A Prediction Rule to Identify Severe Cases among Adult Patients Hospitalized with Pandemic Influenza A (H1N1) 2009

The purpose of this study was to establish a prediction rule for severe illness in adult patients hospitalized with pandemic influenza A (H1N1) 2009. At the time of initial presentation, the baseline characteristics of those with severe illness (i.e., admission to intensive care unit, mechanical ventilation, or death) were compared to those of patients with non-severe illnesses. A total of 709 adults hospitalized with pandemic influenza A (H1N1) 2009 were included: 75 severe and 634 non-severe cases. The multivariate analysis demonstrated that altered mental status, hypoxia (PaO2/FiO2 or = 65 yr) were independent risk factors for severe cases (all P or = 2 risk factors had a higher sensitivity, specificity, positive predictive value and negative predictive value than an APACHE II score of > or = 13. As a prediction rule, the presence of > or = 2 these risk factors is a powerful and easy-to-use predictor of the severity in adult patients hospitalized with pandemic influenza A (H1N1) 2009.

The PPARgamma Agonist Rosiglitazone Inhibits Glioma Cell Proliferation and Migration in vitro and Glioma Tumor Growth in vivo

Peroxisome proliferator-activated receptor-gamma (PPARgamma) has been implicated in the growth inhibition of a number of cancer cells. In the present study, we investigated the antitumor effect of the PPARgamma agonist rosiglitazone in U87MG human glioma cells. Rosiglitazone treatment in vitro reduced cell proliferation without induction of cell death in a dose- and time-dependent manner. Rosiglitazone decreased cell migration and mRNA level of MMP-9. Rosiglitazone treatment also induced marked changes in glioma cell morphology. Oral administration of rosiglitazone in animals with subcutaneous U87MG glioma cells reduced tumor volume. Subsequent tumor tissue analysis showed that rosiglitazone decreased the number of PCNA-positive staining cells and MMP-9 expression and induced apoptosis of tumor cells. These data suggest that rosiglitazone exerts antineoplastic effect in U87MG cells and may serve as potential therapeutic agent for malignant human gliomas.

Alterations in Membrane Transport Function and Cell Viability Induced by ATP Depletion in Primary Cultured Rabbit Renal Proximal Tubular Cells

This study was undertaken to elucidate the underlying mechanisms of ATP depletion-induced membrane transport dysfunction and cell death in renal proximal tubular cells. ATP depletion was induced by incubating cells with 2.5 mM potassium cyanide (KCN)/0.1 mM iodoacetic acid (IAA), and membrane transport function and cell viability were evaluated by measuring Na+-dependent phosphate uptake and trypan blue exclusion, respectively. ATP depletion resulted in a decrease in Na+-dependent phosphate uptake and cell viability in a time-dependent manner. ATP depletion inhibited Na+-dependent phosphate uptake in cells, when treated with 2 mM ouabain, a Na+ pump-specific inhibitor, suggesting that ATP depletion impairs membrane transport functional integrity. Alterations in Na+-dependent phosphate uptake and cell viability induced by ATP depletion were prevented by the hydrogen peroxide scavenger such as catalase and the hydroxyl radical scavengers (dimethylthiourea and thiourea), and amino acids (glycine and alanine). ATP depletion caused arachidonic acid release and increased mRNA levels of cytosolic phospholipase A2 (cPLA2). The ATP depletion-dependent arachidonic acid release was inhibited by cPLA2 specific inhibitor AACOCF3. ATP depletion-induced alterations in Na+-dependent phosphate uptake and cell viability were prevented by AACOCF3. Inhibition of Na+-dependent phosphate uptake by ATP depletion was prevented by antipain and leupetin, serine/cysteine protease inhibitors, whereas ATP depletion-induced cell death was not altered by these agents. These results indicate that ATP depletion-induced alterations in membrane transport function and cell viability are due to reactive oxygen species generation and cPLA2 activation in renal proximal tubular cells. In addition, the present data suggest that serine/cysteine proteases play an important role in membrane transport dysfunction, but not cell death, induced by ATP depletion.

