Angiotensin II Decreases Neuronal Delayed Rectifier Potassium Current: Role of Calcium/Calmodulin-Dependent Protein Kinase II

Mingyan Zhu, Craig H. Gelband, Philip Posner, and Colin Sumners

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Zhu, Mingyan, Craig H. Gelband, Philip Posner, and Colin Sumners. Angiotensin II Decreases Neuronal Delayed Rectifier Potassium Current: Role of Calcium/Calmodulin-Dependent Protein Kinase II. J. Neurophysiol. 82: 1560-1568, 1999. Angiotensin II (Ang II) acts at specific receptors located on neurons in the hypothalamus and brain stem to elicit alterations in blood pressure, fluid intake, and hormone secretion. These actions of Ang II are mediated via Ang II type 1 (AT1) receptors and involve modulation of membrane ionic currents and neuronal activity. In previous studies we utilized neurons cultured from the hypothalamus and brain stem of newborn rats to investigate the AT1 receptor-mediated effects of Ang II on neuronal K+ currents. Our data indicate that Ang II decreases neuronal delayed rectifier (Kv) current, and that this effect is partially due to activation of protein kinase C (PKC), specifically PKCalpha . However, the data also indicated that another Ca2+-dependent mechanism was also involved in addition to PKC. Because Ca2+/calmodulin-dependent protein kinase II (CaM KII) is a known modulator of K+ currents in neurons, we investigated the role of this enzyme in the AT1 receptor-mediated reduction of neuronal Kv current by Ang II. The reduction of neuronal Kv current by Ang II was attenuated by selective inhibition of either calmodulin or CaM KII and was mimicked by intracellular application of activated (autothiophosphorylated) CaM KIIalpha . Concurrent inhibition of CaM KII and PKC completely abolished the reduction of neuronal Kv by Ang II. Consistent with these findings is the demonstration that Ang II increases CaM KII activity in neuronal cultures, as evidenced by increased levels of autophosphorylated CaM KIIalpha subunit. Last, single-cell reverse transcriptase (RT)-PCR analysis revealed the presence of AT1 receptor-, CaM KIIalpha -, and PKCalpha subunit mRNAs in neurons that responded to Ang II with a decrease in Kv current. The present data indicate that the AT1 receptor-mediated reduction of neuronal Kv current by Ang II involves a Ca2+/calmodulin/CaM KII pathway, in addition to the previously documented involvement of PKC.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The octapeptide angiotensin II (Ang II) has various physiological effects mediated by the brain, including stimulation of increased blood pressure, water and sodium intake, vasopressin secretion, and modulation of baroreflex function (Campagnole-Santos et al. 1988; Phillips and Sumners 1998). These actions of Ang II are mediated by specific Ang II type 1 (AT1) receptors that are located on neurons in the hypothalamus and brain stem (Hogarty et al. 1992; Koepke et al. 1990; Qadri et al. 1994). Consistent with these physiological actions are electrophysiological studies that have demonstrated that Ang II elicits specific AT1 receptor-mediated changes in neuronal activity in the hypothalamus and brain stem. For example, selective activation of AT1 receptors elicits increases in neuronal firing rate in the paraventricular nucleus, the subfornical organ, the supraoptic nucleus, and the rostral ventrolateral medulla (Ambuhl et al. 1992; Li and Ferguson 1993; Li and Guyenet 1995; Yang et al. 1992). Similar to the in vivo situation, neurons cultured from the hypothalamus and brain stem of newborn rats contain AT1 receptors (Gelband et al. 1997; Raizada et al. 1993). In previous studies, we have used these cultured neurons to investigate the AT1 receptor-mediated effects of Ang II on neuronal K+ and Ca2+ currents and the intracellular signaling pathways that are involved. The rationale for this approach was that changes in these currents form the basis of changes in neuronal firing rate and ultimately of behavioral and physiological effects that are stimulated by Ang II. We determined that Ang II, via AT1 receptors, increases neuronal firing rate (Wang et al. 1997a). Consistent with this, we have determined that Ang II elicits an AT1 receptor-mediated stimulation of total neuronal calcium current (ICa) and decreases neuronal delayed rectifier K+ current (Kv) and transient (A-type) K+ current (Gelband et al. 1999; Sumners et al. 1996; Wang et al. 1997b). These inhibitory effects of Ang II on Kv current involve a Galpha q/11 protein, stimulation of phosphoinositide (PI) hydrolysis and activation of protein kinase C (PKC), specifically Ca2+-dependent PKCalpha (Pan et al. 1999; Sumners et al. 1996; Zhu et al. 1997). However, our investigations indicated that PKC is only partially responsible for modulation of these K+ currents, and that another Ca2+-dependent mechanism is also involved (Sumners et al. 1996; Zhu et al. 1997). Our present studies are aimed at defining the identity of this Ca2+-dependent pathway. These investigations have centered around calcium/calmodulin-dependent protein kinase II (CaM KII) (Braun and Schulman 1995a; Colbran et al. 1989), for two major reasons. First, several studies have indicated that this enzyme is involved in the AT1 receptor-mediated actions of Ang II in certain cell types (Abraham et al. 1996; Muthalif et al. 1998; Pezzi et al. 1996). Second, even though there is no background literature indicating that CaM KII can modulate neuronal Kv current, this enzyme is a known modulator of neuronal A-type- and calcium-activated K+ currents (Ikeuchi et al. 1996; Pedarzani and Storm 1996; Roeper et al. 1997; Sakakibara et al. 1986; Yamamoto et al. 1997). The data presented here indicate that Ang II, via AT1 receptors, stimulates increases in neuronal intracellular calcium [Ca2+]i and CaM KII activity, the latter indicated by increased levels of autophosphorylated CaM KIIalpha subunit. In addition, the data also indicate that the inhibition of neuronal Kv current by Ang II involves a Ca2+/calmodulin/CaM KII signaling pathway as well as the previously documented PKC pathway. These studies provide the first indication that CaM KII is able to modulate neuronal Kv current.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

