J. M. Wolosin

Regeneration of resistance and ion transport in rabbit corneal epithelium after induced surface cell exfoliation.

SummaryExposure of the rabbit corneal surface to a 20-μm digitonin-0.9% NaCl solution leads to permeabilization of the most superficial cells of the stratified epithelium. The devitalized cells exfoliate spontaneously from the corneal surface. Detergent exposure limited to 4–8 min leads to permeabilization and rapid exfoliation of a monolayer of surface cells. Consistent with the presence of the epithelial paracellular permeability barrier in this cell layer, their permeabilization results in complete loss of transepithelial resistance (Rt). Within minutes after detergent removal an initial recovery ofRt can be noticed indicating generation of a new paracellular permeability barrier by the viable subsurface cells. This recovery proceeds rapidly andRt reaches within 70 min a maximum equal to > 90% of the preexfoliation values (=2.43 kΩ·cm2,n=22). TheRt recovery is fully blocked in a reversible manner by 10 μm dihydrocytochalasin B. The recovery is not affected by inhibition of protein synthesis with 5 μm cycloheximide. When the ocular surface is treated again with digitonin the permeabilization and exfoliation of a monolayer of cells and loss ofRt are repeated. After the second detergent exposure an initial recovery ofRt occurs as before within minutes. However, the pace ofRt recovery is much slower: 4–5 hr are required to reach a stable maximalRt values amounting to about 73% of initial control. This recovery can be fully blocked by 5 μm cycloheximide indicating that protein synthesis is required for generation of tight junctions by the second subcellular layer. With only a fraction ofRt recovered, short-circuit currents amounting to, at least, 50% of control values and attributable in part to cell-to-tear movement of Cl− through the apical surface can be measured. This suggests that apical-type Cl− channels are either present in the apically facing membrane of subsurface cells or that they are rapidly inserted in it from preexisting intracellular pools immediately following the devitalization of the surface cells by digitonin.

Ca2+ mobilization and interlayer signal transfer in the heterocellular bilayered epithelium of the rabbit ciliary body.

1. ‘Ratiometric’ fura‐2 methodology in slice preparations and ‘intensitometric’ fluo‐3 measurements of confocal images were used to simultaneously monitor Ca2+ mobilization in the two distinct, apically joined cell layers which constitute the ciliary body epithelium (CBE): the non‐pigmented (NPE) and pigmented (PE) epithelia. 2. Both methods yielded comparable results regarding Ca2+ responses in the syncytium upon stimulation with adrenergic and cholinergic agonists. 3. The alpha 1‐adrenoceptor agonist phenylephrine elicited a moderate [Ca2+]i increase in the PE, whereas NPE [Ca2+]i remained unchanged or exhibited a slight diminution. 4. In combination with carbachol, the alpha 2‐adrenoceptor agonist brimonidine elicited large Ca2+ increases (> 10‐fold) in both the NPE and PE cell layers, even though previous studies indicated the absence of an alpha 2‐adrenergic effect on [Ca2+]i in the PE. The onset, as well as the peak of the Ca2+ responses in PE cells frequently exhibited a small delay with respect to adjacent NPE cells. No such time difference was observed between adjacent NPE cells. 5. Pre‐incubation of the ciliary body in Ca(2+)‐free solution under conditions known to elicit overt NPE‐PE separation abolished the alpha 2‐adrenocholinergic response in the PE. 6. Addition of heptanol to the perfusate, to block gap‐junctional communication, caused a small [Ca2+]i decrease in the NPE and a slight increase in PE[Ca2+]i. Subsequently, the Ca2+ mobilization in the Pe in response to the brimonidine and carbachol combination was either blocked or showed a substantial delay. The Ca2+ mobilization in the NPE, in contrast, remained unchanged. 7. We conclude that the heterocellular syncytium exhibits rectificatory behaviour with respect to Ca2+ mobilization; responses originating within the NPE are easily transferred to the PE, while the reverse does not occur.