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Internal Anal Sphincter (IAS) plays a critical role in maintaining anal continence, but very little is known about the mechanisms underlying its functioning and physiology (Hashish M. et al., 2010). Malfunctioning of IAS can lead to variety of disorders. Anal incontinence and Hirshsprung diseases are the best known among these disorders and are often correlated to anatomical alterations of the IAS such as traumatic lesions, atrophy, etc (Vaizey C. J. et al., 1997). Fecal incontinence affects 8.6 % of adult population (Whitehead WE. et al., 2009). Despite the high incidence of the fecal incontinence, there are no long-term successful solutions for such patients. Some other pathological conditions, which can directly affect the internal anal sphincter, are fistulas, abscesses, solitary rectal ulcer and anal fissures. To find out the means to correct these IAS disorders, the study of Internal Anal Sphincter (IAS) smooth muscle has great clinical importance (Schiesse l. R. et. al., 2005).
IAS smooth muscle is a tonic tissue. It has unique qualities. It has a quality of sustained contraction without any nerve stimulus. This minimum level of sustained contraction is also known as basal tone. Molecular mechanisms for maintenance of sustained contraction or basal tone in IAS are not fully understood yet. Protein Kinase Cs (PKC) plays an important role in smooth muscle contraction (Sakai H et al., 2010, Xu J et al., 2010, Park SY et al., 2010, Huster M et al., 2010, Kizub IV, et al., 2010). It work by activation and maintaining elevated levels of phospho-MLC20 via inhibition of MLCP by phosphorylation of CPI-17 (the endogenous inhibitory protein of the catalytic subunit of MLCP (Koyama M. et al., 2000; Kitazawa T. et al., 2003). PKC has different isoforms. They are classified into three groups, conventional (cPKC), novel (nPKC) and atypical (aPKC) PKCs. cPKC are Î±, Î²I, Î²II, and Î³, they have binding site for diacylglycerol (DAG), phorbol esters and require Ca2+ for activation. The nPKCs Î´, Îµ, Î· and Î¸ lack the Ca2+ binding domain and therefore do not require Ca2+ for activation. The aPKCs Î¶, Î» and Î¹ have only one cysteine-rich zinc finger like motif and are dependent on phosphatidylserine, but are not affected by DAG, phorbol esters or Ca2+ (Sakai H et al., 2009). The contribution of PKC isoforms in the IAS tone is not fully clear. Many approaches are being used to study the role of PKC in smooth muscle tone. One of them is using inhibitors of these proteins. There are many PKC inhibitors available in the market. Based on the mechanism of action of these inhibitors they can be divided into four classes. 1) Competitive inhibitors, those who act by competing with the ATP binding site of the kinase (1-(5-isoquinolinesulfonyl)-2-methylpiperazine, staurosporine, bisindolylmaleimide, and K252a). However, the specificity of action of these compounds is rather low because the ATP binding site is conserved among different families of protein kinases; most of these compounds also inhibit adenosine 3', 5'-cyclic monophosphate (cAMP)- and guanosine 3', 5'-cyclic monophosphate-dependent protein kinases with inhibitory constants (Ki) only about an order of magnitude larger than the Ki for PKC inhibition. 2) The second class of inhibitors acts by inhibiting the interaction of PKC with phospholipids (polymixin B). The specificity of these drugs seems very low, and these drugs are not widely used as specific PKC inhibitors. 3) The third class of inhibitors includes the PKC pseudosubstrate peptide inhibitors [PKC-(19-36) and PKC-(19-3 l)] they are much more selective than most of the other classes (House C. and Kemp B.E., 1990). These peptides are derived from the regulatory C1 domain of PKC that normally inhibits enzyme activity in the absence of DAG. These inhibitors have the disadvantage that they are not membrane permeable and can only be used with intracellular injection or perfusion techniques. 4) The final class of inhibitors binds to the C1 domain of PKC, which contains a phorbol ester/DAG binding domain. These inhibitors include sphingosine, gossypol, aminoacridines, and calphostin C (Hartzell HC and Rinderknecht., 1996). Calphostin C has been described as one of the most selective inhibitors of PKC available because instead of binding to ATP binding site it binds to DAG binding domain, which is present in conventional and novel PKCs only. calphostin C is a widely used PKC inhibitor and is being used in number of studies to find out the role of PKC in different physiological functions in different cells and organs (Seto S.W. et al., 2010; Zhang Y. et al., 2010, Hong J.Y. et al., 2010, Peng H, et al., 2010) which includes smooth muscle also (Shimamoto H. et al., 1992; Sato K., et al., 2001; Yu J., 2005). Calphostin C inhibits PKC in the presence of light (Bruns, R. F et al., 1991) via site-specific oxidative modification (Singer-Lahat D., 1995). Calphostin C has already been used in human IAS smooth muscle reconstructs and PKCs role had been proved in the maintenance of IAS smooth muscle tone (Somara S et al., 2009). In the intact IAS the contribution of PKC in maintenance of IAS tone is not clear. Moreover, Rho kinase was also proved to be very important in the maintenance of IAS tone (Patel C.A. and Rattan S., 2007) but its contribution in the presence of PKC inhibitor was never studied. In addition, there is phasic activity in IAS and RSM, the phasic activity and the contribution of PKC and Rho Kinase have never been studied in internal anal sphincter.