Newborn Sprague-Dawley rats were obtained from our breeding colony, which originated from Charles River Farms (Wilmington, MA). Dulbeccos modified Eagle's medium (DMEM) and TRIzol reagent were obtained from GIBCO-BRL (Gaithersburg, MD). Plasma derived horse serum (PDHS) came 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 & Co. (Rahway, NJ). PD 123,319 (PD), Calphostin C, KN-93, KN-92, W-7, and CaM KII (281-302) peptide were purchased from Research Biochemicals International (Natick, MA). Tetrodotoxin (TTX) was purchased from Calbiochem (La Jolla, CA). Monoclonal anti-CaM KII antibody was obtained from Transduction Laboratories (Lexington, KY). Anti-ACTIVE CaM KII pAb was obtained from Promega (Madison, WI). Gene-Amp reverse transcriptase-polymerase chain reaction (RT-PCR) kits and all reagents for RT-PCR were purchased from Perkin Elmer Biotechnologies (Norwalk, CT). Autothiophosphorylated CaM KIIalpha was kindly provided by Dr. T. R. Soderling (University of Washington). Ang II, sodium GTP, HEPES, cadmium chloride (CdCl2), fura-2/AM, ethylene glycol-bis (beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), bovine serum albumin, dipotassium ATP, and peroxidase-conjugated affinity purified goat anti-rabbit IgG were purchased from Sigma Chemical (St. Louis, MO). All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA) and were of analytic grade or higher. Oligonucleotide primers for the AT1A receptor (Kakar et al. 1992), CaM KIIalpha (Lin et al. 1987), and PKCalpha (Ono et al. 1988) genes 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:

AT1A receptor

Sense, 5 prime-CCATGCCATCTGTAATCCAC-3 prime

Antisense, 5 prime-AAGGCCACATGAACTGACTC-3 prime

CaM KIIalpha subunit

Sense, 5 prime-GACTCCATGACAGCATCTC-3 prime

Antisense, 5 prime-CATCTGGTGACACTGTAGC-3 prime

PKCalpha

Sense, 5 prime-GGTGTCTCAGAGCTACTCAA-3 prime

Antisense, 5 prime-TGAGGAAGCTGAAGTCAGAG-3 prime

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. 1994).

[Ca 2+]i analyses

Analysis of [Ca 2+]i in cultured neurons was achieved using imaging fluorescence microscopy in cells preloaded with 5 µM fura-2/AM, as detailed by us previously (Wang et al. 1996).

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 A 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 60 ms every 10 s. Under these recording conditions, both the Kv current 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 50 ms after the initiation of the test pulse, at which time they reflect only Kv current (Kang et al. 1994). Current density was derived by dividing transmembrane current (pA) by membrane capacitance (pF). Membrane capacitance was calculated by the equation C = Ac/Delta V, where C is capacitance, Ac is the area of capacitative current, and Delta V is the voltage step. Ac was obtained by the equation Ac = IoRC, which is from Sigma Ic = int Io × e-t/RC, where I is the maximal capacitative current and RC is the time constant, equal to the time when Io is 36%. The average cell capacitance for neurons used in this study was 36.7 ± 10.4 pF (mean ± SE; n = 94 neurons; range, 3.7-70 pF).

