Combination Of Apamin Research Articles

Moderate Intensity Exercise Causes a Shift in the Relative Importance of the Endothelium‐Dependent Relaxing Factors in Mesenteric Arteries of Male UC Davis Type‐2 Diabetes Mellitus (UCD‐T2DM) Rats

Over the past decade obesity and type 2 diabetes (T2D) have reached epidemic levels worldwide. Cardiovascular disease (CVD) is one of the leading causes of mortality and morbidity in diabetic patients. Regular aerobic exercise is a well-characterized lifestyle intervention that improves endothelial dysfunction, insulin resistance, and CVD risk in T2D. However, there are still debates regarding the duration and intensity of exercise required to ameliorate the deleterious metabolic perturbations in patients with T2D. The objective of this study was to investigate the effects of moderate intensity exercise (MIE) on vascular reactivity of mesenteric artery (MA) of male UC Davis Type-2 Diabetes Mellitus Rat (UCD-T2DM) model. Specifically, we examined whether there were changes in the relative contributions of endothelium-derived relaxing factors in modulating vascular reactivity of MA from exercise-trained compared with control sedentary UCD-T2DM rats. Age-matched male control and UCD-T2DM rats underwent forced treadmill exercise (E) or remained sedentary (S) for 8 weeks. Endothelium-dependent vasorelaxation (EDV) to acetylcholine (ACh, 10-8 to 10-5 M) were measured in precontracted MA before and after indomethacin (10µM), a cyclooxygenase (COX) inhibitor; followed by addition of L-NAME (200µM), a nitric oxide synthase (NOS) inhibitor; followed by combination of apamin (1 µM) and TRAM-34 (1 µM) (SKCa and IKCa blockers, respectively). The diabetic exercise trained (DE) rats exhibited decreased visceral adiposity and plasma triglyceride levels compared with the diabetic sedentary (DS) group. However, MIE did not alter plasma glucose or insulin concentrations in either nondiabetic or diabetic UCD-T2DM Rats. Both sensitivity and maximal responses (Emax) to ACh were significantly reduced in MA from DS rats compared with those in control sedentary (CS). MIE improved the Emax to ACh only in MA of DE group. Inhibition of COX enhanced Emax to ACh in DS arteries suggesting the elevation of contractile COX metabolites in the DS group. The addition of L-NAME resulted in a reduction in ACh relaxation of arteries from both CS and DS rats, however the effect was more prominent in DS group, suggesting the contribution of NO was enhanced (or endothelium-dependent hyperpolarizing factor (EDHF)-type relaxation was diminished) in DS arteries. Remarkably, MIE reversed the loss of EDHF-type relaxation in UCD-T2DM Rats. Unlike in the DS group, inhibition of COX had no effect on ACh response in MA of the DE group, suggesting that MIE may cause a shift from contractile COX to NO and EDHF-type relaxation in UCD-T2DM Rats. In conclusion, we provide the first evidence for a positive impact of moderate exercise on mesenteric arterial function in male UCD-T2DM Rats, likely mediated by a contribution of EDHF-type (NO and prostanoid-independent) responses.

Mechanisms underlying the vasorelaxant effect of hydrogen sulfide on human saphenous vein

Hydrogen sulfide (H2 S) represents the third and the youngest member of the gaseous transmitters family. The dominant effect of H2 S on isolated vessels is vasodilation. As the mechanism of H2 S-induced relaxation in human vessels remains unclear, the present study aimed to investigate the effects of H2 S donor, sodium hydrosulfide (NaHS), on isolated human saphenous vein (HSV) and to determine the mechanism of action. Our results showed that NaHS (1µM-3mM) induced a concentration-dependent relaxation of endothelium-intact HSV rings pre-contracted by phenylephrine. Pre-treatment with L-NAME, ODQ and KT5823 significantly inhibited NaHS-induced relaxation, while indomethacin induced partial inhibition. Among K+ channel blockers, the combination of apamin and TRAM-34 significantly affected the relaxation produced by NaHS, while iberiotoxin and glibenclamide only reduced maximal relaxation of HSV. NaHS partially relaxed endothelium-intact rings pre-contracted by high K+ , as well as phenylephrine-contracted rings in the presence of nifedipine. Additionally, the incubation of HSV rings with NaHS increased NO production. These results demonstrate that NaHS produces the concentration- and endothelium-dependent relaxation of isolated HSV. Vasorelaxation to NaHS probably involves activation of NO/cGMP/PKG pathway and partially prostacyclin. In addition, different K+ channels subtypes, especially SKCa and IKCa , as well as BKCa and KATP channels in high concentrations of NaHS, probably participate in the NaHS-induced vasorelaxation.

