Takeo Itoh

Mechanisms of hydrogen peroxide-induced relaxation in rabbit mesenteric small artery.

The effects of hydrogen peroxide were studied on isolated rabbit mesenteric small artery; rabbit superior mesenteric artery and mouse aorta were also studied as reference tissues. For mesenteric small artery, hydrogen peroxide (1 to 100 microM) relaxed a norepinephrine-stimulated artery in a concentration-dependent manner. The relaxation was not significantly affected by removal of the endothelium and was less pronounced in arteries contracted with high-KCl solution plus norepinephrine than in those contracted with norepinephrine alone. The relaxation response to hydrogen peroxide was increased by isobutylmethylxanthine and zaprinast, inhibited by diclofenac, methylene blue and dithiothreitol and unaffected by atropine, tetraethylammonium, superoxide dismutase, deferoxamine, dimethyl sulfoxide or the Rp stereoisomer of adenosine cyclic monophosphothioate. Hydrogen peroxide shifted concentration-contractile response curves for norepinephrine to the right and downwards. Norepinephrine and caffeine elicited a transient, phasic contraction of the mesenteric small artery exposed for 0.5, 1 and 2 min to a Ca2+-free solution. Hydrogen peroxide inhibited the norepinephrine-induced contraction, and to a lesser extent the caffeine-induced contraction, and verapamil did not alter the contraction to norepinephrine. These pharmacological properties of hydrogen peroxide were similar to those of 8-bromo cGMP; 8-bromo cGMP inhibited more potently the norepinephrine-induced than the KCl-induced contraction and the contraction elicited by norepinephrine in Ca2+-free solution. The present results suggest that hydrogen peroxide induces endothelium-independent relaxation of the rabbit mesenteric small artery precontracted with norepinephrine. The effects of hydrogen peroxide may be at least in part mediated by cGMP and cyclooxygenase products in the vascular smooth muscles now used.

Acetylcholine-induced membrane potential changes in endothelial cells of rabbit aortic valve

Using a microelectrode technique, acetylcholine (ACh)‐induced membrane potential changes were characterized using various types of inhibitors of K+ and Cl− channels in rabbit aortic valve endothelial cells (RAVEC). ACh produced transient then sustained membrane hyperpolarizations. Withdrawal of ACh evoked a transient depolarization. High K+ blocked and low K+ potentiated the two ACh‐induced hyperpolarizations. Charybdotoxin (ChTX) attenuated the ACh‐induced transient and sustained hyperpolarizations; apamin inhibited only the sustained hyperpolarization. In the combined presence of ChTX and apamin, ACh produced a depolarization. In Ca2+‐free solution or in the presence of Co2+ or Ni2+, ACh produced a transient hyperpolarization followed by a depolarization. In BAPTA‐AM‐treated cells, ACh produced only a depolarization. A low concentration of A23187 attenuated the ACh‐induced transient, but not the sustained, hyperpolarization. In the presence of cyclopiazonic acid, the hyperpolarization induced by ACh was maintained after ACh removal; this maintained hyperpolarization was blocked by Co2+. Both NPPB and hypertonic solution inhibited the membrane depolarization seen after ACh washout. Bumetanide also attenuated this depolarization. It is concluded that in RAVEC, ACh produces a two‐component hyperpolarization followed by a depolarization. It is suggested that ACh‐induced Ca2+ release from the storage sites causes a transient hyperpolarization due to activation of ChTX‐sensitive K+ channels and that ACh‐activated Ca2+ influx causes a sustained hyperpolarization by activating both ChTX‐ and apamin‐sensitive K+ channels. Both volume‐sensitive Cl− channels and the Na+‐K+‐Cl− cotransporter probably contribute to the ACh‐induced depolarization.

