Abstract
Purpose::
To determine the effects of cAMP on the diffusional water permeability of the isolated rabbit conjunctival epithelium, given our recent finding (ARVO 2006) that the apical membrane expresses aquaporin 5 (AQP5), a water channel homologue regulated by cAMP in other cell systems.
Methods::
Conjunctivae were mounted between Ussing-type hemichambers under short-circuit conditions. Unidirectional water fluxes (Jdw) were measured in either direction by adding 3H2O to one hemichamber and sampling from the other. Electrical parameters were measured simultaneously.
Results::
Jdw were determined under control conditions and after the introduction of either the adenylyl cyclase activator, forskolin (FSK); the lipid permeable cAMP analogue, db-cAMP; and the phosphodiesterase (PDE) inhibitors, IBMX and rolipram (selective for PDE IV). All agents reduced Jdw, with IBMX and rolipram the most potent inhibitors tested. These latter compounds (100 µM) lowered Jdw from 8.07 ± 0.63 to 6.11 ± 0.51 µL/(min cm2) (n= 29, P< 0.001, as paired data), and from 7.91 ± 1.84 to 5.56 ± 1.33 (n= 4, P< 0.03), respectively, representing reductions in water permeability (Pdw) of 24-30%. In contrast, the inhibition in Jdw elicited upon FSK addition (10 µM) was limited to a 4% change, from 5.56 ± 0.72 to 5.37 ± 0.71 µL/(min cm2) (n= 13, P< 0.03, as paired data). An additional 25% reduction in Jdw was obtained upon sequentially introducing IBMX to the FSK-pretreated tissue, suggesting that inhibition of PDE was necessary to evoke salient effects on Jdw. Likewise, IBMX produced succeeding inhibitions of Jdw when applied following relatively limited effects by db-cAMP; and was also a potent inhibitor of Jdw (21% reduction) in conjunctivae pretreated apically with amphotericin B, an ionophore that also serves as an artificial water channel, thereby shunting the native aquaporins present. This latter result implies that cAMP-elicited effects on Jdw are independent of AQP5 activity. Finally, all of the cAMP-elevating agents evoked their established stimulations of the short-circuit current, suggesting that cAMP regulates ionic transport and Pdw independently.
Conclusions::
Pdw may be down regulated by high levels of cAMP (due to PDE inhibition) as a defense mechanism to preserve cell volume in the face of over-stimulated electrolyte transport. Further studies are needed to identify the channels (or gap junctions) involved in the observed phenomena.