Effects of cigarette smoke condensate and 12-0-tetradecanoylphorbol-13-acetage on gap junction structure and function in cultured cells

Abstract

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.

Models and methods for in vitro testing of hepatic gap junctional communication

“…A more sophisticated approach is the local liberation of inositol triphosphate from a caged precursor by flash photolysis, which allows the stimulation of single cells (Decrock et al, 2015; Leybaert and Sanderson, 2001). Metabolic coupling assays, in particular transfer of labelled nucleotides, have been used to demonstrate the inhibitory effects of 12- O -tetradecanoylphorbol-13-acetate on GJIC in co-cultures of primary chick embryo hepatocytes and Chinese hamster V79 lung fibroblasts (van der Zandt et al, 1990).…”

Phosphorylation of Ser-279/282 and Tyr-265 positions on Cx43 as possible mediators of VEGF-165 inhibition of pregnancy-adapted Ca2+ burst function in ovine uterine artery endothelial cells

“…Phosphorylation of Cx43 at multiple c-terminal amino acid residues such as Ser-279/282, Tyr-265, Ser-368, and Ser-262 are targeted by the aforementioned signaling pathways, and have been described as inhibitory phosphorylations (Lampe and Lau 2000). The phorbol ester, PMA (phorbol myristic acid), is commonly used as a receptor-independent inhibitor of gap junction function (Lampe 1994; Sirnes, et al 2008; van der Zandt, et al 1990) and signals through PKC, Src, and ERK in UAEC (Bird et al 2013). Indeed, when P-UAEC are exposed to PMA, sustained phase Ca2+ burst responses to ATP are dramatically reduced (Bird et al 2013; Cale and Bird 2006), and this is associated with the phosphorylation of Cx43 at both positions Ser-279/282 via the ERK-1/2 pathway and Tyr-265 via the Src pathway.…”

Inhibition of intercellular communication by airborne particulate matter

“…The inhibitory effect on IC of cigarette smoke condensate (CSC) is not transient (Jongen et al 1987;Rutten et al 1989;Van Der Zandt et al 1990). In addition, TPA, but not CSC, has been shown to reduce the number of gap junctions in V79 cells (Jongen et al 1987;Van Der Zandt et al 1990) and it has recently been shown that these compounds induce different effects on gap junction morphology and quantity (Van Der Zandt et al 1990). CSC does not seem to activate PKC ( Van der Zandt et al 1990), and this suggests that these compounds act via different mechanisms.…”

“…In addition, TPA, but not CSC, has been shown to reduce the number of gap junctions in V79 cells (Jongen et al 1987;Van Der Zandt et al 1990) and it has recently been shown that these compounds induce different effects on gap junction morphology and quantity (Van Der Zandt et al 1990). CSC does not seem to activate PKC ( Van der Zandt et al 1990), and this suggests that these compounds act via different mechanisms. The mechanism by which APM inhibits IC and the compounds responsible for this inhibition are unknown.…”