Selective stimulation of neuronal AT1 receptors causes a decrease in Kv current (Sumners et al. 1996), whereas selective stimulation of neuronal AT2 receptors causes an increase in neuronal Kv current (Zhu et al. 1998). Some of the neurons in the cultures used here contain both AT1 and AT2 receptors (Gelband et al. 1997). Because the aim of the present studies was to measure AT1 receptor-mediated effects of Ang II on Kv current, all electrophysiological recordings were performed in the presence of 1 µM PD123,319 to block AT2 receptors. PD123,319 did not alter basal Kv current.

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). For the experiments using cells from the whole dish, RT-PCR of the AT1A receptor, CaM KIIalpha , and PKCalpha were performed using Gene-Amp RT-PCR kits essentially as described previously (Huang et al. 1997). In brief, PCR was performed at 95°C for 4 min, followed by 38 cycles at 95°C for 45 s, 61°C for 90 s, and 72°C for 120 s. After final extension at 72°C for 10 min, PCR products were subjected to electrophoresis on a 2% agarose gel containing 1 µg/ml ethidium bromide. Using these conditions, we observed the production of a 169 bp AT1A receptor-specific DNA, a 167 bp CaM KIIalpha -specific DNA, and a 280 bp PKCalpha -specific DNA from the PCR. These correspond to the AT1A receptor and PKCalpha mRNAs, respectively.

For the experiments on single neurons, RT-PCR of the AT1A receptor, CaM KIIalpha , and PKCalpha was performed as detailed by us previously (Zhu et al. 1998). In brief, following recordings of Kv current, 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. A first PCR was performed exactly as described above for neurons from the whole dish. A second PCR was performed (on 20 µl of the 1st PCR products) at 95°C for 4 min followed by 32 cycles at 95°C for 45 s, 61°C for 90 s, and 72°C for 120 s. After final extension at 72°C for 10 min, the PCR products were electrophoresed as above. Using these conditions for single-cell RT-PCR, we observed the production of 169 bp AT1A receptor-, 167 bp CaM KIIalpha -, and 280 bp PCKalpha -specific DNAs, similar to the bands obtained from the whole dish of neurons. In all situations, exclusion of either RNA or MuLV reverse transcriptase resulted in no visible bands after gel electrophoresis.

Analysis of CaM KII subunit proteins

The presence of CaM KIIalpha and CaM KIIbeta subunit proteins in control neuronal cultures was determined by Western Blot analysis using a monoclonal anti-CaM KII antibody (1:250 dilution). Extraction of total cellular protein, SDS polyacrylamide gel electrophoresis, and immunoblotting were performed exactly as detailed previously (Kopnisky et al. 1997). These procedures yielded bands of ~50 and ~60 kDa, corresponding to CaM KIIalpha and CaM KIIbeta subunits, respectively.

The presence of autophosphorylated CaM KIIalpha subunit protein in control or Ang II-treated neuronal cultures was determined by Western Blot analysis using an anti-ACTIVE CaM KII pAb. This antibody preferentially detects CaM KII that is phosphorylated on threonine 286 (pT286) of the alpha -subunit. Total cellular protein was isolated from neuronal cultures as follows. Cells were washed twice with ice-cold PBS (pH 7.2), and 200 µl of ice-cold lysis buffer (1% NP-40, 10% glycerol, 150 mM NaCl, 20 mM Tris-HCl, 1 mM phenylmethyl-sulfonyl fluoride, and 2.5 mg/µl each of aprotinin, leupeptin, antipain, and chymostatin) was added to each dish. Samples were centrifuged at 4°C and 350 × g for 5 min, and the supernatant was removed to a clean tube. A small aliquot was used for analysis of protein concentration (Bradford 1976), and the remainder was stored at -70°C. Proteins were separated by size on a 10% SDS polyacrylamide gel using the system of Laemmli (1970), and transferred to nitrocellulose (Bioblot, Costar) at 100 V for 1 h in Towbin-SDS transfer buffer (25 mM Tris 192 mM glycine, 20 mM methanol, and 0.01% SDS). After transfer the blot was washed once in PBS with 0.05% Tween (PBST) for 10 min. The membrane was blocked in PBST and 1% bovine serum albumin overnight at 37°C. Next, the membrane was incubated for 1 h at room temperature with a 1:5,000 dilution of anti-ACTIVE CaM KII pAb in PBST and 0.1% bovine serum albumin. After this, the membrane was washed three times in PBST, and was then incubated for 1 h at room temperature with a 1:16,000 dilution of peroxidase-conjugated affinity purified goat anti-rabbit IgG in PBST and 0.1% bovine serum albumin. The membrane was then washed three times in PBST at room temperature. Detection of the resulting antigen-antibody complex was performed using the Renaissance ECL kit, according to the manufacturer's directions, and visualized by exposure to Kodak film (Bio Max light) for 90 s.

