The role of K ϩ channels in nitric oxide (NO)-induced vasorelaxation has been largely investigated in resistance vessels where iberiotoxin-sensitive MaxiK channels play a predominant role. However, the nature of the K ϩ channel(s) involved in the relaxation triggered by NO-releasing compounds [nitroglycerin, NTG; NOR 3or atrial natriuretic peptide (ANP) in the conduit vessel aorta has remained elusive. We now demonstrate that, in rat aorta, the relaxation due to these vasorelaxants is not affected by the MaxiK channel blocker iberiotoxin (10 Ϫ7 -10 Ϫ6 M) as was the control vascular bed used (mesenteric artery). The inability of iberiotoxin to prevent NO/ANP-induced aortic relaxations was not due to lower expression of MaxiK in aorta or due to the predominance of iberiotoxin-resistant channels in this conduit vessel. Aortic relaxations were strongly diminished by 4-aminopyridine (4-AP) (Ն5 ϫ 10 Ϫ3 M) or by tetraethylammonium glibenclamide, apamin, charybdotoxin, tertiapin, or E-4031 N-[4-[[1-[2-(6-methyl-2-pyridinyl) ethyl]-4-piperidinyl-]carbonyl]phenyl]methanesulfonamide dihydrochloride). Consistent with a role of K v 2-type channels, K v currents in A7r5 aortic myocytes were stimulated by NTG and inhibited by Ն5 ϫ 10 Ϫ3 M 4-AP. Furthermore, immunocytochemistry, immunoblot, and real-time polymerase chain reaction analyses confirmed the presence of K v 2.1 channels in aorta. K v 2.1 transcripts were ϳ100-fold more abundant than K v 2.2. Our results support low-affinity 4-AP-sensitive K v channels, assembled at least partially by K v 2.1 subunit, as downstream effectors of NO/ANP-signaling cascade regulating aortic vasorelaxation and further demonstrate vessel-specific K ϩ channel involvement in NO/ANP-induced relaxation.Nitroglycerin (NTG), an organic nitrate, has been used for more than 100 years as a remedy for the treatment of cardiovascular diseases, including angina pectoris, myocardial infarction, and congestive heart failure. Nitrovasodilators, including NTG, release nitric oxide (NO) and increase cGMP levels via activation of soluble guanylyl cyclase. As a consequence, cGMP-dependent protein kinase (PKG) activity is enhanced causing modulation (phosphorylation) of various intracellular proteins and vascular relaxation (Waldman and Murad, 1987).Electrophysiological and pharmacomechanical studies in a variety of vascular tissues have pointed out to a pivotal role of plasmalemmal K ϩ channels in PKG-induced relaxation, mainly the large conductance voltage-dependent and Ca 2ϩ -

Docosahexaenoic acid (DHA) is an n-3 type of polyunsaturated fatty acid (w-3 fatty acid). This fatty acid is richly contained as well as eicosapentaenoic acid (EPA) in the fish oil. DHA has been shown to prevent the incidence of cerebral thrombosis, cardiac infarction, arrhythmia, atherosclerosis and hypertension and others.1,2) These circulatory-protective effects of DHA could be partly ascribed to vascular relaxation attained with this fatty acid. In support of this presumption, DHA produces relaxation in the isolated vascular tissue.3-7) However, in these studies, relaxant effects of DHA were mainly examined against the contractions elicited by a-adrenoceptor stimulation with phenylephrine or norepinephrine or by depolarizing high-KCl solution. Furthermore, more than 100 mM of DHA seems to be required to produce a potent vascular relaxation. In the present study, we show that DHA diminishes more strongly thromboxane A 2 (TXA 2 ) receptor (TP receptor)-mediated contraction with less concentration (10 mM) than that elicited through a-adrenoceptor in guinea-pig aorta. This novel action of DHA to inhibit more selectively TP receptor-mediated vasocontraction could substantially contribute to the suppressive effects of this fatty acid on the occurrence of vasospasm. MATERIAL