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

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).