Effect of protein kinase C inhibitors and activators on corneal re-epithelialization in the rat.

PURPOSE: To examine the ability of protein kinase C (PKC) inhibitors and activators to influence the rate of corneal re-epithelialization in the rat. METHOD: Rat corneas with 3 mm diameter central epithelial abrasions were organ-cultured in control medium or in medium with inhibitors or activators of PKC. RESULTS: In control corneas, the defect was completely re-epithelialized by 25 hr. In the presence of the PKC inhibitors staurosporine (100 nM), sphinganine (50 mumol/l), or H-7 (100 mumol/l) there were significantly larger epithelial defects than in controls after 5-25 hr of incubation. Re-epithelialization rates were similar to control corneas when the incubation medium contained HA1004 (100 mumol/l), an analogue of H-7 that is a potent inhibitor of cyclic adenosine monophosphate- and cyclic guanosine monophosphate-dependent protein kinases and a weak inhibitor of PKC. Two PKC activators, 1-oleoyl-2-acetyl-sn-glycerol (OAG) and phorbol 12-myristate 13-acetate (PMA), were unable to enhance the rate of epithelial wound healing. CONCLUSIONS: Our results suggest that PKC activity is an important factor in regulating corneal epithelial wound healing, presumably by influencing cell migration. Moreover, the results with OAG and PMA suggest that PKC is maximally activated during re-epithelialization in this organ-culture assay.

Effect of inhibitors of arachidonic acid metabolism on corneal reepithelialization in the rat.

We examined the ability of inhibitors of arachidonic acid metabolism to influence the rate of healing of organ-cultured rat corneas with 3-mm diameter central epithelial abrasions. In control corneas, and in the presence of the cyclooxygenase inhibitors indomethacin (1 microM), or flurbiprofen (1 microM), the defect was completely reepithelialized by 25 hr. In contrast, corneas cultured with the lipoxygenase inhibitors quercetin (100 microM), esculetin (100 microM), or baicalein (10 microM) or the dual cyclooxygenase/lipoxygenase inhibitors BW 775C (100 microM) or BW A540C (100 microM) had significantly delayed epithelial healing rates when compared with the controls; complete healing of the epithelial defects required 32.5-40 hr. Dose-response studies with esculetin and BW 755C demonstrated that the concentrations for 50% inhibition of reepithelialization (65.3 microM for esculetin, 39.6 microM for BW 755 C) were significantly greater than those for inhibition of 12-lipoxygenase activity (16.6 microM for esculetin, 21.1 microM for BW 755C), the major lipoxygenase activity in normal rat cornea. Addition of 12(S)-5,8,10,14- hydroxyeicosatetraenoic acid [12(S)-HETE, 0.01-10 microM], the main 12-lipoxygenase metabolite of arachidonic acid in normal rat cornea, to the organ cultures did not influence the rate of epithelial wound healing in the absence of presence of 100 microM esculetin. Our results suggest that lipoxygenase activity is an important factor in regulating corneal epithelial wound healing in the rat, presumably by influencing epithelial cell migration. The lipoxygenase enzyme and metabolite(s) responsible for regulating reepithelialization, and the mechanism of action, remain to be determined.

Theoretical investigation of the role of clay edges in prebiotic peptide bond formation. I. Structures of acetic acid, glycine, H2SO4, H3PO4, Si(OH)4, Al(OH)4-.

Activation of amino acids appears to have played a crucial role in prebiotic peptide bond formation. As a model of this process in living systems, phosphates have been used as amino acid activators. The possible role of clay and other minerals has also been investigated. We are presently using ab initio methods to investigate the activation of amino acids by these agents, as an initial step in peptide bond formation. A model of this activation process is described by the reaction: ZCH2COOH + XO4Hn+1 --> ZCH2COOXO3Hn + H2O. The first step in such an investigation, reported here, was to determine the lowest energy structures of a suitable set of reactions. As initial models of amino acids, Z was chosen to be H and NH2, corresponding to acetic acid and glycine, respectively, XO4Hn+1 = H3PO4 represents a phosphate group, while Si(OH)4 describes an edge tetrahedral site of a clay mineral. Al(OH)4- was also included to represent tetrahedral edge site where the silicon is replaced by an aluminum. Finally, to complete the series XO4Hn+1, H2SO4 was added to the set of reactants. All species were optimized using the STO-3G and STO-3G* basis sets. For H3PO4 and Al(OH)4-, STO-3G* full optimizations were not possible. In these cases, certain torsional angles were optimized separately, then held at the optimized value, while the rest of the bond lengths and angles were optimized. All structures were compared to other calculations and to experimental geometries when available.