Junko Kajikuri

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.

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.

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.

Involvement of H2O2 in superoxide-dismutase-induced enhancement of endothelium-dependent relaxation in rabbit mesenteric resistance artery.

The mechanism underlying the enhancement by superoxide dismutase (SOD) of endothelium‐dependent relaxation was investigated in rabbit mesenteric resistance arteries. SOD (200 U ml−1) increased the production of H2O2 in smooth muscle cells (as indicated by the use of an H2O2‐sensitive fluorescent dye). Neither SOD nor catalase (400 U ml−1) modified either the resting membrane potential or the hyperpolarization induced by acetylcholine (ACh, 1 μM) in smooth muscle cells. In arteries constricted with noradrenaline, the endothelium‐dependent relaxation induced by ACh (0.01–1 μM) was enhanced by SOD (200 U ml−1) (P<0.01). This action of SOD was inhibited by L‐NG‐nitroarginine (nitric oxide (NO)‐synthase inhibitor) but not by either charybdotoxin+apamin (Ca2+‐activated‐K+‐channel blockers) or diclofenac (cyclooxygenase inhibitor). Neither ascorbate (50 μM) nor tiron (0.3 mM), superoxide scavengers, had any effect on the ACh‐induced relaxation, but each attenuated the enhancing effect of SOD on the ACh‐induced relaxation. Similarly, catalase (400 U ml−1) inhibited the effect of SOD without changing the ACh‐induced relaxation. In endothelium‐denuded strips constricted with noradrenaline, SOD enhanced the relaxation induced by the NO donor 1‐hydroxy‐2‐oxo‐3‐(N‐methyl‐3‐aminopropyl)‐3‐methyl‐1‐triazene (NOC‐7) (P<0.05). Ascorbate and catalase each attenuated this effect of SOD. H2O2 (1 μM) enhanced the relaxation on the noradrenaline contraction induced by NOC‐7 and that induced by 8‐bromo‐cGMP, a membrane‐permeable analogue of guanosine 3′,5′ cyclic monophosphate (cGMP). SOD had no effect on cGMP production, whether measured in endothelium‐intact strips following an application of ACh (0.1 μM) or in endothelium‐denuded strips following an application of NOC‐7 (0.1 μM). It is suggested that in rabbit mesenteric resistance arteries, SOD increases the ACh‐induced, endothelium‐dependent relaxation by enhancing the action of NO in the smooth muscle via its H2O2‐producing action (rather than via a superoxide‐scavenging action).

Reduced hyperpolarization in endothelial cells of rabbit aortic valve following chronic nitroglycerine administration

This study was undertaken to determine whether long‐term in vivo administration of nitroglycerine (NTG) downregulates the hyperpolarization induced by acetylcholine (ACh) in aortic valve endothelial cells (AVECs) of the rabbit and, if so, whether antioxidant agents can normalize this downregulated hyperpolarization. ACh (0.03–3 μM) induced a hyperpolarization through activations of both apamin‐ and charybdotoxin‐sensitive Ca2+‐activated K+ channels (KCa) in rabbit AVECs. The intermediate‐conductance KCa channel (IKCa) activator 1‐ethyl‐2‐benzimidazolinone (1‐EBIO, 0.3 mM) induced a hyperpolarization of the same magnitude as ACh (3 μM). The ACh‐induced hyperpolarization was significantly weaker, although the ACh‐induced [Ca2+]i increase was unchanged, in NTG‐treated rabbits (versus NTG‐untreated control rabbits). The hyperpolarization induced by 1‐EBIO was also weaker in NTG‐treated rabbits. The reduced ACh‐induced hyperpolarization seen in NTG‐treated rabbits was not modified by in vitro application of the superoxide scavengers Mn‐TBAP, tiron or ascorbate, but it was normalized when ascorbate was coadministered with NTG in vivo. Superoxide production within the endothelial cell (estimated by ethidium fluorescence) was increased in NTG‐treated rabbits and this increased production was normalized by in vivo coadministration of ascorbate with the NTG. It is suggested that long‐term in vivo administration of NTG downregulates the ACh‐induced hyperpolarization in rabbit AVECs, possibly through chronic actions mediated by superoxide.

