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J. Biol. Chem., Vol. 277, Issue 3, 1974-1980, January 18, 2002

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Transforming Growth Factor-₁ Regulates Kir2.3 Inward Rectifier K⁺ Channels via Phospholipase C and Protein Kinase C- in Reactive Astrocytes from Adult Rat Brain^*

Pablo R. Perillan^§, Mingkui Chen^§, Eric A. Potts, and J. Marc Simard^§¶

From the Departments of  Neurosurgery, ^§ Pathology, and ^¶ Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201

Received for publication, August 20, 2001, and in revised form, November 8, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The multifunctional cytokine, transforming growth factor ₁ (TGF-₁), exerts complex effects on astrocytes with early signaling events being less well characterized than transcriptional mechanisms. We examined the effect of TGF-₁ on the 14-pS Kir2.3 inward rectifier K⁺ channel in rat primary cultured reactive astrocytes. Immunofluorescence study showed that cells co-expressed TGF-₁ receptors 1 and 2, Kir2.3, and glial fibrillary acidic protein (GFAP). Patch clamp study showed that TGF-₁ (0.1-100 ng/ml) caused a rapid (<5 min) depolarization because of dose-dependent down-regulation of Kir2.3 channels, which was mimicked by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (10-500 nM) and which was inhibited by the PKC inhibitor calphostin C (100 nM), by PKC desensitization produced by 3 h of exposure to phorbol 12-myristate 13-acetate (100 nM), and by the PKC- isoform-specific inhibitor rottlerin (50 µM). Immunoblot analysis and confocal imaging showed that TGF-₁ caused PKC- translocation to membrane, and co-immunoprecipitation experiments showed that TGF-₁ enhanced association between Kir2.3 and PKC-. Additional electrophysiological experiments showed that Kir2.3 channel down-regulation was blocked by the phospholipase C inhibitors, neomycin (100 µM) and D609 (200 µM). Given the commonality of signaling involving PLC-PKC-, we speculate that TGF-₁-evoked depolarization may be an early signaling event related to gene transcription in astrocytes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Transforming growth factor (TGF)¹ ₁ is a multifunctional, contextually acting cytokine that is neuroprotective and that contributes to tissue repair following brain injury (1-3). TGF-₁ exerts a number of important effects on astrocytes, including growth inhibition, cell aggregation, extracellular matrix production, and chemotaxis, making TGF-₁ an important regulator of reactive astrocytes in brain injury (1).

Astrocytes express two distinct inward rectifier K⁺ channels: the 25-pS weakly rectifying Kir4.1 channel and the 13-pS strongly rectifying Kir2.3 channel, which together maintain the cell stably polarized near the potassium reversal potential (E_K) (4-6). When an astrocyte becomes activated by injury, the Kir4.1 channel appears to be selectively down-regulated (7, 8), leaving only a small residual current caused by the Kir2.3 channel (6). Although small in magnitude, the Kir2.3 current is sufficient to maintain the reactive astrocyte polarized to a resting potential near E_K (9). However, if this channel were also to be down-regulated, one would predict that the cell would no longer maintain a polarized resting potential but would depolarize, presumably to a potential near the chloride reversal potential (E_Cl).

The Kir2.3 channel is a phosphoprotein, with phosphorylation of threonine 53 by protein kinase C (PKC) causing a reduction in open probability (10, 11). Among the signaling pathways identified in astrocytes that could activate PKC and thereby down-regulate Kir2.3, we reasoned that TGF-₁ might serve this role. TGF-₁ signaling involves multiple complex pathways, including Smad proteins, G proteins, and mitogen-activated protein kinases (12, 13). Of particular significance, PLC and the  isoform of PKC (PKC-) have been found to be critical in early aspects of TGF-₁ signaling related to gene transcription (14-17). We thus hypothesized that TGF-₁ might activate this PLC-PKC pathway in reactive astrocytes, resulting in down-regulation of Kir2.3 and depolarization of the cells.

In the present study, we examined this hypothesis by assessing the effect of TGF-₁ on Kir2.3 currents in primary cultured reactive astrocytes isolated from adult rat brain. In previous work with the same preparation of cells, we showed by immunofluorescence and electrophysiological techniques that these cells express 14-pS Kir2.3 channels and that current at potentials negative to E_K is due exclusively to these channels (6). We thus could exploit the simplicity of this system to examine regulation of Kir2.3 channels by an intrinsic receptor-mediated mechanism in a native cell system. Here, we report that TGF-₁ receptors are expressed on primary cultured reactive astrocytes and that application of physiological concentrations of TGF-₁ rapidly depolarizes the cells by down-regulating Kir2.3 channels. In addition, we provide evidence that PLC as well as the PKC isoform PKC- are involved in the signaling pathway mediating cell depolarization. Down-regulation of Kir2.3 channels by TGF-₁ via a PLC-PKC- pathway provides a novel mechanism for regulating the resting membrane potential of reactive astrocytes and provides new insight into early signaling events induced by TGF-₁.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture-- Primary cultures of adult reactive astrocytes were derived from gelatin sponge pellets that had been implanted into a stab wound in the parietal lobe of adult Wistar rats as previously described (6, 9). Gelatin sponge pellets were harvested at 8 days and washed three times in phosphate-buffered saline to remove adherent tissue. Washed pellets were minced into 1-mm lengths in a solution containing serum-free culture medium, yielding 4-9 pieces of 10-15 mg of wet weight each. The end pieces were discarded, and the remaining pieces were placed into individual 35-mm tissue culture dishes (Corning, Corning, NY) with 1.2 ml of culture medium. The pieces of gelatin sponge were cultured in Dulbecco's modified essential medium (Invitrogen), supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), penicillin (100 units/ml) (Sigma), and streptomycin (100 mg/ml) (Biofluids, Rockville, MD). The cultures were maintained in a humidified atmosphere with 10% CO₂ in air. The culture medium was changed every 3 days.

