ABSTRACT
INTRODUCTION: The purpose of this study was to examine and compare the conjunctival irritation (congestion, swelling, and discharge) of topical bimatoprost ophthalmic solution 0.01% and bimatoprost ophthalmic solution 0.03% in rabbits. METHODS: Six healthy New Zealand White rabbits were treated with either bimatoprost 0.01% or bimatoprost 0.03% (3 animals/group). One dose (2 drops/dose) of study medication was administered to the right eye of each animal every 30 minutes for 4.5 hours. Approximately 1 hour after the last dose, conjunctival irritation was assessed using a slit-lamp biomicroscope to individually evaluate conjunctival congestion, swelling, and discharge. RESULTS: The mean conjunctival congestion, swelling and discharge scores for bimatoprost 0.03% were 1.67, 0.33 and 0.33, respectively, and for bimatoprost 0.01% were 2.00, 0.33 and 1.33, respectively. CONCLUSIONS: Despite the lower drug concentration of the 0.01% formulation, bimatoprost 0.01% does not reduce conjunctival irritation, including conjunctival congestion, swelling, and discharge, in rabbits compared to bimatoprost 0.03%. Further studies would be needed to determine whether the increase in the mean conjunctival congestion and discharge scores may be attributed to the increased BAK concentration in the bimatoprost 0.01% formulation.
ABSTRACT
The myocyte enhancer factor-2 (MEF2) family of transcription factors regulates transcription of muscle-dependent genes in cardiac, skeletal and smooth muscle. They are activated by calcium/calmodulin (CaM)-dependent protein kinases I and IV and silenced by CaM KIIdeltaC. MEF2 is held in an inactive form by the class II histone deacetylases (HDAC) until phosphorylated by either CaM kinase I or IV. Upon phosphorylation, HDAC is transported out of the nucleus via a 14-3-3 dependent mechanism freeing MEF2 to drive transcription. The 14-3-3 chaperone protein exists as a homodimer. In the region of homodimerization, there are two canonical CaM kinase II phosphorylation sites (ser60 and ser65). In vitro phosphorylation assay results indicate that 14-3-3beta is indeed a substrate for CaM kinase II. We hypothesize that CaM kinase IIdeltaC phosphorylation of 14-3-3beta will disrupt homodimer formation resulting in the return of HDAC to the nucleus and their reassociation with MEF2. To test this, we mutated serines 60 and 65 of 14-3-3beta to aspartates to mimic the phosphorylated state. In MEF2 enhancer-reporter assays in smooth muscle cells, expression of the 14-3-3beta double mutant attenuated MEF2-enhancer activity driven by CaM kinase I or IV. The intracellular fate of HDAC4 was followed by transfection of smooth muscle cells with an HDAC4-Green Fluorescent Protein fusion hybrid. The 14-3-3beta double mutant prevented HDAC4 cytoplasmic localization in the presence of active CaM kinase I or IV. These data suggest that the mechanism of CaM kinase IIdeltaC silencing of MEF-2-dependent genes is by phosphorylation of 14-3-3beta, which allows HDAC to return to the nucleus to reform a complex with MEF2, thereby silencing MADS box-dependent gene induction in smooth muscle.