Therefore, in the present study, we have tried to understand the contribution of PKC in regulation of IAS and RSM tonic and phasic contraction by using calphostin C and Gö6850 as PKC inhibitors and Y27632 as ROCK inhibitor. In addition, we have tried to find out the relative contribution of PKC vs ROCK on the tonic and phasic activity of IAS and RSM (Rectal Smooth muscle). Effect on tonic and phasic activity was recorded before and after giving the above-mentioned inhibitors in IAS and RSM. PKC and ROCK activity was measured and compared with Phasic and Tonic activity in both IAS and RSM. Western Blot analysis was carried out to check the movement of PKC, ROCK-II from particulate to cytosol. Relative amount of pThr696-MYPT1, pThr18/Ser19-MLC20, and pThr38-CPI-17 was also checked in IAS and RSM to see the effect of inhibition of PKC and ROCK on the level of their phosphorylation.
MATERIALS AND METHODS
Tissue Preparation. Circular smooth muscle strips preparation is already explained in previously published study ( Rattan et al., 2006) but briefly, Male Sprague-Dawley rats (300-350 g) were killed by decapitation, and the anal canal with an adjacent region of the RSM was quickly removed and transferred to oxygenated (95% O2-5% CO2) Krebs physiological solution (KPS) of the following composition (in mM): 118.07 NaCl, 4.69 KCl, 2.52 CaCl2, 1.16 MgSO4 1.01 NaH2PO2, 25 NaHCO3, and 11.10 glucose (37Â°C). Extraneous adventitious blood vessels and skeletal muscle tissues connected to the IAS were removed carefully by sharp dissection. The anal canal was then opened and pinned flat with the mucosal side up on a dissecting tray containing oxygenated KPS. The mucosa was removed carefully by sharp dissection. Circular smooth muscle strips (1x7 mm) of the IAS and RSM (identified as a thickened circular smooth muscle situated at the lowermost part of the anal canal. The experimental protocols of the study were approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University and were in accordance with the recommendations of the American Association for the Accreditation of Laboratory Animal Care.
PKC and ROCK Activity. PKC and ROCK activity was measured in tissue homogenate of IAS smooth muscle and RSM. Tissue strips were hanged on the transducer and treated with different inhibitors used in this study. After treatment with with different concentrations of inhibitors and for different time points, tissue strips were flash frozen in liquid nitrogen and homogenized in ice-cold lysis buffer consisting of 50mM Tris-HCl, pH 7.5, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, supplemented with a protease inhibitor mixture (Pierce) and sodium orthovanadate (Na3VO), a phosphatase inhibitor (Pierce). The protein concentration in IAS and RSM tissue lysates was determined using the BCA protein assay kit (Pierce). Kinase activity was measured using the Nonradioactive Kinase Assay kits (no. EKS-420; Assay Designs for PKC and STA-416; Cell biolabs for ROCK). Tissue lysates from IAS smooth muscle and RSM were titrated to find out the optimal concentration of protein to be used for final activity assay. 5 to 40 ïg (Fig. 1A) of IAS smooth muscle and RSM protein lysates were used for PKC activity and 2 to 32 ïg of protein was used for ROCK activity assay (Fig. 2A). PKC and ROCK standards were diluted and incubated in the same manner to generate a standard curve for quantification of activity. Activity of PKC and ROCK in tissue lysates was compared with PKC and ROCK standards and conc., which felt within the range of interest (in the middle of the standard curve), was used for final activity assay for IAS smooth muscle and RSM (Fig 1B and 2B).