Mechanisms underlying the reduced endothelium-dependent relaxation in human omental resistance artery in pre-eclampsia

1 In pre‐eclampsia, a functional change occurs in the role played by endothelium‐derived nitric oxide (NO) in the regulation of smooth muscle contraction in resistance arteries. We investigated the underlying mechanism in human omental resistance arteries from normotensive pregnant and pre‐eclamptic women in the presence of diclofenac (an inhibitor of cyclo‐oxygenase). 2 In endothelium‐intact strips, the sensitivity to 9,11‐epithio‐11,12‐methano‐thromboxane A2 (STA2) was significantly higher in pre‐eclampsia, and this was not modified by either NG‐nitro‐l‐arginine (l‐NNA, an inhibitor of NO synthase) or removal of the endothelium. 3 Bradykinin and substance P each produced an endothelium‐dependent relaxation of the STA2‐induced contraction in both groups, although the relaxation was significantly smaller for pre‐eclampsia. l‐NNA markedly attenuated the endothelium‐dependent relaxation in the normotensive pregnant group but not in the pre‐eclamptic group. 4 In the presence of l‐NNA, the relaxation induced by sodium nitroprusside (SNP) on the STA2 contraction was significantly smaller for pre‐eclamptic than for normotensive pregnant women. 5 In endothelium‐denuded strips, the relaxation induced by 8‐para‐ chlorophenyl thio‐guanosine‐3′,5′‐cyclic monophosphate (8‐pCPT‐cGMP) on the STA2 contraction was significantly less for pre‐eclampsia. 6 In β‐escin‐skinned strips from both groups of women, 8‐pCPT‐cGMP (1–10 μm) concentration‐dependently attenuated the contraction induced by 0.5 μm Ca2+. However, its relaxing action was significantly weaker in pre‐eclampsia. 7 It is suggested that the weaker responsiveness to NO seen in strips from pre‐eclamptic women may be partly due to a reduced smooth muscle responsiveness to cyclic GMP.

Reduced function of endothelial prostacyclin in human omental resistance arteries in pre‐eclampsia

It remains unclear in pre‐eclampsia whether or not a functional change occurs in the role played by prostacyclin in endothelium‐dependent relaxation in resistance arteries. We examined this using human omental resistance arteries (obtained from pre‐eclamptic or normotensive pregnant women) in the presence of NG‐nitro‐l‐arginine (l‐NNA, an inhibitor of nitric oxide synthase). In endothelium‐intact strips from both groups, 9,11‐epithio‐11,12‐methano‐thromboxane A2 (STA2, a thromboxane A2 mimetic) produced a contraction. Diclofenac (an inhibitor of cyclooxygenase) enhanced the STA2 contraction only in the normotensive pregnant group (1.4 times control, P < 0.01). In the presence of STA2, bradykinin (0.1 μm) produced an endothelium‐dependent relaxation in both groups, the relaxation being significantly smaller for the pre‐eclamptic group (P < 0.002). Diclofenac significantly attenuated the bradykinin‐induced relaxation only for the normotensive pregnant group (31 % inhibition, P < 0.001). The bradykinin‐induced membrane hyperpolarization consisted of diclofenac‐sensitive and ‐insensitive components. The former, but not the latter, was significantly smaller in pre‐eclampsia (‐4.3 vs.−2.6 mV, P < 0.05). The concentrations of 6‐keto‐PGF1α (a stable metabolite of prostacyclin) in these arteries were significantly lower in pre‐eclampsia in both the absence and presence of bradykinin (about 0.2‐0.4 times the normotensive pregnant value in each case, P < 0.01). By contrast, both the relaxation and the membrane hyperpolarization in response to beraprost (10 nm, a stable analogue of prostacyclin) were similar between the two groups. We conclude that, in pre‐eclampsia, a reduced part is played by prostaglandins in the endothelium‐dependent relaxation seen in resistance arteries and that this may be due to a reduced production of prostacyclin by the endothelium.

Effect of cilostazol, a phosphodiesterase type III inhibitor, on histamine‐induced increase in [Ca2+]i and force in middle cerebral artery of the rabbit