A1 Receptor-mediated Protection against Amyloid Beta-induced Injury in Human Neuroglioma Cells

Adenosine has been reported to provide cytoprotection in the central nervous systems as well as myocardium by activating cell surface adenosine receptors. However, the exact target and mechanism of its action still remain controversial. The present study was performed to examine whether adenosine has a protective effect against Abeta-induced injury in neuroglial cells. The astrocyte-derived human neuroglioma cell line, A172 cells, and Abeta25~35 were employed to produce an experimental Abeta-induced glial cell injury model. Adenosine significantly prevented Abeta-induced apoptotic cell death. Studies using various nucleotide receptor agonists and antagonists suggested that the protection was mediated by A1 receptors. Adenosine attenuated Abeta-induced impairment in mitochondrial functional integrity as estimated by cellular ATP level and MTT reduction ability. In addition, adenosine prevented Abeta-induced mitochondrial permeability transition, release of cytochrome c into cytosol and subsequent activation of caspase-9. The protective effect of adenosine disappeared when cells were pretreated with 5-hydroxydecanoate, a selective blocker of the mitochondrial ATP-sensitive K+ channel. In conclusion, therefore we suggest that adenosine exerts protective effect against Abeta-induced cell death of A172 cells, and that the underlying mechanism of the protection may be attributed to preservation of mitochondrial functional integrity through opening of the mitochondrial ATP-sensitive K+ channels.

Cisplatin-induced Alterations of Na+-dependent Phosphate Uptake in Renal Epithelial Cells

Cisplatin treatment increases the excretion of inorganic phosphate in vivo. However, the mechanism by which cisplatin reduces phosphate uptake through renal proximal tubular cells has not yet been elucidated. We examined the effect of cisplatin on Na+-dependent phosphate uptake in opossum kidney (OK) cells, an established proximal tubular cell line. Cells were exposed to cisplatin for an appropriate time period and phosphate uptake was measured using [32P]-phosphate. Changes in the number of phosphate transporter in membranes were evaluated by kinetic analysis, [14C]phosphonoformic acid binding, and Western blot analysis. Cisplatin inhibited phosphate uptake in a time- and dose-dependent manner, and also the Na+-dependent uptake without altering Na+-independent uptake. The cisplatin inhibition was not affected by the hydrogen peroxide scavenger catalase, but completely prevented by the hydroxyl radical scavenger dimethylthiourea. Antioxidants were ineffective in preventing the cisplatin-induced inhibition of phosphate uptake. Kinetic analysis indicated that cisplatin decreased Vmax of Na+-dependent phosphate uptake without any change in the Km value. Na+-dependent phosphonoformic acid binding was decreased by cisplatin treatment. Western blot analysis showed that cisplatin caused degradation of Na+-dependent phosphate transporter protein. Taken together, these data suggest that cisplatin inhibits phosphate transport in renal proximal tubular cells through the reduction in the number of functional phosphate transport units. Such effects of cisplatin are mediated by production of hydroxyl radicals.

Effect of P2 Nucleotide Receptor Activation on Phosphate Transport in Rabbit Proximal Tubular Cells / 대한신장학회잡지

BACKGROUND: Extracellular nucleotides act as agonists to regulate a broad range of physiological processes by interacting with P2 receptors in various tissues including the kidney tubules. This study was undertaken to evaluate the effect of P2 receptor activation on PTH-dependent regulation of phosphate transport in the renal proximal tubular cells. METHODS: Proximal tubular cells were isolated from the rabbit kidney and grown as monolayers on 24 well culture plates. Phosphate uptake was determined by measuring the uptake of radiolabeled phosphate into cell monolayers. Cyclic AMP content was determined by radioimmunoassay using [3H]cAMP assay kit. RESULTS: Activation of P2 receptors with ATP exerted differential effects on phosphate uptake and cAMP generation. In the absence of PTH, it inhibited phosphate uptake and stimulated cAMP generation. In contrast, in the presence of PTH, it attenuated PTH-induced stimulation of cAMP generation and inhibition of phosphate uptake. The profile of the effects of different P2 agonists suggested that P2Y1- and P2Y2-like receptors are involved in the effects of ATP. The effect of ATP to interfere with the PTH-induced regulation was significantly blocked by calphostin C, pertussis toxin or PKC-depletion, whereas, the effects of ATP in the absence of PTH were abolished by indomethacin. CONCLUSION: Our results suggest that PKC-dependent modification of Gi proteins and, subsequently, reduced responsiveness of adenylate cyclases is responsible for the attenuating effect of ATP on the PTH-dependent regulation of phosphate transport in rabbit proximal tubule cells.

