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Evidence for cocaine and methylecgonidine stimulation of M 2 muscarinic receptors in cultured human embryonic lung cells
Author(s) -
Yang Yinke,
Ke Qingen,
Cai Jingbo,
Xiao YongFu,
Morgan James P
Publication year - 2001
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0703819
Subject(s) - muscarinic acetylcholine receptor , methoctramine , carbachol , endocrinology , medicine , muscarinic acetylcholine receptor m2 , forskolin , muscarinic acetylcholine receptor m3 , muscarinic acetylcholine receptor m1 , stimulation , chemistry , adenylyl cyclase , receptor , biology
Muscarinic cholinoceptor stimulation leads to an increase in guanylyl cyclase activity and to a decrease in adenylyl cyclase activity. This study examined the effects of cocaine and methylecgonidine (MEG) on muscarinic receptors by measurement of cyclic GMP and cyclic AMP content in cultured human embryonic lung (HEL299) cells which specifically express M 2 muscarinic receptors. A concentration‐dependent increase in cyclic GMP production was observed in HEL299 cells incubated with carbachol, cocaine, or MEG for 24 h. The increase in cyclic GMP content was 3.6 fold for 1 μ M carbachol ( P <0.01), 3.1 fold for 1 μ M cocaine ( P <0.01), and 7.8 fold for 1 μ M MEG ( P <0.001), respectively. This increase in cyclic GMP content was significantly attenuated or abolished by the muscarinic receptor antagonist atropine or the M 2 blocker methoctramine. In contrast, cocaine, MEG, and carbachol produced a significant inhibition of cyclic AMP production in HEL299 cells. Compared to the control, HEL299 cells treated with 1 μ M cocaine decreased cyclic AMP production by 30%. MEG and carbachol at 1 μ M decreased cyclic AMP production by 37 and 38%, respectively. Atropine or methoctramine at 1 or 10 μ M significantly attenuated or abolished the cocaine‐induced decrease in cyclic AMP production. However, the antagonists alone had neither an effect on cyclic GMP nor cyclic AMP production. Pretreatment of HEL299 cells with pertussis toxin prevented the cocaine‐induced reduction of cyclic AMP production. Western blot analysis showed that HEL299 cells specifically express M 2 muscarinic receptors without detectable M 1 and M 3 . Incubation of HEL299 cells with cocaine, carbachol, and atropine did not alter the expression of M 2 protein levels. However, the inducible isoform of nitric oxide synthase (iNOS) was induced in the presence of cocaine or carbachol and this induction was significantly attenuated after addition of atropine or methoctramine. The present data show that cocaine and MEG significantly affect cyclic GMP and cyclic AMP production in cultured HEL299 cells. Our results also show that these effects result from the drug‐induced stimulation of M 2 muscarinic receptors accompanied with no alterations of receptor expression. However, the induction of iNOS by cocaine may result in the increase in cyclic GMP production.British Journal of Pharmacology (2001) 132 , 451–460; doi: 10.1038/sj.bjp.0703819