Transport of inositol 1,4,5-triphosphate through gap junction channels

Abstract

This study was conducted to assess the effects of chemical carcinogens on the gap junctional intercellular communication in cultured mammalian cells and to prove that gap junction channel provide a pathway for the calcium-mobilizing second messenger, inositol 1,4,5-trisphosphate ($IP_3$). Clone 9 cells derived from rat liver were treated with various carcinogens and anticancer drugs. Gap junctional communication was assessed by measuring the transfer of scrape-loaded lucifer yellow dye. Direct carcinogens such as N-methyl-N``-nitro-N-nitrosoguanidine, hydroxylamine and ethidium bromide exhibited strong inhibition of intercellular communication, while indirect carcinogens such as aflatoxin $B_1$ and ethionine exerted minor effects. The passage of second messenger molecules through gap junction channels was proved by the experiment in which scrape-loaded [$^3H$]-$IP_3$ was transferred into cells along the scraped line. This spread of labled second messenger into neighboring cells was visualized by microautoradiography. To show the direct involvement of gap junction channel in the transport of second messenger, channels were formed in artificial unilamellar liposomes using connexin 32 gap junction protein immunoaffinity purified from rat liver. $[^3H]-IP_3$ was incorporated into vesicles containing open gap junction protein but the vesicles with closed channels failed to incorporate $[^3H]-IP_3$.