Angiotensin II‐induced modulation of endothelium‐dependent relaxation in rabbit mesenteric resistance arteries

The role of local endogenous angiotensin II (Ang II) in endothelial function in resistance arteries was investigated using rabbit mesenteric resistance arteries. First, the presence of immunoreactive Ang II together with Ang II type‐1 receptor (AT1R) and angiotensin converting enzyme (ACE) was confirmed in these arteries. In endothelium‐intact strips, the AT1R‐blocker olmesartan (1 μm) and the ACE‐inhibitor temocaprilat (1 μm) each enhanced the ACh (0.03 μm)‐induced relaxation during the contraction induced by noradrenaline (NA, 10 μm). Similar effects were obtained using CV‐11974 (another AT1R blocker) and enalaprilat (another ACE inhibitor). The nitric‐oxide‐synthase inhibitor NG‐nitro‐l‐arginine (l‐NNA) abolished the above effect of olmesartan. In endothelium‐denuded strips, olmesartan enhanced the relaxation induced by the NO donor NOC‐7 (10 nm). Olmesartan had no effect on cGMP production (1) in endothelium‐intact strips (in the absence or presence of ACh) or (2) in endothelium‐denuded strips (in the absence or presence of NOC‐7). In β‐escin‐skinned strips, 8‐bromoguanosine 3′,5′ cyclic monophosphate (8‐Br‐cGMP, 0.01–1 μm) concentration dependently inhibited the contractions induced (a) by 0.3 μm Ca2+ in the presence of NA+GTP and (b) by 0.2 μm Ca2++GTPγS. Olmesartan significantly enhanced, while Ang II (0.1 nm) significantly inhibited, the 8‐Br‐cGMP‐induced relaxation. We propose the novel hypothesis that in these arteries, Ang II localized within smooth muscle cells activates AT1Rs and inhibits ACh‐induced, endothelium‐dependent relaxation at least partly by inhibiting the action of cGMP on these cells.

Characteristic changes in coronary artery at the early hyperglycaemic stage in a rat type 2 diabetes model and the effects of pravastatin

Background and purpose:  Diabetes is a risk factor for the development of coronary artery disease but it is not known whether the functions of endothelium‐derived nitric oxide (NO) and endothelium‐derived hyperpolarizing factor (EDHF) in coronary arteries are altered in the early stage of diabetes. Such alterations and the effects of pravastatin were examined in left anterior descending coronary arteries (LAD) from Otsuka Long‐Evans Tokushima Fatty (OLETF) rats (type 2 diabetes model) at the early hyperglycaemic stage [vs. non‐diabetic Long‐Evans Tokushima Otsuka (LETO) rats].

Characteristics of attenuated endothelium-dependent relaxation seen in rabbit intrapulmonary vein following chronic nitroglycerine administration.

1 This study was undertaken to determine whether long‐term in vivo administration of nitroglycerine (NTG) downregulates the endothelium‐dependent relaxation induced by acetylcholine (ACh) in the rabbit intrapulmonary vein and, if so, whether the type 1 angiotensin II receptor (AT1R) blocker valsartan normalizes this downregulated relaxation. 2 In strips treated with the cyclooxygenase inhibitor diclofenac, ACh induced a relaxation only when the endothelium was intact. A small part of this ACh‐induced relaxation was inhibited by coapplication of two Ca2+‐activated K+‐channel blockers (charybdotoxin (CTX)+apamin) and the greater part of the response was inhibited by the nitric‐oxide‐synthase inhibitor Nω‐nitro‐L‐arginine (L‐NNA). 3 The endothelium‐dependent relaxation induced by ACh, but not the endothelium‐independent relaxation induced by the nitric oxide donor NOC‐7, was significantly reduced in NTG‐treated rabbits (versus those in NTG‐nontreated control rabbits). The attenuated relaxation was normalized by coapplication of valsartan with the NTG. 4 In the vascular wall, both the amount of localized angiotensin II and the production of superoxide anion were increased by in vivo NTG treatment. These variables were normalized by coapplication of valsartan with the NTG. 5 It is suggested that long‐term in vivo administration of NTG downregulates the ACh‐induced endothelium‐dependent relaxation, mainly through an inhibition of endothelial nitric oxide production in the rabbit intrapulmonary vein. A possible role for AT1R is proposed in the mechanism underlying this effect.