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Activation of BK Channels in GH3 Cells by a c-PLA₂-Dependent G-Protein Signaling Pathway

¹Departments of Anesthesiology and ²Physiology and ³The Center for Cellular and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia

Submitted 23 August 2004; accepted in final form 10 January 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
BK-channels in GH3 cells are activated by arachidonic acid produced by c-PLA₂. -adrenergic agonists also activate BK channels and were presumed to do so via production of cAMP. We, however, show for the first time in GH3 cells that a -adrenergic agonist activates a pertussis-toxin-sensitive G protein that activates c-PLA₂. The arachidonic acid produced by c-PLA₂ then activates BK channels. We examined BK channels in cell-attached patches and in excised patches from untreated GH3 cells and from GH3 cells exposed to c-PLA₂ antisense oligonucleotides. For the cell-attached patch experiments, physiologic pipette and bath solutions were used. For the excised patches, 150 mM KCl was used in both the pipette and bath solutions, and the cytosolic surface contained 1 µM free Ca²⁺ (buffered with 5 mM K₂EGTA). Treatment of GH3 cells with the G protein activator, fluoroaluminate, (AlF₄^–) produced an increase in the P_o of BK channels of 177 ± 41% (mean ± SD) in cell-attached patches. Because G proteins are membrane associated, we also added an activator of G proteins, 100 µM GTP--S, to the cytosolic surface of excised patches. This treatment leads to an increase in P_o of 50 ± 9%. Similar treatment of excised patches with GDP--S had no effect on P_o. Isoproterenol (1 µM), an activator of -adrenergic receptors and, consequently, some G proteins, increased BK channel activity 229 ± 37% in cell-attached patches from cultured GH3 cells. Western blot analysis showed that GH3 cells have -adrenergic receptor protein and that isoproterenol acts through these receptors because the -adrenergic receptor antagonist, propanolol, blocks the action of isoproterenol. To test whether G protein activation of BK channels involves c-PLA₂, we studied the effects of GTP--S on excised patches and isoproterenol on cell attached patches from GH3 cells previously treated with c-PLA₂ antisense oligonucleotides or pharmacological inhibitors of c-PLA₂. Neither isoproterenol nor GTP--S had any effect on P_o in these patches. Similarly, neither isoproterenol nor GTP--S had any effect on P_o in cultured GH3 cells pretreated with pertussis toxin. Isoproterenol also significantly increased the rate of arachidonic production in GH3 cells. These results show that some receptor-linked, pertussis-toxin-sensitive G protein in GH3 cells can activate c-PLA₂ to increase the amount of arachidonic acid present and ultimately increase BK-channel activity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Large-conductance Ca²⁺-activated potassium (BK) channels are widely distributed in many tissues. Six functionally distinct types of BK channels have been described within the CNS (Reinhart et al. 1989). BK channels are intimately associated with such widely diverse CNS functions as neural regulation of the heart originating in the nucleus tractus solitarius and sleep, which is dependent on repetitive rhythmic activity originating in the reticular formation of the thalamus (Golomb et al. 1994). BK channels are also involved in neuropeptide secretion, regulation of presynaptic calcium signals, and neurotransmitter release (Robitaille and Charlton 1992). Because of the physiological importance of BK channels, we have been particularly interested in the cellular signaling mechanisms responsible for controlling the activity of BK channels. As their name suggests, Ca²⁺-activated potassium channel activity is altered by intracellular calcium; however, because of their important role in the CNS function, it is not surprising that other signaling pathways also regulate these channels. In previous work, we have demonstrated that the arachidonic acid produced from cytosolic phospholipase A₂ increases BK channel open probability in GH₃ cells (Denson et al. 1999). Until recently it has been thought that optimal activation of c-PLA₂ requires both increases in intracellular Ca²⁺ and phosphorylation (Gijón et al. 1999). It was also thought that c-PLA₂ resided in the cytosol and translocated to the cell membrane only in response to large increases in intracellular Ca²⁺ (Clark et al. 1991). However, in many cells, including GH3 cells, there is a significant pool of c-PLA₂ constitutively associated with the membrane which is capable of producing arachidonic acid (Denson et al. 2001; Liu et al. 2001; Osterhout and Shuttleworth 2000). Whether membrane associated or not, c-PLA₂ can be further activated by several mechanisms including phosphorylation by protein kinase C (PKC) or mitogen activated protein kinase (MAPK), by reactive oxygen species like H₂O₂, by increases in intracellular Ca²⁺ and by ligand binding to any G-protein-coupled receptor (GPCR) that activates the G protein, G_i or G_o. This activation of cPLA₂ results in an increased production of arachidonic acid and corresponding increase in the P_o of BK channels (Barlow et al. 2000; LaBelle and Polyak 1998; Leslie 1997). Receptor-linked heterotrimeric G proteins have also been reported to activate both BK channels and c-PLA₂ (Kruger et al. 1995; Tong et al. 1998; Yousufzai and Abdel-Latif 1993). For example, isoproterenol, a nonselective adrenergic agonist, activates c-PLA₂ via a GPCR (Chung et al. 1999; LaBelle and Polyak 1998; Nara et al. 1998). Although it is clear that G proteins can activate c-PLA₂ in some systems, the role of G proteins in BK activation remains unclear. In this paper, we investigated whether heterotrimeric G proteins, endogenous to GH3 cells (Gudermann et al. 1996; Paulssen et al. 1992), could activate BK-channels in cultured GH3 cells and the mechanism by which this activation occurs.

