The regulation roles of protein kinase C alpha in enhanced expression of type Ⅰ inositol 1,4,5-trisphophate receptors induced by tumor necrosis factor alpha / 中国医师杂志

Objective To investigate the signal mechanism of protein kinase C alpha(PKC-α)participated in enhanced expression of type Ⅰ inositol 1,4,5-trisphophate receptors (IP3 RI) induced by tumor necrosis factor alpha(TNFα),in order to delineate the mechanisms of decreased glomerular filtration rate (GFR) in hepatorenal syndrome caused by TNFα.Methods The glomerular mesangial cells (GMCs)line from rats was chosen as experimental material.GMCs were divided into control (D),TNFα-2 h,TNFα-4 h,TNFα-8 h,and TNFα-24 h groups.Moreover,another two groups were sanflngol-8h (S),TNFα + Sanfingol-8h(TS)groups.The effect of TNFα on the expression of IP3RI was detected by immunocytochemical staining,Western blotting,teal time-polymerase chain reaction (PCR) assays.Results Immunocytochemical staining demonstrated that IP3 RI was mainly distributed in cytoplasm of GMCs.Enhanced positive staining was determined in all TNFα-treated groups,especially in TNFα-8 h group.Western blotting demonstrated that the expression of IP3RI protein was significantly higher in TNFα-4 h,TNFα-8h and TNFα-24 h groups than control group(4 h:1.82 ± 0.63 ; 8 h:2.95 ± 0.66 ; 24 h:2.48 ± 0.72 ; D:1 ±0.02 ; F =9.24,P ＜ 0.05).The expression of IP3 RI protein was the highest in TNFα-8 h and TNFα-24 h groups(P ＜0.05).No difference was found among S,TS,and control groups(S:1.39 ±0.65; TS:1.35± 0.37 ; P ＞ 0.05).Real time-PCR found the expression of IP3 RImRNA was significantly higher in all TNFα-treated groups than control group(2 h:3.35 ± 1.97; 4 h:3.16 ± 1.35; 8 h:3.70 ± 1.76; 24 h:4.49±1.70; D:1 ±0.01; F =6.167,P ＜0.05).No difference was found among all TNFα-treated groups(P ＞0.05).No difference was found among S,TS,and control groups(S:1.53 ±0.79; TS:1.32 ± 0.38 ; P ＞ 0.05).Conclusions IP3 RI was mainly distributed in cytoplasm of GMCs.TNFa could enhance the expression of IP3 RI protein and IP3 RI mRNA,which could be blocked by sanfingol,a PKCα inhibitor.It might be an important signal in the mechanisms of GFR decrease caused by TNFα in hepatorenal syndrome.

Efffects of Fluoxetine on ATP-induced Calcium Signaling in PC12 Cells

Fluoxetine, a widely used anti-depressant compound, has several additional effects, including blockade of voltage-gated ion channels. We examined whether fluoxetine affects ATP-induced calcium signaling in PC12 cells by using fura-2-based digital calcium imaging and assay for [3H]-inositol phosphates (IPs). Treatment with ATP (100microM) for 2 min induced [Ca2+]i increases. The ATP-induced [Ca2+]i increases were significantly decreased by removal of extracellular Ca2+ and treatment with the inhibitor of endoplasmic reticulum Ca2+ ATPase thapsigargin (1microM). Treatment with fluoxetine for 5 min blocked the ATP-induced [Ca2+]i increase concentration-dependently. Treatment with fluoxetine (30microM) for 5 min blocked the ATP-induced [Ca2+]i increase following removal of extracellular Ca2+ and depletion of intracellular Ca2+ stores. While treatment with the L-type Ca2+ channel antagonist nimodipine for 10 min inhibited the ATP-induced [Ca2+]i increases significantly, treatment with fluoxetine alone blocked the ATP-induced responses. Treatment with fluoxetine also inhibited the 50 mM K+-induced [Ca2+]i increases completely. However, treatment with fluoxetine did not inhibit the ATP-induced [3H]-IPs formation. Collectively, we conclude that fluoxetine inhibits ATP-induced [Ca2+]i increases in PC12 cells by inhibiting both an influx of extracellular Ca2+ and a release of Ca2+ from intracellular stores without affecting IPs formation.

Differential effect of homocysteic acid and cysteic acid on changes of inositol phosphates and (Ca2+)i in rat cerebellar granule cells

The present study was undertaken to characterize homocysteic acid (HCA)-and cysteic acid (CA)mediated formation of inositol phosphates (InsP) in primary culture of rat cerebellar granule cells. HCA and CA stimulated InsP formation in a dose-dependent manner, which was prevented by the N-methyl-D-aspartate (NMDA) receptor antagonist D,L-2-amino-5-phosphopentanoic acid (APV). CA-, but not HCA-, mediated InsP formation was in part prevented by the metabotropic glutamate receptor antagonist alpha-methyl-4-carboxyphenylglycine ((+/-)-MCPG). Both HCA- and CA-mediated increases in intracellular calcium concentration were completely blocked by APV, but were not altered by (+/-)-MCPG. CA-mediated InsP formation was in part prevented by removal of endogenous glutamate. In contrast, the glutamate transport blocker L-aspartic acid-beta-hydroxamate synergistically increased CA responses. These data indicate that in cerebellar granule cells HCA mediates InsP formation wholly by activating NMDA receptor. In contrast, CA stimulates InsP formation by activating both NMDA receptor and metabotropic glutamate receptor, and in part by releasing endogenous glutamate into extracellular milieu.