Briefly, 40 ïg of IAS and RSM tissue lysates for PKC activity assay and 20 ïg for ROCK activity assay were mixed with dilution buffer and incubated on a microtiter plate for 1 h at 300C in the presence of ATP. A proprietary PKC and ROCK substrate was precoated on the wells of the microtiter plate. Next, the microtiter plate was incubated with a phosphospecific IgG antibody to label all the phosphorylated sites resulting from active PKC and ROCK in the samples. Plates were incubated with anti-rabbit IgG: HRP-conjugated antibody, and colorimetric reactions were initiated by adding ABTS substrate solution. Colorimetric reactions were terminated after 30 min, and absorbance was read on an ELISA microplate reader at 450 nm. Fresh lysis buffer (without tissue lysate) was included in the assay as a blank, and tissue lysate from IAS and RSM without ATP served as the negative control. All samples were assayed in quadruplicate and the ELISA was performed two times. Analyses of the standard concentrations confirmed that optical density was linearly related to concentration within the range of interest.
Measurement of Tone and Isometric Tension. The smooth muscle strips were transferred to 2-ml muscle baths containing oxygenated KPS at 37Â°C. One end of the strips was anchored at the bottom of the muscle bath, whereas the other end was connected to a force transducer (model FT03; Grass Instruments, Quincy, MA). Isometric tension was measured by the powerlab/8SP data-acquisition system (AD Instruments, Castle Hill, Australia) and recorded using Chart 4.1.2 (AD Instruments). Each smooth muscle strip was initially stretched to a tension of 1.0 g. The muscle strips were left for 1 h to equilibrate during which they were washed with KPS after every 20 min. For IAS, only smooth muscle strips that developed spontaneous tone and responded by relaxation to EFS and for RSM, only smooth muscle strips that responded by contraction to electrical field stimulation (EFS) were used for the present study (Culver P. J. and Rattan S., 1986; Rattan S., 1995; Schiller L.R., 2005). The RSM strips were characterized by the presence of a low-grade tone with the superimposed phasic contractions. After the equilibration period, the smooth muscle strips were treated with different concentrations of calphostin C (10-8 to 10-4M) Gö6850 (1.0ïM) and Y27632 (1.0ïM) and concentration dose response curve was recorded Graphs were plotted by using Prism 5.3.
Tissue Lysate Preparation and Western Blot Analysis. While the isometric tension was monitored in the IAS and RSM strips. The strips were quick-frozen in the basal state and after pretreatment with calphostin C (1.0ïM), Gö6850 (1.0ïM) and Y27632 (1.0ïM) once the response to above mentioned inhibitors platued, strips were quickly snap frozen into liquid nitrogen and were used for protein extraction for western blot analysis. To freeze the tissues, first the tissue chambers were rapidly lowered, exposing the tissues, and a Wollenberger clamp precooled in liquid N2 was used to snap-freeze the tissues. The frozen tissues were placed in a fresh tube, submerged in liquid N2, and stored at -80Â°C (Rattan S., et al., 2006). Later, the respective tissues were cut into small pieces, and homogenization buffer (1% SDS, 1.0 mM sodium orthovanadate, and 10 mM Tris, pH 7.4) was added to the tissues in a volume equal to five times the weight. The mixture was homogenized on ice. The homogenates were centrifuged (14,000 rpm) for 5 min, and supernatants were collected. Protein concentration in resultant supernatants was determined by using BCA Protein Assay Reagent Kit, using BSA as a standard (Pierce) (Kessler R., 1986). Twenty micrograms of protein in 20ïl of lysates were mixed with 2x Laemmli sample buffer (LSB; with final concentrations of 62.5 mM Tris, 1% SDS, 15% glycerol, 0.005% Bromophenol blue, and 2% -mercaptoethanol) and placed in a boiling water bath for 5 min. Protein in the samples were separated by SDS-polyacrylamide gel. (7.5% gel pThr696-MYPT1; 15% gel for pThr18/Ser19-MLC20, and pThr38-CPI-17). The separated proteins were electrophoretically transferred onto either a nitrocellulose membrane for pThr696- MYPT1 or a polyvinylidene difluoride membrane for pThr18/Ser19-MLC20, pThr38-CPI-17 at 25 V for 14 min. by using iBlotÂ® Dry Blotting System (Invitrogen Corp.) at room temperature. To block nonspecific antibody binding, the membrane was soaked for 1 hour at room temperature in LI-COR buffer. The membrane was then incubated with the specific primary antibodies (1:1,000 for pThr38-CPI-17, pThr696-MYPT1, pThr18/Ser19-MLC20, 1:20,000 for ï¡-actin) diluted in LI-COR buffer containing 0.1% Tween-20 for 1h at room temperature. After washing with TBS-T 3 times (10 min each wash), the membranes were incubated with the IRdye680 and IRdye800 conjugated secondary antibody from LI-COR Biosciences in dark (bovine anti-rabbit 1:10,000 for ROCK-II, MYPT1, and pThr696-MYPT1; bovine anti-goat 1:5,000 pThr38-CPI17). The membranes were washed three times with TBS-T for 10 min each and finally kept in PBS buffer for 10 min. on shaker at room temperature in dark and Scanned by LI-COR Infrared scanner from LI-COR Biosciences.