A cortical collecting duct cell line (CCD cells), M1, obtained from the American Type Culture Collection (Manassas, VA), was used as a positive control for Kir2.3 channel expression (18, 19). The cells were grown in a 1:1 mixture of Ham's F-12 medium and Dulbecco's modified essential medium containing 5 µM dexamethasone (Sigma) supplemented with 5% fetal bovine serum. The cells were maintained in a humidified atmosphere of 5% CO₂/95% air at 37 °C. The culture medium was changed every other day.

Electrophysiology-- The experiments were carried out on cells in primary culture for 1-14 days. Cell cultures were washed three times with extracellular bath solution (see below) at room temperature prior to electrophysiological recording but were not otherwise treated. The membrane currents were amplified (Axopatch 200A, Axon Instruments) and sampled on-line at 5 KHz using a microcomputer equipped with a digitizing board (Digidata 1200A; Axon Instruments) and running Clampex software (version 7.0.42 or version 8.0; Axon Instruments). All of the experiments were performed at room temperature (22-25 °C).

For some experiments (see Figs. 2A and 4, A and B), we used cell-attached patches to measure single channel currents. Pipettes with resistances of 2-3 M were used that were made from borosilicate glass (Kimax; Fisher). Membrane currents were measured during hyperpolarizing step pulses (200 ms), using a pipette potential of 67.5 mV. To measure the membrane potential (E_m) for calculating the reversal potential (E_rev), the recording configuration was converted at the end of single channel recording from the cell-attached configuration to a conventional whole cell configuration.

Macroscopic currents were recorded using a nystatin-perforated whole cell technique (20). Pipettes with resistances of 2-4 M were used. Cells with seal resistances of <3 G and access resistances of >50 M were discarded. The membrane currents were measured during 200-ms step pulses (from 160 to 0 mV in 10-mV steps) or during ramp pulses (140 to +50 mV at 0.32 mV/ms), both from a holding potential of 60 mV.

Recording Solutions-- For cell-attached patch recording of single channels (see Figs. 2D and 4B), we used an extracellular solution that contained 130 mM NaCl, 10 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 32.5 mM Hepes, 12.5 mM glucose, pH 7.4, and a pipette solution that contained 145 mM KCl, 1 mM MgCl₂, 0.2 mM CaCl₂, 10 mM EGTA, 10 mM Hepes, pH 7.3.

To record macroscopic currents using a nystatin-perforated patch technique, the base pipette solution contained 55 mM KCl, 75 mM K₂SO₄, 8 mM MgCl₂, 10 mM Hepes, pH 7.2. Nystatin was solubilized in Me₂SO. Working solutions were made before each experiment by adding 16.5 µl of nystatin stock solution to 5 ml of the base pipette solution to yield a final concentration of nystatin of 165 µg/ml and Me₂SO 3.3 µl/ml (21). The measured osmolarity of the base pipette solution was 290 mosM.

TGF-₁ obtained from Sigma was always placed in solution supplemented with 1 mg/ml albumin (Sigma). Phorbol 12-myristate 13-acetate (PMA), calphostin-C, staurosporin, rottlerin, Go6976, neomycin, U73122, and D609 were obtained from Calbiochem (San Diego, CA).

