1-1-diethyl-2-hydroxy-2-nitrosohydrazine and nitroxyl

Endogenous NO and hydrogen sulfide form HNO, which causes CGRP release via TRPA1 channel activation in sensory nerves. In the present study, stimulation of intact trigeminal afferent neuron preparations with NO donors, Na2S or both was analyzed by measuring CGRP release as an index of mass activation. Combined stimulation was able to activate all parts of the trigeminal system and acted synergistic compared to stimulation with both substances alone. To investigate the contribution of both substances, we varied their ratio and tracked intracellular calcium in isolated neurons. Our results demonstrate that hydrogen sulfide is the rate-limiting factor for HNO formation. CGRP has a key role in migraine pathophysiology and HNO formation at all sites of the trigeminal system should be considered for this novel means of activation.

Topics: Animals; Brain Stem; Calcitonin Gene-Related Peptide; Calcium; Drug Synergism; Female; Hydrazines; Hydrogen Sulfide; Male; Mice, Inbred C57BL; Neurons, Afferent; Nitric Oxide Donors; Nitrogen Oxides; Rats, Wistar; Sulfides; Trigeminal Ganglion; Trigeminal Nucleus, Spinal

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

Topics: Alkenes; Catalysis; Cobalt; Hydrazines; Kinetics; Magnetic Resonance Spectroscopy; Nitric Oxide Donors; Nitrogen Oxides; Nitroso Compounds; Spectrophotometry, Ultraviolet; Vitamin B 12

The nitroxyl anion (HNO) is emerging as a novel regulator of cardiovascular function with therapeutic potential in the treatment of diseases such as heart failure. It remains unknown whether tolerance develops to HNO donors, a limitation of currently used nitrovasodilators. The susceptibility of the HNO donor, Angeli's salt (AS), to the development of vascular tolerance was compared with the NO donors, glyceryl trinitrate (GTN) and diethylamine/NONOate (DEA/NO) in rat isolated aortae. Vasorelaxation to AS was attenuated (P<0.01) by the HNO scavenger l-cysteine, whereas the sensitivity to GTN and DEA/NO was decreased (P<0.01) by the NO. scavenger carboxy-[2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidozoline-1-oxy-3-oxide]. The soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one impaired responses to GTN>or=AS>>DEA/NO. Pretreatment with 10, 30, and 100 micromol/L of GTN for 60 minutes induced a 4- (P<0.05), 13- (P<0.01), and 48-fold (P<0.01) decrease in sensitivity to GTN, demonstrating tolerance development. In contrast, pretreatment with AS or DEA/NO (10, 30, and 100 micromol/L) did not alter their subsequent vasorelaxation. All of the nitrovasodilators (30 micromol/L) displayed a similar time course of vasorelaxation and cGMP accumulation over a 60-minute period. Unlike vasorelaxation, the magnitude of peak cGMP accumulation differed substantially: DEA/NO>>AS>GTN. GTN did not induce cross-tolerance to either AS or DEA/NO. In contrast, pre-exposure to DEA/NO, but not AS, caused a concentration-dependent attenuation (P<0.01) of GTN-mediated relaxation, which was negated by the protein kinase G inhibitor guanosine 3',5'-cyclic monophosphorothioate, 8-(4-chlorophenylthio)-,Rp-isomer, triethylammonium salt. In conclusion, vascular tolerance does not develop to HNO, nor does cross-tolerance between HNO and GTN occur. Thus, HNO donors may have therapeutic advantages over traditional nitrovasodilators.

