Effect of albumin on 14C-alpha-Methyl-D-Glucopyranoside uptake in primary cultured renal proximal tubule cells: involvement of PLC, MAPK, and NF-kappaB

A growing body of evidence implicates albumin has an important regulatory function in renal proximal tubule cells (PTCs). In present study, the effect of bovine serum albumin (BSA) on 14C-alpha-methyl-D-glucopyranoside (alpha-MG) uptake and its related signal molecules were examined in the primary cultured rabbit renal PTCs. BSA significantly increased uptake of alpha-MG, a distinctive proximal tubule marker, as well as expression level of Na+/glucose cotransporters (SGLT1 and SGLT2) proteins. The BSA-induced increase of alpha-MG uptake was completely blocked by actinomycin D and cycloheximide. Neomycin or U 73122 (PLC inhibitors), BAPTA/AM or TMB-8 (intracellular Ca2+ mobilization inhibitors) completely abolished BSA-induced increase of alpha-MG uptake. BSA significantly increased IPs accumulation, but did not affect Ca2+ uptake. Effect of BSA on alpha-MG uptake was blocked by PD 98059, but did not SB 203580. BSA increased phosphorylation of p44/42 mitogen activated protein kinase (MAPK) in a time-dependent manner. NAC or catalase (antioxidants) significantly blocked BSA-induced increase of H2O2 formation and alpha-MG uptake. BSA activated NF-kappaB translocation into nucleus. PDTC, SN50, and TLCK (NF-kappaB inhibitors) also completely blocked BSA-induced increase of alpha-MG uptake, NF-kappaB p65 and phospho IkappaB-alpha activation. In conclusion, BSA stimulates alpha-MG uptake and its action is partially correlated with PLC, MAPK, or NF-kappaB signal molecules in primary cultured renal PTCs.     

High glucose inhibits fructose uptake in renal proximal tubule cells: involvement of cAMP, PLC/PKC, p44/42 MAPK, and cPLA2

The precise signal that regulates fructose transport in renal proximal tubule cells (PTCs) under high glucose conditions is not yet known although fructose has been recommended as a substitute for glucose in the diets of diabetic people. Thus, we investigated that effect of high glucose on fructose uptake and its signaling pathways in primary cultured rabbit renal PTCs. Glucose inhibited the fructose uptake in a time- and dose-dependent manner. A maximal inhibitory effect of glucose on fructose uptake was observed at 25 mM glucose after 48 h, while 25 mM mannitol and l-glucose did not affect fructose uptake. Indeed, 25 mM glucose for 48 h decreased GLUT5 protein level. Thus, the treatment of 25 mM glucose for 48 h was used for this study. Glucose-induced (25 mM) inhibition of fructose uptake was blocked by pertussis toxin (PTX), SQ-22536 (an adenylate cyclase inhibitor), and myristoylated amide 14-22 (a protein kinase A inhibitor). Indeed, 25 mM glucose increased the intracellular cAMP content. Furthermore, 25 mM glucose-induced inhibition of fructose uptake was prevented by neomycin or U-73122 (phospholipase C inhibitors) and staurosporine or bisindolylmaleimide I (protein kinase C inhibitors). In fact, 25 mM glucose increased the total PKC activity and translocation of PKC from the cytosolic to membrane fraction. In addition, PD 98059 (a p44/42 mitogen-activated protein kinase (MAPK) inhibitor) but not SB 203580 (a p38 MAPK inhibitor) and mepacrine or AACOCF3 (phospholipase A2 inhibitors) blocked 25 mM glucose-induced inhibition of fructose uptake. Results of Western blotting using the p44/42 MAPK and GLUT5 antibodies were consistent with the results of uptake experiments. In conclusion, high glucose inhibits the fructose uptake through cAMP, PLC/PKC, p44/42 MAPK, and cytosolic phospholipase A2 (cPLA2) pathways in the PTCs.     

Effect of epinephrine on alpha-methyl-D-glucopyranoside uptake in renal proximal tubule cells.

