Biomedical subjects

K N Bitar

Publications and source records attributed to K N Bitar.

At least 19 recordsLinked to original sources

PubMed2002

HSP27 modulates agonist-induced association of translocated RhoA and PKC-alpha in muscle cells of the colon.

The recruitment of signal transduction molecules to the membrane is crucial for the efficient coupling of extracellular signals and contractile response. The trafficking is dynamic. We have investigated a possible cross talk between agonist-induced association of translocated RhoA and translocated protein kinase C-alpha (PKC-alpha) and a role for heat shock protein 27 (HSP27) in mediating this interaction. Immunoprecipitation with HSP27 monoclonal antibody followed by immunoblotting with either RhoA antibody or PKC-alpha antibody indicated that acetylcholine induced associations of HSP27-RhoA and HSP27-PKC-alpha in the membrane fraction but not in the cytosolic fraction. Immunoprecipitation with anti-RhoA monoclonal antibody followed by immunoblotting with PKC-alpha antibody indicated that acetylcholine induced a significant complexing of RhoA-PKC-alpha in the membrane fraction but not in the cytosolic fraction. In summary, the data indicate that agonist-induced contraction is associated with 1) association of translocated RhoA with HSP27 on the membrane, 2) association of translocated PKC-alpha with HSP27 on the membrane, and 3) association of PKC-alpha with RhoA on the membrane. The data suggest an important role for HSP27 in modulating a multiprotein complex that includes translocated RhoA and PKC-alpha.

Acetylcholine↗

PubMed1999

Differential activation of phosphoinositide 3-kinase by endothelin and ceramide in colonic smooth muscle cells.

We have investigated the hypothesis that different contractile agonists activate distinct catalytic subunits of phosphoinositide (PI) 3-kinase in smooth muscle cells. Endothelin (10(-7) M) induced a sustained increase in PI 3-kinase activity at both 30 s and 4 min of stimulation (151.5 +/- 8.5% at 30 s and 175.8 +/- 8.7% at 4 min, P < 0.005). Preincubation of smooth muscle cells with the tyrosine kinase inhibitor genistein (3 microM) resulted in a significant inhibition of both C2 ceramide-induced and endothelin-induced PI 3-kinase activation and contraction. Preincubation with herbimycin A, an Src kinase inhibitor (3 microM), inhibited only C2 ceramide-induced PI 3-kinase activation and contraction. Western blotting using Src kinase antibody showed that C2 ceramide, not endothelin, stimulated the phosphorylation of Src kinase. Western blotting and immunoprecipitation with PI 3-kinase antibodies to the regulatory subunit p85 and the catalytic subunits p110alpha and p110gamma indicated that both endothelin and C2 ceramide interacted with the regulatory subunit p85; endothelin interacted with the catalytic subunits p110alpha and p110gamma, whereas C2 ceramide interacted only with the catalytic subunit p110alpha. In summary, C2 ceramide activated PI 3-kinase p110alpha subunit by a tyrosine kinase-mediated pathway, whereas endothelin-induced contraction, unlike C2 ceramide, was not mediated by the activation of Src kinase but was mediated by G protein activation of both p110alpha and p110gamma subunits (type IA and IB) of PI 3-kinase.

Animals↗

PubMed1999

Involvement of RhoA and its interaction with protein kinase C and Src in CCK-stimulated pancreatic acini.

