When compared with the rodent colon, nonhuman primate colon shows a closer relationship with human colon in regard to gut size, feces shape, and pattern ( 15 , 21 , 54 , 60 ). Nonhuman primates are widely used for GI studies to understand human intestinal physiology. There are no studies about the effects of PAR agonists on simian colonic motility. Therefore, it is important to understand how PAR agonists influence myogenic motor activity in simian colon to compare with rodent colon. We measured intracellular electrical activities and the corresponding contractile responses and examined MYPT1 and CPI-17 phosphorylation treated with PAR agonists.

Recent studies have shown that in gastric and esophageal SMCs, the contractile response to the PAR2-activating peptide consists of two phases ( 14 , 52 ). The initial contractile responses are mediated by the formation of IP 3 and Ca 2+ /calmodulin-dependent activation of myosin light chain kinase, leading to the phosphorylation of myosin light chain (MLC 20 ). The sustained phase is mediated by a Ca 2+ -independent mechanism that is mostly due to inhibition of MLC phosphatase (MLCP) ( 14 , 52 ). As in any phosphorylation regulated process, the ratio of myosin light chain kinase activity to MLCP activity is a major determinant of the myofilament sensitivity to contractile stimuli. A common mechanism regulating this activity ratio involves MLCP inhibition by protein kinase C (PKC)-mediated CPI-17 (phosphatase inhibitor protein of 17 kDa) phosphorylation, and/or Rho-associated protein kinase 2 (ROCK2) phosphorylation of myosin phosphatase targeting subunit of MLCP (MYPT1) ( 16 , 41 ).

The effects of PAR activation on GI motility are diverse and include relaxation, contraction, or a biphasic response of relaxation followed by contraction ( 11 , 29 , 35 , 46 ). In a previous report, we showed that thrombin (PAR1 agonist) and trypsin (PAR2 agonist) caused biphasic responses in murine colonic muscles: a transient hyperpolarization and relaxation followed by repolarization and excitation. Apamin, a blocker of SK channels, blocks the initial relaxation ( 10 , 25 , 35 , 47 ). Patch-clamp studies showed that the inhibitory response was mediated by activation of SK channels in PDGFRα + cells, whereas the excitatory response was mediated by activation of a Cl − conductance in ICC ( 53 ).

Protease-activated receptors (PARs) are widely distributed in the GI tract and are involved in regulating GI motility ( 28 ). Among the four PAR isoforms (PAR1–4), PAR1 and PAR2 are highly expressed in GI smooth muscles ( 37 , 53 ). The PAR agonists activate the G protein-coupled receptors and initiate intracellular signaling events ( 6 , 12 , 13 , 33 , 55 ). Activation of PAR1 by thrombin or PAR2 by trypsin increases 1,4,5-trisphosphate (IP 3 ) production via G q/11 -mediated activation of phospholipase Cβ. Additionally, PAR1 and PAR2 activation can also reduce cAMP levels because of G i /G o -mediated inhibition of adenylyl cyclase ( 4 , 18 , 39 , 57 ).

Enteric motor neurons innervate gastrointestinal (GI) smooth muscles and regulate GI motility. GI smooth muscles are composed of a SIP syncytium: smooth muscle cells (SMCs), interstitial cells of Cajal (ICCs), and platelet-derived growth factor receptor α positive (PDGFRα + ) cells ( 44 ). Activation of excitatory motor neurons and inhibitory motor neurons causes excitatory junction potentials and inhibitory junctional potentials (IJPs), respectively ( 44 , 45 ). Excitatory junction potentials are a result of activation of muscarinic receptors by release of acetylcholine from cholinergic neurons and activation of Ca 2+ -activated Cl − conductance in ICCs. IJPs are composed of two components. Fast IJPs result from activation of small conductance Ca 2+ -activated K + (SK) channels via purinergic receptor activity (mainly P 2 Y 1 ) on PDGFRα + cells ( 44 ). Release of nitric oxide (NO) by activation of nitrergic neurons induces slow IJPs in ICC. Ion channel candidates for nitric oxide-induced hyperpolarization are not clear. Besides neurotransmitters, many other factors, including humoral or inflammatory mediators can affect GI motility. In this study, we will investigate the role of proteases as inflammatory mediators of colonic motility in the SIP syncytium.