Effect of epinephrine on alpha-methyl-D-glucopyranoside uptake in renal proximal tubule cells. Epinephrine has known to be a very important factor in the regulation of renal sodium excretion. However, the effect of epinephrine on Na+/glucose cotransporter was not fully elucidated. Thus, we examined effect of epinephrine on alpha-methyl-D-glucopyranoside (alpha-MG) uptake and its related signal pathways in the primary cultured rabbit renal proximal tubule cells (PTCs). Epinephrine inhibited alpha-MG uptake in a time- and dose-dependent manner and also decreased SGLT1 and SGLT2 protein level. Both phentolamine and propranolol completely prevented epinephrine-induced inhibition of alpha-MG uptake. The epinephrine-induced inhibition of alpha-MG uptake was blocked by SQ-22536 or myristoylated PKA inhibitor amide 14-22 and epinephrine increased the intracellular cAMP content. In western blotting analysis, epinephrine increases phosphorylation of p44/42 and p38 MAPKs and PD 98059 or SB 203580 blocked the effect of epinephrine. In addition, epinephrine increased AA release and PGE2 production and effects of epinephrine on alpha-MG uptake and AA release were blocked by staurosporine and bisindolylmaleimide I or mepacrine and AACOCF3. Indeed, epinephrine translocated PKC or cPLA2 from cytosol to membrane fraction. In conclusion, epinephrine partially inhibits the alpha-MG uptake through PKA, PKC, p44/42, p38 MAPK, and cPLA2 pathways in the PTCs.     

Effect of adenosine triphosphate on phosphate uptake in renal proximal tubule cells: involvement of PKC and p38 MAPK

ATP has been known to act as an extracellular signal and to be involved in various functions of kidney. Renal proximal tubular reabsorption of phosphate (Pi) contributes to the maintenance of phosphate homeostasis, which is regulated by Na+/Pi cotransporter. However, the effects of ATP on Na+/Pi cotransporters were not elucidated in proximal tubule cells (PTCs). Thus, the effects of ATP on Na+/Pi cotransporter and its related signal pathways are examined in the primary cultured renal PTCs. In the present study, ATP inhibited Pi uptake in a time (> 1 h) and dose (>10(-6)M) dependent manner. ATP-induced inhibition of Pi uptake was correlated with the decrease of type II Na+/Pi cotransporter mRNA. ATP-induced inhibition of Pi uptake may be mediated by P2Y receptor activation, since suramin (non-specific P2 receptor antagonist) and RB-2 (P2Y receptor antagonist) blocked it. ATP-induced inhibition of Pi uptake was blocked by neomycin, U73122 (phospholipase C (PLC) inhibitors), bisindolylmaleimide I, H-7, and staurosporine (protein kinase C (PKC) inhibitors), suggesting the role of PLC/PKC pathway. ATP also increased inositol phosphates (IPs) formation and induced PKC translocation from cytosolic fraction to membrane fraction. In addition, ATP-induced inhibition of Pi uptake was blocked by SB 203580 [a p38 mitogen activated protein kinase (MAPK) inhibitor], but not by PD 98059 (a p44/42 MAPK inhibitor). Indeed, ATP induced phosphorylation of p38 MAPK, which was not blocked by PKC inhibitor. In conclusion, ATP inhibited Pi uptake via PLC/PKC as well as p38 MAPK in renal PTCs.     

High glucose induced translocation of Aquaporin8 to chicken hepatocyte plasma membrane: involvement of cAMP, PI3K/Akt, PKC, MAPKs, and microtubule

Aquaporin8 (AQP8) is a transmembrane water channel that is found mainly in hepatocytes. The direct involvement of AQP8 in high glucose condition has not been established. Therefore, this study examined the effects of high glucose on AQP8 and its related signal pathways in primary cultured chicken hepatocytes. High glucose increased the movement of AQP8 from the intracellular membrane to plasma membrane in a 30 mM glucose concentration and in a time- (> or =10 min) dependent manner. On the other hand, 30 mM mannitol did not affect the translocation of AQP8, which suggested the absence of osmotic effect. Thirty millimolar glucose increased intracellular cyclic adenosine 3, 5-monophosphate (cAMP) level. Moreover, high glucose level induced Akt phosphorylation, protein kinase C (PKC) activation, p44/42 mitogen-activated protein kinases (MAPKs), p38 MAPK, and c-jun NH2-terminal kinase (JNK) phosphorylation. On the other hand, inhibition of each pathway by SQ 22536 (adenylate cyclase inhibitor), LY 294002 (PI3-K phosphatidylinositol 3-kinase inhibitor), Akt inhibitor, staurosporine (PKC inhibitor), PD 98059 (MEK inhibitor), SB 203580 (p38 MAPK inhibitor), or SP 600125 (JNK inhibitor) blocked 30 mM glucose-induced AQP8 translocation, respectively. In addition, inhibition of microtubule movement with nocodazole blocked high glucose-induced AQP8 translocation. High glucose level also increased the level of kinesin light chain and dynein protein expression. In conclusion, high glucose level stimulates AQP8 via cAMP, PI3-K/Akt, PKC, and MAPKs pathways in primary cultured chicken hepatocytes.     

