s-nitro-n-acetylpenicillamine and Hyperplasia

The effect of nitric oxide (NO) on skin barrier recovery rate was evaluated in hairless mouse. Topical application of an NO synthase (NOS) inhibitor and a neuronal nitric oxide synthase (nNOS) inhibitor accelerated the barrier recovery after tape stripping, whereas application of an inducible NOS (iNOS) inhibitor had no effect. After tape stripping, the barrier recovery in nNOS-/- mice was significantly faster than in wild type. Topical application of the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) delayed the barrier recovery in hairless mice. Immediately after barrier disruption on skin organ culture, NO release from the skin was significantly increased. The increase was blocked by nNOS inhibitor, but not by iNOS inhibitor. Topical application of the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) accelerated the barrier recovery, whereas SIN-1 chloride, a guanylyl cyclase activator, delayed the barrier recovery. In cultured human keratinocytes, SNAP increased the intracellular calcium concentration. The increase was blocked by ODQ, but not by the calcium channel-blocker nifedipine. In calcium-free medium, SNAP increased the intracellular calcium concentration. Topical application of both nNOS inhibitor and ODQ also reduced the epidermal hyperplasia induced by barrier disruption under low environmental humidity. These results suggest that NO plays an important signaling role in cutaneous barrier homeostasis and in epidermal hyperplasia induced by barrier disruption.

Topics: Administration, Topical; Animals; Calcium; Cell Membrane Permeability; Cells, Cultured; Enzyme Inhibitors; Homeostasis; Humans; Hyperplasia; Keratinocytes; Male; Mice; Mice, Hairless; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Oxadiazoles; Penicillamine; Quinoxalines; Skin

We compared the effects of NO donors and cGMP analogues on the growth of aortic smooth muscle cells (SMCs) derived from newborn, adult (aged 3 months), and old (aged 2 years) rats. We found that the NO donor S-nitroso-N-acetylpenicillamine failed to block DNA synthesis in SMCs from old rats but was effective in SMCs from newborn and adult rats. However, cGMP analogues were inhibitory in all 3 SMC types. We demonstrated that in SMCs from old rats, NO was unable to increase the concentration of intracellular cGMP, suggesting that either cGMP synthesis was defective or cGMP degradation was enhanced. Western blot analysis revealed that SMCs from old rats do not express the beta subunit of soluble guanylyl cyclase. To confirm the importance of this observation in vivo, we balloon-injured the carotid arteries of adult and old rats. Whereas soluble guanylyl cyclase was expressed at the same level in the media of injured vessels and uninjured vessels of both groups, its expression in the intimas of old rats was reduced by 70% compared with intimas from adult animals. Furthermore, N(omega)-nitro-L-arginine, an inhibitor of NO synthesis, enhanced the intimal thickening in injured vessels in adult rats but not in old rats. We conclude that the loss of NO responsiveness in aged rats is due to the lack of the beta subunit of soluble guanylyl cyclase, and we speculate that this defect contributes to the enhanced intimal thickening in response to injury in old animals.

Topics: Aging; Angioplasty, Balloon; Animals; Aorta; Carotid Artery Injuries; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; DNA; Enzyme Inhibitors; Guanylate Cyclase; Hyperplasia; Male; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Donors; Nitroarginine; Penicillamine; Protein Kinases; Rats; Rats, Inbred F344; Signal Transduction; Solubility; Tunica Intima

Overexpression of the inducible nitric oxide synthase (iNOS) gene inhibits neointimal hyperplasia after arterial injury. The purpose of this study was to examine the mechanism by which nitric oxide (NO) inhibits vascular smooth muscle cell (VSMC) proliferation, specifically focusing on signaling pathways known to be activated by NO, including cyclic guanosine monophosphate (cGMP), p53, and p42/44 mitogen-activated protein kinase (MAPK).. VSMCs that were subjected to iNOS gene transfer demonstrated a reduction in proliferation (80%) that was associated with a marked increase in p21 expression. The antiproliferative and p21 stimulatory effects of NO were not suppressed by the soluble guanylate cyclase inhibitor ODQ, implicating cGMP-independent signaling. The role of p53 in NO-mediated upregulation of p21 and inhibition of proliferation was evaluated using p53 -/- VSMCs. A similar reduction in cellular proliferation and upregulation of p21 expression were achieved with iNOS gene transfer as well as treatment with the NO-donor S-nitroso-N-acetylpenicillamine (SNAP), demonstrating the p53-independent nature of these NO-mediated pathways. The transfer of the iNOS gene activated the p42/44 MAPK, and inhibition of this MAPK pathway with PD98059 partially blocked the antiproliferative effects of NO and completely inhibited the p21 stimulatory effects of NO. For confirmation that iNOS overexpression upregulated p21 in vivo, injured rat carotid arteries were infected with an adenoviral vector carrying the iNOS gene and demonstrated a marked upregulation of p21 expression at three days. However, the ability of NO to inhibit VSMC proliferation does not solely depend on p21 upregulation since the NO-donor SNAP-inhibited VSMC proliferation in p21 -/- VSMCs.. Nitric oxide inhibits VSMC proliferation in association with the upregulation of p21; both occur independent of p53 and cGMP while being partially mediated through the p42/44 MAPK signaling cascade. This represents one potential mechanism by which NO inhibits VSMC proliferation.

