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Small heat shock proteins HSP27 and HSP20 have been implicated in regulation of contraction and relaxation in smooth muscle. Activation of PKC-[alpha] promotes contraction by phosphorylation of HSP27 whereas activation of PKA promotes relaxation by phosphorylation of HSP20 in colonic smooth muscle cells (CSMC). We propose that the balance between the phosphorylation states of HSP27 and HSP20 represents a molecular signaling switch for contraction and relaxation. This molecular signaling switch acts downstream on a molecular mechanical switch [tropomyosin (TM)] regulating thin-filament dynamics. We have examined the role of phosphorylation state(s) of HSP20 on HSP27-mediated thin-filament regulation in CSMC. CSMC were transfected with different HSP20 phosphomutants. These transfections had no effect on the integrity of actin cytoskeleton. Cells transfected with 16D-HSP20 (phosphomimic) exhibited inhibition of acetylcholine (ACh)-induced contraction whereas cells transfected with 16A-HSP20 (nonphosphorylatable) had no effect on ACh-induced contraction. CSMC transfected with 16D-HSP20 cDNA showed significant decreases in 1) phosphorylation of HSP27 (ser78); 2) phosphorylation of PKC-[alpha] (ser657); 3) phosphorylation of TM and CaD (ser789); 4) ACh-induced phosphorylation of myosin light chain; 5) ACh-induced association of TM with HSP27; and 6) ACh-induced dissociation of TM from caldesmon (CAD). We thus propose the crucial physiological relevance of molecular signaling switch (phosphorylation state of HSP27 and HSP20), which dictates 1) the phosphorylation states of TM and CaD and 2) their dissociations from each other. relaxation; heat shock protein; molecular signaling switch; molecular mechanical switch; smooth muscle contraction doi: 10.1152/ajpgi.00479.2009.

We have previously developed bioengineered three-dimensional internal anal sphincter (IAS) rings from circular smooth muscle cells isolated from rabbit and human IAS. We provide proof of concept that bioengineered mouse IAS rings are neovascularized upon implantation into mice of the same strain and maintain concentric smooth muscle alignment, phenotype, and IAS functionality. Rings were bioengineered by using smooth muscle cells from the IAS of C57BL/6J mice. Bioengineered mouse IAS rings were implanted subcutaneously on the dorsum of C57BL/6J mice along with a microosmotic pump delivering fibroblast growth factor-2. The mice remained healthy during the period of implantation, showing no external signs of rejection. Mice were killed 28 days postsurgery and implanted IAS rings were harvested. IAS rings showed muscle attachment, neovascularization, healthy color, and no external signs of infection or inflammation. Assessment of force generation on harvested IAS rings showed the following: 1) spontaneous basal tone was generated in the absence of external stimulation; 2) basal tone was relaxed by vasoactive intestinal peptide, nitric oxide donor, and nifedipine; 3) acetylcholine and phorbol dibutyrate elicited rapid-rising, dose-dependent, sustained contractions repeatedly over 30 min without signs of muscle fatigue; and 4) magnitudes of potassium chloride-induced contractions were 100% of peak maximal agonist-induced contractions. Our preliminary results confirm the proof of concept that bioengineered tings are neovascularized upon implantation. Harvested rings maintain smooth muscle alignment and phenotype. Our physiological studies confirm that implanted rings maintain 1) overall IAS physiology and develop basal tone, 2) integrity of membrane ionic characteristics, and 3) integrity of membrane associated intracellular signaling transduction pathways for contraction and relaxation by responding to cholinergic, nitrergic, and VIP-ergic stimulation. IAS smooth muscle tissue could thus be bioengineered for the purpose of implantation to serve as a potential graft therapy for dysfunctional internal anal sphincter in fecal incontinence. fecal incontinence; tissue engineering; smooth muscle contraction doi: 10.1152/ajpgi.00269.2009.

