Src-mediated phosphorylation regulates subcellular distribution and activity of human inducible nitric oxide synthase.

Inducible nitric oxide synthase (iNOS) expression is regulated at both the transcriptional and post-transcriptional level in epithelial cells. The aim of this study was to characterize the effects of tyrosine phosphorylation on iNOS activity. In a human intestinal epithelial cell line stimulated with cytokines, tyrosine phosphorylation of human iNOS protein was observed after 30 min exposure to pervanadate (PV), an inhibitor of protein tyrosine phosphatases. 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, a specific inhibitor of Src tyrosine kinases, abolished the PV-induced iNOS tyrosine phosphorylation. Cotransfection of Src with iNOS cDNA in human embryonic kidney (HEK) 293 cells resulted in a threefold (P<0.001) increase of iNOS protein levels and tyrosine phosphorylation of iNOS. In the presence of Src, 76% of wild-type (wt) iNOS was redistributed to detergent-insoluble domains and iNOS activity was decreased by 28% (P<0.05) despite increased iNOS protein levels. Analysis of iNOS tyrosine mutants revealed decreased Src-induced effects in Y151F iNOS mutant. Using a GST-fusion protein containing a domain encompassing Y151, we show that Y151 is a direct substrate for active Src in vitro. These findings indicate a role for iNOS tyrosine phosphorylation in the regulation of iNOS activity and the implication of Src tyrosine kinases in this pathway.

Postanoxic functional recovery of the developing heart is slightly altered by endogenous or exogenous nitric oxide.

Nitric oxide synthase (NOS) is strongly and transiently expressed in the developing heart but its function is not well documented. This work examined the role, either protective or detrimental, that endogenous and exogenous NO could play in the functioning of the embryonic heart submitted to hypoxia and reoxygenation. Spontaneously beating hearts isolated from 4-day-old chick embryos were either homogenized to determine basal inducible NOS (iNOS) expression and activity or submitted to 30 min anoxia followed by 100 min reoxygenation. The chrono-, dromo- and inotropic responses to anoxia/reoxygenation were determined in the presence of NOS substrate (L-arginine 10 mM), NOS inhibitor L-NIO (1-5 mM), or NO donor (DETA NONOate 10-100 microM). Myocardial iNOS was detectable by immunoblotting and its activity was specifically decreased by 53% in the presence of 5 mM L-NIO. L-Arginine, L-NIO and DETA NONOate at 10 microM had no significant effect on the investigated functional parameters during anoxia/reoxygenation. However, irrespective of anoxia/reoxygenation, DETA NONOate at 100 microM decreased ventricular shortening velocity by about 70%, and reduced atrio-ventricular propagation by 23%. None of the used drugs affected atrial activity and hearts of all experimental groups fully recovered at the end of reoxygenation. These findings indicate that (1) by contrast with adult heart, endogenously released NO plays a minor role in the early response of the embryonic heart to reoxygenation, (2) exogenous NO has to be provided at high concentration to delay postanoxic functional recovery, and (3) sinoatrial pacemaker cells are the less responsive to NO.

Caveolin-1 down-regulates inducible nitric oxide synthase via the proteasome pathway in human colon carcinoma cells.

To investigate whether caveolin-1 (cav-1) may modulate inducible nitric oxide synthase (iNOS) function in intact cells, the human intestinal carcinoma cell lines HT29 and DLD1 that have low endogenous cav-1 levels were transfected with cav-1 cDNA. In nontransfected cells, iNOS mRNA and protein levels were increased by the addition of a mix of cytokines. Ectopic expression of cav-1 in both cell lines correlated with significantly decreased iNOS activity and protein levels. This effect was linked to a posttranscriptional mechanism involving enhanced iNOS protein degradation by the proteasome pathway, because (i) induction of iNOS mRNA by cytokines was not affected and (ii) iNOS protein levels increased in the presence of the proteasome inhibitors N-acetyl-Leu-Leu-Norleucinal and lactacystin. In addition, a small amount of iNOS was found to cofractionate with cav-1 in Triton X-100-insoluble membrane fractions where also iNOS degradation was apparent. As has been described for endothelial and neuronal NOS isoenzymes, direct binding between cav-1 and human iNOS was detected in vitro. Taken together, these results suggest that cav-1 promotes iNOS presence in detergent-insoluble membrane fractions and degradation there via the proteasome pathway.