Role of Hydroxyl Radicals and Lipid Peroxidation in Cisplatin-induced Acute Renal Failure in Rabbits / 대한신장학회잡지

PURPOSE: This study was undertaken to determine whether lipid peroxidation induced by hydroxyl radicals play a critical role in cisplatin(cis-diamminedichloroplatinum II)-induced acute renal failure. METHODS: Animals received cisplatin at a single i.p. dose of 5 mg/kg, and changes in renal function were measured at 48 hr after cisplatin injection. RESULTS: Cisplatin caused an increase in serum creatinine level, which was accompanied by reduction in GFR. The fractional excretion of Na(+), glucose, and inorganic phosphate was increased in animals treated with cisplatin alone. Cisplatin treatment in vivo inhibited PAH uptake by renal cortical slices and Na(+)-K(+)-ATPase activity in microsomal fraction. Lipid peroxidation was increased in cisplatin-treated kidneys. When animals received the antioxidant N,N'-diphenyl-p-phenylenediamine(DPPD), the iron chelator deferoxamine, and hydroxyl radical scavengers dimethylthiourea and sodium benzoate before cisplatin injection, alterations in renal function and lipid peroxidation induced by cisplatin were significantly prevented. Exposure of renal cortical slices to cisplatin in vitro caused an increase in LDH release and lipid peroxidation, which were completely prevented by DPPD and deferoxamine. By contrast, hydroxyl radical scavengers(dimethylthiourea and thiourea) did not prevent cisplatin-induced LDH release despite they inhibited cisplatin-induced lipid peroxidation. CONCLUSION: These results suggest that the lipid peroxidation resulting from generation of hydroxyl radicals may play a role in cisplatin-induced acute renal failure. In addition, the protective effects of hydroxyl radical scavengers in vivo studies are different from the results obtained from in vitro studies using renal cortical slices.

Extracellular ATP stimulates Na+ and Cl- transport through the activation of multiple purinergic receptors on the apical and basolateral membranes in M-1 mouse cortical collecting duct cells

The mammalian cortical collecting duct (CCD) plays a major role in regulating renal NaCl reabsorption, which is important in Na+ and Cl- homeostasis. The M-1 cell line, derived from the mouse cortical collecting duct, has been used as a mammalian model of the study on the electrolytes transport in CCD. M-1 cells were grown on collagen-coated permeable support and short circuit current (Isc) was measured. M-1 cells developed amiloride-sensitive current 5apprx7 days after seeding. Apical and basolateral addition of ATP induced increase in Isc in M-1 cells, which was partly retained in Na+/-free or Cl--free solution, indicating that ATP increased Na+ absorption and Cl- secretion in M-1 cells. Cl- secretion was mediated by the activation of apical cystic fibrosis transmembrane regulator (CFTR) chloride channels and Ca2+/-activated chloride channels, but Na+ absorption was not mediated by activation of epithelal sodium channel (ENaC). ATP increased cAMP content in M-1 cells. The RT-PCR analysis demonstrated that M-1 cells express P2Y2, P2X3 and P2Y4 receptors. These results showed that ATP regulates Na+ and Cl- transports via multiple P2 purinoceptors on the apical and basolateral membranes in M-1 cells.