Coordinated regulation of smooth muscle contraction and relaxation is required for colonic motility. Contraction is associated with phosphorylation of myosin light chain ([MLC.sub.20]) and interaction of actin with myosin. Thin-filament regulation of actomyosin interaction is modulated by two actin-binding regulatory proteins: tropomyosin (TM) and caldesmon (CAD). TM and CaD are known to play crucial role in actomyosin interaction promoting contraction. Contraction is associated with phosphorylation of the small heat shock protein HSP27, concomitant with the phosphorylation of TM and CaD. Phosphorylation of HSP27 is attributed as being the prime modulator of thin-filament regulation of contraction. Preincubation of colonic smooth muscle cells (CSMC) with the relaxant neurotransmitter vasoactive intestinal peptide (VIP) showed inhibition in phosphorylation of HSP27 (ser78). Attenuation of HSP27 phosphorylation can result in modulation of thin-filament-mediated regulation of contraction leading to relaxation; thus the role of thin-filament regulatory proteins in a relaxation milieu was investigated. Preincubation of CSMC with VIP exhibited a decrease in phosphorylation of TM and CAD. Furthermore, CSMC preincubated with VIP showed a reduced association of TM with HSP27 and with phospho-HSP27 (ser78) whereas there was reduced dissociation of TM from CAD and from phosphoCAD. We thus propose that, in addition to alteration in phosphorylation of [MLC.sub.20], relaxation is associated with alterations in thin-filament-mediated regulation that results in termination of contraction. heat shock protein 27; tropomyosin; caldesmon doi: 10.1152/ajpgi.00201.2009.

Increased airway smooth muscle (ASM) mass, a characteristic finding in asthma, may be caused by hyperplasia or hypertrophy. Cell growth requires increased translation of contractile apparatus mRNA, which is controlled, in part, by glycogen synthase kinase (GSK)-3[beta], a constitutively active kinase that inhibits eukaryotic initiation factor-2 activity and binding of methionyl tRNA to the ribosome. Phosphorylation of GSK-3[beta] inactivates it, enhancing translation. We sought to quantify the contributions of hyperplasia and hypertrophy to increased ASM mass in ovalbumin (OVA)-sensitized and -challenged BALB/c mice and the role of GSK-3[beta] in this process. Immunofluorescent probes, confocal microscopy, and stereological methods were used to analyze the number and volume of cells expressing [alpha]-smooth muscle actin and phospho-[Ser.sup.9] GSK-3[beta] (pGSK). OVA treatment caused a 3-fold increase in ASM fractional unit volume or volume density (Vv) (PBS, 0.006 [+ or -] 0.0003; OVA, 0.014 [+ or -] 0.001), a 1.5-fold increase in ASM number per unit volume (Nv), and a 59% increase in volume per cell (Vv/Nv) (PBS, 824 [+ or -] 76 [micro] [m.sup.3]; OVA, 1,310 [+ or -] 183 [micro][m.sup.3]). In OVA-treated mice, there was a 12-fold increase in the Vv of pGSK (+) ASM, a 5-fold increase in the Nv of pGSK (+) ASM, and a 1.6-fold increase in Vv/Nv. Lung homogenates from OVA-treated mice showed increased GSK-3[beta] phosphorylation and lower GSK-3[beta] activity. Both hyperplasia and hypertrophy are responsible for increased ASM mass in OVA-treated mice. Phosphorylation and inactivation of GSK-3[beta] are associated with ASM hypertrophy, suggesting that this kinase may play a role in asthmatic airway remodeling. ovalbumin; remodeling; stereology

Calponin contributes to the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated Mg-ATPase activity of phosphorylated myosin. Previous studies have shown that the contractile agonist acetylcholine induced a direct association of translocated calponin and PKC-[alpha] in the membrane. In the present study, we have determined the domain of PKC-[alpha] involved in direct association with calponin. In vitro binding assay was carried out by incubating glutathione S-transferase-calponin aa 92-229 with His-tagged proteins of individual domains and different combinations of domains of PKC-[alpha]. Calponin was found to bind directly to the full-length PKC-[alpha]. Calponin bound to C2 and C4 domains but not to C1 and C3 domains of PKC-[alpha]. When incubated with proteins of different combination of domains, calponin bound to C2-C3, C3-C4, and C2-C3-C4 but not to C1-C2 or C1-C2-C3. To determine whether these in vitro bindings mimic the in vivo associations, and in vivo binding assay was performed by transfecting colonic smooth muscle cells with Histagged proteins of individual domains and different combinations of domains of PKC-[alpha]. Coimmunoprecipitation of calponin with Histagged truncated forms of PKC-[alpha] showed that C1-C2, C1-C2-C3, C2-C3, and C3-C4 did not associate with calponin. Calponin associated only with full-length PKC-[alpha] and with C2-C3-C4 in cells in the resting state, and this association increased upon stimulation with acetylcholine. These data suggest that calponin bound to fragments that may mimic the active form of PKC-[alpha] and that the functional association of PKC-[alpha] with calponin requires both C2 and C4 domains during contraction of colonic smooth muscle cells. tropomyosin; smooth muscle; contraction; cytoskeleton; signaling

