Characterization of muscarinic receptors in human lens cells by pharmacologic and molecular techniques.
OBJECTIVE
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 degrees 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 microM 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 microM), 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 microM 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 and sclera.


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 glaucoma.
School of Biological Sciences, University of East Anglia, Norwich, United Kingdom.
D J
DJ
Collison
R A
RA
Coleman
R S
RS
James
J
J
Carey
G
G
Duncan
D000109:Acetylcholine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D016257:Fura-2 / Q000378:metabolism; D006801:Humans; D007908:Lens, Crystalline / Q000187:drug effects / Q000378:metabolism*; D018727:Muscarinic Antagonists / Q000494:pharmacology; D010890:Pirenzepine / Q000031:analogs & derivatives* / Q000494:pharmacology; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018727:Muscarinic Antagonists; D012333:RNA, Messenger; D011976:Receptors, Muscarinic; C065959:AFDX 384; D010890:Pirenzepine; D012313:RNA; D000109:Acetylcholine; D002118:Calcium; D016257:Fura-2