Effect of elevated intracellular cAMP levels on actomyosin contraction in bovine trabecular meshwork cells.
OBJECTIVE
Elevated cAMP in the trabecular meshwork (TM) cells increases the aqueous humor outflow facility. The authors investigated the mechanisms by which elevated cAMP opposes the RhoA-Rho kinase pathway, leading to the relaxation of the actomyosin system in bovine TM cells.


METHODS
Forskolin (Fsk) and rolipram were used to elevate cAMP levels. Changes in the phosphorylation of RhoA at Ser188 (a putative inhibitory site), the regulatory light chain of myosin (pMLC), and the regulatory subunit of myosin phosphatase (MYPT1) were determined by Western blot analysis. The actomyosin contraction was measured by collagen gel contraction (CGC) assay. The impact of cAMP on cell-matrix adhesion was followed by immunostaining of focal adhesion proteins and by electric cell-substrate impedance sensing.


RESULTS
Elevated cAMP led to an increase in the phosphorylation of RhoA at Ser188, to the inhibition of endothelin-1 (ET-1)-induced activation of RhoA, and to the formation of stress fibers. The loss of pMLC along the stress fibers was comparable to that induced by Y-27632 (Rho kinase inhibitor). A concomitant reduction in both MYPT1 phosphorylation and pMLC was observed. Elevated cAMP also reduced (ET-1)-induced CGC and the cell-substrate resistance by >50%.


CONCLUSIONS
In TM cells, elevated cAMP leads to the phosphorylation of RhoA at Ser188. Consequent inhibition of RhoA activity reduces the phosphorylation of MYPT1 at Thr853, leading to a reduction in MLC phosphorylation and actomyosin contraction. These actions, similar to those of the Rho kinase inhibitors, possibly underlie the reported increase in outflow facility in response to Fsk perfusion ex vivo.
School of Optometry, Indiana University, Bloomington, Indiana 47405, USA.
Charanya
C
Ramachandran
Rajkumar V
RV
Patil
Najam A
NA
Sharif
Sangly P
SP
Srinivas
D051339:Actin Depolymerizing Factors / Q000378:metabolism; D000205:Actomyosin / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D048430:Cell Shape; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D017097:Electric Impedance; D004591:Electrophoresis, Polyacrylamide Gel; D019084:Fluorescent Antibody Technique, Indirect; D022001:Focal Adhesions / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D010766:Phosphorylation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020889:Rolipram / Q000494:pharmacology; D012694:Serine / Q000378:metabolism; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D051339:Actin Depolymerizing Factors; D018994:Myosin Light Chains; D005576:Colforsin; D012694:Serine; D000205:Actomyosin; D000242:Cyclic AMP; D043263:Myosin-Light-Chain Phosphatase; D020742:rhoA GTP-Binding Protein; D000262:Adenylyl Cyclases; D020889:Rolipram