We evaluated intracellular pathways responsible for the activation of the small GTP-binding protein Rho p21 in rat pancreatic acini. Intact acini were incubated with or without CCK and carbachol, and Triton X-100-soluble and crude microsomes were used for Western immunoblotting. When a RhoA-specific antibody was used, a single band at the location of 21 kDa was detected. CCK (10 pM-10 nM) and carbachol (0.1-100 microM) dose dependently increased the amount of immunodetectable RhoA with a peak increase occurring at 3 min. High-affinity CCK-A-receptor agonists JMV-180 and CCK-OPE (1-1,000 nM) did not increase the intensities of the RhoA band, suggesting that stimulation of RhoA is mediated by the low-affinity CCK-A receptor. Although an increase in RhoA did not require the presence of extracellular Ca2+, the intracellular Ca2+ chelator 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM abolished the appearance of the RhoA band in response to CCK and carbachol. The Gq protein inhibitor G protein antagonist-2A (10 microM) and the phospholipase C (PLC) inhibitor U-73122 (10 microM) markedly reduced RhoA bands in response to CCK. The protein kinase C (PKC) activator phorbol ester (10-1,000 nM) dose dependently increased the intensities of the RhoA band, which were inhibited by the PKC inhibitor K-252a (1 microM). The pp60(c-src) inhibitor herbimycin A (6 microM) inhibited the RhoA band in response to CCK, whereas the calmodulin inhibitor W-7 (100 microM) and the phosphoinositide 3-kinase inhibitor wortmannin (6 microM) had no effect. RhoA was immunoprecipitated with Src, suggesting association of RhoA with Src. Increases in mass of this complex were observed with CCK stimulation. In permeabilized acini, the Rho inhibitor Clostridium botulinum C3 exoenzyme dose dependently inhibited amylase secretion evoked by a Ca2+ concentration with an IC50 of C3 exoenzyme at 1 ng/ml. We concluded that the small GTP-binding protein RhoA p21 exists in pancreatic acini and appears to be involved in the mediation of pancreatic enzyme secretion evoked by CCK and carbachol. RhoA pathways are involved in the activation of PKC and Src cascades via Gq protein and PLC.

ADP Ribose Transferases↗

PubMed1999

HSP27 in signal transduction and association with contractile proteins in smooth muscle cells.

Sustained smooth muscle contraction is mediated by protein kinase C (PKC) through a signal transduction cascade leading to contraction. Heat-shock protein 27 (HSP27) appears to be the link between these two major events, i.e., signal transduction and sustained smooth muscle contraction. We have investigated the involvement of HSP27 in signal transduction and HSP27 association with contractile proteins (e.g., actin, myosin, tropomyosin, and caldesmon) resulting in sustained smooth muscle contraction. We have carried out confocal microscopy to investigate the cellular reorganization and colocalization of proteins and immunoprecipitation of HSP27 with actin, myosin, tropomyosin, and caldesmon as detected by sequential immunoblotting. Our results indicate that 1) translocation of Raf-1 to the membrane when stimulated with ceramide is inhibited by vasoactive intestinal peptide (VIP), a relaxant neuropeptide; 2) PKC-alpha and mitogen-activated protein kinase translocate and colocalize on the membrane in response to ceramide, and PKC-alpha translocation is inhibited by VIP; 3) HSP27 colocalizes with actin when contraction occurs; and 4) HSP27 immunoprecipitates with actin and with the contractile proteins myosin, tropomyosin, and caldesmon. We propose a model in which HSP27 is involved in sustained smooth muscle contraction and modulates the interaction of actin, myosin, tropomyosin, and caldesmon.

Actins↗

PubMed1998

Origin of molecular species of diacylglycerol induced by bombesin in smooth muscle cells from rabbit rectosigmoid.

