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(Circulation Research. 1999;84:352-359.)
© 1999 American Heart Association, Inc.

Rapid Communication

Angiotensin II Type 1 Receptor–Mediated Inhibition of K⁺ Channel Subunit Kv2.2 in Brain Stem and Hypothalamic Neurons

Craig H. Gelband, John D. Warth, Helen S. Mason, Mingyan Zhu, Jennifer M. Moore, James L. Kenyon, Burton Horowitz, Colin Sumners

From the Department of Physiology (C.H.G., J.D.W., M.Z., J.M.M., C.S.), University of Florida College of Medicine, Gainesville, Fla; Department of Physiology and Cell Biology (H.S.M., J.L.K., B.H.), University of Nevada School of Medicine, Reno, Nev.

Correspondence to Dr Craig H. Gelband, University of Florida School of Medicine, Department of Physiology, PO Box 100274, Gainesville, FL 32610. E-mail gelband{at}phys.med.ufl.edu

	Abstract

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Abstract—Angiotensin II (Ang II) has powerful modulatory actions on cardiovascular function that are mediated by specific receptors located on neurons within the hypothalamus and brain stem. Incubation of neuronal cocultures of rat hypothalamus and brain stem with Ang II elicits an Ang II type 1 (AT₁) receptor–mediated inhibition of total outward K⁺ current that contributes to an increase in neuronal firing rate. However, the exact K⁺ conductance(s) that is inhibited by Ang II are not established. Pharmacological manipulation of total neuronal outward K⁺ current revealed a component of K⁺ current sensitive to quinine, tetraethylammonium, and 4-aminopyridine, with IC₅₀ values of 21.7 µmol/L, 1.49 mmol/L, and 890 µmol/L, respectively, and insensitive to -dendrotoxin (100 to 500 nmol/L), charybdotoxin (100 to 500 nmol/L), and mast cell degranulating peptide (1 µmol/L). Collectively, these data suggest the presence of Kv2.2 and Kv3.1b. Biophysical examination of the quinine-sensitive neuronal K⁺ current demonstrated a macroscopic conductance with similar biophysical properties to those of Kv2.2 and Kv3.1b. Ang II (100 nmol/L), in the presence of the AT₂ receptor blocker PD123,319, elicited an inhibition of neuronal K⁺ current that was abolished by quinine (50 µmol/L). Reverse transcriptase–polymerase chain reaction analysis confirmed the presence of Kv2.2 and Kv3.1b mRNA in these neurons. However, Western blot analyses demonstrated that only Kv2.2 protein was present. Coexpression of Kv2.2 and the AT₁ receptor in Xenopus oocytes demonstrated an Ang II–induced inhibition of Kv2.2 current. Therefore, these data suggest that inhibition of Kv2.2 contributes to the AT₁ receptor–mediated reduction of neuronal K⁺ current and subsequently to the modulation of cardiovascular function.

Key Words: angiotensin II • Kv2.2 • Kv3.1b AT₁ receptor • Xenopus oocyte • cultured neuron

	Introduction

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Mammalian brain contains specific angiotensin II (Ang II) type 1 (AT₁) receptors that are localized mainly in specific nuclei within the hypothalamus and brain stem.^{1 2} Some of these nuclei lie outside of 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 (AVP) release, salt appetite, and drinking behavior,^{3 4 5 6 7} effects that participate in the modulatory role of this peptide on extracellular fluid volume and cardiovascular hemodynamics. All of these effects of Ang II are abolished by pretreatment of the animals with losartan, a selective AT₁ receptor antagonist.⁵ The exact mechanisms by which Ang II exerts its pressor effect have not been fully elucidated, but they appear to involve an increase in sympathetic vasomotor activity and/or an increase in vasopressin release.^{8 9} These actions of Ang II presumably involve the modulation of neuronal activity via changes in the underlying ionic currents and channels.

Data from our group and from other investigators have established that in various cell types, Ang II potentiates both high voltage activated^{10 11} and low voltage¹² activated Ca²⁺ currents and inhibits several K⁺ current subtypes.^{11 13 14} In the brain, these effects could lead to an increase in neuronal excitability and may help to explain the increase in sympathetic vasomotor activity seen with intracerebroventricular injection of Ang II, which would result in stimulation of the cardiovascular system. Therefore, modulation of the effects of Ang II on neuronal Ca²⁺ and K⁺ currents may represent a potential site for therapeutic intervention in the treatment of a variety of cardiovascular disorders including hypertension. However, the specific ion channels that underlie the Ang II–induced alterations in neuronal K⁺ and Ca²⁺ currents are unknown.