Reduced colonic motility has been observed in aged rats with a parallel reduction in acetylcholine (ACh)-induced myosin light chain (ML[C.sub.20]) phosphorylation. ML[C.sub.20] phosphorylation during smooth muscle contraction is maintained by a coordinated signal transduction cascade requiring both PKC-[alpha] and RhoA. Caveolae are membrane microdomains that permit rapid and efficient coordination of different signal transduction cascades leading to sustained smooth muscle contraction of the colon. Here, we show that normal physiological contraction can be reinstated in aged colonic smooth muscle cells (CSMCs) upon transfection with wild-type caveolin-1 through the activation of both the RhoA/Rho kinase and PKC pathways. Our data demonstrate that impaired contraction in aging is an outcome of altered membrane translocation of PKC-[alpha] and RhoA with a concomitant reduction in the association of these molecules with the caveolae-specific protein caveolin-1, resulting in a parallel decrease in the myosin phosphatase-targeting subunit (MYPT) and CPI-17 phosphorylation. Decreased MYPT and CPI-17 phosphorylation activates MLC phosphatase activity, resulting in ML[C.sub.20] dephosphorylation, which may be responsible for decreased colonic motility in aged rats. Importantly, transfection of CSMCs from aged rats with wild-type yellow fluorescent protein-caveolin-1 cDNA restored translocation of RhoA and PKC-[alpha] and phosphorylation of MYPT, CPI-17, and ML[C.sub.20], thereby restoring the contractile response to levels comparable with young adult rats. Thus, we propose that caveolin-1 gene transfer may represent a promising therapeutic treatment to correct the age-related decline in colonic smooth muscle motility. aging; myosin phosphorylation; RhoA; PKC doi:10.1152/ajpgi.00064.2007

Thin-filament regulation of smooth muscle contraction involves phosphorylation, association, and dissociation of contractile proteins in response to agonist stimulation. Phosphorylation of caldesmon weakens its association with actin leading to actomyosin interaction and contraction. Present data from colonic smooth muscle cells indicate that acetylcholine induced a significant association of caldesmon with PKC[alpha] and sustained phosphorylation of caldesmon at set789. Furthermore, acetylcholine induced significant and sustained increase in the association of phosphocaldesmon with heat-shock protein (HSP)27 with concomitant increase in the dissociation of phospho-caldesmon from tropomyosin. At the thin filament level, HSP27 plays a crucial role in acetylcholine-induced association of contractile proteins. Present data from colonic smooth muscle cells transfected with non-phospho-HSP27 mutant cDNA indicate that the absence of phospho-HSP27 inhibits acetylcholine-induced caldesmon phosphorylation. Our results further indicate that the presence of phospho-HSP27 significantly enhances acetylcholine-induced sustained association of phospho-caldesmon with HSP27 with a concomitant increase in acetylcholine-induced dissociation of phospho-caldesmon from tropomyosin. We thus propose a model whereby upon acetylcholine-induced phosphorylation of caldesmon at ser789, the association of phospho-caldesmon (ser789) with phospho-HSP27 results in an essential conformational change leading to dissociation of phospho-caldesmon from tropomyosin. This leads to the sliding of tropomyosin on actin thus exposing the myosin binding sites on actin for actomyosin interaction. acetylcholine; heat-shock protein; protein kinase C