Drug 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. Solvent controls were performed for each protocol. Intracellular application of CaM KII (281-302) peptide and of autothiophosphorylated CaM KIIalpha were achieved as detailed by us previously (Zhu et al. 1997). In brief, a sidearm pipette holder is attached to the head stage of the Axopatch. One side arm is used to apply 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 Kv 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 CaM KIIalpha or CaM KII (281-302) are allowed to diffuse into the neuron, and measurements of Kv current are made 4 min later, at which time a stable peak response is obtained. Care is taken not to overperfuse the neuron, and this is monitored electrically via the Axopatch and on the TV monitor. Thus the concentrations of CaM KIIalpha and CaM KII (281-302) that are given in RESULTS indicate the amounts that were injected at the pipette tip and 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 neuronal cultures. Comparisons were made with the use of a one-way ANOVA followed by Newman-Keuls test to assess statistical significance.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In previous studies, we have determined that the Ang II-induced decrease in neuronal Kv current is partially mediated via activation of a Ca2+-dependent PKC (Pan et al. 1999; Sumners et al. 1996; Zhu et al. 1997). These results also indicated that the inhibitory effect of Ang II on neuronal Kv current was totally abolished by concurrent inhibition of PKC and chelation of [Ca2+]i. Thus it appeared that aside from PKC, an additional Ca2+-dependent mechanism was responsible for mediating the decrease in neuronal Kv current produced by Ang II. Because CaM KII is a known modulator of certain neuronal K+ currents and is activated by AT1 receptor stimulation in certain cell types, in the present studies we decided to investigate the possible role of a Ca2+/calmodulin/CaM KII pathway in the Ang II-induced decrease in neuronal Kv current.

The first series of experiments were performed to establish the effect of Ang II on [Ca2+]i in cultured neurons. The data presented in Fig. 1 demonstrate that superfusion of cultures with normal Tyrode's solution (containing 2 mM CaCl2) in the presence of 100 nM Ang II produces an increase in [Ca2+]i in a representative neuron. The increase in [Ca2+]i elicited by Ang II was abolished by co-superfusion of the AT1 receptor antagonist Los (1 µM; data not shown). The mean resting [Ca2+]i, mean Ang II-stimulated peak [Ca2+]i, and mean steady-state/plateau [Ca2+]i were 116 ± 13, 622 ± 24, and 142 ± 12 nM, respectively (n = 4 neurons). These findings are consistent with our previous demonstration that Ang II elicits on AT1 receptor-mediated increase in PI hydrolysis in neuronal cultures (Sumners et al. 1996). Increases in [Ca2+]i will lead to activation of a number of Ca2+-dependent signaling molecules, including calmodulin, which subsequently activates CaM KII. Thus in the next series of experiments we investigated whether calmodulin was involved in the Ang II-induced decrease in neuronal Kv current. Superfusion of cultures with Ang II (100 nM) caused a significant reduction in neuronal Kv current that was completely inhibited by 1 µM Los (Fig. 2), in agreement with our previous studies (Sumners et al. 1996). Treatment of cultures with a calmodulin antagonist W-7 (10 µM) significantly attenuated the AT1 receptor-mediated inhibition of neuronal Kv current by Ang II (Fig. 2). Higher concentrations of W-7 (20-50 µM) produced no greater attenuation of this Ang II effect (data not shown). Control recordings of neuronal Kv in the presence of W-7 were not significantly different from control recordings in untreated neurons (Fig. 2). Thus the data in Fig. 2 indicate that calmodulin is involved in the AT1 receptor-mediated inhibition of neuronal Kv current by Ang II.



