Robert Jarzyna

Modulation of endothelial cell migration by extracellular nucleotides. Involvement of focal adhesion kinase and phosphatidylinositol 3-kinase - mediated pathways

Extracellular nucleotides bind to type-2 purinergic/pyrimidinergic (P2) receptors that mediate various responses, such as cell activation, proliferation and apoptosis, implicated in inflammatory processes. The role of P2 receptors and their associated signal transduction pathways in endothelial cell responses has not been fully investigated. Here, it is shown that stimulation of human umbilical vein endothelial cells (HUVEC) with extracellular ATP or UTP increased intracellular free calcium ion concentrations ([Ca(2+)](i)), induced phosphorylation of focal adhesion kinase (FAK), p130(cas) and paxillin, and caused cytoskeletal rearrangements with consequent cell migration. Furthermore, UTP increased migration of HUVEC in a phosphatidylinositol 3-kinase (PI3-K)-dependent manner. BAPTA or thapsigargin inhibited the extracellular nucleotide-induced increase in [Ca(2+)](i), a response crucial for both FAK phosphorylation and cell migration. Furthermore, long-term exposure of HUVEC to ATP and UTP, agonists of the G protein-coupled P2Y2 and P2Y4 receptor subtypes, caused upregulation of alpha(v) integrin expression, a cell adhesion molecule known to directly interact with P2Y2 receptors. Our results suggest that extracellular nucleotides modulate signaling pathways in HUVEC influencing cell functions, such as cytoskeletal changes, cellular adhesion and motility, typically associated with integrin-activation and the action of growth factors. We propose that P2Y2 and possibly P2Y4 receptors mediate those responses that are important in vascular inflammation, atherosclerosis and angiogenesis.

Extracellular Nucleotides and Adenosine Independently Activate AMP-Activated Protein Kinase in Endothelial Cells Involvement of P2 Receptors and Adenosine Transporters

AMP-activated protein kinase (AMPK) plays a key role in the regulation of energy homeostasis and is activated in response to cellular stress, including hypoxia/ischemia and hyperglycemia. The stress events are accompanied by rapid release of extracellular nucleotides from damaged tissues or activated endothelial cells (EC) and platelets. We demonstrate that extracellular nucleotides (ATP, ADP, and UTP, but not UDP) and adenosine independently induce phosphorylation and activation of AMPK in human umbilical vein EC (HUVEC) by the mechanism that is not linked to changes in AMP:ATP ratio. HUVEC express NTPDases, as well as 5′-nucleotidase; hence, nucleotides can be metabolized to adenosine. However, inhibition of 5′-nucleotidase had no effect on ATP/ADP/UTP-induced phospho- rylation of AMPK, indicating that AMPK activation occurred as a direct response to nucleotides. Nucleotide-evoked phosphorylation of AMPK in HUVEC was mediated by P2Y1, P2Y2, and/or P2Y4 receptors, whereas P2Y6, P2Y11, and P2X receptors were not involved. The nucleotide-induced phosphorylation of AMPK was affected by changes in the concentration of intracellular Ca2+ and by Ca2+/calmodulin-dependent kinase kinase (CaMKK), although most likely it was not dependent on LKB1 kinase. Adenosine-induced phosphorylation of AMPK was not mediated by P1 receptors but required adenosine uptake by equilibrative nucleoside transporters followed by its (intracellular) metabolism to AMP. Moreover, adenosine effect was Ca2+ and CaMKK independent, although probably associated with upstream LKB1. We hypothesize that P2 receptors and adenosine transporters could be novel targets for the pharmacological regulation of AMPK activity and its downstream effects on EC function.

Inhibition of gluconeogenesis by vanadium and metformin in kidney-cortex tubules isolated from control and diabetic rabbits.

Effect of vanadyl acetylacetonate (VAc) and metformin on gluconeogenesis has been studied in isolated hepatocytes and kidney-cortex tubules of rabbit. Glucose formation from alanine+glycerol+octanoate, pyruvate or dihydroxyacetone was inhibited by 50-80% by 100 microM VAc or 500 microM metformin in renal tubules of control and alloxan-diabetic animals, while the inhibitory action of these compounds in hepatocytes was less pronounced (by about 20-30%). In contrast to VAc, metformin increased the rate of lactate formation by about 2-fold in renal tubules incubated with alanine+glycerol+octanoate. In view of VAc-induced changes in intracellular gluconeogenic intermediates and gluconeogenic enzyme activities, it is likely that this compound may decrease fluxes through pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase and glucose-6-phosphatase. In contrast to VAc, metformin-induced decrease in renal gluconeogenesis may result from a decline of cytosolic oxaloacetate level and consequently PEPCK activity. Following 6 days of VAc administration (1.275 mg Vkg(-1) body weight daily) the blood glucose level in alloxan-diabetic rabbits was normalised while blood glucose changes in control animals were not observed. On the contrary, in diabetic animals treated for 6 days with metformin (200 mg kg(-1) body weight day(-1)) a high blood glucose level was maintained. Unfortunately, VAc-treated control and diabetic rabbits exhibited elevated serum urea and creatinine levels. In VAc-treated animals vanadium was accumulated in kidney-cortex up to 7.6+/-0.6 microg Vg(-1) dry weight. In view of a potential vanadium nephrotoxicity a therapeutic application of vanadium compounds needs a critical re-evaluation.