Agonist-induced activation of the RhoA/Rho kinase (ROCK) pathway results in inhibition of myosin phosphatase and maintenance of myosin light chain ([MLC.sub.20]) phosphorylation. We have shown that RhoA/ROCKII translocates and associates with heat shock protein (HSP)27 in the particulate fraction. We hypothesize that inhibition of the 130-kDa regulatory myosin-binding subunit (MYPT) requires its association with HSP27 in the particulate fraction. Furthermore, it is not certain whether regulation of MYPT by CPI-17 or by ROCKII is due to cross talk between RhoA and PKC-[alpha]. Presently, we examined the cross talk between RhoA and PKC-[alpha] in the regulation of MYPT phosphorylation in rabbit colon smooth muscle cells. Acetylcholine induced 1) sustained phosphorylation of PKC-[alpha], CPI-17, and MYPT; 2) an increase in the association of phospho-MYPT with HSP27 in the particulate fraction; 3) a decrease in myosin phosphatase activity (66.21 [+ or -] 3.52 and 42.19 [+ or -] 3.85%nM/ml lysate at 30 s and 4 min); and 4) an increase in PKC activity (298.12 [+ or -] 46.60% and 290.59 [+ or -] 22.07% at 30 s and 4 min). Inhibition of RhoA/ROCKII by Y-27632 inhibited phosphorylation of MYPT and its association with HSP27. Both Y27632 and a negative dominant construct of RhoA inhibited phosphorylation of MYPT and CPI-17. Inhibition of PKCs or calphostin C or selective inhibition of PKC-[alpha] by negative dominant constructs inhibited phosphorylation of MYPT and CPI-17. The results suggest that 1) acetylcholine induces activation of both RhoA and/or PKC-[alpha] pathways, suggesting cross talk between RhoA and PKC-[alpha] resulting in phosphorylation of MYPT, inhibition of myosin phosphatase activity, and maintenance of MLC phosphorylation; and 2) phosphorylated MYPT is associated with HSP27 and translocated to the particulate fraction, suggesting a scaffolding role for HSP27 in mediating the association of the complex MYPT/RhoA-ROCKII. Thus both pathways (PKC and RhoA) converge on the regulation of myosin phosphatase activities and modulate sustained phosphorylation of [MLC.sub.20]. myosin light chain; contraction; CPI-17; acetylcholine; Rho kinase; colon; phosphatase; protein kinase C; heat shock protein

Previous studies performed at our laboratory have shown that agonist-induced contraction of smooth muscle is associated with translocation of protein kinase C (PKC)-[alpha] and RhoA to the membrane and that this interaction is due to a direct protein-protein interaction. To determine the domains of PKC-[alpha] involved in direct interaction with RhoA, His-tagged PKC-[alpha] proteins of individual domains and different combinations of PKC-[alpha] domains were used to perform in vitro binding assays with the fusion protein glutathione-S-transferase (GST)-RhoA. Coimmunoprecipitation was also performed using smooth muscle cells transfected with truncated forms of PKC-[alpha] in this study. The data indicate that RhoA directly bound to full-length PKC-[alpha], both in vitro (82.57 [+ or -] 15.26% above control) and in transfected cells. RhoA bound in vitro to the C1 domain of PKC-[alpha] [PKC-[alpha] (C1)] (70.48 [+ or -] 20.78% above control), PKC-[alpha] (C2) (72.26 [+ or -] 29.96% above control), and PKC-[alpha] (C4) (90.58 [+ or -] 26.79% above control), but not to PKC-[alpha] (C3) (0.64 [+ or -] 5.18% above control). RhoA bound in vitro and in transfected cells to truncated forms of PKC-[alpha], PKC-[alpha] (C2, C3, and C4), and PKC-[alpha] (C3 and C4) (94.09 [+ or -] 12.13% and 85.10 [+ or -] 16.16% above control, respectively), but not to PKC-[alpha] (C1, C2, and C3) or to PKC-[alpha] (C2 and C3) (0.47 [+ or -] 1.26% and 7.45 [+ or -] 10.76% above control, respectively). RhoA bound to PKC-[alpha] (C1 and C2) (60.78 [+ or -] 13.78% above control) only in vitro, but not in transfected cells, and PKC-[alpha] (C2, C3, and C4) and PKC-[alpha] (C3 and C4) bound well to RhoA. These data suggest that RhoA bound to fragments that may mimic the active form of PKC-[alpha]. The studies using cells transfected with truncated forms of PKC-[alpha] indicate that PKC-[alpha] (C1 and C2), PKC-[alpha] (C1, C2, and C3), and PKC-[alpha] (C2 and C3) did not associate with RhoA. Only full-length PKC-[alpha], PKC-[alpha] (C2, C3, and C4), and PKC-[alpha] (C3 and C4) associated with RhoA. The association increased upon stimulation with acetylcholine. These results suggest that the functional association of PKC-[alpha] with RhoA may require the C4 domain. domains; histidine; fusion proteins

