Vascular endothelial growth factor (VEGF) exerts crucial functions during pathological angiogenesis and normal physiology. We observed increased hematocrit (60-75%) after high-grade inhibition of VEGF by diverse methods, including adenoviral expression of soluble VEGF receptor (VEGFR) ectodomains, recombinant VEGF Trap protein and the VEGFR2-selective antibody DC101. Increased production of red blood cells (erythrocytosis) occurred in both mouse and primate models, and was associated with near-complete neutralization of VEGF corneal micropocket angiogenesis. High-grade inhibition of VEGF induced hepatic synthesis of erythropoietin (Epo, encoded by Epo) >40-fold through a HIF-1alpha-independent mechanism, in parallel with suppression of renal Epo mRNA. Studies using hepatocyte-specific deletion of the Vegfa gene and hepatocyte-endothelial cell cocultures indicated that blockade of VEGF induced hepatic Epo by interfering with homeostatic VEGFR2-dependent paracrine signaling involving interactions between hepatocytes and endothelial cells. These data indicate that VEGF is a previously unsuspected negative regulator of hepatic Epo synthesis and erythropoiesis and suggest that levels of Epo and erythrocytosis could represent noninvasive surrogate markers for stringent blockade of VEGF in vivo.

Mammalian hepatic cytochromes P450 (P450s) are endoplasmic reticulum (ER)-anchored hemoproteins engaged in the metabolism of numerous xeno-and endobiotics. P450s exhibit widely ranging half-lives, utilizing both autophagic-lysosomal (ALD) and ubiquitin-dependent 26S proteasomal (UPD) degradation pathways. Although suicidally inactivated hepatic CYPs 3A and "native" CYP3A4 in S. cerevisiae are degraded via UPD, the turnover of native hepatic CYPs 3A in their physiological milieu has not been elucidated. Herein, we characterize the degradation of native, dexamethasone-inducible CYPs 3A in cultured primary rat hepatocytes, using proteasomal and ALD [NH 4 Cl and 3-methyladenine (3-MA)] inhibitors to examine their specific degradation route. Pulse-chase cum immunoprecipitation analyses revealed a basal 52% 35 S-CYP3A loss over 6 h, which was stabilized by both proteasomal inhibitors. By contrast, no corresponding CYP3A stabilization was detected with either ALD inhibitor NH 4 Cl or 3-MA. Furthermore, MG-262-induced CYP3A stabilization was associated with its polyubiquitylation, thereby verifying that native CYPs 3A were also degraded via UPD. To identify the specific participants in this process, cellular proteins were crosslinked in situ with paraformaldehyde (PFA) in cultured hepatocytes. Immunoblotting analyses of CYP3A immunoprecipitates after PFA-crosslinking revealed the presence of p97, a cytosolic AAA ATPase instrumental in the extraction and delivery of ubiquitylated ER proteins for proteasomal degradation. Such native CYP3A-p97 interactions were greatly magnified after CYP3A suicidal inactivation (which accelerates UPD), and/or proteasomal inhibition, and were confirmed by proteomic and confocal immunofluorescence microscopic † Supported by NIH grants GM44037 (MAC) and DK26506 (MAC), RR012961 (KFM) and NIH grant P30DK26743 (Liver Core Center Cell and Tissue Biology Core). *Corresponding Author: M. A. Correia Dept. of Cellular and Molecular Pharmacology, Mission Bay Campus, Genentech Hall 600 16th Street, N572F/Box 2280 University of California San Francisco, CA 94158−2280 415−476−3992 (TEL) 415−476−5292 (FAX) e-mail: almira.correia@ucsf.edu. Supporting Information: MS/MS data obtained from the proteomic analyses of CYP3A-protein crosslinked complexes after subjection to SDS-PAGE, sequential gel-slicing of each relevant lane, and in situ tryptic digestion followed by LC-MS/MS analyses are provided. The proteins were identified from more than 1 peptide, but only 1 representative spectrum has been provided for each in the supplementary material. This material is available free of charge via the Internet at http://pubs.acs.org NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 15. Published in final edited form as:Biochemistry. analyses. These findings clearly reveal that native CYPs 3A undergo UPD and implicate a role for p97 in this process.The hepatic hemoproteins cytochromes P450 (P450s) 1 are key enzymes in the oxidative metabolism of various endob...