Data were expressed as means ± SE. The Student’s t -test and ANOVA followed by a post hoc test were used where appropriate to evaluate differences in the data. P values less than 0.05 were taken as statistically significant differences. n values refer to the number of recordings from muscle strips in electromechanical and molecular experiments.

Before all experiments the muscle strips were equilibrated in oxygenated KRB solution of the following composition (in mmol/l): 120 NaCl, 5.9 KCl, 1.2 MgCl 2 , 15.5 NaHCO 3 , 1.2 NaH 2 PO 4 , 11.5 dextrose, and 2.5 CaCl 2 . The pH of KRB was 7.3–7.4 when bubbled with 97% O 2 -3% CO 2 . Thrombin, trypsin, tetraethylammonium, TRAM-34, TTX, Gö 6976, and GF 109203X were obtained from Sigma Chemical (St. Louis, MO). Apamin was purchased from Santa Cruz Biotechnology. Y-27632 was obtained from Calbiochem (San Diego, CA) and SAR-1× was donated by Dr. Brian Perrino.

Strips of simian colonic smooth muscles were equilibrated in oxygenated KRB at 37 ± 0.5°C for 1 h with TTX (1 µM). The muscles were then treated with thrombin (50 U/ml) or trypsin (1 µM) in the absence or presence of apamin (300 nM) and at the indicated time points were submerged into ice-cold acetone/10 mM dithiothreitol (DTT)/10% (wt/vol) trichloroacetic acid for 2 min, snap-frozen in liquid N 2 , and stored at −80°C for subsequent Western blot analysis ( 1 ). The muscles were thawed on ice for 5 min, followed by three 1-min washes in ice-cold acetone/DTT, and a 2-min wash in ice-cold lysis buffer, consisting of (in mM) 50 Tris·HCl (pH 8.0), 60 β-glycerophosphate, 100 NaF, 2 EGTA, 25 Na-pyrophosphate, 1 DTT, with 0.5% Nonidet P-40, 0.2% SDS, and protease inhibitor tablet (Roche, Indianapolis, IA)] ( 1 , 23 ). Each tissue was homogenized in 0.20 ml lysis buffer using a Bullet blender (0.01% anti-foam C, 1 stainless steel bead per tube, speed 6, 5 min). The homogenates were centrifuged at 16,000 g , 4°C, 10 min, and the supernatants stored at −80°C. Protein concentrations of the supernatants were determined by Bradford assay, using bovine γ-globulin as the standard. The supernatants were analyzed by SDS-PAGE and Western blotting with rabbit anti-MYPT1, rabbit anti-pT696-MYPT1, rabbit anti-pT853-MYPT1, mouse anti-CPI-17, and rabbit anti-pT38-CPI-17 antibodies (Santa Cruz Biotechnology, Dallas, TX). Protein bands were detected using horseradish peroxidase-conjugated goat-anti rabbit IgG (cat. no. AP-307-P) or goat anti-mouse IgG (cat. no. AP-181-P) secondary antibodies (EMD Millipore, Billerica, MA) and Lumigen TMA-6 (Lumigen, Southfield, MI), and visualized with a CCD camera-based detection system equipped with Visionworks software (Epi Chem II, UVP Laboratory Products, Upland, CA). The tiff images were inverted and adjusted to auto levels and resolution with Adobe Photoshop (CS2, V.9.0.2, Adobe Systems, San Jose, CA,) for densitometry ( 1 ). The ratios from control tissues were normalized to a value of 1.