Fecal incontinence affects people of all ages and social backgrounds and can have devastating psychological and economic consequences. This disorder is largely attributed to decreased mechanical efficiency of the internal anal sphincter (IAS), yet little is known about the pathophysiological mechanisms responsible for the malfunction of sphincteric smooth muscle at the cellular level. The object of this study was to develop a three-dimensional (3-D) physiological model of the IAS bioengineered in vitro from isolated smooth muscle cells. Smooth muscle cells isolated from the IAS of rabbits were seeded in culture on top of a loose fibrin gel, where they migrated and self-assembled in circumferential alignment. As the cells proliferated, the fibrin gel contracted around a 5-mm-diameter SYLGARD mold, resulting in a 3-D cylindrical ring of sphincteric tissue. We found that 1) the bioengineered IAS rings generated a spontaneous basal tone, 2) stimulation with 8-bromo-cAMP (8-Br-cAMP) caused a sustained decrease in the basal tone (relaxation) that was calcium-independent, 3) upon stimulation with ACh, bioengineered IAS rings showed a calcium- and concentration-dependent peak contraction at 30 s that was sustained for 4 min, 4) addition of 8-Br-cAMP induced rapid relaxation of ACh-induced contraction and force generation of IAS rings, and 5) bioengineered sphincter rings show striking functional differences when compared with bioengineered rings made from isolated colonic smooth muscle cells. This is the first report of a 3-D in vitro model of a gastrointestinal smooth muscle IAS. Bioengineered IAS rings demonstrate physiological functionality and may be used in the elucidation of the mechanisms causing sphincter malfunction. extracellular matrix; 8-bromo-adenosine 3',5'-cyclic monophosphate

Smooth muscle contraction regulated by myosin light chain phosphorylation is also regulated at the thin-filament level. Tropomyosin, a thin-filament regulatory protein, regulates contraction by modulating actin-myosin interactions. Present investigation shows that acetylcholine induces PKC-mediated and calcium-dependent phosphorylation of tropomyosin in colonic smooth muscle cells. Our data also shows that acetylcholine induces a significant and sustained increase in PKC-mediated association of tropomyosin with PKC[alpha] in the particulate fraction of colonic smooth muscle cells. Immunoblotting studies revealed that in colonic smooth muscle cells, there is no significant change in the amount of tropomyosin or actin in particulate fraction in response to acetylcholine, indicating that the increased association of tropomyosin with PKC[alpha] in the particulate fraction may be due to acetylcholine-induced translocation of PKC[alpha] to the particulate fraction. To investigate whether the association of PKC[alpha] with tropomyosin was due to a direct interaction, we performed in vitro direct binding assay. Tropomyosin cDNA amplified from colonic smooth muscle mRNA was expressed as GST-tropomyosin fusion protein. In vitro binding experiments using GST-tropomyosin and recombinant PKC[alpha] indicated direct interaction of tropomyosin with PKC[alpha]. PKC-mediated phosphorylation of tropomyosin and direct interaction of PKC[alpha] with tropomyosin suggest that tropomyosin could be a substrate for PKC. Phosphorylation of tropomyosin may aid in holding the slided tropomyosin away from myosin binding sites on actin, resulting in actomyosin interaction and sustained contraction. acetylcholine; fusion proteins