The source of early production of sn-1,2-diacylglycerol (DAG) has for a long time been exclusively linked to hydrolysis of phosphatidylinositol 4,5-diphosphate, which on receptor activation is hydrolyzed into DAG and inositol 1,4,5-trisphosphate. We have investigated the origin of lipid sources of DAG production in smooth muscle cells, in response to contraction induced by peptide agonists. We have performed a quantitative analysis of the molecular species of DAG formed in relation to the known molecular composition of parent phospholipids. The molecular species of phospholipids are sufficiently unique that the phospholipid origin of DAGs and its quantitative contribution to their formation can be measured by HPLC. Cell suspensions (10-15 x 10(6) cells/ml) from the circular muscle of rabbit rectosigmoid were incubated in the presence of the contractile peptide agonist bombesin (BB) at 10(-6) M. Reactions were stopped at different time intervals from 30 s to 4 min. DAGs were extracted, purified by TLC, and benzoylated with benzoic anhydride. The benzoylated DAGs were first purified by TLC and then by normal phase HPLC before they were injected onto a reverse-phase column and eluted isocratically. Furthermore, phospholipids in the lipid extract [phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE)] were purified by TLC and similarly analyzed after hydrolysis to DAGs with phospholipase C (PLC). The DAG molecular species profiles for PI, PC, PS, and PE were all unique. Contraction of cells with BB gave noticeable increases (17-55%) in newly formed DAGs. The major phospholipid source of the newly formed DAGs at 30 s was only approximately 30% from PI, and the remainder was from PC. In contrast, after 4 min of BB stimulation, a decrease was seen in newly formed DAGs in the peak specific for PI hydrolysis. The data suggest that BB-induced contraction by activation of PLCs results in hydrolysis of different phospholipids. The DAGs formed as a result are qualitatively and quantitatively distinct. This could be the basis for the kinetically different pattern of sustained contraction observed with BB.

Animals↗

PubMed1998

Src kinase and PI 3-kinase as a transduction pathway in ceramide-induced contraction of colonic smooth muscle.

Ceramide mediates sustained contraction of smooth muscle cells. C2 ceramide induced a rapid increase in Src kinase activity within 15 s, peaked at 1 min, and was sustained up to 8 min. Contraction and Src kinase activity were inhibited in cells incubated in Ca2+-free medium containing 2 mM EGTA and in cells preincubated with herbimycin A, a Src kinase inhibitor. Immunoblotting using a phosphospecific anti-Src (416Y) antibody showed a ceramide-induced increase in pp60(src) tyrosine phosphorylation. Immunoprecipitation using an anti-phosphotyrosine antibody followed by Western immunoblotting using a monoclonal IgG anti-phosphoinositide 3-kinase NH2 terminal-SH2 domain antibody showed a ceramide-induced increase in phosphoinositide 3-kinase (PI 3-kinase) tyrosine phosphorylation at a protein mass corresponding to 85 kDa, the regulatory subunit of PI 3-kinase, which contains the Src kinase binding site. PI 3-kinase phosphorylation was inhibited by herbimycin A and by the PI 3-kinase inhibitors wortmannin and LY-294002. Preincubation of cells with herbimycin A or PI 3-kinase inhibitors also resulted in an inhibition of mitogen-activated protein (MAP) kinase p42 and p44 activities as seen on Western blots. In summary, we found that 1) the maintenance of sustained contraction is dependent on extracellular Ca2+; 2) ceramide activates a nonreceptor tyrosine kinase pathway through activation of pp60(src) and PI 3-kinase; and 3) the converging signals are probably through activation of MAP kinase.

Amino Acid Sequence↗

PubMed1998

Rho A regulates sustained smooth muscle contraction through cytoskeletal reorganization of HSP27.

The ras-related protein Rho p21 regulates various actin-dependent functions, including smooth muscle contraction. However, the precise mechanism of action of Rho p21 is still not clear. We report here that Rho A is a key regulator of agonist-induced contractile effects in rabbit colonic smooth muscle. Endothelin-1 and C2 ceramide were used. Both seem to activate phosphoinositide 3-kinase (PI 3-kinase) through G protein and pp60(src), respectively. Immunoprecipitation and immunoblotting revealed one form of 21-kDa Rho A that translocated from the cytosol to the membrane in response to stimulation by either endothelin (10(-7) M) or ceramide (10(-7) M) ( approximately 30% increase at 30 s that was sustained at 4 min). The translocation of Rho A to the membrane was confirmed by immunostaining. The translocation of Rho A was inhibited by Clostridium botulinum C3 exoenzyme, which ADP ribosylated Rho A, but was not inhibited by the pp60(src) inhibitor herbimycin A or by the protein kinase C (PKC) inhibitor calphostin C, suggesting that Rho A may be upstream of pp60(src) and PKC or may belong to a different pathway than these proteins. Both ceramide- and endothelin-induced PI 3-kinase activation was inhibited by C3 exoenzyme pretreatment. However, the C3 exoenzyme inhibited endothelin- but not ceramide-induced mitogen-activated protein kinase phosphorylation, indicating that Rho regulates ceramide- and endothelin-induced contraction through different pathways. Furthermore, the dominant negative form of Rho (N19Rho) inhibited the actin binding protein, 27-kDa heat shock protein (HSP27), reorganization in response to ceramide and endothelin observed under confocal microscopy.