Altered cyclooxygenase-1 and enhanced thromboxane receptor activities underlie attenuated endothelial dilatory capacity of omental arteries in obesity

AimsObesity is a risk factor for endothelial dysfunction, the severity of which is likely to vary depending on extent and impact of adiposity on the vasculature. This study investigates the roles of cyclooxygenase isoforms and thromboxane receptor activities in the differential endothelial dilatory capacities of arteries derived from omental and subcutaneous adipose tissues in obesity. Main methodsSmall arteries were isolated from omental and subcutaneous adipose tissues obtained from consented morbidly obese patients (n = 65, BMI 45 ± 6 kg m−2 [Mean ± SD]) undergoing bariatric surgery. Relaxation to acetylcholine was studied by wire myography in the absence or presence of indomethacin (10 μM, cyclooxygenase inhibitor), FR122047 (1 μM, cyclooxygenase-1 inhibitor), Celecoxib (4 μM, cyclooxygenase-2 inhibitor), Nω-Nitro-L-arginine methyl ester (L-NAME, 100 μM, nitric oxide synthase inhibitor) or combination of apamin (0.5 μM) and charybdotoxin (0.1 μM) that together inhibit endothelium-derived hyperpolarizing factor (EDHF). Contractions to U46619 (thromboxane A2 mimetic) were also studied. Key findingsAcetylcholine relaxation was significantly attenuated in omental compared with subcutaneous arteries from same patients (p < 0.01). Indomethacin (p < 0.01) and FR122047 (p < 0.001) but not Celecoxib significantly improved the omental arteriolar relaxation. Cyclooxygenase-1 mRNA and U46619 contractions were both increased in omental compared with subcutaneous arteries (p < 0.05). L-NAME comparably inhibited acetylcholine relaxation in both arteries, while apamin+charybdotoxin were less effective in omental compared with subcutaneous arteries. SignificanceThe results show that the depot-specific reduction in endothelial dilatory capacity of omental compared with subcutaneous arteries in obesity is in large part due to altered cyclooxygenase-1 and enhanced thromboxane receptor activities, which cause EDHF deficiency.

Important role of endothelium-dependent hyperpolarization in the pulmonary microcirculation in male mice: implications for hypoxia-induced pulmonary hypertension.

Endothelium-dependent hyperpolarization (EDH) plays important roles in the systemic circulation, whereas its role in the pulmonary circulation remains largely unknown. Furthermore, the underlying mechanisms of pulmonary hypertension (PH) also remain to be elucidated. We thus aimed to elucidate the role of EDH in pulmonary circulation in general and in PH in particular. In isolated perfused lung and using male wild-type mice, endothelium-dependent relaxation to bradykinin (BK) was significantly reduced in the presence of Nω-nitro-l-arginine by ~50% compared with those in the presence of indomethacin, and the combination of apamin plus charybdotoxin abolished the residual relaxation, showing the comparable contributions of nitric oxide (NO) and EDH in the pulmonary microcirculation under physiological conditions. Catalase markedly inhibited EDH-mediated relaxation, indicating the predominant contribution of endothelium-derived H2O2. BK-mediated relaxation was significantly reduced at day 1 of hypoxia, whereas it thereafter remained unchanged until day 28. EDH-mediated relaxation was diminished at day 2 of hypoxia, indicating a transition from EDH to NO in BK-mediated relaxation before the development of hypoxia-induced PH. Mechanistically, chronic hypoxia enhanced endothelial NO synthase expression and activity associated with downregulation of caveolin-1. Nitrotyrosine levels were significantly higher in vascular smooth muscle of pulmonary microvessels under chronic hypoxia than under normoxia. A similar transition of the mediators in BK-mediated relaxation was also noted in the Sugen hypoxia mouse model. These results indicate that EDH plays important roles in the pulmonary microcirculation in addition to NO under normoxic conditions and that impaired EDH-mediated relaxation and subsequent nitrosative stress may be potential triggers of the onset of PH. NEW & NOTEWORTHY This study provides novel evidence that both endothelium-dependent hyperpolarization and nitric oxide play important roles in endothelium-dependent relaxation in the pulmonary microcirculation under physiological conditions in mice and that hypoxia first impairs endothelium-dependent hyperpolarization-mediated relaxation, with compensatory upregulation of nitric oxide, before the development of hypoxia-induced pulmonary hypertension.

The Janus face of chlorogenic acid on vascular reactivity: A study on rat isolated vessels

BackgroundChlorogenic acid (CGA), the main polyphenol contained in coffee, is a major contributor to dietary polyphenol intake. Few studies reported its anti-hypertensive properties but the mechanisms are still indefinite. PurposeThe present study assessed the direct effect of CGA in endothelium denuded or intact aortic rings from male Wistar rats and the mechanisms involved. Methods/ResultsCGA induced a direct endothelium-dependent relaxation that was significantly reduced by L-NAME (10−4M), indomethacin (10−5M) and combination of apamin (10−7M) and charybdotoxin (10−7M). Incubation of rings with CGA induced a dual effect on agonist-induced vasorelaxation. At 10−6M, it enhanced the relaxant effects of acetylcholine and reduced the contracting effects of phenylephrine due to increased basal and stimulated NOS activity, respectively. At 10−4M, CGA blunted acetylcholine and bradykinin-induced vasorelaxation, reduced phenylephrine-induced vasoconstriction but did not change the response to sodium nitroprusside, a NO-donor. ConclusionIn summary, CGA induces a direct endothelium-dependent vasodilation by increasing NOS, COX and EDHF signalling pathways. However, this new pharmacological action that can explain some positive effects of CGA in case of hypertension has to be modulated at the light of its deleterious impact on vascular relaxation at high concentrations and incite to be cautious when using high doses of CGA in clinical studies.

