Hideki Moritoki

Nonendothelial-derived nitric oxide activates the ATP-sensitive K+ channel of vascular smooth muscle cells

To determine whether endogenous nitric oxide (NO) opens the ATP‐sensitive K+ channel (KATP channel), we investigated the effect of nonendothelial‐derived NO on this channel in cultured smooth muscle cells of the porcine coronary artery by the patch‐clamp technique. In the cells pretreated with endotoxin, the addition of 10−4 M l‐arginine generated NO and activated the KATP channel. Activation of this channel was suppressed by pretreatment with 10−3 M N G‐methyl‐l‐arginine or 10−3 M N x‐nitro‐l‐arginine methyl ester, each of which is a specific antagonist of the l‐arginine‐NO pathway, and by 10−6 M Methylene blue, which blocks guanylate cyclase. The activation of the KATP channel by l‐arginine‐NO pathway is expected to produce hyperpolarization of the cell membrane and relaxation of vascular smooth muscle cells.

Age-related decrease in endothelium-dependent dilator response to histamine in rat mesenteric artery

The effect of aging on the vasodilator responses to histamine, 2-pyridylethylamine and 4-methylhistamine of ring segments of rat mesenteric arteries were investigated. The maximal extent of histamine-induced dilatation of the arteries previously contracted with norepinephrine was greatest for arteries from rats aged 2 and 8 weeks. The maximal response decreased progressively with an increase in age to 13 and 56 weeks. Arteries from 99 week old rats scarcely responded to histamine. Under these conditions, the dilatation induced by papaverine showed no change with age. The vasodilatation caused by 2-pyridylethylamine and 4-methylhistamine also decreased age dependently. The dilatation of the arteries induced by these agents was inhibited by the H1-antagonist chlorpheniramine, but not by the H2-antagonist cimetidine. Removal of the endothelium completely abolished the vasodilator effect of histamine, leaving the effect of papaverine unaffected. Hydroquinone and methylene blue reversed the dilatation induced by histamine, without affecting that caused by papaverine. These results suggest that the age-related decrease in dilatation of rat mesenteric artery in response to histamine is mainly due to a decrease in the ability of the endothelium to liberate a mediator(s).

Evidence for the involvement of cyclic GMP in adenosine-induced, age-dependent vasodilatation.

1 Adenosine‐induced dilatation of rat aorta was present in aorta taken from 4 week‐old rats, attenuated with increase in age of rats to 8 weeks, and was virtually absent in the aorta from 12 week‐old rats. 2 Removal of the endothelium by mechanical rubbing attenuated adenosine‐induced dilatation. 3 Haemoglobin and methylene blue partly reversed the adenosine‐induced endothelium‐dependent dilatation. 4 The order of potency of adenosine derivatives was 5′‐(N‐ethylcarboxamido)adenosine (NECA) > 2‐phenylaminoadenosine (CV‐1808) > 2‐chloroadenosine > N6‐([R]‐[‐]‐phenylisopropyl)adenosine (R‐PIA) > adenosine > N6‐cyclohexyladenosine (CHA) > N6‐([S]‐[+]‐phenylisopropyl)adenosine (S‐PIA), indicating that adenosine receptors mediating the dilatation are of the A2 subtype. 5 [3H]‐NECA bound to preparations of membranes from rats of 4 weeks old; it was displaced more effectively by NECA and the A2 ligand CV‐1808 than by the A1 ligands CHA and S‐PIA. ligands CHA and S‐PIA. 6 The number but not affinity of specific binding sites for NECA decreased considerably with increase in age of rats to 8 weeks, and binding sites for [3H]‐NECA were hardly detected in membrane preparations from rats of 20 weeks old. 7 Adenosine caused a marked increase in cyclic GMP production, but did not induce an increase in the cyclic AMP level. 8 This increase in cyclic GMP production induced by adenosine was abolished by methylene blue or 8‐phenyltheophylline, or by removal of the endothelium. 9 The age‐associated decrease in adenosine‐induced dilatation was found to be associated with a reduction in the formation of cyclic GMP, but not of cyclic AMP. 10 These results suggest that adenosine causes dilatation via A2 receptors by inducing production of an endothelium‐derived relaxing factor (EDRF), which in turn stimulates soluble guanylate cyclase, and so increases production of cyclic GMP. It is also suggested that the main reason for the age‐associated decrease in adenosine‐induced dilatation is a decrease in the number of A2‐receptors or the ability of the endothelium to produce EDRF, leading to decreased production of cyclic GMP.

