DIFFERENT CHANGES IN EXPRESSION AND FUNCTION OF CONNEXIN-26 AND CONNEXIN-32 DURING DNA-SYNTHESIS AND REDIFFERENTIATION IN PRIMARY RAT HEPATOCYTES USING A DMSO CULTURE SYSTEM

In the present study, we determined in detail the changes of liver gap junctions, connexin 26 (Cx26), and connexin 32 (Cx32), during DNA syn thesis and redifferentiation of hepatocytes in vitro. We used primary rat hepatocytes that expressed the liver gap junction proteins, which were cultured in the medium containing epidermal growth factor (EGF) w ith 2% dimethylsulfoxide (DMSO) and 10(-7) mol/L glucagon (a DMSO cult ure system), as we previously reported, In the present cultures, almos t confluent hepatocytes cultured in the medium containing EGF with 2% DMSO and 10(-7) mol/L glucagon, underwent a nearly synchronous wave of DNA synthesis induced by the removal of 2% DMSO and 10(-7) mol/L gluc agon, and the addition of 10 mmol/L nicotinamide, after which the DNA synthesis was completely re-inhibited by the re-addition of 2% DMSO an d 10(-7) mol/L glucagon, During stimulation of DNA synthesis, both Cx2 6 and Cx32 messenger RNA (mRNAs) in hepatocytes transiently increased in the G1 phase and then markedly decreased before the onset of the S phase, while only Cx26 messenger RNA (mRNA) increased slightly in the S/M phase. Furthermore, before the onset of the S phase, a disappearan ce of both Cx26 and Cx32 immunoreactivities and gap junction plaques w ere observed, Gap junctional intercellular communication (GJIC), as me asured by lucifer yellow, which indicated the function of Cx32, decrea sed markedly from before the onset of the S phase, GJIC measured by pr opidium iodide, which indicated the function of Cx26, decreased from b efore the onset of the S phase and then increased slightly in the S/M phase. During the re-inhibition after the stimulation of DNA synthesis , Cx32 mRNA, but not Cx26 mRNA, rapidly returned to the pretreatment c ontrol level. Cx32 immunoreactivity and gap junction plaques also reco vered. However, the recovery of GJIC measured by lucifer yellow was la ter than that of Cx32 expression. These results indicated the differen t changes of expression and function of Cx26 and Cx32 in the hepatocyt es during stimulation and re-inhibition of DNA synthesis. This culture system should be useful as a model in which to study liver gap juncti ons during hepatocyte growth and differentiation in vitro.