The inhibition of gluconeogenesis by chloroquine contributes to its hypoglycaemic action

The effect of chloroquine on gluconeogenesis in isolated hepatocytes and kidney-cortex tubules of rabbit has been studied. The inhibitory action of 200 microM chloroquine was the highest in hepatocytes and renal tubules incubated with glutamine and glutamate+glycerol+octanoate, respectively, while in the presence of other substrates the drug action was less pronounced. With amino acids as substrates, the inhibition of gluconeogenesis was accompanied by a decreased glutamine production, resulting from a decline of glutamate dehydrogenase activity. A decrease in the urea production by hepatocytes incubated with chloroquine in the presence of glutamine but not NH4Cl as the source of ammonium is in agreement with this suggestion. The degree of inhibition by chloroquine of the rate of gluconeogenesis in renal tubules isolated from control rabbits was similar to that determined in diabetic animals. Chloroquine-induced changes in levels of intracellular gluconeogenic intermediates indicate a decrease in phosphoenolpyruvate carboxykinase and glucose-6-phosphatase activities probably due to increased concentration of 2-oxoglutarate, an inhibitor of these two enzymes. In view of the data, it is likely that inhibition by chloroquine of glucose formation in liver and kidney may contribute to the hypoglycaemic action of this drug. The importance of the inhibitory effect of chloroquine on glutamate dehydrogenase activity in the antihyperglycaemic action of the drug is discussed.

The inhibition of gluconeogenesis by gatifloxacin may contribute to its hypoglycaemic action

The action of gatifloxacin, the broad-spectrum fluoroquinolone antibiotic commonly used in the therapy of various bacterial infections, was investigated in isolated rabbit hepatocytes and kidney-cortex tubules by measuring the activity of gluconeogenesis, a process that maintains whole body glucose homeostasis. The data show that in kidney-cortex tubules, application of gatifloxacin at up to 100 microM was followed by a marked accumulation of the drug in the intracellular milieu and a decrease in the rate of glucose formation from pyruvate by 20-50%. Gatifloxacin did not affect the rate of gluconeogenesis from either alanine + glycerol + octanoate or aspartate + glycerol + octanoate. At concentrations between 25 and 200 microM the drug decreased mitochondrial oxygen consumption by 20-45% with pyruvate + malate and ADP. As in the case of alpha-cyano-4-hydroxycinnamate, a well-established inhibitor of the mitochondrial pyruvate transporter, it diminished pyruvate uptake by both renal and hepatic mitochondria. The inhibitory action of gatifloxacin was less pronounced in hepatocytes where reduction in pyruvate-dependent glucose formation and mitochondrial respiration was by no more than 25%. The antibiotic did not influence mitochondrial oxygen consumption with glutamate + malate in either kidney-cortex or liver mitochondria. A differential substrate dependence of gatifloxacin action on gluconeogenesis and mitochondrial respiration combined with a decrease in pyruvate uptake by mitochondria suggest that the inhibitory action of this drug on gluconeogenesis might result from its impairment of pyruvate transport into mitochondria.

ERK1/2 pathway is involved in renal gluconeogenesis inhibition under conditions of lowered NADPH oxidase activity

