SYNERGISTIC RECEPTOR-ACTIVATED CALCIUM INCREASES IN SINGLE NONPIGMENTED EPITHELIAL-CELLS

PURPOSE. To determine whether single nonpigmented ciliary body cells c ontain the signaling mechanism to produce synergistic drug-activated i ncreases in Ca2+, or whether these responses are produced cooperativel y by interaction among groups of cells. METHODS. Suspensions of single nonpigmented cells were plated onto soft collagen gels. Fura-2 fluore scence ratio imaging was used to examine receptor-evoked changes in in tracellular 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 mu M) or e pinephrine (1 mu M) each produced small increases in intracellular Ca2 +, but in combination they produced a Ca2+ increase of more than 10-fo ld. This synergistic Ca2+ increase was a result of activation of musca rinic and alpha(2)-adrenergic receptors because a specific alpha(2)-ad renergic agonist could substitute for epinephrine in producing the res ponse. The response could be blocked by a specific alpha(2)-antagonist and a muscarinic antagonist. An alpha(1)-agonist could not substitute for epinephrine in producing a synergistic increase nor could the syn ergism be blocked by alpha(1)- 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 solutio n containing a low Ca2+ concentration; however, the influx of Ca2+ int o the cell was responsible for maintenance of a steady component of th e Ca2+ increase during maintained drug stimulation and for refilling t he internal stores. CONCLUSIONS. Single nonpigmented cells can produce synergistic increases in Ca2+ on multiple receptor activation, indica ting that the mechanism of synergism does not require the interaction of multiple cells. The Ca2+ increase is a result of release from inter nal stores and Ca2+ entry through an as yet undefined conductance or t ransport system in the plasma membrane.