7-Ethyl-10-hydroxycamptothecin (SN-38) is the pharmacologically active metabolite of irinotecan, in addition to being responsible for severe toxicity. Glucuronidation is the main metabolic pathway of SN-38 and has been shown to protect against irinotecan-induced gastrointestinal toxicity. The purpose of this study was to determine whether common polymorphic UDP-glucuronosyltransferase (UGT) affects SN-38 glucuronidation. First, kinetic characterization of SN-38-glucuronide (SN-38-G) formation was assessed for all known human UGT1A and UGT2B overexpressed in human embryonic kidney 293 cells. To assess the relative activity of UGT isoenzymes for SN-38, rates of formation of SN-38-G were monitored by liquid chromatography/mass spectrometry analysis and normalized by level of UGT cellular expression. Determination of intrinsic clearances predicts that hepatic UGT1A1 and UGT1A9 and the extrahepatic UGT1A7 are major components in SN-38-G formation, whereas a minor role is suggested for UGT1A6, UGT1A8, and UGT1A10. In support of the involvement of UGT1A9, a strong coefficient of correlation was observed in the glucuronidation of SN-38 and a substrate, mainly glucuronidate, by UGT1A9 (flavopiridol) by human liver microsomes (coefficient of correlation, 0.
The first isoform of the Na ؉ -K ؉ -Cl ؊ cotransporter (NKCC1), a widely distributed member of the cation-Cl ؊ cotransporter superfamily, plays key roles in many physiological processes by regulating the ion and water content of animal cells and by sustaining electrolyte secretion across various epithelia. Indirect studies have led to the prediction that NKCC1 operates as a dimer assembled through binding domains that are distal to the amino portion of the carrier. In this study, evidence is presented that NKCC1 possesses self-interacting properties that result in the formation of a large complex between the proximal and the distal segment of the cytosolic C terminus. Elaborate mapping studies of these segments showed that the contact sites are dispersed along the entire C terminus, and they also led to the identification of a critical interacting residue that belongs to a putative forkhead-associated binding domain. In conjunction with previous findings, our results indicate that the uncovered interacting domains are probably a major determinant of the NKCC1 conformational landscape and assembly into a high order structure. A model is proposed in which the carrier could alternate between monomeric and homo-oligomeric units via chemical-or ligand-dependent changes in conformational dynamics.
Purinergic inhibition of Na-K-Cl cotransport has been noted in various renal epithelial cells derived from the collecting tubule, including Madin-Darby canine kidney (MDCK) cells. In recent studies, we have observed purinergic inhibition of Na-K-Cl cotransport in C11-MDCK subclones (alpha-intercalated-like cells). Interestingly, Na-K-Cl cotransport activity was also detected in C7-MDCK subclones (principal-like cells) but was not affected by ATP. In this investigation, we have transfected the human Na-K-Cl cotransporter (huNKCC1) in both C11 and C7 cells to determine whether these differences in NKCC regulation by ATP were due to cell-specific purinoceptor signaling pathways or to cell-specific isoforms/splice variants of the transporter. In both cell lines, we found that endogenous as well as huNKCC1-derived cotransport activity was restricted to the basolateral side. In addition, we were able to show that extracellular application of 100 microM ATP or 100 microM UTP abolished NKCC activity in both mock- and huNKCC1-transfected C11 cells but not in mock- and huNKCC1-transfected C7 cells; in C11 cells, intriguingly, this inhibition was not affected by inhibitors of RNA and protein synthesis and occurred even though expression levels of UTP-sensitive P2Y2-, P2Y4-, and P2Y6-purinoceptors were not different from those observed in C7 cells. These results suggest that C11 cells express an undetermined type of UTP-sensitive P2-purinoceptors or a unique P2Y-purinoceptor-triggered signaling cascade that leads to inhibition of NKCC1.