The membrane potential was measured using intracellular recordings in simian colonic SMCs. Muscle strips (0.5-cm length and 0.5-cm width) were prepared by peeling off the mucosa and submucosa. Oxygenated and prewarmed (37 ± 0.5°C) KRB solution was continuously perfused. Circular muscle was impaled with glass microelectrodes filled with 3 M KCl and having electrical resistances of 80–100 MΩ. Transmembrane potentials were measured with a standard high-input impedance amplifier (WPI Duo 773, Sarasota, FL). Electrical signals were recorded by a computer running AxoScope data acquisition software (Axon Instruments) and analyzed by Clampfit (v.9.02, Axon Instruments) and Graphpad Prism (version 5.0, Graphpad Software, San Diego, CA) software. All experiments were performed in the presence of TTX (1 µM) to eliminate neural involvement in the thrombin- or trypsin-induced responses.

Proximal colons were rinsed with Krebs-Ringer bicarbonate (KRB) solution. The mucosa and submucosa were removed, and the remnant tunica muscularis was circumferentially cut by 1-cm length and 0.4-cm width. Organ bath techniques were applied to measure motility generated by muscle strips of proximal colon. The strips were suspended in a 5-ml organ bath chamber containing oxygenated (97% O 2 -3% CO 2 ) KRB solution. One end of a muscle strip was tied to a fixed mount, and the opposite end was connected to an isometric force transducer (Fort 10, WPI, Sarasota, FL). Bath temperature was maintained at 37 ± 0.5°C and KRB solution was changed every 15 min. Muscle strips were stabilized for 30 min without a force followed by equilibrating for 60–90 min under a resting force of 0.5–1 g. Mechanical responses were recorded on a computer running Axoscope (Axon Instruments, Foster City, CA). The amplitude, frequency, and the area under the curve (AUC) for 2-min recordings of spontaneous contractions were measured. The change in parameters after drug application was compared with the parameters before drug application. Tetrodotoxin (TTX) (1 µM) was added to the bath for 10 min before the application of thrombin or trypsin to eliminate neural involvement in thrombin- or trypsin-induced responses in all experiments.

Fig. 9. Apamin effect on MYPT1 T853 phosphorylation by thrombin or trypsin. Representative Western blots of MYPT1 T853 or T696 phosphorylation in simian colonic muscles treated with thrombin (50 U/ml, A ) or trypsin (1 µM, B ) for 2 or 5 min in the absence or presence of apamin (300 nM). Summary data of the pT853/MYPT1 (open box) and pT696/MYPT1 (closed box) ratios from simian colonic muscles treated with thrombin (50 U/ml, C ) or trypsin (1 µM, D ) for 2 or 5 min in the absence or presence of apamin (300 nM). Some muscle strips were treated with thrombin or trypsin for 5 min in the presence of the ROCK inhibitor, Y-27632 (3 µM). *** P < 0.001 when compared with control. ### P < 0.001 when compared with thrombin or trypsin in the presence of apamin at 5 min. C, control; MYPT1, myosin phosphatase targeting subunit of MLCP; ROCK, Rho-associated protein kinase.

We investigated the effect of apamin (300 nM) on the increase in MYPT1 T853 phosphorylation by thrombin or trypsin to compare with the effect of thrombin and trypsin alone. Apamin did not affect the increase in T853 phosphorylation observed with 5-min treatment of thrombin or trypsin alone [fold changes compared with control; 2.1 ± 0.1 in thrombin alone vs. 2.0 ± 0.2 in thrombin in presence of apamin ( P = 0.58); 2.1 ± 0.1 in trypsin alone vs. 1.8 ± 0.1 in trypsin in the presence of apamin) ( P = 0.12) Fig. 9 ]. However, the ROCK inhibitor, Y-27632 (3 µM) completely blocked the increase in MYPT1 T853 phosphorylation by thrombin or trypsin and reduced the phosphorylation of both T853 and T696 below the control basal levels ( Fig. 9 ).