ADP Ribose Transferases↗

PubMed1998

Access to intracellular calcium during development in the feline gastric antrum.

Circular smooth muscle cells from the feline newborn antrum, unlike the adult, are unable to respond to myogenic agonists in the absence of extracellular calcium or to exogenous inositol 1,4,5-trisphosphate (IP3). This study examined the reasons behind the relative inaccessibility of intracellular calcium stores in the newborn period. IP3 binding was determined in antral smooth muscle homogenates from adult cats and newborns by evaluating the competitive binding of D-myo-[3H]IP3 and unlabeled IP3. Receptor density (Bmax) (fmol/mg of protein) and binding affinity (Kd) were determined. The Kd was similar in adults (31 +/- 4 nM) and newborns (28 +/- 7 nM); however, the Bmax was markedly decreased in the newborn (647 +/- 181.0 fmol/mg) compared with the adult (1755 +/- 275 fmol/mg). In adult and newborn antral cells, thapsigargin, which causes a net release of Ca2+ from intracellular stores by inhibiting Ca(2+)-ATPase-dependent reuptake activity, caused an early contraction at 30 s that was maintained for at least 20 min. We conclude that, in the newborn, dynamic intracellular calcium stores are present in the smooth muscle of the feline antrum and that differences in accessibility of intracellular calcium stores may be related to changes in the release of calcium from IP3-sensitive stores.

Animals↗

PubMed1997

Signal transduction pathways associated with contraction during development of the feline gastric antrum.

BACKGROUND & AIMS: Unlike adult antral cells, feline newborn antral cells are unable to contract in response to agonists in the absence of extracellular calcium or in response to exogenous inositol 1,4,5-triphosphate (IP3) after permeabilization. Changes in intracellular pathways that are associated with these differences were examined. METHODS: In adult and kitten antrum isolated smooth muscle cell contraction, levels of 1,2-diacylglycerol (DAG) and IP3 were assessed in response to cholecystokinin (CCK). RESULTS: CCK-induced contraction was transient in the adult and sustained in the kitten. U73122 blocked contraction in adult antral cells but not kitten antral cells. In adult antral tissue, CCK (10(-7) mol/L) caused an early transient increase in the level of DAG, whereas in the newborn antrum, CCK (10(-7) mol/L) caused a sustained increase in the DAG level for up to 4 minutes. IP3 showed an early increase in both age groups. Newborn contraction is associated with an initial increase in IP3 and sustained elevation of DAG levels, whereas in adult antral cells, there is a transient increase in both IP3 and DAG. CONCLUSIONS: The relative inaccessibility of intracellular calcium stores in the newborn is associated with age-related differences in signal transduction pathways.

Aging↗

PubMed1997

Bombesin-stimulated ceramide production and MAP kinase activation in rabbit rectosigmoid smooth muscle cells.

We have investigated the hypotheses that 1) bombesin activation of protein kinase C (PKC) results in the hydrolysis of sphingolipids and the production of ceramide and that 2) ceramide produced on activation by bombesin mediates sustained contraction of smooth muscle cells by activation of PKC and mitogen-activated protein (MAP) kinase. Ceramide production was assessed using a technique that involved benzoylation of purified ceramide extracts, followed by reverse-phase high-performance liquid chromatography. Contraction of smooth muscle cells isolated from the rabbit rectosigmoid and stimulated with bombesin gave a significant increase in newly formed ceramide (38 +/- 3.5%). 12-O-tetradecanoylphorbol-13-acetate also induced production of ceramide, which was blocked by calphostin C. The short-chain permeable C2 ceramide induced a sustained contraction and activation of MAP kinase, which was blocked by calphostin C. The increase in MAP kinase activity was maximal at 30 s and declined at 2 min. The data suggest that stimulation of smooth muscle cells by bombesin results in a functional coupling between sn-1,2-diacylglycerol (DAG)/ PKC and a sphingomyelinase, whereby DAG activates the hydrolysis of sphingomyelin to produce ceramide. Ceramide in turn activates PKC, which then activates MAP kinase. This could be the basis for the sustained contraction observed with bombesin.

