Bradykinin Activation of Extracellular Signal-Regulated Kinases in Human Trabecular Meshwork Cells

Abstract

Purpose: : Bradykinin (BK) acts on B₂ kinin receptors to promote matrix metalloproteinase (MMP) secretion from trabecular meshwork (TM) cells and to increase conventional outflow facility. Because acute secretion of specific MMPs can be dependent on the activity of extracellular signal-regulated MAP kinases (ERK 1/2), experiments were performed to determine BK effects on ERK 1/2 activation in human TM cells.

Methods: : BK signaling was examined in primary cultures of human TM cells studied at passages 2 and 3. Activation of ERK 1/2 was determined by immunoblot of cellular lysates using anti-phospho-ERK 1/2 and anti-ERK 1/2 antibodies.

Results: : Incubation of human TM cells with BK produced a concentration-dependent increase in ERK 1/2 phosphorylation compared to unstimulated cells. The response maximum occurred at 100 nM BK and the EC₅₀ was approximately 1.6 nM. ERK 1/2 phosphorylation was increased four to eight-fold relative to basal activity. BK stimulation of ERK 1/2 activity peaked within 2 to 10 min of exposure and then declined to control levels by 60 min. Treatment of TM cells with the selective B₂ kinin receptor agonist, Tyr⁸-BK, also stimulated ERK 1/2 phosphorylation while the B₁ selective agonist, Lys-[Des-Arg⁹]-BK had no significant effect. In addition, activation of ERK 1/2 by BK was blocked by pretreatment of cells with the B₂ receptor antagonist, Hoe-140. BK treatment failed to activate either JNK or p38 MAP kinase. Downregulation of protein kinase C (PKC) by persistent exposure of cells to phorbol 12-myristate 13-acetate prevented BK activation of ERK 1/2. Pretreatment of cells with the nonselective PKC inhibitor, GF109203, had an equivalent effect. Finally, incubation of TM cells with U0126, an inhibitor of MAP kinase kinase (MEK), was also observed to block BK-induced ERK 1/2 phosphorylation.

Conclusions: : These results indicate that BK acts on TM cells to rapidly activate the extracellular signal-regulated MAP kinases, ERK 1/2. This effect is dependent on signaling through the PKC pathway and MEK activation. ERK 1/2 stimulation by BK, like BK actions to increase MMP secretion and enhance outflow facility, is mediated by B₂ kinin receptors. The composite of the data supports the idea that BK activates ERK 1/2 in TM cells to promote secretion of constitutively-expressed MMPs and increase aqueous outflow.

Keywords: signal transduction: pharmacology/physiology • receptors: pharmacology/physiology • trabecular meshwork