Fyn is a downstream target of the pleiotrophin/receptor protein tyrosine phosphatase beta/zeta-signaling pathway: regulation of tyrosine phosphorylation of Fyn by pleiotrophin

Pleiotrophin (PTN the protein, Ptn the gene) signals downstream targets through inactivation of its receptor, the transmembrane receptor protein tyrosine phosphatase (RPTP)beta/zeta, disrupting the balanced activity of RPTPbeta/zeta and the activity of a constitutively active tyrosine kinase. As a consequence of the inactivation of RPTPbeta/zeta, PTN stimulates a sharp increase in the levels of tyrosine phosphorylation of the substrates of RPTPbeta/zeta in PTN-stimulated cells. We now report that the Src family member Fyn interacts with the intracellular domain of RPTPbeta/zeta in a yeast two-hybrid system. We further demonstrate that Fyn is a substrate of RPTPbeta/zeta, and that tyrosine phosphorylation of Fyn is sharply increased in PTN-stimulated cells. In previous studies, we demonstrated that beta-catenin and beta-adducin are targets of the PTN/RPTPbeta/zeta-signaling pathway and defined the mechanisms through which tyrosine phosphorylation of beta-catenin and beta-adducin disrupts cytoskeletal protein complexes. We conclude that Fyn is a downstream target of the PTN/RPTPbeta/zeta-signaling pathway and suggest that PTN coordinately regulates tyrosine phosphorylation of beta-catenin, beta-adducin, and Fyn through the PTN/RPTPbeta/zeta-signaling pathway and that together Fyn, beta-adducin, and beta-catenin may be effectors of the previously described PTN-stimulated disruption of cytoskeletal stability, increased cell plasticity, and loss of cell-cell adhesion that are characteristic of PTN-stimulated cells and a feature of many human malignant cells in which mutations have established constitutive expression of the Ptn gene.     

The receptor protein tyrosine phosphatase (RPTP)beta/zeta is expressed in different subtypes of human breast cancer

Increasing evidence suggests mutations in human breast cancer cells that induce inappropriate expression of the 18-kDa cytokine pleiotrophin (PTN, Ptn) initiate progression of breast cancers to a more malignant phenotype. Pleiotrophin signals through inactivating its receptor, the receptor protein tyrosine phosphatase (RPTP)beta/zeta, leading to increased tyrosine phosphorylation of different substrate proteins of RPTPbeta/zeta, including beta-catenin, beta-adducin, Fyn, GIT1/Cat-1, and P190RhoGAP. PTN signaling thus has wide impact on different important cellular systems. Recently, PTN was found to activate anaplastic lymphoma kinase (ALK) through the PTN/RPTPbeta/zeta signaling pathway; this discovery potentially is very important, since constitutive ALK activity of nucleophosmin (NPM)-ALK fusion protein is causative of anaplastic large cell lymphomas, and, activated ALK is found in other malignant cancers. Recently ALK was identified in each of 63 human breast cancers from 22 subjects. We now demonstrate that RPTPbeta/zeta is expressed in each of these same 63 human breast cancers that previously were found to express ALK and in 10 additional samples of human breast cancer. RPTPbeta/zeta furthermore was localized not only in its normal association with the cell membrane but also scattered in cytoplasm and in nuclei in different breast cancer cells and, in the case of infiltrating ductal carcinomas, the distribution of RPTPbeta/zeta changes as the breast cancer become more malignant. The data suggest that the PTN/RPTPbeta/zeta signaling pathway may be constitutively activated and potentially function to constitutively activate ALK in human breast cancer.     

Anaplastic lymphoma kinase is expressed in different subtypes of human breast cancer

Pleiotrophin (PTN, Ptn) is an 18kDa cytokine expressed in human breast cancers. Since inappropriate expression of Ptn stimulates progression of breast cancer in transgenic mice and a dominant negative PTN reverses the transformed phenotype of human breast cancer cells that inappropriately express Ptn, it is suggested that constitutive PTN signaling in breast cancer cells that inappropriately express Ptn activates pathways that promote a more aggressive breast cancer phenotype. Pleiotrophin signals by inactivating its receptor, the receptor protein tyrosine phosphatase (RPTP)beta/zeta, and, recently, PTN was found to activate anaplastic lymphoma kinase (ALK) through the PTN/RPTPbeta/zeta signaling pathway in PTN-stimulated cells, not through a direct interaction of PTN with ALK and thus not through the PTN-enforced dimerization of ALK. Since full-length ALK is activated in different malignant cancers and activated ALK is a potent oncogenic protein, we examined human breast cancers to test the possibility that ALK may be expressed in breast cancers and potentially activated through the PTN/RPTPbeta/zeta signaling pathway; we now demonstrate that ALK is strongly expressed in different histological subtypes of human breast cancer; furthermore, ALK is expressed in both nuclei and cytoplasm and, in the ;;dotted" pattern characteristic of ALK fusion proteins in anaplastic large cell lymphoma. This study thus supports the possibility that activated ALK may be important in human breast cancers and potentially activated either through the PTN/RPTPbeta/zeta signaling pathway, or, alternatively, as an activated fusion protein to stimulate progression of breast cancer in humans.     