GH3 cells are a continuous neurosecretory cell line originally derived from the anterior pituitary. They have been used as a neuronal model and a model of cellular secretion (Kushmerick et al. 1999). These electrically excitable neurosecretory cells have spontaneous action potentials that are dependent on both K⁺ and Ca²⁺ and have a complete GPCR signal transduction cascade linking agonist stimulation by thyrotropin releasing hormone (TRH) to prolactin release (Gershengorn 1986). Neurophysiologically, this cell line serves as a cellular model for the study of pituitary autocrine and paracrine function (Vankelecom and Denef 1997).

We used GH3 to examine the effect of a -adrenergic agonist on BK channels using cell-attached patches and then examined the mechanism for activation of the BK channels in excised, inside-out patches. This meant we could examine the effects of G protein activation at constant cytosolic calcium and without complications from other signaling pathways. Based on our experiments, we conclude that isoproterenol activates a pertussis-toxin-sensitive G protein that activates BK channels by activation of cPLA₂ and subsequent production of arachidonic acid.

	METHODS

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Cell culture

The GH3 cell line was obtained from American Type Culture Collection (ATCC; Rockville, MD) and were grown at 37° in a 5% C0₂ atmosphere in Dulbecco's minimum essential media (DMEM) supplemented with 15% heat-activated horse serum, 2.5% fetal bovine serum, and 2 mM glutamine. Cells for electrophysiologic experiments were plated on polylysine-coated petri dishes to which a polycarbonate recording chamber with a volume of 0.2 ml had been previously affixed with silicone elastomer (Sylgard). Cells were used 1–3 days after plating, and cells from passages 25–45 were used in the experiments described in this manuscript. Previous studies have demonstrated a consistent P_o versus [Ca_i²⁺] and P_o versus mV relationship as well as the extent of BK-channel expression exists over this range of cell passages (Denson et al. 1996, 1999, 2001).

c-PLA₂ sense and antisense oligonucleotides

Sense and antisense oligonucleotides targeting two possible translation start codons of rat c-PLA₂ were synthesized by the Microchemical Facility of Emory University (Atlanta, GA) as previously described (Denson et al. 1999, 2001). We have previously reported that treatment of cultured GH3 cells with 5 µM of each antisense oligonucleotide in serum-free media for 20–24 h results in ca 80% decrease in c-PLA₂ by both Western blot and direct biochemical assay (Denson et al. 1999).

Drug exposure paradigm

For all experiments, drug exposure was effected using a gravity perfusion/suction removal technique with a perfusion rate of 2.0 ml/min and a dead volume of 1.0 ml. Previous experiments showed that exchange was 90 ± 7% complete after 0.5 min (Denson and Eaton 1994). After obtaining a high resistance (>25 G) seal, patches were excised in a 1 µM Ca_i²⁺ solution. Control recordings were obtained in K₂EGTA-buffered solutions containing the desired Ca_i²⁺ concentration as described in the following text.

Electrophysiologic recordings

All experiments in this study used either the cell-attached or the excised (inside-out) patch configuration of the patch-clamp technique. Electrodes were fabricated from Corning 7052 glass (Garner Glass, Fullerton, CA) in two steps on a Narishige PP-83 electrode puller (Narishige, Tokyo, Japan). Electrodes were fire polished to a final tip resistance between 3 and 5 M. Recordings were performed at room temperature with a Dagan Model 3900 patch-clamp amplifier (Dagan, Minneapolis, MN). All experiments were conducted with the patch depolarized to +20 mV. Single-channel data were digitized using Axoscope-7 software (Axon Instruments; Foster City, CA) at a sampling rate of 5 kHz and filtered at 2 kHz using a 4-pole low-pass Bessel filter.

Data analysis

The digitized single-channel data were analyzed in 1-min segments to generate NP_o versus time plots using Fetchan and P-Stat software programs (Axon Instruments). NP_o versus time plots were used to determine the time course for reaching a stable maximum effect for each series of experiments. Open probabilities were determined from the amplitude histograms by fitting each amplitude histogram to the appropriate sum of Gaussian distribution functions using iterative nonlinear regression software (PeakFit 4; SPSS, Chicago, IL) after correction of the baseline to make 0 current coincident with the state in which all channels were closed. NP_o values were first calculated from the amplitude histogram. The open probability (P_o) was then calculated as NP_o/N. The number of channels in each patch (N) was estimated by dividing the total conductance obtained during exposure of the patch to 100 µM Ca_i²⁺ by the unit conductance associated with a one-state change. Because the duration of open and closed intervals varied from very short to very long durations, data obtained from a patch, which contained a single channel, were binned logarithmically and analyzed according to the method of Sigworth and Sine (1987).

Solutions

The solutions used in all patch-clamp experiments were (in mM) 150 KCl, 2 MgCl₂, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.30, or 140 KCl, 5 HEPES, 5 K₂EGTA, and 4.55 CaCl₂ (10 µM free ionized Ca²⁺), pH 7.30, for the pipette and 140 KCl, 15 HEPES, 5 K₂EGTA, and 0.1, 1, or 100 µM Ca_i²⁺ pH 7.4 for the bath. AlF₄^– solutions were prepared immediately before use by adding AlCl₃ and NaF to the standard bath solution as previously described (Susa et al. 1997). Solutions of GTP--S, GDP--S, pertussis toxin (PTX), and isoproterenol were all prepared at the desired concentration in bath solution immediately prior to use.

