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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Nitrotyrosylation of Ca²⁺ Channels Prevents c-Src Kinase Regulation of Colonic Smooth Muscle Contractility in Experimental Colitis

Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia (G.R.R., M.K., M.D., H.I.A.); and Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma (N.S., A.P.M.)

Received March 19, 2007; accepted June 4, 2007.

	Abstract

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Basal levels of c-Src kinase are known to regulate smooth muscle Ca²⁺ channels. Colonic inflammation results in attenuated Ca²⁺ currents and muscle contraction. Here, we examined the regulation of calcium influx-dependent contractility by c-Src kinase in experimental colitis. Ca²⁺-influx induced contractions were measured by isometric tension recordings of mouse colonic longitudinal muscle strips depolarized by high K⁺. The E_max to CaCl₂ was significantly less in inflamed tissues (38.4 ± 7.6%) than controls, indicative of reduced Ca²⁺ influx. PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine], a selective Src kinase inhibitor, significantly reduced the contractile amplitude and shifted the pD₂ from 3.88 to 2.44 in controls, whereas it was ineffective in inflamed tissues (3.66 versus 3.43). After pretreatment with a SIN-1 (3-morpholinosydnonimine)/peroxynitrite combination, the maximal contraction to CaCl₂ was reduced by 46 ± 7% in controls but unaffected in inflamed tissues (13 ± 11%). Peroxynitrite also prevented the inhibitory effect of PP2 in control tissues. In colonic single smooth muscle cells, PP2 inhibited Ca²⁺ currents by 84.1 ± 3.9% in normal but only 36.2 ± 13% in inflamed tissues. Neither the Ca²⁺ channel Ca_v1.2b, gene expression, nor the c-Src kinase activity was altered by inflammation. Western blot analysis showed no change in the Ca²⁺ channel protein expression but increased nitrotyrosylated-Ca²⁺ channel proteins during inflammation. These data suggest that post-translational modification of Ca²⁺ channels during inflammation, possibly nitrotyrosylation, prevents c-Src kinase regulation resulting in decreased Ca²⁺ influx.

	Materials and Methods

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All animal protocols followed the guidelines and approval by the Virginia Commonwealth University Institutional Animal Care and Use Committee.

Induction of Colitis. Colonic inflammation was induced by intracolonic instillation of TNBS (2.5% in 50% ethanol) in male Balb/C mice through a 3.5-French catheter carefully inserted into the rectum. The catheter tip was inserted 4 cm proximal to the anal verge. Mice were carefully held in a vertical position (head down) for 60 s after TNBS instillation to ensure distribution of the TNBS within the entire colon. Control animals received the same volume (100 µl) of vehicle solution. The mice were weighed daily and inspected for diarrhea and rectal bleeding for assessing the "disease activity index" as described previously (Jin et al., 2004). Tissues were used for isometric tension recording, real time-PCR, enzyme assay, or electrophysiological experiments 3 days post-TNBS. The time adopted (3 days after TNBS challenge) is consistent with several other studies in mice (Daniel et al., 2006; Beck et al., 2007) and correlated well with the disease activity index, as we have described previously (Akbarali et al., 2000; Jin et al., 2004).