Topics: Animals; Carotid Arteries; Cell Division; Cyclic GMP; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Gene Expression Regulation, Enzymologic; Gene Transfer Techniques; Guanylate Cyclase; Humans; Hyperplasia; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; Nitric Oxide Donors; Nitric Oxide Synthase; Penicillamine; Proto-Oncogene Proteins p21(ras); Rats; Rats, Sprague-Dawley; Signal Transduction; Tumor Suppressor Protein p53; Tunica Intima; Up-Regulation

Airway smooth muscle (ASM) hypertrophy and hyperplasia are important determinants of bronchial responsiveness in asthma, and agents that interfere with these processes may prevent airway remodeling. We tested the hypothesis that activators of soluble and particulate guanylyl cyclases would inhibit human ASM cell (HASMC) proliferation. We report that the nitric oxide (NO) donors S-nitroso-N-acetylpenicillamine (SNAP; 10(-6) to 10(-4) M) and sodium nitroprusside (10(-5) to 10(-3) M) and human atrial natriuretic peptide [ANP-(1-28); 10(-8) to 10(-6) M], which activate soluble and particulate guanylyl cyclases, respectively, inhibited serum- and thrombin-induced proliferation of cultured HASMCs. The antimitogenic effect of SNAP was reversed by hemoglobin (10(-5) M), an NO scavenger, suggesting that NO donation was involved. The antiproliferative effects of SNAP and ANP-(1-28) were potentiated by the cGMP-specific phosphodiesterase zaprinast and mimicked by 8-bromo-cGMP (10(-6) to 10(-3) M), suggesting that cGMP-dependent mechanisms were involved. However, first, ANP-(1-28) produced a smaller antiproliferative effect than SNAP in contrast to their abilities to elevate cGMP, and second, rat ANP-(104-126), which binds selectively to ANP clearance receptors without elevating cGMP, had a small antiproliferative effect, suggesting that cGMP-independent mechanisms were also involved. These results provide evidence for a novel antiproliferative effect of NO and ANP in HASMCs mediated through cGMP-dependent and cGMP-independent mechanisms.

Topics: Asthma; Atrial Natriuretic Factor; Blood Proteins; Cell Division; Cells, Cultured; Coloring Agents; Cyclic GMP; Diuretics; Hemoglobins; Hemostatics; Humans; Hyperplasia; Lung; Mitogens; Muscle, Smooth; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Penicillamine; Peptide Fragments; Phosphodiesterase Inhibitors; Purinones; Tetrazolium Salts; Thiazoles; Thrombin; Vasodilator Agents

The universal response of vein grafts after insertion into the arterial circulation is the development of intimal hyperplasia; smooth muscle cell proliferation and connective tissue deposition, which may be modulated in part by dysfunctional endothelial nitric oxide (NO) metabolism. This study examines the effects of single dose, local application by pluronic gel of a NO donor, S-nitroso-N-acetylpenicillamine (SNAP) and an NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME) on the formation of intimal hyperplasia.. Forty New Zealand white rabbits underwent jugular vein interposition grafting of the common carotid artery.. Ten animals were controls, 10 animals had the outer surface of the vein graft coated with 30% pluronic gel (2.5 ml), and 10 each were immersed for 15 min prior to insertion in Ringer lactate containing 10(-3) M of SNAP or L-NAME and then had their vein grafts coated with 2.5 ml of gel containing either SNAP (10(-3) M) or L-NAME (10(-3) M), which allows for sustained delivery for up to 6 h. On the 28th post operative day, the animals were sacrificed and vein grafts were harvested for morphology by electron microscopy (SEM and TEM) and dimensional analysis by videomorphometry.. All vein grafts developed intimal hyperplasia. On SEM the vein grafts had a confluent layer of endothelial cells with multiple layers of smooth muscle cells representing intimal hyperplasia in TEM. There were no demonstrable morphological differences between the four groups. Local treatment with SNAP produced a significant 36% decrease in mean intimal thickness (72 +/- 4 microns vs. 45 +/- 4 microns; mean +/- S.E.M.; p < 0.01) without a change in medial thickness compared to gel-only treated groups (58 +/- 6 microns vs. 61 +/- 7 microns; p = ns). Inhibition of NO synthase by L-NAME had no effect on the development of intimal hyperplasia (72 +/- 4 microns vs. 79 +/- 10 microns; p = ns); medial thickness was also unchanged.. These data confirm the protective effect of NO in vascular injury and suggest that NO synthase activity is either absent or reduced to such a level that further inhibition in this short time course is not relevant to the pathophysiology of vein graft intimal hyperplasia.

Topics: Animals; Carotid Artery, Common; Cell Division; Cells, Cultured; Collagen; Cytoplasm; Endothelium, Vascular; Female; Hyperplasia; Microscopy, Electron, Scanning; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Penicillamine; Rabbits; Vascular Patency; Veins