Drugs and chemicals

c-PLA₂ sense and antisense oligonucleotides (described in the preceding text) were synthesized by the Microchemical Facility of Emory University (Atlanta, GA). GTP--S, GDP--S, pertussis toxin (PTX), and isoproterenol were all obtained from Sigma-Aldrich, St. Louis, MO. The c-PLA₂ inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3) was obtained from Biomol (Plymouth Meeting, PA). A polyclonal antibody (host:rabbit) for the ₁-receptor was obtained from Novus Biologicals (Littleton, CO). A polyclonal antibody (host:chicken) for the ₂-receptor was obtained from Chemicon International (Temecula, CA). Corresponding secondary antibodies were obtained from KPL (Gaithersburg, Maryland) and Chemicon International.

Arachidonic acid efflux protocol

The efflux of labeled arachidonic acid from cells in response to agonists has been used as a measure of cellular production of arachidonic acid in GH3 cells (Denson et al. 1996). Efflux experiments were conducted as previously reported. Briefly, sufficient ³H-arachidonic acid in ethanol to result in a final concentration of 1 µCi/ml media was evaporated to dryness under dry nitrogen, serum-free GH3 media containing 0.2% BSA added, and the mixture vortexed. GH3 cells plated at a density of 1 x 10⁶ cells/dish in a six-well plate were incubated in 2 ml GH3 media containing labeled arachidonic acid. After an overnight exposure, sample was counted and the remaining "hot" media was carefully removed. The cells were then washed three times with 2 ml arachidonic-acid-free media and returned to the incubator. At each time point of interest, sample of the media was removed and counted and the sample volume replenished with fresh arachidonic-acid-free media. Following the last sample, media was removed and 1 N NaOH was added to lyse the cells. After 2 min, 1 N HC1 was added to neutralize the NaOH. The cells were scraped and vortexed, and a sample was removed for counting the remaining radioactivity in the cells. Rates of arachidonic acid release were calculated at each time point from the fraction of total counts remaining at the corresponding time.

Western blot analysis

Western blot analysis was completed as previously described (Denson et al. 1999). Briefly, 10⁷ GH3 cells were grown in a T-75 tissue culture flask. Media was removed, the cells washed with PBS and scraped in fresh PBS and centrifuged at 4°C. The pellet was resuspended in 300 µl of RIPA buffer (150 mM NaCl, 10 mM NaPO4, pH 7.2, 1% NP-40, 0.1% SDS, and 0.25% deoxycholate) containing protease inhibitors (antipan, leupeptin, pmsf, tpck/tlck). After cell lysis (1 h at 4°C), the suspension was centrifuged at 2000 g for 10 min to precipitate unlysed cells. Samples were prepared for SDS-polyacrylamide gel electrophoresis by diluting the supernatant with sample buffer (Tris, pH 6.8; 0.1%SDS, 10% glycerol, and 0.025% Bromphenol Blue) and heating at 95°C for 10 min. The resultant solution (45 µl) was loaded in each lane on a 7.5% polyacrylamide gel. After electrophoresis (1 h at 150 V), the proteins were transferred to nitrocellulose. The nitrocellulose blots were then probed with primary antibodies for either the ₁- or ₂-adrenergic receptor. For detection of the ₁-receptor, a rabbit polyclonal antibody at a dilution of 1:500 was used. For detection of the ₂-receptor, a chicken polyclonal antibody at a dilution of 1:500 was used. Blots were then washed and incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:10,000) for 1 h at room temperature for the ₁-receptor or a goat anti-chicken antibody (1:5000) for the ₂-receptor. Receptors were visualized by chemiluminescence using an ECL kit from Sigma (EPS-1, Sigma-Aldrich). The ₁-receptor was assigned to the band at 64 kDa, whereas the ₂-receptor was assigned to a band between 50 and 75 kDa.

Statistical analysis

Within group comparisons for two treatments were accomplished using a t-test for repeated measures. In all cases, a P value of <0.05 was required to reject the null hypothesis. Intergroup comparisons for a single treatment were made using an ANOVA followed either by a post hoc Scheffe or Tukey test for multiple comparisons as appropriate. All data are presented as means ± SD unless otherwise specified.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Single BK-channel characteristics

Single BK channel currents were recorded with two patch-clamp configurations: cell attached patches to measure the effects of the nonselective -adrenergic agonist, isoproterenol, and the G protein activator, AlF₄^–. Excised, inside-out patches were used for all other measurements. Unit conductance for all channels regardless of configuration was 288 ± 13 pS. However, as might be expected, there was a large difference in the open probability (P_o) between channels recorded with the two different patch-clamp configurations (cell attached and excised inside-out). The cell-attached configuration, in the absence of a calcium ionophore, results in a much lower P_o than an excised patch. In addition, groups of cells treated with antisense oligonucleotides directed against c-PLA₂ have inherently lower P_o values for either configurations when compared with their untreated counterparts. Despite these differences in P_o between different treatment groups, the within-group variability is consistently small (ca 30%) for each experimental condition studied. The two intracellular Ca²⁺ concentrations of 0.1 and 1 µM were chosen because this is the normal range encountered before, during, and after an action potential in GH3 cells (Schlegel et al. 1987).

