Regulation of ATP-sensitive K+ channels by protein kinase C in murine colonic myocytes

Jae Yeoul Jun, In Deok Kong, Sang Don Koh, Xuan Yu Wang, Brian A. Perrino, Sean M. Ward, and Kenton M. Sanders

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the regulation of ATP-sensitive K+ (KATP) currents in murine colonic myocytes with patch-clamp techniques. Pinacidil (10-5 M) activated inward currents in the presence of high external K+ (90 mM) at a holding potential of -80 mV in dialyzed cells. Glibenclamide (10-5 M) suppressed pinacidil-activated current. Phorbol 12,13-dibutyrate (PDBu; 2 × 10-7 M) inhibited pinacidil-activated current. 4-alpha -Phorbol ester (5 × 10-7 M), an inactive form of PDBu, had no effect on pinacidil-activated current. In cell-attached patches, the open probability of KATP channels was increased by pinacidil, and PDBu suppressed openings of KATP channels. When cells were pretreated with chelerythrine (10-6 M) or calphostin C (10-7 M), inhibition of the pinacidil-activated whole cell currents by PDBu was significantly reduced. In cells studied with the perforated patch technique, PDBu also inhibited pinacidil-activated current, and this inhibition was reduced by chelerythrine (10-6 M). Acetylcholine (ACh; 10-5 M) inhibited pinacidil-activated currents, and preincubation of cells with calphostin C (10-7 M) decreased the effect of ACh. Cells dialyzed with protein kinase C varepsilon -isoform (PKCvarepsilon ) antibody had normal responses to pinacidil, but the effects of PDBu and ACh on KATP were blocked in these cells. Immunofluorescence and Western blots showed expression of PKCvarepsilon in intact muscles and isolated smooth muscle cells of the murine proximal colon. These data suggest that PKC regulates KATP in colonic muscle cells and that the effects of ACh on KATP are largely mediated by PKC. PKCvarepsilon appears to be the major isozyme that regulates KATP in murine colonic myocytes.

potassium channel openers; glibenclamide; protein kinase C inhibitors; protein kinase C varepsilon -isoform


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SINCE THE IDENTIFICATION OF a K+ current in cardiac myocytes that was inhibited by intracellular ATP (24), there have been many reports that ATP-sensitive K+ (KATP) channels play important roles in regulating resting membrane potential and membrane excitability in a variety of tissues. The common properties of KATP channels are that they are 1) activated by K+ channel agonists such as lemakalim, diazoxide, and pinacidil, 2) inhibited by glibenclamide, and 3) modulated by intracellular ATP and nucleotide diphosphates (3, 6, 13, 30).

In previous studies of murine colonic muscle cells, KATP channels were found to be activated under resting conditions (17). Application of glibenclamide depolarized membrane potential and increased excitability. K+ channel openers, such as lemakalim and pinacidil, hyperpolarized intact muscles and blocked the spontaneous discharge of action potentials. The single-channel conductance activated by K+ channel openers was 27 pS in symmetrical K+ gradients. Molecular studies showed the expression of the ionic channel of KATP (Kir 6.2) and the sulfonylurea receptor type 2B (SUR2B) in murine colonic myocytes. Intracellular ATP regulates the open probability of KATP channels, but ATP is normally in the millimolar range in smooth muscle myocytes (1, 26). Nucleotide diphosphate also contributes to the regulation of open probability (13). For example, we found that after rundown of KATP channels in patches excised from colonic myocytes, either high concentrations of ADP (1 mM) or low concentrations of ATP (0.1 mM) applied to the intracellular surface could restore openings of KATP channels. Therefore, the ratio of ADP to ATP may be an important factor regulating these channels. Agonists and second messengers may also regulate KATP channels in colonic myocytes.

