Research Article
Abstract
The ability of cells to tolerate hypoxia is critical to their survival, but varies greatly among different cell types. Despite alterations in many cellular responses during hypoxic exposure, pulmonary arterial endothelial cells (PAEC) retain their viability and cellular integrity. Under similar experimental conditions, other cell types, exemplified by renal tubular epithelial cells, are extremely hypoxia sensitive and are rapidly and irreversibly damaged. To investigate potential mechanisms by which PAEC maintain cellular and functional integrity under these conditions, we compared the turnover of adenine and guanine nucleotides in hypoxia tolerant PAEC and in hypoxia-sensitive renal tubular endothelial cells under various hypoxic conditions. Under several different hypoxic conditions, hypoxia-tolerant PAEC maintained or actually increased ATP levels and the percentage of these nucleotides found in the high energy phosphates, ATP and GTP. In contrast, in hypoxia-sensitive renal tubular endothelial cells, the same high energy phosphates were rapidly depleted. Yet, in both cell types, there were minor alterations in the uptake of the precusor nucleotide and its incorporation into the appropriate purine nucleotide phosphates and marked decreases in ATPase and GTPase activity. This maintenance of high energy phosphates in hypoxic PAEC suggests that there exists tight regulation of ATP and GTP turnover in these cells and that preservation of these nucleotides may contribute to the tolerance of PAEC to acute and chronic hypoxia.
Authors
A V Tretyakov, H W Farber
Abstract
Sodium-dependent bile acid transport in the rat ileum is abruptly expressed at weaning. Degenerate oligonucleotides, based on amino acid sequence identities between the rat liver and hamster ileal transporters, were used to amplify a rat ileal probe. A 1.2-kb cDNA clone, which contains the full coding region (348 amino acids, 38 kD), was isolated by hybridization screening. In vitro translation yielded a 38-kD protein which glycosylated to 48 kD. Sodium-dependent uptake of taurocholate was observed in oocytes injected with cRNA. Northern blot analysis revealed a 5.0-kb mRNA in ileum, kidney, and cecum. A 48-kD protein was detected in ileal brush border membranes and localized to the apical border of villus ileal enterocytes. mRNA and protein expression, which were negligible before weaning, increased dramatically at weaning. Nuclear transcription rates for the transporter increased 15-fold between postnatal days 7 and 28. The apparent molecular weight of the transporter also increased between days 19 and 28. In summary, the developmental regulation of the rat ileal sodium-dependent bile acid cotransporter is characterized by transcriptionally regulated increases in mRNA and protein levels at the time of weaning with changes in apparent molecular weight of the protein after weaning.
Authors
B L Shneider, P A Dawson, D M Christie, W Hardikar, M H Wong, F J Suchy
Abstract
The mechanism(s) of insulin resistance in non-insulin-dependent diabetes mellitus remains ill defined. The current studies sought to determine whether non-insulin-dependent diabetes mellitus is associated with (a) a delay in the rate of onset of insulin action, (b) impaired hepatic and extrahepatic kinetic responses to insulin, and (c) an alteration in the contribution of gluconeogenesis to hepatic glucose release. To answer these questions, glucose disappearance, glucose release, and the rate of incorporation of 14CO2 into glucose were measured during 0.5 and 1.0 mU/kg-1 per min-1 insulin infusions while glucose was clamped at approximately 95 mg/dl in diabetic and nondiabetic subjects. The absolute rate of disappearance was lower (P < 0.05) and the rate of increase slower (P < 0.05) in diabetic than nondiabetic subjects during both insulin infusions. In contrast, the rate of suppression of glucose release in response to a change in insulin did not differ in the diabetic and nondiabetic subjects during either the low (slope 30-240 min:0.02 +/- 0.01 vs 0.02 +/- 0.01) or high (0.02 +/- 0.00 vs 0.02 +/- 0.00) insulin infusions. However, the hepatic response to insulin was not entirely normal in the diabetic subjects. Both glucose release and the proportion of systemic glucose being derived from 14CO2 (an index of gluconeogenesis) was inappropriately high for the prevailing insulin concentration in the diabetic subjects. Thus non-insulin-dependent diabetes mellitus slows the rate-limiting step in insulin action in muscle but not liver and alters the relative contribution of gluconeogenesis and glycogenolysis to hepatic glucose release.