Epidermal growth factor inhibits 14C-alpha-methyl-D-glucopyranoside uptake in renal proximal tubule cells: involvement of PLC/PKC, p44/42 MAPK, and cPLA2

The effect of EGF on (14)C-alpha-methyl-D-glucopyranoside (alpha-MG) uptake and its related signaling pathways were examined in primary cultured rabbit renal proximal tubule cells (PTCs). Epidermal growth factor (EGF) (50 ng/ml) was found to inhibit alpha-MG uptake, a distinctive proximal tubule marker. The EGF effect was blocked by AG1478 (an EGF receptor antagonist) or genistein and herbimycin (tyrosine kinase inhibitors), respectively. In addition, the EGF-induced inhibition of alpha-MG uptake was blocked by neomycin and U73122 (phospholipase C inhibitors) as well as staurosporine, H-7, and bisindolylmaleimide I (protein kinase C inhibitors). EGF was also observed to increase inositol phosphate formation. Furthermore, both the EGF-induced inhibition of alpha-MG uptake and increase of arachidonic acid (AA) release were blocked by AACOCF(3) (a cytosolic phospholipase A(2) inhibitor), indomethacin (a cyclooxygenase inhibitor), and econazole (a cytochrome P-450 epoxygenase inhibitor). We examined the involvement of mitogen-activated protein kinases (MAPKs) in mediating the effect of EGF on alpha-MG uptake. Indeed, EGF increased phosphorylation of p44/p42 MAPK and the EGF-induced inhibition of alpha-MG uptake as well as the stimulatory effect of EGF on AA release was blocked by PD 98059 (a p44/42 MAPK inhibitor), suggesting a causal relationship. However, inhibitors of PKC also prevented the EGF-induced increase of AA release. In conclusion, EGF partially inhibited alpha-MG uptake via PLC/PKC, p44/42 MAPK, and PLA(2) signaling pathways.     

IL-1beta signaling in cat lower esophageal sphincter circular muscle

In a cat model of acute experimental esophagitis, resting in vivo lower esophageal sphincter (LES) pressure and in vitro tone are lower than in normal LES, and the LES circular smooth muscle layer contains elevated levels of IL-1beta that decrease the LES tone of normal cats. We now examined the mechanisms of IL-1beta-induced reduction in LES tone. IL-1beta significantly reduced acetylcholine-induced Ca(2+) release in Ca(2+)-free medium, and this effect was partially reversed by catalase, demonstrating a role of H(2)O(2) in these changes. IL-1beta significantly increased the production of H(2)O(2), and the increase was blocked by the p38 MAPK inhibitor SB-203580, by the cytosolic phospholipase A(2) (cPLA(2)) inhibitor AACOCF3, and by the NADPH oxidase inhibitor apocynin, but not by the MEK1 inhibitor PD-98059. IL-1beta significantly increased the phosphorylation of p38 MAPK and cPLA(2). IL-1beta-induced cPLA(2) phosphorylation was blocked by SB-203580 but not by AACOCF3, suggesting sequential activation of p38 MAPK-phosphorylating cPLA(2). The IL-1beta-induced reduction in LES tone was partially reversed by AACOCF3 and by the Ca(2+)-insensitive PLA(2) inhibitor bromoenol lactone (BEL). IL-1beta significantly increased cyclooxygenase (COX)-2 and PGE(2) levels. The increase in PGE(2) was blocked by SB-203580, AACOCF3, BEL, and the COX-2 inhibitor NS-398 but not by PD-98059 or the COX-1 inhibitor valeryl salicylate. The data suggested that IL-1beta reduces LES tone by producing H(2)O(2), which may affect Ca(2+)-release mechanisms and increase the synthesis of COX-2 and PGE(2). Both H(2)O(2) and PGE(2) production depend on sequential activation of p38 MAPK and cPLA(2). cPLA(2) activates NADPH oxidases, producing H(2)O(2), and may produce arachidonic acid, converted to PGE(2) via COX-2.     