Chemicals and Drugs. calphostin C, Gö6850 and Y27632 were purchased from Biomol (Plymouth Meeting, PA). The following antibodies were used in this study: ï¡-actin and MLC20 antibodies were from Sigma (St. Louis, MO) pThr696-MYPT1, pThr38-CPI-17, pThr18/Ser19-MLC20 from Santa Cruz Biotechnology (Santa Cruz, CA), IRdye680 and IRdye800 conjugated mouse, goat, and rabbit secondary antibodies were obtained from LI-COR Biosciences.
Data Analysis. All the calculations for force experiments were done with Graphpad Prism 5.3. Values are means ï‚±SE from at least three independent experiments. Relative densities of western blots were calculated by using Image J software from NIH. For the comparison of IAS and RSM, the relative densities for IAS were normalized to 1. The changes in phosphorylation were normalized to basal phosphorylation, and basal phosphorylation was plotted as 1.00. One-way ANOVA followed by a Bonferroni post hoc test was used (Pï€¼ 0.05) to calculate statistical significance.
Effect of calphostin C on PKC and ROCK activity. PKC and ROCK activity was measured in tissue lysates and compared it with enzyme activity in standards (active PKC and active ROCK) and choosing the conc. of tissue lysates, which falls in the linear range of standards (Fig 1A and 2A). Tissue strips were treated with different concentrations (10-8 M to 10-5 M) of calphostin C for 2, 4, 6, 8, 10 minutes and snap frozen in liquid nitrogen with wollenbergers clamp. Lysates were made and enzyme activity was measured. Maximum effect reported was of 10-4 M calphostin C for 10 minutes incubation in muscle bath (Fig. 1B). Enzyme activity was checked by loading equal amount of protein from each sample. PKC activity was significantly higher in IAS (0.003 ï‚± pmol/ug of tissue lysate than IAS smooth muscle (0.002 ï‚± pmol /ug of tissue lysate) in basal state (Fig. 1C). Calphostin C significantly decreased PKC activity in IAS smooth muscle and RSM in dose dependent manner (Fig. 1B). On an average the effect of each concentration of calphoatin C reached platue after 10 minutes in IAS smooth muscle and RSM strips. Calphostin C had no effect on ROCK activity in IAS smooth muscle and RSM (Fig. 1E) and IC50s of calphostin C for ROCK and PKC activity for IAS smooth muscle and RSM are given in table 1.
Effect of Y27632 on PKC and ROCK activity. IAS smooth muscle and RSM strips were hanged on muscle transducer in muscle bath and were treated with 10-5M Y27632. Once the effect of Y27632 plateau muscle strips were quickly snap frozen in liquid nitrogen and PKC and ROCK activity was measured. In basal state, ROCK activity was significantly higher in IAS smooth muscle (0.0037ï‚±pmol /ïg of tissue lysate) than RSM (0.0017ï‚±pmol /ïg of tissue lysate) and it was significantly inhibited by Y27632 in IAS smooth muscle and RSM strips (Fig. 2, A and C) Effect of different conc. (10-8 to 10-5M) of Y27632 were checked on PKC and ROCK activity in IAS smooth muscle and RSM (Fig. 2, C and D). Y27632 significantly inhibited the ROCK activity but had no effect of PKC activity in both IAS smooth muscle and RSM (Fig. 2E).
