Synergistic receptor-activated calcium increases in single nonpigmented epithelial cells.
OBJECTIVE
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.
Cilluffo
M C
MC
Department of Physiological Science, University of California, Los Angeles 90095-1527, USA.
Xia
S L
SL
Farahbakhsh
N A
NA
Fain
G L
GL
D000109:Acetylcholine / Q000494:pharmacology; D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002924:Ciliary Body / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D003094:Collagen; D004338:Drug Combinations; D004357:Drug Synergism; D004837:Epinephrine / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D005782:Gels; D018727:Muscarinic Antagonists / Q000494:pharmacology; D011817:Rabbits; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism*
D000317:Adrenergic alpha-Antagonists; D004338:Drug Combinations; D005456:Fluorescent Dyes; D005782:Gels; D018727:Muscarinic Antagonists; D018341:Receptors, Adrenergic, alpha-2; D011976:Receptors, Muscarinic; D003094:Collagen; D000109:Acetylcholine; D002118:Calcium; D016257:Fura-2; D004837:Epinephrine