The aim of this study was to elucidate the mechanisms involved in the inhibition of renal gluconeogenesis occurring under conditions of lowered activity of NADPH oxidase (Nox), the enzyme considered to be one of the main sources of reactive oxygen species in kidneys. The in vitro experiments were performed on primary cultures of rat renal proximal tubules, with the use of apocynin, a selective Nox inhibitor, and TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a potent superoxide radical scavenger. In the in vivo experiments, Zucker diabetic fatty (ZDF) rats, a well established model of diabetes type 2, were treated with apocynin solution in drinking water. The main in vitro findings are the following: (1) both apocynin and TEMPOL attenuate the rate of gluconeogenesis, inhibiting the step catalyzed by phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of the process; (2) in the presence of the above-noted compounds the expression of PEPCK and the phosphorylation of transcription factor CREB and ERK1/2 kinases are lowered; (3) both U0126 (MEK inhibitor) and 3-(2-aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione (ERK inhibitor) diminish the rate of glucose synthesis via mechanisms similar to those of apocynin and TEMPOL. The observed apocynin in vivo effects include: (1) slight attenuation of hyperglycemia; (2) inhibition of renal gluconeogenesis; (3) a decrease in renal PEPCK activity and content. In view of the results summarized above, it can be concluded that: (1) the lowered activity of the ERK1/2 pathway is of importance for the inhibition of renal gluconeogenesis found under conditions of lowered superoxide radical production by Nox; (2) the mechanism of this phenomenon includes decreased PEPCK expression, resulting from diminished activity of transcription factor CREB; (3) apocynin-evoked inhibition of renal gluconeogenesis contributes to the hypoglycemic action of this compound observed in diabetic animals. Thus, the study has delivered some new insights into the recently discussed issue of the usefulness of Nox inhibition as a potential antidiabetic strategy.

Effect of polyamines on glutamate dehydrogenase within permeabilized kidney-cortex mitochondria and isolated renal tubules of rabbit

The effect of polyamines on glutamate dehydrogenase [L-glutamate: NAD(P) oxidoreductase (deaminating) [EC 1.4.1.3]) activity has been studied in both permeabilized kidney-cortex mitochondria and isolated renal tubules of rabbit. Spermidine was the most potent inhibitor of glutamate synthesis in permeabilized mitochondria resulting in about 80% decrease of the enzyme activity at 5 mM concentration. Putrescine, alpha-monofluoromethylputrescine (MFMP) and (R,R)-delta-methyl-alpha-acetylenic-putrescine (MAP) were more efficient than spermine. The inhibitory action of polyamines was potentiated by an elevated NADH content in the reaction mixture. Increasing concentrations of either NH4Cl, KCl or NaCl in the incubation medium resulted in a decrease of polyamine-induced inhibition of the enzyme activity, indicating that monovalent cations can compete with polyamines for the binding site at glutamate dehydrogenase. The inhibitory action of spermidine on glutamate synthesis was abolished by 2 mM ADP or 10 mM L-leucine, allosteric activators of the enzyme, as well as on the addition of either oxalate or sulphate at 20 mM concentrations. Spermidine did not affect glutamate formation when NADH was substituted by NADPH, suggesting an importance of the NADH binding to the inhibitory site of the enzyme for a decrease of reductive amination of 2-oxoglutarate by polyamine. Although spermidine did not influence glutamate deamination in the presence of NAD+, it stimulated this process by about 70% when NAD+ was substituted by NADP+. In the presence of ADP the stimulatory effect of polyamine was not significant. The data indicate that in permeabilized rabbit kidney-cortex mitochondria the effect of polyamines on both glutamate formation and glutamate deamination via the reaction catalysed by glutamate dehydrogenase is dependent upon the coenzyme utilized by the enzyme. In the presence of NADH their inhibitory effect on the glutamate formation may be alleviated by allosteric activators of the enzyme, and concentrations of potassium, sodium, sulphate and oxalate. In isolated rabbit renal tubules incubated with 5 mM methionine sulfoximine and aminooxyacetate, in order to inhibit glutamine synthetase and aminotransferases, respectively, 5 mM spermidine decreased glutamate formation by about 30%, while putrescine and spermine did not significantly diminish the enzyme activity. In the presence of octanoate glutamate formation was reduced by about 30% by naturally occurring polyamines as well as MFMP and MAP, indicating that under these conditions NADH rather than NADPH is utilized as the coenzyme. In view of these data it is possible to suggest that polyamines may be of importance to control glutamate dehydrogenase activity under physiological conditions.

Differentialin vivo andin vitro effect of gentamicin on glutamate synthesis and glutamate deamination in rabbit kidney-cortex tubules and mitochondria