Induction of cyclooxygenase-2 by lipopolysaccharide in canine tracheal smooth muscle cells: involvement of p42/p44 and p38 mitogen-activated protein kinases and nuclear factor-kappaB pathways

Lipopolysaccharide (LPS) was found to induce inflammatory responses in the airways and exerted as a potent stimulus for PG synthesis. This study was to determine the mechanisms of LPS-enhanced cyclooxygenase (COX)-2 expression associated with PGE(2) synthesis in tracheal smooth muscle cells (TSMCs). LPS markedly increased the expression of COX-2 and release of PGE(2) in a time- and concentration-dependent manner, whereas COX-1 remained unaltered. Both the expression of COX-2 and the generation of PGE(2) in response to LPS were attenuated by a tyrosine kinase inhibitor genistein, a phosphatidylcholine-phospholipase C inhibitor D609, a phosphatidylinositol-phospholipase C inhibitor U73122, protein kinase C inhibitors, GF109203X and staurosporine, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and phosphatidylinositol 3-kinase (PI3-K) inhibitors, LY294002 and wortmannin. Furthermore, LPS-induced NF-kappaB activation correlated with the degradation of IkappaB-alpha, COX-2 expression, and PGE(2) synthesis, was inhibited by transfection with dominant negative mutants of NIK and IKK-alpha, but not by IKK-beta. LPS-induced COX-2 expression and PGE(2) synthesis were completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK inhibitor), but these two inhibitors had no effect on LPS-induced NF-kappaB activation, indicating that NF-kappaB is activated by LPS independently of activation of p42/p44 MAPK and p38 MAPK pathways in TSMCs. Taken together, these findings suggest that the increased expression of COX-2 correlates with the release of PGE(2) from LPS-challenged TSMCs, at least in part, independently mediated through MAPKs and NF-kappaB signalling pathways. LPS-mediated responses were modulated by PLC, Ca(2+), PKC, tyrosine kinase, and PI3-K in these cells.     

ATP stimulates Na+-glucose cotransporter activity via cAMP and p38 MAPK in renal proximal tubule cells

Extracellular ATP plays an important role in the regulation of renal function. However, the effect of ATP on the Na⁺-glucose cotransporters (SGLTs) has not been elucidated in proximal tubule cells (PTCs). Therefore, this study was performed to examine the action of ATP on SGLTs and their related signal pathways in primary cultured rabbit renal PTCs. ATP increased [¹⁴C]--methyl-D-glucopyranoside (-MG) uptake in a time-dependent (>1 h) and dose-dependent (>10^–6 M) manner. ATP stimulated -MG uptake by increasing in V_max without affecting K_m. ATP-induced increase of -MG uptake was correlated with the increase in both SGLT1 and SGLT2 protein expression levels. ATP-induced stimulation of -MG uptake was blocked by suramin (nonspecific P2 receptor antagonist), RB-2 (P2Y receptor antagonist), and MRS-2179 (P2Y₁ receptor antagonist), suggesting a role for the P2Y receptor. ATP-induced stimulation of -MG uptake was blocked by pertussis toxin (PTX, a G_i protein inhibitor), SQ-22536 (an adenylate cyclase inhibitor), and PKA inhibitor amide 14-22 (PKI). ATP also increased cAMP formation, which was blocked by PTX and RB-2. However, pretreatment of adenosine deaminase did not block ATP-induced cAMP formation. In addition, ATP-induced stimulation of -MG uptake was blocked by SB-203580 (p38 MAPK inhibitor), but not by PD-98059 (p44/42 MAPK inhibitor) or SP-600125 (JNK inhibitor). Indeed, ATP induced phosphorylation of p38 MAPK. In conclusion, ATP increases -MG uptake via cAMP and p38 MAPK in renal PTCs. adenosine 5'-triphosphate; mitogen-activated protein kinase     

Involvement of p38 MAPK and ERK/MAPK pathways in staurosporine-induced production of macrophage inflammatory protein-2 in rat peritoneal neutrophils.