Authors
D Turk, A Alzaid, S Dinneen, K S Nair, R Rizza
Abstract
We have previously suggested that hyperglycemia per se may contribute to diabetic hypertensive and vascular disease by altering cellular ion content. To more directly investigate the potential role of glucose in this process, we measured cytosolic free calcium in primary cultures of vascular smooth muscle cells isolated from Sprague-Dawley rat tail artery before and after incubation with 5 (basal), 10, 15, and 20 mM glucose. Glucose significantly elevated cytosolic free calcium in a dose- and time-dependent manner, from 110.0 +/- 5.4 to 124.5 +/- 9.0, 192.7 +/- 20.4, and 228.4 +/- 21.9 nM at 5, 10, 15, and 20 mM glucose concentrations, respectively. This glucose-induced cytosolic free calcium elevation was also specific, no change being observed after incubation with equivalent concentrations of L-glucose or mannitol. This glucose effect was also dependent on extracellular calcium and pH, since these calcium changes were inhibited in an acidotic or a calcium-free medium, or by the competitive calcium antagonist lanthanum. We conclude that ambient glucose concentrations within clinically observed limits may alter cellular calcium ion homeostasis in vascular smooth muscle cells. We suggest that these cellular ionic effects of hyperglycemia may underlie the predisposition to hypertension and vascular diseases among diabetic subjects and/or those with impaired glucose tolerance.
Authors
M Barbagallo, J Shan, P K Pang, L M Resnick
Abstract
Elevations of plasma total or LDL cholesterol are major risk factors for cardiovascular disease. Efforts directed at preventing and treating cardiovascular disease have often focused on reducing the levels of these substances in the blood. The Watanabe Heritable Hyperlipidemic Rabbit, which has exceedingly high plasma cholesterol levels resulting from an LDL receptor deficiency, provides an excellent animal model for testing new treatments. A recombinant adenoviral vector containing the rabbit LDL receptor cDNA was administered to Watanabe rabbits. Plasma total cholesterol levels in the treated animals were reduced from 825.5 +/- 69.8 (mean +/- SD) to 247.3 +/- 61.5 mg/dl 6 d after infusion. These animals also demonstrated a 300-400% increase in plasma levels of HDL cholesterol and apo AI 10 d after treatment. As a result, the LDL:HDL ratio exhibited a dramatic decrease. Because only the rabbit LDL receptor gene was used for treatment, the results strongly suggest that the elevations of plasma HDL cholesterol and apo AI were secondary to a reduction in plasma total cholesterol in the treated animals. These results suggest an inverse relationship between plasma LDL and HDL cholesterol levels and imply that reduction of LDL cholesterol levels may have a beneficial effect on plasma HDL cholesterol.
Authors
J Li, B Fang, R C Eisensmith, X H Li, I Nasonkin, Y C Lin-Lee, M P Mims, A Hughes, C D Montgomery, J D Roberts
Abstract
Mildly oxidized low density lipoprotein (MM-LDL) produced by oxidative enzymes or cocultures of human artery wall cells induces endothelial cells to produce monocyte chemotactic protein-1 and to bind monocytes. HDL prevents the formation of MM-LDL by cocultures of artery wall cells. Using albumin treatment and HPLC we have isolated and partially characterized bioactive oxidized phospholipids in MM-LDL. Platelet activating factor-acetylhydrolase (PAF-AH), a serine esterase, hydrolyzes short chain acyl groups esterified to the sn-2 position of phospholipids such as PAF and particular oxidatively fragmented phospholipids. Treatment of MM-LDL with PAF-AH (2-4 x 10(-2) U/ml) eliminated the ability of MM-LDL to induce endothelial cells to bind monocytes. When HDL protected against the formation of MM-LDL by cocultures, lysophosphatidylcholine was detected in HDL; whereas when HDL was pretreated with diisopropyl fluorophosphate, HDL was no longer protective and lysophosphatidylcholine was undetectable. HPLC analysis also revealed that the active oxidized phospholipid species in MM-LDL had been destroyed after PAF-AH treatment. In addition, treatment of MM-LDL with albumin removed polar phospholipids that, when reisolated, induced monocyte binding to endothelial cells. These polar phospholipids, when treated with PAF-AH, lost biological activity and were no longer detected by HPLC. These results suggest that PAF-AH in HDL protects against the production and activity of MM-LDL by facilitating hydrolysis of active oxidized phospholipids to lysolipids, thereby destroying the biologically active lipids in MM-LDL.
