is to determine whether ANG II elicits inhibition of Kv current via PKC-mediated phosphorylation of Kv2.2; to accomplish this goal, an understanding of which isozyme of PKC is involved is essential.

The data presented here show that DAG as well as the Ca2+-dependent PKC-alpha are involved in the regulation of neuronal Kv current, changes that will ultimately lead to altered cardiovascular function. This is the first demonstration of a specific PKC isozyme that is responsible for modulating ANG II-induced changes in neuronal Kv current. The identification of PKC-alpha as a key intermediate will allow for more discrete analysis of the role of this serine/threonine kinase in the modulation of Kv channel activity.


    METHODS
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INTRODUCTION
METHODS
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Materials. Newborn Sprague-Dawley rats were obtained from our breeding colony, which originated from Charles River Farms (Wilmington, MA). DMEM was obtained from GIBCO-BRL (Gaithersburg, MD). Losartan potassium was generously provided by William Henckler (Merck, Rahway, NJ). PD-123319 and calphostin were purchased from Research Biochemicals International (Natick, MA). Tetrodotoxin was purchased from Calbiochem (La Jolla, CA). U-73122, plasma-derived horse serum, ANG II, sodium GTP, HEPES, CdCl2, BSA, dipotassium ATP, and peroxidase-conjugated affinity-purified goat anti-rabbit IgG were purchased from Sigma Chemical (St. Louis, MO). PKC antibodies were purchased from Santa Cruz Biochemicals. All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA) and were of analytic grade or higher.

Preparation of cultured neurons. Neuronal cocultures were prepared from the hypothalamus and brain stem of newborn Sprague-Dawley rats exactly as described previously (33). Cultures consist of 90% neurons and 10% astrocyte glia and microglia.

For the PKC antisense (AS) oligodeoxynucleotide experiments, PKC-alpha -AS, -beta -AS, and -gamma -AS and PKC-alpha sense (S) were dissolved in water to an initial concentration of 200 µM, as previously described by our research group (20). For each transfection, 10 µl of AS solution combined with 5 µl of Lipofectin reagent were added to the culture dish. Cells were incubated at 37°C in a CO2 incubator for 24-72 h before use. During this time, the medium was not changed, nor was the AS replenished at any time. AS and S oligonucleotides for the PKC-alpha , -beta , and -gamma genes (23) were synthesized in the DNA core facility of the Interdisciplinary Center for Biotechnology Research, University of Florida. The sequences of these primers are as follows
PKC-&agr;-S: 5′-GAACCATGGCTGACGTTTACC-3′

PKC-&agr;-AS: 5′-GGTAAACGTCAGCCATGGTTC-3′

PKC-&bgr;-AS: 5′-CGCAGCCGGGTGACCCGGCCGC-3′

PKC-&ggr;-AS: 5′-GCGGCCGGGTCAGCCATCTTG-3′

Electrophysiological recordings. Kv current was recorded using the whole cell patch-clamp technique (9). The superfusate solution contained (in mmol/l) 140 NaCl, 5.4 KCl, 2.0 CaCl2, 2.0 MgCl2, 0.3 NaH2PO4, 0.001 tetrodotoxin, 0.3 CdCl2, 0.001 PD-123319 (angiotensin type 2 receptor antagonist), 10 HEPES, and 10 dextrose, with pH adjusted to 7.4 with NaOH. The recording electrode had resistances of 2-4 MOmega when filled with an internal pipette solution containing (in mmol/l) 140 KCl, 2 MgCl2, 4 ATP, 0.1 GTP, 10 dextrose, and 10 HEPES, with pH adjusted to 7.2 with KOH.

Kv current was recorded by stepping from a holding potential of -80 to +10 mV for 80 ms every 10 s. Under these recording conditions, Kv current and a transient Kv (A-type) current were recorded. However, because of normal cell-to-cell variation in the cultures, some neurons contain Kv and A-type K+ currents while other cells may have only one type of Kv current. For this reason, the current measurements from which mean current densities were derived were made 50 ms after the initiation of the test pulse, at which time they reflect only Kv current (9). Current density is reported as picoamperes per picofaraday.