Immunofluorescence and Confocal Microscopic Imaging-- Cultures grown on chamber slides (LAB-TEK, Naperville, IL) were rinsed with phosphate-buffered saline and then fixed with a 4% paraformaldehyde/phosphate-buffered saline solution at 4 °C for 10 min. Fixed cultures were incubated 1 h at room temperature, followed by incubation overnight at 4 °C with anti-TGF-₁-R1 and anti-TGF-₁-R2 rabbit polyclonal antibodies (1:400 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), anti-GFAP monoclonal antibodies (1:5000 dilution; Sigma), or anti-Kir2.3 polyclonal antibodies (1:800 dilution; a kind gift from Dr. David S. Bredt, University of California, San Francisco, CA) in phosphate-buffered saline supplemented with 1% bovine serum albumin. After several washes, primary antibodies were detected using Alexa 546-conjugated and Alexa 488-conjugated species-appropriate secondary antibody for GFAP and Kir2.3, respectively (1:200 dilution; Molecular Probes, Eugene, OR). The cultures were mounted using ProLong antifade mounting medium (Molecular Probes). For epifluorescence imaging, immunolabeled cultures were examined on a Nikon Diaphot microscope equipped with a 10× PL FLUOTAR (Leitz Wetzlar), a 63× PL APO (Leitz Wetzlar), and other objective lenses. Images were captured using a SenSys digital camera (Roper Scientific Inc.) and processed using a personal computer (Dell, Pentium III) running IPLab software (version 8.0; Scanalytics, Inc.). For confocal imaging, the samples were examined using a Zeiss LSM510 confocal microscope. Post-processing of images was performed using Adobe Photoshop (version 5.0; Adobe Systems Inc.). The controls for the immunostaining procedures included kidney, lung, and smooth muscle cells, as well as omission of primary antibodies.

Western Blot Analysis and Co-immunoprecipitation-- Cultured astrocytes were rinsed with 0.25% trypsin-0.03% EDTA solution and incubated at 37 °C for 15 min until cells detached. The cell suspension was aspirated and centrifuged at 1,000 × g for 3 min, and the pellet was lysed in liquid nitrogen, then thawed, and solubilized in lysis buffer, which contained 50 mM Tris-HCl, pH 7.4, 250 mM NaCl, and 0.1% Triton X-100, supplemented with the phosphatase and protease inhibitors, 5 mM NaF, 0.1 mM sodium orthovanadate, 5 mg/ml leupeptin, 10 mg/ml aprotinin, 50 mg/ml phenylmethylsulfonyl fluoride, and 5 mg/ml pepstatin A. Cytosolic and membrane fractions were separated by centrifugation at 16,000 × g for 10 min at 4 °C. Following normalization of protein content, 30-µg samples of total protein extract were fractionated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes using standard techniques. Immunoblotting was performed using anti-Kir2.3 (1:400 dilution; Alomone, Jerusalem, Israel), anti-GFAP (1:1000; Sigma), and isoform-specific anti-PKC (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies, with detection carried out using the ECL system (Amersham Biosciences, Inc.). Levels of immunoreactive proteins were determined by densitometric scanning using an ImageMaster video documentation system, with image acquisition and analysis carried out using ImageMaster video documentation system software, version 2.0 (Amersham Biosciences, Inc.).

For co-immunoprecipitation, membrane fractions were preincubated with 20 µl/ml protein A-agarose beads (Amersham Biosciences, Inc.) plus preimmune rabbit serum for 1 h to remove nonspecific binding. Protein was then immunoprecipitated for 2 h at 4 °C with 2 µg/ml rabbit anti-Kir2.3 antibody (Alomone) together with 20 µl/ml protein A-agarose. The beads were subsequently washed four times in 0.5 ml of immunoprecipitation buffer (100 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.2% SDS, 2 mM EDTA, 10 mM Hepes, 1 mM sodium orthovanadate, pH 7.5) and boiled in 30 µl of 2× sample buffer. The supernatant was immunoblotted as described above.

Data Analysis-- Holding currents were not subtracted from recordings. Difference currents were obtained simply by subtracting currents recorded at two different times during ramp pulses with no other processing employed. To quantify activity of Kir2.3 channels in macroscopic recordings as a function of time (see Figs. 3D, 5D, 6A, and 7A), we measured the current at 130 mV (I₁₃₀) during 200-ms pulses from holding potential of 60 mV; no further processing was required, because no other channel but Kir2.3 is active at this potential (6). Fractional block of Kir2.3 was determined by dividing I₁₃₀ after treatment by I₁₃₀ before treatment. Concentration-response data were fit to a standard logistic equation (see Fig. 3E) using the Levenberg-Marquardt algorithm as implemented in Origin 6.1 (Microcal Software, Northampton, MA). The data are given as the means ± S.D. Statistical analysis was carried out using Student's t test for unpaired samples or analysis of variance (see Figs. 6D and 7B).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Identification of TGF-₁ Receptors and Kir2.3 Channels-- Immunofluorescence study of primary cultured reactive astrocytes was performed to demonstrate expression of TGF-₁-receptors (TGF-₁-R1 and TGF-₁-R2). In agreement with previous observations (22, 23), the cells studied at 5-7 days in culture demonstrated abundant label for both TGF-₁-R1 and TGF-₁-R2 (Fig. 1). Double labeling of cultures for either Kir2.3 or GFAP showed positive signal (Fig. 1) (6), confirming that primary cultured GFAP-positive reactive astrocytes express both TGF-₁-R1 and TGF-₁-R2, as well as Kir2.3 channels.