Asymmetrical distribution of P2Y nucleotide receptors in rabbit inner medullary collecting duct cells

We cultured the rabbit inner medullary collecting duct (IMCD) cells as monolayers on collagen-coated membrane filters, and investigated distribution of the P2Y receptors by analyzing nucleotide-induced short circuit current (Isc) responses. Exposure to different nucleotides of either the apical or basolateral surface of cell monolayers stimulated Isc. Dose-response relationship and cross-desensitization studies suggested that at least 3 distinct P2Y receptors are expressed asymmetrically on the apical and basolateral membranes. A P2Y2-like receptor, which responds to UTP and ATP, is expressed on both the apical and basolateral membranes. In addition, a uracil nucleotide receptor, which responds to UDP and UTP, but not ATP, is expressed predominantly on the apical membrane. In contrast, a P2Y1-like receptor, which responds to ADP and 2-methylthio-ATP, is expressed predominantly on the basolateral membrane. These nucleotides stimulated intracellular cAMP production with an asymmetrical profile, which was comparable to that in the stimulation of Isc. Our results suggest that the adenine and uracil nucleotides can interact with different P2Y nucleotide receptors that are expressed asymmetrically on the apical and basolateral membranes of the rabbit IMCD cells, and that both cAMP- and Ca2+-dependent signaling mechanisms underlie the stimulation of Isc.

Activation of SAPK and increase in Bak levels during ceramide and indomethacin-induced apoptosis in HT29 cells

It has been reported that activation of sphingomyelin pathway and nonsteroidal anti-inflammatory drugs (NSAIDS) inhibit the promotion of colon carcinoma. Ceramide, a metabolite of sphingomyelin, and indomethacin were shown to induce apoptosis in colon carcinoma cells. However, the mechanisms of ceramide- and indomethacin-induced apoptosis in the colon carcinoma cells are not clearly elucidated. Recent studys showed that indomethacin-induced apoptosis in colon cancer cells through the cyclooxygenase-independent pathways, and that may be mediated by generation of ceramide. In this study, we compared effects of ceramide and indomethacin on important modulators of apoptotic processes in HT29 cells, a human colon cancer cell line. Ceramide and indomethacin induced apoptosis dose- and time-dependently. Ceramide and indomethacin increased stress-activated protein kinase (SAPK) activity, and decreased mitogen-activated protein kinase (MAPK) activity. The expression of Bak was increased by the treatment of ceramide and indomethacin. The expression of other Bcl-2 related proteins (Mcl-1, Bcl-XL, Bax) which were known to be expressed in colon epithelial cells was not changed during the ceramide- and indomethacin-induced apoptosis. Our results suggest that ceramide and indomethacin share common mechanisms for induction of apoptosis in HT29 cells.

Role of phospholipase A2 in hypoxia-induced renal cell injury

The present study was designed to assess the roles of PLA2 activation and arachidonic acid (AA) metabolites in hypoxia-induced renal cell injury. Hypoxia increased LDH release in a dose-dependent manner in rabbit renal cortical slices, and this increase was significant after 20-min hypoxia. The hypoxia-induced LDH release was prevented by amino acids, glycine and alanine, and extracellular acidosis (pH 6.0). Buffering intracellular Ca2+ by a chelator, but not omission of Ca2+ in the medium produced a significant reduction in hypoxia-induced LDH release. The effect of hypoxia was blocked by PLA2 inhibitors, mepacrine, butacaine, and dibucaine. A similar effect was observed by a 85-kD cPLA2 inhibitor AACOCF3. AA increased hypoxia-induced LDH release, and albumin, a fatty acid absorbent, prevented the LDH release, suggesting that free fatty acids are involved in hypoxia-induced cell injury. These results suggest that PLA2 activation and its metabolic products play important roles in pathogenesis of hypoxia-induced cell injury in rabbit renal cortical slices.

Effects of ethanol on Na+-dependent solute uptake in rabbit renal brush-border membrane vesicles

This study was undertaken to examine the effect of ethanol on Na+-dependent transport systems (glucose, phosphate, and dicarboxylate) in renal brush-border membrane vesicles (BBMV). Ethanol inhibited Na+-dependent uptakes of glucose, phosphate, and succinate in a dose-dependent manner, but not the uptakes of Na+-independent. The H+/TEA antiport was reduced by 8% ethanol. Kinetic analysis showed that ethanol caused a decrease in Vmax of three transport systems, leaving Km values unchanged. Ethanol decreased phlorizin binding, which was closely correlated with the decrease in Vmax of Na+-glucose uptake. These results indicate that ethanol inhibits Na+-dependent uptakes of glucose, phosphate, and dicaboxylate and that the reduction in Vmax of Na+-glucose uptake is caused by a decrease in the number of active carrier proteins in the membrane.