Fig. 8. CPI-17 T38 phosphorylation by thrombin or trypsin in the presence of apamin. Representative Western blots of CPI-17 T38 phosphorylation in simian colonic muscles treated with thrombin (50 U/ml, A ) or trypsin (1 µM, B ) for 2 min or 5 min in the absence or presence of apamin (300 nM). Summary data of the pT38/CPI-17 ratios from simian colonic muscles treated with thrombin (50 U/ml, C ) or trypsin (1 µM, D ) for 2 min or 5 min in the absence or presence of apamin (300 nM). Some muscle strips were treated with thrombin or trypsin for 5 min in the presence of the PKC inhibitors, Gö 6976 (3 µM) or GF 109203X (3 µM). *** P < 0.001 when compared with control. ### P < 0.001 when compared with thrombin or trypsin in the presence of apamin at 5 min. C, control; PKC, protein kinase C; p-CPI-17, phosphorylated CPI-17.

Fig. 7. Effects of thrombin or trypsin on CPI-17 T38 phosphorylation. Representative Western blots of CPI-17 T38 phosphorylation in simian colonic muscles treated with thrombin (50 U/ml, A ) or trypsin (1 µM, B ) for 2 to 20 min. Summary data of the pT38/CPI-17 ratios from simian colonic muscles treated with thrombin (50 U/ml, C ) or trypsin (1 µM, D ) for 2 to 20 min. C, control.

Because an increase in CPI-17 phosphorylation is typically associated with Ca 2+ influx, we examined the effects of thrombin or trypsin treatment on CPI-17 T38 phosphorylation in simian colonic smooth muscles. Thrombin or trypsin treatment of simian colonic smooth muscles had no effect on CPI-17 T38 phosphorylation at each time point ( Fig. 7 ). Because apamin inhibited the hyperpolarization induced by thrombin or trypsin, we tested the effect of apamin on CPI-17 T38 phosphorylation. Thrombin or trypsin treatment at 2 min or 5 min, in the presence of apamin (300 nM), significantly increased CPI-17 T38 phosphorylation (fold changes compared with control; 2.1 ± 0.2 at 2 min, 2.2 ± 0.1 at 5 min in thrombin treatment; 2.1 ± 0.1 at 2 min, 2.2 ± 0.1 at 5 min in trypsin treatment; Fig. 8 ). CPI-17 can be phosphorylated by PKC ( 41 , 44 ). Thus, we tested the effects of PKC inhibitors on CPI-17 T38 phosphorylation. Gö 6976 (3 µM), the inhibitor of Ca 2+ -dependent PKCs, blocked the portion of CPI-17 T38 phosphorylation increased by apamin in the presence of thrombin or trypsin ( Fig. 8 ). The pan-PKC inhibitor, GF 109203X (3 µM), inhibited CPI-17 T38 phosphorylation even below the control basal level. These data suggest that thrombin or trypsin induces Ca 2+ influx in the presence of apamin, which inhibits SK channels and induces depolarization, consequentially activating PKC, inducing CPI-17 T38 phosphorylation, and in turn contracting simian colonic muscles. In addition, these findings suggest that basal CPI-17 phosphorylation can be controlled by a Ca 2+ -independent PKC.

Fig. 6. The effect of thrombin or trypsin on MYPT1 T853 and T696 phosphorylation. Representative Western blots of MYPT1 phosphorylation in simian colonic muscles treated with thrombin (50 U/ml, A ) or trypsin (1 µM, B ) for 2 to 20 min. Summary data of the pT853/MYPT1 (open box) and pT696/MYPT1 (closed box) ratios from simian colonic muscles treated with thrombin (50 U/ml, C ) or trypsin (1 µM, D ) for 2 to 20 min. *** P < 0.001 when compared with control. MYPT1, myosin phosphatase targeting subunit of MLCP. C, control; MYPT1, myosin phosphatase targeting subunit of MLCP; p-MYPT1, phosphorylated MYPT1.

We tested whether PAR activation by thrombin or trypsin increases MYPT1 phosphorylation by ROCK, as seen with Ca 2+ sensitization mechanisms ( 14 ). Figure 6 showed that thrombin (50 U/ml) or trypsin (1 µM) treatment of simian colonic smooth muscles significantly increased MYPT1 T853 phosphorylation (e.g., fold changes compared with control; 2.0 ± 0.1 at 2 min, 1.8 ± 0.1 at 5 min, 1.9 ± 0.1 at 10 min, and 1.8 ± 0.1 at 20 min in thrombin treatment; 1.9 ± 0.1 at 2 min, 2.0 ± 0.1 at 5 min, 1.8 ± 0.1 at 10 min, and 1.9 ± 0.1 at 20 min in trypsin treatment) but had no effect on T696 phosphorylation.