In vascular and visceral smooth muscle, vasodilating substances such as adenosine, isoproterenol, and calcitonin gene-related peptide activate KATP channels via cAMP-dependent mechanisms (22, 23). On the other hand, excitatory agonists such as angiotensin II and carbachol inhibit KATP channels in vascular, tracheal, bladder and esophageal smooth muscles via protein kinase C (PKC)-dependent mechanisms (1, 6a, 7, 18, 25). In the present study, we have characterized the regulation of KATP channels in gastrointestinal muscle cells by PKC varepsilon -isoform (PKCvarepsilon ), which has not been previously investigated. These studies were performed on isolated murine colonic myocytes by using patch-clamp techniques.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell isolation. Colonic smooth muscle cells were isolated from 20- to 30-day-old BALB/C mice of either sex. Mice were anesthetized with chloroform and killed by cervical dislocation, and the proximal colon was quickly removed. The colon was opened along the myenteric border, and mucosa and submucosa were removed in Ca2+-free Hanks' solution containing (in mM) 125 NaCl, 5.36 KCl, 15.5 NaOH, 0.336 Na2HCO3, 0.44 KH2PO4, 10 glucose, 2.9 sucrose, and 11 HEPES. Strips of colonic muscle were transferred to the same buffer solution containing 230 units of collagenase (Worthington Biochemical), 2 mg of fatty acid-free bovine serum albumin (Sigma Chemical), 2 mg of trypsin inhibitor (Sigma Chemical), and 0.1 mg of papain (Sigma Chemical). Incubation in the enzyme solution was carried out at 37°C for ~10-12 min, and then the tissues were washed with Ca2+-free Hanks' solution. Single cells were obtained by gentle agitation with a wide-bored glass pipette. Isolated cells were kept at 4°C until they were used. Before electrophysiological studies were begun, a drop of the cell suspension was pipetted into a small chamber (0.3 ml) on the stage of an inverted microscope. The experiments were carried out within 6 h of dispersing the cells. All electrophysiological recordings were performed at room temperature (22-25°C).

Voltage-clamp experiment. Patch-clamp experiments using the dialyzed whole cell, perforated whole cell, and cell-attached patch configurations were performed on colonic smooth muscle cells. Glass pipettes with resistance of ~3-5 MOmega for whole cell configuration and 5-10 MOmega for cell-attached single-channel recordings were used. Membrane currents were amplified by an Axopatch 1-D (Axon instruments) or an Axopatch 200B amplifier and CV-4 head stage (Axon Instruments, Foster City, CA). Command pulses were applied with an IBM-compatible personal computer and pCLAMP software (version 5.5 or 6.1; Axon instruments). The data were filtered at 5 kHz and displayed on an oscilloscope, a computer monitor, and a pen recorder (Gould, Valley View, OH). To minimize activities of voltage-dependent K+ channels and Ca2+-activated K+ channels, we performed the whole cell experiments at a holding potential of -80 mV in a solution containing elevated extracellular K+ concentration (see below).

The single-channel conductance responsible for KATP channels in murine colonic myocytes was previously shown to be due to 27-pS K+ channels in a symmetrical K+ gradient (17). In the present study, asymmetrical K+ gradients were used (5 mM to 140 mM), and under these conditions the amplitude of the 27-pS KATP channels would be ~0.6 pA at 0 mV as fit by the Goldman-Hodgkin-Katz (GHK) equation. To further confirm that the single-channel currents observed in on-cell patches in the current study were due to KATP channels, we excised the patches at the end of the experiments and noted channel rundown. Open probability was restored by ADP (1 mM), as we have shown previously for KATP channels in murine colonic myocytes (17).

Solutions and drugs. For dialyzed whole cell recordings, the internal pipette solution contained (in mM) 10 NaCl, 102 KCl, 1 CaCl2, 1 GTP, 10 HEPES, 10 EGTA, 0.1 ATP, and 1 MgCl2, adjusted to pH 7.2 with KOH (38 mM). The normal external solution contained (in mM) 135 NaCl, 5 KCl, 1 MgCl2, 10 HEPES, and 0.2 CaCl2, adjusted to pH 7.4 with Tris. The high-K+ external solution contained K+ (90 mM) and Na+ (50 mM). For amphotericin B perforated patches, the composition of pipette solution (in mM) was 110 K-gluconate, 30 KCl, 5 MgCl2, and 5 HEPES, adjusted to pH 7.2 with Tris. Final concentration of amphotericin B was 270 µg/ml.

