IBMX-elicited inhibition of water permeability in the isolated rabbit conjunctival epithelium.

Abstract Agents expected to increase intracellular cAMP levels were tested on the diffusional water permeability ( P dw ) of isolated rabbit conjunctival epithelia given recent indications of the apical expression of AQP5, a water channel homologue regulated by cAMP in other cell systems. For these experiments, segments of conjunctivae were mounted between Ussing-type hemichambers under short-circuit conditions. Unidirectional water fluxes ( J dw ) were measured by adding 3 H 2 O to one hemichamber and sampling from the other, while the electrical parameters ( I sc and R t ) were recorded simultaneously. J dw were determined under control conditions and after the introduction of forskolin, dibutyryl-cAMP, rolipram and IBMX. All agents reduced J dw , with rolipram and IBMX the most effective inhibitors (≈28% reduction), while simultaneously evoking stimulations of the I sc ; suggesting that cAMP regulates ionic transport and P dw independently. This observation was consistent with the elimination of the IBMX-elicited I sc stimulations by the PKA inhibitor, H89, and the ineffectiveness of the sulfonamide in preventing the J dw reductions produced by the xanthine. Data from mannitol fluxes and Arrhenius plots indicated that the IBMX-elicited P dw reduction occurred at the level of water-transporting channels, but the specific moiety was not identified. Instead it was observed that lipophiles commonly used in other systems to uncouple cellular communication precluded the effects of IBMX on J dw , but the mechanism for these results was not directly linked to gap-junction blockade in the conjunctiva, as assessed by the transepithelial electrical parameters. Putatively, agents such as heptanol, by also fluidizing the bilayer, may have changed the conformation of a water channel in a manner preventing down-regulation by IBMX. Nevertheless, this study uncovered an apparently unique response to cAMP elevation exhibited by the conjunctiva, namely that P dw declines via an H89-insensitive pathway under conditions whereby PKA-dependent electrolyte transport might be over stimulated due to excessive cAMP levels (e.g., PDE inhibition).