Characterization of relaxant mechanism of H2S in mouse corpus cavernosum

The aim of this study was to investigate the mechanism of H2 S-induced relaxation in mouse corpus cavernosal tissue. l-cysteine (10(-6) × 10(-3) mol/L) and exogenous H2 S (NaHS; 10(-6) to 10(-3) mol/L) induced concentration-dependent relaxation. l-cysteine-induced relaxations was reduced by d,l-propargylglycine, a cystathionine gamma lyase (CSE) inhibitor but not influenced by aminooxyacetic acid, a cystathionine beta synthase (CBS) inhibitor. l-cysteine induced relaxations, but not of those of H2 S diminished in endothelium-denuded tissues. N(ω) -nitro-l-arginine (l-NA; 10(-4) mol/L), a nitric oxide synthase inhibitor, and ODQ (10(-4) mol/L), a guanylyl cyclase inhibitor, increased the H2 S-induced relaxation. Zaprinast (5 × 10(-6) mol/L) and sildenafil (10(-6) mol/L), phosphodiesterase inhibitors, inhibited H2 S-induced relaxation. Adenylyl cyclase inhibitors N-ethylmaleimide (2.5 × 10(-5) mol/L) and SQ22536 (10(-4) mol/L) reduced relaxation to H2 S. Also, H2 S-induced relaxation was reduced by KCl (50 mmol/L), 4-aminopyridine (10(-3) mol/L), a Kv inhibitor, glibenclamide (10(-5) mol/L), a KATP inhibitor or barium chloride (10(-5) mol/L), a KIR inhibitor. However, H2 S-induced relaxation was not influenced by apamin (10(-6) mol/L), a SKC a (2+) inhibitor, charybdotoxin (10(-7) mol/L), an IKC a (2+) and BKC a (2+) inhibitor or combination of apamin and charybdotoxin. Nifedipine (10(-6) mol/L), an L-type calcium channel blocker and atropine (10(-6) mol/L), a muscarinic receptor blocker, inhibited H2 S-induced relaxation. However, H2 S-induced relaxation was not influenced by ouabain (10(-4) mol/L), a Na(+) /K(+) -ATPase inhibitor. This study suggests that H2 S endogenously synthesizes from l-cysteine by CSE endothelium-dependent in mouse corpus cavernosum tissue, and exogenous H2 S may cause endothelium-independent relaxations via activation of K channels (KATP channel, KV channels, KIR channels), L-type voltage-gated Ca(2+) channels, adenylyl cyclase/cAMP pathway and muscarinic receptor, and there is the interaction between H2 S and NO/cGMP.

Impaired endothelium‐dependent vasorelaxation contributes to hypertension in leukocyte‐specific protein 1 (LSP1)‐deficient mice

LSP1 is an intracellular, Ca2+‐and F‐actin‐binding protein expressed in leukocytes and endothelial cells. Adult male LSP1‐deficient (Lsp1−/−) mice had higher blood pressure than their WT counterparts. By Western blot, LSP1 was found present in aorta and mesenteric artery tissues in WT mice but not in Lsp1−/− mice. The role of LSP1 in regulating arterial vascular tone was studied. The contraction of aorta and mesenteric arteries in response to phenylephrine and angiotensin II was similar in WT and Lsp1−/− mice. The relaxation of aorta artery was more sensitive to sodium nitroprusside and methacholine in Lsp1−/− mice than in WT mice. The phenylephrine‐constricted mesenteric arteries showed identical relaxation curve to sodium nitroprusside in these two strains of mice, but had no relaxation to methacholine in Lsp1−/− mice or in denuded WT mesenteric arteries. Methacholine‐induced mesenteric arterial relaxation in WT mice was suppressed partially by the pretreatment of L‐NAME or the combination of apamin + TRAM‐34, abolished completely by the combination of L‐NAME + apamin + TRAM‐34, but not changed by indomethacin. Our results demonstrate the defects in endothelium‐dependent vasorelaxation in resistance arteries in mice deficient in LSP1 revealing an important role for LSP1 in endothelium‐dependent vasodilation in small resistance arteries and blood pressure regulation.