Effect of calphostin C on phasic activity in IAS smooth muscle and RSM (rate of contraction). Phasic activity or response was divided into two different rhythms i.e "high speed and low amplitude" and "slow speed and high amplitude" contractions. In IAS smooth muscle, only one pattern of phasic activity exists i.e. "high speed and low amplitude" and RSM had both the patterns of phasic activity. Rate of phasic contractions in IAS smooth muscle and RSM was noted down after adding 1ÂµM of calphostin C, Very little effect of all these inhibitors was reported on "high speed and low amplitude" in IAS smooth muscle and RSM. calphostin C and Go6850 (1ÂµM) had significantly greater effect on "slow speed and high amplitude" in RSM. There was 53.27% decrease in these contractions in RSM. They were 16.7 per minute in basal state and went down to 8.23 per minute after adding calphostin C or Gö6850 (Fig. 3 A and B). There were further decrease (20.0%) in slow speed and high amplitude contractions on the addition of Y27632 (1ÂµM) after calphostin C and Go6850. IC50s of calphostin C for rate of IAS smooth muscle and RSM are given in table 1.
Effect of calphostin C on Tonic and Phasic activity in IAS smooth muscle and RSM.
Tissue strips of IAS smooth muscle and RSM were treated with calphostin C (10-8 to 10-4M) in the muscle bath and its effect on tonic and phasic response of IAS smooth muscle and RSM was recorded. In each experiment, resting phasic and tonic response in IAS smooth muscle and RSM was calculated by giving zero calcium (KPS without CaCl2) in each experiment and resulting responses was considered as 100% of IAS smooth muscle and RSM. calphostin C 10-5 M had its maximum effect in IAS smooth muscle and RSM phasic and tonic response. calphostin C produced concentration dependent decrease in IAS smooth muscle and RSM tonic and phasic response. Decrease in tonic response was 20 Â± 4.12 % in IAS and 9.6 Â± 4.26 % in RSM (Fig. 4 A). calphostin C had effect on phasic response both in IAS smooth muscle and RSM but the effect was significantly higher in RSM than IAS smooth muscle i.e. 41.85 Â± 4.17 % decrease in phasic activity in RSM and 12.75 Â± 4.24 % decrease in phasic activity in IAS smooth muscle respectively. Original tracings are given in (Fig 6 A and B).
Cumulative effect of calphostin C, Gö6850, Y27632 on Tonic response in IAS smooth muscle and RSM. Cumulative effect of calphostin C, Gö6850, Y27632 on IAS smooth muscle and RSM activity was checked by adding inhibitor into the 2ml muscle bath one after the other, without washing the previous one, and by making sure that the effect of previously added inhibitor had reached the plateau before adding the next one. calphostin C and Gö6850 had similar effect on IAS smooth muscle and RSM. There was very little further decrease in IAS smooth muscle and RSM tonic response when Gö6850 (10-5M) was added to muscle bath in the presence of calphostin C (10-5M). Tonic response in IAS smooth muscle further changed from 20.0 Â± 3.12% to 33 Â± 2.12%, In RSM it went down from 18.3 Â± 4.12% to 22.4 Â± 4.54 % (Fig. 5A). Once effect of Gö6850 plateau, Y27632 (10-5) was added and its effect was noted. Tonic response went down significantly only after the addition of Y27632. In IAS smooth muscle, it went down from 33 Â± 2.12% to 97.2 Â± 3.12% and in RSM, it went down from 22.4 Â± 4.54 % to 64 Â± 3.27 % (Fig. 5A).
Cumulative effect of calphostin C, Gö6850, Y27632 on phasic activity in IAS smooth muscle and RSM. calphostin C (10-5 M) has significantly greater effect on phasic activity in RSM than IAS smooth muscle i.e 41.21 Â± 4.45 % and 12.27 Â± 5.22 %, respectively. Gö6850 (10-5 M) did not produce any further significant effect on IAS smooth muscle and RSM phasic response. Phasic response with Gö6850 shifted from 12.27 Â± 5.22 % and 47.47 Â± 4.55 % in IAS smooth muscle and RSM, respectively. Y27632 did not make any significant change in phasic response in IAS smooth muscle, when added after calphostin C and Gö6850. With Y27632 (10-5M) phasic response decreased from 12.27 Â± 5.22 % to 12.90 Â± 5.11 % in IAS smooth muscle. Y27632 when added on the top of calphostin C and Gö6850, had slightly greater effect on phasic response in RSM, than its effect on phasic response in IAS smooth muscle i.e. 47.47 Â± 4.55 % with calphostin C +Gö6850 and 49.52 Â± 4.21 % calphostin C +Gö6850+Y27632 (Fig. 5B).