Authors
A D Watson, M Navab, S Y Hama, A Sevanian, S M Prescott, D M Stafforini, T M McIntyre, B N Du, A M Fogelman, J A Berliner
Abstract
Hepatic glycogen concentration was measured in six subjects with insulin-dependent diabetes mellitus (IDDM) and nine weight-matched control subjects using 13C nuclear magnetic resonance spectroscopy during a day in which three isocaloric mixed meals were ingested. The relative fluxes of the direct and indirect (3 carbon units-->-->glycogen) pathways of hepatic glycogen synthesis were also assessed using [1-13C]glucose in combination with acetaminophen to noninvasively sample the hepatic UDP-glucose pool. Mean fasting hepatic glycogen content was similar in the two groups. After each meal, hepatic glycogen content increased, peaking 4-5 h after the meal in both groups. By 11:00 p.m. the IDDM subjects had synthesized only 30% of the glycogen that was synthesized by the control group [IDDM subjects, net increment = 44 +/- 20 (mean +/- SE) mM; control subjects, net increment = 144 +/- 14 mM; P < 0.05]. After breakfast the flux through the gluconeogenic pathway relative to the direct pathway of hepatic glycogen synthesis was 1.7-fold greater in the IDDM subjects (59 +/- 4%) than in the control subjects (35 +/- 4%, P < 0.0003). In conclusion, under mixed meal conditions, subjects with poorly controlled IDDM have a major defect in net hepatic glycogen synthesis and augmented hepatic gluconeogenesis. The former abnormality may result in an impaired glycemic response to counterregulatory hormones, whereas both abnormalities may contribute to postprandial hyperglycemia.
Authors
J H Hwang, G Perseghin, D L Rothman, G W Cline, I Magnusson, K F Petersen, G I Shulman
Abstract
Authors
J S Videen, T Michaelis, P Pinto, B D Ross
Abstract
Authors
M Kirstein, M Rivet-Bastide, S Hatem, A Bénardeau, J J Mercadier, R Fischmeister
Abstract
Compensatory hepatic regeneration after partial hepatectomy (PH) is dependent upon the extent of resection. This study analyzes the regulation of the AP-1 transcription factor c-Jun during hepatic regeneration. There is a progressive increase in c-jun mRNA levels after sham operation, one-third PH, and two-thirds PH. A concomitant increase in AP-1 binding activity is also observed. The c-Jun protein is a major constituent of the AP-1 complex in quiescent and early regenerating liver. The activity of c-Jun nuclear kinase (JNK), which phosphorylates the activation domain of the c-Jun protein, is markedly stimulated after one-third PH. JNK1 or an immunologically related kinase is a constituent of this stimulated JNK activity after PH. When primary cultures of adult rat hepatocytes are incubated with epidermal growth factor or transforming growth factor-alpha, AP-1 transcriptional activity is increased and the activation domain of the c-Jun protein is further potentiated. Phosphopeptide mapping of the endogenous c-Jun protein in proliferating cultured hepatocytes demonstrates phosphorylation of the c-Jun activation domain. Combining the results of these in vivo and culture studies, we conclude that the minimal stimulation of one-third PH activates JNK, which phosphorylates the c-Jun activation domain in hepatocytes, resulting in enhanced transcription of AP-1-dependent genes.
Authors
J K Westwick, C Weitzel, H L Leffert, D A Brenner
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