Topics: Animals; Aorta, Thoracic; Benzoates; Cyclic GMP-Dependent Protein Kinases; Cysteine; Drug Tolerance; Enzyme Inhibitors; Free Radical Scavengers; Hydrazines; Imidazoles; In Vitro Techniques; Male; Nitric Oxide; Nitric Oxide Donors; Nitrites; Nitrogen Oxides; Nitroglycerin; Oxadiazoles; Quinoxalines; Rats; Rats, Inbred WKY; Time Factors; Vasodilation

Donors of nitroxyl (HNO), the reduced congener of nitric oxide (NO), exert positive cardiac inotropy/lusitropy in vivo and in vitro, due in part to their enhancement of Ca(2+) cycling into and out of the sarcoplasmic reticulum. Here we tested whether the cardiac action of HNO further involves changes in myofilament-calcium interaction. Intact rat trabeculae from the right ventricle were mounted between a force transducer and a motor arm, superfused with Krebs-Henseleit (K-H) solution (pH 7.4, room temperature) and loaded iontophoretically with fura-2 to determine [Ca(2+)](i). Sarcomere length was set at 2.2-2.3 microm. HNO donated by Angeli's salt (AS; Na(2)N(2)O(3)) dose-dependently increased both twitch force and [Ca(2+)](i) transients (from 50 to 1000 microm). Force increased more than [Ca(2+)](i) transients, especially at higher doses (332 +/- 33% versus 221 +/- 27%, P < 0.01 at 1000 microm). AS/HNO (250 microm) increased developed force without changing Ca(2+) transients at any given [Ca(2+)](o) (0.5-2.0 mm). During steady-state activation, AS/HNO (250 microm) increased maximal Ca(2+)-activated force (F(max), 106.8 +/- 4.3 versus 86.7 +/- 4.2 mN mm(-2), n = 7-8, P < 0.01) without affecting Ca(2+) required for 50% activation (Ca(50), 0.44 +/- 0.04 versus 0.52 +/- 0.04 microm, not significant) or the Hill coefficient (4.75 +/- 0.67 versus 5.02 +/- 1.1, not significant). AS/HNO did not alter myofibrillar Mg-ATPase activity, supporting an effect on the myofilaments themselves. The thiol reducing agent dithiothreitol (DTT, 5.0 mm) both prevented and reversed HNO action, confirming AS/HNO redox sensitivity. Lastly, NO (from DEA/NO) did not mimic AS/HNO cardiac effects. Thus, in addition to reported changes in Ca(2+) cycling, HNO also acts as a cardiac Ca(2+) sensitizer, augmenting maximal force without altering actomyosin ATPase activity. This is likely to be due to modulation of myofilament proteins that harbour reactive thiolate groups that are targets of HNO.

Topics: Animals; Ca(2+) Mg(2+)-ATPase; Calcium; Heart; Hydrazines; In Vitro Techniques; Intracellular Membranes; Myocardial Contraction; Myocardium; Myofibrils; Nitric Oxide Donors; Nitrites; Nitrogen Oxides; Osmolar Concentration; Rats; Rats, Sprague-Dawley

Diazeniumdiolates, more commonly referred to as NONOates, have been extremely useful in the investigation of the biological effects of nitric oxide (NO) and related nitrogen oxides. The NONOate Angeli's salt (Na(2)N(2)O(3)) releases nitroxyl (HNO) under physiological conditions and exhibits unique cardiovascular features (i.e., positive inotropy/lusitropy) that may have relevance for pharmacological treatment of heart failure. In the search for new, organic-based compounds that release HNO, we examined isopropylamine NONOate (IPA/NO; Na[(CH(3))(2)CHNH(N(O)NO]), which is an adduct of NO and a primary amine. The chemical and pharmacological properties of IPA/NO were compared to those of Angeli's salt and a NO-producing NONOate, DEA/NO (Na[Et(2)NN(O)NO]), which is a secondary amine adduct. Under physiological conditions IPA/NO exhibited all the markers of HNO production (e.g., reductive nitrosylation, thiol reactivity, positive inotropy). These data suggest that primary amine NONOates may be useful as HNO donors in complement to the existing series of secondary amine NONOates, which are well-characterized NO donors.

Topics: Animals; Azo Compounds; Calcitonin Gene-Related Peptide; Cardiovascular System; Cell Survival; Cells, Cultured; Cricetinae; Cricetulus; Cyclic GMP; Dogs; Glutathione; Hemodynamics; Hydrazines; Lethal Dose 50; Male; Nitric Oxide Donors; Nitrites; Nitrogen Oxides; Uric Acid