Midkine, a newly discovered regulator of the renin-angiotensin pathway in mouse aorta: significance of the pleiotrophin/midkine developmental gene family in angiotensin II signaling

We previously demonstrated that pleiotrophin (PTN the protein, Ptn the gene) highly regulates the levels of expression of the genes encoding the proteins of the renin-angiotensin pathway in mouse aorta. We now demonstrate that the levels of expression of these same genes are significantly regulated in mouse aorta by the PTN family member midkine (MK the protein, Mk the gene); a 3-fold increase in expression of renin, an 82-fold increase in angiotensinogen, a 6-fold decrease in the angiotensin converting enzyme, and a 6.5-fold increase in the angiotensin II type 1 and a 9-fold increase in the angiotensin II type 2 receptor mRNAs were found in Mk-/- mouse aorta in comparison with the wild type (WT, +/+). The results in Mk-/- mice are remarkably similar to those previously reported in Ptn-/- mouse aorta, with the single exception of that the levels of the angiotensinogen gene expression in Ptn-/- mice are equal to those in WT+/+ mouse aorta, and thus, in contrast to Mk gene expression unaffected by levels of Ptn gene expression. The data indicate that MK and PTN share striking but not complete functional redundancy. These data support potentially high levels importance of MK and the MK/PTN developmental gene family in downstream signals initiated by angiotensin II either in development or in the many pathological conditions in which MK expression levels are increased, such as atherosclerosis and many human neoplasms that acquire constitutive endogenous Mk gene expression by mutation during tumor progression and potentially provide a target through the renin-angiotensin pathway to treat advanced malignancies.     

Tumstatin, the NC1 domain of alpha3 chain of type IV collagen, is an endogenous inhibitor of pathological angiogenesis and suppresses tumor growth

Angiogenesis, the formation of new blood vessels, is required for physiological development of vertebrates and repair of damaged tissue, but in the pathological setting contributes to progression of cancer. During tumor growth, angiogenesis is supported by up-regulation of angiogenic stimulators (pro-angiogenic) and down-regulation of angiogenic inhibitors (anti-angiogenic). The switch to the angiogenic phenotype (angiogenic switch) allows the tumors to grow and facilitate metastasis. The bioactive NC1 domain of type IV collagen alpha3 chain, called tumstatin, imparts anti-tumor activity by inducing apoptosis of proliferating endothelial cells. Tumstatin binds to alphaVbeta3 integrin via a mechanism independent of the RGD-sequence recognition and inhibits cap-dependent protein synthesis in the proliferating endothelial cells. The physiological level of tumstatin is controlled by matrix metalloproteinase-9, which most effectively cleaves it from the basement membrane and its physiological concentration in the circulation keeps pathological angiogenesis and tumor growth in check. These findings suggest that tumstatin functions as an endogenous inhibitor of pathological angiogenesis and functions as a novel suppressor of proliferating endothelial cells and growth of tumors.     

Pleiotrophin stimulates tyrosine phosphorylation of beta-adducin through inactivation of the transmembrane receptor protein tyrosine phosphatase beta/zeta

