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C. Ramachandran, R. V. Patil, N. Sharif, S. P. Srinivas; Cross-Talk Between cAMP and RhoA-Rho Kinase in Bovine Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3234.
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Mechanisms that oppose actomyosin contraction of the trabecular meshwork (TM) cells increase aqueous humor outflow facility. We have investigated mechanisms by which elevated cAMP affects actomyosin contraction via regulation of the Rho kinase-dependent activity of the myosin light chain (MLC) phosphatase complex in cultured bovine TM cells.
Forskolin (FSK; 10 µM), in combination with rolipram (50 µM), was used to elevate intracellular cAMP which was measured by ELISA. As a biochemical measure of actomyosin contraction, phosphorylated MLC (ppMLC) was assessed. Impact of cAMP on the activation of RhoA and phosphorylation of its PKA target site, Ser188 was assessed by western blot.Effect of Rho kinase on MLCP was followed by phosphorylation of its regulatory component MYPT1 (130 kDa) using phospho-specific antibodies. As a direct indicator of loss of actomyosin contraction, collagen gel contraction (CGC) was assessed in response to endothelin-1 (ET-1; 100 nM) with and without FSK. As an indirect measure, cell-matrix adhesion in terms of cell-substrate impedance was measured using ECIS. Phosphorylation of MLC and Ser188 are expressed as % increase over untreated cells.
Treatment with FSK plus rolipram led to 10-fold increase in cAMP, and also a time-dependent increase in the phosphorylation of RhoA at Ser188 (217 +15; n=4). Similar treatment led to inhibition of (ET-1)-induced activation of RhoA, formation of stress fibers, and MLC phosphorylation (ET-1: 192+9, ET-1+Fsk: 3+1.4, ET-1+Y: 23+6.7). The effects of forskolin on stress fibers and associated ppMLC were similar to those of Y-27632 (5 µM), a selective Rho kinase inhibitor. Elevated cAMP also reduced MYPT1 phosphorylation at Rho kinase target site of Thr853 but not at Thr696. In the CGC assay, elevated cAMP prevented basal and (ET-1)-induced contractions by >50% (n=9). The relaxation was comparable to that produced by Myosin II ATPase inhibitor (blebbistatin, 10 µM, n=4) and greater than Y-27632 (n=6). Elevated cAMP also reduced cellular impedance of TM cells by 50% with the response similar to Y-27632.
Elevated cAMP opposes RhoA activation. This inhibition, presumably through PKA phosphorylation of its Ser188 residue, underlies the reduced phosphorylation of MYPT1 at Thr853 and consequent reduction in ppMLC. The reduction in actomyosin contraction and loss of cell-matrix interaction, mimicking the effect of Rho kinase inhibitors, may underlie the apparent increase in outflow facility in response to FSK perfusion.
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