PubMed · 7776462
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T F Lue, J D Levine. 1995. Impotence.. https://doi.org/10.1016/s0022-5347(01)67234-3
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Human embryonic kidney (HEK) 293 cells stably transfected with the His-tagged thromboxane receptor alpha (TPalpha) were used to study the phosphorylation and desensitization of the receptor induced by prostaglandin E1 (PGE1) or forskolin. These agents are known to increase the intracellular level of cyclic AMP (cAMP) and activate cAMP-dependent protein kinase (PKA). Pretreatment of cells with either agent significantly attenuated Ca2+ release induced by the agonist [1S-[1alpha,2alpha(Z),3beta(1E,3S),4alpha]]-7-[3-[3-hydroxy-4-(4-indophenoxy)-1-butenyl]-7-oxabicyclo[2,2,1]hept-2-yl]-5-heptenoic acid (I-BOP). These agents also induced concentration-dependent phosphorylation of TPalpha as demonstrated by increased 32P-labeling of the receptor from cells prelabeled with 32P(i). To facilitate the identification of the intracellular domains involved in phosphorylation, glutathione S-transferase (GST)-intracellular domain fusion proteins were used as substrates for purified PKA. It was found that only the C-terminal tail fusion protein could serve as a substrate for PKA. To identify the specific serine/threonine residues in the C-terminal tail that are involved in phosphorylation, various alanine mutants of these residues were checked for their ability to serve as substrates. Ser-331 was found to be involved in PKA-mediated phosphorylation. The S331A mutant receptor overexpressed in HEK 293 cells was not phosphorylated significantly following stimulation by PGE1 or forskolin, indicating that Ser-331 was the major site of phosphorylation. Furthermore, cells overexpressing the mutant receptor became responsive to I-BOP-induced Ca2+ mobilization even after pretreatment with PGE1 or forskolin. These results indicate that Ser-331 is the primary site responsible for the phosphorylation and desensitization of the human TPalpha induced by agents that activate PKA.
Administration of methotrexate to rats results in severe enterocolitis and death. Previous our studies showed that a synthetic analog of prostaglandin E(1), OP-1206 [17S, 20-dimethyl-trans-Delta(2)-prostaglandin E(1)] ameliorated the anticancer agent-induced enterocolitis of rats. In the current study, we have focused on the biochemical effect of OP-1206 on the methotrexate-induced intestinal inflammation implicating reactive oxygen species (ROS). Methotrexate (15 mg/kg body weight) was orally administered to rats once daily for 5 days. OP-1206 (0.5 microg/kg body weight) was orally administered to rats twice a day for 5 days. On the 6th day, the chemiluminescence from the jejunum was measured to evaluate the generation of ROS. Spontaneous chemiluminescence from the jejunum of the methotrexate-treated rats increased significantly, compared with the control. Luminol-enhanced chemiluminescence from inflamed mucosal scrapings from the jejunum of the methotrexate-treated rats indicated more remarkable enhancement than the control rats. The treatment of OP-1206 with methotrexate showed significantly lower chemiluminescence of both the jejunum and mucosal scrapings than those of the methotrexate-treated rats. The alkaline phosphatase (ALP) activity, as a marker of small intestinal differentiation, in the intestinal mucosa of the methotrexate-treated rats decreased remarkably, but that of the methotrexate and OP-1206-treated rats was significantly higher than that of the methotrexate-treated rats. Thus, OP-1206 may possibly help the anticancer chemotherapy by protecting the small intestine from the methotrexate-induced damage.