Dominance of flow‐mediated constriction over flow‐mediated dilatation in the rat carotid artery

The shearing forces generated by flow generally evoke dilatation in systemic vessels but constriction in the cerebral circulation. The aim of this study was to determine the effects of flow on the conduit artery delivering blood to the brain in the rat, that is, the carotid artery. Carotid artery segments were mounted in a pressure myograph and pressurized to 100 mmHg. Changes in vessel diameter to flow (0.5-10 mL·min⁻¹ for 2-10 min) at constant pressure were then measured using a video dimension analyser. Following the induction of tone, the onset of flow evoked a transient dilatation followed by a powerful constriction that was sustained until the termination of flow. Endothelial denudation or treatment with indomethacin, N(G)-nitro-L-arginine methyl ester, or the combination of apamin and TRAM-34 showed that the initial flow-mediated dilatation arose from the combined actions of endothelium-derived NO and endothelium-derived hyperpolarizing factor (EDHF). The flow-mediated constriction, which increased in magnitude with increasing flow rate and duration of flow, was also endothelium dependent, but was unaffected by treatment with superoxide dismutase, BQ-123, indomethacin, HET0016 or carbenoxolone. Flow-mediated constriction therefore appeared not to involve superoxide anion, endothelin-1, a COX product, 20-HETE or gap-junctional communication. Although a weak, transient flow-mediated dilatation is observed in the rat carotid artery, the dominant response to flow is a powerful and sustained constriction. Whether this flow-mediated constriction in the carotid artery serves as an extracranial mechanism to regulate cerebral blood flow remains to be determined.

The Contribution of d-Tubocurarine–sensitive and Apamin-sensitive K-channels to EDHF-mediated Relaxation of Mesenteric Arteries From eNOS−/− Mice

The nature of the potassium channels involved in determining endothelium-derived hyperpolarizing factor-mediated relaxation was investigated in first-order small mesenteric arteries from male endothelial nitric oxide synthase (eNOS-/-)-knockout and control (+/+) mice. Acetylcholine-induced endothelium-dependent relaxation of small mesenteric arteries of eNOS-/- was resistant to N-nitro-L-arginine and indomethacin and the guanylyl cyclase inhibitor, 1H-(1,2,4) oxadiazolo (4,3-a) quinoxalin-1-one. Apamin and the combination of apamin and iberiotoxin or apamin and charybdotoxin induced a transient endothelium-dependent contraction of small mesenteric arteries from both eNOS-/- and +/+ mice. Acetylcholine-induced relaxation in eNOS-/- mice was unaffected by charybdotoxin or apamin alone but significantly inhibited by the combination of these agents. However, the combination of scyllatoxin and iberiotoxin did not mimic the inhibitory effect of the apamin/charybdotoxin combination. Tubocurarine alone completely blocked acetylcholine-induced relaxation in eNOS-/- mice. Single channel analysis of myocytes from small mesenteric arterioles revealed a large conductance calcium-activated potassium channel that was sensitive to iberiotoxin, charybdotoxin, and tetraethylammonium. Tubocurarine blocked this channel from the cytosolic side but not when applied extracellularly. Solutions of nitric oxide (NO) gas also relaxed small mesenteric arteries that had been contracted with cirazoline in a concentration-dependent manner, and the sensitivity to NO was reduced by iberiotoxin and the combination of apamin, scyllatoxin, or tubocurarine with charybdotoxin but not by apamin, charybdotoxin, scyllatoxin, or tubocurarine alone. These data indicate that acetylcholine-induced endothelium-derived hyperpolarizing factor-mediated relaxation in small mesenteric arteries from eNOS-/- involved the activation of tubocurarine and apamin-/charybdotoxin-sensitive K-channels. In eNOS+/+ mice, the acetylcholine-induced response was primarily mediated by NO and was sensitive to iberiotoxin and the combination of apamin and charybdotoxin.

Potentiation of EDHF-mediated relaxation by chloride channel blockers

To investigate the involvement of Cl⁻ channels in endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation in rat mesenteric arteries. Cl⁻ channel and K(ir) channel activities were studied using whole-cell patch clamping in rat mesenteric arterial smooth muscle cells. Isometric tension of arterial rings was measured in organ chambers. The volume-activated Cl⁻ current in rat mesenteric arterial smooth muscle cells was abolished by Cl⁻ channel blockers NPPB or DIDS. The EDHF-mediated vasorelaxation was potentiated by NPPB and DIDS. The EDHF response was diminished by a combination of apamin and charybdotoxin, which agreed with the hypothesis that EDHF response involves the release of K(+) via the Ca²(+)-activated K(+) channels in endothelial cells. The elevation of K(+) concentration in bathing solution from 1.2 mmol/L to 11.2 mmol/L induced an arterial relaxation, which was abolished by the combination of BaCl₂ and ouabain. It is consistent to the hypothesis that K(+) activates K(+)/Na(+)-ATPase and inward rectifier K(+) (K(ir)) channels, leading to the hyperpolarization and relaxation of vascular smooth muscle. The K(+)-induced relaxation was augmented by NPPB, DIDS, or withdrawal of Cl⁻ from the bathing solution, which could be reversed by BaCl₂, but not ouabain. The potentiating effect of Cl⁻ channel blockers on K(+)-induced relaxation was probably due to the interaction between Cl⁻ channels and K(ir) channels. Moreover, the K(+)-induced relaxation was potentiated when the arteries were incubated in hyperosmotic solution, which is known to inhibit volume-activated Cl⁻ channels. The inhibition of Cl⁻ channels, particularly the volume-activated Cl⁻ channels, may potentiate the EDHF-induced vasorelaxation through the K(ir) channels.

