Hiromasa Araki

Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

BackgroundProteinase-activated receptors (PARs; PAR1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT) which contributes to the increase in myofibroblast population.MethodsEMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA) for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells).ResultsStimulation of PAR with thrombin (1 U/ml) or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM) for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β). Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR) kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor.ConclusionPAR4 stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT) as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation.

Pressor effect of L‐threo‐3,4‐dihydroxyphenylserine in rats

The pressor effect of L‐threo‐3,4‐dihydroxyphenylserine (L‐threo‐DOPS) in rats and its decarboxylation in vivo have been examined. On i.v. administration, it produces a slow‐onset and long‐lasting pressor response, but no significant change in heart rate or e.c.g. The pressor effect was markedly reduced by inhibition of peripheral decarboxylase and by blockade of α‐adrenoceptors. The slow‐onset and long‐acting pressor effect was also evident when the drug was given orally, while intracerebroventricular administration produced a long‐lasting decrease in blood pressure. Noradrenaline (NA) concentrations in the plasma were significantly increased by both i.v. and oral administration of L‐threo‐DOPS. Elevation of plasma NA concentration by L‐threo‐DOPS given i.v. was suppressed by inhibition of decarboxylase. The plasma concentration of the drug was highest immediately after its i.v. administration. Its pressor effect was enhanced in rats made hypotensive by chemical sympathectomy with 6‐hydroxydopamine (6‐OHDA), compared with control rats, nevertheless, L‐threo‐DOPS produced the same increase in plasma NA concentrations in sympathectomized rats as in the controls. These results indicate that L‐threo‐DOPS is gradually converted to NA by L‐aromatic amino acid decarboxylase in vivo. These findings suggest that L‐threo‐DOPS may be clinically useful as an oral pressor agent for the treatment of certain disorders related to hypotension.

Positive chronotropic effect of threo-3,4-dihydroxyphenylserine as a precursor of noradrenaline in rat isolated atria.

whereas K+ acts primarily by facilitating the entry of membrane to inhibit the influx of extracellular CaZ+ membranal or extracellular lightly bound Ca2+ (the experiments performed in Ca2+-free high-K+ (Hudgins & Weiss, 1968). The inhibition of norsolution provided additional evidence for a membrane adrenaline and K+ responses induced by bunaphtide site of action of bunaphtide), (2) the intracellular Ca2+ suggests that although the experiments do not clearly storage sites to block the release of Ca2+ from the identify the sites of bunaphtide action, the results could stores or (3) at both sites, decreasing the avilability of be interpreted as possible action at three sites: (1) the Ca2+ at the contractile apparatus. January 17, 1978 COMMUNICATIONS, J. Pharm. Pharmac., 1978,30, 456

Age-related changes in the chronotropic effect and the enzymic decarboxylation of L-threo-3,4-dihydroxyphenylserine in the rat heart

The cardiac effect of L‐threo‐3,4‐dihydroxyphenylserine (L‐threo‐DOPS) and the enzymatic decarboxylation of the drug by L‐aromatic amino acid decarboxylase (AADC) were studied in atria isolated from rats ranging in age from newborn to adults and the findings compared with the cardiac effect of noradrenaline (NA). L‐threo‐DOPS produced dose‐dependent, slow‐onset and positive chronotropic effects in atria from rats of different ages. Its effect was inhibited in atria from benserazide‐treated rats, suggesting that the effects are due to NA formed from L‐threo‐DOPS by enzymic decarboxylation rather than to the compound itself. Chronotropic sensitivity to L‐threo‐DOPS was highest in the newborn and decreased with age during the first 3 weeks of life. The development of cardiac response to it correlated well with the development of enzymic decarboxylation of the drug but did not correlate with the developments of chronotropic sensitivity to NA and of NA concentrations in the heart. These findings suggest that in newborn rats, L‐threo‐DOPS is effectively converted by AADC to NA which in turn acts on β‐receptors in the pacemaker cell membrane.