Isometric Tension Recording. Approximately 1.5-cm strips of distal colon were suspended in the longitudinal direction in an organ bath containing 15 ml of Krebs solution, bubbled continuously with carbogen (95% O₂ and 5% CO₂) at 37°C under a resting tension of 1 g and equilibrated for a period of 1 h. Isometric contractions were recorded by a force transducer (model GR-FT03; Radnoti, Monrovia, CA) connected to a personal computer using Acqknowledge 382 software program (BIOPAC Systems, Santa Barbara, CA). Preliminary experiments were conducted to examine the length-tension characteristics of normal and inflamed colonic muscle, as described earlier (Vallance et al., 1997). Both normal and inflamed muscle strips produced their respective maximal contraction to carbachol at a resting tension of 1g, and the muscle length was not different at this optimal tension. Hence, we kept constant the passive tension applied and the tissue strip length for further experiments. After equilibration in Krebs solution, tissues were incubated for 30 min in Ca²⁺-free high-potassium solution (80 mM) in which equimolar NaCl was replaced by KCl containing 0.1 mM EGTA and changed every 15 min. Cumulative dose-dependent contraction responses to CaCl₂ (10 µM to 1 mM) were performed in distal colon strips depolarized by Calcium-free high-K⁺ (80 mM)-physiological saline solution (without EGTA), both in the presence and absence of 10 µM PP2, a Src kinase inhibitor. Moreover, cumulative dose-dependent contraction to sodium orthovanadate (1 µM to 1 mM), a tyrosine phosphatase inhibitor, was also performed in distal colon, equilibrated in Krebs physiological saline solution both in the presence and absence of PP2 (10 µM). The contractions were normalized to tissue cross-sectional area for analysis (Vallance et al., 1997). To determine whether nitrosylation mimics inflammation-induced changes, both normal and inflamed distal colon strips were incubated in SIN-1 (500 µM) for 1 h followed by sodium peroxynitrite (150 µM) twice at 10-min interval and washed with calcium-free high-K⁺-physiological saline solution. CaCl₂ (1 mM)-induced contractile responses were recorded before and after peroxynitrite treatment and also after incubation in PP2 (10 µM) for 15 min.

Electrophysiological Recording. Single smooth muscle cells from mouse distal colon were obtained enzymatically as described previously (Hu et al., 1998). Ca²⁺ currents were recorded at room temperature using EPC 10 patch-clamp amplifier (HEKA, Lambrecht/Pfalz, Germany). Patch micropipettes with resistances of 3 to 5 M were pulled from borosilicate glass capillaries on a Flaming-Brown P97 (Sutter Instruments Company, Novato, CA) electrode puller. The cells were continuously perfused with HEPES-buffered physiologic salt solution containing 20 mM Ba²⁺ as the charge carrier, and the internal pipette solution contained CsCl to block K⁺ currents. Pulse generation and data acquisition were performed using Patchmaster version 2.15 software (HEKA). The cells were voltage-clamped at –60 mV, and membrane currents were recorded at a voltage step to +20 mV over a period of 500 ms. After basal recording, the cells were perfused with SIN-1 (10 µM) and sodium peroxynitrite (150 µM) for 15 min and washed with HEPES-buffered physiologic salt solution before the Ca²⁺ currents were recorded again. The inhibition of Ca²⁺ currents were calculated as a percentage of the peak current.

RNA Extraction and Real Time-Polymerase Chain Reaction. Total RNA was purified from colon using the Invitrogen Purelink Micro to Midi RNA purification system (Invitrogen, Carlsbad, CA). Purified RNA was eluted in 70 µl of total volume with diethyl pyrocarbonate-treated water. Equal concentrations of RNA were used in all of the samples as determined by UV spectrometer; 1 µg of RNA/reaction was used in all of the reactions. Quantace SensiMix One-Step mix was used with changes to adapt the protocol to TaqMan (Roche Diagnostics, Mannheim, Germany) multiplex use. Real time reverse-transcribed PCR was performed on MJ MiniOpticon (Bio-Rad, Hercules, CA) in a multiplex assay for Ca_v1.2b (FAM) and GAPDH (HEX) as the internal control. All primers and probes were designed using Beacon Designer (Premier Biosoft Int., Palo Alto, CA). The multiplex primer sequences used were 5'-CAT CCT TGC TGA ACT CAG TGC-3' (forward) and 5'-TCG AAA TTG AAC TTC CCT CCA AAG-3' (reverse) for Ca_v (NM_009781 [GenBank] ), 5'-GCT GCC CAG AAC ATC ATC CC-3' (forward), and 5'-AGA TCC ACG ACG GAC ACA TTG-3' (reverse) for GAPDH (NM _001001303). The multiplex TaqMan probe sequences were 5'-56-FAM/CCT CCC TGC TGC TGC TCC TCT TCC/3BHQ1–3' for Ca_v, 5'-/5HEX/ATC CAC TGG TGC TGC CAA GGC TGT/3BHQ1/-3' for GAPDH. Real time-PCR assay for IL-1 was also performed separately using the primers 5'-GCA GAC AGC TCA ATC TCT AGG AG-3' (forward) and 5'-TCT CTT TGA ACA GAA TGT GCC ATG-3' (reverse) and 5'-/56-FAM/TGA CCC TGA GCG ACC TGT CTT GGC/3BHQ1/-3' (TaqMan probe) (NM_008361 [GenBank] ) to confirm inflammatory changes.

