Tumor necrosis factor-α and matrix metalloproteinase-9 cooperatively exacerbate neurovascular degeneration in the neonatal rat retina.

Matrix metalloproteinases (MMPs) and tumor necrosis factor (TNF)-α contribute to the pathogenesis of several ocular diseases. Previous studies have shown that MMP-9 activation plays an important role in capillary degeneration in injured retinas. In this study, we aimed to determine the roles of TNF-α in capillary degeneration and MMP-9 activation in the injured retina. In rats, retinal injury was induced by intravitreal injection of N-methyl-D-aspartic acid (NMDA, 200 nmol) at postnatal day 7. We examined (1) the effects of blocking MMP-9 and TNF-α signaling pathway on capillary degeneration, (2) changes in protein levels and distribution of MMP-9 and TNF-α, and (3) the interaction between MMP-9 and TNF-α in regulating the expression level of each protein in retinas of NMDA-injected eyes. Intravitreal injection of GM6001, an MMP inhibitor, or TNF-α neutralizing antibody (anti-TNF-α Ab) attenuated capillary degeneration in retinas of NMDA-injected eyes. Protein levels of TNF-α increased 2 h after NMDA injection, whereas those of MMP-9 increased 4 h after the injection. Anti-TNF-α Ab suppressed activation of MMP-9 in retinas of NMDA-injected eyes, whereas GM6001 diminished the TNF-α protein expression. Incubation of recombinant TNF-α with supernatants of homogenized retina increased protein levels and activity of MMP-9. These results suggest that TNF-α and MMP-9 collaboratively increase their expression levels in the retina following neurodegeneration, thus leading to retinal capillary degeneration. The cooperative interaction between MMP-9 and TNF-α could be involved in the exacerbation of retinal neurovascular degeneration.

Reduced Relaxant Response to Adenine in the Superior Mesenteric Artery of Spontaneously Hypertensive Rats.

We investigated the vascular response to nucleobase adenine using freshly isolated superior mesenteric arteries of spontaneously hypertensive rats (SHR) and its control, Wistar Kyoto (WKY) rats. Endothelium-dependent and endothelium-independent relaxations were assessed in isolated segments in an organ bath. The releases of the metabolites of thromboxane A2 and prostaglandin I2 were also detected. Adenine induced vasorelaxation in both the endothelium-intact and endothelium-denuded arteries in a concentration-dependent manner. In the SHR group, the adenine-induced relaxation was slightly but significantly reduced in the endothelium-intact rings when compared with that in the WKY group. However, the relaxation in the endothelium-denuded rings were similar between the two groups. The difference in the adenine-mediated relaxation in the superior mesenteric arteries between the SHR and WKY groups was eliminated by endothelial denudation and a nitric oxide (NO) synthase inhibitor. In the absence and presence of adenine, SHR tended to have higher levels of metabolites of thromboxane A2 and prostaglandin I2 compared with WKY. However, adenine did not induce the release of these substances in the arteries in both the SHR and WKY groups. These results suggest that the reduced adenine-mediated relaxation in the superior mesenteric arteries in SHR is due to a lack of contribution from the endothelium-derived NO and not from the release of prostanoids.

Extracellular Uridine Nucleotides-Induced Contractions Were Increased in Femoral Arteries of Spontaneously Hypertensive Rats.

