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Abstract
PURPOSE: To determine whether single nonpigmented ciliary body cells contain the signaling mechanism to produce synergistic drug-activated increases in Ca2+, or whether these responses are produced cooperatively by interaction among groups of cells. METHODS: Suspensions of single nonpigmented cells were plated onto soft collagen gels. Fura-2 fluorescence ratio imaging was used to examine receptor-evoked changes in intracellular Ca2+ concentration. RESULTS: Nonpigmented cells plated on soft collagen gels retained a rounded shape with membrane evaginations visible on their surface. Application of acetylcholine (10 microM) or epinephrine (1 microM) each produced small increases in intracellular Ca2+, but in combination they produced a Ca2+ increase of more than 10-fold. This synergistic Ca2+increase was a result of activation of muscarinic and alpha2-adrenergic receptors because a specific alpha2-adrenergic agonist could substitute for epinephrine in producing the response. The response could be blocked by a specific alpha2-antagonist and a muscarinic antagonist. An alpha1-agonist could not substitute for epinephrine in producing a synergistic increase nor could the synergism be blocked by alpha1- or beta-antagonists. The Ca2+ increase was largely produced by release from internal stores, because the peak amplitude of the response was nearly the same in the external solution containing a low Ca2+ concentration; however, the influx of Ca2+ into the cell was responsible for maintenance of a steady component of the Ca2+ increase during maintained drug stimulation and for refilling the internal stores. CONCLUSIONS: Single nonpigmented cells can produce synergistic increases in Ca2+ on multiple receptor activation, indicating that the mechanism of synergism does not require the interaction of multiple cells. The Ca2+ increase is a result of release from internal stores and Ca2+ entry through an as yet undefined conductance or transport system in the plasma membrane.