c-Src Kinase Activity Assay. Src kinase activity of cell lysates from distal colon smooth muscle from control and inflamed mice was determined according to the manufacturer's instructions (Upstate Biotechnology, Charlottesville, VA). In brief, 200 µg of protein from cell lysates was incubated with 1 µg of anti-c-Src-specific antibody (SRC2; Santa Cruz Biotechnology Inc., Santa Cruz, CA) at 4°C overnight in a final volume of 500 µl. Immunocomplexes were collected by incubation with 30 µl of protein A/G plus agarose for 1 h and then washed three times with ice-cold phosphate-buffered saline buffer. Ten microliters (150 µM final concentration) of the substrate peptide, 10 µl of Src reaction buffer, and 10 µlof[-³²P]ATP stock were added to a microcentrifuge tube and incubated for 10 min at 30°C with agitation. Twenty microliters of 40% trichloroacetic acid was then added to precipitate peptides, and a 25-µl aliquot was transferred onto the center of a numbered P81 paper square. The assay squares were washed three times for 5 min, each with 0.75% phosphoric acid and once with acetone. The assay squares were transferred to a scintillation vial, 5 ml of scintillation mixture was added, and the level of radioactivity was determined in a scintillation counter. The presence of active c-Src was also detected by Western blots using anti-Src-specific antibody (Tyr416; Upstate Biotechnology Inc.).

Quantitative Analysis of Inflammation-Induced Nitrotyrosylation of Ca²⁺ Channels in Distal Colon by Immunoblotting. Colons were excised from mice, cleaned, and flash-frozen in liquid nitrogen and used immediately. Tissues were homogenized in lysis buffer solution (10 ml/g tissue). The buffer contained 1x radioimmunoprecipitation assay buffer, 1x Complete protease inhibitor cocktail (Roche Diagnostics), 30 mM NaF, 4 mM Na₃VO₄, 5 µg/ml calpain inhibitor II, and 5 mM phenylmethylsulfonyl fluoride (added just before use). The tissues with buffer were kept on ice, dounced for 75 to 120 strokes, and sonicated for 5 to 10 s at 30% power (Fisher Sonic Dismembrator model 100; Thermo Fisher Scientific, Waltham, MA). The sonication was repeated after a 1-min rest. The samples were rotated at 4°C for 15 min and centrifuged at 14,000 rpm at 4°C for 15 min. Lysis buffer (500 µl) was added to 1 mg of protein [determined by BCA assay kit (Pierce, Rockford, IL)], combined with 30 µl of protein A/G beads (Santa Cruz Biotechnology, Inc.) and 1 µg of Ca_V1.2 antibody (BD Biosciences PharMingen, San Jose, CA) and incubated at 4°C overnight. Immunoprecipitation samples were centrifuged at 14,000 rpm at 4°C for 30 s, and the buffer was removed. Beads were washed four to five times for 5 min each at 4°C in lysis buffer with 0.5 M NaCl. After last wash, 30 µl of sample loading buffer was added to the beads. Samples were run on a 7% SDS-polyacrylamide gel electrophoresis semi-dry and transferred on a 0.2-mm polyvinylidene difluoride membrane at 17 V for 3.5 h. Membranes were blocked for 1 h at room temperature with Odyssey blocking buffer (LI-COR, Lincoln, NE) and incubated overnight at 4°C with primary antibodies diluted in Odyssey blocking buffer with 0.1% Tween 20/rabbit polyclonal anti-Ca_V1.2 antibody (1:500) (BD Biosciences PharMingen) and mouse monoclonal anti-nitrotyrosine antibody (1:1000) (Calbiochem, San Diego, CA). The membranes were then incubated for 1 h at room temperature with secondary antibodies, each of anti-mouse IRDye 800 and anti-rabbit IRDye 680, diluted (1:4000) in Odyssey blocking buffer with 0.1% Tween 20. After rinsing with phosphate-buffered saline twice, the membranes were visualized using LI-COR Odyssey Infrared imaging system (LI-COR).