Sustained activation of N-methyl-d-aspartate (NMDA) -type glutamate receptors leads to excitotoxic neuronal death in stroke, brain trauma, and neurodegenerative disorders. Superoxide production by NADPH oxidase is a requisite event in the process leading from NMDA receptor activation to excitotoxic death. NADPH oxidase generates intracellular H + along with extracellular superoxide, and the intracellular H + must be released or neutralized to permit continued NADPH oxidase function. In cultured neurons, NMDAinduced superoxide production and neuronal death were prevented by intracellular acidification by as little as 0.2 pH units, induced by either lowered medium pH or by inhibiting Na + /H + exchange. In mouse brain, superoxide production induced by NMDA injections or ischemia-reperfusion was likewise prevented by inhibiting Na + /H + exchange and by reduced expression of the Na + /H + exchanger-1 (NHE1). Neuronal intracellular pH and neuronal Na + /H + exchange are thus potent regulators of excitotoxic superoxide production. These findings identify a mechanism by which cell metabolism can influence coupling between NMDA receptor activation and superoxide production.any metabolic processes generate hydrogen ions, and hydrogen ions in turn influence cell metabolism and survival (1). Cerebral ischemia in particular produces acidosis of variable degree, depending upon blood glucose levels, degree of blood flow reduction, and other factors. Severe acidosis, below pH 6.4, exacerbates ischemic injury (2) by mechanisms involving protein denaturation, acid-sensing calcium channels, and release of ferrous iron (3-5). Conversely, lesser degrees of acidosis, in the range of 7.0-6.5, reduce both ischemic injury (6) and glutamateinduced neuronal death (7). These neuroprotective effects have been attributed to an inhibitory effect of hydrogen ions on NMDA receptor activation (8-10), but a causal link has not been demonstrated.Excessive activation of N-methyl-D-aspartate (NMDA) type glutamate receptors leads to excitotoxic cell death in stroke and other neurological disorders (11,12). Superoxide production by NADPH oxidase is a requisite event in the process leading from NMDA receptor activation to excitotoxic cell death (13)(14)(15)(16)(17)(18)(19) Together, the pH sensitivity of NOX2 and the role of NOX2 in NMDA receptor-mediated cell death suggest the possibility that reduced intracellular pH might limit neurotoxicity by dissociating NMDA receptor activation from superoxide production. Findings presented here confirm that both the superoxide production and cell death resulting from neuronal NMDA receptor activation are highly pH sensitive. We show that neurons use Na + /H + exchange as a major route of proton efflux during NOX2 activation, and either genetic or pharmacologic inhibition of neuronal Na + /H + exchange prevent both excitotoxic superoxide production and cell death. ResultsMild Acidosis Blocks NMDA-Induced Superoxide Production and Cell Death. We first performed cell culture studies at a physiological medium pH ...

We examined the effects of two exercise training modalities, i.e., low-intensity endurance and sprint running, on in vitro, isovolumic myocardial performance following ischemia and reperfusion. Rats ran on a treadmill 5 d.wk-1 for 6 wk at the following levels: endurance; 20 m.min-1, 0% grade, 60 min.d-1 and sprint; five 1-min runs at 75 m.min-1, 15% grade interspersed with 1-min active recovery runs at 20 m.min-1, 15% grade. Both endurance and sprint training significantly improved exercise tolerance relative to control (P < 0.05) on two graded exercise tests. Buffer perfused hearts of control (N = 18), endurance (N = 20), and sprint (N = 13) trained animals underwent no-flow ischemia (20 min) and reperfusion (30 min) in a Langendorff mode. During reperfusion, left ventricular developed pressure and its first derivative were 20% higher in sprint (P < 0.05) than either endurance or control hearts. Left ventricular end-diastolic pressure was lowest in sprint during reperfusion (sprint, 10 +/- 1 mm Hg vs endurance, 14 +/- 2 mm Hg; and control, 14 +/- 2 mm Hg, at 30 min reperfusion). Hearts were then used for biochemical studies or dissociated into single cells for measurement of contraction, cell calcium, and action potential duration. Single cell contractions were greatest in sprint despite similar calcium transients in all groups. Ischemia/reperfusion caused action potential prolongation in control but not trained myocytes. Hearts from sprint had the greatest glyceraldehyde-3-phosphate dehydrogenase activity (P < 0.05) and a tendency towards increased superoxide dismutase activity. These results suggest that sprinting increases myocardial resistance to ischemia/reperfusion. This protection may be secondary to increased myofilament calcium sensitivity and/or myocardial expression of glyceraldehyde-3-phosphate dehydrogenase.