Values are means ± SE. Statistical significance was evaluated with the use of a paired t-test and ANOVA. Differences were considered significant at P < 0.05.

Analysis of PKC-AS effectiveness. The presence of Ca2+-dependent PKC subunit isoforms was determined by Western blot analysis exactly as described previously (14, 42). Protein was extracted from triplicate culture dishes for each experimental data point. All lanes were loaded with the same amount of protein (20 µg).


    RESULTS
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INTRODUCTION
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Role of phospholipase C in ANG II-mediated inhibition of neuronal Kv current. Although it has been previously shown that ANG II-mediated inhibition of neuronal Kv current was coupled to the AT1 receptor and activation of Galpha q/11 (35), the role of phospholipase C (PLC) has yet to be determined. In all the experiments presented, Kv current was recorded in the presence of PD-123319 (1 µmol/l), a selective blocker of angiotensin type 2 receptors. Under these recording conditions, ANG II (100 nmol/l) elicited a significant inhibitory effect on Kv current (Fig. 1). The effect of ANG II was completely reversed by superfusion of the AT1 receptor antagonist losartan (1 µmol/l; Fig. 2A; see Fig. 5). U-73122 has recently been shown to be a specific PLC inhibitor (32). When the neurons were pretreated with the PLC inhibitor U-73122 (1 µmol/l) for 15 min, the effect of ANG II was abolished. Therefore, these data show that PLC is involved in the AT1 receptor-dependent inhibition of Kv current.


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Fig. 1.   Effects of U-73122 on ANG II-mediated inhibition of neuronal Kv current. A: representative current traces (top) and mean data (bottom, n = 6) showing that U-73122 (1 µmol/l) blocks ANG II (100 nmol/l)-mediated inhibition of neuronal Kv current (voltage step -80 to +10 mV). Con, control. *P < 0.05.



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Fig. 2.   Effects of ANG II, 1-oleoyl-2-acetyl-sn-glycerol (OAG), 1,2-dioctanoyl-sn-glycerol (1,2-diC8), and 1,3-dioctanoylglycerol (1,3-diC8) on neuronal Kv current. A: representative current traces (top) and time course (bottom) showing effects of ANG II (100 nmol/l) and losartan (Los, 1 µmol/l) on Kv current (voltage step -80 to +10 mV). B: representative current traces and time course showing effects of OAG (1 µmol/l) on Kv current. C: representative current traces and time course showing effects of 1,2-diC8 (1 µmol/l) on Kv current. D: representative current traces and time course showing effects of 1,3-diC8 (10 µmol/l) on Kv current.

Effects of DAG analogs and intracellular Ca2+ on neuronal Kv current. It was demonstrated in our previous studies that the AT1 receptor-mediated inhibition of neuronal Kv current involves a signaling cascade that requires changes in intracellular free Ca2+ concentration and activation of PKC (35). However, there are no data that show that the inhibition of neuronal K+ current is via activation of DAG and/or a specific PKC isoform. Cell-permeable DAG analogs 1,2-dioctanoyl-sn-glycerol (1 µmol/l) and 1-oleoyl-2-acetyl-sn-glycerol (OAG, 1 µmol/l) were used to examine whether these agents produce effects similar to ANG II. Superfusion of 1,2-dioctanoyl-sn-glycerol (1 µmol/l, n = 7) or OAG (1 µmol/l, n = 6) inhibited Kv current (Fig. 2, B and C; see Fig. 5). Complete current-voltage relationships for the effects of ANG II or OAG on Kv current are shown in Fig. 3. The inactive DAG analog 1,3-dioctanoylglycerol (10 µmol/l, n = 5) was without effect (Fig. 2D; see Fig. 5). Because DAG activates PKC, we tested the effects of OAG on Kv current in the presence of the PKC blocker chelerythrine. Pretreatment of neurons with chelerythrine (1 µmol/l) for 20 min completely abolished the effect of OAG on Kv current (Figs. 4A and 5). Finally, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (10 mmol/l), a Ca2+ chelating agent, predissolved in pipette solution, also abolished the effect of OAG on Kv current, indicating the involvement of Ca2+ and/or a Ca2+-dependent PKC isoform (Figs. 4B and 5).