Western Blot Data. Tissue strips from IAS smooth muscle and RSM were snap frozen in liquid nitrogen in basal state and after treatment with calphostin C (10-5M), Gö6850 (10-5). and Y27632 (10-5 M). Membrane and cytosol fractions were separated for PKC and ROCK-II inhibition study. Whole tissue lysate was used for all other proteins. Western blots were run for different proteins. The ratio of total amount of these proteins by Î±-actin in 20Âµg of tissue lysate in basal state of IAS was normalized as 1.00 and considered as 100%. Western blot data of pThr38-CPI-17, pThr696-MYPT1 and pSer18/Thr19-MLC20 is given in figures 7 to 9. ï¡-actin was used as internal control for all the western blots.
Effect of calphostin C, Gö6850, Y27632 on PKC and ROCK-II. PKC and ROCK-II inactivation is marked by its movement from membrane to cytosol. There was significant amount of inactivation of PKC with calphostin C and Gö6850. About 90% of the protein moved to cytosol after treatment with calphostin C. Y27632 (10-5 M) had no effect on the movement of PKC but it had significant effect on the movement of ROCK-II form membrane to cytosol in IAS and RSM (Fig. 7 and 8, A,B,C).
Effect of calphostin C, Gö6850 and Y27632 on pThr38-CPI-17. CPI-17 can be phosphorylated by PKC or Rho Kinase and can be an important link between PKC and ROCK (Pang H, et al., 2005; Niiro N, et al., 2003; Kitazawa T, et al., 2000). pThr38-CPI-17 was plotted against ï¡-actin. pThr38-CPI-17 expression was higher in IAS smooth muscle than RSM as reported in our previous study. There was decrease in pThr38-CPI-17 in both IAS smooth muscle (20 ï‚± 2.5%) smooth muscle and RSM (50 ï‚± 2.7%) after calphostin C. There was slight further decrease in pThr38-CPI-17 in both IAS smooth muscle (22 ï‚± 2.4%) smooth muscle and RSM (54 ï‚± 1.2%) with Gö6850 treatment. Y27632 significantly reduced the amount of pThr38-CPI-17 in IAS (90ï‚± 4.0%) smooth muscle and in RSM (87 ï‚± 2.5%) (Fig. 7G and 8G).
Effect of calphostin C, Gö6850 and Y27632 on pThr696-MYPT1. pThr696-MYPT1 was significantly higher in basal state in IAS smooth muscle than RSM whereas MYPT1 was significantly lower in IAS smooth muscle than RSM. calphostin C and Gö6850 decreased the level of pThr696-MYPT1 in IAS smooth muscle and RSM but it was not as significant as it was done by Y27632. There was 15 ï‚± 2.3%, 17 ï‚± 2.4% and 95 ï‚± 1.4% decrease in pThr696-MYPT1in IAS with 1ÂµM of calphostin C, Gö6850 and Y27632, respectively in IAS. In case of RSM it was 07ï‚± 2.1%, 10ï‚±2.2% and 90ï‚±1.2% decrease in pThr696-MYPT1in RSM respectively (Fig. 7H and 8H).
Effect of calphostin C, Gö6850 and Y27632 on pThr18/Ser19-MLC20. calphostin C (10-5M) and Gö6850 (10-5M) had decreased the level of pThr18/Ser19-MLC20 in IAS smooth muscle and RSM, but the effect was much greater in RSM as compared to IAS smooth muscle. There were 20ï‚± 1.4% in IAS and 30ï‚± 1.6% decrease with Gö6850 In comparison Y27632 (10-5) had significant effect on the level of pThr18/Ser19-MLC20 both in IAS smooth muscle and RSM. The level of pThr18/Ser19-MLC20 significantly went down after the treatment with Y27632 (10-5) in IAS smooth muscle and RSM (Fig. 7I and 8I).