Direct association and translocation of PKC-[alpha] with calponin. Am J Physiol Gastrointest Liver Physiol 286:G954-G963, 2004. First published January 15, 2004; 10.l152/ajpgi.00477.2003.--Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgAT-Pase activity of phosphorylated myosin. Calponin has also been shown to interact with PKC. We have studied the interaction of calponin with PKC-[alpha] and with the low molecular weight heat-shock protein (HSP)27 in contraction of colonic smooth muscle cells. Particulate fractions from isolated smooth muscle cells were immunoprecipitated with antibodies to calponin and Western blot analyzed with antibodies to HSP27 and to PKC-[alpha]. Acetylcholine induced a sustained increase in the immunocomplexing of calponin with HSP27 and of calponin with PKC-[alpha] in the particulate fraction, indicating an association of the translocated proteins in the membrane. To examine whether the observed interaction in vivo is due to a direct interaction of calponin with PKC-[alpha], a cDNA of 1.3 kb of human calponin gene was PCR amplified. PCR product encoding 622 nt of calponin eDNA (nt 351-972 corresponding to amino acids 92-229) was expressed as fusion glutathione S-transferase (GST) protein in the vector pGEX-KT. We have studied the direct association of GST-calponin fusion protein with recombinant PKC-[alpha] in vitro. Western blot analysis of the fractions collected after elution with reduced glutathione buffer (pH 8.0) show a coelution of GST-calponin with PKC-[alpha], indicating a direct association of GST-calponin with PKC-[alpha]. These data suggest that there is a direct association of translocated calponin and PKC-[alpha] in the membrane and a role for the complex calponin-PKC-[alpha]-HSP27, in contraction of colonic smooth muscle cells. heat-shock protein 27; smooth muscle; contraction; cytoskeleton

Tropomyosin interacts with phosphorylated HSP27 in agonist-induced contraction of smooth muscle. Am J Physiol Cell Physiol 286: C1290-C1301, 2004. First published January 28, 2004; 10.1152/ajpcell.00458.2003.--Displacement of the contractile protein tropomyosin from actin filament exposes the myosin-binding sites on actin, resulting in actin-myosin interaction and muscle contraction. The objective of the present study was to better understand the interaction of tropomyosin with heat shock protein (HSP)27 in contraction of smooth muscle cells of the colon. We investigated the possibility of a direct protein-protein interaction of tropomyosin with HSP27 and the role of phosphorylated HSP27 in this interaction, Immunoprecipitation studies on rabbit smooth muscle cells indicate that upon acetylcholine-induced contraction tropomyosin shows increased association with HSP27 phosphorylated at Ser82 and Ser78. Transfection of smooth muscle cells with HSP27 phosphorylation mutants indicated that the association of tropomyosin with HSP27 could be affected by HSP27 phosphorylation. In vitro binding studies with glutathione S-transferase (GST)tagged HSP27 mutant proteins show that tropomyosin has greater direct interaction to phosphomimic HSP27 mutant compared with wild-type and nonphosphomimic HSP27. Our data suggest that, in response to a contractile agonist, HSP27 undergoes a rapid phosphorylation that may strengthen its interaction with tropomyosin. acetylcholine; fusion proteins; serine

IGF-I is a potent fibrogenic growth factor that stimulates proliferation of intestinal smooth muscle cells and increases synthesis of collagen and IGF-l-binding proteins by the cells. These processes contribute to intestinal fibrosis that develops in patients with Crohn's disease and in Lewis-strain rats with experimental Crohn's disease. The aim of this study was to determine which early docking proteins are associated with IGF-I receptor signal transduction and which transduction pathway is involved in IGF-I-mediated gene regulation in intestinal smooth muscle cells. Primary cultures of smooth muscle cells isolated from the muscularis externa of the distal colon of Lewis rats were treated with IGF-I (100 ng/ml). Immunoprecipitation studies demonstrated that IGF-I stimulation resulted in tyrosine phosphorylation of IRS-1, IRS-2, and Shc. Coimmunoprecipitation demonstrated a close association between the IGF-I receptor and these three early docking proteins. Concurrent treatment with the MAPK inhibitor PD98059 (10 [micro]M) resulted in an inhibition of the IGF-I-mediated increase in IGFBP-5 and collagen [[alpha].sub.1](I) mRNAs, while concurrent treatment with the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin (100 nM) had no effect. In additional experiments, cells were transiently transfected with adenoviral vectors dominantly expressing inactive mutant Akt or constitutively expressing wild-type Akt. In both cases, the IGF-I-mediated increase in collagen I protein did not differ from that observed in control cultures that had been transfected with an adenoviral vector carrying the LacZ reporter gene. These results suggest that the MAPK pathway is key to IGF-I-mediated gene regulation in intestinal smooth muscle cells, whereas data do not suggest a role for the Akt-dependent pathway in our system. insulin-like growth factor I; signal transduction; Crohn's disease; inflammatory bowel disease

