Net Fluorescein Flux Across Corneal Endothelium Strongly Suggests Fluid Transport is due to Electro-osmosis.

We have presented prior evidence suggesting that fluid transport results from electro-osmosis at the intercellular junctions of the corneal endothelium. Such phenomenon ought to drag other extracellular solutes. We have investigated this using fluorescein-Na2 as an extracellular marker. We measured unidirectional fluxes across layers of cultured human corneal endothelial (HCE) cells. SV-40-transformed HCE layers were grown to confluence on permeable membrane inserts. The medium was DMEM with high glucose and no phenol red. Fluorescein-labeled medium was placed either on the basolateral or the apical side of the inserts; the other side carried unlabeled medium. The inserts were held in a CO2 incubator for 1 h (at 37 °C), after which the entire volume of the unlabeled side was collected. After that, label was placed on the opposite side, and the corresponding paired sample was collected after another hour. Fluorescein counts were determined with a (Photon Technology) DeltaScan fluorometer (excitation 380 nm; emission 550 nm; 2 nm bwth). Samples were read for 60 s. The cells utilized are known to transport fluid from the basolateral to the apical side, just as they do in vivo in several species. We used 4 inserts for influx and efflux (total: 20 1-h periods). We found a net flux of fluorescein from the basolateral to the apical side. The flux ratio was 1.104 ± 0.056. That difference was statistically significant (p = 0.00006, t test, paired samples). The endothelium has a definite restriction at the junctions. Hence, an asymmetry in unidirectional fluxes cannot arise from osmosis, and can only point instead to paracellular solvent drag. We suggest, once more, that such drag is due to electro-osmotic coupling at the paracellular junctions.

Effects of DIDS, a disulfonic stilbene derivative, on chloride movements in toad skeletal muscles.

In order to investigate the characteristics of the movement of Cl- ions in toad skeletal muscles we decided to study the relative membrane permeabilities of chloride and nitrate and the effects of DIDS (4,4'-diisothyocyanatostilbene-2,2'-disulphonate) upon the hyperpolarizations produced in muscle fibers when chloride or nitrate ions rapidly replace impermeant sulphate ions in the external solution. For experiments where membrane potential changes were recorded in response to sudden changes in extracellular solutions, small bundles from the semitendinosus muscles were used. We showed that DIDS reduced in a reversible manner the Cl- permeability (pCl) in toad skeletal muscle fibers. The results supporting this conclusion were the following. First, a diminished hyperpolarization in response to a sudden exposure of the fibers to a solution containing Cl-. In these experiments DIDS reduced the pCl/pK ratio to 5.5 from a control value of 12. Second, a smaller transient of the resting potential when [Cl]o was changed from 120 to 30 mM and vice versa.