Similar effects of cis-diamminedichloroplatinum(II) and cis-diammine-1,1-cyclobutanedicarboxylatoplatinum(II) on sodium-coupled glucose uptake in renal brush-border membrane vesicles.

cis-Diamminedichloroplatinum(II) (cDDP) has been shown to interfere with reabsorption processes in renal tubular epithelia, leading to polyuria, magnesium and sodium wasting and glucosuria. cDDP inhibits the Na+-coupled uptake of methyl-alpha-D-glucopyranoside (MGP) in renal proximal tubular cells in primary culture. cis-Diammine-1,1-cyclobutane dicarboxylatoplatinum(II) (CBDCA) produces tubular injury qualitatively similar to that of cDDP with a reduced severity. CBDCA inhibits Na+-coupled MGP uptake in renal proximal tubular cells in primary culture at concentrations 20- to 30-times higher than those of cDDP. The Na+/glucose cotransport protein possesses sulphydryl groups (SH) essential for its activity. Platinum complexes have strong affinity for SH groups. We compared the direct effects of cDDP (0.04-1.0 mM) and CBDCA (1-30 mM) on Na+-coupled MGP uptake in rabbit renal brush-border membrane (BBM) vesicles. cDDP and CBDCA inhibited Na+-coupled MGP uptake in a concentration-dependent manner, mainly through a decrease in Vmax of the cotransport protein. These effects were associated with platinum binding to BBM and decreases in protein-bound SH groups. CBDCA altered Na+-coupled MGP uptake at concentrations 30-times higher than those of cDDP. When BBM vesicles were preincubated with cDDP or CBDCA, diethyldithiocarbamate (an antidote against cDDP-induced nephrotoxicity) partly restored Na+-coupled MGP uptake and reduced the amount of platinum bound to BBM, but did not restore protein-bound SH groups. These findings strongly suggest that the inhibition of Na+-coupled MGP uptake by cDDP and CBDCA is mainly mediated by direct chemical binding of platinum to essential SH groups of the cotransport protein but may also involve other nucleophilic groups, such as the SCH3 group of methionine residues.

Role of protein thiols in inhibition of sodium-coupled glucose uptake by cisplatin in renal brush-border membrane vesicles.

The potent anticancer drug cis-diamminedichloroplatinum (II) (cDDP) impairs glucose reabsorption by renal proximal tubular cells, which leads to glucosuria. We investigated the direct effect of cDDP (0.04-2 mM) on the Na+/glucose cotransport system in brush-border membrane (BBM) vesicles from the rabbit renal cortex. cDDP induced 1) concentration-dependent inhibition of the Na+/glucose cotransport system, by decreasing its Vmax value and, to a lesser extent, its affinity, and 2) platinum binding to BBM vesicles, associated with decreases in protein-bound thiols. cDDP produced weaker inhibition of the Na+/glucose cotransport system and platinum binding to BBM vesicles than did highly reactive cDDP hydrated derivatives, with similar decreases in protein-bound thiols. Treatment with diethyldithiocarbamic acid (a drug protecting against cDDP nephrotoxicity), immediately after cDDP exposure, 1) partially lifted the cDDP-induced inhibition of the Na+/glucose cotransporter, 2) reduced platinum binding to BBM vesicles, but 3) did not modify the cDDP-induced decrease in protein-bound thiols. Our findings strongly suggest that cDDP-induced inhibition of the Na+/glucose cotransport system is mainly mediated by direct chemical binding of cDDP and/or its hydrated derivatives to essential sulfhydryl groups of the transport protein and may also involve other nucleophilic groups (e.g., the -SCH3 group of methionines).

Activity and inducibility of drug-metabolizing enzymes in immortalized hepatocyte-like cells (mhPKT) derived from a L-PK/Tag1 transgenic mouse.

This report describes the establishment and characterization of the mhPKT cell line derived from the liver of a transgenic mouse harboring the simian virus (SV40) large T and small t antigens placed under the control of the 5' regulatory sequence of the rat L-type pyruvate kinase (L-PK) gene. mhPKT cells had a prolonged life span, expressed the SV40-encoded nuclear large T antigen when grown in glucose-enriched medium, and induced tumors when injected subcutaneously into athymic (nu-nu) mice. Growth on petri dishes or filters yielded multiple layers of cuboid cells, with numerous spaces between adjacent cells that were closed by junctional complexes. These bile canaliculi-like structures exhibited numerous microvilli in which villin, an actin-binding brush-border protein, colocalized with actin. These bile canaliculi-like structures appeared to be functional as they accumulated fluorescein. mhPKT cells conserved the expression of the liver-specific transcription factors HNF1, HNF3, HNF4, and DBP together with substantial levels of L-PK and albumin but not alpha-fetoprotein mRNA transcripts. mhPKT cells mainly metabolized testosterone into androstenedione and 6beta-hydroxytestosterone, as in vivo. 3-Methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) markedly increased ethoxyresorufin-O-deethylase activity and the related cytochrome P450 (CYP) 1A1/2 protein, whereas alpha-naphtoflavone antagonized the TCDD-elicited induction. Phenobarbital slightly increased the CYP2B-mediated activities of pentoxyresorufin-O-depentylase, 2beta- and 16beta-testosterone hydroxylase. mhPKT cells also had substantial sulfotransferase, UDP-glucuronyltransferase, and glutathione S-transferase activities. This model may serve as a tool for long-term in vitro studies of xenobiotic metabolism, potent CYP inducers, and hepatocyte damage due to drugs and other factors.

Consecutive use of hormonally defined serum-free media to establish highly differentiated human renal proximal tubule cells in primary culture.

