IERG Abstracrt Book.indd - LV Prasad Eye Institute

30 Oral PresentationPurpose: Agents that decrease actomyosin contraction of trabecular meshwork (TM) cellsincrease aqueous humor outflow facility. In this study, we investigated the mechanisms by whichelevated intracellular cAMP opposes RhoA-Rho kinase pathway, leading to relaxation of theactomyosin system in TM cells.Methods: Forskolin (FSK) and rolipram were used to elevate cAMP levels. As a biochemicalmeasure of actomyosin contraction, myosin light chain phosphorylation (pMLC) was assessed.Impact of cAMP on the activation of RhoA and phosphorylation of its PKA target site, Ser188was assessed by western blot. Inhibitory phosphorylation of the regulatory subunit (MYPT1) ofMLC-Phosphatase by Rho kinase was followed using phospho-specific antibodies. Actomyosincontraction was assessed using collagen gel contraction (CGC) and its impact on cell-matrixadhesion was measured in terms of cell-substrate impedance using ECIS.Results: Treatment with FSK plus rolipram led to 10-fold increase in cAMP, and also a timedependentincrease in the phosphorylation of RhoA at Ser188. Similar treatment led to theinhibition of agonist-induced RhoA activation, formation of stress fibers, and pMLC. ElevatedcAMP reduced MYPT1 phosphorylation at the Rho kinase target site of Thr853 but not atThr696. In the CGC assay, elevated cAMP prevented basal and agonist-induced contractionsby >50%. It also reduced TM cellular impedance by 50%. These effects of FSK were similar tothose induced by Y-27632, a selective Rho kinase inhibitor.Conclusions: Elevated cAMP inhibits RhoA activation. This inhibition, presumably throughthe phosphorylation of its Ser188 residue, underlies the reduced phosphorylation of MYPT1at Thr853 and consequent reduction in pMLC. The reduction in actomyosin contraction andloss of cell-matrix interaction, mimicking the effect of Rho kinase inhibitors, may underlie theincrease in outflow facility in response to FSK perfusion.IPT 003Structure and Stability of a Single Betagamma-crystallin Domain of a ProteinBrainillin from Mouse Brain Resemble the Lens Gamma-CrystallinV Rajanikanth, P Aravind, Aditya K Singh, Shanti Swaroop Srivastava, R Sankarnarayanan,Yogendra SharmaCentre for Cellular and Molecular Biology, Hyderabad, IndiaPurpose: The purpose of this investigation was to understand the structural features ofvertebrate non-lens homologous βγ-Crystallins. This is particularly important to understandhow structural homologues of vertebrate are similar or dissimilar to the lens crystallins andtheir recruitment in non-lenticular tissues.Methods: With the known signature sequence of the members of the βγ-crystallin superfamily,we identified a protein in human genome possessing five βγ-crystallin domains. Total RNA wasisolated from the mouse brain and typical secondβ γ-crystallin domain was amplified usingthe gene specific primers. The recombinant protein was prepared and compared with variouscrystallin domains for its structure, stability and folding properties.Results: The second βγ-crystallin domain (90 residues) was more typical to lens crystallin.