In the present study, we have dissected, using biophysical and pharmacological methods, the total outward K⁺ current in hypothalamus and brain stem neuronal cocultures prepared from the newborn rat. Results from these studies revealed that a major component of neuronal K⁺ current was sensitive to low doses of quinine, tetraethylammonium (TEA), and 4-aminopyridine (4-AP) and insensitive to -dendotoxin (-DTX), charybdotoxin, and mast cell degranulating peptide (MCDP), suggesting the presence of a "Kv2.2- or Kv3.1b-like" current.^{15 16} Ang II, which normally inhibits neuronal K⁺ current,¹¹ had no significant effects on K⁺ current in the presence of quinine. Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis of the neuronal cocultures demonstrated the presence of mRNA for Kv2.2 and Kv3.1b. However, Western blot analyses showed that these cultures expressed Kv2.2 and not Kv3.1b protein. Coexpression of mRNA for Kv2.2 and the AT_1A receptor in Xenopus oocytes resulted in an Ang II–dependent inhibition of Kv2.2 current. Therefore, these data suggest that inhibition of Kv2.2 contributes to the Ang II–induced reduction of neuronal outward K⁺ current.

	Materials and Methods

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Preparation of Neuronal Cultures
Neuronal cocultures were prepared from brain stem and a hypothalamic block of newborn Sprague-Dawley rats as previously described.^{17 18} Cultures were grown for 10 to 14 days. At this time, the cultures consisted of 90% neurons and 10% astroglia.

Whole-Cell Patch Clamp of Cultured Neurons
Membrane currents in isolated neurons were measured using the whole-cell variation of the patch-clamp technique.¹⁹ Patch electrodes were pulled from glass capillary tubing (Corning 7052), fire-polished on a microforge, and had resistances of 2 to 4 M when filled with an internal solution that contained (in mmol/L) KCl 110, MgCl₂ 4.5, EGTA 9, Li-GTP 0.3, Tris-ATP 4, creatine phosphate 14, and HEPES 9 (pH 7.4) with KOH. The standard external solution contained (in mmol/L) NaCl 134, KCl 5.4, CaCl₂ 1.35, MgCl₂ 2, CdCl₂ 0.3, tetrodotoxin 0.0001, glucose 10, and HEPES 10 (pH 7.4) with NaOH. In experiments using TEA of 4-AP to block the K⁺ current, an equimolar concentration of NaCl was replaced with the blocking agent. Whole-cell currents were recorded with an Axopatch 200B patch-clamp amplifier (Axon Instruments). Data were collected using a P5-133 computer and pCLAMP 6.02 software. All current measurements were made at the end of the test pulse unless otherwise stated. All experiments were performed at room temperature (19°C to 22°C).

Isolation and Voltage Clamp of Xenopus Oocytes
Ovarian lobes were removed from anesthetized adult female Xenopus laevis frogs (Xenopus 1, Ann Arbor, Mich), mechanically opened, and defolliculated with collagenase (1 mg/mL) in Ca²⁺-free ND96 solution (see below) at room temperature for 2 to 3 hours. The oocytes were then collected, rinsed, and stored in ND96 solution containing (in mmol/L) sodium pyruvate 2.5, NaCl 96, KCl 2, CaCl₂ 1.8, MgCl₂ 1, HEPES 5 (pH 7.4) with NaOH plus gentamicin (50 mg/mL) at 19°C for up to 24 hours before injection. Stage V and VI oocytes were selected and injected with mRNA (1 µg/µL) for Kv2.2 and/or the AT_1A receptor to a total volume of 50 nL. In the oocytes expressing Kv2.2 and/or the AT_1A receptor, the mRNA was mixed in a ratio of 1:1, and a total volume of 50 nL was injected. The oocytes were then stored for 3 to 4 days until electrophysiological assay.

The 2-electrode voltage-clamp technique was used to measure the K⁺ currents from Xenopus oocytes with a GeneClamp 500 amplifier (Axon Instruments). Microelectrodes were pulled from a glass capillary (Kimax-51, Kimble Products) with resistance of 1 to 3 M when filled with 3 mol/L potassium aspartate. Recordings were made at room temperature (24°C to 28°C) in low-chloride, Ca²⁺-free ND96 solution that contained (in mmol/L) sodium isethionate 96, KCl 2, MgCl₂ 2.8, HEPES 5 (pH 7.4) with NaOH plus 50 µmol/L niflumic acid to inhibit the endogenous Ca²⁺-activated Cl^- current in oocytes. Data were collected using a DigiData 1200 interface and pCLAMP 6.0 software (Axon Instruments). Membrane capacitance was measured in every experiment and did not change with drug treatments.