For cell-attached patch recordings, the external solution contained (in mM) 140 KCl, 1 EGTA, 0.2 CaCl2, and 10 HEPES, adjusted to pH 7.4 with Tris. The pipette solution contained (in mM) 135 NaCl, 5 KCl, 1 MgCl2, and 10 HEPES, adjusted to pH 7.4 with Tris. Charybdotoxin (200 nM) was included in the pipette solution in the majority of cell-attached patch experiments to inhibit large-conductance Ca2+-activated K+ channels.

Pinacidil, calphostin C, and chelerythrine were purchased from RBI. Glibenclamide, 4-alpha -phorbol ester, and phorbol 12,13-dibutyrate (PDBu) were purchased from Sigma. All drugs except chelerythrine were dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO was <0.05%. PKCvarepsilon (1:150, Boehringer Mannheim, Mannheim, Germany; or 1:200, PanVera, Madison, WI) antibody was introduced into the pipette. Heat-inactivated PKCvarepsilon antibody was prepared by boiling at 100°C for 20 min.

Immunohistochemistry. The proximal colon segments were opened along the mesenteric border, and luminal contents were washed away with Krebs Ringer bicarbonate solution (KRB). The opened segments were pinned onto the base of a Sylgard dish with the mucosal side facing up. The mucosa was removed by sharp dissection, and the remaining tunica muscularis was fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 15 min at 4°C. After fixation, preparations were washed for 30 min in phosphate-buffered saline (PBS; 0.05 M, pH 7.4). Nonspecific antibody binding was reduced by incubation of the tissues in 10% normal goat serum for 1 h at room temperature. Tissues were incubated with the primary antibody (polyclonal rabbit antiserum against PKCvarepsilon ; 1:150; Boehringer Mannheim) for 48 h at 4°C. The tissues were then exposed to FITC-coupled goat anti-rabbit IgG (1:100; Vector Laboratories, Burlingame, CA) for 1 h at room temperature. Control tissues were prepared by omitting either primary or secondary antibodies from incubation solutions. All the antisera were diluted with 0.3% Triton X-100 in 0.05 M PBS (pH 7.4). Tissues were examined with a Bio-Rad MRC 600 confocal microscope (Hercules, CA) with an excitation wavelength appropriate for FITC (494 nm).

Indirect immunofluorescence of PKCvarepsilon in isolated smooth muscle cells. Murine proximal colon smooth muscle cells were enzymatically dispersed and incubated in physiological solution. After attachment to glass coverslips for 2-3 h, the cells were rinsed twice with PEM (100 mM PIPES, pH 7.4, 2 mM EGTA, and 1 mM Mg-acetate) and then incubated in PEM/3.7% formaldehyde for 30 min. The PEM/3.7% formaldehyde was gently aspirated, and the cells were rinsed twice with PBS and then incubated for 5 min in PBS/100 mM glycine to quench any remaining formaldehyde. The cells were then extracted in PEM/0.3% Nonidet P-40 for 10 min. The detergent extraction was stopped by two rinses with PBS, and the cells were stored in PBS at 4°C before indirect immunofluorescence staining.

For staining PKCvarepsilon , the glass coverslips were drained of excess PBS and placed in a humidified chamber. The coverslips were incubated for 1 h at 37°C with either 30 µl of PanVera anti-PKCvarepsilon antibody (1:25 dilution in PBS) or 30 µl of Alexa 488-conjugated goat anti-rabbit IgG antibody (Molecular Probes) diluted 1:200 in PBS. The coverslips were then washed in PBS and incubated for 1 h at 37°C with 30 µl of Alexa 488-conjugated goat anti-rabbit IgG antibody diluted 1:200 in PBS. The coverslips were washed in PBS, rinsed with distilled H2O, and mounted cell-side down onto glass slides with Aquamount. PKCvarepsilon staining of individual dispersed smooth muscle cells was visualized by confocal microscopy.