Involvement of nitric oxide pathway in the PAF-induced relaxation of rat thoracic aorta

1 The mechanism of the vasorelaxant effect of platelet activating factor (PAF) on rat thoracic aorta and the effect of aging on the PAF‐induced relaxation were investigated. 2 PAF at concentrations causing relaxation induced marked increases in guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) production, but did not induce an increase in adenosine 3′:5′‐cyclic monophosphate (cyclic AMP). 3 Removal of the endothelium by mechanical rubbing, and treatment with the PAF antagonists CV‐3988, CV‐6209 and FR‐900452, the nitric oxide biosynthesis inhibitor, NG‐nitro l‐arginine, the radical scavenger, haemoglobin, and the soluble guanylate cyclase inhibitor, methylene blue, inhibited PAF‐induced relaxation and abolished or attenuated PAF‐stimulated cyclic GMP production. 4 The relaxation was greatest in arteries from rats aged 4 weeks. With an increase in age, the response of the arteries to PAF was attenuated. 5 Endothelium‐dependent cyclic GMP production also decreased with increase in age of the rats. 6 These results suggest that PAF stimulates production of nitric oxide from l‐arginine by acting on the PAF receptors in the endothelium, which in turn stimulates soluble guanylate cyclase in the smooth muscle cells, and so increases production of cyclic GMP, thus relaxing the arteries. Age‐associated decrease in PAF‐induced relaxation may result from a reduction of cyclic GMP formation.

Age-associated decrease in histamine-induced vasodilatation may be due to reduction of cyclic GMP formation

1 The effects of aging on histamine‐induced vasodilatation and cyclic GMP production in rat thoracic aorta were investigated. 2 This histamine‐induced dilatation of the aorta was mediated by H1‐receptors and was dependent on the endothelium. 3 Histamine induced the greatest dilatation of arteries of 3–4 week old rats, progressively less of those of rats of 8 to 56 weeks old, and scarcely detectable dilatation of those of 100 week old rats. 4 Histamine induced cyclic GMP production in aorta from rats of 4 weeks old, with no change in the cyclic AMP level. This increase in the cyclic GMP level induced by histamine also decreased with age, being about 70% as great at 8 weeks, 50% as great at 50–60 weeks, and 10% as great at 130 weeks of age. 5 Removal of the endothelium completely abolished the histamine‐induced increase in cyclic GMP. 6 The dilator effect of nitroprusside, which enhances cyclic GMP production by stimulating guanylate cyclase directly (not indirectly via the endothelium derived relaxing factor, EDRF), also snowed age‐related attenuation. 7 The dilator effect of 8‐bromo cyclic GMP, which stimulates cyclic GMP‐dependent protein kinase, also decreased during aging. 8 These results suggest that aging reduces the ability of the endothelium to produce EDRF, which stimulates guanylate cyclase, and so decreases cyclic GMP production. Thus the decreased dilator response of the arteries to histamine during aging is probably due to both loss of endothelial function and reduction of guanylate cyclase activity. Alteration of cyclic GMP‐dependent protein kinase activity may also participate in the age‐related changes.

Possible mechanisms of age-associated reduction of vascular relaxation caused by atrial natriuretic peptide.