Polymerase Chain Reaction
Extraction of total RNA was performed with TRIzol exactly as previously described.²⁰ Specific oligonucleotides (20 bp) were synthesized against the 5'-untranslated region of rat Kv2.2 and Kv3.1b.^{21 22 23} The sense sequence for Kv2.2 corresponded to nucleotides 1–20, 5'-ACGTGGCAGAACCGGAGTGA-3', and the antisense sequence corresponded to nucleotides 531–550, 5'-TCCAGGAGACTCTCGTGCGT-3', with a resultant amplification product of 550 bp. The sense sequence for Kv3.1b corresponded to nucleotides 1–20, 5'-GCGAGCGCATCGTGATCAAC-3', and the antisense sequence corresponded to nucleotides 573–592, 5'-GGTCATCTCCAGCTCGTCCT-3', with a resultant amplification product of 437 bp. PCR was performed with use of a Perkin-Elmer GeneAmp PCR kit (Applied Biosystems) according to the manufacturer's instructions in a reaction volume of 50 µL with 1 mmol/L MgCl₂ and 10 pmol/L of each (sense and antisense) primer. Total RNA of 1 µg was used in the reaction. The reaction was performed with an initial denaturation of 5 minutes followed by 35 cycles with the following profile: 94°C for 1 minute, 57°C for 1 minute, 72°C for 1 minute, with a final extension step of 8 minutes at 72°C in a thermal cycler (Biometra). Reaction products (10 µL) were electrophoresed through a 1% agarose/TBE gel (90 mmol/L Tris base, 90 mmol/L boric acid, and 2 mmol/L Na₂ EDTA) and visualized with ethidium bromide.

Western Analysis
Total cell protein was extracted from neuronal cocultures grown in 35-mm Nunc dishes. Cells were washed once with ice-cold PBS, and 200 µL of boiling 2x electrophoresis sample buffer (1x 125 mmol/L Tris HCl [pH 6.8], 2% SDS, 5% glycerol, and 1% ß-mercaptoethanol) was added to each dish. The cells were scraped with rubber policemen and transferred to a microfuge tube. The samples were boiled for 5 minutes, briefly sonicated (Cole-Parmer 4710 series ultrasonic homogenizer, setting 40% duty cycle, output control 4), and spun at 1300g for 5 minutes, and the supernatant was removed to a new tube. A small aliquot was used for protein concentration analysis (Bradford assay), and the remainder of samples were stored at -20°C.

Proteins were separated by size on a 10% SDS polyacrylamide gel using the system of Laemmli and transferred to nitrocellulose (BioBlot, Costar) at 100V for 1 hour in Towbin-SDS transfer buffer (25 mmol/L Tris, 192 mmol/L glycine, 20% methanol, and 0.01% SDS). After transfer, the blot was washed once in PBS with 0.05% Tween 20 (PBST) for 10 minutes. The membrane was blocked in 10% nonfat dry milk (Carnation) in PBST and 1% BSA for 1 hour at room temperature with shaking. For detection of Kv3.1b protein, the membrane was incubated overnight at 4°C with a final concentration of 3 µg/mL of anti–Kv3.1b antibody (Alomone Labs) in PBST containing 10% nonfat dry milk and 1% BSA. For detection of Kv2.2 protein, the membrane was incubated overnight at 4°C with a final concentration of 1 µg/mL of anti–Kv2.2 antibody (gift of J. Trimmer, SUNY-Stony Brook, Stony Brook, NY) in PBST containing 10% nonfat dry milk and 1% BSA. After this incubation, the membrane was then washed 1 time for 15 minutes and 4 times for 5 minutes in PBST followed by a 1-hour incubation with gentle agitation at room temperature in the presence of peroxidase-conjugated, affinity-purified goat anti-mouse (Jackson Immuno Research Labs, Inc) diluted 1:200 in PBST containing 10% nonfat dry milk and 1% BSA. The membrane was then washed 1 time for 15 minutes and 4 times for 5 minutes in PBST followed by a 1-hour incubation with gentle agitation at room temperature in the presence of peroxidase-conjugated, affinity-purified goat anti-rabbit (Sigma) diluted 1:16 000 in PBST containing 10% nonfat dry milk and 1% BSA.