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Fig. 1. Angiotensin II (Ang II) increases [Ca2+]i in cultured neurons. Spatial average changes in [Ca2+]i (top) and pseudocolor images (bottom) of a cultured neuron during Ang II (100 nM) superfusion. The increase in [Ca2+]i elicited by Ang II was present until wash out. This is representative of 4 neurons. Images were taken from an area that represents the cell body.



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Fig. 2. Inhibition of calmodulin attenuates the reduction of neuronal Kv current produced by Ang II. K+ current was recorded during 100-ms voltage steps from a holding potential of -80 to +10 mV. Top: representative current tracings showing the effects of superfused Ang II (100 nM) on K+ current (left) in untreated neurons (± 1 µM Los) and (right) in neurons pretreated with the calmodulin antagonist W-7 (10 µM) for 30 min. Control recordings (Con) in all sets of traces were made before application of Ang II. All recordings were made in the presence of 1 µM PD 123,319 to block AT2 receptors. Bottom: bar graphs showing means ± SE of Kv current densities obtained in each treatment situation. Sample sizes were 15 and 8 neurons for the untreated and W-7 groups, respectively. *P < 0.001 compared with the respective control. #P < 0.005 compared with Ang II alone.

Calmodulin (in combination with Ca2+) activates CaM KII, so the next aim was to investigate the role of this kinase in the reduction of neuronal Kv current produced by Ang II. However, it was first important to determine whether Ang II stimulates CaM KII activity in neuronal cultures. Immunoblot analyses using a specific anti-CaM KII antibody revealed the presence of an ~50 kDa CaM KIIalpha subunit and an ~60-kDa CaM KIIbeta subunit in neuronal cultures (Fig. 3A). This is consistent with the picture in rat brain, where these are the predominant CaM KII isoforms (Braun and Schulman 1995a). Incubation of neuronal cultures with 100 nM Ang II (in the presence of 1 µM PD123,319 to block AT2 receptors), elicited an increase in the levels of phosphorylated CaM KIIalpha subunit protein (Fig. 3B). Because the glia within these cultures do not contain AT1 receptors (Sumners, unpublished results), the present findings are indicative of an AT1 receptor-mediated increase in neuronal CaM KII activity. Treatment of cultures with CaM KII inhibitors significantly attenuated the AT1 receptor-mediated inhibition of neuronal Kv current by Ang II. For example, inclusion of CaM KII inhibitory peptide [CaM KII (281-302) 2 µM (Braun and Schulman 1995b; Smith et al. 1992)] in the pipette solution or pretreatment of cultures with the CaM KII inhibitor KN-93 (10 µM; 10 min) (Sumi et al. 1991) caused a significant attenuation of Ang II-inhibited Kv current (Fig. 4). By contrast, pretreatment of cultures with KN-92 (10 µM; 10 min), an inactive analogue of KN-93, did not alter this Ang II effect on neuronal Kv current (Fig. 4). The role of CaM KII was further demonstrated by experiments in which the inhibitory effect of Ang II on neuronal Kv current was partially reversed by acute administration of 2 µM CaM KII (281-302) via intracellular application (Fig. 5). Full reversal of the inhibitory effect of Ang II on neuronal Kv current was obtained with subsequent superfusion of 1 µM Los (Fig. 5). Control recordings of neuronal Kv current in the presence of these CaM KII inhibitors were not significantly different from control recordings in untreated neurons (Fig. 4). In addition, higher concentrations of these CaM KII inhibitors (e.g., 20 µM KN-93) produced no further attenuation of Ang II-modulated Kv current (data not shown). The data presented in Figs. 4 and 5 therefore indicate that CaM KII is involved in the AT1 receptor-mediated inhibition of neuronal Kv current by Ang II. If this is the case, it follows that selective activation of CaM KII may mimic the effects of Ang II on neuronal Kv current. To simulate activation of CaM KII, we utilized activated (autothiophosphorylated) CaM KIIalpha . This has been used previously by others to modulate neuronal K+ currents (Lledo et al. 1995). Intracellular application of 200 nM autothiophosphorylated CaM KIIalpha produced a significant decrease in neuronal Kv current of 7.8 ± 1.4% (n = 3 neurons, P < 0.005) compared with control recordings (see representative tracings in Fig. 6). In contrast, similar application of boiled (denatured) autothiophosphorylated CaM KII (200 nM) did not significantly alter neuronal Kv current (decrease of 1.3 ± 0.6%, n = 5 neurons). Although small, the reduction of neuronal Kv current produced by the autothiophosphorylated CaM KIIalpha is reasonable. This is explained because the maximal reduction of Kv current produced by Ang II is ~20% (Fig. 7), and our present data indicate that this effect of Ang II is only partially due to activation of CaM KII (Figs. 4 and 5).