Pleiotrophin (PTN the protein, Ptn the gene) signals through a unique mechanism; it inactivates the tyrosine phosphatase activity of its receptor, the transmembrane receptor protein tyrosine phosphatase (RPTP)beta/zeta, and increases tyrosine phosphorylation of the substrates of RPTPbeta/zeta through the continued activity of a yet to be described protein tyrosine kinase(s) in PTN-stimulated cells. We have now found that the cytoskeletal protein beta-adducin interacts with the intracellular domain of RPTPbeta/zeta in a yeast two-hybrid system, that beta-adducin is a substrate of RPTPbeta/zeta, that beta-adducin is phosphorylated in tyrosine in cells not stimulated by PTN, and that tyrosine phosphorylation of beta-adducin is sharply increased in PTN-stimulated cells, suggesting that beta-adducin is a downstream target of and regulated by the PTN/RPTPbeta/zeta signaling pathway. beta-Catenin was the first downstream target of the PTN/RPTPbeta/zeta signaling pathway to be identified; these data thus also suggest that PTN coordinately regulates steady state levels of tyrosine phosphorylation of the important cytoskeletal proteins beta-adducin and beta-catenin and, through PTN-stimulated tyrosine phosphorylation, beta-adducin may contribute to the disruption of cytoskeletal structure, increased plasticity, and loss of homophilic cell-cell adhesion that are the consequences of PTN stimulation of cells and a characteristic feature of different malignant cells with mutations that activate constitutive expression of the endogenous Ptn gene.     

The effect of pleiotrophin signaling on adipogenesis

Pleiotrophin (PTN) plays diverse roles in cell growth and differentiation. In this investigation, we demonstrate that PTN plays a negative role in adipogensis and that glycogen synthase kinase 3beta (GSK-3beta) and beta-catenin are involved in the regulation of PTN-mediated preadipocyte differentiation. Knocking down the expression of PTN using siRNA resulted in an increase in phospho-GSK-3beta expression, and the accumulation of nuclear beta-catenin, which are critical downstream signaling proteins for both the PTN and Wnt signaling pathways. Our investigation suggests that there is a PTN/PI3K/AKT/GSK-3beta/beta-catenin signaling pathway, which cross-talks with the Wnt/Fz/GSK-3beta/beta-catenin pathway and negatively regulates adipogenesis.     

Roles of sphingosine-1-phosphate signaling in angiogenesis

Sphingosine-1-phosphate (S1P) is a blood-borne lipid mediator with pleiotropic biological activities. S1P acts via the specific cell surface G-protein-coupled receptors, S1P(1-5). S1P(1) and S1P(2) were originally identified from vascular endothelial cells (ECs) and smooth muscle cells, respectively. Emerging evidence shows that S1P plays crucial roles in the regulation of vascular functions, including vascular formation, barrier protection and vascular tone via S1P(1), S1P(2) and S1P(3). In particular, S1P regulates vascular formation through multiple mechanisms; S1P exerts both positive and negative effects on angiogenesis and vascular maturation. The positive and negative effects of S1P are mediated by S1P(1) and S1P(2), respectively. These effects of S1P(1) and S1P(2) are probably mediated by the S1P receptors expressed in multiple cell types including ECs and bone-marrow-derived cells. The receptor-subtype-specific, distinct effects of S1P favor the development of novel therapeutic tactics for antitumor angiogenesis in cancer and therapeutic angiogenesis in ischemic diseases.     

CORKSCREW1 defines a novel mechanism of domain specification in the maize shoot

In higher plants, determinate leaf primordia arise in regular patterns on the flanks of the indeterminate shoot apical meristem (SAM). The acquisition of leaf form is then a gradual process, involving the specification and growth of distinct domains within the three leaf axes. The recessive corkscrew1 (cks1) mutation of maize (Zea mays) disrupts both leaf initiation patterns in the SAM and domain specification within the mediolateral and proximodistal leaf axes. Specifically, cks1 mutant leaves exhibit multiple midribs and leaf sheath tissue differentiates in the blade domain. Such perturbations are a common feature of maize mutants that ectopically accumulate KNOTTED1-like homeobox (KNOX) proteins in leaf tissue. Consistent with this observation, at least two knox genes are ectopically expressed in cks1 mutant leaves. However, ectopic KNOX proteins cannot be detected. We therefore propose that CKS1 primarily functions within the SAM to establish boundaries between meristematic and leaf zones. Loss of gene function disrupts boundary formation, impacts phyllotactic patterns, and leads to aspects of indeterminate growth within leaf primordia. Because these perturbations arise independently of ectopic KNOX activity, the cks1 mutation defines a novel component of the developmental machinery that facilitates leaf-versus-shoot development in maize.     

