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David J. Collison, Robert A. Coleman, Rowena S. James, Jae Carey, George Duncan; Characterization of Muscarinic Receptors in Human Lens Cells by Pharmacologic and Molecular Techniques. Invest. Ophthalmol. Vis. Sci. 2000;41(9):2633-2641.
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purpose. Activation of muscarinic receptors has been implicated in an increased
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 other ocular cells.
methods. Human lens cells were perfused with artificial aqueous humor (35°C)
after fura-2 incorporation, and calcium levels were measured using a
fluorometric single-cell digital imaging system. Acetylcholine was the
primary muscarinic agonist, and the receptor subtypes were elucidated
by determining the relative effectiveness of pirenzepine and AF-DX 384
in blocking the agonist-induced response. The levels of expression of
mRNA for the receptor subtypes M1 through M5 were determined by
quantitative reverse transcription–polymerase chain reaction (QRT-PCR)
using a sequence detection system (ABI Prism 7700; Perkin–Elmer,
Foster City, CA). This was performed using total RNA extracted from
native lens, retina, iris, and sclera and also cultured lens cells.
results. Acetylcholine induced a similar concentration-dependent increase in
peak-amplitude cytosolic calcium in the range 100 nM to 100 μM 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 μM), a second phase
appeared in the HLE-B3 cells that was absent in the NHLEC response. The
50% inhibitory concentration (IC50) values for blockade of
a 1 μM acetylcholine response by pirenzepine 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 more than
90% of the total muscarinic receptor mRNA from NHLEC was of M1 origin.
In the HLE-B3 cells, however, more than 95% of the mRNA was of M3
origin. mRNA for M3 was also in greatest abundance in other eye
tissues, although there was a significant contribution from M1 in iris
conclusions. Both NHLECs and HLE-B3 cells express muscarinic receptors that produce
significant changes in cytosolic calcium in response to acetylcholine.
Both pharmacologic and QRT-PCR evidence shows that whereas the M1
subtype predominates in NHLECs, M3 is the major contributor in HLE-B3
cells. In all other eye 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
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