Nuclear muscarinic acetylcholine receptors in corneal cells from rabbit.

PURPOSE: Previous studies have indicated that muscarinic acetylcholine receptors (mAChR) may be present in an unexpected, unique location and play a singular role in cellular growth regulation of rabbit corneal epithelium that may be of general physiologic significance if found in other cells. The purpose of this study was to examine rabbit corneas and corneal cells in culture to determine mAChR location and tissue distribution. METHODS: Using [3H]-propylbenzilylcholine mustard ([3H]PrBChM), which binds covalently to the active site of mAChR, rabbit corneal cross-sections, cultured corneal keratocytes, epithelial and endothelial cells, as well as nuclei isolated from these cultured corneal cells were labeled, stained, and autoradiographed. Nuclei labeled with [3H]PrBChM were further analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. RESULTS: Direct visual confirmation of the localization of mAChRs was obtained. MAChR were found in epithelial and endothelial layers of fresh-frozen corneal cross-sections, in cultured rabbit epithelial and endothelial cells, and on isolated rabbit epithelial and endothelial cell nuclei. mAChR were not detectable in keratocytes with these techniques. When [3H]PrBChM-labeled nuclei from cultured corneal cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, epithelial and endothelial samples showed specific mAChR binding, whereas binding to keratocyte nuclei was not detectable. CONCLUSIONS: As a result of these findings, a revised hypothesis is suggested for the locations and possible functions of mAChR in regulation of growth in corneal and other cells.

Identification and subcellular distribution of muscarinic acetylcholine receptor-related proteins in rabbit corneal and Chinese hamster ovary cells.

PURPOSE: The authors examined the muscarinic acetylcholine receptor (mAChR) subtypes in rabbit corneal epithelial and endothelial cells and in subcellular fractions of these cell types. A Chinese hamster ovary (CHO) cell line (nontransfected CHO K1), expected to be a negative control, also was investigated. METHODS: Whole cell homogenate and subcellular fractions were labeled with the covalent-binding, mAChR-specific ligand [3H]propylbenzilylcholine mustard ([3H]PrBChM) and were analyzed by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, or SDS-PAGE, and autoradiography. RESULTS: A pattern of multiple PrBChM-binding proteins was detected in homogenates of corneal epithelial and endothelial cells and, surprisingly, in the CHO cells. Ligand binding to all of these proteins is inhibited by the mAChR antagonists atropine sulfate and quinculidinyl benzilate. The sizes of four of the labeled protein bands are the same as the molecular masses deduced from mAChR sequence data for subtypes m3, m4, m5, and either m1 or m2. One band of 47 kd, smaller than any reported sequence, was also observed. Two of the [3H]PrBChM-binding proteins, one at 59 to 62 kd (corresponding to m5 in size) and another at 47 kd, clearly were present when highly purified nuclei were analyzed. CONCLUSIONS: The presence of multiple mAChR-like proteins at low concentrations in these disparate cell types suggests the possibility of a more general regulatory role for this type of receptor than was considered previously. Combined with other reports, the identification of proteins with the characteristics of mAChRs in purified nuclei adds support to data indicating the likelihood of G-protein-coupled signaling across the nuclear envelope.

Muscarinic acetylcholine receptor antagonists inhibit chick scleral chondrocytes.

PURPOSE: Muscarinic acetylcholine receptors (mAChRs) have been implicated in the control of myopia in humans and in animal models. This study was conducted to determine whether mAChRs influence the growth of the chick sclera and, if so, which mAChR subtypes are involved. METHODS: Sclera and scleral chondrocytes from normal and form-deprived eyes of 10- to 14-day-old chicks were treated with a total of seven ligands: two agonists, carbachol (nonselective) and McN-A-343 (selective for the M1 mAChR subtype); and five antagonists, atropine (nonselective), pirenzepine and telenzepine (M1), gallamine (M2), and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; M1 and M3). Incorporation of sulfate into glycosaminoglycans and of thymidine into DNA were quantified and normalized to sample DNA content. Possible toxicity of ligands at high doses was examined by analysis of cell number (by cell counting), viability (by trypan blue exclusion), and cellular metabolic activity (by dehydrogenase activity). RESULTS: Cellular proliferation and extracellular matrix production were inhibited by atropine in whole sclera and in its cartilaginous layer. Sulfate incorporation by chondrocytes from normal and form-deprived eyes was inhibited by mAChR antagonists with a rank order of potency (atropine > pirenzepine = 4-DAMP >> gallamine) consistent with regulation by M1, rather than M3 or M2 mAChR subtypes. Pirenzepine inhibited sulfate incorporation by chondrocytes from form-deprived eyes more effectively than those from normal eyes. Chondrocyte cultures were not viable when grown in high doses of any of the ligands used except gallamine. CONCLUSIONS: In chick scleral chondrocytes, synthesis of DNA and glycosaminoglycans was inhibited by mAChR antagonists. This inhibition was probably mediated by the M1 subtype mAChR. Therefore in vivo the sclera may be a site of action for the mAChR antagonists previously used to influence myopia. Although at high concentrations mAChR antagonists tested seemed to be toxic to chondrocytes, at lower doses inhibition occurred without toxic effects.

