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 α₁-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 α₂-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 a-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 Ca²⁺-free solution under conditions known to elicit overt NPE-PE separation abolished the α₂-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.