Confirmation of in Vitro Nitrosylation of Ca²⁺ Channels by ONOO^– Treatment. HEK 293 cells were transfected by _1c subunit of Ca²⁺ channel from human jejunum smooth muscle and treated with SIN-1 (100 µM) for 15 min and sodium peroxynitrite (150 µM) twice at 5-min intervals for nitrosylation. Immunoblotting of transfected cell lysates with anti-nitrotyrosine antibody after immunoprecipitation with anti-Ca²⁺-channel antibody was performed to confirm successful nitrosylation of Ca²⁺ channels by in vitro treatment with these nitrosylating agents.

Solutions. The Krebs physiological saline solution contained: 118 mM NaCl, 4.6 mM KCl, 1.3 mM NaH₂PO₄, 1.2 mM MgSO₄, 25 mM NaHCO₃, 11 mM glucose, and 2.5 mM CaCl₂. Stock solutions of 100 mM EGTA were made in 0.2 N NaOH. The pipette internal solution contained 100 mM L-aspartic acid, 30 mM cesium chloride, 1 mM MgCl₂, 5 mM HEPES, 2 mM adenosine triphosphate (disodium salt), and 5 mM EGTA, with pH adjusted to 7.2 using cesium hydroxide. The HEPES-buffered physiologic salt solution contained 135 mM NaCl, 5.4 mM KCl, 0.33 mM NaH₂PO₄, 5 mM HEPES, 1 mM MgCl₂, 20 mM BaCl₂, 5 mM D-glucose, with pH adjusted to 7.4 using NaOH.

Drugs. PP2 (10 mM in dimethyl sulfoxide) was purchased from Calbiochem. Sodium orthovanadate and picryl sulfonic acid (TNBS) were purchased from Sigma-Aldrich (St. Louis, MO); SIN-1 chloride was from Tocris Cookson Inc. (Ellisville, MO); and sodium peroxynitrite was from Cayman Chemical (Ann Arbor, MI).

Data Analysis. All data are shown as mean ± S.E.M. Cumulative dose-dependent responses to CaCl₂ or Na₃VO₄ in the presence and absence of PP2 were analyzed by repeated measures-ANOVA followed by Bonferroni's post-test. Data from different groups were analyzed by either unpaired t test or two way-ANOVA followed by Bonferroni's post-test. Data from peroxynitrite treatment experiments were analyzed by one-way ANOVA followed by Bonferroni's post-test. P 0.05 was considered significant.

	Results

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Effect of Inflammation on Calcium-Induced Contraction in Murine Distal Colon. Cumulative addition of CaCl₂ induced a concentration-dependent contraction in both control and inflamed distal colon depolarized by Ca²⁺-free high potassium (80 mM) physiological saline solution. Inflammation significantly reduced (P 0.01) the calcium-induced contraction to 0.22 ± 0.04 g/mm² (E_max) from 0.56 ± 0.05 g/mm² (E_max) of control tissues (Fig. 1, A and B), representing a 59% reduction in contraction to 1 mM Ca²⁺. The pD₂ values for the calcium were unaffected by inflammation (control: 3.8 ± 0.2; inflamed: 3.6 ± 0.1). However, colonic inflammation did not alter the calcium-induced contractions in the ileum (Fig. 1C).

View larger version (21K): [in this window] [in a new window]   Fig. 1. Representative tracings of CaCl2-induced dose-dependent contractions in isolated distal colon from control (left) and inflamed (right) mice (A). Graph showing the summarized data on the effect of inflammation on the cumulative concentration-response curves to CaCl2 in mouse distal colon (B) and ileum (C), depolarized by Ca2+-free high-potassium (80 mM) physiological saline solution. Each point represents the mean ± S.E.M. *, P 0.05, **, P 0.01 versus control; unpaired t test; n = 6–8.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Quantitative comparison of gene expression for Cav1.2b and IL-1 from control and inflamed distal colon tissues. A, representative raw data of real time-PCR from control and inflamed tissues. Alphabets in parentheses are C for control and I for inflamed groups. B, average Ct for each sample was normalized against the Ct value of corresponding GAPDH and presented as Ct. Data represent mean ± S.E.M., analyzed by unpaired t test, *, P 0.05 versus control.