Both fluoroaluminate (AlF₄^–) and GTP--S increase BK-channel activity in cultured GH3 cells

Cell-attached patches were used for experiments with the G protein activator, AlF₄^–. AlF₄^– is quite membrane permeable so that G protein activation by external application of AlF₄^– is almost as rapid and complete as is application to the cytosolic surface of excised patches (Susa et al. 1997; Yousufzai and Abdel-Latif 1993). After we made control recordings of BK-channel activity with the patch depolarized to +20 mV, a freshly prepared solution of 10 µM AlF₄^– was added, and BK-channel activity was recorded continuously for 10 min. Treatment of cultured GH3 cells with extracellular AlF₄^– resulted in a highly significant increase (P < 0.001) in BK-channel activity (177 ± 41%; n = 8, mean ± SD) and was significantly greater (P < 0.01) than the increase with GTP--S. This may be due to the fact that GTP--S is hydrolyzed, albeit slowly, whereas AlF₄^– binds to and locks the G protein into a persistently activated state. Treatment of excised patches from GH3 cells, depolarized to +20 mV and exposed to 0.1 µM intracellular Ca²⁺, with 100 µM GTP--S also resulted in a consistent and significant (P < 0.001) increase in BK-channel activity of 50 ± 9% (n = 8; see Fig. 1). There was no significant change in the voltage dependence of P_o over the range of 0 to +60 mV after GTP--S treatment (data not shown).

View larger version (42K): [in this window] [in a new window]   FIG. 1. Activation of G proteins results in BK-channel activation in cultured GH3 cells. A: a comparison of BK-channel activity in untreated cell-attached patches and patches treated with 0.5 mM AlF4–. B: untreated excised patches and excised patches treated with 100 µM GTP--S. All experiments were conducted with VH = +20 mV. Solutions used in these experiments are described in METHODS. For the single-channel records in this figure, the ‘C’ represents level when all channels are closed. The numerical values (e.g., 1, 2,...) represent the number of channels open. A illustrates a representative experiment with AlF4–. The unit conductance of the channels in this patch is 235 pS. AlF4– resulted in an increase in Po from 0.09 (untreated cells; top) to 0.19 (cells treated with AlF4–, bottom). B illustrates a representative experiment with GTP--S with a [Cai2+] of 0.1 µM. The unit conductance of the channels in this patch is 345 pS. GTP--S resulted in an increase in Po from 0.22 (untreated cells; top) to 0.35 (cells treated with GTP--S; bottom). C and D provide a summary of these experiments. C: treatment of cultured GH3 cells with AlF4–, resulted in a highly significant increase (P < 0.001) in BK-channel activity of 177 ± 41% (n = 8, mean ± SD) and was significantly greater (*P < 0.01) than the increase with GTP--S. D: treatment of excised patches from cultured GH3 cells, with 100 µM GTP--S also resulted in a consistent and significant (P < 0.001) increase in BK-channel activity of 50 ± 9% (n = 8).

The effect of AlF₄^– and GTP--S suggested that activation of G proteins could activate BK channels. Therefore we examined the effect of isoproterenol, an agonist for one type of GPCR, -adrenergic receptors. We chose isoproterenol because it stimulates the release of growth hormone in GH3 cells (Gabriel et al. 1989). Because BK channels also regulate the release of growth hormone in GH3 cells, we sought to determine whether treatment of GH3 cells with isoproterenol would result in BK-channel activation. We used cell-attached patches depolarized to +20 mV. After control recordings had been made, we added 1 µM isoproterenol to the bath and recorded single-channel activity continuously for an additional 10 min. The P_o of channels in the cell-attached patches was relatively low presumably because of low intracellular calcium; nonetheless, treatment of GH3 cells with isoproterenol resulted in a significant increase in BK-channel activity (P < 0.001). P_o increased from 0.029 ± 0.010 in control patches to 0.092 ± 0.030 after treatment with isoproterenol (see Fig. 2, A and C). The effects of isoproterenol could be reversed by perfusing the cells with control bath solution without isoproterenol (data not shown).

View larger version (31K): [in this window] [in a new window]   FIG. 2. One G protein coupled receptor ligand, isoproterenol, activates BK-channels in GH3 cells. A: single-channel recordings were made using the cell-attached patch configuration with a bath [Ca2+] of 2 mM and a VH of +20 mV. After control recordings were made in untreated cells (top 2 traces), a solution containing 1 µM isoproterenol was introduced and the single-channel activity continuously recorded for 10 min (bottom 2 traces). Isoproterenol (1 µM), increased BK-channel activity in this patch from a Po of 0.015 to a Po of 0.039. The unit conductance for the channels in this patch was 216 pS. For the single-channel records in this figure, the C represents the state in which all channels are closed. The numerical values (e.g., 1, 2,...) represent the number of channels in an open state. B, inset: Western blot demonstrating the presence of both 1- and 2-adrenergic receptors in untreated GH3 cells. C: for these experiments, the cell-attached configuration was used with a bath [Ca2+] of 2 mM and a VH of +20 mV. After control recordings were made in untreated cells, a solution containing 1 µM isoproterenol was introduced and the single-channel activity continuously recorded for 10 min. Isoproterenol treatment resulted in a highly significant increase in Po (P < 0.001 by paired t-test) from 0.029 ± 0.004 in control patches to 0.092 ± 0.015 (n = 6). D: for these experiments, the cell-attached configuration was used with a bath [Ca2+] of 2 mM and a VH of +20 mV. After control recordings were made in cells pretreated with 20 µM propranolol, a solution containing propranolol and 1 µM isoproterenol was introduced and the single-channel activity continuously recorded for 10 min. Isoproterenol treatment did not result in a significant increase in Po (P < 0.06 by paired t-test) from 0.037 ± 0.027 in control patches to 0.022 ± 0.011 (n = 8).

