Sustained Intracellular Acidosis Triggers the Na⁺/H⁺ Exchager-1 Activation in Glutamate Excitotoxicity

The Na⁺/H⁺ exchanger-1 (NHE-1) is a ubiquitously expressed pH-regulatory membrane protein that functions in the brain, heart, and other organs. It is increased by intracellular acidosis through the interaction of intracellular H⁺ with an allosteric modifier site in the transport domain. In the previous study, we reported that glutamate-induced NHE-1 phosphorylation mediated by activation of protein kinase C-β (PKC-β) in cultured neuron cells via extracellular signal-regulated kinases (ERK)/p90 ribosomal s6 kinases (p90RSK) pathway results in NHE-1 activation. However, whether glutamate stimulates NHE-1 activity solely by the allosteric mechanism remains elusive. Cultured primary cortical neuronal cells were subjected to intracellular acidosis by exposure to 100 μM glutamate or 20 mM NH₄Cl. After the desired duration of intracellular acidosis, the phosphorylation and activation of PKC-β, ERK1/2 and p90RSK were determined by Western blotting. We investigated whether the duration of intracellular acidosis is controlled by glutamate exposure time. The NHE-1 activation increased while intracellular acidosis sustained for >3 min. To determine if sustained intracellular acidosis induced NHE-1 phosphorylation, we examined phosphorylation of NHE-1 induced by intracellular acidosis by transient exposure to NH₄Cl. Sustained intracellular acidosis led to activation and phosphorylation of NHE-1. In addition, sustained intracellular acidosis also activated the PKC-β, ERK1/2, and p90RSK in neuronal cells. We conclude that glutamate stimulates NHE-1 activity through sustained intracellular acidosis, which mediates NHE-1 phosphorylation regulated by PKC-β/ERK1/2/p90RSK pathway in neuronal cells.

Influence of chronic fluorosis on protein kinase Cβ/p66shc signal pathway in the brain of rats / 中华地方病学杂志

Objective To investigate the influence of chronic fluorosis on protein kinase Cβ (PKCβ)/p66shc signal pathway in the brain of rats,and reveal the molecular mechanism of brain damage.Methods According to body weight by the random number table method thirty SD rats were divided into three groups of 10 each (half females and half males),the normal control group [less than 0.5 mg/L of fluorine (prepared with NaF) in drinking water],low fluoride exposure group (10.0 mg/L fluorine),and high fluoride exposure group (50.0 mg/L fluoride).The experiment period was 6 months.The protein level of PKCβ,p66shc,phospho-p66shc and preserved ammonia acyl isomerase (Pin1) in rat brain was detected by Western blotting.The level of neuron nuclear antigen (NeuN),p66shc and phospho-p66sh in brain of rats was detected by immunohistochemistry.Results By Western blotting,the levels of PKCβ,Pin1 and phospho-p66shc protein in brain tissue in high fluoride exposure group [(193.00 ± 57.53)％,(228.21 ± 71.14)％,(201.54 ±:50.86)％] were higher than those of the normal control groups [(100.00 ± 21.24)％,(100.00 ± 40.55)％,(100.00 ± 13.35)％,all P ＜ 0.05].By immunohistochemistry,the numbers of NeuN staining in brain tissue of the rats in both high and low fluoride exposure groups [(49.50 ± 12.57)％,(65.66 ±14.58)％] were lower than that of the control group [(100.00 ± 18.32)％,all P ＜ 0.01].The level of phospho-p66shc protein in brain tissue in high fluoride exposure group [(242.66 ± 93.01)％] was higher than those of the low fluoride exposure and the normal control groups [(152.53 ± 60.65)％,(100.00 ± 25.63)％,all P ＜ 0.01].Conclusion Chronic fluorosis has increased the expressions of PKCβ,Pin1 and phospho-p66shc at protein level in brain of rats,which may be related to the molecular mechanism of brain damage resulted from chronic fluorosis.

Protein kinase Cβ_2 mediated high glucose-induced human umbilical vein endothelial cells injury via regulation of peroxisome proliferator-activated receptor α / 中华内分泌代谢杂志

Objective To investigate the roles of protein kinase C(PKC)β_2 and PPARα in the mRNA expression of vascular endothelial growth factor(VEGF)and vascular cell adhesion molecule-1(VCAM-1)in human umbilical vein endothelial cells(HUVECs)exposed to high glucose,and to explore their relationship.Methods The HUVECs were divided into eight groups:normal glucose(NG,5 mmoL/L D-glucose)group,high glucose(HG,25 mmol/L D-ucose)group,osmotic control(L,NG+20 mmol/L L-glucose)group,normal glucose transfected with empty vector(NN,NG+AdS-null)group,high glucose transfected with PKCβ_2(HB,HG+Ad5-PKCβ_2)group,high glucose plus fenofibrate(HF,HG+40 μmol/L fenofibrate)group,and HB plus fenofibrate (HBF,HB+40μmol/L fenofibrate)group.HUVECs were incubated with fenofibrate for 20 minutes as HF20 group.All cells in various groups were cultured for 6 days.The expressions of VEGF and VCAM-1 mRNA were detected by RT-PCR.PPARα protein expression was tested by Western blot.The expression and traaslocation of PKCβ_2 protein were observed by confocal laser scanning microscopo.Results (1)HG increased VEGF and VCAM-1 mRNA expressions,with 1.91 and 1.56 folds of NG group,respectively(both P<0.05).VEGF and VCAM-1 mRNA expressions in HB group further increased,with 2.59 and 2.07 folds of NG group,respectively(both P<0.05).Fenofibrate significantly decreased VEGF and VCAM-1 mRNA expressions,with 68%and 74%of HG group,respectively(both P<0.05).There were no significant difierences in the expressiong of VEGF,VCAM-1 mRNA between HF20 and HG groups.(2)The protein expression of PPARα decreased by 20%in HG group compared with NG group,and further decreased in HB group,being 78%of HG group.Compared with HG group,PPARαexpression increased by 13%in HF group(P<0.05).(3)HG induced PKCβ_2 translocation from cytosol to nucleus and quantitative analysis showed the ratio of plasma/nuclear fluorescence intensity in HG group decreased by 37% compared with NG group(P<0.05).The PKCβ_2 translocation was more obvious in HB group than in HG group.Conclusion Hiigll glucose stimulates VEGF and VCAM-1 mRNA expressions in HUVECs via PKCβ_2 activationdependent PPARα pathway.