View larger version (79K): [in this window] [in a new window]   Fig. 1.   TGF-1 receptors, Kir2.3, and GFAP co-localize on primary cultured reactive astrocytes. Immunofluorescence images show label for TGF-1 receptors, TGF-1-R1 and TGF-1-R2, Kir2.3, and GFAP, as indicated. The upper panels show a single cell double-labeled for GFAP and Kir2.3. Different cells are shown in the lower panels.

Cell-attached recordings were used to demonstrate single channel activity consistent with Kir2.3 inward rectifier channels. As previously reported (6), inward currents were recorded at negative potentials (Fig. 2A) that exhibited a slope conductance of 14.2 pS (Fig. 2B), which is characteristic for Kir2.3 (6, 10, 11). At potentials positive to the K⁺ reversal potential (E_K), however, no single channel activity was observed (Fig. 2A), consistent with a strongly rectifying channel.

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Identification of Kir2.3 in reactive astrocytes. A, single channel records from a cell-attached patch showing channel openings at negative potentials but not at positive potentials. The channel openings are plotted upward, with the open state denoted by dotted lines. B, plot of single channel amplitude (means ± S.E.) versus membrane potential with fit to linear equation indicating a slope conductance of 14.2 pS; the data were from seven patches. C, immunoblot showing Kir2.3 signal in primary cultured reactive astrocytes. CCD-M1 cells were used as positive control.

The immunofluorescence experiments on Kir2.3 reported above utilized the same antibody that we had previously used (6). To further confirm the presence of Kir2.3, we performed immunoblots utilizing a different antibody raised against a different epitope of Kir2.3. As shown in Fig. 2C, this antibody recognized, in primary cultured reactive astrocytes, the same molecular weight protein band as in the CCD-M1 cells, giving further evidence that these cells express Kir2.3 channels.

Regulation of Kir2.3-- In initial experiments, we confirmed that the resting potential of primary cultured reactive astrocytes was sensitive to Ba²⁺, consistent with the interpretation that the resting potential of these cells is maintained by a Ba²⁺-sensitive inward rectifier channel (9). Cells studied under current clamp exhibited a negative resting potential (65.1 ± 3.8 mV, 11 cells), only slightly more positive than E_K, 67.5 mV. As previously shown (9), the addition of 1 mM Ba²⁺ to the bath solution caused a rapid, reversible depolarization of the cells (Fig. 3A).

View larger version (32K): [in this window] [in a new window]   Fig. 3.   TGF-1 depolarization is due to down-regulation of Kir2.3 channel. A, current clamp recording showing reversible depolarization after the addition of 1 mM Ba2+ to the bath, followed by depolarization after addition of 10 ng/ml TGF-1 to the bath, as indicated by the bars. B, currents measured during step pulses under control conditions (panel a) and after the addition of 10 ng/ml TGF-1 to the bath (panel b); the currents during pulses to 130 mV are shown separately (panel c). C, current-voltage curves for the data in B, with data from the control family denoted by a, data after TGF-1 denoted by b, and the difference current denoted by a-b. D, magnitude of I130 (circles) and of current at the holding potential (squares), before and after the addition of 10 ng/ml TGF-1 to the bath, as indicated by the bar. E, concentration-response data (means ± S.E.) for fractional inhibition of I130 by TGF-1; one concentration per cell; four to nine cells at each concentration; fit to logistic equation indicated a half-maximum effect at 6 ng/ml.

We also measured the resting potential after exposure to TGF-₁. Application of 10 ng/ml TGF-₁ at room temperature caused the cells to depolarize within min of addition of the cytokine (Fig. 3A). Although the response to TGF-₁ was somewhat slower than that with Ba²⁺, it occurred over a time frame inconsistent with a transcriptional mechanism. In 15 cells, a depolarization of 22.3 ± 8.4 mV was observed 10 min after application of 10 ng/ml TGF-₁.

We used a nystatin-perforated patch technique and voltage clamp recordings to examine whether TGF-₁-induced depolarization might be due to an effect on inward rectifier current. Currents recorded at various potentials during step pulses (Fig. 3B, panel a) showed the pattern of inward current at negative potentials, outward current at positive potentials, and a flat plateau region in between (Fig. 3C, a), as previously observed (6). Application of 10 ng/ml TGF-₁ caused a strong diminution of the Kir2.3 current within moments of addition of the cytokine (Fig. 3, B, panel b, and C, b). The effect was readily appreciated at 130 mV (Fig. 3B, panel c) and occurred in <5 min after cytokine application (Fig. 3D). A plot of the concentration-response relationship for the effect of TGF-₁ on Kir2.3 current at 130 mV is shown in Fig. 3E, showing that the effect of TGF-₁ was concentration-dependent and occurred at physiologically relevant concentrations of the cytokine.

In some cells, the outward current was noticeably decreased by TGF-₁ (Fig. 3C, b), whereas in other cells, it was essentially unaffected. The response of outward currents to TGF-₁ was not further characterized in the present study.

