Enhancement of nitric oxide production by methylecgonidine in cultured neonatal rat cardiomyocytes.
1. In the present experiments, we investigated the effects of methylecgonidine (MEG) on nitric oxide (NO) production in cultured neonatal rat cardiomyocytes. Incubation of cultured cardiomyocytes with carbachol or MEG for 48 h significantly enhanced NO production. No release was increased from 1.48+/-0.13 microM (mg protein)(-1) for control to 5.73+/-0.19 microM (mg protein)(-1) for 1 microM carbachol treated cells (P<0.001). In addition, incubation with 1 microM MEG enhanced NO production to 5.55+/-0.28 microM (mg protein)(-1). The effects of MEG on NO production were concentration-dependent. The muscarinic antagonist atropine prevented the enhancement of NO production induced by carbachol or MEG. Compared to MEG-induced NO production, cocaine was much less potent. 2. The enhancement of NO production by carbachol or MEG was even greater in cultured cardiomyocytes transfected with the M(2) cDNA. After 48-h incubation with 1 microM carbachol or 1 microM MEG, NO production was increased by 6.5 and 6.7 fold, respectively, in cardiomyocytes overexpressing M(2) receptors. Coincubation with atropine or N(G)-nitro-L-arginine methyl ester abolished the enhancement of NO production. In contrast, NO production enhanced by carbachol or MEG in M(1)- or M(3)-transfected cardiomyocytes was similar to the level in non-transfected cells. 3. Western blot analysis showed that the protein levels of M(1), M(2), and M(3) were significantly increased in cardiomyocytes transfected with the receptor cDNAs, but MEG had no effect on the expressions. It is interesting that both carbachol and MEG caused a significant increase in constitutive endothelial NO synthase (eNOS) only in M(2)-transfected cardiomyocytes, not in non-transfected, M(1)- or M(3)-transfected cells. Again, atropine blocked the MEG-produced induction of eNOS. 4. Our data demonstrate that MEG significantly enhanced NO production in cultured cardiomyocytes and that the enhancement of NO production may result from MEG stimulation of muscarinic M(2) receptors.
Yang
Yinke
Y
The Charles A. Dana Research Institute and Harvard-Thorndike Laboratory, Cardiovascular Division, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, MA 02215, USA.
Liao
Haisun
H
Ke
Qingen
Q
Cai
Jingbo
J
Xiao
Yong-Fu
YF
Morgan
James P
JP
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D018679:Cholinergic Agonists / Q000494:pharmacology; D003042:Cocaine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000242:Cyclic AMP / Q000096:biosynthesis; D006152:Cyclic GMP / Q000096:biosynthesis; D004790:Enzyme Induction / Q000187:drug effects; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D051381:Rats; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D018679:Cholinergic Agonists; D011976:Receptors, Muscarinic; D009569:Nitric Oxide; C068464:anhydroecgonine methyl ester; D002217:Carbachol; D000242:Cyclic AMP; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496318:Nos2 protein, rat; C496316:Nos3 protein, rat; D006152:Cyclic GMP; D003042:Cocaine