Effect of c-Src Inhibition on Calcium-Induced Contraction in Inflamed Tissues. Smooth muscle calcium channels are regulated by c-Src kinase under basal conditions (Wijetunge and Hughes, 1996), which is attenuated following inflammation. In single smooth muscle cells from both vascular and nonvascular tissues, Src kinase inhibition suppresses Ca²⁺ channel currents (Wijetunge and Hughes, 1996; Hu et al., 1998). To determine whether calcium influx-induced contraction is sensitive to the Src kinase inhibitor, tissues were preincubated with PP2 (10 µM), a c-Src kinase inhibitor. In the presence of 10 µM PP2, calcium-induced contractions were significantly reduced in both control and inflamed colonic tissues (Fig. 3, A and B). However, the extent of inhibition was significantly greater in controls compared with the inflamed tissues. At 0.1 mM Ca²⁺, the contractions were reduced by 46.2 ± 9.21% compared with 19.67 ± 5.07% after inflammation (P = 0.03) (Fig. 3C). The effect of inflammation on Src regulation may be related to changes in the kinase activity. Therefore, we measured Src kinase activity from the distal colon of control and inflamed tissues. As shown in Fig. 4, phosphate incorporation was not significantly altered by inflammation (n = 3). To determine the possibility that altered tyrosine phosphatase activity may account for the differences in the calcium-induced contractions after inflammation, the effect of sodium orthovanadate, a tyrosine phosphatase inhibitor, was examined. Cumulative addition of Na₃VO₄ (1 µM to 1 mM) produced a concentration-dependent contraction in distal colon from both control and inflamed mice. Inflammation significantly (P 0.01) reduced the vanadate-induced contraction. The E_max values of Na₃VO₄ were 1.5 ± 0.15 g/mm² in control and 0.42 ± 0.14 g/mm² in inflamed tissues (Fig. 5).

View larger version (9K): [in this window] [in a new window]   Fig. 3. Effect of PP2 (10 µM), a c-Src kinase inhibitor on the cumulative CaCl2-induced concentration-dependent contractions in isolated distal colon from control (A) and inflamed (B) mice. Maximal contractions of respective control group were considered as 100%. The percent inhibition of CaCl2 (0.1 mM)-induced contraction by PP2 is significantly less in distal colon from inflamed mice (C). The CaCl2 contractions were elicited in tissues depolarized by high-potassium (80 mM) physiological saline solution. Points represent mean ± S.E.M. Data were analyzed by either repeated measures-ANOVA followed by Bonferroni's post-test (A and B) or unpaired t test (C). *, P 0.05, **, P 0.01 versus respective controls.

View larger version (11K): [in this window] [in a new window]   Fig. 4. Graphical representation of c-Src kinase activity data from normal and inflamed distal colon tissues showing no difference in their phosphate incorporation function. Data were analyzed by unpaired t test, n = 3.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Representative tracings of Na3VO4, a tyrosine phosphatase inhibitor-induced dose-dependent contractions in isolated distal colon from control (A) and inflamed (B) mice. C, graphical representation of tensions developed, normalized to tissue cross-sectional area. Points represent mean ± S.E.M. Data were analyzed by two-way ANOVA followed by Bonferroni's post-test. **, P 0.01 versus control, n = 5.

Preincubation with PP2 (10 µM) for 15 min significantly (P 0.01) reduced the Na₃VO₄-induced contraction in control tissues, whereas it did not affect the Na₃VO₄-induced contraction in inflamed tissues. The respective E_max and pD₂ values of Na₃VO₄ in control and PP2 treatment were 100, 4.13 ± 0.03 (control), and 68.3 ± 9.2, and 3.44 ± 0.16% (PP2) in control tissues, whereas in the inflamed tissues, the corresponding values were 100, 4.37 ± 0.12 (inflamed-control), 119.3 ± 14.3, and 4.40 ± 0.10% (inflamed-PP2) (n = 5). The percent inhibition by PP2 of E_max of vanadate was significantly (P 0.05) higher in control tissues (36.79 ± 6.05%) compared with inflamed tissues (–19.98 ± 18.4%) (Fig. 6). These data suggest that an altered phosphatase activity does not account for the decreased Src-mediated regulation of calcium entry during inflammation.

