title: Comparison of nitric oxide production in response to carbachol between macrovascular and microvascular cardiac endothelial cells.
abstract: Cardiac microvascular endothelial cells (EC) play an important role in the physiological regulation of coronary blood flow, but their function has not been rigorously examined, because suitable in vitro models have not been available. Cardiac macrovascular and microvascular EC were isolated and cultured from 14-16-week-old Sprague-Dawley rats to examine the pharmacological responses of carbachol-induced nitric oxide (NO) production using a Griess method. Carbachol-induced NO production was only detected in cardiac macrovascular EC, which suggests that endothelial production of NO differs between macrovascular and microvascular EC. Next, cardiac microvascular EC was treated with either vehicle, angiotensin-converting enzyme (ACE) inhibitor (captopril, 10 micromol/L) or angiotensin II type 1 (AT1) receptor antagonist (CV11974, 10 micromol/L) for 4 days. Carbachol-induced NO production was improved by captopril (136+/-45nmol, p<0.01 vs vehicle) and CV11974 (146+/-30nmol, p<0.01 vs vehicle). Angiotensin II concentration in the culture medium and protein expressions of endothelial nitric oxide synthase and AT1 receptor in the EC were similar among the 3 groups. Interestingly, the level of muscarinic subtype 3 (M3) receptor was significantly increased in the EC treated with captopril (214%, p<0.01) and CV11974 (296%, p<0.01). When cardiac microvascular EC were treated with neomycin (non-selective phospholipase C inhibitor), carbachol-induced NO production was also improved (146+/-35nmol, p<0.01, neomycin I mmol/L) together with increased expression of M3 receptor (p<0.01). These data suggest that the upregulation of the M3 receptor by captopril or CV11974 occurs via a phospholipase C-dependent pathway. Cardiac microvascular EC also produced NO constitutively, as did the macrovascular EC, but carbachol-induced NO production was decreased. The present data suggest that the upregulation of the M3 receptor by the ACE inhibitor and AT1 receptor antagonist is a new beneficial effect of these drugs on microvascular endothelial function.
authors.affiliation: First Department of Internal Medicine, Yamagata University School of Medicine, Japan. tmiyasit@med.id.yamagata-u.ac.jp
authors.forename: Takehiko
authors.initials: T
authors.lastname: Miyashita
authors.forename: Yasuchika
authors.initials: Y
authors.lastname: Takeishi
authors.forename: Hiroki
authors.initials: H
authors.lastname: Takahashi
authors.forename: Takuya
authors.initials: T
authors.lastname: Miyamoto
authors.forename: Satoshi
authors.initials: S
authors.lastname: Fujii
authors.forename: Tetsuhiko
authors.initials: T
authors.lastname: Yoshimura
authors.forename: Hitonobu
authors.initials: H
authors.lastname: Tomoike
authors.forename: Shuichi
authors.initials: S
authors.lastname: Kato
authors.forename: Isao
authors.initials: I
authors.lastname: Kubota
mesh_terms: D000804:Angiotensin II / Q000378:metabolism; D057911:Angiotensin Receptor Antagonists; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D001713:Biphenyl Compounds; D002216:Captopril / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology*; D002478:Cells, Cultured; D003326:Coronary Circulation / Q000187:drug effects*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008833:Microcirculation / Q000187:drug effects; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D009994:Osmolar Concentration; D051381:Rats; D017207:Rats, Sprague-Dawley; D044140:Receptor, Angiotensin, Type 1; D043587:Receptor, Muscarinic M3; D011945:Receptors, Angiotensin / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism; D013777:Tetrazoles / Q000494:pharmacology; D010738:Type C Phospholipases / Q000502:physiology
chemical_list: D057911:Angiotensin Receptor Antagonists; D000806:Angiotensin-Converting Enzyme Inhibitors; D001562:Benzimidazoles; D001713:Biphenyl Compounds; D044140:Receptor, Angiotensin, Type 1; D043587:Receptor, Muscarinic M3; D011945:Receptors, Angiotensin; D011976:Receptors, Muscarinic; D013777:Tetrazoles; D000804:Angiotensin II; D009569:Nitric Oxide; D002217:Carbachol; D002216:Captopril; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D010738:Type C Phospholipases; C081643:candesartan