Decreases in splanchnic vascular resistance contribute to hypotensive effects ofl-serine in hypertensive rats

l-Serine administration reduces mean arterial pressure (MAP) in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats rendered hypertensive by chronic oral treatment with the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME). To determine if the fall in MAP was due to decreases in vascular resistance or cardiac output (CO), and to record regional hemodynamic effects, we measured the distribution of fluorescent microspheres to single bolus intravenous injections of l-serine (1 mmol/kg) in 14-wk-old male WKY, SHR, and l-NAME-treated WKY rats. MAP and total peripheral resistance (TPR) were significantly higher (P < 0.01), whereas CO was lower in l-NAME-treated WKY (P < 0.01) and SHR (P < 0.05). l-Serine administration led to a rapid fall in MAP (WKY 22%, l-NAME-WKY 46%, SHR 34%,) and TPR (WKY 24%, l-NAME-WKY 68%, SHR 53%), whereas CO was elevated. In WKY rats, l-serine induced an increase in blood flow only in the small intestine (53%) while it was more profound in several vascular beds of hypertensive rats [l-NAME-WKY: small intestine (238%), spleen (184%), diaphragm (85%), and liver (65%); SHR: small intestine (217%), spleen (202%), diaphragm (116%), large intestine (105%), pancreas (96%), and liver (93%)]. Pretreatment with a combination of apamin (a small calcium-activated potassium channel inhibitor) and charybdotoxin (an intermediate calcium-activated potassium channel inhibitor) abolished the l-serine-induced changes in blood flow and TPR. l-Serine acts predominantly on apamin- and charybdotoxin-sensitive potassium channels in the splanchnic circulation to increase blood flow, thereby contributing to the fall in TPR and the pronounced blood pressure-lowering effect of l-serine in hypertensive rats.

Mechanisms of endothelium-dependent vasodilation in male and female, young and aged offspring born growth restricted

Numerous epidemiological studies have shown that cardiovascular dysfunction in adult life may be programmed by compromised growth in utero. Aging is a risk factor for vascular endothelial-dependent dysfunction. After birth, the impact of intrauterine growth restriction (IUGR) on normal aging mechanisms of vascular dysfunction is not known. We hypothesized that IUGR would cause changes in vascular function that would affect the mechanisms of endothelium-dependent vasodilation later in life in an age- or sex-dependent manner. To create an IUGR model, pregnant Sprague-Dawley rats were placed in a hypoxic (12% O(2)) or control (room air, 21% O(2)) environment from days 15 to 21 of the pregnancy, and both male and female offspring were investigated at 4 or 12 mo of age. Endothelial function was assessed in small mesenteric arteries using methacholine (MCh)-induced vasodilation in a wire myograph system. The involvement of nitric oxide (NO), prostaglandins, and endothelium-derived hyperpolarizing factor (EDHF) was assessed using the inhibitors N(omega)-nitro-l-arginine methyl ester hydrochloride, meclofenamate, or a combination of apamin and TRAM-34 (SK(Ca) and IK(Ca) blockers), respectively. EDHF-induced vasodilation was further investigated by using inhibitors of P450 epoxygenases [N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide] and gap junctions (18alpha-glycyrrhetinic acid). NO-mediated vasodilation was significantly reduced in aged controls and both young and aged IUGR females. EDHF-mediated vasodilation was maintained in all groups; however, an additional involvement of gap junctions was found in females exposed to hypoxia in utero, which may represent a compensatory mechanism. A change in the mechanisms of vasodilation occurring at an earlier age in IUGR offspring may predispose them to adult cardiovascular diseases.