After either incubation, the membrane was washed 1 time for 15 minutes and 4 times for 5 minutes in PBST at room temperature. Detection of the resulting antigen-antibody-peroxidase complex was done with use of the Renaissance (Du Pont–NEN) enhanced chemiluminescence (ECL) kit, according to the manufacturer's directions, and visualized by exposure to Kodak film (BioMax Light) for 30 seconds.

Data Analysis
All results are expressed as mean±SEM and were obtained from combining data from individual experiments. Comparisons of multiple means were made using an analysis of variance, followed by a Newman-Keuls test to assess statistical significance. A value of P<0.05 was determined to be significant. Dose-response relationships were fit to the following equation: I/I_max=[1+([drug]/IC₅₀)]^-1.

	Results

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As a first step toward elucidating the underlying K⁺ conductances that contribute to the outward current in these neuronal cocultures, we characterized basic biophysical properties of the total outward K⁺ current using the whole-cell, patch-clamp technique. Figure 1 shows the voltage dependence of activation (Figure 1A and 1B) and inactivation (Figure 1C and 1D) of the outward current. The activation curve was obtained from analysis of the peak amplitude of tail current elicited after depolarization of the membrane to potentials ranging from -80 to +40 mV for 500 ms followed by a step to -50 mV for 150 ms from a holding potential of -80 mV to elicit tail currents. Peak tail current was then plotted against the membrane potential of the test pulse and fit to a single Boltzmann function. The smooth line through the data points reveals a threshold for activation of approximately -20 mV, half-maximal activation at -0.23±1.88 mV (n=7), and a slope factor of 9.4±0.6 (n=7). The voltage-dependent inactivation of this current was determined using 10-second conditioning pulses to various potentials followed by 2.5-second test steps to +20 mV. Inactivation during the conditioning pulse was not complete at the end of the 10-second pulse, and, therefore, only approximates the steady-state inactivation. The magnitude of the current was measured at the end of the voltage pulse to avoid any contamination of the A-type K⁺ current, which has been shown to inactivate with a time constant of approximately 14 ms.¹⁴ The data were normalized to the maximal current and then plotted as a function of the conditioning step potential. The resulting curve was then fit with a single Boltzmann function with half inactivation at -29.6±0.34 mV (n=5) and a slope factor of -8.4±0.9 (n=5).

View larger version (23K): [in this window] [in a new window]   Figure 1. Voltage-dependent activation and inactivation of the total K+ outward current. A, Raw current traces recorded with voltage-clamp step depolarizations to potentials ranging from -80 to +40 mV for 500 ms from a holding potential of -80 mV followed by repolarization to -50 mV. B, I-V relationship obtained by plotting the magnitude of the tail current, normalized to the maximum at +40 mV, versus the step potential (n=7). C, Voltage-dependent inactivation of the total outward current measured using a double-pulse protocol. The cells were initially depolarized with a conditioning pulse to potentials ranging from -80 to +20 mV (VH=-80 mV) for 10 seconds. After a brief (10 ms) repolarization to -80 mV, the cells were then stepped to +20 mV for 2.5 seconds. D, Plot of the I-V relationship for which the magnitude of the current, measured at the end of the 2.5-second test pulse and normalized to the maximum test current obtained, was plotted as a function of the step potential of the conditioning pulse (n=7).

Recovery from inactivation of the total outward current was also examined using a protocol in which neurons were held at -80 mV and then stepped for 10 seconds to +20 mV as a conditioning pulse, back to -80 mV for recovery of various duration, and then to +20 mV for 2-second test pulses. A plot of the magnitude of the current at the end of the test pulse versus the recovery interval shows that there are at least 2 phases to the recovery, including a relatively rapid initial phase during which 50% of the current recovers (15±3 ms, n=6) followed by a prolonged phase requiring more than 2 seconds for the current to fully recover (2123±23 ms, n=6). These 2 phases fit well with the recovery kinetics of inactivating and noninactivating Kv channel subtypes.¹⁵

As an additional step in characterizing the outward K⁺ current in these neurons, various pharmacological agents were used. Figures 2A, 2C, and 2E show raw current traces obtained under control conditions and after various doses of TEA, 4-AP, or quinine, respectively. The current before and after increasing concentrations of blocker was measured at +20 mV (end of the pulse), and the data were plotted as the percentage of current remaining versus the concentration of blocker. The apparent IC₅₀ values for block by these various agents were calculated as TEA 1.49±0.21 mmol/L (Figure 2B), 4-AP 0.89±0.10 mmol/L (Figure 2D), and quinine 21.7±2.66 µmol/L (Figure 2F). Kv gene products that may underlie this current may be Kv1.1, Kv1.3, Kv2.2, and Kv3.1b.¹⁵ To further pharmacologically dissect the K⁺ current, charybdotoxin (100 to 500 nmol/L), -DTX (100 to 500 nmol/L), and MCDP (1 µmol/L) were also tested, because these agents have been shown to inhibit Kv1.1 and Kv1.3. These agents were without any significant effect on total K⁺ current (data not shown).

