Nitric oxide (NO) generated by inducible NO synthase (iNOS) plays crucial roles in inflammation and host defense. With an intrinsically bound calmodulin, iNOS is fully active once expressed in cells. Thus, regulation of NO production from iNOS was thought to primarily occur at the enzyme transcriptional level. Here we show that NO synthesis from iNOS can be profoundly modulated by heat shock protein 90 (hsp90) through proteinprotein interaction. To study whether hsp90 affects iNOS function, recombinant murine iNOS was purified from an Escherichia coli expression system by affinity chromatography. Hsp90, at physiological concentrations (10 -500 nM), dose-dependently increased iNOS activity. This was a specific effect because neither denatured hsp90 nor irrelevant bovine serum albumin affected iNOS function. Overexpression of hsp90 enhanced NO production in iNOS-transfected cells. On the contrary, hsp90 inhibition dramatically decreased NO formation from iNOS in macrophages. Co-immunoprecipitation studies showed that hsp90 and iNOS associated with each other in cells. Overexpression of iNOS resulted in NO-mediated cellular injury. Hsp90 inhibition markedly attenuated NO formation and prevented cellular injury. These results demonstrated that hsp90 is an allosteric enhancer of iNOS. iNOS is coupled with hsp90 in cells, and this coupling facilitates NO synthesis. In light of the critical role of hsp90 in iNOS-mediated cytotoxic action, modulating the interaction between hsp90 and iNOS may be a new approach to intervene inflammation and immune response.
Rat brain membrane fractions obtained using Triton X-100 were applied to a D-myo-inositol 1,4,5-trisphosphate [D-Ins(1,4,5)P3] immobilized column, followed by gel filtration and anion-exchange chromatography. Two proteins with molecular masses of 130 and 85 kDa, as assessed by SDS-polyacrylamide gel electrophoresis, were purified to apparent homogeneity as D-[3H]Ins(1,4,5)P3-binding proteins with no D-Ins(1,4,5)P3-metabolizing activity. Partial amino acid sequence determinations of these proteins revealed that the 130 kDa protein appears to be a new D-Ins(1,4,5)P3-binding protein and the 85 kDa protein is a delta 1-isozyme of phospholipase C. We have previously purified 130 and 85 kDa proteins as D-[3H]Ins(1,4,5)P3-binding proteins, from rat brain cytosol fraction. Antibodies against the 130 kDa protein from the cytosol cross-reacted with the membrane 130 kDa protein purified in this study, suggesting that the membrane 130 kDa protein is likely to be the same as the protein from the cytosol fraction. The inhibition of D-[3H]Ins(1,4,5)P3 binding by D-isomers of inositol phosphates available clarified that the 130 kDa protein has a similar affinity for D-Ins(1,4,5,6)P4 to that for D-Ins(1,4,5)P3, while the 85 kDa protein is specific to D-Ins(1,4,5)P3.
The aim of the present study has been to confirm the existence of a transport pathway for a drug (cephalexin) to the cerebrospinal fluid (CSF) directly from the nasal cavity, by comparing the drug's concentrations in CSF after intranasal (i.n.), intravenous (i.v.) and intraduodenal (i.d.) administration. Higher levels of the drug were found in CSF following i.n. administration compared with the i.v. and i.d. routes, even though its plasma concentrations were similar. These findings suggest the existence of a direct transport pathway for cephalexin from the nasal cavity to the CSF. The concentration of drug in CSF at 15 min after i.n. administration was higher than that at 30 min. In contrast, its concentrations in CSF at 15 min after i.v. and i.d. administration were not significantly different from those at 30 min. The results confirm the presence of a direct transport pathway to CSF from the nasal cavity. This pathway may represent a new delivery route to CSF and possibly to brain parenchyma.
Ca2؉ -activated Cl ؊ channels are inhibited by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P 4 ) (Xie, W., Kaetzel, M. A., Bruzik, K. S., Dedman, J. R., Shears, S. B., and Nelson, D. J. (1996) J. Biol. Chem. 271, 14092-14097), a novel second messenger that is formed after stimulusdependent activation of phospholipase C (PLC). In this study, we show that inositol 1,3,4-trisphosphate (Ins(1,3,4)P 3 ) is the specific signal that ties increased cellular levels of Ins(3,4,5,6)P 4 to changes in PLC activity. We first demonstrated that Ins(1,3,4)P 3 inhibited Ins(3,4,5,6)P 4 1-kinase activity that was either (i) in lysates of AR4 -2J pancreatoma cells or (ii) purified 22,500-fold (yield ؍ 13%) from bovine aorta. Next, we incubated [ 3 H]inositol-labeled AR4 -2J cells with cell permeant and non-radiolabeled 2,5,6-tri-O-butyryl-myo-inositol 1,3,4-trisphosphate-hexakis(acetoxymethyl) ester. This treatment increased cellular levels of Ins(1,3,4)P 3 2.7-fold, while [ 3 H]Ins(3,4,5,6)P 4 levels increased 2-fold; there were no changes to levels of other 3 H-labeled inositol phosphates. This experiment provides the first direct evidence that levels of Ins(3,4,5,6)P 4 are regulated by Ins(1,3,4)P 3 in vivo, independently of Ins(1,3,4)P 3 being metabolized to Ins(3,4,5,6)P 4 . In addition, we found that the Ins(1,3,4)P 3 metabolites, namely Ins(1,3)P 2 and Ins(3,4)P 2 , were >100-fold weaker inhibitors of the 1-kinase compared with Ins(1,3,4)P 3 itself (IC 50 ؍ 0.17 M). This result shows that dephosphorylation of Ins(1,3,4)P 3 in vivo is an efficient mechanism to "switch-off " the cellular regulation of Ins(3,4,5,6)P 4 levels that comes from Ins(1,3,4)P 3 -mediated inhibition of the 1-kinase. We also found that Ins(1,3,6)P 3 and Ins(1,4,6)P 3 were poor inhibitors of the 1-kinase (IC 50 ؍ 17 and >30 M, respectively). The non-physiological trisphosphates, D/LIns(1,2,4)P 3 , inhibited 1-kinase relatively potently (IC 50 ؍ 0.7 M), thereby suggesting a new strategy for the rational design of therapeutically useful kinase inhibitors. Overall, our data provide new information to support the idea that Ins(1,3,4)P 3 acts in an important signaling cascade.
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.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.