We investigated the effect of aging on atrial natriuretic peptide (ANP)-induced relaxation and cyclic GMP (cGMP) formation in the rat thoracic aorta. In the aorta from young rats (4 weeks old), removal of the endothelium, and treatment with the nitric oxide synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), the radical scavenger, hemoglobin (Hb), and the soluble guanylate cyclase inhibitor, methylene blue (MB), attenuated ANP-induced relaxation and considerably reduced ANP-stimulated cGMP formation. With increasing age of the rats, the ANP-induced relaxation and cGMP formation in endothelium-intact aorta decreased, and Hb, L-NAME and MB no longer inhibited the ANP-induced effects, irrespective of whether the endothelium was present or absent. In the arteries without endothelium, the age-associated reduction in ANP-induced relaxation was less than in arteries with endothelium. Aging also decreased the relaxation induced by the soluble guanylate cyclase activator, nitroprusside. Potentiation due to the cGMP-phosphodiesterase (cGMP-PDE) inhibitor, M&B 22948, of the ANP-induced relaxation was greater in aortas from old rats than in those from young rats, suggesting that the degradation of cGMP may be accelerated in old rats. These results suggest that the relaxant action of ANP on the thoracic aorta from young rats is in part modulated by endothelium-derived relaxing factor (EDRF/nitric oxide), which in turn activates soluble guanylate cyclase, thus elevating the cGMP level. Aging may decrease the ANP-induced relaxation and ANP-stimulated increase in cGMP level by decreasing the ability of endothelial cells to produce EDRF, by decreasing guanylate cyclase activity, and by enhancing cGMP-PDE activity.

L-arginine induces relaxation of rat aorta possibly through non-endothelial nitric oxide formation

1 The relaxation of rings of rat thoracic aorta induced by l‐arginine and its derivatives was investigated. 2 l‐Arginine (0.3–100 μm), but not d‐arginine, induced relaxation of the arteries, which was detectable after 2 h and maximal after 4–6 h on its repeated application; it was endothelium‐independent. 3 l‐Arginine methyl ester, Nα‐benzoyl l‐arginine and l‐homo‐arginine had essentially similar effects to those of l‐arginine. 4 NG‐nitro l‐arginine methyl ester (l‐NAME, 3 μm), NG‐nitro l‐arginine (l‐NNA, 1 μm) and NG‐monomethyl l‐arginine (l‐NMMA, 10 μm), inhibitors of nitric oxide (NO) formation from l‐arginine, inhibited or reversed the l‐arginine‐induced relaxation, irrespective of the presence or absence of the endothelium. In contrast, NG‐nitro d‐arginine was without effect. 5 Haemoglobin (Hb, 10 nm) and methylene blue (MB, 0.3 μm) inhibited or reversed the l‐arginine‐induced relaxation. 6 l‐Arginine (1–100 μm), but not d‐arginine, increased guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) levels in the tissues that relaxed in response to l‐arginine. This effect of l‐arginine was suppressed by Hb (3 μm), MB (1 μm) and l‐NAME (100 μm). Removal of the endothelium did not significantly alter the l‐arginine‐induced cyclic GMP production. 7 These results suggest that l‐arginine itself caused a slowly developing relaxation of rat aorta, possibly via formation of NO by an endothelium‐independent mechanism.

Nitric oxide synthase responsible for l-arginine-induced relaxation of rat aortic rings in vitro may be an inducible type

1 Characteristics of l‐arginine‐induced non‐endothelial nitric oxide (NO) formation in rat isolated thoracic aorta were investigated. 2 Relaxation to l‐arginine in arterial rings devoid of endothelium developed about 2 h after the first challenge with l‐arginine and reached a maximum after a further 4 h of incubation. 3 After the arteries had relaxed in response to l‐arginine, guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) production was stimulated. In fresh arteries that had not yet relaxed in response to l‐arginine, l‐arginine failed to elevate cyclic GMP levels. 4 Glucocorticoids, actinomycin D and polymyxin B prevented the development of l‐arginine‐induced relaxation and l‐arginine‐stimulated increase in cyclic GMP formation. 5 Once l‐arginine‐induced relaxation developed, these agents no longer suppressed the relaxation and increase in cyclic GMP formation to l‐arginine. 6 From these results, it is suggested that in the isolated thoracic aorta of the rat, endotoxin in the medium triggers induction of a non‐endothelial NO synthase during prolonged incubation, which accelerates production of NO from added l‐arginine to cause relaxation of the arteries via cyclic GMP. Glucocorticoids and protein synthesis inhibitors may prevent induction of NO synthase. It is suggested that the NO synthase mediating the production of muscle‐derived NO from l‐arginine is an inducible type.