Patil, Suresh B., Mercy D. Pawar, and Khalil N. Bitar. Phosphorylated HSP27 essential for acetylcholine-induced association of RhoA with PKC[alpha]. Am J Physiol Gastrointest Liver Physiol 286: G635-G644, 2004. First published October 30, 2003; 10.1152/ajpgi.00261.2003.--Reorganization of the cytoskeleton and association of contractile proteins are important steps in modulating smooth muscle contraction. Heat shock protein (HSP) 27 has significant effects on actin cytoskeletal reorganization during smooth muscle contraction. We investigated the role of phosphorylated HSP27 in modulating acetylcholine-induced sustained contraction of smooth muscle cells from the rabbit colon by transfecting smooth muscle cells with phosphomimic (3D) or nonphosphomimic (3G) HSP27. In 3G cells, the initial peak contractile response at 30 s was inhibited by 25% (24.0 [+ or -] 4.5% decrease in cell length, n = 4). The sustained contraction was greatly inhibited by 75% [9.3 [+ or -] .9% decreases in cell length (n = 4)]. Furthermore, in 3D cells, translocation of both PKC[alpha] and of RhoA was greatly enhanced and resulted in a greater association of PKC[alpha]-RhoA in the membrane fraction. In 3G transfected cells, PKC[alpha] and RhoA failed to translocate in response to stimulation with acetylcholine, resulting in an inhibition of association of PKC[alpha]-RhoA in the membrane fraction. Studies using GST-RhoA fusion protein indicate that there is a direct association of RhoA with PKC[alpha] and with HSP27. The results suggest that phosphorylated HSP27 plays a crucial role in the maintenance of association of PKC[alpha]-RhoA in the membrane fraction and in the maintenance of acetylcholine-induced sustained contraction. phosphorylation; cytoskeleton

Esophageal (ESO) circular muscle contraction and lower esophageal sphincter (LES) tone are PKC dependent. Because MAPKs may be involved in PKC-dependent contraction, we examined ERK1/ERK2 and p38 MAPKs in ESO and LES. In permeabilized LES muscle cells, ERK1/2 antibodies reduced 1,2-dioctanoylglycerol (DG)- and threshold ACh-induced contraction, which are PKC dependent, but not maximal ACh, which is calmodulin dependent. LES tone was reduced by the ERK1/2 kinase inhibitor PD-98059 and by the p38 MAPK inhibitor SB203580. In permeable ESO cells, ACh contraction was reduced by ERK1/ERK2 and p38 MAPK antibodies and by PD-98059 and SB-203580. ACh increased MAPK activity and phosphorylation of MAPK and of p38 MAPK. The 27-kDa heat shock protein (HSP27) antibodies reduced ACh contraction. HSP27 and p38 MAPK antibodies together caused no greater inhibition than either one alone, p38 MAPK and HSP27 coprecipitated after ACh stimulation, suggesting that HSP27 is linked to p38 MAPK. These data suggest that PKC-dependent contraction in ESO and LES is mediated by the following two distinct MAPK pathways: ERK1/2 and HSP27-1inked p38 MAPK. second messenger system; signal transduction; mitogen-activated protein kinase; protein kinase C; lower esophageal sphincter