1 The effect of cilostazol, an inhibitor of phosphodiesterase type III (PDE III), on the contraction induced by histamine was studied by making simultaneous measurements of isometric force and the intracellular concentration of Ca2+ ([Ca2+]i) in endothelium‐denuded muscle strips from the peripheral part of the middle cerebral artery of the rabbit. 2 High K+ (80 mM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force. Cilostazol (10 μM) did not modify the resting [Ca2+]i, but it did significantly decrease the tonic contraction induced by high K+ without a corresponding change in the [Ca2+]i response. 3 Histamine (3 μM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force. Cilostazol (3 and 10 μM) significantly reduced both the phasic and tonic increases in [Ca2+]i and force induced by histamine, in a concentration‐dependent manner. 4 Rp‐adenosine‐3′ : 5′‐cyclic monophosphorothioate (Rp‐cAMPS, 0.1 mM), a PDE‐resistant inhibitor of protein kinase A (and as such a cyclic AMP antagonist), did not modify the increases in [Ca2+]i and force induced by histamine alone, but it did significantly decrease the cilostazol‐induced inhibition of the histamine‐induced responses. 5 In Ca2+‐free solution containing 2 mM EGTA, both histamine (3 μM) and caffeine (10 mM) transiently increased [Ca2+]i and force. Cilostazol (1–10 μM) (i) significantly reduced the increases in [Ca2+]i and force induced by histamine, and (ii) significantly reduced the increase in force but not the increase in [Ca2+]i induced by caffeine. 6 In ryanodine‐treated strips, which had functionally lost the histamine‐sensitive Ca2+ storage sites, histamine (3 μM) slowly increased [Ca2+]i and force. Cilostazol (3 and 10 μM) lowered the resting [Ca2+]i, but did not modify the histamine‐induced increase in [Ca2+]i, suggesting that functional Ca2+ storage sites are required for the cilostazol‐induced inhibition of histamine‐induced Ca2+ mobilization. 7 The [Ca2+]i‐force relationship was obtained in ryanodine‐treated strips by applying ascending concentrations of Ca2+ (0.16–2.6 mM) in Ca2+‐free solution containing 100 mM K+. Histamine (3 μM) shifted the [Ca2+]i‐force relationship to the left and increased the maximum Ca2+‐induced force. Under the same conditions, whether in the presence or absence of 3 μM histamine, cilostazol (3–10 μM) shifted the [Ca2+]i‐force relationship to the right without producing a change in the maximum Ca2+‐induced force. 8 It is concluded that, in smooth muscle of the peripheral part of the rabbit middle cerebral artery, cilostazol attenuates the histamine‐induced contraction both by inhibiting histamine‐induced Ca2+ mobilization and by reducing the myofilament Ca2+ sensitivity. It is suggested that the increase in the cellular concentration of cyclic AMP that will follow the inhibition of PDE III may play an important role in the cilostazol‐induced inhibition of the histamine‐contraction.

Randomized Controlled Trial of the Effect of Short-term Coadministration of Methylcobalamin and Folate on Serum ADMA Concentration in Patients Receiving Long-term Hemodialysis

BACKGROUND Serum asymmetric dimethylarginine (ADMA) levels are increased in maintenance hemodialysis patients, and this abnormality may increase cardiovascular risk. We investigated whether combined administration of oral folate and intravenous methylcobalamin in such patients is more beneficial than oral folate alone at decreasing circulating ADMA levels. STUDY DESIGN Randomized controlled trial. SETTING & PARTICIPANTS Patients undergoing hemodialysis. INTERVENTION 40 patients were randomly assigned to 1 of 2 groups. For 3 weeks, they received supplementation with either folate alone (15 mg/d; n = 20; folate group) or coadministered folate (15 mg/d) and methylcobalamin (500 mug after each hemodialysis treatment 3 times weekly; n = 20; methylcobalamin group). OUTCOMES PRIMARY OUTCOMES normalization of plasma homocysteine levels (<15 mumol/L), decrease in serum ADMA levels. SECONDARY OUTCOMES change in augmentation index in the carotid artery and ratios of S-adenosylmethionine to S-adenosylhomocysteine (as a transmethylation indicator) and dimethylamine to ADMA (as an indicator of ADMA hydrolysis). MEASUREMENTS Blood samples were collected under fasting conditions during the prehemodialysis procedure. RESULTS The proportion showing normalization of plasma homocysteine levels was much greater in the methylcobalamin group (18 of 20 patients; 90%) than in the folate group (6 of 20; 30%; P < 0.001). The percentage of decrease in ADMA levels was greater in the methylcobalamin than folate group (25.4% +/- 10.2% vs 13.2% +/- 11.2%; P < 0.001). The increase in ratio of S-adenosylmethionine to S-adenosylhomocysteine was not different between the 2 groups; however, the ratio of dimethylamine to ADMA was increased in only the methylcobalamin group (P = 0.04). Augmentation index was decreased in only the methylcobalamin group (P = 0.03). LIMITATIONS This study had an open-label nature and did not examine long-term effects of homocysteine-normalizing therapy (no clinical end points). CONCLUSION Coadministration of intravenous methylcobalamin and oral folate in hemodialysis patients normalized hyperhomocysteinemia and decreased ADMA levels and arterial stiffness. We suggest that this regimen may have greater potential than folate alone to decrease cardiovascular risk in such patients.