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Fig. 3.   Current-voltage relationship for the ANG II- or OAG-mediated inhibition of neuronal Kv current. Each point represents >= 6 experiments.



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Fig. 4.   Effect of OAG on neuronal Kv current in the presence of chelerythrine (Che) or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). A: representative current traces (top) and time course (bottom) showing effect of OAG on Kv current after chelerythrine (1 µmol/l) pretreatment (20 min, voltage step -80 to +10 mV). B: representative current traces and time course showing effect of OAG on Kv current. The Ca2+ chelator BAPTA (10 mmol/l) was predissolved in the pipette solution.



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Fig. 5.   Mean inhibitory effect of angiotensin type 1 (AT1) receptor activation or diacylglycerol analogs on neuronal Kv current. Values are means ± SE of 6, 7, 5, 6, 5, and 8 cells for ANG II, 1,2-diC8, 1,3-diC8, OAG, Che + OAG, and BAPTA + OAG, respectively. **P < 0.01 compared with control.

Effect of PKC-AS oligodeoxynucleotides on Kv current. To determine which Ca2+-dependent isozyme of PKC was responsible for the AT1 receptor-mediated effect of ANG II and OAG on neuronal Kv current, PKC-alpha -AS, -beta -AS, and -gamma -AS were used to elicit the "knockdown" of PKC-alpha , -beta , and -gamma protein to reduce the expression of the corresponding isozyme. We previously used this technique to selectively reduce various PKC isozymes (20). Western blot analysis using neurons that were pretreated for 24 h with the various PKC-AS (2 µmol/l) showed that the AS for each PKC isozyme only "knocked down" the corresponding PKC protein while having no effect on the others; i.e., PKC-alpha -AS decreased the protein levels of PKC-alpha and not -beta or -gamma (Fig. 6). Each PKC-AS oligodeoxynucleotide reduced its target protein by ~60-70%. Therefore, we used these AS constructs in electrophysiological experiments. In the PKC-alpha -AS-pretreated neurons (24-72 h), the inhibitory effects of ANG II and OAG on Kv current were abolished (Figs. 7A, 8A, and 9). In neurons pretreated with PKC-beta -AS or -gamma -AS, the effects of ANG II or OAG were still present and resembled control responses (Figs. 7, B and C, 8, B and C, and 9). Furthermore, in PKC-alpha -S-pretreated neurons as controls, in which PKC-alpha isozyme expression would be unaffected by the pretreatment, superfusion of ANG II (100 nM) elicited a significant inhibitory effect on Kv current that was reversed by losartan (1 µmol/l; Figs. 7D and 9). These data suggest that PKC-alpha plays a crucial role in the AT1 receptor-mediated inhibitory effect of ANG II on neuronal Kv current.


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Fig. 6.   Protein kinase C (PKC) antisense (AS) knockdown of Ca2+-dependent PKC isozymes. Cultured neurons were incubated with AS and sense (S) constructs (2 µmol/l) for 24 h. Western blots show specificity of each PKC-AS for isozyme of PKC. Each experiment was performed in triplicate and repeated 3 times. C, control.



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Fig. 7.   Effect of PKC-AS on AT1 receptor-mediated inhibition of neuronal Kv current. A: representative current traces (top) and time course (bottom) showing effect of ANG II (100 nmol/l) on Kv current after PKC-alpha -AS pretreatment (voltage step -80 to +10 mV). B, C, and D: representative current traces and time course showing effects of ANG II (100 nmol/l) and Los (1 µmol/l) on Kv current after pretreatment with PKC-beta -AS, PKC-gamma -AS, and PKC-alpha -S, respectively.