The first isoform of the Na ؉ -K ؉ -Cl ؊ cotransporter (NKCC1) is of central importance for the control of cellular ion concentration and epithelium-mediated salt secretion. Several studies have established that a change in intracellular [Cl ؊ ] (Cl ؊ i ) represents a key signaling mechanism by which NKCC1-induced Cl ؊ movement is autoregulated and by which Cl ؊ entry and exit on opposite sides of polarized cells are coordinated. Although this signaling mechanism is coupled to a pathway that leads to post-translational modification of the carrier, no unifying model currently accounts for the ion dependence of NKCC1 regulation. In this paper, evidence is presented for the first time that hsp90 associates with the cytosolic C terminus of NKCC1, probably when the carrier is predominantly in its unfolded form during early biogenesis. Evidence is also presented that the Cl ؊ i -dependent regulatory pathway can be activated by a thermal stress but that it is no longer operational if NKCC1-expressing cells are pretreated with geldanamycin, an antibiotic that inhibits hsp90, albeit nonspecifically. Taken together, our data indicate that binding of hsp90 to NKCC1 may be required for Na؊ cotransport to occur at the cell surface and that it could play an important role in ion-dependent signaling mechanisms, insofar as the maneuvers that were used to alter the expression or activity of the chaperone do not exert their main effect by inducing other cellular events such as the unfolded protein response. Further studies will be required to elucidate the functional relevance of this novel interaction. Cation-ClϪ cotransporters (CCCs) 1 are polytopic membrane proteins that couple the movement of Cl Ϫ to that of Na ϩ and/or K ϩ ions in or out of cells (1-4). Seven such proteins have been identified to date; the Na ϩ -K ϩ -Cl Ϫ cotransporters (NKCCs) isoform 1 and 2 (5, 6), the Na ϩ -Cl Ϫ cotransporter isoform 1 (7), and the K ϩ -Cl Ϫ cotransporters isoform 1-4 (8 -11). Other proteins that are homologous to the CCCs have also been identified (12); they are termed orphan CCCs because their ionic substrates have not been identified to date.The NKCC1 is considered a housekeeping carrier that is responsible for the bumetanide-sensitive component of Na ϩ -K ϩ -Cl Ϫ cotransport across cellular membranes (1-4). One of its main functions is to regulate cell volume (V cell ) and intracellular ClBecause of its basolateral localization in polarized cells, this carrier also promotes fluid and electrolyte secretion through a variety of epithelia by cooperating with apically disposed ion transport systems including Cl Ϫ channels (13-17). Several lines of evidence suggest that NKCC1 activity is increased by phosphorylation of its cytosolic termini (see Fig. 1) in response to Cl Ϫ i reduction or cell shrinkage. To this effect, recent studies have reported binding of proline-alanine-rich stress-related kinase (PASK) to NKCC1 and provided strong evidence that this interaction leads to phosphorylation-dependent activation of Na 18 -20). Along the sa...
OBJECTIVE: To examine some cellular mechanisms which regulate adipose cell metabolism in ovariectomized (OVX) and intact rats subjected or not to dehydroepiandrosterone (DHEA). DESIGN: Rats were assigned to one of four treatment groups for 27 weeks. The main effects tested were castration (À Àor ) and DHEA treatment (À Àor ) which consisted of a single daily percutaneous application of DHEA cream (30 mgaml in 50% ethanol ± 50% propyleneglycol). SUBJECTS: Forty female Sprague ± Dawley rats (sixteen-month old). MEASUREMENTS: Body weight and fat mass (by dual-energy X-ray absorptiometry), retroperitoneal (RP) fat pad weight; plasma insulin and triglyceride levels, and HDL-cholesterol (C) concentrations; lipoprotein lipase (LPL) and hormone-sensitive lipase (HSL) activities. RESULTS: No difference in body composition and RP fat pad weight was observed between the intact and intact-DHEA groups. LPL and HSL activities were also similar in both groups. The increased weight of OVX rats was paralleled by a higher adiposity and greater RP adipose tissue mass, which was associated with both a marked rise in LPL activity and a slight diminution in HSL activity in this depot, compared to intact animals. OVX-DHEA rats displayed a reduced adiposity and a lighter RP fat depot, which was associated with a decrease in LPL and an increase in HSL activities, compared to untreated OVX animals. Fasting plasma insulin and TG levels were also decreased whereas plasma HDL-C concentrations were increased in intact-DHEA and OVX-DHEA rats. CONCLUSION: These results show that the antiobesity effects of DHEA are dependent upon the ovarian status of the animal. These effects may involve changes in the lipid storage and the mobilization capacity of adipose tissue.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.