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The inward L-dihydroxyphenylalanine (L-dopa) transport supplies renal proximal tubule cells (PTCs) with the precursor for dopamine synthesis. We have previously described insulin-induced stimulation of L-dopa uptake into PTCs. In the present paper we examined insulin-related signaling pathways involved in the increase of L-dopa transport into isolated rat PTCs. Insulin (50-500 [micro]U/ml) increased L-dopa uptake by PTCs, reaching the maximal increment (60% over the control) at 200 [micro]U/ml. At this concentration, insulin also increased insulin receptor tyrosine phosphorylation. Both effects were abrogated by the tyrosine kinase inhibitor genistein (5 [micro]M). In line, inhibition of the protein tyrosine phosphatase by pervanadate (0.2-100 [micro]M) caused a concentration-dependent increase in both the uptake of L-dopa (up to 400%) and protein tyrosine phosphorylation. A synergistic effect between pervanadate and insulin on L-dopa uptake was observed only when threshold (0.2 [micro]M), but not maximal (5 [micro]M), concentrations of pervanadate were assayed. Insulin-induced stimulation of L-dopa uptake was also abolished by inhibition of phosphatidylinositol 3-kinase (PI3K; 100 nM wortmannin, and 25 [micro]M LY-294002) and protein kinase C (PKC; 1 [micro]M RO-318220). Insulin-induced activation of PKC-[zeta] was confirmed in vitro by its translocation from the cytosol to the membrane fraction, and in vivo by immunohistochemistry studies. Insulin caused a wortmannin-sensitive increase in Akt/protein kinase B (Akt/PKB) phosphorylation and a dose-dependent translocation of Akt/PKB to the membrane fraction. Our findings suggest that insulin activates PKC-[zeta], and Akt/PKB downstream of PI3K, and that these pathways contribute to the insulin-induced increase of L-dopa uptake into PTCs. amino acid transport; second messengers; dopamine

Mucosal changes in inflammatory bowel disease (IBD) are characterized by ulcerative lesions accompanied by a prominent infiltrate of inflammatory cells including lymphocytes, macrophages, and neutrophils and alterations in 5-hydroxytryptamine (5-HT)-producing enterochromaffin (EC) cells. Mechanisms involved in recruiting and activating these cells are thought to involve a complex interplay of inflammatory mediators. Studies in clinical and experimental IBD have shown the upregulation of various chemokines including monocyte chemottractant protein (MCP)-1 in mucosal tissues. However, precise information on the roles of this chemokine or the mechanisms by which it takes part in the pathogenesis of IBD are not clear. In this study, we investigated the role of MCP-1 in the development of hapten-induced experimental colitis in mice deficient in MCP-1. Our results showed a significant reduction in the severity of colitis both macroscopically and histologically along with a decrease in mortality in MCP-l-deficient mice compared with wild-type control mice. This was correlated with a downregulation of myeloperoxidase activity, IL-1[beta], IL-12p40, and IFN-[gamma]/production, and infiltration of CD[3.sup.+] T cells and macrophages in the colonic mucosa. In addition, we observed significantly lower numbers of 5-HT-expressing EC cells in the colon of MCP-1-deficient mice compared with those in wild-type mice after dinitrobenzenesulfonic acid. These results provide evidence for a critical role of MCP- 1 in the development of colonic inflammation in this model in the context of immune and enteric endocrine cells. monocyte chemottractant protein-1; dinitrobenzenesulfonic acid; CD3-positive cells; 5-hydroxytryptamine; serotonin

Cultures of a cell line derived from pig proximal tubule epithelium, LLC-PK1, were investigated for their uptake of L-3,4-dihydroxyphenylalanine (L-dopa) and the decarboxylation of L-dopa to dopamine. The kinetics of absorption was determined for both the apical and basal membranes. The kinetics of dopamine formation was also determined based on the source of the substrate whether basal or apical. Dopamine secretion from this cell line was also investigated.

A defect in the renal dopamine production in 12-mo-old rats results when there is a decreased uptake of dopa by tubular brush-border membrane. In 12-mo-old rats, the level of dopa and norepinephrine(NE) in the renal cortex are higher (P

Eldrup, Ebbe, and Erik A. Richter. DOPA, dopamine, and DOPAC concentrations in the rat gastrointestinal tract decrease during fasting. Am J Physiol Endocrinol Metab 279: E815-E822, 2000.--The aim of the present study was to test the hypothesis that 3,4-dihydroxyphenylalanine (DOPA) and dopamine (DA) in the gastrointestinal tract are to a large extent of exogenous origin and derived from food. Tissue concentrations of norepinephrine (NE), epinephrine (Epi), DA, DOPA, and 3,4-dihydroxyphenylacetic acid (DOPAC), as measured by reverse-phase HPLC with electrochemical detection, were studied in fed and 4-day-fasted Wistar rats as well as in sympathectomized and adrenodemedullated rats. Sympathectomy and adrenal demedullectomy decreased tissue concentrations of NE and Epi, respectively, but had no effect on the level of tissue DOPA. Large amounts of DOPA and DA were present in the gastrointestinal tract. Fasting decreased DOPA and DA in the stomach and DOPA concentrations in the quadriceps muscle but no concentrations in other organs. DOPAC in the heart decreased both in response to sympathectomy and to fasting, whereas DOPAC decreased in plasma after fasting and in skeletal muscle after sympathectomy. We conclude that the food content of DOPA and DA is of major importance for the metabolism of DA and, thus, for the dopamine-sulfate content in the gastrointestinal tract and in plasma. The decrease in muscle DOPA after fasting may be explained by less insulin being available during fasting for stimulation of DOPA uptake in the muscle depot. DOPAC in the organism seems to be of a dual origin, derived partly from DA in the food and partly from DA synthesized in sympathetic nerves. 3,4-dihydroxyphenylalanine; 3,4-dihydroxyphenylacetic acid; 6-hydroxydopamine; sympathectomy; adrenodemedullectomy; norepinephrine; epinephrine; plasma