Although much effort has been generated to identify the active platelet factor, there is a paucity of information concerning the cellular mechanism by which the active factor functions to decrease endothelial permeability. Two signaling pathways have been studied in depth with regard to endothelial permeability. It is well known that the cAMP/ protein kinase A pathway prevents and reverses an increase in vascular permeability induced by a variety of mediators, diseases, and syndromes. The effect of the cGMP/protein kinase G pathway on endothelial permeability is controversial and may be cell, tissue, and/or organ specific. Considering the overwhelming list of publications involving the above two signaling pathways, Gainor and coworkers  asked whether one of these two pathways is responsible for the permeability-decreasing activity of PCM. Two approaches were taken, using a desensitization protocol with three different cAMP/protein kinase A-enhancing agents and using inhibitors of protein kinases A and G. The cAMP-enhancing agents isoproterenol, forskolin, and 8-bromo-cAMP were incubated individually with endothelial cell monolayers as two separate challenges that were 45 minutes apart. Cells respond to the first challenge of each of these agents with an increase in electrical resistance. Following the second challenge with each agent, electrical resistance does not increase, indicative of desensitization. In contrast, desensitization is not observed when PCM is administered as the second challenge. Pharmacological inhibition of protein kinase A with KT-5720 blocks the increased endothelial electrical resistance induced by 8-bromo-cAMP but has no effect on the activity of PCM, LPA, or S1P. Similar findings are obtained upon inhibition of protein kinase G with KT-5823. The conclusion is that PCM, LPA, and S1P tighten the endothelial barrier via a cellular mechanism independent of protein kinases A and G.
Since cAMP/protein kinase A and G are not involved in the platelet activity, the next logical approach was to turn to the proposed signaling pathways elicited by LPA. LPA as well as S1P activate endothelial differentiation gene (Edg now LPA or S1P) receptors with nanomolar affinities. LPA and S1P bind to a number of Edg receptors, most notably Edg 2 (LPA 1) and Edg 4 (LPA 2) and Edg 1 (S1P 1), Edg 3 (S1P 3), and Edg 5 (S1P 2), respectively. In neurites and fibroblasts, LPA initiates signaling via activation of the G proteins, Gi, Gq, and G12/13. The mitogenic response induced by LPA in NIH 3T3 fibroblasts is pertussis toxin sensitive, indicating involvement of the Gi protein, and also includes Ras and mitogen-activated protein kinase. LPA also profoundly influences the actin cytoskeleton via activation of G12/13, Rho, and Rho kinase. Rho signaling has been shown to increase actin stress fibers. LPA as well as thrombin reorganizes the cortical actin and causes cell rounding of N1E-115 mouse neuroblastoma cells via a signaling pathway independent of calcium mobilization, activation of protein kinase C, or altered levels of intracellular cAMP. Inactiva-tion of Rho by C3 exoenzyme or Clostridium difficile toxin B and subsequent disruption of actin causes an increase in endothelial and epithelial permeability. These observations indicate that novel pathways independent of cAMP/protein kinase A exist that influence mitogenesis, cell shape, cell motility, and endothelial permeability, and that the Gi and G12/13 signaling pathways are prime signaling candidates.
Based upon the foregoing literature, initial studies with PCM, LPA, and S1P focused on the Gj signaling pathway because the pathway is well known, it can be manipulated with selective pharmacological inhibitors, and the Gq signaling pathway has been implicated in increasing endothe-lial permeability. Endothelial cell monolayers were treated first with genistein to generally inhibit tyrosine kinases because there are a number of proteins in the Gi signaling pathway that require tyrosine phosphorylation. Genistein prevents the increase in endothelial electrical resistance induced by PCM and S1P. Next, the Edg-1 receptor was depleted with antisense oligonucleotide methodology and the Gi protein was inhibited with pertussis toxin. Edg 1 couples primarily with Gi and Edg 3 and Edg 5 with Gq and G12/13. Endothelial cells appear to express Edg 1 more abundantly than Edg 3, and Edg 5 is undetectable in human umbilical veins. Depletion of Edg-1 and Edg-3 receptors inhibits the activity of S1P, and pertussis toxin inhibits the activities of both LPAand S1P. Interestingly, pertussis toxin delayed by 10 to 15 minutes the characteristic, rapid rise in endothelial electrical resistance induced by PCM. These different findings with pertussis toxin would suggest that there are other active components in PCM, other than LPA and S1P. Also, pertussis toxin does not prevent the increase in endothelial electrical resistance induced by isoproterenol, providing additional evidence that S1P- and cAMP-enhancing agents function via different cellular targets.
Phosphatidylinositol 3-kinase (PI-3 kinase) is a downstream effector of Gi in many cells and has been linked to other cellular responses induced by LPA and S1P. Inhibition of PI-3 kinase with two mechanistically different inhibitors, wortmannin and LY-294002, also causes a delay of 10 to 15 minutes in the initial, rapid rise in endothelial electrical resistance induced by PCM, LPA, and S1P. Because inhibitors of PI-3 kinase only delay the increase in electrical resistance, other G proteins and signaling pathways could be involved. One possible explanation is that the active platelet factor initiates a fast signaling pathway dependent on PI-3 kinase and a slower-acting pathway independent of this kinase. Another very plausible explanation is that the rapid signaling pathway elicited by S1P requires the interaction of the lipid by-products of PI-3 kinase with pleckstrin homology domains of the signaling proteins. In other words, activation of PI-3 kinase positions the essential signaling proteins in a complex that facilitates a rapid response. Inac-tivation of PI-3 kinase somewhat uncouples the signaling proteins and temporally delays the signaling process. Taken together these results indicate that PCM, LPA, and S1P rapidly increase endothelial electrical resistance via a novel signaling pathway involving tyrosine kinases, the Gi protein, and PI-3 kinase.
Since inhibitors of Gi and PI-3 kinase significantly modify the permeability-decreasing activity of PCM, LPA, and S1P, the next logical step was to determine if the downstream extracellular signal-regulated kinase (ERK) was involved. PCM and LPA rapidly and transiently increase ERK phosphorylation, indicative of an increase in ERK activity. Inhibition of MEK, the upstream kinase of ERK, with U-0126 blocks the increase in ERK phosphorylation but has no effect on the activity of PCM. Therefore, platelets do not function via ERK to increase endothelial electrical resistance.
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