Western blotting. Colon smooth muscle tissues were obtained as described in Immunohistochemistry. Smooth muscle lysates were obtained by ground glass homogenization in 40 mM Tris · HCl, pH 7.5, 6 mM MgSO4, 1 mM EGTA, 0.1% Triton X-100, 0.5 mM dithiothreitol, and protease inhibitors. The crude homogenate was centrifuged at 10,000 g at 4°C for 15 min. The supernatant was analyzed for protein content by using the Bradford assay, with bovine gamma globulin as standard. The supernatant was separated by SDS-PAGE (10%) and electroblotted onto nitrocellulose. The blots were incubated in blocking buffer (PBS, 0.2% casein, and 0.1% Tween 20) for 45 min, followed by incubation with rabbit anti-PKCvarepsilon antibody (1:200 dilution; PanVera) in blocking buffer for 90 min. To neutralize the antibody, we incubated a 10-fold molar excess of antigenic peptide with the antibody for 1 h before the blots were incubated. The blots were washed with two 5-min washes in blocking buffer minus casein, followed by incubation with alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (1:5,000 dilution; Tropix) in blocking buffer for 90 min. The blots were washed as before and then washed with two 2-min washes in assay buffer (20 mM Tris · HCl, pH 9.8, and 1 mM MgCl2). Immunodetection was carried out by using the Western-Star enhanced chemiluminescence kit (Tropix).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To avoid contamination from voltage-dependent currents expressed by murine colonic myocytes, cells were held at -80 mV, and the external bathing solution was changed from 5 to 90 mM K+. Activation of a K+ conductance under these conditions results in inward current. Switching from 5 to 90 mM external K+ resulted in a steady-state inward current that averaged -58.2 ± 8.4 pA (n = 14 experiments). Pinacidil (10-5 M) activated additional inward currents with a mean amplitude of -429 ± 13 pA (n = 14), and glibenclamide (10-5 M) suppressed 90 ± 3% of the pinacidil-activated currents (n = 7, Fig. 1A). PDBu (2 × 10-7 M), an activator of PKC, inhibited pinacidil-activated currents by 67 ± 6% (n = 8) slowly in dialysis patch, and the remaining currents were returned to baseline by 5 mM K+-containing solution (Fig. 1B). On the other hand, 4-alpha -phorbol (5 × 10-7 M), an inactive form of PDBu, had no effect on the pinacidil-activated currents in four cells (Fig. 1C).


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Fig. 1.   Activation of ATP-sensitive K+ (KATP) channels in dialyzed murine colonic myocytes. A: changing the external solution from 5 mM K+ to 90 mM K+ elicited an inward current in cells. Addition of pinacidil (10-5 M) activated a large steady-state current that was blocked by glibenclamide (10-5 M). B: the current activated was reduced by phorbol 12,13-dibutyrate (PDBu; 2 × 10-7 M). C: the inactive phorbol ester (4-alpha -phorbol; 5 × 10-7 M) did not inhibit the pinacidil-activated current. Dotted lines denote 0 current in all traces.

We reported the single-channel conductance of KATP in murine colonic myocytes previously (i.e., 27 pS in symmetrical 140/140 mM K+) (17). In the present study the open probability of KATP channels was measured in cell-attached patches. Pinacidil activated K+ channels at 0 mV in 5 mM extracellular/140 mM intracellular K+ concentrations (Fig. 2, A and B). The control open probability was 0.10 ± 0.05 (Fig. 2D). After treatment with pinacidil, open probability increased to 0.38 ± 0.10 (Fig. 2E). Addition of PDBu (5 × 10-7 M) to the same cells suppressed the openings of KATP channels (0.08 ± 0.09; n = 3) (Fig. 2, C and F).


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Fig. 2.   Single KATP channels are activated by pinacidil and inhibited by phorbol ester. A: under the conditions of an asymmetrical K+ gradient, single KATP channels opened spontaneously in cell-attached patches. Addition of pinacidil (10 -5 M) increased the open probability of KATP channels (B). Addition of PDBu (5 × 10-7 M) in the continued presence of pinacidil reduced the open probability of KATP channels (C). Histograms show amplitude under control conditions (D) and in the presence of pinacidil (E) and pinacidil plus PDBu (F). c, Closed state (solid lines); o, open state (dotted lines).

We tested whether activation of PKC was responsible for the inhibitory effects of PDBu on KATP by pretreating cells with PKC inhibitors (Fig. 3). Inhibitors with different mechanisms of action were chosen. Chelerythrine is a potent PKC inhibitor that binds in a noncompetitive manner to the ATP binding site (8). Calphostin C inhibits PKC by binding to the phorbol ester binding site (4). Pretreatment with either chelerythrine (10-6 M) (Fig. 3A) and calphostin C (10-7 M) (Fig. 3B) for 20 min had no effect on the activation of current by pinacidil, but these compounds reduced the effects of PDBu on the current [i.e., to 17 ± 5% (n = 5) and 14 ± 4% (n = 5)], respectively (Fig. 3C).