We also carried out single channel experiments using the cell attached configuration to confirm that the inward rectifier current that was down-regulated by TGF-₁ was due to Kir2.3 channels. A typical record, shown at low temporal resolution (Fig. 4A) and at higher temporal resolution (Fig. 4B), illustrates numerous channel openings under control conditions (Fig. 4, A and B, sections 1), with fewer openings after the addition of 10 ng/ml TGF-₁ (Fig. 4, A and B, sections 2) and partial return of activity after washout of cytokine (Fig. 4, A and B, sections 3). Quantitative analysis of the probability of channel opening (n·P_o) in a different patch showed a large reduction, from 0.32 to 0.06, with 10 ng/ml TGF-₁ (Fig. 4C). Similar results were observed in six other patches. A decrease in activity of a 14-pS conductance following addition of TGF-₁ is consistent with specific down-regulation of Kir2.3 channels. In addition, demonstration of this effect in a cell-attached configuration confirms that loss of channel activity was not due to a direct extracellular block of the channel but was likely mediated by an intracellular second messenger.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   TGF-1 down-regulates 14-pS Kir2.3 channel. A and B, single channel records obtained at 130 mV in a cell-attached patch at low temporal resolution (A) and at higher temporal resolution (B), before (section 1) and after (section 2) the addition of 10 ng/ml TGF-1 to the bath and after the washout of cytokine (section 3). The channel openings are plotted upward, with the open state denoted by dotted lines. C, all points histogram of records before (upper panel) and after (lower panel) the addition of 10 ng/ml TGF-1 to the bath, with data fit to a double-Guassian function, indicating the values of n·Po of 0.32 and 0.06 for the two sets of data, respectively.

Kir2.3 channels are known to be down-regulated by PKC-mediated phosphorylation (10, 11). We thus sought to determine whether the effect of TGF-₁ could be mimicked by stimulation of PKC using the phorbol ester, PMA. When studied using a whole cell, perforated patch technique, application of 10-500 nM PMA caused a strong diminution of the Kir2.3 current, similar to that observed with Ba²⁺ or TGF-₁ (Fig. 5, A and B). In six cells, application of 500 nM PMA caused a 92.7 ± 7.4% diminution of Kir2.3 current over the course of 5-6 min, an effect that was significant (p < 0.01, by t test) (Fig. 5D). Also, when studied at the single channel level using a cell-attached configuration, application of 500 nM PMA caused a strong diminution of the 14-pS channel, similar to that observed with TGF-₁ (Fig. 5C). Thus, at both the macroscopic and single channel levels, the down-regulatory effect of TGF-₁ on Kir2.3 current was mimicked precisely by activation of PKC.

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Phorbol ester mimics effect of TGF-1. A, voltage clamp recordings showing the current during step pulses under control conditions (top panel), after the addition of 10 nM PMA to the bath (middle panel), and after the addition of 500 nM PMA to the bath (bottom panel). B, current-voltage curves for the data in A, with data from the control family denoted by a, data after 500 nM PMA denoted by b, and the difference current denoted by a-b. C, single channel current before (a) and after (b) 500 nM PMA; cell-attached patch with pipette potential = 0 mV. D, magnitude of normalized I130 (means ± S.E.) in four control cells (squares) and in six cells exposed to 500 nM PMA (circles).

Confirming involvement of PKC in signaling induced by TGF-₁ requires that the effect of TGF-₁ be prevented by blockers of PKC. We thus assessed the effect of pretreatment of cells with the pan-specific PKC inhibitors, staurosporin (25 nM; Fig. 6A) and calphostin C (100 nM; not shown), on the response to TGF-₁. In six and five cells pretreated with these two agents, respectively, application of 10 ng/ml TGF-₁ had no effect on the Kir2.3 current recorded under voltage clamp (Fig. 6A).

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Protein kinase C is required for TGF-1 signaling. A, magnitude of I130 (circles) and of current at the holding potential (squares), in the presence of 25 nM staurosporin, before and after the addition of 10 ng/ml TGF-1 to the bath, as indicated by the bars. B, voltage clamp recordings showing current during step pulses after 3 h of pretreatment with 100 nM PMA (top panel), after the addition of 10 ng/ml TGF-1 to the bath (second panel), after the addition of 1 mM Ba2+ to the bath (third panel), and after the washout of Ba2+ (bottom panel). C, current clamp recording of a cell after 3 h of pretreatment with 100 nM PMA, before and after the addition of 10 ng/ml TGF-1 and 1 mM Ba2+ to the bath, as indicated by the bars. D, bar graph showing inhibitory effect of 10 ng/ml TGF-1 on Kir2.3 current at 130 mV under control conditions (14 cells) and in the presence of 50 µM rottlerin (14 cells) or 100 nM Go6976 (13 cells); pairwise multiple comparison indicated a significant difference (p < 0.05) with rottlerin but not with Go6976.

