PubMed · 14264022
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D E PATERSON, R DAVID, S J BAKER. 1965. RADIODIAGNOSTIC PROBLEMS IN MALABSORPTION.. https://doi.org/10.1259/0007-1285-38-447-181
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Ischemia is the central pathogenic factor underlying a spectrum of intestinal disorders. The study of the cellular signaling responses to ischemic stress in nonepithelial cells has progressed substantially in the previous several years, but little is known about the response in epithelial cells. Unique features of the epithelial response to ischemic stress suggest differential regulation with regards to signaling. The PKC family of proteins has been implicated in ischemic stress in nonepithelial systems. The role of PKC isoforms in chemical ischemia in intestinal epithelial cells is evaluated in this study. Additionally, the phosphorylation of the F-actin cross-linking protein myristoylated alanine-rich C kinase substrate (MARCKS) is also studied. Chemical ischemia resulted in the transient activation of only the isoform PKC-epsilon as detected by translocation employing the subcellular fractionation technique. The pharmacological agonists phorbol 12-myristate 13-acetate and carbachol also led to the translocation of PKC-epsilon. By immunofluoresence, MARCKS is noted to be located at the lateral membrane under control conditions. In response to carbachol, MARCKS translocates to the cytosol, indicating its phosphorylation, which is additionally confirmed biochemically. Consistent with this observation, carbachol induces the translocation of PKC-epsilon to proximity with MARCKS at the lateral membrane. In response to chemical ischemia, MARCKS fails to translocate and phosphorylation does not increase. Additionally, the translocation of PKC-epsilon is not to the lateral membrane but rather basally. The data suggest that the differential translocation of PKC-epsilon in response to pharmacological agonists versus ischemic stress may lead to different effects on downstream targets.
We have compared the regulation of M(2) and M(3) muscarinic receptors heterologously expressed in HEK-293 cells upon long-term exposure towards the agonist carbachol. Carbachol time- and concentration-dependently reduced M(2) receptor density with a maximum reduction of about 60%. Treatment with 1mM carbachol for 24hr was accompanied by desensitisation of carbachol-induced Ca(2+) elevations (maximum response reduced by 70%) but not by alterations in the expression of various G-protein alpha-subunits. Consistently, heterologous desensitisation of Ca(2+) elevations by the purinergic receptor agonist ATP or by sphingosine-1-phosphate was not detected. In contrast, carbachol time- and concentration-dependently up-regulated M(3) receptors with maximum increases to about 350% of control values. The up-regulation was fully blocked by cycloheximide indicating that it was dependent on protein synthesis. Concomitant with the up-regulation of the M(3) receptor was a reduction in the expression of the alpha-subunit of G(q/11). The net effect of these two opposite regulatory mechanisms was a lack of alteration of carbachol-stimulated Ca(2+) elevation. However, the reduction of G(q/11) was accompanied by a heterologous desensitisation of Ca(2+) elevations by ATP and sphingosine-1-phosphate. Levels of M(2) and M(3) receptor mRNA as assessed by real-time PCR were not significantly altered by carbachol exposure for either receptor, suggesting that alterations of mRNA stability did not contribute to the observed changes in receptor number. We conclude that M(2) and M(3) receptor expression within the same cell undergoes differential agonist-induced regulation being accompanied by distinct regulation of G-protein expression leading to differential effects on signal transduction by other receptor systems.
PURPOSE: G-protein-coupled receptors are known to mediate cell growth via divergent signaling pathways. It has been reported that colon cancer cells express muscarinic acetylcholine receptor (mAChR) although their functional role is largely unknown. The aim of this study is to elucidate possible mechanisms responsible for the growth-promoting effect of mAChRs in colon cancer cells by using colon cancer cell line T84. METHODS: Carbachol, a stable mAChR agonist, dose-dependently induced cell growth with a maximal effect observed at 100 microM, equipotent with 1 nM EGF. 4-DAMP, a specific antagonist of subtype 3 mAChR, inhibited the stimulatory effect by carbachol, suggesting that the growth-promoting effect was receptor-mediated. Carbachol also dose-dependently stimulated extracellular signal-regulated protein kinase (ERK) activation. This effect was inhibited by PD98059, an inhibitor of extracellular signal-regulated protein kinase kinase, which also blocked carbachol activation of cell proliferation, indicating that the p21Ras-ERK pathway is an important signaling cascade in the mitogenic effect. To investigate how mAChR activated the p21Ras-ERK pathway, transactivation of epidermal growth factor receptor (EGFR) was examined. RESULTS: Carbachol induced tyrosine phosphorylation of EGFR, which was abolished by an EGFR tyrosine kinase inhibitor AG1478. Transactivation by carbachol was also abrogated by a metalloproteinases (MMPs) inhibitor GM6001 or an EGFR-blocking antibody (LA-1), suggesting that binding of EGFR ligand(s) produced by MMPs may initiate transactivation in a manner dependent on EGFR tyrosine kinase. The tyrosine-phosphorylated EGFR was immunoprecipitated together with GRB2 and tyrosine-phosphorylated Shc, indicating that transactivated EGFR is able to generate downstream signals. AG 1478 and LA-1 inhibited carbachol stimulation of cell growth. CONCLUSIONS: Taken together, our results indicate that the growth-promoting effect of subtype 3 mAChR in colon cancer cells may depend on transactivated EGFR-ERK pathways. EGFR not only receives external stimuli but also serves as a scaffold for downstream signaling molecules.