Effect of ethanol on Na+-Pi uptake in opossum kidney cells: Role of membrane fluidization and reactive oxygen species

This study was undertaken to examine the effect of ethanol on Na+-dependent phosphate (Na+-Pi) uptake in opossum kidney (OK) cells, an established renal proximal tubular cell line. Ethanol inhibited Na+-dependent component of phosphate uptake in a dose-dependent manner with I50 of 8.4%, but it did not affect Na+-independent component. Similarly, ethanol inhibited Na+-dependent uptakes of glucose and amino acids (AIB, glycine, alanine, and leucine). Microsomal Na+-K+-ATPase activity was not significantly altered when cells were treated with 8% ethanol. Kinetic analysis showed that ethanol increased Km without a change in Vmax of Na+-Pi uptake. Inhibitory effect of n-alcohols on Na+-Pi uptake was dependent on the length of the hydrocarbon chain, and it resulted from the binding of one molecule of alcohol, as indicated by the Hill coefficient (n) of 0.8-1.04. Catalase significantly prevented the inhibition, but superoxide dismutase and hydroxyl radical scavengers did not alter the ethanol effect. A potent antioxidant DPPD and iron chelators did not prevent the inhibition. Pyrazole, an inhibitor of alcohol dehydrogenase, did not attenuate ethanol-induced inhibition of Na+-Pi uptake, but it prevented ethanol-induced cell death. These results suggest that ethanol may inhibit Na+-Pi uptake through a direct action on the carrier protein, although the transport system is affected by alterations in the lipid environment of the membrane.

Apoptotic Cell Death Induced by Disruption of Epithelial Cell and Extracellular Matrix Interactions in Mouse Inner Medullary Collection Duct Cells / 대한신장학회잡지

Cell-matrix interactions have major effects upon phenotypic features such as gene regulation, cytoskeletal structure, differentiation, and aspects of cell growth control. Upon detachment from the matrix epithelial cells enter into programmed cell death and this cell detachment-induced apoptosis has been referred to as "anoikis". This study was undertaken to determine whether apoptosis is induced by inhibition of contact with extracellular matrix in mouse inner medullary collecting duct cells (mIMCD-3), what are signaling mechanisms of the process and whether EGF protects detachment-induced apoptosis. Upon detachment from the extracellular matrix, MDCK and mIMCD-3, which were derived from inner medulla of SV40 transgenic mouse, entered into programmed cell death as a time-dependent manner. Apoptotic cell death induced by cell detachment increased in serum-free medium, which was partly protected by the addition of epidermal growth factor (EGF). Ly294002 and wortmannin, inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase), negated the EGF effect, whereas PD98059, a MEK inhibitor, did not. The addition of SB203580, an inhibitor of p38 kinase, did not protect apoptosis in suspended mIMCD-3 cells. These results indicate that apoptosis is induced by inhibition of contact with extracellular matrix in mouse inner medullary collecting duct cells and that PI 3-kinase, not MAPK, is a key mediator of the EGF-induced survival of renal epithelial cells in the absence of attachment.

Effect of endothelin-1 on proliferation and differentiation of rat tracheal epithelial cells

A number of substances involved in the proliferation and differentiation of the tracheobronchial epithelium have been identified. The defects in the control of the proliferation and differentiation of tracheobronchial epithelial cells appear to constitute crucial steps in the transition of normal cells to neoplastic ones. Endothelin-1 is produced by tracheal epithelial cells, and its receptors are present in tracheal epithelial cells. However, the effect of endothelin-1 on the proliferation and differentiation of tracheal epithelial cells has not been clearly elucidated. This study was undertaken to investigate these actions of endothelin-1 in primary cultured cells of rat tracheal epithelia. Endothelin-1 stimulated proliferation of tracheal epithelial cells 1.5-fold when compared with that of control cells. Endothelin-1 increased mitogen-activated protein kinase (MAPK) activity. Herbimycin A, a tyrosine kinase inhibitor, inhibited endothelin-1-induced proliferation of epithelial cells. The treatment of endothelin-1 during the primary culture of tracheal epithelial cells increased AB-PAS-stained cell population and ciliated cell population 6.5 fold and 1.5 fold, respectively, when compared with those in control cells. The responsiveness to carbachol and forskolin in the Cl- secretion was increased 1.7 and 1.9 fold, respectively, in the endothelin-treated epithelial cells. These results indicated that endothelin-1 increases proliferation via MAPK pathway and stimulates differentiation to secretory and ciliated cells in rat tracheal epithelial cells.