INTRODUCTION: Femoral arterial dysfunction including abnormal vascular responsiveness to endogenous ligands was often seen in arterial hypertension. Extracellular nucleotides including uridine 5'-diphosphate (UDP) and uridine 5'-triphosphate (UTP) play important roles for homeostasis in the vascular system including controlling the vascular tone. However, responsiveness to UDP and UTP in femoral arteries under arterial hypertension remains unclear. The aim of this study was to investigate if hypertension has an effect of vasoconstrictive responsiveness to UDP and UTP in femoral arteries of spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats (WKYs) after 7 and 12 months old. METHODS: Organ baths were conducted to determine vascular reactivity in isolated femoral arterial rings. RESULTS: In femoral arteries obtained from 12-month-old rats, augmented contractile responses to UDP and UTP were seen in femoral arteries of SHR than in those of WKY under situations not only intact but also nitric oxide synthase inhibition, whereas no difference of extracellular potassium-induced vasocontraction was seen in both SHR and WKY groups. Similar contraction trends occurred in femoral arteries obtained from 7-month-old rats. Moreover, contractions induced by UDP and UTP were increased in endothelium-denuded arteries. Cyclooxygenase inhibition decreased the contractions induced by these nucleotides and abolished the differences in responses between the SHR and WKY groups. CONCLUSIONS: This study demonstrates the importance of regulation of extracellular uridine nucleotides-induced contractions in hypertension-associated peripheral arterial diseases.

Methylglyoxal augments uridine diphosphate-induced contraction via activation of p38 mitogen-activated protein kinase in rat carotid artery.

The methylglyoxal elicits diverse adverse effects on the body. Uridine diphosphate, an extracellular nucleotide, plays an important role as a signaling molecule controlling vascular tone. This study aimed to evaluate the relationship between methylglyoxal and uridine diphosphate-induced carotid arterial contraction in rats. Additionally, we examined whether p38 mitogen-activated protein kinase (MAPK) would involve such responses. Organ baths were conducted to determine vascular reactivity in isolated carotid arterial rings, and western blotting was used for protein analysis. Treatment with methylglyoxal to carotid arterial rings showed concentration-dependent augmentation to uridine diphosphate-induced contraction in the absence and presence of NG-nitro-L-arginine, which is a nitric oxide synthase inhibitor, whereas, methylglyoxal did not affect serotonin- or isotonic high K+-induced contraction in the presence of a nitric oxide synthase inhibitor. Under nitric oxide synthase inhibition, SB203580, which is a selective p38 MAPK inhibitor, suppressed uridine diphosphate-induced contraction in both the control and methylglyoxal-treated groups, and the difference in uridine diphosphate-induced contraction was abolished by SB203580 treatment. The levels of phosphorylated p38 MAPK were increased by methylglyoxal in carotid arteries, not only under the basal condition but also under uridine diphosphate stimulation. The suppression of uridine diphosphate-induced contraction by a highly selective cell-permeable protein kinase C inhibitor bisindolylmaleimide I was observed in the methylglyoxal-treated group but not in the controls. Moreover, methylglyoxal-induced augmentation of uridine diphosphate-induced contraction was prevented by N-acetyl-L-cysteine. These results suggest that methylglyoxal could enhance uridine diphosphate-induced contraction in rat carotid arteries and may be caused by activation of p38 MAPK and protein kinase C and increased oxidative stress.

Indoxyl sulfate enhances endothelin-1-induced contraction via impairment of NO/cGMP signaling in rat aorta.

The microbiome-derived tryptophan metabolite, indoxyl sulfate, is considered a harmful vascular toxin. Here, we examined the effects of indoxyl sulfate on endothelin-1 (ET-1)-induced contraction in rat thoracic aortas. Indoxyl sulfate (10-3 M, 60 min) increased ET-1-induced contraction but did not affect isotonic high-K+-induced contraction. The ET-1-induced contraction was enhanced by endothelial denudation in both control and indoxyl sulfate-treated groups. BQ123 (10-6 M), an ETA receptor antagonist, reduced the ET-1-induced contraction in both control and indoxyl sulfate groups. BQ788 (10-6 M), an ETB receptor antagonist, increased the contraction in the control group but had no effect on the indoxyl sulfate group. Conversely, indoxyl sulfate inhibited relaxation induced by IRL1620, an ETB receptor agonist. L-NNA, an NO synthase (NOS) inhibitor, increased the ET-1-induced contractions in both the control and indoxyl sulfate groups, whereas L-NPA (10-6 M), a specific neuronal NOS inhibitor, did not affect the ET-1-induced contraction in both groups. However, ODQ, an inhibitor of soluble guanylyl cyclase, increased the ET-1-induced contraction in both groups. Organic anion transporter (OAT) inhibitor probenecid (10-3 M) and antioxidant N-acetyl-L-cysteine (NAC; 5 × 10-3 M) inhibited the effects of indoxyl sulfate. A cell-permeant superoxide scavenger reduced the ET-1-induced contraction in the indoxyl sulfate group. The aortic activity of SOD was reduced by indoxyl sulfate. The present study revealed that indoxyl sulfate augments ET-1-induced contraction in rat aortae. This enhancement may be due to the impairment of NO/cGMP signaling and may be attributed to impairment of the antioxidant systems via cellular uptake through OATs.

