REGULATION OF GAP JUNCTION INTERCELLULAR COMMUNICATION BY PH IN MC3T3-E1 OSTEOBLASTIC CELLS

Gap junction intercellular communication (GJIC) may be related to coor dinating the function of osteoblasts during bone mineralization, Since an alkaline pH supports mineral deposition while an acidic pH promote s mineral dissolution, it was investigated whether GJIC is altered by changes in extracellular pH (pH(0)) Functional GJIC was assessed by fl uorescent dye transfer after microinjection, and connexin protein abun dance was examined by immunoprecipitation and immunoblotting in MC3T3- E1 cells, a model of osteoblast-like cells, The percent of cells coupl ed 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 pH(0) 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. De termination of intracellular pH (pH(i)) showed that it was similar to pH(0) after 2.75 h of incubation at each ambient pH. When pH, was clam ped at 6.9 or 7.2, there was a time-dependent decrease in the gap junc tion coupling frequency at a pH(i) of 6.9 when pH(0) was 7.2. Steady-s tate mRNA levels were decreased at pH(0) 6.9 but were unchanged at eit her pH(0) 7.2 or 7.6. Our conclusions are that (1) longer incubations (greater than or equal to 2.75 h) at low pH(0) 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 pH(0), respectiv ely, suggests that gating of the GJ channel by pH may also occur; (3) pH(0)-induced alterations in GJIC in the MC3T3-E1 osteoblastic model a re related to concomitant changes in pH(i).