cAMP-dependent signalling is involved in adenosine-stimulated Cl- secretion in rabbit colon mucosa

An important property of the intestine is the ability to secrete fluid. The intestinal secretion is regulated by a number of substances including vasoactive intestinal peptide (VIP), ATP and different inflammatory mediators. One of the most important secretagogues is adenosine during inflammation. However, the controversy concerning the underlying mechanism of adenosine-stimulated Cl- secretion in intestinal epithelial cells still continues. To investigate the effect of adenosine on Cl- secretion and its underlying mechanism in the rabbit colon mucosa, we measured short circuit current (ISC) under automatic voltage clamp with DVC-1000 in a modified Ussing chamber. Adenosine, when added to the basolateral side of the mucosa, increased ISC in a dose-dependent manner. The adenosine-stimulated ISC response was abolished when Cl- in the bath solution was replaced completely with gluconate. In addition, the ISC response was inhibited by a basolateral Na-K-Cl cotransporter blocker, bumetanide, and by apical Clchannel blockers, dephenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenyl-propylamino)-benzoate (NPPB), glibenclamide. Amiloride, an epithelial Na+ channel blocker, and 4,4-diisothiocyanato-stilbene-2,2-disulphonate (DIDS), a Ca2+-activated Cl- channel blocker, had no effect. In the mucosa pre-stimulated with forskolin, adenosine did not show any additive effect, whereas carbachol resulted in a synergistic potentiation of the ISC response. The adenosine response was inhibited by 10 micrometer H-89, an inhibitor of protein kinase A. These results suggest that the adenosine-stimulated ISC response is mediated by basolateral to apical Cl- secretion through a cAMP-dependent Cl- channel. The rank order of potencies of adenosine receptor agonists was 5'-(N-ethylcarboxamino)adenosine(NECA) > N6-(R-phenylisopropyl)adenosine(R-PIA)>2-(p-(2-carbonylethyl)-phenyl-et hylamino)-5'-N-ethylcarboxaminoadenosine(CGS21680). From the above results, it can be concluded that adenosine interacts with the A2b adenosine receptor in the rabbit colon mucosa and a cAMP-dependent signalling mechanism underlies the stimulation of Cl- secretion.

Role of phospholipase A2 in oxidant-induced alteration in phosphate transport in primary cultured rabbit renal proximal tubule cells

The present study was undertaken to examine the role of phospholipase A2 (PLA2) in oxidant-induced inhibition of phosphate transport in primary cultured rabbit renal proximal tubule cells. Uptakes of phosphate and glucose were dose-dependently inhibited by an oxidant t-butylhydroperoxide (tBHP), and the significant inhibition appeared at 0.025 mM of tBHP, whereas tBHP-induced alterations in lipid peroxidation and cell viability were seen at 0.5 mM. tBHP stimulated arachidonic acid (AA) release in a dose-dependent fashion. A PLA2 inhibitor mepacrine prevented tBHP-induced AA release, but it did not alter the inhibition of phosphate uptake and the decrease in cell viability induced by tBHP. tBHP-induced inhibition of phosphate transport was not affected by a PKC inhibitor, staurosporine. tBHP at 0.1 mM did not produce the inhibition of Na+-K+-ATPase activity in microsomal fraction, although it significantly inhibited at 1.0 mM. These results suggest that tBHP can inhibit phosphate uptake through a mechanism independent of PLA2 activation, irreversible cell injury, and lipid peroxidation in primary cultured rabbit renal proximal tubular cells.