Differential Contractile Reactivity to Nucleotides in Femoral Arteries of OLETF and LETO Rats.

Extracellular nucleotides play an important role in the regulation of vascular function, and an abnormal vascular function is an important participant in the development and progression of diabetic vascular complications. The purpose of this study was to determine whether contractile responses induced by extracellular nucleotides and a dinucleotide, uridine adenosine tetraphosphate (Up4A), in femoral arteries would be altered at the chronic stage of type 2 diabetes. We determined the changes in contractile reactivity induced by ATP, uridine triphosphate (UTP), uridine diphosphate (UDP), and Up4A in the femoral arteries of Otsuka Long-Evans Tokushima Fatty (OLETF) rats (aged male type 2 diabetic rats) and, Long-Evans Tokushima Otsuka (LETO) rats (controls for OLETF rats). ATP-induced contractions were greater in OLETF rats than in LETO rats. UTP-induced contractions were lower in OLETF rats than in LETO rats. UDP- and Up4A-induced contractions were similar between OLETF and LETO rats. The femoral artery contractile changes induced by the extracellular nucleotides and dinucleotide were similar when nitric oxide synthase was inhibited. These results suggest that the extent of femoral artery contractile reactivity to nucleotides/dinucleotides differs during long-term duration of type 2 diabetes.

Toll-Like Receptor 4 Inhibitor TAK-242 Augments Acetylcholine-Induced Relaxation in Superior Mesenteric Arteries of the Streptozotocin-Induced Diabetic Rat.

Although vascular dysfunction is a key event in the development of diabetic complications, and abnormal toll-like receptor 4 (TLR4) may contribute to the pathophysiology of vascular diseases, the direct relationships between TLR4 and vascular function in diabetic arteries are still poorly understood. Thus, to investigate whether pharmacological blockade of TLR4 affects vascular function in the superior mesenteric artery (SMA) of streptozotocin (STZ)-induced diabetic rats, the SMA was isolated from male Wistar rat injected once with STZ (65 mg/kg, 27-34 weeks) which was treated with TAK-242 (10-6 M), a TLR4 inhibitor, for approximately 1 d using organ culture techniques. After incubation, functional and biochemical studies were performed. In the functional study, treatment with TAK-242 increased acetylcholine (ACh)-induced relaxation of the diabetic SMA in the intact condition. Sodium nitroprusside (SNP)-induced relaxation was also increased in the TAK-242-treated group compared with the vehicle-treated group. Under cyclooxygenase (COX) blockade by indomethacin (10-5 M), ACh-induced relaxation was similar in the vehicle- and TAK-242-treated groups. In addition, ACh-induced relaxation in the combined presence of the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine (L-NNA) (10-4 M), and indomethacin (10-5 M) was similar in the vehicle- and TAK-242-treated groups. The productions of thromboxane (TX) B2 in cultured medium in the presence of ACh (10-5 M) were lower in the TAK-242-treated group than in the vehicle-treated group. These data suggested that TAK-242 could augment endothelium-dependent relaxation by partly suppressing vasoconstrictor TXA2 or increasing NO signaling. TLR4 inhibition may be a novel therapeutic strategy to assist in the management of diabetes-associated vascular complications.