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Fig. 3.   Effects of PDBu on KATP are reduced by inhibitors of protein kinase C (PKC). Addition of pinacidil (10-5 M) activated a large steady-state current that was reduced by PDBu (2 × 10-7 M). When cells were pretreated with chelerythrine (10-6 M) (A) or calphostin C (10-7 M) (B), the effects of PDBu on KATP were significantly reduced. Dotted lines denotes 0 current. C: summary of experiments with PDBu and PKC inhibitors (n = no. of experiments). *P < 0.05.

We also performed experiments by using the perforated patch technique to test the effects of PDBu on the pinacidil-activated current in nondialzyed cells. The amplitude of pinacidil-activated currents was -288 ± 20 pA (n = 4). PDBu inhibited pinacidil-activated currents by 92 ± 4% (n = 5) in cells studied with the perforated patch technique (Fig. 4, A and C). This was a greater degree of inhibition than observed in dialyzed cells, suggesting that diluting the soluble components of the cytoplasm with the pipette solution reduced the effectiveness of PKC regulation. Chelerythrine (10-6 M) reduced the inhibition of the pinacidil-activated current by PDBu to 16 ± 5% (n = 5) (Fig. 4, B and C).


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Fig. 4.   Effects of PDBu on KATP in cells with perforated patches. A: addition of pinacidil (10-5 M) activated a large steady-state current that was reduced by PDBu (2 × 10-7 M). The effects of PDBu on cells with perforated patches were larger than on cells that were dialyzed with the pipette solution (compare with Fig. 3). B: pretreatment with chelerythrine (10-6 M) inhibited the effects of PDBu on KATP. Dotted lines denote 0 current. C: summary of experiments with PDBu and chelerythrine in cells with permeabilized patch technique (n = no. of experiments). *P < 0.05.

Acetylcholine (ACh), a primary excitatory neurotransmitter in the gastrointestinal (GI) tract, has been shown to activate PKC in some smooth muscles (28). Therefore, we exposed dialyzed cells to ACh (10-5 M) and found that this treatment reduced the pinacidil-activated current by 58 ± 5% (n = 5) (Fig. 5, A and C). Preincubation of the cells with calphostin C (10-7 M) decreased the inhibitory effects of ACh (to 10 ± 5%, n = 5) (Fig. 5, B and C).


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Fig. 5.   Effects of acetylcholine (ACh) on KATP. A: addition of pinacidil (10-5 M) activated a large steady-state current that was blocked by ACh (10-5 M). B: pretreatment with calphostin C (10-7 M) inhibited the effects of ACh on KATP. Dotted lines denote 0 current. C: summary of experiments with ACh and calphostin C (n = no. of experiments). *P < 0.05.

PKCvarepsilon has been implicated in mediating responses to several agonists, including ACh, in smooth muscles (9, 28). Therefore, we performed experiments in which cells were dialyzed with an antibody raised against a PKCvarepsilon -specific epitope to test whether this isozyme participates in regulation of KATP. After dialysis with anti-PKCvarepsilon , PDBu inhibited the pinacidil-activated current by only 12 ± 3% (n = 5) (Fig. 6, A and D). When cells were dialyzed with anti-PKCvarepsilon antibody that had been heat inactivated, PDBu inhibited the pinacidil-activated current by 59 ± 7% (n = 5) (Fig. 5, B and D), which was not significantly different from the effect of PDBu in control studies. Similarly, when cells were dialyzed with PKCvarepsilon antibody, ACh had only a small effect on KATP current (reduced ACh inhibition to 10 ± 5%, n = 5) (Fig. 6, C and D).


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Fig. 6.   PKCvarepsilon antibody blocked inhibition of KATP by PDBu and ACh. Cells were dialyzed with anti-PKCvarepsilon antibody. Pinacidil (10-5 M) activated a large steady-state current that was not blocked by PDBu (2 × 10-7 M) (A) or ACh (10-5 M) (B). When cells were dialyzed with heat-inactivated anti-PKCvarepsilon antibody, PDBu had an inhibitory effect on KATP (C). D: summary of the effects of PDBu and ACh on KATP in cells dialyzed with PKCvarepsilon antibody (n = no. of experiments). *P < 0.05.