Effects of H2O2 on membrane potential of smooth muscle cells in rabbit mesenteric resistance artery.

The effects of H(2)O(2) on the membrane potential of smooth muscle cells of rabbit mesenteric resistance arteries were investigated. H(2)O(2) (3-30 microM) concentration-dependently hyperpolarized the membrane; this was inhibited by catalase but not by superoxide dismutase or the hydroxyl-radical scavenger dimethylthiourea. The cyclooxygenase inhibitor diclofenac partly inhibited the responses; the subsequent addition of the 5-lipoxygenase inhibitor 2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-p-benzoquinone (AA-861) (but not the cytochrome P(450) inhibitor 17-octadecynoic acid) further attenuated H(2)O(2)-induced hyperpolarizations. The sarcolemmal ATP-sensitive K(+) (K(ATP)) channel inhibitor 1-[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenylsulfonyl]-3-methylthiourea, sodium salt (HMR-1098), blocked the H(2)O(2)-induced hyperpolarization in the absence and presence of diclofenac. H(2)O(2) increased the production of prostaglandin E(2) and prostacyclin (estimated from its stable metabolite 6-keto-prostaglandin F(1alpha)), both of which produce a HMR-1098-sensitive hyperpolarization in the smooth muscle cells. It is concluded that, in smooth muscle cells of rabbit mesenteric artery, H(2)O(2) increases the synthesis of vasodilator prostaglandins and possibly 5-lipoxygenase products, which produce a hyperpolarization by activating sarcolemmal K(ATP) channels.

Role of endothelium in regulation of smooth muscle membrane potential and tone in the rabbit middle cerebral artery

The characteristic features of the endothelium‐mediated regulation of the electrical and mechanical activity of the smooth muscle cells of cerebral arteries were studied by measuring membrane potential and isometric force in endothelium‐intact and ‐denuded strips taken from the rabbit middle cerebral artery (MCA). In endothelium‐intact strips, histamine (His, 3–10 μM) and high K+ (20–80 mM) concentration‐dependently produced a transient contraction followed by a sustained contraction. Noradrenaline (10 μM), 5‐hydroxytryptamine (10 μM) and 9,11‐epithio‐11, 12‐methano‐thromboxane A2 (10 nM) each produced only a small contraction (less than 5% of the maximum K+‐induced contraction). NG‐nitro‐L‐arginine (L‐NOARG, 100 μM), but not indomethacin (10 μM), greatly enhanced the phasic and the tonic contractions induced by His (1–10 μM) in endothelium‐intact, but not in endothelium‐denuded strips, suggesting that spontaneous or basal release of nitric oxide (NO) from endothelial cells potently attenuates the His‐induced contractions. Acetylcholine (ACh, 0.3–3 μM) caused concentration‐dependent relaxation (maximum relaxation by 89.7±7.5%, n=4, P<0.05) when applied to endothelium‐intact strips precontracted with His. L‐NOARG had little effect on this ACh‐induced relaxation (n=4; P<0.05). Apamin (0.1 μM), but not glibenclamide (3 μM), abolished the relaxation induced by ACh (0.3–3 μM) in L‐NOARG‐treated strips (n=4, P<0.05). In endothelium‐intact tissues, His (3 μM) depolarized the smooth muscle membrane potential (by 4.4±1.8 mV, n=12, P<0.05) whereas ACh (3 μM) caused membrane hyperpolarization (−20.9±3.0 mV, n=25, P<0.05). The ACh‐induced membrane hypepolarization persisted after application of L‐NOARG (−23.5±5.9 mV, n=8, P<0.05) or glibenclamide (−20.6±5.4 mV, n=5, P<0.05) but was greatly diminished by apamin (reduced to −5.8±3.2 mV, n=3, P<0.05). Sodium nitroprusside (0.1–10 μM) did not hyperpolarize the smooth muscle cell membrane potential (0.2±0.3 mV, n=4, P>0.05) but it greatly attenuated the His‐induced contraction in endothelium‐denuded strips (n=4, P<0.05). These results suggest that, under the present experimental conditions: (i) spontaneous or basal release of NO from endothelial cells exerts a significant negative effect on agonist‐induced contractions in rabbit MCA, and (ii) ACh primarily activates the release of endothelium‐derived hyperpolarizing factor (EDHF) in rabbit MCA.