Relaxation of rat thoracic aorta induced by the Ca2+‐ATPase inhibitor, cyclopiazonic acid, possibly through nitric oxide formation

1 The effect of the Ca2+‐ATPase inhibitor, cyclopiazonic acid (CPA), was studied on rat thoracic aortic ring preparations. 2 At concentrations above 0.3 μm, CPA induced relaxation in the arteries precontracted with phenylephrine. Removal of the endothelium abolished CPA‐induced relaxation. 3 The nitric oxide (NO) synthase inhibitor NG‐nitro l‐arginine (3–300 μm), the free radical scavenger haemoglobin (0.1–3 μm), the soluble guanylate cyclase inhibitor, LY83583 (0.1–10 μm), each inhibited the endothelium‐dependent relaxation to CPA. The potassium channel blocker, glibenclamide (10 μm) and cyclo‐oxygenase inhibitor, indomethacin (100 μm for 60 min and then washed out) did not alter the action of CPA. 4 The calmodulin inhibitors calmidazolium (3–10 μm) and W‐7 (100 μm) also abolished CPA‐induced relaxation. 5 CPA (10 μm) increased guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) levels in arteries with an intact endothelium, without affecting adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) levels. 6 The inhibitors of NO synthesis and actions, the calmodulin inhibitor and removal of the endothelium abolished the CPA‐stimulated increase in the levels of cyclic GMP. 7 In Ca2+‐free solution, CPA failed to induce relaxation or to stimulate cyclic GMP production. Relaxation to nitroprusside was not affected under these conditions. 8 These results suggest that CPA can stimulate NO synthesis, possibly by inhibiting a Ca2+‐ATPase, which replenishes Ca2+ in the intracellular storage sites in endothelial cells. Depletion of the Ca2+ store in the endothelium may then trigger influx of extracellular Ca2+, contributing to an increase in free Ca2+ in the endothelial cells, which activates NO synthase and NO formation.

Endothelin-3-induced relaxation of rat thoracic aorta: a role for nitric oxide formation.

1 Endothelin‐3 (ET‐3) at concentrations below those which caused contraction (30 nm) elicited endothelium‐dependent relaxation followed by rebound contraction in rat isolated thoracic aorta. 2 Endothelin‐1 also relaxed the rat aorta with a similar potency. 3 The nitric oxide (NO) synthase inhibitor, NG‐nitro l‐arginine, the radical scavenger, haemoglobin and the soluble guanylate cyclase inhibitor, methylene blue, each inhibited the ET‐3‐induced relaxation. 4 The calmodulin inhibitor, calmidazolium, considerably attenuated the relaxation caused by ET‐3 without affecting that to nitroprusside. 5 Concentrations of ET‐3 that were necessary to induce the relaxation also caused concentration‐dependent elevation of guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) levels. 6 NG‐nitro l‐arginine, haemoglobin, methylene blue, calmidazolium and removal of the endothelium completely abolished ET‐3‐stimulated cyclic GMP production. 7 These results suggest that ET‐3 triggers NO formation possibly via ETB receptors on the endothelium to activate soluble guanylate cyclase, which in turn stimulates cyclic GMP production and smooth muscle relaxation. The enzyme contributing to the NO formation may be of the calcium/calmodulin‐dependent, constitutive type.