View larger version (9K): [in this window] [in a new window]   Fig. 6. Effect of PP2 (10 µM), a c-Src kinase inhibitor, on the cumulative Na3VO4-induced concentration-dependent contraction in isolated distal colon from control (A) and inflamed (B) mice. Maximal contractions of respective control group were considered as 100%. The percent inhibition of Na3VO4-induced maximal contraction (1 mM) by PP2 is significantly reduced in distal colon from inflamed mice (C). Points represent mean ± S.E.M. Data were analyzed by repeated measures-ANOVA followed by Bonferroni's post-test (A and B) or unpaired t test (C). *, P 0.05, **, P 0.01 versus corresponding controls.

Effect of Nitrosylation on the Calcium-Induced Contraction and PP2 Inhibitory Effects in Normal Tissues. After preincubation of normal distal colon strips in nitrosylating agents (see Materials and Methods), the CaCl₂ (1 mM)-induced contraction was reduced by 45.6 ± 7.3% (n = 5). It is noteworthy that there was no significant further decrease in the calcium-induced contraction after inhibition of c-Src kinase. The PP2-induced inhibition of CaCl₂ contraction after nitrosylation remained at approximately 60 ± 3.97%; n = 5 (Fig. 7A).

View larger version (18K): [in this window] [in a new window]   Fig. 7. Effect of nitrosylation on CaCl2-induced contractions in Ca2+-free high-K+ (80 mM)-depolarizing solution. A, CaCl2 (1 mM)-induced contractions were reduced after nitrosylation of normal distal colon tissues, and c-Src kinase inhibition by PP2 after nitrosylation did not further reduce the CaCl2 contraction. B, nitrosylation of inflamed tissues did not affect the CaCl2-induced contractions, and further c-Src kinase inhibition was also ineffective on the CaCl2 contractions. Initial contractions of each group were considered as 100%. C, ACh-induced contractions in the absence of extracellular calcium were not affected by nitrosylation. Data were analyzed by one-way ANOVA followed by Bonferroni's post-test. a, P 0.05 versus respective control, n = 5. D, immunoblots with anti-Cav1.2 antibody (left) and anti-nitrotyrosine antibody (right) from control and inflamed colonic smooth muscle samples. Protein samples were immunoprecipitated by the anti-Cav1.2 antibody before immunoblot. There was significantly greater nitrotyrosylated protein in inflamed tissues compared with controls.

Quantitative Analysis of Inflammation-Induced Nitrotyrosylation of Ca²⁺ Channels in Distal Colon by Immunoblotting. To confirm whether inflammation increases the nitrotyrosylation of Ca²⁺ channels, we examined the protein expression of the Ca²⁺ channel in normal and inflamed distal colon smooth muscle by immunoprecipitation with anti-Ca_v1.2 antibody followed by immunoblot with anti-Ca_v12 and anti-nitrotyrosine antibodies. Visualization by infrared imaging showed distinct bands of approximately 240 kDa, which corresponds to the Ca_v1.2 protein in the same proportions in both the normal and inflamed tissues. The nitrotyrosine antibody also revealed a band of a protein similar in size (240 kDa), both in normal and inflamed tissues but significantly abundant in the inflamed samples (Fig. 7D). Similar results were obtained from three different colon samples. Although isometric tension experiments gave evidence for decreased functional ability of Ca²⁺ channel and its attenuated c-Src kinase regulation following nitrosylation or inflammation, Western blotting confirmed increased nitrotyrosylation of Ca²⁺ channels during inflammation.

The Effect of Nitrotyrosylation on Calcium Channels Was Further Confirmed by Voltage-Clamp Studies. Figure 8 shows that calcium currents were reduced by 84.1 ± 3.9% (n = 3) by ONOO^– in control colonic muscle cells. As reported previously (Akbarali et al., 2000), calcium currents were markedly reduced after inflammation. In these cells, ONOO^– reduced basal currents by 36.2 ± 13% (n = 5), which was significantly less than in controls (P = 0.03) (Fig. 8B).

View larger version (14K): [in this window] [in a new window]   Fig. 8. Effect of nitrosylation on Ca2+ currents in distal colonic smooth muscle cells. A, representative recording of Ca2+ currents obtained at +20 mV from –60-mV holding potential before and after perfusion with ONOO– from control and inflamed cells. B, bar graph showing percentage inhibition of peak Ca2+ currents after ONOO– treatment. Bars represent mean ± S.E.M; n = 3 (control) and 5 (inflamed). Data were analyzed by unpaired t test. *, P = 0.03 compared with normal cells.