Making the cut: protease-mediated regulation of angiogenesis

Angiogenesis is an integral element of normal physiologic development as well as of wound healing and a variety of pathologic conditions. Since the earliest studies of the cellular processes required for the formation of new capillaries from preexisting vessels, proteolysis has been recognized as one of the earliest and most sustained activities involved in these events. Several proteases including matrix metalloproteases (MMPs), and the closely related ADAM (a disintegrin and metalloprotease domain) and ADAMTS (a disintegrin and metalloprotease domain with thrombospondin motifs) families, as well as cysteine and serine proteases, have been implicated in this regulation. The current review addresses the contribution of these proteases in the positive and negative regulation of angiogenesis as mediated by degradation of the endothelial basement membrane and extracellular matrix proteins, release of angiogenic factors, processing of cytokines, growth factors and growth factor receptors, and the production of endogenous inhibitors.     

Inhibition of the mitogenic, angiogenic and tumorigenic activities of pleiotrophin by a synthetic peptide corresponding to its C-thrombospondin repeat-I domain

Pleiotrophin (PTN), is a heparin-dependent growth factor involved in angiogenesis and tumor growth. PTN contains a thrombospondin repeat-I (TSR-I) motif in its two beta-sheet domains that are involved in its binding to heparin and its neurite outgrowth activity. Based on the importance of the binding of PTN to heparin in its dimerization and biological activities, we have designed two synthetic peptides, P(13-39) and P(65-97) corresponding to a part of the N-terminal and C-terminal TSR-I motif of PTN, respectively. P(65-97) inhibited the mitogenic, tumorigenic and angiogenic activities of PTN, as well as the mitogenic and an angiogenic activity of fibroblast growth factor-2 (FGF-2). However, P(65-97) had no effect on the mitogenic activity of epidermal growth factor, which does not bind heparin. P(65-97) but not P(13-39) inhibited the binding of PTN and to a lesser extent of FGF-2 to heparin using an immunoassay and an optical biosensor assay and bound directly to heparin with a K(d) of 120 nM. These findings suggest that P(65-97), containing amino acids 65-97 of the TSR-I motif of the C-terminal domain of PTN, inhibits the activities of PTN and FGF-2 by virtue of its ability to bind heparin very effectively and so compete with the growth factors for their polysaccharide co-receptor.     

Identification of a redox-sensitive switch within the JAK2 catalytic domain

Four cysteine residues (Cys866, Cys917, Cys1094, and Cys1105) have direct roles in cooperatively regulating Janus kinase 2 (JAK2) catalytic activity. Additional site-directed mutagenesis experiments now provide evidence that two of these residues (Cys866 and Cys917) act together as a redox-sensitive switch, allowing JAK2's catalytic activity to be directly regulated by the redox state of the cell. We created several variants of the truncated JAK2 (GST/(NΔ661)rJAK2), which incorporated cysteine-to-serine or cysteine-to-alanine mutations. The catalytic activities of these mutant enzymes were evaluated by in vitro autokinase assays and by in situ autophosphorylation and transphosphorylation assays. Cysteine-to-alanine mutagenesis revealed that the mechanistic role of Cys866 and Cys917 is functionally distinct from that of Cys1094 and Cys1105. Most notable is the observation that the robust activity of the CC866,917AA mutant is unaltered by pretreatment with dithiothreitol or o-iodosobenzoate, unlike all other JAK2 variants previously examined. This work provides the first direct evidence for a cysteine-based redox-sensitive switch that regulates JAK2 catalytic activity. The presence of this redox-sensitive switch predicts that reactive oxygen species can impair the cell's response to JAK-coupled cytokines under conditions of oxidative stress, which we confirm in a murine pancreatic β-islet cell line.     

Trask phosphorylation defines the reverse mode of a phosphotyrosine signaling switch that underlies cell anchorage state

Phosphotyrosine signaling in anchored epithelial cells constitutes a spacially ordained signaling program that largely functions to promote integrin-linked focal adhesion complexes, serving to secure cell anchorage to matrix and as a bidirectional signaling hub that coordinates the physical state of the cell and its environment with cellular functions including proliferation and survival. Cells release their adhesions during processes such as mitosis, migration or tumorigenesis, but the fate of signaling through tyrosine phosphorylation in unanchored cells remains poorly understood. In an examination of epithelial cells in the unanchored state, we find abundant phosphotyrosine signaling, largely recommitted to an anti-adhesive function mediated through the Src family phosphorylation of their transmembrane substrate Trask/CDCP1/gp140. Src-Trask phosphorylation inhibits integrin clustering and focal adhesion assembly and signaling, defining an active phosphotyrosine signaling program underlying the unanchored state. Src-Trask signaling and Src-focal adhesion signaling inactivate each other, constituting two opposing modes of phosphotyrosine signaling that define a switch underline cell anchorage state. Src kinases are prominent drivers of both signaling modes, identifying their position at the helm of adhesion signaling capable of specifying anchorage state through substrate selection. These experimental studies along with concurring phylogenetic evidence suggest that phosphorylation on tyrosine is a signaling function fundamentally linked with the regulation of integrins.Key words: Trask, CDCP1, gp140, tyrosine phosphorylation, integrin, Src     