Beneficial Effect of Verapamil Against Ischemic Acute Renal Failure in Rabbits / 대한신장학회잡지

This study was undertaken to determine whether verapamil protects renal function in rabbits with ischemic acute renal failure. Renal ischemia was induced by clamping bilateral renal arteries for 60 min. One group received intravenously an infusion of verapamil (lmg/kg) for 30 min prior to initiation of renal artery clamping and the other group received equal volume of saline. Renal blood flow was measured with flowmeter before (basal) and 24 hr after ischemia. Serum creatinine level increased 24 hr after ischemia and remained high to 72 hr. When verapamil was pretreated, the level 48 and 72 hr after ischemia was significantly decreased compared with saline insusion. Urine flow was markedly decreased 24 hr after ischemia and remained depressed to 72 hr, but it was significantly increased 72 hr after ischemia in verapa- mil-pretreatment animals as compared with the saline-infusion animals. GFR were markedly reduced 24 hr after ischemia and remained depressed to 72 hr, which was significantly prevented by verapamil pretreatment. Ischemia caused a significant increase in FEVa and a reduction in Uosm, and TcH2O, indicating impairment in urine concentrating ability of tubules, and the impairment was significantly attenuated by verapamil. The uptake of p-aminohippurate in cortical slices was depressed by ischemia, which was significantly prevented by verapamil pretreatment. In salineinfusion animals, renal blood flow was not significantly different between the basal value and that after 24 hr of reflow. Renal blood flow was not significantly altered by verapamil pretreatment. Anoxia/reoxygenation injury in the control renal slices was not significantly prevented by Ca channel blockers. These results suggest that verapamil exerts a protective effect in ichemic acute renal failure, and the beneficial effects may be attributed to effects other than vasodilation. These data also indicate that a reduction in GFR following ischemia does not result from change in renal blood flow.

Effect of t-butylhydroperoxide on Na+/-dependent glutamate uptake in rabbit brain synaptosome

The effect of an organic peroxide, t-butylhydroperoxide (t-BHP), on glutamate uptake was studied in synaptosomes prepared from cerebral cortex. t-BHP inhibited the Na+/-dependent glutamate uptake with no change in the Na+/-independent uptake. This effect of t-BHP was not altered by addition of Ca2+ channel blockers (verapamil, diltiazem and nifedipine) or PLA2 inhibitors (dibucaine, butacaine and quinacrine). However, the effect was prevented by iron chelators (deferoxamine and phenanthroline) and phenolic antioxidants (N,N'-diphenyl-phenylenediamine, butylated hydroxyanisole, and butylated hydroxytoluene). At low concentrations (< 1.0 mM), t-BHP inhibited glutamate uptake without altering lipid peroxidation. Moreover, a large increase in lipid peroxidation by ascorbate/Fe2+ was not accompanied by an inhibition of glutamate uptake. The impairment of glutamate uptake by t-BHP was not intimately related to the change in Na+/-K+/-ATPase activity. These results suggest that inhibition of glutamate uptake by t-BHP is not totally mediated by peroxidation of membrane lipid, but is associated with direct interactions of glutamate transport proteins with t-BHP metabolites. The Ca2+ influx through Ca2+ channel or PLA2 activation may not be involved in the t-BHP inhibition of glutamate transport.

Functional characteristics of neutral amino acid transporter in opossum kidney (OK) cells

The characteristics of Na+/-dependent cycloleucine uptake was investigated in OK cells with regard to substrate specificity and regulation by protein kinase C (PKC). Inhibition studies with different synthetic and natural amino acids showed a broad spectrum affinity to neutral amino acids regardless of their different side chains including branched or aromatic, indicating that the Na+/-dependent cycloleucine uptake in OK cells is mediated by System B-o or System B degree -like transporter rather than the classical System A or ASC. Phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate, but not 4 alpha-PMA elicited a time-dependent biphasic stimulation of Na+/-dependent cycloleucine uptake, which produced early transient peak at 30 min and late sustained peak at 180 min. Both the early and late stimulations by PMA were due to an increase in Vmax and not due to a change in Km. PKC inhibitors blocked both the early and late stimulation by PMA, while protein synthesis inhibitors blocked the late stimulation only. These results suggest the existence and regulation by PKC of System B degree or System B degree -like broad spectrum transport system for neutral amino acids in OK cells.