PubMed · 10948037
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D Ralph, T McNicholas. UK management guidelines for erectile dysfunction.. https://doi.org/10.1136/bmj.321.7259.499
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Mast cells are implicated in the pathogenesis of a broad spectrum of immunological disorders. These cells release inflammatory mediators in response to a number of stimuli, including IgE-Ag complexes. The degranulation of mast cells is modified by PGs. To begin to delineate the pathway(s) used by PGs to regulate mast cell function, we examined bone marrow-derived mast cells (BMMC) cultured from mice deficient in the EP(1), EP(2), EP(3), and EP(4) receptors for PGE(2). Although BMMCs express all four of these PGE(2) receptors, potentiation of Ag-stimulated degranulation and IL-6 cytokine production by PGE(2) is dependent on the EP(3) receptor. Consistent with the coupling of this receptor to G(alphai), PGE(2) activation of the EP(3) receptor leads to both inhibition of adenylate cyclase and increased intracellular Ca(2+). The magnitude of increase in intracellular Ca(2+) induced by EP(3) activation is similar to that observed after activation of cells with IgE and Ag. Although PGE alone is not sufficient to initiate BMMC degranulation, stimulation of cells with PGE along with PMA induces degranulation. These actions are mediated by the EP(3) receptor through signals involving Ca(2+) mobilization and/or decreased cAMP levels. Accordingly, these studies identify PGE(2)/EP(3) as a proinflammatory signaling pathway that promotes mast cell activation.
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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.