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Fig. 3. Stimulation of CaM KII activity by Ang II in neuronal cultures. A: representative Western immunoblot showing the presence of CaM KIIalpha - and beta -subunit proteins in neuronal cultures. Data were obtained using a monoclonal anti-CaM KII antibody as detailed in METHODS. From left to right, lanes represent Jurkat lymphocyte control cells (5 µg), 20-µg neuronal cultures (NC 20) and 40-µg neuronal cultures (NC 40). B: representative Western immunoblot showing the effects of Ang II (100 nM, 1 min; in the presence of 1 µM PD 123,319) on the activated (phosphorylated) form of CaM KIIalpha in neuronal cultures. Data were obtained using the anti-ACTIVE CaM KIIalpha pAb, as detailed in METHODS. This was repeated 3 times with similar results.



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Fig. 4. Pretreatment of neurons with CaM KII inhibitors attenuates the reduction of neuronal Kv current produced by Ang II. K+ current was recorded as described in Fig. 1. Top: from left to right, representative current tracings showing the effects of superfused Ang II (100 nM) on K+ current in untreated neurons, or in neurons treated with the CaM KII inhibitors CaM KII (281-302) peptide (2 µM, included in pipette solution) or KN-93 (10 µM added to DMEM for 10 min and to superfusate). Also presented are representative current tracings showing the effects of KN-92 (10 µM, added to DMEM for 10 min and to superfusate), an inactive analogue of KN-93, on the Ang II-induced decrease in K+ current. Control (Con) recordings were made before the application of Ang II, and all recordings were made in the presence of 1 µM PD 123,319 to block AT2 receptors. Bottom: bar graphs showing means ± SE of Kv current densities obtained in each treatment situation. Sample sizes were 15, 11, 8, and 8 neurons for the untreated, CaM KII (281-302), KN-93, and KN-92 groups respectively. *P < 0.001 compared with the respective control. #P < 0.005 compared with Ang II alone. ++P < 0.05 compared with Ang II/KN-93 group.



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Fig. 5. Ang II-induced decreases in neuronal Kv current: effects of acute treatment with a CaM KII inhibitor. K+ current was recorded as described in Fig. 1. A: representative time course showing the effects of intracellular application of 2 µM CaM KII (281-302) on the reduction of Kv current produced by superfusion of 100 nM Ang II. Once the effects of CaM KII (281-302) had stabilized, 1 µM Los was added to the superfusate. B: bar graphs are means ± SE of Kv current densities recorded during the application of Ang II, CaM KII (281-302) and Los. Data are from 3 neurons, *P < 0.005 compared with control.



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Fig. 6. Activated CaM KIIalpha decreases neuronal K+ current. K+ current was recorded as in Fig. 1. A: current tracings showing the effect of intracellular application of activated (autothiophosphorylated) CaM KIIalpha (200 nM) on neuronal K+ current. B: current tracings showing the effect of intracellular perfusion of boiled (denatured) CaM KIIalpha (200 nM) on neuronal K+ current. These data are representative of 3 (A) and 5 (B) neurons.



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Fig. 7. Concurrent inhibition of CaM KII and PKC completely reverses the Ang II-induced decrease in neuronal Kv current. K+ current was recorded as in Fig. 1. Top: representative current tracings showing the effects of superfusion of Ang II (100 nM) on K+ current in untreated neurons, and in neurons treated with either the PKC inhibitor Calphostin C (Cal, added to DMEM for 30 min, and to superfusate) or with Cal plus KN-93 (10 µM, added to DMEM for 10 min, and to superfusate). Control (Con) recordings were made before application of Ang II. All recordings were made in the presence of 1 µM PD 123,319 to block AT2 receptors. Bottom: bar graphs showing means ± SE of current densities (plotted as % of control) in each treatment situation. Sample sizes were 14, 9, and 7 neurons for the untreated, Cal, and Cal/KN-93 groups, respectively. *P < 0.001 compared with the respective control. #P < 0.001 compared with Ang II effect in control (untreated) neurons.

