Effects of cigarette smoke condensate and 12-O-tetradecanoylphorbol-13-acetate on gap junction structure and function in cultured cells.
The effects of cigarette smoke condensate (CSC) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on gap junction structure, quantity and function were investigated. Gap junction morphology was studied in rotary-shadowed freeze-fracture replicas of primary chick embryo hepatocytes. CSC (24 micrograms/ml) induced a strong decrease of gap junction areas; within 6 h the areas were reduced by greater than 60%. In the first 3 h of exposure, TPA (100 ng/ml) also reduced gap junction areas, but in the next 3 h a partial recovery was observed. Protoplasmic fracture face centre-to-centre particle spacings were used as a measure for gap junction coupling. CSC had a slow (although not significant) reducing effect on particle spacings, while TPA induced a reduction from 10.6 nm (control) to 10.0 nm within 3 h, indicating a reduction of coupling. Gap junctions were quantified in thin sections of cultured chick embryo hepatocytes, V79 fibroblasts, and co-cultivated hepatocytes and V79 cells. CSC did not influence gap junction numbers in any of these cultures, while TPA treatment caused a disappearance of gap junctions between hepatocytes and between hepatocytes and V79 cells in the first 12 h of cultivation. In the following 36 h a slow recovery could be observed. Gap junctions between V79 cells had already disappeared within 30 min. Metabolic co-operation between hepatocytes and hypoxanthine-guanine phosphoribosyltransferase-deficient V79 cells was quickly and continuously blocked by CSC over 27 h, whereas the phorbol ester induced a transient block. The dissimilar effects of these compounds on both gap junction structure and function indicate that they act via different mechanisms. The finding that CSC did not inhibit phorbol ester protein kinase C binding and did not activate this protein kinase in vitro supports this hypothesis.
van der Zandt
P T
PT
Department of Toxicology, Agricultural University, Wageningen, The Netherlands.
de Feijter
A W
AW
Homan
E C
EC
Spaaij
C
C
de Haan
L H
LH
van Aelst
A C
AC
Jongen
W M
WM
D000818:Animals; D029742:Caenorhabditis elegans Proteins; D002122:Calcium Chloride / Q000494:pharmacology; D002352:Carrier Proteins; D002478:Cells, Cultured; D002642:Chick Embryo; D004075:Diglycerides / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D005614:Freeze Fracturing; D007365:Intercellular Junctions / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure*; D007700:Kinetics; D008099:Liver / Q000201:enzymology / Q000648:ultrastructure; D008854:Microscopy, Electron; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D011955:Receptors, Drug / Q000378:metabolism; D012906:Smoke; D012907:Smoking; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D004075:Diglycerides; D011955:Receptors, Drug; D012906:Smoke; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D015240:Phorbol 12,13-Dibutyrate; D004533:Egtazic Acid; D011493:Protein Kinase C; D002122:Calcium Chloride; D013755:Tetradecanoylphorbol Acetate; C013965:diolein