Mechanism of development of tone in IAS smooth muscle is not fully understood yet. Several possible pathways are being suggested to produce sustained contraction in different tissues. Most important among them are acting through PKC and RhoA/ROCK. Direct activation of PKC by phorbol esters causes sustained contraction of vascular smooth muscle, suggesting a role for PKC in regulating smooth muscle contraction. Agonist induced contraction via PKC involves the activation of enzyme phospholipase C which hydrolysis of phosphatidylinositol 4, 5-bisphosphate into inositol 1, 4, 5-trisphosphate and DAG. Inositol 1, 4, 5-trisphosphate stimulates Ca2+ release from the intracellular stores, and DAG stimulates PKC (Jiang M.J. and Morgan K.G. 1987; Khalil RA and van Breemen C. 1988; Kanashiro C.A. and Khalil R.A. 1998a,b ). PKC can phosphorylate CPI 17 (Eto M et al. 2007). PKC can cross talk with ROCK via CPI17 (Niiro N. et al., 2003). PKC can activates Rho A and ROCK pathway which can inhibit MLCP and hence can cause sustained contraction (Kimura K. et al., 1996; Kandabashi T. et al., 2003). Rho-kinase, as well as protein kinase C (PKC), was shown to phosphorylate and activate a MLCP inhibitor phosphoprotein, CPI-17, to induce Ca2+sensitization (Somlyo and SomlyoÂ 2003; WebbÂ R.C. 2003; Ratz et al.Â 2005; Cogolludo et al.Â 2007). calphostin C is a cPKCs and nPKCs inhibitor and has been used in number of studies to check the physiological function of PKC (Poole D.P, Furness J.B. 2007; Mizuno Y., et al., 2008; Somara S. et al., 2009; Sakai H et al., 2009). calphostin C had substantially reduced the phasic and tonic contractions caused by carbachol (Amemiya T et al., 2005). PKC had shown to play an important role in lowering esophageal tone (Karen M. et al., 2005). The role PKC in IAS tone was never determined. In our previous studies, we have proved that RhoA/ ROCK pathway is involved in the maintenance of tone (Rattan et al., 2006) but the role of PKC and its effect on IAS tone was never evaluated. In the present study, we have tried to find out the comparative role of PKC and Rho kinase on phasic and tonic response in IAS and RSM by using PKC and Rho kinase inhibitors.
Effect of calphostin C and Gö6850 on tonic response in IAS smooth muscle and RSM. We have found that inhibition of PKC by calphostin C and Gö6850 had a very little effect on tonic response in IAS smooth muscle and RSM. There were only 20.0 Â± 3.12% decrease in IAS smooth muscle tone and 9.6 Â± 4.26 % in RSM tone (Fig 4A), from the above observation it is clear that PKC is not a primary target for maintenance of tone in IAS smooth muscle. Study by sim et al., (2008) had reported similar results in lower esophageal sphincter (LES) muscles. They had proved that PKC inhibitors had negligible effects, contrasting with that reported for cats, where RhoA kinase does not play a major role but PKC signaling is thought to be critical for LES tone (Harnett et al., 2005) but they have proved that RhoA-kinase inhibitors Y-27632 and HA-1077 abolish both spontaneous tonic contraction and additional nerve-evoked contractions of lower esophageal sphincter muscles. These characteristics of human muscles are similar to those reported for LES of rats (Rattan et al., 2003). However, it had been proved that inhibiton of PKC by calphostin C had had abolished tone in human IAS reconstructs (Somara et al., 2009). Preincubation with PKC inhibitor calphostin C had significantly reduced the tone (9.00 ï‚± 2.00ÂµN; n= ; Pâ‰¤0.05) than Rho/ROCK inhibitor Y-27632 did show reduction (18.50 ï‚±2.08 ÂµN; n= 2; P â‰¤ 0.05)That might be due to dedifferentiation of smooth muscle in culture media (camley-campbell et al., 1979) without adding additional additives such as ascorbic acid (Arkawa et al., 2003).
Decrease in phosphorylation of CPI17 and MYPT1 by calphostin C shows the PKC induced inhibition of MLCP via CPI17 in IAS and RSM but compared to PKC, ROCK induced phosphorylation of CPI17 was very high IAS smooth muscle than RSM (Fig 7G and 8G). Moreover, the level of CPI17 is very high in basal state in IAS smooth muscle than RSM that might be one reason of 25 % decrease of tonic response in IAS smooth muscle and RSM by PKC. However, the effect of inhibition of CPI17 and its effect on MLCP and IAS tone is not clear yet.