The Ang II-induced reduction of neuronal Kv current is partially mediated by PKC (Sumners et al. 1996). Considering that the present data also indicate a role for CaM KII in this response, we tested the effects of co-inhibition of PKC and CaM KII on the reduction of neuronal Kv current produced by Ang II. Treatment of cultures with the PKC inhibitor Calphostin C (Cal; 750 nM, 30 min) produced a partial inhibition of Ang II's effects on Kv current (Fig. 7), in agreement with our previous data (Sumners et al. 1996). In cultures that had been co-treated with Cal (750 nM) and KN-93 (10 µM), Ang II failed to produce a significant reduction in neuronal Kv current (Fig. 7). Collectively, the present studies suggest that the Ang II-induced reduction of neuronal Kv current via AT1 receptors is mediated via dual intracellular messenger pathways, namely PKC and CaM KII. To further establish these signaling molecules as mediators of the Ang II-induced decrease in neuronal Kv current, it is necessary to demonstrate their presence within the responsive neurons. The RT-PCR data presented in Fig. 8 demonstrate the presence of AT1 receptor, PKCalpha , and CaM KIIalpha mRNAs in a whole dish of neuronal cultures. Further, the data indicate the presence of these mRNAs in a single neuron that responded to Ang II with an AT1 receptor-mediated reduction of Kv current (Fig. 8). Thus these RT-PCR data provide strong support for a role of PKC and CaM KII in the Ang II-induced decrease in neuronal Kv current.



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Fig. 8. Localization of CaM KIIalpha and PKCalpha mRNAs within Ang II responsive neurons. A: current tracing from a representative neuron showing the reduction of K+ current (AT1 receptor-mediated) produced by Ang II (100 nM) in the presence of 1 µM PD 123,319. 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. B: ethidium bromide-stained gel showing the RT-PCR DNA products that correspond to the AT1A receptor, CaM KIIalpha , and PKCalpha mRNAs. Leftmost lane is 100 bp ladder. Lanes 2, 4, and 6 are AT1A receptor, PKCalpha , and CaM KIIalpha mRNAs, respectively, from the responsive neuron in A. Lanes 1, 3, and 5 are the AT1A receptor, PKCalpha , and CaM KIIalpha mRNAs from a whole dish of neuronal cultures. This analysis was repeated 6 times with similar results.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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The physiological and behavioral events that are stimulated by Ang II binding to its AT1 receptors in brain involve depolarization and activation of specific neuronal pathways. In previous studies, we have utilized primary neuronal cultures from newborn rat hypothalamus and brain stem to investigate the effects of Ang II on changes in neuronal activity and the underlying ionic currents. Our data indicated that Ang II acting via AT1 receptors increases neuronal firing rate (Wang et al. 1997a), increases total Ca2+ current and reduces Kv and A-type K+ currents (Gelband et al. 1999; Sumners et al. 1996; Wang et al. 1997b). In further studies, we investigated the intracellular signaling pathways that mediate the effects of Ang II on these currents. The Ang II-induced decrease in neuronal Kv current involves a signaling pathway that begins with Galpha q/11-mediated activation of phospholipase C (PLC) and increased PI hydrolysis (Sumners et al. 1996). The products of PI hydrolysis, diacylglycerol and inositol 1,4,5-triphosphate (IP3), activate PKC and increase intracellular Ca2+, respectively (Berridge 1997). Because [Ca2+]i transients are usually short in duration, the Ang II-stimulated increase in [Ca2+]i probably sets off a cascade of biochemical events that involves a number of Ca2+-dependent kinases. Our data have indicated that a calcium-dependent PKC isozyme, PKCalpha , is partially responsible for the Ang II-induced decrease in neuronal Kv current (Pan et al. 1999; Sumners et al. 1996). However, our data also indicated that another Ca2+-dependent mechanism was responsible for the residual inhibitory action of Ang II on neuronal Kv current. The present studies clearly indicate that this other Ca2+-dependent mechanism involves activation of CaM KII, because the inhibitory effect of Ang II on neuronal Kv current is attenuated by a calmodulin antagonist (Fig. 2), or by inhibition of CaM KII (Figs. 4 and 5). Furthermore, the inhibitory effect of Ang II on neuronal Kv current is mimicked by intracellular application of activated (autothiophosphorylated) CaM KIIalpha (Fig. 6). The demonstration that Ang II activates CaM KII in neuronal cultures (Fig. 3) also supports the conclusion that this enzyme mediates the actions of Ang II on neuronal Kv current. Concurrent inhibition of CaM KII and PKC resulted in no significant inhibitory effect of Ang II on neuronal Kv current (Fig. 7).

