cAMP delays disappearance of gap junctions between pairs of rat hepatocytes in primary culture

J. C. Saez, W. A. Gregory, T. Watanabe, R. Dermietzel, E. L. Hertzberg, L. Reid, Michael V. L. Bennett, David C. Spray

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Abstract

Freshly isolated adult rat hepatocytes were found to be coupled through gap junctions, but coupling decreased abruptly 5-8 h after plating the cells on plastic culture dishes in physiological saline containing insulin and fetal cell serum. Loss of intercellular coupling was associated with disappearance of 27-kDa gap junction protein and of gap junctions seen by electron microscopy or immunocytochemistry. This disappearance of coupling was delayed ~8 h by treatment of the cultures with membrane permeant adenosine 3',5'-cyclic monophosphate (cAMP) [but not guanosine 3',5'-cyclic monophosphate (cGMP)] derivatives. Levels of gap junction protein and anatomically identified junctions were also maintained by 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP). Level of mRNA encoding the gap junction protein was maintained longer in cells treated with 8-BrcAMP than in untreated cells, but 8-BrcAMP did not detectably increase the transcription rate. Thus prolongation of coupling must result at least partially from extension of the lifetime of gap junction mRNA, allowing translation of message and assembly of channels for a longer period after plating. Treatment of cells with mRNA or protein synthesis inhibitors (α-amanitin and cycloheximide) prolonged coupling to a similar extent as did treatment with 8-BrcAMP. α-Amanitin blocked transcription of gap junction mRNA, but levels of cytoplasmic mRNA encoding the 27-kDa gap junction protein were maintained, presumably by block of transcription of an mRNA degrading factor. The factor is probably a protein, since a similar effect of mRNA level was produced in cycloheximide-treated cells. Cells cultured in control medium were also observed to flatten as they became uncoupled, whereas cells cultured for as long as 16 h in elevated 8-BrcAMP remained round and well coupled. The correlation between shape and coupling strength was not obtained after treatment with the microtubule stabilizing agent, taxol, which maintained the spherical shape of the cells but did not delay the disappearance of dye coupling. Nocodazole, which blocks microtubule formation, also maintained the spherical shape of the cells but delayed the disappearance of dye coupling. In addition to gating by covalent modification or other mechanisms, hormones and drugs that alter the intracellular cAMP concentration may affect intracellular communication by changing the lifetime of the mRNA encoding the main gap junction protein, thereby decreasing or increasing its synthesis. In addition, cAMP may act by decreasing removal of junctions from appositional membranes.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume257
Issue number1
StatePublished - 1989

Fingerprint

Gap Junctions
Connexins
Rats
Hepatocytes
Messenger RNA
Amanitins
Transcription
Cell Shape
Cycloheximide
Microtubules
Plating
Cultured Cells
Coloring Agents
Nocodazole
8-Bromo Cyclic Adenosine Monophosphate
Cells
Protein Synthesis Inhibitors
Membranes
Excipients
Cyclic GMP

ASJC Scopus subject areas

  • Cell Biology
  • Clinical Biochemistry
  • Physiology

Cite this

cAMP delays disappearance of gap junctions between pairs of rat hepatocytes in primary culture. / Saez, J. C.; Gregory, W. A.; Watanabe, T.; Dermietzel, R.; Hertzberg, E. L.; Reid, L.; Bennett, Michael V. L.; Spray, David C.

In: American Journal of Physiology - Cell Physiology, Vol. 257, No. 1, 1989.

Research output: Contribution to journalArticle

Saez, J. C. ; Gregory, W. A. ; Watanabe, T. ; Dermietzel, R. ; Hertzberg, E. L. ; Reid, L. ; Bennett, Michael V. L. ; Spray, David C. / cAMP delays disappearance of gap junctions between pairs of rat hepatocytes in primary culture. In: American Journal of Physiology - Cell Physiology. 1989 ; Vol. 257, No. 1.
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T1 - cAMP delays disappearance of gap junctions between pairs of rat hepatocytes in primary culture

AU - Saez, J. C.

AU - Gregory, W. A.

AU - Watanabe, T.

AU - Dermietzel, R.

AU - Hertzberg, E. L.

AU - Reid, L.

AU - Bennett, Michael V. L.

AU - Spray, David C.

PY - 1989

Y1 - 1989

N2 - Freshly isolated adult rat hepatocytes were found to be coupled through gap junctions, but coupling decreased abruptly 5-8 h after plating the cells on plastic culture dishes in physiological saline containing insulin and fetal cell serum. Loss of intercellular coupling was associated with disappearance of 27-kDa gap junction protein and of gap junctions seen by electron microscopy or immunocytochemistry. This disappearance of coupling was delayed ~8 h by treatment of the cultures with membrane permeant adenosine 3',5'-cyclic monophosphate (cAMP) [but not guanosine 3',5'-cyclic monophosphate (cGMP)] derivatives. Levels of gap junction protein and anatomically identified junctions were also maintained by 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP). Level of mRNA encoding the gap junction protein was maintained longer in cells treated with 8-BrcAMP than in untreated cells, but 8-BrcAMP did not detectably increase the transcription rate. Thus prolongation of coupling must result at least partially from extension of the lifetime of gap junction mRNA, allowing translation of message and assembly of channels for a longer period after plating. Treatment of cells with mRNA or protein synthesis inhibitors (α-amanitin and cycloheximide) prolonged coupling to a similar extent as did treatment with 8-BrcAMP. α-Amanitin blocked transcription of gap junction mRNA, but levels of cytoplasmic mRNA encoding the 27-kDa gap junction protein were maintained, presumably by block of transcription of an mRNA degrading factor. The factor is probably a protein, since a similar effect of mRNA level was produced in cycloheximide-treated cells. Cells cultured in control medium were also observed to flatten as they became uncoupled, whereas cells cultured for as long as 16 h in elevated 8-BrcAMP remained round and well coupled. The correlation between shape and coupling strength was not obtained after treatment with the microtubule stabilizing agent, taxol, which maintained the spherical shape of the cells but did not delay the disappearance of dye coupling. Nocodazole, which blocks microtubule formation, also maintained the spherical shape of the cells but delayed the disappearance of dye coupling. In addition to gating by covalent modification or other mechanisms, hormones and drugs that alter the intracellular cAMP concentration may affect intracellular communication by changing the lifetime of the mRNA encoding the main gap junction protein, thereby decreasing or increasing its synthesis. In addition, cAMP may act by decreasing removal of junctions from appositional membranes.

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