Impaired UTP-induced relaxation in the carotid arteries of spontaneously hypertensive rats.

Uridine 5'-triphosphate (UTP) has an important role as an extracellular signaling molecule that regulates inflammation, angiogenesis, and vascular tone. While chronic hypertension has been shown to promote alterations in arterial vascular tone regulation, carotid artery responses to UTP under hypertensive conditions have remained unclear. The present study investigated carotid artery responses to UTP in spontaneously hypertensive rats (SHR) and control Wistar Kyoto rats (WKY). Accordingly, our results found that although UTP promotes concentration-dependent relaxation in isolated carotid artery segments from both SHR and WKY after pretreatment with phenylephrine, SHR exhibited significantly lower arterial relaxation responses compared with WKY. Moreover, UTP-induced relaxation was substantially reduced by endothelial denudation and by the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine in both SHR and WKY. The difference in UTP-induced relaxation between both groups was abolished by the selective P2Y2 receptor antagonist AR-C118925XX and the cyclooxygenase (COX) inhibitor indomethacin but not by the thromboxane-prostanoid receptor antagonist SQ29548. Furthermore, we detected the release of PGE2, PGF2α, and PGI2 in the carotid arteries of SHR and WKY, both at baseline and in response to UTP. UTP administration also increased TXA2 levels in WKY but not SHR. Overall, our results suggest that UTP-induced relaxation in carotid arteries is impaired in SHR perhaps due to impaired P2Y2 receptor signaling, reductions in endothelial NO, and increases in the levels of COX-derived vasoconstrictor prostanoids.

Role of S-Equol, Indoxyl Sulfate, and Trimethylamine N-Oxide on Vascular Function.

Gut microbiota have been emerging as important contributors to the regulation of host homeostasis. Accordingly, several substances converted by gut microbiota can have beneficial or adverse effects on human health. Among them, S-equol, which is produced from the isoflavone daidzein in the human and animal gut by certain microbiota, exerts estrogenic and antioxidant activities. Indoxyl sulfate, which is metabolized in the liver from indole converted from dietary tryptophan by bacterial tryptophanases in the colon, is known as a protein-bound uremic toxin. Trimethylamine N-oxide, which is generated via the oxidization of gut microbiota-derived trimethylamine by hepatic flavin monooxygenases, is known as an accelerator of atherosclerosis. The aforementioned gut-derived substances could be potential regulators of systematic tissue/organ function, including the vascular system. Macro- and microvascular complications of cardiovascular and metabolic diseases, including atherosclerosis, hypertension, and diabetes, occur systemically and represent the principal cause of morbidity and mortality. Vascular endothelial and smooth muscle dysfunction play pivotal roles in the development and progression of vasculopathies. We herein review the link between the aforementioned gut-derived substances and endothelial and vascular smooth muscle cell function. This information will provide a conceptual framework that would allow the development of novel preventive and/or therapeutic approaches against vasculopathies.

Trimethylamine-N-oxide Specifically Impairs Endothelium-Derived Hyperpolarizing Factor-Type Relaxation in Rat Femoral Artery.

