Yamaguchi D T, Huang J T, Ma D
Geriatrics Research, Education, and Clinical Center, Veterans Administration Medical Center, West Los Angeles, California, USA.
J Bone Miner Res. 1995 Dec;10(12):1891-9. doi: 10.1002/jbmr.5650101208.
Gap junction intercellular communication (GJIC) may be related to coordinating the function of osteoblasts during bone mineralization. Since an alkaline pH supports mineral deposition while an acidic pH promotes mineral dissolution, it was investigated whether GJIC is altered by changes in extracellular pH (pHo) Functional GJIC was assessed by fluorescent dye transfer after microinjection, and connexin protein abundance was examined by immunoprecipitation and immunoblotting in MC3T3-E1 cells, a model of osteoblast-like cells. The percent of cells coupled by GJIC was found to be 40.7% (24 of 59 injected cells) at pH 6.9, 72.2% (26 of 36) at pH 7.2, and 92.8% (26 of 28) at pH 7.6. A decrease in GJIC was detectable by 30-60 minutes of exposure to a pHo of 6.9. Decreased gap junction communication was also found in cells after 3, 8, and 24 h of incubation in a bicarbonate-CO2 system at an ambient pH of 6.9. Connexin protein abundance experiments showed that at after exposure to a pH of 6.9 for 2.75 h, the specific band(s) at 41-43 kD were fainter compared with these same band(s) at pH 7.2 and 7.6. There was no significant difference in band densities at pH 7.2 and 7.6. Determination of intracellular pH (pHi) showed that it was similar to pHo after 2.75 h of incubation at each ambient pH. When pHi was clamped at 6.9 or 7.2, there was a time-dependent decrease in the gap junction coupling frequency at a pHi of 6.9 when pHo was 7.2. Steady-state mRNA levels were decreased at pHo 6.9 but were unchanged at either pHo 7.2 or 7.6. Our conclusions are that (1) longer incubations ( > or = 2.75 h) at low pHo decrease GJIC which in part may be due to a decrease in connexin protein abundance perhaps as a result of a decrease in connexin steady-state mRNA expression; (2) GJIC inhibition or augmentation found at low and high pHo, respectively, suggests that gating of the GJ channel by pH may also occur; (3) pho-induced alterations in GJIC in the MC3T3-E1 osteoblastic model are related to concomitant changes in pHi.
缝隙连接细胞间通讯（GJIC）可能与骨矿化过程中协调成骨细胞的功能有关。由于碱性pH值有利于矿物质沉积，而酸性pH值促进矿物质溶解，因此研究了细胞外pH值（pHo）的变化是否会改变GJIC。通过显微注射后荧光染料转移评估功能性GJIC，并通过免疫沉淀和免疫印迹检测成骨样细胞模型MC3T3-E1细胞中连接蛋白的丰度。发现在pH 6.9时，通过GJIC连接的细胞百分比为40.7%（59个注射细胞中的24个），在pH 7.2时为72.2%（36个中的26个），在pH 7.6时为92.8%（28个中的26个）。暴露于pHo 6.9 30 - 60分钟后可检测到GJIC降低。在pH 6.9的碳酸氢盐 - CO2系统中孵育3、8和24小时后，细胞中的缝隙连接通讯也降低。连接蛋白丰度实验表明，在暴露于pH 6.9 2.75小时后与pH 7.2和7.6时相比，41 - 43 kD处的特异性条带更淡。在pH 7.2和7.6时条带密度无显著差异。细胞内pH值（pHi）的测定表明，在每个环境pH值下孵育2.75小时后，pHi与pHo相似。当pHi钳制在6.9或7.2时，当pHo为7.2且pHi为6.9时，缝隙连接耦合频率随时间下降。在pHo 6.9时稳态mRNA水平降低，但在pHo 7.2或7.6时保持不变。我们的结论是：（1）在低pHo下较长时间孵育（≥2.75小时）会降低GJIC，这部分可能是由于连接蛋白丰度降低，可能是连接蛋白稳态mRNA表达降低的结果；（2）分别在低pHo和高pHo下发现的GJIC抑制或增强表明，pH可能也会对GJ通道进行门控；（3）在MC3T3-E1成骨细胞模型中，pHo诱导的GJIC改变与pHi的伴随变化有关。