Fig. 5. The effect of Rho kinase inhibitors, Y-27632 and SAR-1x on thrombin- or trypsin-induced delayed contractile responses in simian colonic muscles. Representative traces illustrating that Y-27632 (3 µM) decreased thrombin (50 U/ml, A )- or trypsin (1 µM, B )-induced delayed contractile responses. SAR-1x (2 µM) also decreased thrombin (50 U/ml, C )- or trypsin (1 µM, D )-induced delayed responses. Summary data of AUC of Y-27632 and SAR-1x on thrombin (open box)- or trypsin (closed box)-induced delayed contractile responses ( E and F ). *** P < 0.001 when compared with thrombin or trypsin alone. AUC, area under the curve.

In contrast to murine colonic responses to thrombin or trypsin ( 53 ), thrombin or trypsin induced delayed contractions without depolarization in simian colonic muscles. Therefore, we hypothesized that the thrombin- or trypsin-induced delayed contractile responses could be mediated by Ca 2+ -independent pathways. Because PAR1 and PAR2 can be coupled to G 12/13 , which can activate RhoA and its downstream signaling pathways ( 22 , 49 ), we investigated the role of ROCK on thrombin- or trypsin-induced delayed contractile responses. The ROCK inhibitors, Y-27632 and SAR-1x, were applied during the steady state of delayed contractile periods induced by thrombin or trypsin treatment in simian colon. Y-27632 (3 µM) significantly reduced the AUC of thrombin-induced contraction to 34.3 ± 5.4% ( Fig. 5, A and E; P < 0.001; n = 6) and AUC of trypsin-induced contraction to 29.0 ± 5.5% ( Fig. 5, B and E; P < 0.001; n = 6). Another ROCK inhibitor, SAR-1x (2 µM) also reduced thrombin-induced contractile response to 10.5 ± 3.5% ( Fig. 5, C and F; P < 0.001; n = 5) or trypsin-induced contractile response to 5.9 ± 2.4% ( Fig. 5, D and F; P < 0.001; n = 5). These results suggest that the delayed contractile responses to thrombin or trypsin could be mediated by ROCK-dependent pathways in simian colon.

Fig. 3. The effects of tetraethylammonium (TEA) and TRAM-34 on the initial relaxation responses by thrombin or trypsin in simian colonic muscles. Representative mechanical traces showing that TEA (1 mM) did not show any effect on thrombin (50 U/ml, A )- or trypsin (1 µM, B )-induced initial relaxation in simian colonic muscles. TRAM-34 (10 µM) also had no effect on the thrombin (50 U/ml, C )- or trypsin (1 µM, D )-induced initial relaxation.

There are many reports that initial hyperpolarization by PAR activators is due to activation of Ca 2+ -activated K + currents in many GI regions ( 26 , 27 , 35 , 46 , 47 , 53 ). Thus, we tested the effect of Ca 2+ -activated K + channels on the initial relaxation in response to thrombin or trypsin. K + channel blockers were applied for 10 min before thrombin or trypsin application. Tetraethylammonium (1 mM) for blocking large conductance Ca 2+ -activated K + channel or TRAM-34 (10 µM) for blocking intermediate Ca 2+ -activated K + channel had no effect on thrombin- or trypsin-induced initial relaxation ( Fig. 3 ). However, apamin (300 nM) pretreatment significantly reduced the initial relaxation of spontaneous contractions from 3.6 ± 0.5 to 1.0 ± 0.2 min by thrombin ( P < 0.01, n = 7) and from 3.1 ± 0.3 to 1.0 ± 0.2 min by trypsin ( P < 0.001, n = 7, Fig. 4, A and B ). These results suggest that the major cause of the initial relaxation by thrombin or trypsin is activation of SK channels. In microelectrode recoding experiments, pretreatment of apamin significantly decreased thrombin-induced hyperpolarization from 13.8 ± 1.5 to 3.9 ± 0.7 ΔmV ( P < 0.001; n = 5) or trypsin-induced hyperpolarization from 7.0 ± 0.8 to 3.1 ± 0.6 ΔmV ( P < 0.001; n = 5) compared with control ( Fig. 2, A and B in control, Fig. 4, C and D in the presence of apamin). These results suggest that thrombin or trypsin elicited hyperpolarization mainly via activation of SK channels.

