endothelin-1 and 1-1-diethyl-2-hydroxy-2-nitrosohydrazine

Nitroxyl (HNO) is a redox congener of NO. We now directly compare the antihypertrophic efficacy of HNO and NO donors in neonatal rat cardiomyocytes and compare their contributing mechanisms of actions in this setting. Isopropylamine-NONOate (IPA-NO) elicited concentration-dependent inhibition of endothelin-1 (ET1)-induced increases in cardiomyocyte size, with similar suppression of hypertrophic genes. Antihypertrophic IPA-NO actions were significantly attenuated by l-cysteine (HNO scavenger), Rp-8-pCTP-cGMPS (cGMP-dependent protein kinase inhibitor), and 1-H-(1,2,4)-oxodiazolo-quinxaline-1-one [ODQ; to target soluble guanylyl cyclase (sGC)] but were unaffected by carboxy-PTIO (NO scavenger) or CGRP8-37 (calcitonin gene-related peptide antagonist). Furthermore, IPA-NO significantly increased cardiomyocyte cGMP 3.5-fold (an l-cysteine-sensitive effect) and stimulated sGC activity threefold, without detectable NO release. IPA-NO also suppressed ET1-induced cardiomyocyte superoxide generation. The pure NO donor diethylamine-NONOate (DEA-NO) reproduced these IPA-NO actions but was sensitive to carboxy-PTIO rather than l-cysteine. Although IPA-NO stimulation of purified sGC was preserved under pyrogallol oxidant stress (in direct contrast to DEA-NO), cardiomyocyte sGC activity after either donor was attenuated by this stress. Excitingly IPA-NO also exhibited acute antihypertrophic actions in response to pressure overload in the intact heart. Together these data strongly suggest that IPA-NO protection against cardiomyocyte hypertrophy is independent of both NO and CGRP but rather utilizes novel HNO activation of cGMP signaling. Thus HNO acutely limits hypertrophy independently of NO, even under conditions of elevated superoxide. Development of longer-acting HNO donors may thus represent an attractive new strategy for the treatment of cardiac hypertrophy, as stand-alone and/or add-on therapy to standard care.

Topics: Animals; Animals, Newborn; Antioxidants; Cardiomegaly; Cardiovascular Agents; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type I; Dose-Response Relationship, Drug; Endothelin-1; Enzyme Inhibitors; Gene Expression Regulation; Guanylate Cyclase; Hydrazines; Myocytes, Cardiac; Nitric Oxide Donors; Nitrogen Oxides; Pyrogallol; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Second Messenger Systems; Soluble Guanylyl Cyclase; Time Factors

Circulating levels of endothelin (ET)-1 and endogenous ET(A)-mediated constriction are increased in human aging. The mechanisms responsible are not known.. Investigate the storage, release, and activity of ET-1 system in arteries from young and aged Fischer-344 rats.. After NO synthase inhibition (L-NAME), thrombin contracted aged arteries, which was inhibited by endothelial denudation, ET(A) receptor antagonism (BQ123), and ECE inhibition (phosphoramidon, SM19712) or by inhibiting exocytosis (TAT-NSF, N-ethylmaleimide-sensitive factor inhibitor). Thrombin did not cause endothelium-dependent contraction of young arteries. In aged but not young arteries, thrombin rapidly increased ET-1 release, which was abolished by endothelium denudation or TAT-NSF. L-NAME did not affect ET-1 release. ET-1 immunofluorescent staining was punctate and distinct from von Willebrand factor (VWF). VWF and ET-1 immunofluorescent intensity was similar in young and aged quiescent arteries. Thrombin rapidly increased ET-1 staining and decreased VWF staining in aged but had no effect in young aortas. After L-NAME, thrombin decreased VWF staining in young aortas. NO donor DEA-NONOate (1 to 100 nmol/L) reversed thrombin-induced exocytosis in young (VWF) but not aged L-NAME-treated aortas (VWF, ET-1). Expression of preproET-1 mRNA and ECE-1 mRNA were increased in aged compared to young endothelium. BigET-1 levels and contraction to exogenous BigET-1 (but not ET-1) were also increased in aged compared to young arteries.. The stimulated exocytotic release of ET-1 is dramatically increased in aged endothelium. This reflects increased reactivity of exocytosis, increased expression and storage of ET-1 precursor peptides, and increased expression of ECE-1. Altered endothelial exocytosis of ET-1 and other mediators may contribute to cardiovascular pathology in aging.

Topics: Age Factors; Aging; Animals; Aorta, Thoracic; Aspartic Acid Endopeptidases; Dose-Response Relationship, Drug; Endothelin A Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Enzyme Inhibitors; Exocytosis; Fluorescent Antibody Technique; Glycopeptides; Hydrazines; In Vitro Techniques; Mesenteric Arteries; Metalloendopeptidases; N-Ethylmaleimide-Sensitive Proteins; NG-Nitroarginine Methyl Ester; Nitric Oxide Donors; Nitric Oxide Synthase; Peptides, Cyclic; Rats; Rats, Inbred F344; Receptor, Endothelin A; RNA, Messenger; Sulfonamides; Sulfonylurea Compounds; Thrombin; Up-Regulation; Vasoconstriction; von Willebrand Factor

The effect of previous nitrate therapy on vascular responses to endothelin-1 (ET-1) and NO was investigated in human internal mammary artery (IMA) in vitro. Cumulative concentration-response curves to ET-1 were constructed in rings of IMA and the data grouped into IMA from patients given nitrates prior to the bypass graft operation (nitrate group) and IMA from patients who were not prescribed nitrates (control group). No significant differences were observed between the two groups, either in EC(50) value [P>0.05; 3.5 nM (2.4-5.3 nM; 95% confidence interval) and 4.8 (2.2-10 nM), nitrate and control groups respectively] or E(max) (P>0.05; 78+/-7.5% and 85+/-9.5%, nitrate and control group respectively). No significant differences in concentration-response curves to the NO-donor diethylamine NONOate (DEA/NO) in rings of IMA pre-constricted with 10 nM ET-1 were observed between control and nitrate groups [P>0.05; EC(50) values 0.59 (0.21-1.7) microM and 0.17 (0.03-0.87) microM; E(max) 110+/-5.7% and 112+/-4.5%, nitrate and control groups respectively]. Concentration-response curves to DEA/NO constructed in normal coronary artery were not significantly different from those in coronary artery obtained from patients with ischaemic heart disease (IHD) [P>0.05; E(max) 124+/-11% and 138+/-20%; EC(50) 0.08 (0.02-0.30) microM and 0.23 (0.02-24) microM, normal and IHD respectively]. These data indicate that nitrate therapy does not induce long-term changes in the ET signalling pathway. Furthermore, the tolerance to nitrate therapy is likely to be because of impaired bio-transformation of the drug rather than reduced sensitivity of the media to NO. The similar responses to DEA/NO in normal and atherosclerotic coronary artery suggests that the reduced vasodilator responses in IHD is because of a dysfunctional endothelium and is not mediated by changes in the NO signalling pathway of the smooth muscle.

Topics: Arteriosclerosis; Coronary Vessels; Dose-Response Relationship, Drug; Endothelin-1; Humans; Hydrazines; In Vitro Techniques; Mammary Arteries; Muscle, Smooth, Vascular; Nitrates; Nitric Oxide Donors; Nitrogen Oxides; Statistics, Nonparametric; Vasoconstrictor Agents