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Fig. 8.   Effect of PKC-AS on OAG-mediated inhibition of neuronal Kv current. Representative current traces (top, voltage step -80 to +10 mV) and time course (bottom) show effects of OAG (1 µmol/l) on Kv current after pretreatment with PKC-alpha -AS (A), PKC-beta -AS (B), and PKC-gamma -AS (C).



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Fig. 9.   Mean effect of PKC-AS on AT1 receptor or diacylglycerol analog inhibition of neuronal Kv current. Values are means ± SE of 4, 3, and 5 cells for PKC-alpha -AS, -beta -AS, and -gamma -AS, respectively, in OAG-treated group and 4, 4, 3, and 4 cells for PKC-alpha -AS, -beta -AS, and -gamma -AS and PKC-S, respectively, in ANG II-treated group. *P < 0.05 compared with control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our previous data indicate that ANG II acting via AT1 receptors increases neuronal firing rate and reduces Kv and A-type K+ currents (9, 33, 35, 38, 39, 42). One signaling pathway that all the above effects of ANG II had in common was the activation of PKC. However, there are no data that show that the inhibition of neuronal Kv current is dependent on the activation of DAG and/or a specific PKC isoform. The data presented here clearly show that PLC and DAG are involved in the regulation of neuronal Kv current, since membrane-permeant forms of DAG inhibit Kv current in a manner similar to ANG II (Figs. 1-5). The present studies also clearly indicate that the Ca2+-dependent PKC-alpha is also involved in AT1 receptor- and DAG-mediated inhibition of neuronal Kv current (Figs. 7-9). These conclusions are primarily based on the fact that PKC-AS directed against the alpha -isoform, and not the beta - or gamma -isoform, of PKC blocked the inhibitory effects of ANG II and DAG analogs. This is an important finding, since it could be argued that ANG II acts in a paracrine manner to release a substance that, in turn, causes inhibition of Kv current in another neuron.

It is apparent from the results that the effects of ANG II on Kv current were completely abolished by PKC-alpha -AS treatments, which, according to Western blot analysis, reduced PKC-alpha protein by only 60-70%. There may be a number of reasons for this discrepancy. The most obvious possibility is that the effects of the AS are heterogeneous, causing complete depletion of PKC-alpha in some cells, along with abolition of electrophysiological responses, while only reducing PKC-alpha expression in other cell populations. Thus, by analysis of PKC-alpha levels in the whole dish, only an overall reduction of this protein after AS treatment would be detected. Another possibility is that the PKC-alpha -AS has selective actions on the active and inactive pools of PKC present in these cells. For example, it may completely deplete the activated PKC-alpha that is required for signal transduction and have minimal effects on the inactive pool of this kinase. The result would be complete inhibition of ANG II effects on Kv current but only an overall reduction in PKC-alpha expression. This is, of course, speculation, and analyses of PKC-alpha activity would be required to prove or disprove this idea.

The hormonal regulation of ion channels plays an important role in the second-to-second regulation of the brain. There is a great deal of evidence that phosphorylation and/or dephosphorylation of Kv channel proteins is important in the regulation of their activity (17). PKC has been shown to modulate in vitro neuronal K+ currents (5, 7, 11, 31). With the use of expression systems, Kv1.1, Kv1.2, Kv1.3, Kv1.5, and Kv3.1 channels were inhibited by PKC in a similar manner. Namely, current amplitudes are reduced irreversibly over a long time course with little or no change in kinetics (1, 3, 6, 8, 21, 22, 25, 38). Using cultured neurons and the Xenopus oocyte expression system, we recently showed that the AT1 receptor-mediated inhibition of neuronal Kv currents is due to inhibition of Kv2.2 (9). Therefore, considering the fact that ANG II stimulates PKC activity in cultured neurons, it is reasonable to speculate that the reduction in Kv current caused by ANG II is mediated via direct phosphorylation of Kv2.2 by PKC or indirectly by PKC phosphorylating an auxiliary protein in a signaling cascade.