View larger version (24K): [in this window] [in a new window]   Figure 2. Pharmacology of the total outward K+ current. A, C, and E, Representative current traces obtained with a repetitive voltage pulse to +20 mV (VH=-80 mV) under control conditions and after application of the indicated concentrations of TEA, 4-AP, and quinine, respectively. B, D, and F, Dose-response curves for which the magnitude of the current after drug application (Idrug) was normalized to the magnitude of the current measured under control conditions (Icontrol) for TEA (n=5), 4-AP (n=5), and quinine (n=4), respectively. The IC50 values are as follows: TEA 1.49±0.21 mmol/L, 4-AP 0.89±0.10 mmol/L, and quinine 21.7±2.66 µmol/L.

It has recently been reported that both Kv2.2 and Kv3.1b display a unique sensitivity to low doses of quinine,^{15 16} suggesting that either Kv2.2, Kv3.1b, or a combination of these channels may underlie the quinine-sensitive current component, because other K⁺ channels show a sensitivity to quinine that is at least a log unit higher than that of Kv2.2.¹⁵ To test for this possibility, an IC₅₀ dose of quinine (25 µmol/L) was used to isolate the quinine-sensitive K⁺ current component. This dose was chosen because it significantly inhibits the outward K⁺ current in our neuronal cultures, but it should have minimal effects on other known K⁺ channels.¹⁵ Outward currents were recorded in the absence and presence of quinine using activation and inactivation protocols, and then the currents in the presence of quinine were subtracted from the control currents, allowing for the isolation of the quinine-sensitive K⁺ current. Figure 3 shows the activation and inactivation curves for the quinine-sensitive K⁺ current in these neurons. The curves were fit as previously described for the total outward current. For the activation curve (Figure 3B), the smooth line through the data shows a voltage-dependent outward current that activates at -30 mV, is half maximal at 2.33±4.73 mV, and has a slope factor of 10.2±0.87. The inactivation curve (Figure 3D) is half inactivated at -38.6±0.55 mV (n=3) and has a slope factor of -8.8±0.93 (n=3). A comparison of the properties of Kv2.2, Kv3.1b, and the quinine-sensitive K⁺ current is presented in the Table and demonstrates that these 3 conductances share several biophysical and pharmacological similarities.^{15 16 21 22 23 24}

View larger version (28K): [in this window] [in a new window]   Figure 3. Voltage-dependent activation and inactivation of the quinine-sensitive K+ current. A and C, Raw current traces for voltage-dependent activation and inactivation, respectively. Voltage-clamp currents were recorded using identical protocols to those outlined in Figure 1. The quinine-sensitive current was defined as the difference current obtained by subtracting the current in the presence of quinine (25 µmol/L) from the current obtained under control conditions. B and D, I-V relationships for the activation (n=4) and inactivation (n=3) of the quinine-sensitive current, respectively. Voltage-dependent activation was obtained by plotting the magnitude of the tail current, normalized to the maximum at +40 mV, versus the step potential. Voltage-dependent inactivation was plotted as the magnitude of the current at the end of the 2.5-second test pulse (normalized to the maximum test current obtained) and plotted as a function of the step potential of the conditioning pulse.

View this table: [in this window] [in a new window]   Table 1. Biophysical Characteristics of Neuronal Kv current, Kv2.2, and Kv3.1b

We have previously reported that Ang II decreases delayed rectifier K⁺ current in cultured neurons in a Gq/protein kinase C (PKC)/calcium/calmodulin protein (CAM) kinase II–dependent manner.^{11 25} Therefore, the effect of Ang II on the quinine-sensitive K⁺ current was next examined. Figure 4 illustrates that Ang II (100 nmol/L), in the presence of the AT₂ receptor blocker PD123,319 (1 µmol/L), is capable of decreasing K⁺ current, an effect that is reversible on washout. Quinine (50 µmol/L) also inhibits K⁺ current but to a greater extent. In the presence of quinine, Ang II produced no significant reduction in K⁺ current. These results suggest that the regulation of Kv current by Ang II is via a quinine-sensitive Kv channel(s).