Immunohistochemical experiments were also performed to determine whether murine colonic muscle cells express PKCvarepsilon immunoreactivity. With the Boehringer Mannheim anti-PKCvarepsilon antibody, prominent immunoreactivity was observed in smooth muscle cells and enteric neurons (Fig. 7). Circular and longitudinal muscle cells were immunopositive (Fig. 7A). In testing the specificity of the Boehringer Mannheim anti-PKCvarepsilon antibody (i.e., Western blots of purified PKC isozymes; data not shown), it was recognized that the Boehringer Mannheim anti-PKCvarepsilon antibody demonstrated cross-reactivity with PKCdelta . Therefore, we tested immuoreactivity of smooth muscle cells by using a second anti-PKCvarepsilon antibody (PanVera; highly selective for PKCvarepsilon ) and found isolated colonic myocytes to be immunopositive (Fig. 7, B and C). To further investigate the expression of PKCvarepsilon in murine proximal colon smooth muscle, we carried out Western blot analysis of colonic smooth muscle lysates using the PanVera anti-PKCvarepsilon antibody. As shown in the Western blot in Fig. 8, the purified PKCvarepsilon migrates in SDS-PAGE to its expected molecular mass of ~90 kDa. In addition, a single immunoreactive band between 75 and 100 kDa that comigrates with purified PKCvarepsilon in SDS-PAGE was detected in proximal colon smooth muscle lysates. The immunodetection of this band was blocked by preincubation of the anti-PKCvarepsilon antibody with the antigenic peptide (Fig. 8, lane 3).


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Fig. 7.   A: cross section of tunica muscularis of the murine colon labeled with the Boehringer Mannheim anti-PKCvarepsilon antibody showing prominent immunoreactivity in circular (CM) and longitudinal (LM) muscle layers. Cells within the myenteric plexus (MP) were also immunopositive with this antibody. B: isolated smooth muscle cells were also immunopositive for PKCvarepsilon when a highly selective antibody from PanVera was used (see METHODS). C: a negative control in which the PanVera primary antibody was omitted. Scale bars are as indicated.



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Fig. 8.   Immunodetection of PKCvarepsilon in murine proximal colon smooth muscles. Purified PKCvarepsilon (lane 1, 0.5 µg) and proximal colon smooth muscle tissue lysate (lanes 2 and 3; 50 µg) were separated by SDS-PAGE and immunoblotted as described in METHODS. Lane 3: preincubation of anti-PKCvarepsilon antibody with antigenic peptide.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In a previous study we found that KATP contributes to the regulation of membrane potential and excitability in murine colonic muscles (17). Glibenclamide blocked resting KATP conductance, produced depolarization, and increased action potential generation. Lemakalim and pinacidil, which are activators of KATP, increased the open probability of single KATP channels, hyperpolarized muscles, and inhibited spontaneous action potentials. Activation of KATP channels under basal conditions was also observed in pig proximal urethra (31). Thus modulation of the open probability of KATP channels could provide a means of regulating excitability in some smooth muscles. The fact that KATP contributes to the resting conductance of smooth muscle cells suggests that inhibition of this current could be a mechanism by which excitatory transmitters or hormones could act.

Regulation of KATP channels in smooth muscles occurs by several mechanisms. Cytoplasmic ATP levels are usually at millimolar levels (1, 26) and fall substantially only under conditions of severe metabolic inhibition. Cytoplasmic ATP may therefore set a relatively low open probability for the channels under basal conditions, against which other regulator factors may modulate channel openings (27). Studies have also shown that the open probability of KATP in smooth muscles is regulated by nucleotide diphosphate concentrations (13), and this property of the isoform(s) of KATP expressed by smooth muscle may enhance open probability over that predicted from the high levels of ATP. KATP channels are also known to be activated by a variety of vasodilators through protein kinase A (23, 26, 27). In contrast, several vasoconstrictors are coupled through G proteins to phospholipases, generation of inositol 1,4,5-trisphosphate and diacylglycerol, and activation of PKC. Previous studies of vascular, tracheal, and bladder smooth muscle cells have shown that stimulation by excitatory agonists inhibits KATP via activation of PKC (1, 2, 6a, 18, 25). The present study sought to determine whether PKC regulates KATP in GI smooth muscle cells and whether this mechanism might mediate some of the excitatory effects of cholinergic stimulation. In rabbit esophageal smooth muscle, KATP was suppressed by phorbol 12-myristate 13-acetate, and these effects were significantly reduced by PKC inhibitors (7). Our results have extended previous studies by showing that a specific isoform of PKC (PKCvarepsilon ) is expressed in colonic muscles and appears to be largely responsible for regulation of KATP.