Fig. 2. The effects of thrombin or trypsin on the membrane potential in simian colonic muscles. Representative traces illustrating that thrombin (50 U/ml, A ) or trypsin (1 µM, B ) caused hyperpolarization in the presence of TTX (1 µmol/l) in simian colonic muscles. The summarized data of thrombin (open box) or trypsin (closed box) effects on the RMP at 2 min and 10 min application in simian colon ( C ). * P < 0.05 and ** P < 0.01 when compared with control, respectively. RMP, resting membrane potential; TTX, tetrodotoxin.

According to our previous study in murine colon, thrombin or trypsin induces an initial hyperpolarization followed by repolarization ( 53 ). Therefore, we performed intracellular microelectrode recordings to examine the effects of thrombin or trypsin on the membrane potential in simian colon. Both thrombin (50 U/ml) and trypsin (1 µM) initially hyperpolarized simian colonic muscle from −54 ± 2.9 to −68 ± 3.6 mV ( Fig. 2, A and C ; P < 0.05; n = 5) and from −52 ± 0.9 to −59 ± 1.0 mV, respectively ( Fig. 2, B and C ; P < 0.01; n = 5). Membrane potentials were restored to the control resting membrane potential level after the initial hyperpolarization without delayed depolarization.

Fig. 1. The effects of thrombin or trypsin on the spontaneous contractility in simian colonic muscles. Representative mechanical traces illustrating that thrombin (50 U/ml, A ) or trypsin (1 µM, B ) caused an initial relaxation followed by delayed contractions in the presence of TTX (1 µmol/l) in simian colonic muscles. Summary data of amplitude ( C ), frequency ( D ) and area under the curve (AUC) for 2 min recording ( E ) of spontaneous contractility following application of thrombin (open box) or trypsin (closed box) at 2, 5, and 10 min. * P < 0.05, ** P < 0.01, and *** P < 0.001 when compared with control. BPM, beats/min.

The amplitude, frequency, and AUC of spontaneous contractions for 2 min were analyzed to investigate the effects of thrombin or trypsin in simian colon. Both thrombin and trypsin initially inhibited the spontaneous contractions followed by augmented contractions ( Fig. 1 ). Thrombin (50 U/ml) and trypsin (1 µM) abolished the spontaneous contractions for 3.6 ± 0.5 and 3.1 ± 0.3 min in colon, respectively ( Fig. 1, A and B , n = 7 each). Following the initial inhibitory response, continuous perfusion of thrombin or trypsin induced augmented contractile responses. The amplitudes of spontaneous contractions were increased to 281.5 ± 26.6% after 5 min and 229.2 ± 34.5% after 10 min by thrombin (both P < 0.001; n = 7) and 231.1 ± 14.7% after 5 min ( P < 0.001; n = 8) and 161.6 ± 16.6% after 10 min ( P < 0.01; n = 8) by trypsin ( Fig. 1 C ). The frequencies of contractions were increased from 1.8 ± 0.2 cycle/min to 2.8 ± 0.1 cycle/min after 5 min by thrombin ( Fig. 1 D ; P < 0.001; n = 6) and from 1.6 ± 0.3 to 2.9 ± 0.2 cycle/min after 5 min by trypsin ( Fig. 1 D ; P < 0.001; n = 7). AUC was also significantly increased to 507.8 ± 129.2% after 5 min ( P < 0.05, n = 9) and 241.1 ± 50.7% after 10 min ( P < 0.05, n = 9) by thrombin and 462.2 ± 83.4% after 5 min ( P < 0.01, n = 8) and 185.7 ± 35.3% after 10 min ( P < 0.05, n = 8) by trypsin ( Fig. 1 E ). These results show a dual effect of thrombin or trypsin; initial relaxation followed by contractions in simian colonic muscles.