View larger version (17K): [in this window] [in a new window]   Fig. 9. In vitro treatment with nitrosylating agents, SIN-1 (100 µM) and sodium peroxynitrite (150 µM), nitrosylate Ca2+ channels. HEK 293 cells were transfected with human 1c subunit of Ca2+ channel and treated with the nitrosylating agents. Immunoblots with anti-Cav1.2 antibody (left) and anti-nitrotyrosine antibody (right) from transfected HEK 293 cell lysate samples. M refers to the marker. Protein samples were immunoprecipitated by the anti-Cav1.2 antibody before immunoblot. There was significantly greater nitrotyrosylated Ca2+ channel protein after ONOO– treatment.

	Discussion

Top Abstract Materials and Methods Results Discussion References

The calcium-induced contractions in a depolarizing solution can be attributed largely to calcium entry through L-type calcium channels. As expected, the contractions were significantly reduced during inflammation, consistent with decreased calcium channel currents identified previously in patch-clamp studies. Although it has been clearly demonstrated in several different models of colonic inflammation that the amplitude of calcium currents are reduced, we have previously shown (Kang et al., 2004) that calcium channel protein and gene expression remain unaltered in the dextran-sulfate sodium (DSS)-treated mouse. Liu et al. (2001) initially demonstrated down-regulation of calcium channel protein expression in canine colon with acetic acid/ethanol-induced inflammation, and Shi et al. (2005) recently showed that tumor necrosis factor-, a primary proinflammatory cytokine, can result in transcriptional regulation of human smooth muscle calcium channel (Shi et al., 2005). In the rat TNBS model of inflammation, Kinoshita et al. (2003) did not find changes in protein or gene expression of the calcium channel. Likewise, in the present study, we did not detect differences in the gene expression of Ca_v1.2b, although IL-1 was enhanced with inflammation. These differences may relate to species differences or, more likely, the extent and induction of inflammation. Nevertheless, the decreased contractions are consistent with inflammation-induced decreases in calcium channel currents.

Several lines of evidence indicate that the _1c calcium channel subunit is under the basal regulation of Src kinase (Akbarali, 2005). Direct association of _1c and c-Src has been demonstrated in colonic smooth muscle (Hu et al., 1998), whereas Src kinase inhibition attenuates calcium currents and tyrosine phosphatase inhibitors enhance it. The precise biophysical mechanisms by which Src can enhance calcium currents are not fully understood but may involve conversion of the channel to a second open state resulting in increased availability (Nakayama et al., 2006), which may occur as a result of direct phosphorylation of the tyrosine residues in the C-terminal segments. In the colonic circular smooth muscle, Src association was demonstrated with the _1c C-terminal fusion protein (Jin et al., 2002), and Tyr2122 has been shown as the critical tyrosine residue for the Src-mediated potentiation for integrins (Gui et al., 2006) and insulin-like growth factor-1 (Bence-Hanulec et al., 2000) in rat smooth muscle and neurons, respectively. It is noteworthy that, even at high concentrations of Src kinase inhibitors, the calcium currents are only inhibited by approximately 60 to 70%. This would suggest that the tyrosine phosphorylation of the calcium channel plays a modulatory role on the voltage-dependent activation of the calcium channel. This is consistent with inflammation resulting in a 60% reduction of calcium-induced contractions.