A 1 receptors mediate adenosine inhibitory effects in mouse ileum via activation of potassium channels

Aims We investigated the effects induced by exogenous adenosine on the spontaneous contractile activity of the longitudinal muscle of a mouse ileum, the receptor subtypes activated, the involvement of enteric nerves and whether opening of K + channels was a downstream event leading to the observed effects. Main methods Mechanical responses of the mouse ileal longitudinal muscle to adenosine were examined in vitro as changes in isometric tension. Key findings Adenosine caused a concentration-dependent reduction of the spontaneous contraction amplitude of the ileal longitudinal muscle up to its complete disappearance. This effect induced was markedly reduced by an A 1 receptor antagonist, but not by A 2 and A 3 receptor antagonists and mimicked only by the A 1 receptor agonist. Adenosine uptake inhibitors did not change adenosine potency. A 1 receptor expression was detected at the smooth muscle level. Adenosine responses were insensitive to tetrodotoxin, atropine or nitric oxide synthase inhibitor. Tetraethylammonium and iberiotoxin, BK Ca channel blockers, significantly reduced adenosine effects, whilst 4-aminopyridine, a K v blocker, apamin, a small conductance Ca 2+-activated K + (SK Ca) channel blocker, charybdotoxin, an intermediate conductance Ca 2+-activated K + (IK Ca) and BK Ca channel blocker, or glibenclamide, an ATP-sensitive K + channel blocker, had no effects. The combination of apamin plus iberiotoxin caused a reduction of the purinergic effects greater than iberiotoxin alone. Significance Adenosine acts as an inhibitory modulator of the contractility of mouse ileal longitudinal muscle through postjunctional A 1 receptors, which in turn would induce opening of BK Ca and SK Ca potassium channels. This study would provide new insight in the pharmacology of purinergic receptors involved in the modulation of the gastrointestinal contractility.

C007 Redox-sensitive activation of endothelial nitric oxide synthase by catechins: role of hydroxyl moieties

Objectives Several rich sources of polyphenols have been shown to strongly increase the endothelial formation of nitric oxide (NO), a potent vasoprotecting factor, via the redox-sensitive activation of the PI3-kinase/Akt pathway leading to the phosphorylation of endothelial NO synthase. The purpose of the present study was to investigate the molecular mechanisms underlying the stimulatory effect of catechins on the endothelial formation of NO using different catechins (flavan-3-ols). Methods Vascular reactivity studies were performed using porcine coronary artery rings, which were suspended in organ chambers for the measurement of changes in isometric tension. All experiments were performed in the presence of indomethacin (an inhibitor of cyclooxygenases), and the combination of apamin and charybdotoxin (two inhibitors of endothelium-derived hyperpolarizing factor-mediated effects) to assess only the NO component of the relaxation. Cultures of porcine coronary artery endothelial cells (P1) were used to determine the phosphorylation level of Akt and endothelial NO synthase by Western blot analysis. Both natural and synthetic catechins were evaluated. Results (-)-Epigallocatechin-3-O-gallate (EGCg) induced potent endothelium-dependent relaxations in porcine coronary artery rings. The EGCg-induced relaxation was inhibited by MnTMPyP (a membrane permeant analogue of superoxide dismutase, SOD) whereas extracellular SOD had no effect, indicating a major role of the intracellular formation of ROS. Relaxations to EGCg were minimally affected by rotenone (an inhibitory of the mitochondrial respiratory chain), sulphenazol (an inhibitor of cytochrome P450), apocynin (an inhibitor of NADPH oxidase) or allopurinol (an inhibitor of xanthine oxidase). The replacement of all hydroxyl groups of EGCg by O-methyl groups resulted in the total loss of the relaxing activity whereas partial replacement decreased the relaxing activity. Conclusions EGCg caused endothelium-dependent relaxations of coronary arteries via the redox-sensitive formation of NO in endothelial cells. The stimulatory effect does not involve major intracellular sources of ROS including the mitochondrial respiratory chain, xanthine oxidase, NADPH oxidase and cytochrome P450 but is critically dependent on the presence of hydroxyl groups possibly leading to auto-oxidation of the polyphenol.

L-Serine lowers while glycine increases blood pressure in chronic L-NAME-treated and spontaneously hypertensive rats

To determine the acute hemodynamic effects of the nonessential amino acid, glycine, and its precursor, L-serine, in normotensive and hypertensive rats. Changes in mean arterial pressure and heart rate evoked by comparable intravenously administered doses (0.3-3.0 mmol/kg) of L-serine, D-serine and glycine were examined in anaesthetized normotensive 14-week-old male Sprague-Dawley, Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats and WKY rats subjected to chronic nitric oxide synthase inhibition by treatment with NG nitro L-arginine methyl ester (0.7 mg/ml in drinking water for 5 days). L-Serine evoked a greater maximal fall in mean arterial pressure [L-serine vs. D-serine in Sprague-Dawley rats, mean +/- standard error of the mean values (mmHg): 30 +/- 3 vs. 20 +/- 5, P < 0.05; in control WKY rats: 46 +/- 3 vs. 30 +/- 4, P < 0.05; in NG nitro L-arginine methyl ester-treated WKY rats: 93 +/- 6 vs. 41 +/- 5, P < 0.01; in spontaneously hypertensive rats: 81 +/- 7 vs. 39 +/- 5 P < 0.01]. The effects of L-serine were significantly reduced in rats pretreated with a combination of apamin and charybdotoxin, inhibitors of the small conductance and intermediate conductance calcium-activated potassium (KCa) channels. Glycine elicited a dose-dependent fall in mean arterial pressure in normotensive WKY rats (25 +/- 4; P < 0.01) and evoked pressor responses in both spontaneously hypertensive rats (29 +/- 3; P < 0.01) and NG nitro L-arginine methyl ester-pretreated hypertensive WKY (39 +/- 5; P < 0.01) rats. Both the depressor and pressor responses to glycine were abolished by pretreatment with the N-methyl D-aspartate receptor antagonist, MK-801. The profound stereo-selective antihypertensive effect of L-serine is neither mediated nor mimicked by glycine. It does not require N-methyl D-aspartate receptor activation by glycine but likely involves activation of endothelial KCa channels. L-Serine is a potential antihypertensive agent.