DISCUSSION

In the present study, the effects of thrombin (PAR1 agonist) and trypsin (PAR2 agonist) on the contractile and electrical activities and MYPT1 and CPI-17 phosphorylation of simian colonic muscles were examined. The physiological concentration of thrombin or trypsin can be variable depending on the extent of inflammation or trauma. In previous reports, maximal responses in mouse colon or cat esophagus were obtained with thrombin (50 U/ml) and trypsin (1 µM) (14, 46, 53). We tested the effects of thrombin and trypsin using these concentrations for this study.

Both thrombin and trypsin induced biphasic responses in simian colonic muscles comprised of an initial transient hyperpolarization and relaxation followed by repolarization and contractions. Similar biphasic responses by PAR activators have been reported in murine colonic muscles (25, 26, 35, 36, 47, 53). Many reports suggest that the initial hyperpolarization is caused by Ca2+-activated K+ currents by both PAR activators in GI smooth muscles (26, 27, 35, 46, 47, 53). We found that apamin (an SK blocker) significantly inhibits the initial hyperpolarization by thrombin or trypsin in simian colonic muscles. Other Ca2+-activated K+ channel blockers, tetraethylammonium (a large conductance Ca2+-activated K+ channel blocker) and TRAM-34 (an intermediate conductance Ca2+-activated K+ blocker) had no effect on the thrombin- or trypsin-induced hyperpolarization suggesting that the initial hyperpolarization is mainly mediated by activation of SK channels in simian colonic muscles. SK channels are activated by an increase in intracellular Ca2+. PAR agonists are coupled to G q/11 , which increases intracellular Ca2+ by activation of phospholipase Cβ-IP 3 pathways and in turn activating SK channels. Because SK channels are highly expressed in PDGFRα+ cells (19, 32, 40), the initial hyperpolarization by thrombin or trypsin is likely due to activation of SK channels in PDGFRα+ cells.

Our previous study showed that Ca2+-dependent pathways in ICCs and SMCs were responsible for the excitatory response to PAR activators in murine colon (53). However, the delayed contractile responses to thrombin or trypsin in simian colon were significantly augmented compared with the control contractions. These responses did not correspond to the thrombin- or trypsin-induced electrical responses, which did not induce depolarization in simian colon. These observations suggested that the thrombin- or trypsin-induced delayed contractile responses could be mediated by Ca2+ sensitization pathways, which typically result in enhanced contractile responses. However, the mechanisms underlying the subsequent contractile phase have not been clarified (25, 26, 35, 36, 53, 61, 62). In feline esophageal muscles, the sustained contraction evoked by PAR2 activation is mediated by PKA and ROCK-dependent phosphorylation of MYPT1 but not CPI-17 phosphorylation (52). In rat duodenum, the contractile responses to PAR1 and PAR2 activation are mediated, in part, by activation of L-type calcium channels, PKC, and tyrosine kinase activities (25). We determined here that thrombin or trypsin treatment results in an initial inhibitory phase via SK channel activation, and the delayed excitatory contractile phase is due to activation of the Ca2+ sensitization pathways in simian colonic muscles. Indeed, a major component of the inhibitory neuromuscular transmission is mediated by SK channels in mouse colon (17, 44, 50, 51).