Anal Canal↗

PubMed1997

IGF-I induces collagen and IGFBP-5 mRNA in rat intestinal smooth muscle.

Insulin-like growth factor (IGF) binding protein 5 (IGFBP-5) mRNA was studied in intestines of rats with peptidoglycan-polysaccharide enterocolitis by Northern analysis and in situ hybridization. IGFBP-5 mRNA was increased 2.4 +/- 0.5-fold in inflamed rat colon compared with controls and was highly expressed in smooth muscle. Cultured rat intestinal smooth muscle cells were used to study the regulation of IGFBP-5 and type I collagen synthesis. IGF-I (100 ng/ml) increased IGFBP-5 mRNA (1.9 +/- 0.1-fold) and collagen type alpha1(I) mRNA (1.6 +/- 0.2-fold) in cultured smooth muscle cells. IGF-I induced a dose- and time-dependent increase in IGFBP-5 in conditioned medium by Western ligand blot and by immunoblot. IGF-I did not affect the IGFBP-5 mRNA decay rate after transcriptional blockade. Cycloheximide abolished IGFBP-5 mRNA. In conclusion, IGFBP-5 mRNA is expressed by intestinal smooth muscle and is increased during chronic inflammation. IGF-I increases IGFBP-5 and collagen mRNAs in intestinal smooth muscle cells.

Animals↗

PubMed1996

Protein kinase C mediates spontaneous tone in the cat lower esophageal sphincter.

The intracellular pathways responsible for maintenance of tone in the lower esophageal sphincter (LES) are not well understood. We show that the protein kinase C (PKC) antagonists (1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride) and calphostin C reduce spontaneous resting tone in LES muscle strips, whereas the calmodulin antagonist N-(6-aminohexyl-5-chloro-1-naphthalenesulfonamide hydrochloride) has no effect, which suggests that LES tone is maintained by a PKC-mediated mechanism. In addition, U73122, an inhibitor of phosphatidylinositol-4,5-bisphosphate (PIP2)-specific phospholipase C, and D609, an inhibitor of phosphatidylcholine-specific phospholipase C, reduced diacylglycerol formation and LES tone in a concentration-dependent manner. Finally diacylglycerol levels and PKC activity were reduced during relaxation of the LES induced by the inhibitory neurotransmitter vasoactive intestinal peptide. These data suggest that resting LES tone is associated with elevated diacylglycerol levels and PKC activity, which are reduced during relaxation. Diacylglycerol is derived from at least two different sources. Hydrolysis of PIP2 by PIP2-specific phospholipase C produces equimolar amounts of inositol 1,4,5-triphosphate and diacylglycerol, which may interact synergistically to activate PKC and develop tone. Furthermore, PKC-mediated contraction may be augmented by additional diacylglycerol production arising from the hydrolysis of phosphatidylcholine by phosphatidylcholine-specific phospholipase C.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1996

Somatostatin inhibits bombesin-stimulated Gi-protein via its own receptor in rabbit colonic smooth muscle cells.