Isoproterenol activates BK channels via -adrenergic receptors

Although isoproterenol is generally considered a specific agonist for -adrenergic receptors (Hoffman 2001) that is incapable of activating -adrenergic receptors and we had demonstrated the presence of both 1- and 2-adrenergic receptor protein, we also demonstrated that a specific -adrenergic antagonist (Hoffman 2001) blocked the action of isoproterenol on BK channels. For these experiments, we pretreated GH3 cells 20 µM propranolol (Chen et al. 2002) for 15 min and then formed a cell-attached patch. Single-channel activity was recorded for 10 min after which the bath solution was replaced with one containing 20 µM propranolol and 1 µM isoproterenol, and BK-channel activity was recorded for an additional 10 min. The V_H, bath and electrode solutions were identical to those described above (Fig. 2, A and C) in which the effect of isoproterenol was examined. Propranolol completely eliminated any stimulatory effect of isoproterenol (Fig. 2D). The mean P_o in the presence of propranolol alone was 0.037 ± 0.021, whereas the P_o for cells treated with propranolol and isoproterenol together was 0.022 ± 0.011, not significantly different from propranolol alone (by 2-tailed t-test for repeated measures: P < 0.06, n = 8). These data further support the idea that the effect of isoproterenol is mediated through -adrenergic receptors.

Isoproterenol activates BK channels via a pertussis-toxin-sensitive G protein

Although -adrenergic receptors are traditionally thought to couple to G_s proteins, there are several recent reports of the receptors coupling to G_i (Gosmanov et al. 2002; Gudermann et al. 1996, 1997; LaBelle and Polyak 1998; Wellner-Kienitz et al. 2001; Xiao 2001). Chung et al. (1999) reported that isoproterenol could activate BK-channels via a G_i-mediated pathway in rabbit mesenteric arterial smooth muscle cells. Which G protein is involved (G_s or G_i/o) in -adrenergic-mediated stimulation of BK channels can be determined by treatment with pertussis toxin (PTX); PTX only blocks the G_i/G_o family of G proteins and has no effect on G_s activation. Therefore we pretreated GH3 cells with 5 µg/ml PTX for 3 h. PTX completely eliminated the stimulatory effect of isoproterenol on BK-channels (see Fig. 3A). P_o in control cells in these experiments was 0.028 ± 0.011, and in cells pretreated with PTX, P_o was 0.028 ± 0.014. Although both G_i and G_o proteins are present in GH3 cells (Paulssen et al. 1992), Chung et al. (1999) have shown that only G_i appears to be coupled to the isoproterenol receptor in native cells; therefore the PTX inhibition of the -adrenergic response is most consistent with inhibition of G_i.

View larger version (15K): [in this window] [in a new window]   FIG. 3. Isoproterenol activates BK-channels in GH3 cells via a PTX-sensitive G protein receptor coupled to c-PLA2. A: all experiments were conducted using the cell-attached patch configuration with a bath [Ca2+] of 2 mM and the patches depolarized to +20 mV. Po in cells pretreated with PTX (5 µg/ml) for 3 h was 0.028 ± 0.011, and after treatment with isoproterenol, the Po was 0.028 ± 0.014 (P < 0.5; n = 6). The effect of isoproterenol on BK channels in cultured GH3 cells was also inhibited by pretreating cells with c-PLA2 antisense oligonucleotides. Po for control cells in these experiments was 0.011 ± 0.005 and was unchanged after isoproterenol (0.011 ± 0.005; n = 6).

In other types of cells, isoproterenol has been reported to activate c-PLA₂ via a GPRC mechanism involving G_i (LaBelle and Polyak 1998). We have shown in several previous publications that the expression of cPLA₂ can be strongly inhibited by treatment with specific antisense oligonucleotides. We used this approach again to show that the effect of isoproterenol on BK channels was also inhibited by pretreating cells with c-PLA₂ antisense oligonucleotides. P_o for control cells in these experiments was 0.011 ± 0.005 and was unchanged after isoproterenol (0.011 ± 0.005; see Fig. 3B). These data provide additional support for our hypothesis that activation of c-PLA₂ (with a corresponding increase in BK-channel activity) can arise via a GPRC mechanism involving G_i/o.

Isoproterenol increases c-PLA₂-mediated arachidonic acid release from GH3 cells

We have previously reported that there is tonic c-PLA₂-dependent arachidonic acid release in GH3 cells and that compounds that either inhibit or stimulate c-PLA₂ result in a corresponding change in arachidonic acid production (Denson et al. 1996). To verify our hypothesis that the increase in BK-channel activity observed with isoproterenol treatment was through stimulation of c-PLA₂, we studied the effect of isoproterenol on arachidonic acid efflux. Treatment of GH3 cells with 1 µM isoproterenol resulted in a highly significant increase in the rate of arachidonic acid release when compared with control. Specifically, the tonic rate of arachidonic acid release was 0.032 ± 0.016 h^–1 (n = 4) and was 0.074 ± 0.009 h^–1 (n = 4) after isoproterenol (P < 0.005; see Fig. 4).

View larger version (19K): [in this window] [in a new window]   FIG. 4. Isoproterenol increases c-PLA2 mediated arachidonic acid release from GH3 cells. For these experiments, GH3 cells were treated with either control media or media containing 1 µM isoproterenol. Samples were withdrawn a 2, 4, 8, 12, 20, and 30 min and at 1, 2, and 3 h for determination of the rate of arachidonic acid efflux as described in the preceding text. Each point in A is the means ± SE for 4 replicates. Isoproterenol () resulted in a significant stimulation of arachidonic acid efflux when compared with control (; P < 0.005). B: the mean rate of arachidonic acid efflux (as the fraction of total arachidonic acid counts released per hour). Isoproterenol produces a significant increase in efflux (means ± SE for 4 experiments).