Chronic clofibrate administration prevents salineinduced endothelial dysfunction and oxidative stress in young Sprague-Dawley rats

High salt intake causes hypertension and endothelial dysfunction in young Sprague-Dawley rats. Clofibrate (clof) prevents this salt induced hypertension. We asked whether clof can prevent salt-induced endothelial dysfunction, and if so, its mechanism. We also questioned whether high salt intake can induce endothelial dysfunction without hypertension in older animals. Young (Y, 5 weeks) and old (O, 53 weeks) male Sprague-Dawley rats were given either vehicle (Con, 20 mM Na2CO3) or 0.9% NaCl (Sal) to drink for three weeks. Some young rats received clof (80 mg/d) in their drinking fluid. After three weeks, we measured mean arterial pressure (MAP), endothelial function, by comparing hypotensive responses to acetylcholine (ACh, endothelium dependent) and sodium nitroprusside (SNP, endothelium independent), plasma total nitrite+nitrate levels (PNOx), by the Griess reaction, and aortic superoxide production by lucigenin chemiluminescence. Carotid artery MAP did not change in O. Sal-Y developed hypertension: 133+/-3 vs. 114+/-2 mmHg, P < 0.001, which was prevented by clof: 105+/-2 mmHg. ACh induced a similar dose dependent hypotensive response in Con-O and Sal-O that was inhibited by L-NAME (100mg/kg i.v.). Responses to ACh were blunted in Sal-Y but not in Con-Y. Further, L-NAME inhibited ACh responses only in Con-Y. The response to SNP was similar in all animals. Importantly, the ACh-induced hypotensive response was potentiated in clof+Sal-Y, an effect which was attenuated by blocking calcium-activated potassium channels (KCa) with a combination of apamin (50 ug/kg i.v.) + charybdotoxin (50 ug/kg i.v.), but not by L-NAME. PNOx was reduced in Sal-Y compared to Con-Y (2.09+/-0.26 vs. 4.8+/-0.35 microM, P < 0.001), but not in Sal-O. Aortic superoxide production was higher (P < 0.001) in Sal-Y (2388+/-40 milliunits/mg/min) than Sal-O (1107+/-159 milliunits/mg/min), but was reduced by clof (1378+/-64 milliunits/mg/min; P < 0.001). High salt intake increases oxidative stress in young animals, leading to impaired nitric oxide activity and endothelial dysfunction. Clofibrate prevents endothelial dysfunction partly through reduced O2 - formation but mainly via selective activation of KCa channels. Older animals are resistant to both salt induced hypertension and oxidative stress.

Developmental changes in endothelium‐dependent relaxation of the chicken ductus arteriosus

We tested the hypothesis that endothelium‐dependent relaxation in the chicken ductus arteriosus (DA) is developmentally regulated. Isolated DA rings from 15‐, 19‐ and 21‐day‐old chicken embryos relaxed to acetylcholine (ACh). This relaxation was unaffected by indomethacin but impaired by endothelium removal, by the NO synthase inhibitor L‐NAME, and by the soluble guanylate cyclase inhibitor ODQ, suggesting the involvement of NO. This NO production was confirmed with the fluorescent probe DAF‐2DA. The combination of apamin and charybdotoxin with L‐NAME produced a further inhibition of ACh‐induced relaxation, suggesting the participation of a putative EDHF. In the 21‐day DA, the relaxations induced by ACh and sodium nitroprusside SNP were markedly reduced and scanning electron microscopy demonstrated an irregular endothelial lining with protrusion and detachment of endothelial cells. The relaxations induced by BAY 41‐2272 and 8‐Br cGMP were not affected by age. In summary, we found that ACh induces endothelium‐dependent relaxation of the chicken DA and that NO and EDHF are involved in this response. During chicken DA closure, endothelial cells undergo morphologic and functional alterations that result in the lack of endothelium‐dependent relaxation.Supported by “Fundación de Investigación Médica Mutua Madrileña”

