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Astrocyte gap junction communication (GJC) is thought to contribute to death signal propagation following central nervous system injury, noteworthy in some ischemia/anoxia models. The inhibition of p38/stress-activated protein kinase 2 (p38/SAPK2) by a pyrimidyl imidazole derivative has been reported to reduce the extent of the lesion area after cerebral ischemia. Therefore, interleukin-1beta (IL-1beta), which contributes to stroke-induced brain injury and activates p38/SAPK2, and hyperosmolarity induced by sorbitol, a potent stimulus of p38/SAPK2 in non-neuronal cells, were used to investigate a possible involvement of p38/SAPK2 in GJC modulation in mouse cultured astrocytes. Both stimuli inhibited dye coupling within minutes. The IL-1beta effect was transient, while that of sorbitol lasted up to 90 min. Both stimuli induced a rapid p38/SAPK2 activation, the kinetic of which matched that of induction of dye coupling inhibition. Immunocytochemical studies showed that IL-1beta and sorbitol induced a p38/SAPK2 translocation from the nucleus to the cytoplasm. The pharmacological agent SB203580 specifically blocked p38/SAPK2 activation, cytoplasmic translocation and reversed the IL-1beta and sorbitol-induced inhibition of GJC. Further characterization of the p38/SAPK2 mode of action on GJC, performed with sorbitol, revealed an increased phosphorylation of protein kinase C (PKC) substrates abolished by both PKC inhibitors and SB203580. Expression and serine phosphorylation of connexin 43, the main component of astrocyte gap junctions, were unchanged, suggesting the existence of additional intracellular signaling mechanisms modulating the channel gating. Altogether, these results demonstrate that p38/SAPK2 is a central mediator of IL-1beta and sorbitol inhibitory actions on GJC and establish PKC among the distal effectors of p38/SAPK2., (Copyright 2004 Wiley-Liss, Inc.)

The present work was designed to investigate the effects of oscillating fluid flow on gap junctional intercellular communication (GJIC) and the gap junction protein connexin (Cx) 43 in osteocyte-like MLOY-4 cells. Cells were exposed for 1 h to oscillating fluid flow at a shear stress of +/-10 dyn/cm(2) and a frequency of 1 Hz in a parallel plate flow chamber. Control cells were incubated in the chamber but were not exposed to oscillating fluid flow. Functional analysis of GJIC indicated that MLOY-4 cells exposed to oscillating fluid flow established more gap junctions with an independent population of dye-labeled cells than did control cells. Phosphorylation of Cx43 was quantified by immunoprecipitation with an anti-Cx43 antibody followed by immunoblot analysis using an anti-phosphoserine antibody. Phosphoserine was normalized to Cx43 in each sample. Compared to control cells, phosphoserine content of Cx43 increased approximately twofold in cells exposed to oscillating fluid flow. The possible role of the extracellular signal regulated kinase (ERK1/2) in the flow-induced upregulation of GJIC was also investigated. The ERK1/2 inhibitor PD-98059 significantly attenuated the effects of oscillating fluid flow on MLOY-4 cells GJIC. These results indicate that oscillating fluid flow regulates GJIC in MLOY-4 cells via the ERK1/2 MAP kinase. In addition, increased serine phosphorylation of Cx43 correlates with the flow-induced increase in GJIC.

1. Cannabinoids are potent inhibitors of endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxations. We set out to study the mechanism underlying this effect and the possible role of cannabinoid-induced changes in intercellular gap junction communication. 2. In cultured endothelial cells, Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and the cannabinoid receptor agonist HU210, increased the phosphorylation of extracellular regulated kinases 1/2 (ERK1/2) and inhibited gap junctional communication, as determined by Lucifer Yellow dye transfer and electrical capacity measurements. 3. Delta(9)-THC elicited a pronounced increase in the phosphorylation of connexin 43, which was sensitive to PD98059 and U0126, two inhibitors of ERK1/2 activation. Inhibition of ERK1/2 also prevented the Delta(9)-THC-induced inhibition of gap junctional communication. 4. Delta(9)-THC prevented both the bradykinin-induced hyperpolarization and the nitric oxide and prostacyclin-independent relaxation of pre-contracted rings of porcine coronary artery. These effects were prevented by PD98059 as well as U0126. 5. In the absence of Delta(9)-THC, neither PD98059 nor U0126 affected the NO-mediated relaxation of coronary artery rings but both substances induced a leftward shift in the concentration - relaxation curve to bradykinin when diclofenac and N(omega)nitro-L-arginine were present. Moreover, PD98059 and U0126 prolonged the bradykinin-induced hyperpolarization of porcine coronary arteries, without affecting the magnitude of the response. 6. These results indicate that the cannabinoid-induced activation of ERK1/2, which leads to the phosphorylation of connexin 43 and inhibition of gap junctional communication, may partially account for the Delta(9)-THC-induced inhibition of EDHF-mediated relaxation. Moreover, the activation of ERK1/2 by endothelial cell agonists such as bradykinin, appears to exert a negative feedback inhibition on EDHF-mediated responses.

Gap junction intercellular communication (GJIC) is involved in the regulation of many cellular processes. The gap junction channels are made up of connexins and the flow of polar low molecular weight molecules through these channels is inhibited by several groups of substances, such as tumour promoters and growth factors. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), chlordane and the growth factor epidermal growth factor (EGF) are potent inhibitors of GJIC in several cell types, including the rat liver epithelial cell line IAR6.1. The induced inhibition of communication by TPA and EGF in IAR6.1 cells is associated with hyperphosphorylation of connexin43, the connexin responsible for GJIC. Two enzyme inhibitors, PD98059, a specific inhibitor of MEK kinase, and GF109203X, a selective inhibitor of protein kinase C (PKC), were used to study the signalling pathways involved in the effect of EGF and TPA on GJIC, with the following conclusions. The inhibition of cell communication in IAR6.1 cells by EGF is likely to be mediated by direct phosphorylation of connexin43 by MAP kinase. TPA blocks GJIC mainly by the direct action of PKC, but also partly through cross-talk with the MAP kinase pathway. Connexin43 hyperphosphorylation induced by TPA is, as for EGF, mediated through MAP kinase, while PKC seems to block GJIC either through other substrates or induces a type of connexin43 phosphorylation that causes no significant electrophoresis mobility shift.