Our previous work has shown that there is significant cPLA₂ production of arachidonic acid at normal intracellular calcium concentrations but that this activity can be substantially enhanced by increases in the calcium concentration of the bath bathing the cytosolic surface of excised patches. The tonic activity could either be due to calcium or some other tonically active signaling pathway. If there was tonic activity of G_i/o protein, then inhibition should reduce BK-channel activity. Therefore we compared the effect of the G protein activator, GTP--S, with the G protein inhibitor, GDP--S. For this series of comparative experiments, we used excised patches from GH3 cells depolarized to +20 mV and exposed to 1 µM intracellular Ca²⁺. As expected from the results in Fig. 1B, treatment of cultured GH3 cells with 100 µM GTP--S resulted in an increase in BK-channel activity from a control P_o of 0.40 ± 0.04 to 0.60 ± 0.11 (n = 8) within 10 min (Fig. 5A). On the other hand, treatment of cultured GH3 cells with 100 µM GDP--S had no significant (P < 0.5) effect on BK-channel activity (n = 6). For this series of experiments, the control P_o was 0.47 ± 0.05, whereas the P_o after 10 min of treatment with GDP--S was 0.45 ± 0.045. (see Fig. 5B).

View larger version (14K): [in this window] [in a new window]   FIG. 5. Inactivation of G-proteins does not reduce BK-channel activity. For this series of comparative experiments, excised patches from cultured GH3 cells with the patch depolarized to +20 mV and exposed to 1 µM [Cai2+] were used. As noted previously, treatment of cultured GH3 cells with 100 µM GTP--S resulted in an increase in BK-channel activity from a control Po of 0.40 ± 0.04 to 0.60 ± 0.11 (n = 8) within 10 min (A). On the other hand, treatment of cultured GH3 cells with 100 µM GDP--S had no significant (P < 0.5) effect on BK-channel activity (n = 6). For this series of experiments, the control Po was 0.47 ± 0.05, whereas the Po after 10 min of treatment with GDP--S was 0.45 ± 0.045 (B).

G protein activation of BK channels by GTP--S requires c-PLA₂

We had already shown that inhibiting expression of cPLA₂ could block the effect of isoproterenol. We presumed that this result meant that a G protein activated by the adrenergic receptor (G_i/o) required cPLA₂ activation. To test the hypothesis that the increase in activity observed with GTP--S involved a G-protein signaling pathway in which c-PLA₂ was an important component, we exposed excised patches from GH3 cells, which had been pretreated with antisense oligonucleotides against c-PLA₂ to 100 µM GTP--S. All excised patches in this set of experiments were exposed to 1 µM intracellular Ca²⁺ and depolarized to +20 mV. Unlike cultured cells in which the P_o of BK channels increased significantly after treatment with GTP--S, P_o in patches from antisense-treated cells did not change significantly (0.18 ± 0.02 vs. 0.17 ± 0.03; P < 0.5; see Fig. 6A). Although we and others (Denson et al. 2001; Liu et al. 2001; Osterhout and Shuttleworth 2000) have previously reported that a significant portion of the c-PLA₂ pool in GH3 cells normally resides at or near the plasma membrane, we sought to further validate this observation by conducting experiments with the c-PLA₂ inhibitorAACOCF3 (Street et al. 1993) in excised patches. All excised patches in this set of experiments were exposed to 1 µM intracellular Ca²⁺ and depolarized to +20 mV. After we obtained control recordings, the patches were exposed to a bath solution containing 15 µM AACOCF3 after which channel activity was continuously recorded for 10 min to ensure any effect of the inhibitor had stabilized. The patches were then exposed to a bath solution containing 15 µM AACOCF3 and 100 µM GTP--S. Channel activity was continuously recorded for an additional 10 min. Like pretreatment with c-PLA₂ antisense, addition of AACOCF3 by itself to excised patches resulted in a significant reduction in P_o from 0.37 ± 0.08 to 0.21 ± 0.04 (P < 0.001; n = 6). Further addition of GTP--S after AACOCF3 did not result in a significant change in P_o (0.21 ± 0.04 vs. 0.20 ± 0.06; P < 0.3; n = 6; see Fig. 6B). These results are essentially identical to those obtained using GH3 cells exposed to antisense oligonucleotides (Fig. 6A) and further demonstrate that a significant amount of active c-PLA₂ is available in excised patches in untreated GH3 cells.

View larger version (13K): [in this window] [in a new window]   FIG. 6. G protein activation of BK-channels by GTP--S requires c-PLA2. A: these experiments utilized excised patches from GH3 cells, which had been pretreated with antisense oligonucleotides against c-PLA2. All excised patches were exposed to 1 µM [Cai2+] and depolarized to +20 mV. After control recordings were completed, patches were exposed to a bath solution containing 100 µM GTP--S. Po in patches from antisense-treated cells did not change significantly (0.18 ± 0.02 vs. 0.17 ± 0.03; n = 6; P < 0.5) after treatment with GTP--S. B: these experiments utilized excised patches from GH3 cells that had been treated with the c-PLA2 inhibitor, AACOCF3 All excised patches were exposed to 1 µM [Cai2+] and depolarized to +20 mV. After control recordings were completed, patches were exposed to a bath solution containing 15 µM AACOCF3. Po in patches from AACOCF3-treated cells decreased significantly (0.37 ± 0.08 vs. 0.21 ± 0.04; P < 0.001; n = 6). After the effect of AACOCF3 had stabilized, patches were exposed to a solution containing 15 µM AACOCF3 and 100 µM GTP--S. Continuous single-channel recordings were made for 10 min. Treatment of the patches with GTP--S did not result in any significant change in Po (0.21 ± 0.04 vs. 0.20 ± 0.06; P < 0.5; n = 6).

