H-1152P is a very potent and specific ROCK inhibitor with a
K i of 1.6 nM for Rho-kinase, 630 nM for protein kinase A, 9.270 μM for protein kinase C, and 10.1 μM for myosin light-chain kinase in cell-free assays. However, higher concentrations of this inhibitor ranging between 0.1 and 10 μM are necessary for the suppression of ROCK in NT-2 cells,
29 possibly because of competition with intracellular ATP present at the micromolar range.
49 Our observations on Tenon’s capsule fibroblasts support these findings and underscore the need for high doses of this molecule for ROCK-inhibition in cell-based assays. However, this increases the risk of unintentionally targeting other kinases, with PKA being the most likely candidate owing to the relatively lower
K i of H-1152P for this kinase. PKA is a ubiquitously expressed intracellular signaling molecule that regulates ion channel conductivity, gene transcription, cell metabolism, actin cytoskeletal dynamics, and migration.
50 PKA can also directly phosphorylate and inactivate RhoA.
51 52 This broad spectrum of functions, some of which antagonize the activity of ROCK, may therefore give rise to complications or weaken the effects when an inhibitor showing differential affinity for both kinases is applied at high concentrations. To ascertain that the PKA pathway was not influenced by H-1152P in Tenon’s capsule fibroblasts, we analyzed the phosphorylation level of adducin isoforms at S726/S662 as an indicator of PKA activity. The PKA mediated phosphorylation mainly at S726 causes its dissociation from the F-actin cytoskeleton in vitro. The supernatants of the cell lysates we collected after centrifugation at 12,000
g are expected to contain both the cytoskeleton-bound and the dissociated forms of adducin.
53 Our immunoblots revealed no significant change in the levels of p-α-adducin and only a slight decrease in the level of p-γ-adducin, which did not become more pronounced at increasing concentrations of H-1152P. Though the reason for this decrease remains to be elucidated, these findings favor the view that H-1152P exerted its effects on Tenon’s capsule fibroblasts without considerably interfering with PKA activity at the concentrations tested.