Highly differentiated human proximal tubule (HPT) cells in primary culture were established from heterogeneous suspension of tubules prepared from the human renal cortex by an original two-step procedure. First, gluconeogenic-competent HPT cells were selected by using a hormonally defined serum-free medium without glucose or insulin; then, the selected HPT cells were grown in a medium containing a low concentration of glucose (1 mM) and insulin (0.5 micrograms/mL) but no antibiotics. HPT cells grown on plastic support formed confluent, cobblestone-like monolayers with numerous mitochondria and pinocytosis vacuoles, solitary cilia, junctional complexes, and a well-developed brush border consisting of densely packed microvilli. Compared with cell monolayers on plastic support, HPT cells grown on porous filter membranes showed better morphologic differentiation. HPT cell monolayers expressed the following differentiated functions of the proximal tubule in situ: a low-affinity, high-capacity Na(+)-dependent glucose transport system inhibited by phlorizin, a high-affinity Na(+)-dependent phosphate transport system, a basolateral organic cation uptake inhibited by mepiperphenidol, parathyroid hormone-sensitive cAMP synthesis, brush-border hydrolase activities, gluconeogenesis-associated enzymes, glutathione-S-transferases and N-acetyl-beta-D-glucosaminidase. The medium containing low glucose and insulin concentrations markedly limited the increase in glycolysis but did not prevent the falls in gluconeogenesis and brush-border hydrolase activity at any time of the culture period. Similar decreases of brush border enzyme activities were obtained for HPT cells grown either on plastic or on porous filter membrane. A thorough characterization study demonstrated that this simple and preparative experimental approach makes it possible to establish highly differentiated HPT cells in primary culture suitable for investigating human renal proximal tubular cell function.

Modulation of sodium-coupled uptake and membrane fluidity by cisplatin in renal proximal tubular cells in primary culture and brush-border membrane vesicles.

The proximal tubule appears to be the main target for the adverse effects of cis-diamminedichloroplatinum (II) (cDDP). We evaluated the early effects of cDDP at concentrations (3 to 67 microM) lower that those which alter cell viability, on three apical transport systems and on the physical state of the brush border membrane (BBM) in rabbit proximal tubule (RPT) cells in primary culture. The maximal effect, corresponding to a 30% decrease in Na(+)-coupled uptake of phosphate (Pi) and alpha-methylglucopyranoside (MGP) and a twofold increase in Na(+)-coupled alanine uptake, was obtained at 17 microM (5 micrograms/ml) cDDP and occurred through a modification of their affinity. At this concentration, cDDP increased BBM fluidity and decreased the BBM cholesterol content by 28%, without increasing the permeability of tight junctions. To clarify the role of cDDP-induced increase in BBM fluidity on alterations of Na(+)-coupled uptake, these parameters were also investigated in BBM vesicles isolated from rabbit renal cortex directly exposed to cDDP. cDDP induced a concentration-dependent inhibition of Na(+)-coupled uptake of MGP, Pi and alanine in BBM vesicles from the renal cortex, associated with a decrease in protein sulfhydryl content, without modifying BBM fluidity. Our findings strongly suggest that the cDDP-induced increase in BBM fluidity in RPT cells results from an indirect mechanism, possibly an alteration of cholesterol metabolism, and did not play a major role in the cDDP-induced inhibition of Na+/Pi and Na+/glucose cotransport systems that may be mainly mediated through a direct chemical interaction with essential sulfhydryl groups of the transporters.

Dissimilar alterations of sodium-coupled uptake by platinum-coordination complexes in renal proximal tubular cells in primary culture.

The potent anticancer drug cis-diamminedichloroplatinum (II) (CDDP) interferes early with electrolyte transport by the renal proximal tubule. To study the early effects of platinum coordination complexes on apical Na(+)-coupled transport systems, we examined the effect of increasing concentrations of CDDP, trans-diamminedichloroplatinum (II) (TDDP) and cis-diammine-1,1-cyclobutane-dicarboxylate platinum (II) (CBDCA) on Na(+)-coupled uptake of P(i), methyl-alpha-D-glucopyranoside (MGP) and L-alanine by rabbit proximal tubule cells in primary culture. At 17 microM CDDP and 540 microM CBDCA, 1) cell viability (lactate dehydrogenase release) and ATP content were unaffected, 2) Na(+)-K(+)-ATPase activity was reduced by 40%, 3) Na(+)-coupled uptake of MGP and P(i) was reduced, whereas 4) Na(+)-coupled uptake of alanine rose to twice the control value. Alterations of Na(+)-coupled uptake of P(i), MGP and alanine were due to changes in Km, with no significant change in Vmax. At 333 microM TDDP, Na(+)-dependent P(i) and MGP uptake decreased, whereas Na(+)-independent uptake increased markedly and was associated with a decline in cell viability and ATP content. We conclude that 1) the TDDP-induced decrease in Na+/P(i) and Na+/glucose cotransport was associated with reduced cell viability, 2) both CDDP and CBDCA had different effects on Na+/P(i), Na+/glucose and Na+/alanine cotransport, arguing against an alteration of the Na+ gradient due to reduced Na(+)-K(+)-ATPase activity and 3) CBDCA induced alterations of Na(+)-coupled uptake similar to those of CDDP at concentrations 20 to 30 times higher.(ABSTRACT TRUNCATED AT 250 WORDS)