GTP--S activation of BK channels involves G_i/o

Although PTX blocks the action of isoproterenol implying an involvement of G_i/o protein, GTP--S could activate any G protein available on the cytosolic surface of excised patches. To determine whether G_i/o was the G protein, which activates c-PLA₂ in excised patches, we conducted a series of experiments using excised patches from cells that had been pretreated for 3 h with 5 µg/ml PTX. PTX ADP-ribosylates members of the G_i/o family of G proteins. In so doing, PTX prevents receptor-G protein interaction. In addition, pretreatment with PTX also prevents binding and activation of G_i/o by GTP--S or GTP (Katada et al. 1984; O'Neill et al. 1990; Sariban-Sohraby et al. 1999; Xiao et al. 1999; Yassin et al. 1996). Excised patches from these cells were exposed to a bath solution containing 5 µg/ml PTX and 1 µM intracellular Ca²⁺. All patches were depolarized to +20 mV. After control recordings were obtained, GTP--S (100 µM) was added to the cytosolic bath and single-channel activity was continuously recorded for an additional ten minutes. Pretreatment of cells with 5 µg/ml PTX completely inhibited the stimulatory effect of GTP--S in cultured GH3 cells. P_o for the control cells was 0.38 ± 0.05 and 0.41 ± 0.05 after treatment with GTP--S. These results suggest that G_i/o is the G protein coupled to c-PLA₂.

All of the signaling elements required for BK-channel activation by isoproterenol are contained in excised patches

If our hypothesis that isoproterenol binding to -adrenergic receptors activates c-PLA₂ and subsequently BK channels via a G_i/o-dependent pathway, then it should be possible to stimulate BK channels in excised patches by incorporating isoproterenol in the pipette solution (see schematic in Fig. 8B). For these experiments, the bath (intracellular) solution contained 0.1 µM Ca²⁺ and the patches were depolarized to +20 mV. The tips of the pipettes were filled with saline containing no isoproterenol, and the remainder of the pipette was backfilled with the saline that contained 1 µM isoproterenol. We have previously shown that low-molecular-weight solutes (like isoproterenol will equilibrate with the end of the pipette in 7–10 min. Channel activity was continuously recorded for 15 min. Repeated measures analysis on data from seven excised patches showed a statistically significant increase in P_o from the first 5 min compared with the last 5 (from 0.31 ± 0.06 to 0.38 ± 0.08, P < 0.013; n = 7; see Fig. 7). Interestingly, 4/7 patches increased 28 ± 3%, 2/7 patches increased 8 ± 2% and 1/7 decreased 5%. The activity of untreated patches or patches with no response (compare the effect of GDP--S) generally decrease with time. It is statistically unlikely (P = 0.011, z test) in a group of seven patches, that six should increase P_o and that four should increase by >25%. These data suggest that all of the transduction elements shown in Fig. 8B are present in many (4/7) if not most (6/7) excised patches from GH3 cells.

View larger version (30K): [in this window] [in a new window]   FIG. 8. Schematic model of G protein activation of BK channels. This diagram shows the cellular signaling pathways involved in BK channel activation in a cell-attached patch (left) or and excised, inside-out patch (right). On the left, isoproterenol activates -adrenergic receptors that activate the G protein, Gi/o. Pertussis toxin can block the activation of Gi/o. Activation of Gi/o stimulates cPLA2 to produce arachidonic acid (AA) that has been previously shown to activate BK channels and lysophospholipids (not shown) that could also contribute to the activation. Antisense oligonucleotides that inhibit expression of cPLA2 that inhibit expression of cPLA2 block the action of isoproterenol. Activation of -adrenergic receptors may also activate other signaling pathway, in particular, increased intracellular calcium. An increase in intracellular calcium would strongly reinforce the stimulation by Gi/o and cPLA2. On the other hand, in excised patches, calcium is held constant and therefore cannot contribute to the activation of BK channels. However, direct stimulation of Gi/o with GTP--S stimulates the channels, an effect that is completely blocked by pertussis toxin. Pretreatment of cells with antisense oligonucleotides against c-PLA2 also completely inhibited the activation of BK channels precluding any direct effect of GTP--S on the channels.

View larger version (10K): [in this window] [in a new window]   FIG. 7. All of the signaling elements required for BK-channel activation by isoproterenol are contained in excised patches. For these experiments, the bath (intracellular) solution contained 0.1 µM Ca2+, and the patches were depolarized to +20 mV. The tips of the pipettes were filled with saline containing no isoproterenol, whereas the remainder of the pipette was backfilled with the saline that contained 1 µM isoproterenol. We have previously shown that low molecular weight solutes (like isoproterenol) will equilibrate with the end of the pipette in 7–10 min. Channel activity was continuously recorded for 15 min. Repeated-measures analysis on data from 7 excised patches showed a statistically significant increase in Po from the 1st 5 min compared with the last 5 (from 0.31 ± 0.06 to 0.38 ± 0.08, P = 0.013; n = 7).

	DISCUSSION

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GH3 cells contain -adrenergic receptors

Although there is one anecdotal report suggesting no endogenous expression of adrenergic receptors in GH3 cells (Guerrero and Minneman 1999), others have shown that isoproterenol can stimulate growth hormone release in GH3 cells through both ₁- and ₂-receptors (Gabriel et al. 1989). In the present investigation, Western blot analysis of cell lysates from untreated GH3 cells revealed easily detectable amounts of both the ₁- and ₂-adrenergic receptors, further demonstrating our data are consistent with this observation.