After exposure to peroxynitrite, the contractile responses in the normal colon were markedly decreased and were not further affected by the Src kinase inhibitors. NO is an important factor in the pathophysiology of inflammatory bowel disease (IBD) because increased NO production and iNOS activity were observed in both clinical and experimental IBD (Boughton-Smith et al., 1993; Middleton et al., 1993; Singer et al., 1996). During intestinal inflammation, mononuclear cells and granulocytes infiltrate the mucosa with increased levels of myeloperoxidase that play a central role in tissue oxidant formation. Activated macrophages and neutrophils reduce oxygen to superoxides (Zhu et al., 1992). NO and superoxide react rapidly to form the highly cytotoxic oxidant peroxynitrite (Beckman et al., 1990). Peroxynitrite through formation of intermediary secondary species incorporates a nitro group on tyrosine residues, resulting in primarily 3-nitrotyrosine. Kong et al. (1996) showed that nitrotyrosylation of the synthetic peptide corresponding to tyrosine kinase phosphorylation site for cyclin-dependent kinase prevents tyrosine phosphorylation. Hence, we hypothesized that tyrosine nitrosylation of Ca²⁺ channel proteins occur during inflammation, making the channels poor substrates to c-Src kinase. Thus, nitrosylation of normal tissues with exogenous peroxynitrite reduced the initial calcium-induced contraction and prevented the effects of the c-Src kinase inhibitor. In contrast, inflamed tissues were most probably nitrotyrosylated and hence presented with decreased contractions, whereas the effect of Src kinase inhibition was abrogated. This was further supported by the decreased effect of nitrotyrosylation on calcium currents in inflamed cells. The tyrosine phosphatase inhibitor sodium orthovanadate also failed to revert the decreased contractions during colonic inflammation and remained unresponsive to inhibition by PP2. On the other hand, in normal tissues, the sodium vanadate contractions were sensitive to PP2. Taken together, these data would suggest that Src-mediated phosphorylation is blunted following nitrotyrosylation. Further biochemical studies will be necessary to define the primary site(s) corresponding to phosphorylation and nitrotyrosylation. It is noteworthy that, in biological systems, the levels of nitrotyrosylation are low and that one to five 3-nitrotyrosine residues per 10,000 were detected (Radi, 2004). This may be attributed to requirement of consensus sequences in the target protein and repair mechanisms operative under inflammatory conditions. Nonetheless, the fact that acetylcholine-induced contractions in the absence of calcium entry were unaffected by peroxynitrite treatment indicates that the contractile proteins remain viable under these conditions. Moreover, Western blotting following immunoprecipitation using Ca_v1.2 and nitrotyrosine antibodies confirmed no change in the protein expression of Ca²⁺ channels but increased nitrotyrosylated Ca²⁺ channels during inflammation. Several studies have demonstrated the involvement of induction of iNOS leading to the formation of nitrotyrosines during inflammation. Selective inhibition of iNOS suppresses acute experimental colitis (Kankuri et al., 2001), and iNOS-deficient mice are less sensitive genetically to TNBS-induced changes and even prevent nitrotyrosine formation, although the massive neutrophil infiltration is unaffected in these mice (Zingarelli et al., 1999). It is interesting that iNOS produced by bone marrow-derived cells is shown to play a critical role in mediating the inflammatory response during colitis, suggesting that cell-specific regulation of iNOS may represent a novel form of therapy of patients with inflammatory bowel disease (Beck et al., 2007). Nonetheless, further studies are required to comprehensively examine the respective contribution of iNOS, reactive oxygen/nitrogen species, all induced during inflammation to gain more clear insight into the basis of nitrotyrosylation of the calcium channel, preventing its regulation by c-Src kinase.

In conclusion, these data suggest that altered regulation of smooth muscle calcium channels by c-Src kinase may underlie the decreased motility during colonic inflammation. These studies also provide evidence of a critical modulatory role for basal Src kinase activity. A better understanding of these processes will lead to valuable insights into the potential therapeutic approaches in inflammatory conditions, such as IBD.

	Acknowledgements

 
We thank Dr. Gianrico Farrugia for providing human jejunal _1c construct.

	Footnotes

 
This work was supported by National Institutes of Health Grant DK46367.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.123075.

ABBREVIATIONS: DSS, dextran sulfate sodium; TNBS, trinitrobenzene sulfonic acid; SH, Src homology; ONOO^–, peroxynitrite; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; iNOS, inducible nitric-oxide synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEK, human embryonic kidney; ANOVA, analysis of variance; IL, interleukin; ACh, acetylcholine; Ct, cycle threshold; IBD, inflammatory bowel disease; FAM, carboxyfluorescein; HEX, hexachlorofluorescein; BHQ, Black Hole Quencher.

Address correspondence to: Dr. Hamid I. Akbarali, Professor, Department of Pharmacology and Toxicology, McGuire Hall, 316, P.O. Box 980524, Virginia Commonwealth University, 1112 E. Clay Street, Richmond, VA 23298. E-mail: hiakbarali{at}vcu.edu

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