Bradykinin activation of extracellular signal-regulated kinases in human trabecular meshwork cells

Abstract

Bradykinin stimulation of B 2 kinin receptors has been shown to promote matrix metallo-proteinase (MMP) secretion from trabecular meshwork cells and to increase conventional outflow facility. Because acute secretion of MMPs can be dependent on the activity of extracellular signal-regulated MAP kinases (ERK1/2), experiments were performed to determine bradykinin effects on ERK1/2 in cultured human trabecular meshwork cells and the relationship of these effects to MMP-9 release. Treatment of cells with bradykinin produced a rapid 4-to 6-fold increase in ERK1/2 phosphorylation. Stimulation of ERK1/2 activity peaked within 2 min and then declined to control levels by 60 min. The response maximum occurred with 100 nM bradykinin and the estimated EC 50 was 0.7 nM. Treatment of cells with the B 2 kinin receptor agonist, Tyr 8- bradykinin, also stimulated ERK1/2 phosphorylation while the B 1 agonist, Lys- [Des-Arg 9]- bradykinin had no significant effect. In addition, activation of ERK1/2 by bradykinin or Tyr 8- bradykinin was blocked by the selective B 2 receptor antagonist, Hoe-140. Inhibition of MAP kinase kinase (MEK) with U0126 also blocked bradykinin-induced ERK1/2 phosphorylation. Suppression of protein kinase C activity with the nonselective inhibitor, GF109203X, or by down-regulation with phorbol ester, diminished, but did not eliminate, bradykinin activation of ERK1/2. A similar decrease of ERK1/2 stimulation was observed when Src kinase was inhibited by 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Finally, blockade of bradykinin-induced ERK1/2 activation substantially reduced the peptide’s action to stimulate MMP-9 release into the extracellular environment. The data demonstrate that bradykinin promotes ERK1/2 activation in human trabecular meshwork cells. The effect is mediated by B 2 kinin receptors, involves two different signaling pathways, and results in increased secretion of MMP-9.