Characterization of muscarinic receptors in human lens cells by pharmacologic and molecular techniques

PURPOSE. Activation of muscarinic receptors has been implicated in an incre ased risk of cataract after anticholinesterase treatment for glaucoma. The purpose of the present study was to determine the acetylcholine muscarinic receptor subtype(s) present in native human lens epithelial cells (NHLECs) and a human lens cell line, HLE-B3, and to compare the distribution in othe r ocular cells. METHODS. Human lens cells were perfused with artificial aqueous humor (35 d egrees C) after fura-2 incorporation, and calcium levels were measured usin g a fluorometric single-cell digital imaging system. Acetylcholine was the primary muscarinic agonist, and the receptor subtypes were elucidated by de termining the relative effectiveness of pirenzepine and AF-DX 384 in blocki ng the agonist-induced response. The levels of expression of mRNA for the r eceptor subtypes M1 through M5 were determined by quantitative reverse tran scription-polymerase chain reaction (QRT-PCR) using a sequence detection sy stem (ABI Prism 7700; Perkin-Elmer, Foster City, Ci). This was performed us ing total RNA extracted from native lens, retina, iris, and sclera and also cultured lens cells. RESULTS. Acetylcholine induced a similar concentration-dependent increase i n peak-amplitude cytosolic calcium in the range 100 nM to 100 mu M in both native and HLE-B3 cells. However, the kinetics of the response waveforms to 30-second pulses of acetylcholine were different in the two cell types. At higher concentrations (>1 mu M), a second phase appeared in the HLE-B3 cel ls that was absent in the NHLEC response. The 50% inhibitory concentration (IC50) values for blockade of a 1 mu M acetylcholine response by pirenzepin e and AF-DX 384 were 30 nM and 230 nM, respectively, for NHLECs, and 300 nM and 92 nM, respectively, for HLE-B3 cells. The QRT-PCR data showed that mo re than 90% of die total muscarinic receptor mRNA from NHLEC was of M1 orig in. In the HLE-B3 cells, however, more than 95% of the mRNA was of M3 origi n. mRNA for M3 was also in greatest abundance In other eye tissues, althoug h there was a significant contribution from MZ in iris and sclera. CONCLUSIONS. Both NHLECs and HLE-B3 cells express muscarinic receptors that produce significant changes in cytosolic calcium in response to acetylchol ine. Both pharmacologic and QRT-PCR evidence shows that whereas the M1 subt ype predominates in NHLECs, M3 is the major contributor in HLE-B3 cells. In all other eve tissues, M3 appears to be the major contributor. These data should be taken into account when choosing particular models to investigate cataract mechanisms and also when designing muscarinic agonists to treat g laucoma.