Ca2+-mobilization and cell contraction after muscarinic cholinergic stimulation of the chick embryo lens.
OBJECTIVE
In the embryonic lens, cells of the anterior epithelium proliferate, migrate through the equatorial zone, and elongate to form primary lens fibers at the posterior pole. During this stage of development, cholinesterase (ChE) activity has been described as in other embryonic tissues implicated in morphogenesis. The purpose of the study was to demonstrate in addition to ChE the presence of muscarinic acetylcholine receptors (mAChR) and choline acetyltransferase (ChAT) and to test whether the muscarinic cholinergic system is involved in the regulation of cellular movements.


METHODS
In the chick embryo lens (Hamburger-Hamilton [HH] stage 21), the expression of mAChR and ChAT were demonstrated by immunohistochemistry. Isolated whole embryonic lenses were loaded with fura-2/AM, and changes of cytosolic calcium were measured after muscarinic stimulation. Size changes were assessed by morphometry with the use of time-lapse videos.


RESULTS
mAChR was present in the equatorial zone of the lens and in the elongating primary lens fibers. Anti-ChAT immunoreactivity was restricted to the primary lens fibers. Addition of carbachol induced an intracellular Ca2+ peak followed by a plateau phase of extracellular Ca2+ influx. The plateau phase was reversed by addition of atropine. Concomitant with the calcium release, a contraction of the lens and an increase in opacity was observed.


CONCLUSIONS
The localization of ChAT in the differentiating fibers of the lens indicates that acetylcholine is synthesized during differentiation and modulates morphogenesis and elongation of mAChR-positive progenitor cells in the equatorial zone. The carbachol-induced contraction indicates that the embryonic muscarinic system may be involved in the regulation of cellular movements.
Oppitz
Matthias
M
Institute of Anatomy, Department of Experimental Embryology, Department of Cellular Neurobiology, University of Tübingen, Tübingen, Germany.
Mack
Andreas
A
Drews
Ulrich
U
D000818:Animals; D001285:Atropine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology*; D002454:Cell Differentiation; D002465:Cell Movement / Q000187:drug effects*; D002642:Chick Embryo; D002795:Choline O-Acetyltransferase / Q000378:metabolism*; D018679:Cholinergic Agonists / Q000494:pharmacology*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000031:analogs & derivatives* / Q000378:metabolism; D007124:Immunoenzyme Techniques; D007908:Lens, Crystalline / Q000187:drug effects / Q000196:embryology*; D008856:Microscopy, Fluorescence; D009024:Morphogenesis; D018727:Muscarinic Antagonists / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000378:metabolism*
D018679:Cholinergic Agonists; D005456:Fluorescent Dyes; D018727:Muscarinic Antagonists; D011976:Receptors, Muscarinic; C049925:fura-2-am; D001285:Atropine; D002217:Carbachol; D002795:Choline O-Acetyltransferase; D002118:Calcium; D016257:Fura-2