Corneal keratocytes: in situ and in vitro organization of cytoskeletal contractile proteins.

PURPOSE: Recent studies of corneal wound healing suggest that activated corneal keratocytes develop myofibroblast-like characteristics including a putative contractile apparatus comprised, in part, of intracellular microfilament bundles (i.e., stress fibers) containing f-actin, myosin, and alpha-actinin; extracellular fibronectin fibrils; and fibronectin surface membrane receptors (alpha 5 beta 1 integrin). The purpose of this study was to determine the expression and organization of specific components of the contractile apparatus in normal, quiescent (in situ) corneal keratocytes, and to compare the in situ organization with that of activated, tissue culture (in vitro) corneal keratocytes that potentially mimic wound healing fibroblasts. METHODS: Cat corneal tissue was obtained immediately after sacrifice and was either fixed for in situ studies or cultured with MEM supplemented with 10% fetal calf serum for in vitro studies. Keratocytes (in situ and in vitro) were stained with the following probes: phalloidin, a mushroom toxin that specifically binds to f-actin; rabbit anti-bovine aortic myosin; monoclonal anti-human alpha-actinin; monoclonal anti-human vimentin; rabbit anti-human alpha 5 beta 1 integrin; monoclonal anti-human alpha 5 integrin; monoclonal anti-human connexin 43; and goat anti-human fibronectin. The cytoskeletal organization and co-localization were evaluated using epifluorescent and confocal microscopy. RESULTS: Normal, quiescent corneal keratocytes were distributed within the cornea as a lattice network, interconnected by broad, cellular processes extending from a flattened cell body. The f-actin distribution of in situ keratocytes was predominantly cortical and appeared to be closely associated with the plasma membrane. In addition, punctate areas that appeared to correlate with the localization of adhesion sites were identified. These punctate regions appeared to stain with antibodies to alpha 5 beta 1 but to not alpha 5. These data suggest that the fibronectin receptor, alpha 5 beta 1 integrin, is not present on normal corneal keratocytes. Based on co-localization studies, rabbit anti-bovine aortic myosin and monoclonal anti-alpha-actinin staining had similar distributions to FITC-phalloidin. Interconnections between keratocytes also showed staining for connexin 43, indicating the presence of gap junctions. By contrast, activated, cultured (in vitro) keratocytes showed an FITC-phalloidin staining pattern localized predominantly along intracellular stress fibers not detected in normal, quiescent keratocytes. Myosin and alpha-actinin staining had a similar stress fiber distribution, arranged in alternating bands and suggesting a sarcomeric distribution. Associated with stress fibers there was both anti-alpha 5 beta 1 and anti-alpha 5 staining, indicating the presence of focal adhesions. CONCLUSIONS: This study demonstrates that there are major structural differences in the organization of contractile cytoskeletal proteins between normal, quiescent (in situ), and activated (in vitro) keratocytes. In situ, contractile proteins appear to be associated with the cortical f-actin network, probably related to maintenance of cell shape and interconnectivity. Alternatively, activated keratocytes were characterized by the presence of a putative contractile apparatus comprised of f-actin, myosin, and alpha-actinin organized into sarcomeric, muscle-like bundles (stress fibers) associated with focal contacts containing alpha 5 beta 1 integrin. These data suggest that activation of keratocytes, i.e. myofibroblast transformation, must involve the reorganization of cytoplasmic contractile proteins as well as the expression of alpha 5 beta 1 integrin and the formation of focal contacts.