Another method for assessing the involvement of PKC involves prolonged exposure to PMA to desensitize PKC. This method is also useful for evaluating involvement of one of three classes of PKC isoforms, based on PKC sensitivity to PMA-induced desensitization (24, 25). Primary cultures were incubated with 0.1 µM PMA for 3 h (n = 3) and for 26 h (n = 3). In both cases, voltage clamp recordings showed the typical current-voltage curves, including a strong inward rectifier current (Fig. 6B, top panel). Application to cells pretreated for 3 h (Fig. 6B, second panel) or to cells pretreated for 26 h (not shown) of 10 ng/ml TGF-₁ had only a very weak or no appreciable effect on the current recorded under voltage clamp. In the same cells, however, 1 mM Ba²⁺ exerted its expected reversible block of Kir2.3 current (Fig. 6B, third and fourth panels), showing that absence of down-regulation by TGF-₁ was not due an abnormal inward rectifier but likely reflected a true down-regulation of PKC by chronic PMA. Also, in cells pretreated 3 h with PMA and studied under current clamp, 10 ng/ml TGF-₁ failed to induce cell depolarization, although Ba²⁺ readily produced a strong reversible depolarization (Fig. 6C).

Loss of TGF-₁-induced down-regulation of Kir2.3 current after only 3-h incubation with PMA excluded involvement of atypical ( and ) PKC isoforms and suggested involvement of a member of the rapidly desensitized group ( or ) rather than a member of the slowly desensitized group (I, II, , , and ) (24). To further evaluate involvement of  versus  PKC isoforms, we performed experiments with the isoform-specific inhibitors, Go6976 and rottlerin, which selectively inhibit PKC- and PKC-, respectively (26, 27). In 14 control cells in this series, 10 ng/ml TGF-₁ produced a 64.6 ± 17.5% inhibition of the Kir2.3 current (Fig. 6D). Pretreatment with the PKC--specific agent, Go6976 (100 nM; 13 cells) at 40 times its EC₅₀ value did not significantly reduce the inhibitory effect of 10 ng/ml TGF-₁, with 56.6 ± 22.2% inhibition of Kir2.3 current being observed (Fig. 6D). In contrast, the PKC--specific agent, rottlerin (50 µM; 14 cells), at 10 times its EC₅₀ value, significantly reduced the inhibitory effect of 10 ng/ml TGF-₁, with only 28.9 ± 17.9% inhibition of Kir2.3 current being observed (Fig. 6D), consistent with involvement of PKC- in TGF-₁-mediated inhibition of Kir2.3.

Involvement of PKC in TGF-₁-induced down-regulation of Kir2.3 current implies that the intrinsic PKC activator, diacylglycerol, must be produced by phospholipid hydrolysis. Previous reports have shown that TGF-₁-induced signaling is likely to involve phosphatidylcholine-specific PLC, based on sensitivity to D609, and is unlikely to involve phosphatidylinositide-specific PLC, based on absence of effect of U73122 (14, 17). We thus tested these two compounds, as well as another phosphatidylinositide-specific PLC inhibitor, neomycin sulfate (28). U73122 proved not to be useful because it rapidly caused cell lysis. Both neomycin (100 µM) and D609 (200 µM), however, were effective inhibitors of TGF-₁-induced down-regulation of Kir2.3 current. As shown in Fig. 7A, pretreatment of cells with neomycin greatly reduced down-regulation of current expected with application of 10 ng/ml TGF-₁, although in the same cell, subsequent application of PMA (500 nM) resulted in the expected reduction in current, consistent with an effect of neomycin upstream of PKC. In three groups of cells, one pretreated with control vehicle (9 cells), one pretreated with neomycin (8 cells), and one pretreated with D609 (9 cells), subsequent application of 10 ng/ml TGF-₁ resulted in 67.4 ± 13.9, 1.2 ± 4.7, and 18.4 ± 9.2% inhibition of Kir2.3 current (p < 0.05 by analysis of variance) (Fig. 7B), confirming involvement of PLC in TGF-₁-induced signaling.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Phospholipase C is required for TGF-1 signaling. A, magnitude of I130 (circles) and of current at the holding potential (squares) in the presence of 100 µM neomycin, before and after the addition of 10 ng/ml TGF-1 and 500 nM PMA to the bath, as indicated by the bars. B, bar graph showing fractional down-regulation of I130 by 10 ng/ml TGF-1 in untreated cells, in cells pretreated with 100 µM neomycin, and in cells pretreated with 200 µM D609, as indicated; the data (means ± S.E.) are for 11, 6, and 5 cells, respectively, in the three groups.

Translocation of PKC--- In the previous experiments, pharmacological evidence for involvement of PKC- was presented, based on rapidity of desensitization and effect of isoform-specific inhibitors (25). We also performed Western immunoblots to determine which of the rapidly desensitized PKC isoforms was translocated from cytosol to membrane fractions by TGF-₁. The cells were incubated for 10 min without or with 10 ng/ml TGF-₁ and then were lysed, and the cytosolic and membrane fractions were studied separately. In unstimulated control cells, both the cytosolic and membrane fractions exhibited signal for PKC- at 78 kDa (Fig. 8B, TGF-₁). In TGF-₁-stimulated cells, a 28.3% reduction of cytosolic levels and a 73.2% increase in membrane levels for PKC- was observed (Fig. 8B, +TGF-₁), an effect that was significant (p < 0.05, by t test). By contrast, TGF-₁ stimulation had no effect on PKC- (Fig. 8A). Translocation of PKC- from cytosol to plasma membrane was corroborated using confocal imaging, which showed an increase in membrane localization of PKC- 10 min after exposure to TGF-₁ compared with control (Fig. 8C, panel b versus panel a).