AND METHODSHartley guinea-pigs of either sex were housed under controlled conditions (temperature 21-22°C, relative air humidity 50Ϯ5%). Food and water were available ad libitum to all animals. This study was conducted in accordance with the Guideline for the Care and Use of Laboratory Animals adopted by the Committee on the Care and Use of Laboratory Animals of Toho University School of Pharmaceutical Sciences (accredited by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan).Preparation of Thoracic Aortic Rings and Measurement of Tension Changes Preparation of thoracic aortic rings and tension change measurements followed our previous procedures. 8,9) Briefly, a section of the thoracic aorta was removed from guinea-pigs and placed in normal Tyrode's solution (mM): NaCl, 158.3; KCl, 4.0; CaCl 2 , 2.0; MgCl 2 , 1.05; NaH 2 PO 4 , 0.42; NaHCO 3 , 10.0 and glucose, 5.6. The aorta was cleaned of loosely adhering fat and connective tissues, and cut into ring segments of about 2 mm in length. The endothelium was removed by rubbing gently the intimal surface of ring preparations with a manicure painting brush. Subsequently, the ring segments were mounted using stainless steel hooks (outer diameter, 200 mm) under the optimal resting tension of 1.0 g in a 5-ml organ bath (UC-5; UFER Medical Instrument, Kyoto, Japan) containing normal Tyrode's solution. Muscle tension changes were isometrically recorded with a force-displacement transducer (TB-611T: Nihon Kohden, Tokyo, Japan) connected to a minipolygraph (RM-6100; Nihon Kohden, Tokyo, Japan). Ring preparations were equilibrated for 60-90 min prior contraction with isotonic high-KCl (80 mM) Tyrode's solution (mM): NaCl, 82.3; KCl, 80.0; CaCl 2 , 2.0; MgCl 2 , 1.05; NaH 2 P...

We examined the relaxant effects of natriuretic peptide family on the isolated guinea-pig aorta to determine the receptor subtype which primarily mediates this vascular relaxation, with particular attention to the apparent contribution of voltage-dependent and Ca2+-activated KS (BK(Ca)) channels to the response. Three endogenous natriuretic peptide ligands (natriuretic peptide, ANP; brain natriuretic peptide, BNP; C-type natriuretic peptide, CNP) produced a concentration-dependent relaxation in de-endothelialized guinea-pig aorta pre-contracted by noradrenaline (NA), with a potency order of ANP > or = BNP >> CNP. Although the relaxations elicited by these three natriuretic peptide ligands were significantly diminished by iberiotoxin (IbTx, 10(-7) M), a selective BK(Ca) channel blocker, the inhibitory effect of IbTx was most pronounced for the CNP-induced relaxation; when estimated at 10(-7) M of each peptide, the apparent extent of BK(Ca) channel contribution to the total relaxant response was approximately 60% for CNP > approximately 20% for either ANP or BNP. Supporting the substantial role of BK(Ca) channels in the vascular responses, high-KCl (80 mM) potently suppressed the relaxations induced by these natriuretic peptide ligands. The relaxant response to 8-Bromo-cyclic GMP, a membrane permeable cyclic GMP analogue, was also diminished by IbTx (10(-7) M) and high-KCl (80 mM), which indicates the key role of cyclic GMP in the BK(Ca) channel-mediated, natriuretic peptide-elicited vascular relaxation. These results indicate that the A-type receptor (NPR-A, which is more selective for ANP and BNP) rather than the B-type receptor (NPR-B, which is more selective for CNP) predominates in the guinea-pig aorta as the natriuretic peptide receptor which mediates this vascular smooth muscle relaxation. Although activation of BK(Ca) channels substantially contributes to both NPR-A- and NPR-B-activated relaxations, particularly in the NPR-B-activated relaxation, this K channel may function as a primary relaxant mediator in this conduit artery.