Stimulation of rat peritoneal neutrophils with staurosporine (64 nM) induced production of macrophage inflammatory protein-2 (MIP-2) and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase/MAP kinase (ERK/MAPK). The staurosporine-induced MIP-2 production at 4 h was inhibited by the highly specific p38 MAPK inhibitor SB 203580 and the MAPK/ERK kinase (MEK-1) inhibitor PD 98059 in a concentration-dependent manner. By treatment with SB 203580 (1 microM) or PD 98059 (50 microM), the staurosporine-induced increase in the levels of mRNA for MIP-2 was only partially lowered, although the staurosporine-induced MIP-2 production was completely inhibited. Consistent with the inhibition by the protein synthesis inhibitor cycloheximide, SB 203580 and PD 98059 inhibited MIP-2 production at 4 h either when added simultaneously with staurosporine or 2 h after stimulation with staurosporine. In contrast, the DNA-dependent RNA polymerase inhibitor actinomycin D did not inhibit MIP-2 production at 4 h when it was added 2 h after staurosporine stimulation. Dot blot analysis demonstrated that treatment with SB 203580 or PD 98059 down-regulates the stability of MIP-2 mRNA. These results suggested that p38 MAPK and ERK/MAPK pathways are involved in translation of MIP-2 mRNA to protein and stabilization of MIP-2 mRNA.     

Interleukin-1beta-induced cyclooxygenase-2 expression is mediated through activation of p42/44 and p38 MAPKS,and NF-kappaB pathways in canine tracheal smooth muscle cells

Interleukin-beta (IL-1beta) was found to induce inflammatory responses in the airways, which exerted a potent stimulus for PG synthesis. This study was to determine the mechanisms of IL-1beta-enhanced cyclooxygenase (COX)-2 expression associated with PGE(2) synthesis in tracheal smooth muscle cells (TSMCs). IL-1beta markedly increased COX-2 expression and PGE(2) formation in a time- and concentration-dependent manner in TSMCs. Both COX-2 expression and PGE(2) formation in response to IL-1beta were attenuated by a tyrosine kinase inhibitor, genistein, a phosphatidylcholine-phospholipase C inhibitor, D609, a phosphatidylinositol-phospholipase C inhibitor, U73122, protein kinase C inhibitors, GF109203X and staurosporine, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and phosphatidylinositol 3-kinase (PI3-K) inhibitors, LY294002 and wortmannin. IL-1beta-induced activation of NF-kappaB correlated with the degradation of IkappaB-alpha in TSMCs. IL-1beta-induced NF-kappaB activation, COX-2 expression, and PGE(2) synthesis were inhibited by the dominant negative mutants of NIK and IKK-alpha, but not by IKK-beta. IL-1beta-induced COX-2 expression and PGE(2) synthesis were completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 inhibitor), but these two inhibitors had no effect on IL-1beta-induced NF-kappaB activation, indicating that activation of p42/44 and p38 MAPK and NF-kappaB signalling pathways were independently required for these responses. These findings suggest that the increased expression of COX-2 correlates with the release of PGE(2) from IL-1beta-challenged TSMCs, at least in part, independently mediated through MAPKs and NF-kappaB signalling pathways in canine TSMCs. IL-1beta-mediated responses were modulated by PLC, Ca(2+), PKC, tyrosine kinase, and PI3-K in these cells.     

PKC and MAPKs pathways mediate EGF-induced stimulation of 2-deoxyglucose uptake in mouse embryonic stem cells.

It has been reported that epidermal growth factor (EGF) and EGF receptor were highly expressed in embryo, suggesting that the EGF system is related to early embryo development in an autocrine and/or paracrine manner. Glucose becomes the preimplantation exogenous energy substrate and enters the blastocyst via glucose transporters. Thus, the effect of EGF on [3H]-2-deoxyglucose (2-DG) uptake and its related signaling pathways were examined in mouse embryonic stem (ES) cells. EGF significantly increased 2-DG uptake in time- and concentration- dependent manner (>12 hr, >10 ng/ ml) and increased mRNA and protein level of glucose transporter 1 (GLUT1) compared to control, respectively. Actinomycin D and cycloheximide completely blocked the effect of EGF on 2-DG uptake. EGF-induced increase of 2-DG uptake was blocked by AG1478 (EGF receptor tyrosine kinase blocker), genistein or herbimycin (tyrosine kinase inhibitors). In addition, EGF effect was blocked by neomycin and U 73122 [phospholipase C (PLC) inhibitors] as well as staurosporine and bisindolylmaleimide I [protein kinase C (PKC) inhibitors]. EGF was also observed to increase inositol phosphates (IPs) formation and activate a PKC translocation from the cytosolic to membrane fraction, suggesting a role of PLC and PKC. SB 203580 [p38 mitogen activated protein kinase (MAPK) inhibitor] or PD 98059 (p44/42 MAPKs inhibitor) blocked EGF-induced increase of 2-DG uptake. EGF also increased phosphorylation of p38 MAPK and p44/42 MAPKs, which was blocked by genistein or bisindolylmaleimide I, respectively. In conclusion, EGF partially increased 2-DG uptake via PKC, p38 MAPK, and p44/42 MAPKs in mouse ES cells.     

