1 An 'atypical' 5-HT2 receptor which is located on the endothelium of rat jugular vein has been described. In the present study we 4 These data are consistent with the presence of 5-HT2B receptors mediating endothelium-dependent relaxation of rat jugular vein.
Nitric oxide (NO) synthase activity, which converts arginine to citrulline and NO, is present in homogenates of rat gastric mucosal cells. The aims of this study were to identify the form of NO synthase expressed in gastric cells isolated from fed rats, and to investigate the metabolism of arginine by suspensions of intact mucosal cells. Antibodies directed against the neuronal form of NO synthase recognised a protein of 160 kDa on immunoblots of extracts of gastric cells, and stained isolated cells of approx. 8 microm in diameter. NO synthase was enriched in a cell fraction which banded at high-density in a Percoll gradient, and was inhibited (IC50) by N(G)-nitro-L-arginine (0.8 microM), N(G)-monomethyl-L-arginine (12.6 microM), L-canavanine (147 microM), trifluoperazine (140 microM) and by phosphorylation involving protein kinase C. Intact gastric cells converted exogenous arginine to ornithine and citrulline. Arginase was present in the cells, and was predominantly responsible for arginine metabolism because formation of ornithine and citrulline was reduced by the arginase inhibitors, N(G)-hydroxy-L-arginine and L-ornithine, but not by NO synthase inhibitors such as N(G)-nitro-L-arginine. In conclusion, NO synthase that resembles the neuronal isoform is present in gastric mucosal cells, but a pathway involving arginase seems to be largely responsible for citrulline formation from exogenous arginine in intact mucosal cells.
In the gastric mucosa nitric oxide (NO) is implicated in the regulation of blood flow, in the maintenance of the integrity of the gastric mucosa and in the control of mucus secretion [ 11. Ca'+-dependent NO synthase, which converts arginine to citrulline and NO, is present in the mucosa and in suspensions of isolated mucosal cells [l]. Arginine may also be metabolised by arginase to ornithine and urea. Arginase and NO synthase have been found together in rat coronary endothelial cells [2], in small intestinal mucosal cells [3], and are co-induced by lipopolysaccharide in macrophages [4]. Furthermore, interactions between the NO synthase and the arginase metabolic pathways have been suggested [5, 61. In this study the formation of labelled metabolites from L-['Hlarginine was used to investigate pathways metabolising arginine under basal conditions in suspensions of rat gastric mucosal cells. Cells were prepared from everted sacs of corpus by pronase digestion coupled with intermittent Ca" chelation [7]. The incubation medium was Krebs bicarbonate buffer (pH 7.4) containing: 118 mM NaCI, 4.7 mM KC1, 1.2 mM MgSO,, 1.2 mM KH,PO,, 25 mM NaHCO,, 1.25 mM CaCl,, 11 mM glucose, and O.lg/lOO ml bovine serum albumin. Cells (5x106 cells/ml) were preincubated for 15 min at 37OC in medium alone, or with the addition of the arginase inhibitors [8] NC-hydroxy-L-arginine (4 mM) and Lornithine (1 mM), or the NO synthase inhibitor, N"-nitro-Larginine (0.3 mM) [l]. Aliquots of cells were then exposed to L-[2,3,4,5-,H]arginine (3 pCi/ml, 43 nM) and the extracellular volume marker ["C]-PEG 4000 (0.4 pCi/ml) in 20 ml polyethylene vials for between 5 and 15 min. The suspensions were centrifuged at 10,000 x g for 2 sec and cell pellets were extracted overnight at 4OC with 200 p1 of 65% ethanol containing 0.5 mg/ml of each of the unlabelled amino acids arginine, ornithine and citrulline. Extracts were analysed by ion-exchange chromatography on Dowex AG 50-W8, or by thin-layer chromatography on silica gel 60 plates with chloroform : methanol : ammonium hydroxide (2 : 2 : 1 by volume) as the solvent. Radioactivity associated with arginine, ornithine and citrulline was determined by liquid scintillation counting. The formation of metabolites was expressed as a percentage of the total 3H associated with the pellet, or as a percentage of intracellular radioactivity. Arginase activity was determined by radiochemical assay [6]. After 15 min incubation with [)H]arginine analysis of cell pellets by thin-layer chromatography gave the following results (Means f S. E. M. for label associated with the pellet for n=4 separate cell preparations): 67 f 4.4 YO arginine, 8.5 f 1.1 Yo ornithine and 15 f 2.1 % citrulline. 13 f 1.1 YO of the label was identified as citrulline when extracts were analysed by ion-exchange chromatography. Preincubation with L-ornithine (1 mM) and NG-hydroxy-Larginine (4 mM) reduced (P< 0.01) the total intracellular 'H in the pellet after 5 min incubation with pH]-arginine by 75Abbreviation used NO, nitric oxide f 4.4% and 56 f 5....
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