View larger version (20K): [in this window] [in a new window]   Figure 4. Ang II inhibition of the quinine-sensitive K+ current. A, Representative current traces illustrating the effect of Ang II (100 nmol/L) in control conditions and in the presence of quinine (50 µmol/L). K+ currents were evoked by a voltage step from a holding potential of –80 mV to +50 mV followed by repolarization to –50 mV. B, Effects of Ang II and quinine on K+ current (n=4). *P<0.05, **P<0.01.

To test for the possibility that Kv2.2 and/or Kv3.1b might be contributing to the outward current in these neurons, RT-PCR analysis was performed. Amplification of total RNA followed by separation of amplification products on agarose gels revealed products of the expected size for Kv2.2 (550 bp) and Kv3.1b (437 bp) when RT was included in the reaction (Figure 5A, lanes 3 and 9) but not when this enzyme was omitted (Figure 5A, lanes 2 and 8), suggesting the presence of Kv2.2 and Kv3.1b mRNA. Given that these neuronal cocultures are contaminated by a small population (10%) of astroglial cells, it was possible that the observed product was amplified from the glial cells and not from the neurons. This possibility was tested in 2 ways. Total RNA was isolated from neonatal rat astroglial cultures and subjected to RT-PCR with identical primers to those used in the neuronal cocultures. Amplification of RNA isolated from these pure astroglial cultures failed to produce a band for Kv2.2 or Kv3.1b when separated on an agarose gel, despite the fact that the RNA was intact as evidenced by the amplification of ß-actin from the same RNA sample (data not shown). Because these astroglial cultures must be grown under different serum conditions than the neuronal cocultures, an argument could be made that this could be affecting the expression of Kv2.2 and Kv3.1b. To rule out this possibility, neuronal cocultures were treated with 100 mmol/L KCl for 10 minutes to kill the neurons. The KCl was washed out, and the remaining glial cells were allowed to grow under identical serum conditions as the neuronal cocultures for a period of 1 week. At this time, there were no neurons present as determined by visual inspection. RNA was isolated from these cultured glial cells and subjected to RT-PCR. Again, no expression of Kv2.2 or Kv3.1b could be detected in the glial cultures (Figure 5A, lanes 5 and 11), although the RNA was intact as shown by amplification of ß-actin (Figure 5A, lanes 6 and 12).

View larger version (42K): [in this window] [in a new window]   Figure 5. Expression of Kv2.2 and Kv3.1b in cultured neurons. A, Agarose gel showing RT-PCR products for Kv2.2 and Kv3.1b. Lanes 1 and 7 show a 100-bp DNA ladder with the high-intensity band representing 600 bp. Lanes 2, 3, 8, and 9 represent RNA isolated from neurons, and lanes 4, 5, 10, and 11 represent RNA isolated from glial cells after the neurons were killed with KCl. The 550- and 437-bp products depicted in lanes 2 and 9 correspond to the predicted size of the amplification product based on the specific primers used for Kv2.2 and Kv3.1b. Lanes 1, 3, 8, and 10 are negative controls in which RT was omitted from the reaction mix using cultured neurons or glial cells. Lanes 6 and 12 represent amplification of ß-actin from the glial cultures. B, Representative Western blot analysis showing the presence of Kv2.2 in hypothalamus and brain stem neuronal cultures and rat brain regions. C, Representative Western blot analysis showing the presence of Kv3.1b in selective rat brain regions but not in cultured neurons. Experiments were repeated at least 3 times.

We then performed Western blot analyses to investigate whether Kv2.2 and Kv3.1b proteins were present in the cultured neurons. Figure 5B illustrates that Kv2.2 (125 kDa) is present in the cultured neurons as well as adult cortex, hypothalamus/brain stem, cerebellum, and hippocampus. However, Kv3.1b (97 or 85 kDa) is not present in either the cultured neurons or in the adult hypothalamus/brain stem but is present in cortex, hippocampus, and cerebellum. Taken together with the RT-PCR data, these data show that the mRNAs for both Kv2.2 and Kv3.1b are present in neurons from rat hypothalamus and brain stem, but only protein for Kv2.2 is expressed.