Role of p38 MAPK in CYP2E1-dependent arachidonic acid toxicity

Polyunsaturated fatty acids such as arachidonic acid (AA) play an important role in alcohol-induced liver injury. AA promotes toxicity in rat hepatocytes with high levels of cytochrome P4502E1 (CYP2E1) and in HepG2 E47 cells, which express CYP2E1. The possible role of mitogen-activated protein kinase (MAPK) members in this process was evaluated. SB203580, a p38 MAPK inhibitor, and PD98059, an ERK inhibitor, but not wortmannin a phosphatidylinositol 3-kinase (PI3K) inhibitor, prevented AA toxicity in pyrazole hepatocytes and E47 cells. SB203580 prevented the enhancement of AA toxicity by salicylate. SB203580 neither lowered the levels of CYP2E1 nor affected CYP2E1-dependent oxidative stress. The decrease in mitochondrial membrane potential produced by AA was prevented by SB203580. Treating CYP2E1-induced cells with AA activated p38 MAPK but not ERK or AKT. This activation was blocked by antioxidants. AA increased the translocation of NF-kappaB to the nucleus. Salicylate blocked this translocation, which may contribute to the enhancement of AA toxicity by salicylate. SB203580 restored AA-induced NF-kappaB translocation, which may contribute to protection against toxicity. In conclusion, AA toxicity was related to lipid peroxidation and oxidative stress, and to the activation of p38 MAPK, as a consequence of CYP2E1-dependent production of reactive oxygen species. Activation of p38 MAPK by AA coupled to AA-induced oxidative stress may synergize to cause cell toxicity by affecting mitochondrial membrane potential and by modulation of NF-kappaB activation.     

High glucose-induced inhibition of alpha-methyl-D-glucopyranoside uptake is mediated by protein kinase C-dependent activation of arachidonic acid release in primary cultured rabbit renal proximal tubule cells

Abnormal glucose handling in the proximal tubule may play an important role in the development of diabetic nephropathy. Thus, the present study was designed to examine the effect of high glucose on alpha-methyl-D-glucopyranoside (alpha-MG) uptake and its signaling pathways in the primary cultured rabbit renal proximal tubule cells (PTCs). When PTCs were preincubated with 25 or 50 mM glucose for 4 h, 25 or 50 mM glucose significantly inhibited alpha-MG uptake, while 25 or 50 mM mannitol and L-glucose did not affect. Actinomycin D and cycloheximide did not block the effect of high glucose on alpha-MG uptake. Twenty-five millimoles glucose-induced inhibition of alpha-MG uptake was blocked by mepacrine and AACOCF(3), phospholipase A(2) (PLA(2)) inhibitors. Twenty-five millimoles of glucose, not mannitol or L-glucose, significantly increased the [(3)H]-arachidonic acid (AA) release compared to control. In addition, the 25 mM glucose-induced [(3)H]-AA release was completely blocked by mepacrine or AACOCF(3). Indomethacin, a cyclooxygenase inhibitor, blocked the high glucose-induced inhibition of alpha-MG uptake, although econazole, cytochrome P-450 a epoxygenase inhibitor, and nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, did not. On the other hand, staurosporine and bisindolylmaleimide I, protein kinase C (PKC) inhibitors, blocked 25 mM glucose-induced increase of [(3)H]-AA release and inhibition of alpha-MG uptake. However, neomycin, U 73122, and phospholipase c(PLC) inhibitors did not block the effect of 25 mM glucose on [(3)H]-AA release and alpha-MG uptake. Pretreatment of methoxyverapamil, an L-type Ca(2+) channel blocker, abolished 25 mM glucose-induced increase of [(3)H]-AA release. Indeed, 25 mM glucose increased translocation of cPLA(2) from cytosolic fraction to membrane fraction. In conclusion, the present results demonstrate that high glucose inhibits alpha-MG uptake by the increase of AA release via the activation of PKC.