The effect of somatostatin on Bombesin-induced contraction of isolated rabbit colonic smooth muscle cells was examined. Preincubation of muscle cells with somatostatin 10(-6) M inhibited bombesin-induced contraction. To characterize somatostatin receptors, muscle cells (10(5) cells/tube) were incubated at 24 degrees C with 125I-Tyr0-SS-28. Binding reached a plateau at 60 sec and was reversible by addition of excess synthetic SS-28. Scatchard analysis revealed high and low affinity bindings sites (Ka = 0.48 +/- 0.01 and 40 +/- 13 (nM +/- S.E.), 1830 +/- 433 and 65820 +/- 13183 receptors/cell +/- S.E.). Inhibition of 125I-Tyr0-SS-28 binding was possible with biologically active analogs of somatostatin, indicating the specificity of the receptors to somatostatin. Binding of 125I-Tyr0-SS-28 was inhibited by GTP gamma s, a nonhydrolysable analog of guanosine 5'-triphosphate, whereas adenosine 5'-triphosphate at a high concentration (100 microM) slightly inhibited the binding. Further, pretreatment of muscle cells with pertussis toxin at 37 degrees C abolished binding of 125I-Tyr0-SS-28, although pretreatment of cells with cholera toxin had no effect. Inasmuch as Gi protein is postulated as a signal protein, muscle cells were labeled with 3H-methionine, before stimulation with Bombesin (10(-6) M), in the presence and absence of somatostatin (10(-6) M). The cells were then lysed and Gi was precipitated by a Gi specific antibody. Gi synthesis was stimulated by bombesin at 60 sec and somatostatin inhibited it (6114 +/- 986 vs. 2998 +/- 841 cpm +/- S.E., P < .05). These data suggest that colonic smooth muscle cells contain specific receptor for somatostatin-28 and that somatostatin reverses bombesin-induced contraction regulated by Gi-type G protein.

Animals↗

PubMed1995

Phospholipase A2-activating peptide-induced contraction of smooth muscle is mediated by protein kinase C--MAP kinase cascade.

The mammalian phospholipase A2-activating protein (PLAP) affects of smooth muscle cells isolated from the rabbit rectosigmoid. PLAP (10(6) M)-induced contraction peaked at 30 sec and was sustained at 4 min. MAP kinase was activated by PLAP (10(-6) M), as measured using myelin basic protein (MBP) as substrate. The increase in MAP kinase activity was rapid at 30 sec (159 +/- 2.5%) and remained at a sustained level (162 +/- 7.9%) at 4 min. Preincubation of the cells with the PLA2 inhibitor ONO-RS-082 (10(-6) M) or with the PKC inhibitor calphostin C (10(-6) M) resulted in inhibition of contraction, as well as inhibition of the associated increase in MAP kinase activation. The data indicates that PLAP-specific contractile effect on isolated smooth muscle cells is mediated by an activation of a PKC-MAP kinase cascade and suggests a putative role for PLA2-coupled G protein activation of PKC-MAP kinase as an alternate transduction pathway in smooth muscle contraction.

Animals↗

PubMed1995

Antiserum to tumor necrosis factor and failure to prevent murine colitis.

Cytokines regulate many aspects of disease and have been implicated as mediators of the inflammatory reactions in patients with both ulcerative (UC) and Crohn's colitis. We examined the local and systemic appearance of tumor necrosis factor (TNF) and interleukin 6 (IL-6) in an experimental animal model of inflammatory bowel disease. Colitis was induced in CBA/J mice by adding dextran sulfate sodium (DSS), 5% (wt/vol), to their water. DSS-induced colitis is a reproducible animal model for evaluating the role of cytokines in the pathology of colitis. Animals were weighed daily, and stools were checked for the presence of blood. Groups of mice were killed daily, blood samples were taken for measurement of plasma cytokine levels, and colonic samples were taken for histology and measurement of TNF and IL-6 bioactivity. Mice fed DSS developed colitis with bloody diarrhea, weight loss, and colonic inflammation by days 5-9. Histologic examination of the colons showed focal crypt destruction and ulceration. In mice with DSS-induced colitis no TNF was detectable in colonic tissue extracts or in plasma. In contrast, plasma IL-6 was detectable from days 4 to 9 and was detectable in colonic tissue in only a few (two of four) terminally ill animals on day 9. Animals were injected with a neutralizing, polyclonal anti-TNF antiserum that maintained high in vivo neutralizing titers for > or = 48 h. This anti-TNF antiserum failed to block or modify the severity of colitis induced by DSS. Failure to detect local or systemic TNF and failure to prevent colonic inflammation with anti-TNF antiserum showed that TNF is not an inflammatory mediator in DSS-induced murine colitis.