View larger version (24K): [in this window] [in a new window]   Fig. 8.   TGF-1 activates protein kinase C- and not protein kinase C-. A and B, immunoblots (upper panels) of cytosolic fraction (lanes C) and of membrane fraction (lanes M) of primary cultured astrocytes exposed to control vehicle (TGF-1) or to 10 ng/ml TGF-1 (+TGF-1), developed with anti-PKC- (A) and anti-PKC- (B) antibodies; densitometric analysis (lower panels) of three separate blots for the corresponding lanes shown in the upper panels. *, p < 0.05. C, confocal image of control cells (panel a) and of cells 10 min after exposure to 10 ng/ml TGF-1 immunolabeled for PKC- (panel b). D, co-immunoprecipitation experiment showing enhanced immunoreactivity for PKC- but not PKC- of membrane fraction isolated with Kir2.3 antibody after exposure to 10 ng/ml TGF-1.

Finally, to further establish the association between PKC- and Kir2.3, we performed co-immunoprecipitation experiments on cell membrane fractions using Kir2.3 antibody for protein isolation. Subsequent immunoblots of isolated protein showed that PKC- immunoreactivity was strongly enhanced 10 min after exposure to TGF-₁ (10 ng/ml) compared with controls (Fig. 8D). By contrast, TGF-₁ had no effect on PKC- immunoreactivity (Fig. 8D).

	DISCUSSION

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In this study, we confirmed expression of Kir2.3 inward rectifier K⁺ channels in primary cultured reactive astrocytes from adult rat brain using immunofluorescence, immunoblotting, and single channel measurements. For immunofluorescence, we used an affinity purified, polyclonal antibody raised against the peptide, CRMQAATLPLDNISY, corresponding to the 14-amino acid C-terminal domain of Kir2.3 plus an additional N-terminal cysteine residue (6, 29, 30). For immunoblotting, we used the commercially available, affinity-purified polyclonal antibody raised against the peptide, EFGSHLDLERMQAAYLPLDNY, corresponding to amino acid residues 418-437 of rat Kir2.3 plus an additional N-terminal cysteine residue (31, 32). Both antibodies have been well characterized, with evidence indicating that both demonstrate high specificity for Kir2.3. The immunoblot data obtained with the commercial antibody, which was raised against a different epitope of the Kir2.3 molecule than the original antibody we used, reaffirm our previous finding that the 14-pS, strongly rectifying, inward rectifier K⁺ channel expressed in reactive astrocytes from adult rat brain is the Kir2.3 gene product.

The principal finding of this study is that TGF-₁ depolarizes reactive astrocytes by down-regulating Kir2.3 channels. Despite the widespread effects reported for the TGF-s in central nervous system cells including astrocytes, relatively little has been reported on effects related to ion channels and even less on early signaling events involving nontranscriptional mechanisms. In murine microglia, delayed rectifier K⁺ channels caused by Kv1.3 are up-regulated after 24 h of exposure to TGF-₁ (33). In chick ciliary ganglion neurons, the avian isoform of TGF-₁ exerts complex effects on K_Ca channels, including an alteration of Ca²⁺-gating properties of an intermediate conductance K_Ca channel and biphasic stimulation of K_Ca channels via activation of a MEK1-Erk pathway and is responsible for normal developmental expression of K_Ca channels (34-36). In Aplysia, TGF-₁ increases firing of nociceptive neurons, decreases the firing threshold, and induces long term facilitation (37, 38). Thus, the rapid TGF-₁-mediated down-regulation of Kir2.3 that we observed here is an uncommon observation for this cytokine. By contrast, other cytokines, especially TNF-, have been shown in several studies to have rapid effects on ion channel regulation (39-41).

A second important finding of the present study is that TGF-₁-induced down-regulation of Kir2.3 current is mediated by PKC-. Regulation of Kir2.3 channels has been extensively investigated, especially in expression systems, in which these channels have been found to be regulated by polyamines (42), by G proteins (43), by H⁺ (44-46), by internal Mg²⁺ (47), and by PKC-mediated phosphorylation (10, 11). PKC-mediated down-regulation of inward rectifier current elicited by an intrinsic receptor mechanism has been reported (48-50), but the channels involved were not identified, either by molecular or single channel methods, and may have been Kir family members other than Kir2.3. As for Kir2.3 channels specifically, down-regulation by PKC has been shown previously only in oocyte expression systems. The present study is the first to show a PKC-mediated effect on Kir2.3 channels elicited by an intrinsic receptor mechanism in native cells, affirming the biological importance of this mechanism. The rich repertoire of regulatory mechanisms currently recognized for the Kir2.3 inward rectifier channel, including cytokine-induced regulation as shown here, suggests that this channel plays an important role in the functioning of reactive astrocytes.

