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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.