Animals↗

PubMed1995

Modulation of smooth muscle contraction by sphingosylphosphorylcholine.

We have investigated the effect of sphingosylphosphorylcholine (SPC), a synthetic product that was implicated in the sphingomyelin cycle, and have assessed its role in intracellular signaling. SPC induced a dose-dependent contractile effect of smooth muscle cells isolated from the rectosigmoid of the rabbit. Maximal contraction occurred at 10(-6) M. The response started early, 25.4 +/- 6% shortening at 15 s, peaked at 30 s (32.5 +/- 2%), and remained sustained at 8 min (30.0 +/- 3.5%). Preincubation of the cells with thapsigargin had no effect on contraction induced by SPC. The response to a combination of threshold concentrations of inositol 1,4,5-trisphosphate (IP3) and SPC was additive and was significantly different from the maximal response elicited by each agent alone. SPC also induced activation of mitogen-activated protein kinase (MAP kinase). This study demonstrates that SPC is important in cellular signaling of gastrointestinal smooth muscle cells through a mechanism that is independent of IP3-sensitive calcium release and probably through activation of a protein kinase C-mediated activation of MAP kinase.

Animals↗

PubMed1995

Activation of MAP kinase and translocation with HSP27 in bombesin-induced contraction of rectosigmoid smooth muscle.

We have investigated whether mitogen-activated protein (MAP) kinase cascade is essential for sustained contraction of smooth muscle cells of the rabbit rectosigmoid. We have identified MAP kinase as one of the enzymes activated by bombesin, performed immunologic studies blocking the activation of MAP kinase, and conducted confocal localization of MAP kinase in relation to heat-shock protein (HSP27), postulated to be involved in the sustained contraction of smooth muscle. Immunoblotting revealed two forms of MAP kinase (42 and 44 kDa). Activation of MAP kinase by bombesin was rapid, reaching a maximum in 30 s and subsequently declining. [D-Phe6,Leu13,psi(CH2NH),Phe14]BN-(6-14), a potent bombesin antagonist, and protein kinase C (PKC) inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, calphostin C, and chelerythrine inhibited the increase in MAP kinase induced by bombesin. Immunofluorescent dual labeling and confocal microscopy indicate that these two proteins are closely distributed in resting cells and that during bombesin-induced contraction MAP kinase translocates accompanied by HSP27. In conclusion, a series of events involving PKC activation, MAP kinase activation, and MAP kinase-HSP27 translocation could be the signaling pathway involved in bombesin-induced sustained contraction.

Animals↗

PubMed1995

Characterization of actin and myosin in the developing stomach.

During growth and development, dietary intake changes from being predominantly liquid in the newborn period to mixed solid liquid meals. These alterations in diet vary the functional demands placed on the stomach. It has been shown that, during development, smooth muscle of the stomach undergoes changes in the mechanism responsible for the contractile process. In this study, we have investigated the possibility that there are structural changes in two of the major proteins that are responsible for generation of force during smooth muscle contraction: actin and myosin. Actin and myosin were identified in newborn kittens (1 wk old) and adult gastric smooth muscle using one-dimensional SDS-PAGE. Although both the antrum and fundus of the kitten have significantly smaller total amounts of actin and myosin per mg protein than the adult, the ratio of actin to myosin is not significantly different between the age groups. Two different myosin heavy chain (MHC) isoforms, MHC1 (205 kD) and MHC2 (200 kD), were identified in all tissues. The relative amount of MHC1 remained constant during maturation of the stomach. We observed an increase in the amount of MHC2 in the adult, which resulted in a decreased ratio of MHC1 to MHC2 in the adult. We postulate that the decreased quantity of actin and myosin in the kitten stomach and the observed changes in the ratio of the MHC isoforms are related to changes in the gastric motor that occur during growth and development.

Actins↗