Apart from showing the involvement of PKC, we also obtained evidence that it is specifically the  isoform of PKC that is involved in TGF-₁-induced down-regulation of Kir2.3. The PKC family is comprised of at least 12 members, many of which are expressed in astrocytes (51). PKC isoforms can be classified into three groups, based on sensitivity to PMA desensitization (24, 25). The first group, with isoforms  and , is desensitized rapidly by PMA; the second group, with isoforms I, II, , , and , requires longer exposure to PMA for desensitization; and the third group, with isoforms  and , exhibits no desensitization with prolonged exposure to PMA. In our experiments, we found that a brief 3-h exposure to PMA desensitized reactive astrocytes to TGF-₁-induced down-regulation of Kir2.3 current, suggesting involvement of either the  or  isoforms of PKC. Additional electrophysiological experiments with isoform-specific inhibitors confirmed involvement of PKC- and not PKC-. Subsequent immunoblot and confocal experiments showed that TGF-₁ caused translocation from cytosol to membrane of the  but not of the  isoform, and co-immunoprecipitation experiments showed a strong increase in association between Kir2.3 and PKC- but not PKC- after exposure to TGF-₁. Together, these data provide strong evidence that PKC- was involved in the rapid effect of TGF-₁. Our data in astrocytes on involvement of PKC- are in agreement with previous reports in other systems on involvement of PKC- in TGF-₁-induced signaling (17).

As expected, given the involvement of PKC, we also showed that upstream PLC was involved in TGF-₁-induced down-regulation of Kir2.3 current. We found that both the phosphatidylinositide-specific PLC selective agent, neomycin, as well as the phosphatidylcholine-specific PLC-selective agent, D609, were highly effective in blocking TGF-₁-induced down-regulation of Kir2.3, but we could not assess the effectiveness of the phosphatidylinositide-specific PLC-selective agent, U73122, because of cell damage that it caused. Block by D609 accords with previous observations implicating phosphatidylcholine-specific PLC (14, 16, 17), but efficacy of neomycin was surprising, given that U73122 has previously been found to be ineffective in inhibiting TGF-₁-induced signaling (14, 17). Our data clearly implicate PLC activity as being required for production of diacylglycerol and activation of PKC. However, our data are not as conclusive as those of the previous authors (14, 17) as to whether phosphatidylcholine- or phosphatidylinositide-specific PLC is exclusively involved as a source of intrinsic diacylglycerol in TGF-₁-induced signaling.

The membrane potential of reactive astrocytes appears to be carefully regulated under a variety of complex signaling situations. TGF-₁ is a multifunctional cytokine that exerts complex effects on astrocytes, including growth inhibition, cell aggregation, extracellular matrix production, and chemotaxis (1, 3). It is unlikely that depolarization effected by TGF-₁ subserves any of these complex effects in a specific manner. For example, depolarization of astrocytes is associated with proliferation (52, 53), whereas TGF-₁ activation, which produces depolarization, appears to favor growth inhibition (1). However, all of the complex cellular effects initiated by TGF-₁ possess the common feature that they all involve activation of gene transcription at one or more points during execution. We speculate that depolarization may be integral to the initiation of gene transcription as part of the larger complex effects induced by TGF-₁. In support of this hypothesis, TGF-₁-induced gene transcription is known to require PLC and PKC--signal transduction (14-17), exactly as shown here for TGF-₁-mediated depolarization. Moreover, transcription has previously been linked to cell depolarization (54). It appears from the current experiments that membrane depolarization of reactive astrocytes is an early signaling event following activation by TGF-₁ and possibly by other cytokines as well (40). Cytokine-mediated regulation of membrane potential represents a new, potentially important aspect in understanding the function of reactive astrocytes.

	ACKNOWLEDGEMENTS

We thank Dr. David S. Bredt (University of California, San Francisco, CA) for the kind gift of anti-Kir2.3 antibody used for the immunofluorescence experiments and Jia Bi Yang and Qiang Wang for expert technical assistance.

	FOOTNOTES

^* This work was supported by a Merit Review Grant from the Department of Veterans Affairs (Baltimore, MD).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.

To whom correspondence should be addressed: Dept. of Neurosurgery, 22 South Greene St., Baltimore, MD 21201. Tel.: 410-328-0850; Fax: 410-328-0756; E-mail: msimard@surgery1.umaryland.edu.

Published, JBC Papers in Press, November 16, 2001, DOI 10.1074/jbc.M107984200

	ABBREVIATIONS

The abbreviations used are: TGF, transforming growth factor; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate; TGF-₁-R, TGF-₁ receptor; GFAP, glial fibrillary acidic protein.

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