Crystal structures and molecular mechanism of a light-induced signaling switch: The Phot-LOV1 domain from Chlamydomonas reinhardtii

Phot proteins (phototropins and homologs) are blue-light photoreceptors that control mechanical processes like phototropism, chloroplast relocation, or guard-cell opening in plants. Phot receptors consist of two flavin mononucleotide (FMN)-binding light, oxygen, or voltage (LOV) domains and a C-terminal serine/threonine kinase domain. We determined crystal structures of the LOV1 domain of Phot1 from the green alga Chlamydomonas reinhardtii in the dark and illuminated state to 1.9 A and 2.8 A resolution, respectively. The structure resembles that of LOV2 from Adiantum (Crosson, S. and K. Moffat. 2001. PROC: Natl. Acad. Sci. USA. 98:2995-3000). In the resting dark state of LOV1, the reactive Cys-57 is present in two conformations. Blue-light absorption causes formation of a proposed active signaling state that is characterized by a covalent bond between the flavin C4a and the thiol of Cys-57. There are differences around the FMN chromophore but no large overall conformational changes. Quantum chemical calculations based on the crystal structures revealed the electronic distribution in the active site during the photocycle. The results suggest trajectories for electrons, protons, and the active site cysteine and offer an interpretation of the reaction mechanism.     

Identification of a switch in neurotrophin signaling by selective tyrosine phosphorylation

Neurotrophins, such as nerve growth factor and brain-derived neurotrophic factor, activate Trk receptor tyrosine kinases through receptor dimerization at the cell surface followed by autophosphorylation and recruitment of intracellular signaling molecules. The intracellular pathways used by neurotrophins share many common protein substrates that are used by other receptor tyrosine kinases (RTK), such as Shc, Grb2, FRS2, and phospholipase C-gamma. Here we describe a novel RTK mechanism that involves a 220-kilodalton membrane tetraspanning protein, ARMS/Kidins220, which is rapidly tyrosine phosphorylated in primary neurons after neurotrophin treatment. ARMS/Kidins220 undergoes multiple tyrosine phosphorylation events and also serine phosphorylation by protein kinase D. We have identified a single tyrosine (Tyr(1096)) phosphorylation event in ARMS/Kidins220 that plays a critical role in neurotrophin signaling. A reassembled complex of ARMS/Kidins220 and CrkL, an upstream component of the C3G-Rap1-MAP kinase cascade, is SH3-dependent. However, Tyr(1096) phosphorylation enables ARMS/Kidins220 to recruit CrkL through its SH2 domain, thereby freeing the CrkL SH3 domain to engage C3G for MAP kinase activation in a neurotrophin dependent manner. Accordingly, mutation of Tyr(1096) abolished CrkL interaction and sustained MAPK kinase activity, a response that is not normally observed in other RTKs. Therefore, Trk receptor signaling involves an inducible switch mechanism through an unconventional substrate that distinguishes neurotrophin action from other growth factor receptors.     

A network of conserved intramolecular contacts defines the off-state of the transmembrane switch mechanism in a seven-transmembrane receptor

Activation of the rhodopsin-like 7-transmembrane (7-TM) receptors requires switching interhelical constraints that stabilize the inactive state to a new set of contacts in the activated state, which binds the cognate G-protein. The free energy to drive this is provided by agonist binding, which has higher affinity to the active than to the inactive conformation. We have sought specific interhelical constraint contacts, using the M(1) muscarinic acetylcholine receptor as a model. Histidine substitutions of particular groups of amino acids, in transmembrane domains 3, 6, and 7, created high-affinity Zn(2+) binding sites, demonstrating the close proximity of their side chains in the inactive state. Alanine point substitutions have shown the effect of weakening the individual intramolecular contacts. In each case, the acetylcholine affinity was increased, implying promotion of the activated state. These amino acids are highly conserved throughout the 7-TM receptor superfamily. We propose that they form an important part of a network of conserved interhelical contacts that defines the off-state of a general transmembrane switch mechanism.     

