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A. Enriquez De Salamanca, Y. Diebold, M. Calonge, K. Siemasko, J. Gao, M.E. Stern; Western Blot Analysis of Muscarinic and Adrenergic Receptors in a Normal Human Conjunctival (NHC) Epithelial Cell Line . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3783.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The normal human conjunctiva (NHC) cell line developed in our laboratory has been previously shown to express M2- and M3 muscarinic, and α- and ß-adrenergic receptors by immunofluorescence and FACS. This study investigates the presence of M1-, M2-, and M3- muscarinic and all α- and ß-adrenergic receptor subtypes by western blot analysis in the NHC cell line. Methods: Passages 70-75 of the NHC were subjected to SDS-PAGE electrophoresis and western blotting. Specific polyclonal anti-muscarinic (M1, M2, M3), anti α- (α1A-, α1B-, α1C-, α2A-, α2B-, α2C) and anti ß-adrenergic (ß1-, ß2-, ß3-) receptor subtype antibodies were used. Results: Western blot analysis of NHC cells homogenates subjected to SDS-PAGE electrophoresis confirmed the presence of immunoreactive bands for M2- M3-muscarinic and α1A-, α1B-, α1C-, α2A-, α2B-, α2C-, ß1-, ß2-, ß3- adrenergic receptors. Additionally, immunoreactivity was also detected for M1- muscarinic receptor, which was not previously detected by immunofluorescence or FACS. Conclusions: The NHC cell line expresses all muscarinic and adrenergic receptor subtypes previously reported to be present in the normal human conjunctiva. Although it remains to be proven whether these receptors are in a functional state, these findings offer further support to the use of the NHC cell line as a valid in vitro system to study neural regulation of conjunctival epithelium. Support: Allergan Inc., Irvine, CA. USA.
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