We directly tested the possibility that Ang II is inhibiting Kv2.2 using the Xenopus oocyte expression system. The mRNAs for the AT_1A receptor and for Kv2.2 (Figure 6) were injected into oocytes. One to 4 days after injection, a family of currents was elicited in the oocytes from a holding potential of -80 mV with steps to potentials ranging from -50 to +50 mV followed by repolarization to -40 mV. Application of Ang II (100 nmol/L) for 15 minutes resulted in a slowly developing inhibition of the outward current in oocytes coinjected with the AT_1A receptor (0.73±0.03 of control at +50 mV, P<0.001; n=6) but not in oocytes injected only with Kv2.2 (0.99±0.02 of control at +50 mV, n=7) (Figure 6A through 6C). Pretreatment of the oocytes with losartan (1 µmol/L) prevented the Ang II–induced inhibition of Kv2.2 (0.93±0.02 of control at +50 mV, n=8), but after washout of losartan, a second application of Ang II decreased the outward current (0.67±0.09 of control at +50 mV, n=4) (Figure 6D). The time course of inhibition of Kv2.2 and lack of washout by Ang II were similar to inhibition of Kv1.5 and Kv1.2 by acetylcholine when coexpressed with the human M3 receptor in oocytes or COS cells.²⁶ These experiments demonstrate that the inhibition of Kv2.2 is mediated through an AT₁ receptor and not through the endogenous oocyte angiotensin receptor, because the endogenous receptor is not inhibited by losartan.²⁷

View larger version (25K): [in this window] [in a new window]   Figure 6. AT1A receptor–mediated inhibition of Kv2.2 in Xenopus oocytes. A, Representative traces demonstrating inhibition of Kv2.2 current by Ang II (100 nmol/L) in oocytes coinjected with mRNA encoding Kv2.2 and the AT1A receptor. K+ currents were evoked by a voltage step from a holding potential of –80 mV to +50 mV followed by repolarization to –40 mV. A slowly developing inhibition of Kv2.2 can be seen after 15 minutes of exposure to Ang II, which continued to develop over a 10-minute wash period. B, Peak I-V relationship recorded from the oocytes in panel A. , control; , 15 minutes of Ang II; and , 10 minutes of wash. C, Time course of Ang II–induced inhibition of Kv2.2 in oocytes injected with either mRNA encoding Kv2.2 alone () or Kv2.2 plus the AT1A receptor (). Kv2.2 currents were measured at a test potential of +50 mV and normalized to the magnitude of current measured under control conditions (n=6 to 9). Ang II was applied for 15 minutes as indicated, followed by a 30-minute wash. D, Pretreatment of 6 oocytes with the AT1 receptor antagonist losartan (1 µmol/L) prevented the Ang II–induced inhibition of Kv2.2 current. In 4 oocytes, reexposure to Ang II after washout of losartan inhibited the current.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The existence of a local renin-angiotensin system in the brain is now well established.²⁸ In addition, specific nuclei within the brain, collectively known as the circumventricular organs, lie outside the blood-brain barrier and possess AT₁ receptors that sense circulating levels of Ang II.²⁹ Centrally acting Ang II has been shown to induce release of AVP via a catecholaminergic pathway in the brain,^{30 31} increase sympathetic vasomotor activity,^{8 9} and therefore regulate a number of cardiovascularly relevant physiological events. Support for this idea comes from recent evidence demonstrating that AT₁ and AT₂ receptors are colocalized on catecholaminergic neurons.³² Both the increase in sympathetic activity and release of AVP have been implicated as mediators of the vasopressor, elevated dipsogenic, and Na⁺ appetite responses and the increase in fluid volume observed with intracerebroventricular. injection of Ang II. However, the underlying cellular mechanism for these responses is unclear.

Ang II, via AT₁ receptors, results in an inhibition of neuronal K⁺ current in several cell types.^{11 13 33} This inhibition would depolarize or alter the refractoriness of the neuronal membrane, resulting in an increase in neuronal firing rate. Therefore, modulation of the specific K⁺ channel(s) inhibited by Ang II represents a potential therapeutic target site in the treatment of a variety of cardiovascular diseases including hypertension. However, the specific K⁺ channel(s) that is inhibited by Ang II has not been elucidated.