Chronic blockade of the AT2 receptor with PD123319 impairs insulin signaling in C57BL/6 mice

The renin-angiotensin system modulates insulin action. Angiotensin type 1 receptor exerts a deleterious effects while the angiotensin type 2 receptor (AT2R) appears to have beneficial effects providing protection against insulin resistance and type 2 diabetes. Although recent reports indicate that agonism of AT2R ameliorates diabetes and insulin resistance, the phenotype of AT2R-knockout mice seems to be controversial relating this aspect. Thus, in this study we have explored the role of AT2R in the control of insulin action. To that end, C57Bl/6 mice were administered the synthetic AT2R antagonist PD123319 for 21 days (10 mg/kg/day ip); vehicle treated animals were used as control. Glucose tolerance, metabolic parameters, in vivo insulin signaling in main insulin-target tissues as well as levels of adiponectin, TNF-α, MCP-1 and IL-6 in adipose tissue were assessed. AT2R blockade with PD123319 induced a marginal effect on glucose homeostasis but an important reduction in the insulin-induced phosphorylation of the insulin receptor and Akt in both liver and adipose tissue. Insulin signaling in skeletal muscle remained unaltered after treatment with PD123319, which could explain the minimal effect on glucose homeostasis induced by PD123319. Our current results reinforce the notion that the AT2R has a physiological role in the conservation of insulin action.     

Effects of mechanical loading on the expression of pleiotrophin and its receptor protein tyrosine phosphatase beta/zeta in a rat spinal deformity model

Mechanical loading of the spine is a major causative factor of degenerative changes and causes molecular and structural changes in the intervertebral disc (IVD) and the vertebrae end plate (EP). Pleiotrophin (PTN) is a growth factor with a putative role in bone remodeling through its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ). The present study investigates the effects of strain on PTN and RPTPβ/ζ protein expression in vivo. Tails of eight weeks old Sprague-Dawley rats were subjected to mechanical loading using a mini Ilizarov external apparatus. Rat tails untreated (control) or after 0 degrees of compression and 10°, 30° and 50° of angulation (groups 0, I, II and III respectively) were studied. PTN and RPTPβ/ζ expression were evaluated using immunohistochemistry and Western blot analysis. In the control group, PTN was mostly expressed by the EP hypertrophic chondrocytes. In groups 0 to II, PTN expression was increased in the chondrocytes of hypertrophic and proliferating zones, as well as in osteocytes and osteoblast-like cells of the ossification zone. In group III, only limited PTN expression was observed in osteocytes. RPTPβ/ζ expression was increased mainly in group 0, but also in group I, in all types of cells. Low intensity RPTPβ/ζ immunostaining was observed in groups II and III. Collectively, PTN and RPTPβ/ζ are expressed in spinal deformities caused by mechanical loading, and their expression depends on the type and severity of the applied strain.     

The alteration of intracellular signaling on the smooth muscle cells contraction in cat esophagitis

We investigated the alteration of signal transduction after acute esophagitis in cat lower esophageal sphincter (LES). Acute esophagitis (AE) was induced by perfusion with 0.1N HCl at a rate of 1 ml/min for 45 min over three consecutive days. Acetylcholine (ACh)-induced contraction was inhibited by M3> M1 or M2 antagonists in normal LES. In AE, inhibition by M2 antagonists increased significantly, so that contraction was inhibited by M3> M2> M1 antagonists and the expression of M2 and M3 receptors were increased when compared to normal LES. In normal cells, ACh-induced contractions were antagonized by antibody against G(q/11) and the phosphatidylinositol-specific phospholipase C (PI-PLC) antagonist, U73122. The phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor, D609, or the phospholipase D inhibitor, propranolol had no effects on contraction in normal LES. However, in AE, G(q/11), and G(i3) antibodies reduced ACh-induced contraction and U73122, propranolol and D609 also reduced the contraction. In AE, we found that the expressions of G protein subtypes were increased but the expression of PLCbeta1, and PLCgamma1 were decreased when compared to normal LES. In conclusion, experimental esophagitis may alter the signal transduction by ACh in LES. ACh-induced contraction is mediated by M3 receptor, G(q/11) and PI-PLC in normal LES. However, in AE, the contractions are mediated by M2, M3 receptor, G(q/11) and G(i3). PC-PLC and PLD as PI-PLC are also involved in ACh-induced cell contraction in AE.