PKC comprises one of the major second messenger systems that mediate the responses to agonists in smooth muscles. There are several isozymes of PKC, some of which are translocated from the cytosol to the cell membrane upon stimulation (14, 15). PKC isozymes can be activated by diacylglycerol, intracellular Ca2+, or phospholipids under physiological conditions (29). PKCalpha and PKCbeta undergo agonist-induced translocation from the cytosol to the plasma membrane in vascular smooth muscle cells (15, 20, 28). Activation and translocation of the Ca2+-dependent isozyme, PKCalpha , in vascular smooth muscle cells occurs at basal or near basal levels of intracellular Ca2+ concentration (15). In contrast PKCvarepsilon , which has been implicated in contraction of ferret aorta smooth muscle cells, translocates from the cytosol to the surface membrane under low-Ca2+ conditions and may mediate Ca2+-independent contractions in these cells (9, 14, 16, 33). PKCvarepsilon also contributes to the mediation of agonist responses in GI muscles (28); however, the full range of substrates for PKCvarepsilon in GI muscle cells has not been described. In the present study we have provided data showing that Kir 6.2 (17), SUR2B (17), or a subsidiary regulatory protein may be a substrate for PKCvarepsilon in colonic muscle cells. Dialysis of cells with a specific antibody against PKCvarepsilon blocked most of the inhibitory effects of PDBu on KATP. In addition, immunohistochemical and Western analyses demonstrate the presence of PKCvarepsilon in murine proximal colon smooth muscles.

The inhibitory effects of PKC on KATP open probability may be related to the specific isoforms of Kir 6 or sulfonylurea receptors (SUR) expressed in smooth muscles. For example, in rabbit and human ventricular myocytes, which express Kir 6.2 and SUR2A (18, 19), PKC activated KATP by reducing channel sensitivity to intracellular ATP (10). In contrast, in epithelial cells of the renal proximal tubule, KATP channels were inhibited by phorbol ester in cell-attached patches, and PKC applied to excised patches decreased open probability (21). Similar regulation was observed in the present study in cells that express Kir 6.2 and SUR2B (17). Because the primary difference between colonic KATP and cardiac KATP appears to be the isoform of SUR associated with the channels, it may be that the primary regulation by PKC occurs via this subunit.

The present study provides a new mechanism by which cholinergic stimulation might enhance the excitability of GI smooth muscles. ACh stimulates nonselective cation channels in GI muscles (5, 11, 12, 32), and this effect would be expected to result in depolarization and an increase in excitability. Inhibition of K+ channels that are open under basal conditions could also provide excitatory input in these cells. Many of the K+ channels expressed by GI muscle cells are activated by depolarization and have low open probabilities at resting potentials. Previous studies have demonstrated basal activation of KATP (17), so suppression of these channels would have a depolarizing influence on colonic cells. Together with the nonselective cation conductance expressed by many GI muscles and activated by excitatory neurotransmitters, inhibition of KATP by PKC may be an important excitatory mechanism.


    ACKNOWLEDGEMENTS

We thank Dr. Mike Walsh (Univ. of Calgary) for helpful discussions.


    FOOTNOTES

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-41315 and DK-57168 (to B. Perrino). J. Y. Jun was supported by Chosun University (Republic of Korea), the Korean Ministry of Science and Technology, and the Korean Science and Engineering Foundation through the Research Center for Proteinous Materials.

Address for reprint requests and other correspondence: K. M. Sanders, Dept. of Physiology and Cell Biology, Univ. of Nevada School of Medicine, Reno, NV 89557 (E-mail: kent{at}physio.unr.edu).

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

Received 12 November 2000; accepted in final form 24 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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