Model for G protein activation of BK channels in GH3 cells

Figure 8 shows a schematic drawing of the mechanism for G protein activation of BK channels. In this model in cell-attached patches (A), isoproterenol activates -adrenergic receptors that activate the G protein, G_i/o (as others have recently described (Gosmanov et al. 2002; Gudermann et al. 1996, 1997; Kushmerick et al. 1999; LaBelle and Polyak 1998; Wellner-Kienitz et al. 2001; Xiao 2001). G_i/o activates cPLA₂, which produces arachidonic acid and lysophospholipids. We have shown that arachidonic acid can bind to and activate BK channels, but lysophospholipids might also contribute to the activation. In cell-attached patches, it is possible that other signaling pathways are also activated (by isoproterenol or AlF₄). In fact, it would be surprising if G_s proteins were not activated. Nonetheless, only G_i/o appears to be involved in BK channel activation because pertussis toxin can inhibit isoproterenol-mediated BK stimulation. As suggested by Jelsema and Axelrod (1987), G protein activation can occur via an interaction between an effector and either the or subunits after dissociation of the heterotrimeric complex. Because PTX acts by preventing this dissociation, we cannot unequivocally determine whether the activation occurs via an action of the or subunits. Along similar lines, both G_i and G_o are endogenously expressed in GH3 cells (Gudermann et al. 1996; Paulsson et al. 1992). Because PTX can inhibit dissociation of both G proteins, we cannot unequivocally identify G_i as the G protein activated by isoproterenol. As suggested by Paulssen et al. (1992), activation of G_i and G_o in GH3 cells was agonist specific. However, G_i does appear to be the most likely candidate because it is preferentially activated by isoproterenol (compared with G_o) in other cell types (Chung et al. 1999; LaBelle and Polyak 1998).

In intact cells, if intracellular calcium increases in response to isoproterenol, then this will reinforce the action of cPLA₂ to produce an even larger response than G_i/o activation alone. Of course, these alternative pathways cannot contribute to BK activation in excised, inside-out patches (Fig. 8B) where GTPS directly activates G_i/o. The effect of GTPS cannot be to activate cPLA₂ directly because the G_i/o inhibitor, pertussis toxin, completely inhibits the action of GTPS. Moreover, G_i/o is not directly activating BK channels because inhibiting cPLA₂ expression with antisense oligonucleotides also completely blocks the action of GTPS. cPLA₂ inhibitors are generally competitive inhibitors that may not produce complete block except at very high inhibitor concentrations; therefore we used antisense oligonucleotides in addition to a pharmacologic inhibitor (AACOCF3) in an effort to minimize any nonspecific effects of the pharmacologic inhibitors on the signal transduction pathways we studied. As anticipated, the results from experiments using either technique were identical.

The smaller response and larger variability in the GTP--S response compared with AlF₄ is probably a reflection of the fact that AlF₄ was applied to intact cells so that even G proteins outside the patch can contribute to the activation of cPLA₂ and production of arachidonic acid that subsequently activates BK channels. We and others have previously reported the activation of BK channels by the direct association of BK-channel proteins with arachidonic acid (and other cis-fatty acids), suggesting that there is a stereo-specific arachidonic acid binding site on the -subunit of BK-channels (Clark et al. 1991; Denson et al. 2000). This implies that association of arachidonic acid with BK channels stabilizes the channel in an open state. When GTPS is applied to excised patches, the magnitude of the response will depend on how many G_i/o and cPLA₂ proteins are present in the excised patch of membrane. The character of excised patches probably also explains the result of applying GDPS. GDPS will inactivate already active G proteins (that are activating BK channels) or inhibit activation of initially inactive G proteins. In an excised patch, there is no reason to expect that G proteins will already be active so application of GDPS, not surprisingly, will have no effect. Because the application of a pharmacologic inhibitor of c-PLA₂ to an excised patch always results in a decrease in P_o, we reasoned that there is a close association between c-PLA₂ and BK channels in GH3 cells. If the pathway depicted in the cartoon in Fig. 8B is correct, then a significant number of excised patches would contain all of the transduction elements required for BK channel activation by isoproterenol. If this is true, adding isoproterenol to the pipette solution would result in an increase in P_o in a significant number of excised patches. Our results showing P_o increased in 6/7 patches where isoproterenol had been backfilled in the patch pipette supports this signal transduction pathway.

In summary, we have demonstrated that in addition to the well-documented activation of c-PLA₂ by tyrosine phosphorylation or increases in [Ca_i²⁺] and subsequent calcium-dependent translocation to the membrane, cPLA₂ can be activated by a GPCR, which activates G_i/o. This activation of cPLA₂ results in an increased production of arachidonic acid and lysophospholipids and a corresponding increase in the P_o of BK channels.

	GRANTS

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This work was supported by National Science Foundation Grant IBN-0091964 to D. D. Denson and D. C. Eaton and National Institute of Diabetes and Digestive and Kidney Diseases Grant R37DK-37963 to D. C. Eaton.

	ACKNOWLEDGMENTS

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The authors gratefully acknowledge B. J. Duke for skillful technical assistance in the plating and maintenance of the cells used in this investigation. The authors also gratefully acknowledge S. Ramosevac for conducting the Western blot analyses.

	FOOTNOTES

 
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.

Address for reprint requests and other correspondence: D. D. Denson, Dept. of Anesthesiology, Emory University School of Medicine, 3B-South Emory University Hospital, 1364 Clifton Rd., Atlanta, GA 30322 (E-mail: Don_Denson{at}emoryhealthcare.org)

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