The object of this theme is to offer new perspectives on the effect of aging on signal-transduction pathways associated with agonist-induced contraction of smooth muscle cells from the colon. Smooth muscle cells from old rats (32 mo old) exhibit limited cell length distribution and diminished contractility. The observed reduced contractile response may be due to the effect of aging on signal-transduction pathways, especially an inhibition of the tyrosine kinase-Src kinase pathway, a reduced activation of the PKC pathway, and a reduced association of contractile proteins [heat shock protein 27 (HSP27)-tropomyosin, HSP27-actin, actin-myosin]. Levels of HSP27 phosphorylation are also reduced compared with adult rats. heat shock protein 27; actin; tropomyosin; myosin; phosphatidylinositol 3-kinase; pp[60.sup.src] kinase

We have investigated the role of heat shock protein 27 (HSP27) phosphorylation and the association of HSP27 with contractile proteins actin, myosin, and tropomyosin. Smooth muscle cells were labeled with [[sup.32]P]orthophosphate. C2-ceramide (0.1 [micro]M), an activator of protein kinase C (PKC), induced a sustained increase in HSP27 phosphorylation that was inhibited by calphostin C. C2-ceramide-induced (0.1 [micro]M) sustained colonic smooth muscle cell contraction was accompanied by significant increases in the association of HSP27 with tropomyosin and in the association of HSP27 with actin. The significant increases occurred at 30 s after stimulation and were sustained at 4 min. Contraction was also associated with strong colocalization of HSP27 with tropomyosin and with actin as observed after immunofluorescent labeling of tropomyosin, actin, and HSP27 followed by confocal microscopy. Transfection of smooth muscle cells with HSP27 phosphorylation mutants indicated that phosphorylation of HSP27 could affect myosin association with actin. In conclusion 1) HSP27 phosphorylation appears to be necessary for reorganization of HSP27 inside the cell and seems to be directly correlated with the PKC signal transduction pathway, and 2) agonist-induced phosphorylation of HSP27 modulates actin-myosin interaction through thin-filament regulation of tropomyosin. heat shock protein; tropomyosin; protein kinase C; mitogen-activated protein kinase

The intracellular pathways involved in the activation of the small GTP-binding protein Rho p21 in rat pancreatic acini have been investigated. Intact pancreatic acini were incubated in the presence or absence of cholecystokinin and carbachol, and Triton X-100-soluble and crude microsomes were utilized for Western immunoblotting. Results show that the small GTP-binding protein RhoA p21 is present in pancreatic acini and may be involved in the mediation of pancreatic enzyme secretion induced by cholecystokinin and carbachol.

The role of endothelin and ceramide in the differential activation of phosphoinositide 3-kinase in colonic smooth muscle cells has been investigated. Endothelin was found to induce a sustained rise in phosphoinositide 3-kinase activity at both 30 seconds and four minutes of stimulation. Preincubation of smooth muscle cells with the tyrosine kinase inhibitor genistein, on the other hand, significantly restricted both C(sub 2) ceramide-induced and endothelin-induced phosphoinositide 3-kinase activation and contraction.

A study was conducted to determine how the ras-related protein Rho-A regulates agonist-induced signal transduction cascades in smooth muscle contraction and how it regulates actin binding proteins. Results indicate that Rho A is found in smooth muscle cells and that it is activated during ceramide- and endothelin-induced phosphoinositide 3-kinase-mediated sustaine contraction. Also, Rho may control muscle cell contraction through various pathways via reorganization of the actin binding protein, 27-kDa heat shock protein.

The effects of C2 ceramide on Src kinase activity were analyzed. Ceramide-induced smooth muscle contraction was calcium dependent, in which the sustained contraction induced by ceramide is inhibited in the absence of extracellular calcium. Ceramide seemed to activate a nonreceptor tyrosine kinase pathway through the activation of pp060(supra src) and phosphoinositide 3-kinase. Ceramide activation of Src was also found to be calcium dependent.

A study was conducted to examine the origin of lipid sources of diacylglycerol production in rabbit smooth muscle cells based on contraction induced by peptide agonists. The animals' internal anal sphincter was removed by sharp dissection while smooth muscle cells were mixed with aliquots in glass tubes. Results indicated that bombesin-induced contraction by activation of phospholiase C led to the hydrolysis of various phospholipids.