Noradrenaline (NA) produces sustained contractions in conduit arteries such as aorta isolated from various animal species. In guinea-pig aorta, NA-produced sustained contraction is largely dependent upon the influx of extracellular Ca2+, but is refractory to the treatment with organic Ca2+ entry blockers. In the present study, we attempted to characterize pharmacologically the Ca2+ entry channel responsible for NA-produced sustained contraction of guinea-pig aorta using SK&F 96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenylethyl]-1H-imi dazole) and LOE 908 ((R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2- (2,3,4-trimethoxyphenyl)ethyl]-acetamide), both of which block voltage-independent Ca2+ channels. The effects of SK&F 96365 and LOE 908 on NA-produced contraction were compared with those on extracellular Ca2+-dependent contractile and endothelium-dependent relaxant responses to thapsigargin (TSG), an inhibitor of Ca2+-pump Ca2+-ATPase. NA (3x10(-6) M)-produced sustained contraction of guinea-pig aorta without endothelium exhibited a strong dependency on the extracellular Ca2+. Nicardipine (10(-7) M), diltiazem (10(-5) M) and verapamil (10(-5) M) did not show any appreciable inhibitory effects on NA-produced sustained contraction. SK&F 96365 concentration-dependently (10(-6)-10(-4) M) attenuated the NA-produced sustained contraction whereas LOE 908 did not affect it at concentrations up to 10(-4) M. Similarly, extracellular Ca2+-dependent contraction of guinea-pig aorta without endothelium in response to TSG was also diminished by SK&F 96365 but was unaffected by LOE 908. In fura-PE3-loaded vascular preparations, SK&F 96365 decreased both cytoplasmic Ca2+ level ([Ca2+]i) and muscle tension elevated by NA and TSG. Both SK&F 96365 and LOE 908 did not affect an endothelium-dependent relaxation of guinea-pig aorta in response to TSG. These findings suggest that in guinea-pig aortic smooth muscle cells, NA activates Ca2+ influx across the plasma-membrane through the Ca2+-permeable channel which is identical with or has similar properties to the store-operated Ca2+ channel (SOCC) stimulated by TSG, but is distinct from endothelial cell SOCC.

The vasorelaxant profile of a novel azulene-1-carboxamidine derivative, HNS-32 [N1,N1-dimethyl-N2-(2-pyridylmethyl)-5-isopropyl-3,8-dimethyl-azulene-1-carboxamidine, CAS 186086-10-2], was investigated in the isolated rabbit aorta precontracted with high KCl, noradrenaline (NA) or phorbol 12, 13-dibutyrate (PDBu) and compared with those of nifedipine and nitroglycerin. In preparations without endothelium, HNS-32 elicited concentration-dependent, full inhibition of contractions elicited by high KCI (80 mM), NA (3x10(-6) M) or PDBu (10(-6) M). In contrast, nifedipine inhibited only the contraction elicited by membrane depolarization with high KCl. Nitroglycerin also attenuated high-KCl-, NA- and PDBu-elicited contractions effectively, although full suppression was obtained only for NA-elicited contraction. Whilst the relaxant effect of HNS-32 was not affected by the presence of endothelium, the relaxant response to acetylcholine was endothelium dependent. Addition of excess Ca2+ restored both the HNS-32-reduced tension in muscle precontracted with high KCI and the nifedipine-mediated tension decrease. Relaxation elicited by HNS-32 was not affected by the adenylate cyclase inhibitor, 9-(tetrahydro-2'-furyl)adenine (SQ 22,536, 10(-4) M), the soluble guanylate cyclase inhibitor, 1H-(1,2,4)-oxadiazolo-(4,3-a)-quinoxalin-1-one (ODQ, 10(-5) M) or a cocktail of K+ channel blockers (glybenclamide 10(-6) M, tetraethylammonium 2x10(-3) M, apamin 10(-7) M, 4-aminopyridine 10(-4) M and Ba2+ 10(-5) M). These findings indicate that HNS-32 inhibits both L-type Ca2+ channel-dependent and -independent vascular contraction. Blockade of Ca2+ entry through L-type Ca2+ channels may be involved in the inhibitory effect of HNS-32 on the contraction due to membrane depolarization with high KCl. On the other hand, HNS-32 seems to inhibit Ca2+ channel-independent contraction via mechanism(s) other than elevation of cyclic nucleotides (cAMP and cGMP) and opening of K+ channels.