Effect of Y27632 on tonic response in IAS smooth muscle and RSM. Y27632 is a common ROCK inhibitor and abolishing of tonic response with Y27632 shows the involvement of RhoA/ROCK pathway in the maintenance of tone in IAS and RSM. In case of Tonic, response Y27632 is significantly more effective in abolishing the tone than calphostin c and Gö6850 (Fig. 6). 10-5M Y27632 almost abolishes 100% of the tone response in IAS smooth muscle and RSM. Y27632 had very little effect on the amplitude of phasic activity in IAS. These results are consistant with many other studies in different tissues from different organisms (Rattan et al., 2003; Somlyo and Somlyo, 2003; Shabir et al., 2004; Gokina et al., 2005; Harnett et al., 2005). Western blots data (Fig. 7 A, B, C and 8 A, B, C) shows the movement of ROCK-II from membrane to cytosol after treatment of muscle strips with Y27632. Western blots results also prove the significant decrease in the phosphorylation of MYPT1 and MLC20. These results prove the involvement of RhoA/ROCK and MLCP pathway in the maintenance of tone in IAS smooth muscle (Rattan et al., 2003). Y-27632 significantly attenuated contraction than that by PKC inhibitor calphostin C or Gö6850, is consistent with the selective inhibition of RhoA kinase in other smooth muscles (Rattan et al., 2003; Somlyo and Somlyo, 2003; Shabir et al., 2004; Gokina et al., 2005; Harnett et al., 2005).
Effect of calphostin C and Gö6850 on Phasic response in IAS and RSM.
calphostin C (10-5 M) has significant effect on phasic response in RSM. calphostin C (10-5 M) produced 12..34 Â± 5.32 % of decrease in phasic response in IAS smooth muscle and 41.21 Â± 4.45 % of decrease in phasic response in RSM. Gö6850 (10-5 M) has similar effect like calphostin C both in IAS and RSM phasic response. calphostin C has effect on the phasic activity in IAS smooth muscle and RSM. Two types of phasic activity were observed. In case of IAS smooth muscle the phasic response was noted down as "fast speed and low amplitude" which was not effected by calphostin C. In case of RSM "fast speed and low amplitude" and "slow speed and high amplitude" phasic activity was noted. calphostin C had very little effect on "fast rate and low amplitude" in RSM too but it has significant effect (p â‰¤ 0.05) on "slow rate and high amplitude". The above results prove the involvement of PKC in the "slow rate and high amplitude" phasic activity in RSM. Phasic activity was inhibited by Gö6850 in newborn mouse bladders. carbachol-induced force by approximately inhibited by 60% in the newborn mouse bladders by inhibiting PKC (Ekman M et al., 2009). The above results prove that there are two different pathways responsible for tonic and phasic responses in IAS and RSM, further studies are required to confirm these results.
Effect of Y27632 on Phasic response in IAS smooth muscle and RSM. Y27632 had no effect on "fast rate and low amplitude" in IAS smooth muscle and RSM. Y27632 had effect on the "slow rate and high amplitude" in RSM (Fig. 3 and 6). The effect of Y27632 on the phasic response cannot be ignored. These observations shows the importance of ROCK in both phasic and tonic response, but how PKC and ROCK interacts with each other in phasic response in RSM is not clear yet and need to be studied. In the end, we can say that MLCP is very important anc cannot be ignored in tonic and phasic smooth muscle contraction.
A study by sabir et al., 2004 demonstrated that blockade of ROCK did decrease phasic contraction of ureter smooth muscle, in part due to reduction of voltage-dependent Ca2+ current. This effect of RhoA-kinase blockers was evident in rats, but not guinea pig ureters, indicating species differences.
In the end PKC has little role to play in the maintaince of IAS tone but it is involved in "slow rate and high amplitude" phasic response in RSM, as it is clear from the above results that decrease in rate of amplitude of phasic response in RSM after PKC inhibition by calphostin C and Gö6850, where as tonic response is not significantly affected by PKC inhibition both in IAS smooth muscle and RSM. So, it is proved that PKC is not required for the maintenance of tone in IAS; it might be involved during the initial stages of the development of tone (which is not clear yet and needs to be studied) but do not play very significant role, once the tone is developed, The decrease in phasic activity with the inhibition of PKC suggests that ca2+/calmodulin/MLCK pathway might be involved in phasic response in RSM. Interaction of PKC and ROCK can also not be ruled out in RSM because of the effect of both calphostin C and Y27632 on the phasic response in RSM.