Effect of Propofol on Norepinephrine-induced Increases in [Ca2+](i) and Force in Smooth Muscle of the Rabbit Mesenteric Resistance Artery

Background Propofol (2,6‐diisopropylphenol) possesses vasodilating activity in vivo and in vitro. The propofol‐induced relaxation of agonist‐induced contractions in small resistance arteries has not been clarified. Methods The effect of propofol was examined on the contractions induced by norepinephrine and high K+ in endothelium‐denuded rabbit mesenteric resistance artery in vitro. The effects of propofol on the [Ca2+]i mobilization induced by norepinephrine and high K+ were studied by simultaneous measurement of [Ca2+]i using Fura 2 and isometric force in ryanodine‐treated strips. Results Propofol attenuated the contractions induced by high K+ and norepinephrine, the effect being greater on the high K+‐induced contraction than on the norepinephrine‐induced contraction. In Ca2‐free solution, norepinephrine produced a transient contraction resulting from the release of Ca2+ from storage sites that propofol attenuated. In ryanodine‐treated strips, propofol increased the resting [Ca2+]i but attenuated the increases in [Ca2+]i and force induced by both high K+ and norepinephrine. In the presence of nicardipine, propofol had no inhibitory action on the residual norepinephrine‐induced [Ca2+]i increase, whereas it still modestly increased resting [Ca2+]i, as in the absence of nicardipine. Conclusions In smooth muscle of the rabbit mesenteric resistance artery, propofol attenuates norepinephrine‐induced contractions due to an inhibition both of Ca2+ release and of Ca2+ influx through L‐type Ca2+ channels. Propofol also increased resting [Ca2+]i, possibly as a result of an inhibition of [Ca2+]i removal mechanisms. These results may explain in part the variety of actions seen with propofol in various types of vascular smooth muscle.

Sarpogrelate hydrochloride reduced intimal hyperplasia in experimental rabbit vein graft

OBJECTIVES The selective 5-HT(2A) receptor antagonist sarpogrelate has been clinically used for treatment in atherosclerotic diseases. However, it remains unknown whether administration of sarpogrelate inhibits intimal hyperplasia seen in autologous vein grafts. Therefore, we sought to clarify this question using an experimental rabbit vein graft model. METHODS Male rabbits were divided into two groups: a control group and a sarpogrelate-treated group. The jugular vein was interposed in the carotid artery in reversed fashion for 4 weeks and intimal hyperplasia of the grafted vein was measured (n = 8, in each group). Acetylcholine (ACh)-induced endothelium-dependent relaxation was tested by precontraction with prostaglandin F(2alpha) (PGF(2alpha), 5 muM) (n = 5, in each). endothelial nitric oxide synthase (eNOS) protein expression and superoxide production of these veins were also assessed. RESULTS The suppression of intimal hyperplasia was significantly greater in the sarpogrelate-treated group than in the control group. ACh induced an endothelium-dependent relaxation in the sarpogrelate-treated group (but not in the control group). In endothelium-intact strips from the sarpogrelate-treated group, the nitric oxide (NO) synthase inhibitor nitroarginine enhanced the PGF(2alpha)-induced contraction and blocked the ACh-induced relaxation. Immunoreactive eNOS protein expression was similar between the two groups but superoxide production (estimated from ethidium fluorescence) in endothelial cells was significantly smaller in the sarpogrelate-treated group. CONCLUSION The present results indicate that in vivo blockade of 5-HT(2A) receptors leads to an inhibition of intimal hyperplasia in rabbit vein graft. It is suggested that an increased function of endothelium-derived NO through a reduction in endothelial superoxide production may be a possible underlying mechanism for this. These novel findings support the clinical usefulness of sarpogrelate for preventing intimal hyperplasia in vein graft after bypass grafting.