Collectively, the data from our present and previous studies indicate that the reduction of neuronal Kv current elicited by Ang II results from Galpha q/11/PLC- mediated activation of two distinct Ca2+-dependent/enzymes, CaM KII and PKC. Importantly, single-cell RT-PCR data also demonstrate the presence of these signaling components within the same (Ang II-responsive) neuron. This is essential, because it might be argued that superfusion of Ang II activates neuronal AT1 receptor at one locus, causing release of a paracine factor, which diffuses to the recording neuron and inhibits Kv current (via CaM KII and PKC).

The fact that Ang II reduces neuronal Kv current via a dual modulatory pathway has a number of important implications. Because activation of both PKC and CaM KII is needed for the full inhibitory action of Ang II on Kv current, it is possible that the magnitude of Ang II's action can be modified by pathways or factors that interrupt the activation of these enzymes. For example, inhibition of either PKC or CaM KII would blunt, but not eliminate the effect of Ang II on Kv current. By contrast, a factor that inhibits the stimulation of PLC by Ang II would abolish the actions of this peptide on neuronal Kv current. Therefore these may be potential mechanisms through which other neurotransmitters/hormones can modify the physiological and behavioral actions of Ang II in the brain. Other important points concern the mechanisms through which Kv current is modulated by PKC and CaM KII. It is well-known from studies in other systems that both PKC and CaM KII can modulate neuronal K+ currents (Doerner et al. 1988; Grega et al. 1987; Ikeuchi et al. 1996; Pedarzani and Storm 1996; Roeper et al. 1997; Shearman et al. 1989), although few of these studies have addressed the molecular mechanisms involved. There is now a great deal of evidence that phosphorylation/dephosphorylation of K+ channel proteins is important in the regulation of their activity, and in the regulation of K+ currents (Levitan 1994). With respect to AT1 receptor activation, we have determined that the reduction in neuronal Kv currents is due to inhibition of the Kv2.2 K+ channel subunit (Gelband et al. 1999). Inspection of the amino acid sequence of the rat Kv2.2 channel reveals the presence of multiple consensus PKC (S/T-X-R/K) and CaM KII (R-X-X-S/T) phosphorylation sites within the cytoplasmic domains (Chandy and Gutman 1995; Hwang et al. 1992; Luneau et al. 1991; Rettig et al. 1992). Therefore considering that Ang II stimulates both PKC (Sumners et al. 1996) and CaM KII activities in cultured neurons, it is reasonable to speculate that the reduction in Kv current caused by Ang II is mediated via phosphorylation of Kv2.2 by these enzymes. It is also possible that through selective phosphorylation events, PKC and CaM KII can inhibit neuronal Kv current via modulation of different biophysical properties of the underlying K+ channels (e.g., channel open and closed times, activation and inactivation kinetics, time of 1st latency, etc.). The relationships between channel phosphorylation via PKC and CaM KII, channel activity, biophysical properties, and changes in Kv current will be the subject of our future studies.

Clearly, many questions remain to be answered. For example, do the observed changes in neuronal Kv current include direct channel phosphorylation by PKC and CaM KII? Or, are the effects of these serine/threonine kinases mediated via activation of other enzymes (e.g., tyrosine kinases) that subsequently modulate channel activity via phosphorylation? The latter situation is a realistic possibility because in preliminary studies we have shown that the tyrosine kinase inhibitor genistein attenuates Ang II-induced decreases in neuronal Kv current (unpublished observations). Other questions concern the functional or cellular role of the reduction in neuronal Kv current produced by Ang II. It is established that some of the physiological and behavioral actions of Ang II in the brain involve modulation of central noradrenergic neurons (Sumners et al. 1994). Indeed, our previous studies indicate that AT1 receptors are present on noradrenergic neurons in the cultures used here, and that stimulation of these receptors results in an increase in neuronal firing rate and release of norepinephrine (Gelband et al. 1997; Richards et al. 1999; Wang et al. 1997a). Thus it is tempting to speculate that the reduction in Kv current caused by Ang II via PKC and CaM KII contributes to membrane depolarization and ultimately, the release of norepinephrine. This speculation will be the subject of our further studies.


    ACKNOWLEDGMENTS

The authors thank J. Moore for technical assistance and M. Fancey for typing this manuscript.

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


    FOOTNOTES

Address for reprint requests: C. Sumners, Dept. of Physiology, Box 100274, 1600 SW Archer Rd., Gainesville, FL 32610.

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 23 March 1999; accepted in final form 25 May 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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