Because PAR1 and PAR2 can be coupled to G 12/13 , which can activate RhoA and its downstream effectors, we examined the involvement of ROCK on thrombin- or trypsin-induced delayed contractile responses. The ROCK inhibitors, Y-27632 and SAR-1x, significantly reduced the AUCs of thrombin- or trypsin-induced contractions suggesting that the delayed contractile responses to thrombin or trypsin are mediated by ROCK-dependent pathways in simian colonic muscles. Because increased MYPT1 phosphorylation by Rho-kinase is characteristic of enhanced smooth muscle contractile responses, we examined the effects of thrombin or trypsin treatment on MYPT1 phosphorylation. As shown in Fig. 6, thrombin or trypsin significantly increased MYPT1 T853 phosphorylation at each time point but had no effect on T696 phosphorylation. These findings are similar to other findings reporting that only MYPT1 T853 phosphorylation is typically increased by G protein-coupled receptor (GPCR) activation in GI smooth muscles (2, 5, 20, 30, 34, 59). The ROCK inhibitors Y-27632 and SAR-1x blocked the increase in MYPT1 T853 phosphorylation and also reduced the levels of MYPT1 T853 and T696 phosphorylation below the basal control levels. These findings are similar to our previous findings in mouse colonic muscles (2) and indicate the thrombin- or trypsin-induced increase in MYPT1 T853 phosphorylation is mediated by ROCK activation. Apamin had no effect on the increase in MYPT1 T853 phosphorylation by thrombin or trypsin, further indicating that the augmented contractile responses of simian colonic muscles evoked by thrombin or trypsin treatment are Ca2+-independent. In addition, these findings suggest that the basal or constitutive phosphorylation of MYPT1 T853 and T696 is maintained by ROCK, similar to murine colonic muscles (1). Although these findings suggest that basal MYPT1 T696 phosphorylation is mediated by ROCK, T696 phosphorylation was not increased by thrombin or trypsin treatment. Previous findings suggest that MYPT1 T696 phosphorylation in resting or unstimulated smooth muscles may already be maximally phosphorylated (2) and may also be maximally phosphorylated in simian colonic muscles.

Because an increase in CPI-17 phosphorylation is typically associated with Ca2+ influx, we examined the effects of thrombin or trypsin treatment on CPI-17 T38 phosphorylation in simian colonic muscles. The results in Fig. 7 show that neither thrombin nor trypsin treatment of simian colonic muscles increased CPI-17 T38 phosphorylation at each time point including the delayed contractile phase. However, in the presence of apamin, thrombin or trypsin significantly increased CPI-17 T38 phosphorylation. These data suggest that initial hyperpolarization by thrombin or trypsin is due to activation of SK channels and in turn decreases Ca2+ influx, and thus does not affect Ca2+-dependent PKC phosphorylation with no effect on CPI-17 phosphorylation.

To prove this hypothesis, we performed experiments to explore the role of PKC in CPI-17 phosphorylation. The PKC inhibitors Gö 6976 (3 µM) or GF 109203X (3 µM) each completely blocked the apamin-induced increase in CPI-17 T38 phosphorylation in the presence of thrombin or trypsin. The pan-PKC inhibitor GF 109203X reduced T38 phosphorylation significantly below the control basal level whereas Gö 6976, which targets the Ca2+-dependent PKC isoforms only inhibited the increase in T38 phosphorylation by apamin in the presence of thrombin or trypsin, further suggesting that Ca2+ influx is increased by apamin and is activating the Ca2+-dependent PKC isoforms.

Thrombin (PAR1 agonist), which is an essential enzyme in the coagulation cascade, is potentially present as an inflammatory mediator in the GI tract during inflammation or tissue trauma (56, 58). Trypsin, which is a digestive protease, and mast cell-derived tryptase (PAR2 agonists) are both widely expressed by several cell types in the GI tract (24). These endogenous proteases are massively increased in various diseases, including inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and ischemia (7, 8, 31, 43, 56). In addition, the molecular expression of both PAR1 and PAR2 are altered in patients with IBD and IBS (3, 9, 38, 42, 48). Therefore, PAR activation may affect colonic dysmotility in patients with IBD and IBS.

In conclusion, these studies show that in nonhuman primate colonic muscles, the PAR1 and PAR2 activators, thrombin and trypsin, caused biphasic responses: initial hyperpolarization and relaxation followed by repolarization and contraction. The initial hyperpolarization and relaxation in response to thrombin or trypsin is mainly mediated by activation of SK channels in simian colonic muscles. Ca2+ sensitization pathways, including CPI-17 and ROCK mediate the contractile responses in simian colonic muscles to thrombin or trypsin.