A number of questions remain to be answered. For example, do the observed changes in neuronal Kv current include direct channel phosphorylation by PKC, or are the effects of these serine/threonine kinases indirect and mediated via activation of other enzymes (e.g., tyrosine kinases) that subsequently modulate channel activity via phosphorylation? The identification of PKC-alpha as the PKC isozyme that is responsible for mediating the inhibitory effects of ANG II on Kv current will enable us to determine whether this serine/threonine kinase directly phosphorylates Kv2.2. There is evidence that PKC activates cAMP-dependent protein kinase (24, 28), protein tyrosine kinases (15, 30), mitogen-activated protein kinases (16), and tyrosine phosphatase (4, 36), and one or more of these enzymes might link AT1A receptors and PKC to the inhibition of Kv2.2. In light of this, the tyrosine kinase inhibitor genistein attenuates the ANG II-induced inhibition of neuronal Kv current (unpublished observations). Finally, what is the physiological consequence between ANG II-dependent inhibition of neuronal Kv current and the actions of ANG II in the brain? We previously showed that AT1 receptor activation increases neuronal firing rate and the release of norepinephrine (29, 38). Thus it is tempting to speculate that the reduction in Kv current caused by ANG II via PKC contributes to the release of norepinephrine.


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants HL-49130 and NS-19441.


    FOOTNOTES

Address for reprint requests and other correspondence: C. H. Gelband, Dept. of Physiology, Box 100274, 1600 SW Archer Rd., Gainesville, FL 32610 (E-mail: gelband{at}phys.med.ufl.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 14 August 2000; accepted in final form 1 November 2000.


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DISCUSSION
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34.   Sumners, C, Tang W, Zelezna B, and Raizada MK. Angiotensin II receptor subtypes are coupled with distinct signal transduction mechanisms in neurons and astroglia from rat brain. Proc Natl Acad Sci USA 88: 7567-7571, 1991[Abstract].

35.   Sumners, C, Zhu M, Gelband CH, and Posner P. Angiotensin II type 1 receptor modulation of neuronal K+ and Ca2+ currents: intracellular mechanisms. Am J Physiol Cell Physiol 271: C154-C163, 1996[Abstract/Free Full Text].

36.   Tsai, W, Morielli AD, Cachero TG, and Peralta EG. Receptor protein tyrosine phosphatase-alpha participates in the M1 muscarinic acetylcholine receptor-dependent regulation of Kv1.2 channel activity. EMBO J 18: 109-118, 1999[Abstract/Free Full Text].

37.   Vogalis, F, Ward M, and Horowitz B. Suppression of two cloned smooth muscle-derived delayed rectifier potassium channels by cholinergic agonists and phorbol esters. Mol Pharmacol 48: 1015-1023, 1995[Abstract].

38.   Wang, D, Sumners C, Gelband CH, and Posner P. Mechanisms underlying the chronotropic effect of angiotensin II on cultured neurons from rat hypothalamus and brainstem. J Neurophysiol 78: 1013-1020, 1997[Abstract/Free Full Text].

39.   Wang, D, Sumners C, Posner P, and Gelband CH. A-type K+ current in neurons cultured from neonatal rat hypothalamus and brainstem: modulation by angiotensin II. J Neurophysiol 78: 1021-1029, 1997[Abstract/Free Full Text].

40.   Yang, CR, Phillips MI, and Renaud LP. Angiotensin II receptor activation depolarizes rat supraoptic neurons in vitro. Am J Physiol Regulatory Integrative Comp Physiol 263: R1333-R1338, 1992[Abstract/Free Full Text].

41.   Zhu, M, Gelband CH, Moore JM, Posner P, and Sumners C. Angiotensin II type 2 receptor stimulation of neuronal delayed rectifier potassium current involves phospholipase A2 and arachidonic acid. J Neurosci 18: 679-686, 1998[Abstract/Free Full Text].

42.   Zhu, M, Neubig RR, Wade SM, Posner P, Gelband CH, and Sumners C. Modulation of K+ and Ca2+ currents in cultured neurons by an angiotensin II type 1A receptor peptide. Am J Physiol Cell Physiol 273: C1040-C1048, 1997[Abstract/Free Full Text].


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