The effect of gentamicin on both glutamate synthesis and glutamate deamination was studied in kidney-cortex mitochondria and tubules isolated from both control and gentamicin-treated animals. In kidney-cortex mitochondria which were permeabilized in order to make a free access of substrates and antibiotic to the glutamate dehydrogenase, gentamicin appeared to be a very potent inhibitor of glutamate synthesis, resulting in about 60% decrease of the enzyme activity at 5 mM concentration. Other aminoglycoside antibiotics decreased the enzymatic activity, in the following order: gentamicin > neomycin = tobramycin = kanamycin > biodacyna > amikacin > streptomycin. This, in principle, corresponds to their known nephrotoxic potential observed in vivo. The inhibitory action of antibiotics was abolished by neither ADP nor leucine, allosteric activators of glutamate dehydrogenase. Surprisingly, gentamicin did not decrease the rate of ammonia formation from glutamate when added to both renal tubules and mitochondria isolated from control rabbits. This indicates that the antibiotic exerts its inhibitory effect on glutamate dehydrogenase activity in the direction of glutamate synthesis only. In contrast, the rate of both glutamate deamination and glutamate synthesis was about 40% lower in renal tubules and mitochondria isolated from kidney-cortex of animals which were given antibiotics for 10 days. In view of these results it seems that (i) the depression of ammoniagenesis in gentamicin-treated animals may be due to a decrease of glutamate dehydrogenase content and (ii) under conditions in vitro the aminoglycoside inhibits the enzyme activity in the direction of glutamate synthesis while it does not affect the glutamate deamination.

Extracellular Nucleotides and Adenosine Independently Activate AMP-Activated Protein Kinase in Endothelial Cells

AMP-activated protein kinase (AMPK) plays a key role in the regulation of energy homeostasis and is activated in response to cellular stress, including hypoxia/ischemia and hyperglycemia. The stress events are accompanied by rapid release of extracellular nucleotides from damaged tissues or activated endothelial cells (EC) and platelets. We demonstrate that extracellular nucleotides (ATP, ADP, and UTP, but not UDP) and adenosine independently induce phosphorylation and activation of AMPK in human umbilical vein EC (HUVEC) by the mechanism that is not linked to changes in AMP:ATP ratio. HUVEC express NTPDases, as well as 5′-nucleotidase; hence, nucleotides can be metabolized to adenosine. However, inhibition of 5′-nucleotidase had no effect on ATP/ADP/UTP-induced phospho- rylation of AMPK, indicating that AMPK activation occurred as a direct response to nucleotides. Nucleotide-evoked phosphorylation of AMPK in HUVEC was mediated by P2Y1, P2Y2, and/or P2Y4 receptors, whereas P2Y6, P2Y11, and P2X receptors were not involved. The nucleotide-induced phosphorylation of AMPK was affected by changes in the concentration of intracellular Ca2+ and by Ca2+/calmodulin-dependent kinase kinase (CaMKK), although most likely it was not dependent on LKB1 kinase. Adenosine-induced phosphorylation of AMPK was not mediated by P1 receptors but required adenosine uptake by equilibrative nucleoside transporters followed by its (intracellular) metabolism to AMP. Moreover, adenosine effect was Ca2+ and CaMKK independent, although probably associated with upstream LKB1. We hypothesize that P2 receptors and adenosine transporters could be novel targets for the pharmacological regulation of AMPK activity and its downstream effects on EC function.

Extracellular Nucleotides and Adenosine Independently Activate AMP-Activated Protein Kinase in Endothelial Cells. Involvement of P2 Receptors and Adenosine Transporters: AMPK activation by nucleotides and adenosine

AMP-activated protein kinase (AMPK) plays a key role in the regulation of energy homeostasis and is activated in response to cellular stress, including hypoxia/ischemia and hyperglycemia. The stress events are accompanied by rapid release of extracellular nucleotides from damaged tissues or activated endothelial cells (EC) and platelets. We demonstrate that extracellular nucleotides (ATP, ADP, and UTP, but not UDP) and adenosine independently induce phosphorylation and activation of AMPK in human umbilical vein EC (HUVEC) by the mechanism that is not linked to changes in AMP:ATP ratio. HUVEC express NTPDases, as well as 5′-nucleotidase; hence, nucleotides can be metabolized to adenosine. However, inhibition of 5′-nucleotidase had no effect on ATP/ADP/UTP-induced phospho- rylation of AMPK, indicating that AMPK activation occurred as a direct response to nucleotides. Nucleotide-evoked phosphorylation of AMPK in HUVEC was mediated by P2Y1, P2Y2, and/or P2Y4 receptors, whereas P2Y6, P2Y11, and P2X receptors were not involved. The nucleotide-induced phosphorylation of AMPK was affected by changes in the concentration of intracellular Ca2+ and by Ca2+/calmodulin-dependent kinase kinase (CaMKK), although most likely it was not dependent on LKB1 kinase. Adenosine-induced phosphorylation of AMPK was not mediated by P1 receptors but required adenosine uptake by equilibrative nucleoside transporters followed by its (intracellular) metabolism to AMP. Moreover, adenosine effect was Ca2+ and CaMKK independent, although probably associated with upstream LKB1. We hypothesize that P2 receptors and adenosine transporters could be novel targets for the pharmacological regulation of AMPK activity and its downstream effects on EC function.