In the present study, we characterized the outward K⁺ current in neuronal cocultures of rat brain stem and hypothalamus in an attempt to identify the specific K⁺ conductance(s) modulated by Ang II. Electrophysiological experiments were used to define the biophysical properties of activation, inactivation, and recovery from inactivation for the total outward K⁺ current. However, the results from these studies are consistent with the properties of several different types of Kv channels.¹⁵ Pharmacological dissection of Kv current was then used to try and show which Kv channel subunit(s) was modulated by Ang II. It was first determined that a large portion (80%) of total outward K⁺ current was sensitive to low doses of quinine (IC₅₀ 21.7 µmol/L), TEA, and 4-AP. These data narrowed the candidate K⁺ channel subunits to Kv1.1, Kv1.3, Kv2.2, and Kv3.1b. Additional pharmacological analyses showed that total Kv current was insensitive to charybdotoxin, -DTX, and MCDP. Because Kv2.2 and Kv 3.1b show a unique sensitivity to low doses of quinine when expressed in Xenopus oocytes,^{15 16 23} have similar sensitivities to TEA and 4-AP, and are relatively insensitive to charybdotoxin, -DTX, and MCDP, this suggested the presence of Kv2.2 and Kv 3.1b in these neuronal cultures. Analysis of the quinine-sensitive K⁺ current revealed a conductance with similar biophysical properties to those reported for Kv2.2 and Kv3.1b, and in the presence of low doses of quinine, which is relatively specific for Kv2.2 and Kv3.1b, Ang II produced no inhibitory effect on neuronal K⁺ current. RT-PCR analyses confirmed the presence of Kv2.2 and Kv3.1b mRNA, lending additional support to the idea that Kv2.2 and/or Kv3.1b are components of total outward K⁺ current in these neurons. However, Western blot analyses demonstrated that of these 2 subunits, only Kv2.2 is present in the cultures.

The pharmacological and molecular data provided in the present study suggest that total outward K⁺ current is composed of a nonhomogeneous population of channels, because 20% to 30% of the current remained, despite the use of maximum concentrations of several inhibitors. Pharmacological dissection of total outward K⁺ current is made even more difficult by the fact that there are no specific inhibitors or blocking antibodies against Kv2.2, and, therefore, it was not possible to determine whether Ang II was solely inhibiting Kv2.2. Because the initial goal of the present study was to determine the specific K⁺ channel(s) present in these neurons that are inhibited by Ang II, we chose to use the Xenopus oocyte expression system to determine if Kv2.2 was inhibited by Ang II. The observed losartan-sensitive inhibition of Kv2.2 by Ang II suggests that these channels may be, at least partially, responsible for the observed Ang II–induced inhibition in cocultures of rat hypothalamus and brain stem. However, definitive in vitro proof of this observation will have to await the development of a specific inhibitor of Kv2.2.

Although the results from the present study have demonstrated the inhibition of Kv2.2 by Ang II, they have not ruled out the possibility that Ang II modulates other K⁺ channels. The present study also has not addressed the second messenger system through which this inhibition occurs. The AT₁ receptor is traditionally believed to be coupled through a G-protein transduction mechanism involving the G_q/G₁₁ family. This family of G proteins typically activates phospholipase C-ß (PLC-ß), which catalyzes the hydrolysis of phosphatidylinositol-4,5-bisphosphate to inositol-1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). IP₃ promotes Ca²⁺ release from the endoplasmic reticulum, and DAG activates PKC. Both of these pathways have been shown to be important in neuronal AT₁ receptor signaling,¹¹ but exactly how these mediators ultimately modulate neuronal K⁺ channels is unknown. Kv2.2 has a well-conserved PKC phosphorylation site between the S4 and S5 transmembrane segments.¹⁵ We have recently shown,³⁴ using the oocyte expression system, that the inhibition of Kv2.2 by Ang II is blocked by PKC inhibitors and intracellular BAPTA, similar to that which was shown by Sumners et al¹¹ in hypothalamic/brain stem neurons in culture. Thus, it is possible that the Ang II–induced inhibition of Kv current is via a Ca²⁺-dependent, PKC-mediated phosphorylation of Kv2.2. In neurons, there is also evidence that AT₁ receptors couple to a stimulation of mitogen-activated protein (MAP) kinase.^{35 36} However, it is unclear if MAP kinases are also involved in the modulation of neuronal Kv current.

The discovery in the present study of a specific Kv channel inhibited by Ang II will allow for a more detailed examination of the intracellular signaling mechanism of the AT₁ receptor in neurons that represent important targets for the regulation of cardiovascular events. It will also allow for a more direct assessment of whether cross-talk occurs between AT₁ and AT₂ receptors. The answers to these questions will be the focus of future research.

	Acknowledgments

 
This work was supported by grants from the National Institutes of Health HL-49130 and NS-19441 (to C.S. and C.H.G.); DK-41315 (to B.H. and J.L.K.); American Heart Association (Florida Affiliate), Postdoctoral Fellowship (to J.D.W.); and the Council for Tobacco Research (to C.H.G.). We would like to thank Dr Jeffrey K. Harrison for the AT₁ receptor cDNA.

Received August 27, 1998; accepted December 20, 1998.

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