Blood Pressure Reduction, Potassium Channels, and the Endothelium

In this issue of Hypertension , Mishra et al1 compared the in vitro effects of the amino acid l-serine, a precursor of central neurotransmitters, and acetylcholine in phenylephrine-constricted mesenteric arterioles in N G-nitro-l-arginine methyl ester (l-NAME)-pretreated hypertensive rats, as well as in normotensive rats.1 l-Serine evoked concentration-dependent vasodilatation in endothelium-intact but not in endothelium-denuded vessels. This response to l-serine was abolished by a combination of apamin (a small conductance Ca2+-activated K+ channel inhibitor) and TRAM-34 (an intermediate conductance Ca2+-activated K+ channel inhibitor), ouabain (Na+ pump inhibitor) and barium chloride (an inward-rectifier K+ channel inhibitor), or when the vessels were constricted by potassium chloride. In addition, the authors determined in vivo changes in mean arterial blood pressure and heart rate induced by acute intravenous infusion of either l-serine or acetylcholine in anesthetized rats. The maximal response to l-serine was higher in the l-NAME treatment group in contrast to the maximal response to acetylcholine. l-Serine evoked a rapid, reversible, dose-dependent fall in mean arterial pressure without increasing heart rate, which was more pronounced in l-NAME-treated hypertensive rats than in the control rats. This acute hypotensive effect of l-serine was significantly inhibited by apamin and charybdotoxin pretreatment, a combination that blocks Ca-activated K channels or endothelium-derived hyperpolarizing factor (EDHF). The authors discussed that the acute dose-dependent response to l-serine may be because of activation of vascular/endothelial KCa channels (exaggerated when the NO system is blunted in the chronic l-NAME-pretreated …

C‐type natriuretic peptide and endothelium‐dependent hyperpolarization in the guinea‐pig carotid artery

C-type natriuretic peptide (CNP) has been proposed to make a fundamental contribution in arterial endothelium-dependent hyperpolarization to acetylcholine. The present study was designed to address this hypothesis in the guinea-pig carotid artery. The membrane potential of vascular smooth muscle cells was recorded in isolated arteries with intracellular microelectrodes. Acetylcholine induced endothelium-dependent hyperpolarizations in the presence or absence of N (G)-nitro-L-arginine, indomethacin and/or thiorphan, inhibitors of NO-synthases, cyclooxygenases or neutral endopeptidase, respectively. Acetycholine hyperpolarized smooth muscle cells in resting arteries and produced repolarizations in phenylephrine-stimulated arteries. CNP produced hyperpolarizations with variable amplitude. They were observed only in the presence of inhibitors of NO-synthases and cyclooxygenases and were endothelium-independent, maintained in phenylephrine-depolarized carotid arteries, and not affected by the additional presence of thiorphan. In arteries with endothelium, the hyperpolarizations produced by CNP were always significantly smaller than those induced by acetylcholine. Upon repeated administration, a significant tachyphylaxis of the hyperpolarizing effect of CNP was observed, while consecutive administration of acetycholine produced sustained responses. The hyperpolarizations evoked by acetylcholine were abolished by the combination of apamin plus charybdotoxin, but unaffected by glibenclamide or tertiapin. In contrast, CNP-induced hyperpolarizations were abolished by glibenclamide and unaffected by the combination of apamin plus charybdotoxin. In the isolated carotid artery of the guinea-pig, CNP activates K(ATP) and is a weak hyperpolarizing agent. In this artery, the contribution of CNP to EDHF-mediated responses is unlikely.

Increased Contractility to Noradrenaline and Normal Endothelial Function in Mesenteric Small Arteries from the Goto-Kakizaki Rat Model of Type 2 Diabetes

Type 2 diabetes is associated with many circulatory manifestations, including alteration in endothelial function and hypertension. In this study we investigate the morphology and contractile response as well as the endothelial function of resistance arteries from the spontaneously diabetic Goto-Kakizaki (GK) rat, a model of lean type 2 diabetes expressing glucose intolerance. Isolated mesenteric small arteries were investigated under isometric conditions in a wire myograph system using noradrenaline (NA) and the endothelium-dependent vasorelaxant acetylcholine (ACh). Media thickness was measured and media lumen ratio calculated. No apparent morphological difference was noted between the arteries from GK rats and control Wistar (CW) rats. When exposed to the maximal NA concentration used (30 microM), arteries from GK rats developed significantly more tension than arteries from CW rats. In the presence of indomethacin (a specific blocker of the COX synthase) and of L-NAME (an inhibitor of eNOS), the response to NA was still significantly greater in GK rat arteries. Under control conditions, arteries from both groups showed intact relaxation to ACh. After incubation with indomethacin and L-NAME, both groups showed a non-NO nonprostaglandin-dependent relaxation to ACh. This relaxation could be blocked by a combination of apamin and charybdotoxin. This study shows that mesenteric small arteries from the diabetic GK rat have increased contractile response to NA, along with a normal endothelial function and unaltered morphology.