Although substantial evidence suggests that an increase in the level of trimethylamine-N-oxide (TMAO) is associated with the risk of cardiovascular diseases, including atherosclerosis, chronic kidney diseases, and hypertension, the direct effect of TMAO on vascular endothelial function remains unclear. Therefore, we investigated the acute effects of TMAO on endothelium-dependent relaxation induced by acetylcholine (ACh) in the superior mesenteric arteries and femoral arteries of rat. In endothelium-intact preparations, it was observed that TMAO (300 µmol/L for 60 min) did not affect ACh-induced relaxation in either of the two arteries. In endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation under nitric oxide synthase (NOS) and cyclooxygenase (COX) inhibitions by Nω-nitro-L-arginine (L-NNA) and indomethacin, respectively, TMAO specifically impairs the relaxation in femoral arteries but not in the superior mesenteric arteries. Under the inhibitory actions of NOS and as well as blockade of intermediate-conductance calcium-activated potassium channel (IKCa) (by TRAM-34) and small-conductance calcium-activated potassium channel (SKCa) (by apamin), which are putative sources of EDHF, ACh-induced relaxation was low, and there were no differences between the control and TMAO-treated groups with respect to both arteries. In femoral arteries, TMAO slightly reduces ACh-induced relaxation in the presence of indomethacin (preserved NO and EDHF signals) but does not affect ACh-induced NO-mediated relaxation under the combined presence of indomethacin, TRAM-34, and apamin. These results suggest that acute treatment with TMAO specifically impairs EDHF-mediated relaxation in the femoral arteries but not in the superior mesenteric arteries. These novel observations show that TMAO is a causative factor in the development of peripheral arterial disease.

Mechanisms underlying suppression of noradrenaline-induced contraction by prolonged treatment with advanced glycation end-products in organ-cultured rat carotid artery.

We investigated the direct effects of prolonged exposure to advanced glycation end-products (AGEs) on noradrenaline-induced contraction of rat carotid artery smooth muscle. Noradrenaline-induced contraction of endothelium-denuded carotid artery rings was suppressed by AGE-bovine serum albumin (AGE-BSA) pretreatment (0.01 and 0.1 mg/mL for 23 ± 1 h) compared with vehicle pretreatment (control), whereas isotonic-K+-induced contraction was not significantly altered by AGE-BSA pretreatment. This reduction in noradrenaline-induced contraction by AGE-BSA (0.1 mg/mL) was reversed by iberiotoxin, an inhibitor of large-conductance calcium-activated potassium (BKCa) channels, but not by inhibitors of other K channels [4-AP (Kv inhibitor), TRAM-34 (IKCa inhibitor), or glibenclamide (KATP inhibitor)]. Acute incubation of carotid arterial rings with H2O2 had also reduced noradrenaline-induced contraction in control arteries, but it had no effect on noradrenaline-induced contraction in AGE-BSA-pretreated arteries. Alternatively, acute incubation with the H2O2 scavenger catalase increased noradrenaline-induced contraction of AGE-BSA-pretreated arteries but had no effect on noradrenaline-induced contraction of control arteries. Noradrenaline-induced contraction in the presence of H2O2 was increased by co-treatment with iberiotoxin. The AGE-BSA-mediated suppression of noradrenaline-induced contraction was prevented by the organic cation transporter 3 (OCT3) inhibitor corticosterone, whereas the expression of OCT3 protein was similar between control and AGE-BSA-treated endothelium-denuded carotid arteries. These findings suggest that noradrenaline-induced arterial contraction is reduced by prolonged AGE-BSA exposure due to activation of BKCa channels via H2O2 generation and increased OCT3-mediated noradrenaline transport activity.

Amplification of the COX/TXS/TP receptor pathway enhances uridine diphosphate-induced contraction by advanced glycation end products in rat carotid arteries.

Advanced glycation end products (AGEs) play a pivotal role in vascular functions under various pathophysiological conditions. Although uridine diphosphate (UDP) is an important extracellular nucleotide, the relationship between AGEs and UDP regarding their effect on vascular functions remains unclear. Therefore, we investigated the effects of AGE-bovine serum albumin (AGE-BSA) on UDP-mediated responses in rat thoracic aorta and carotid arteries. In rat thoracic aorta, UDP-induced relaxation was observed and this relaxation was similar between control (1.0 v/v% PBS) and AGE-BSA-treated (0.1 mg/mL for 60 min) groups. In contrast, contraction but not relaxation was obtained following UDP application to carotid arteries with and without endothelia; contraction was greater in the AGE-BSA-treated group than in the control group. The difference in UDP-induced contraction between the two groups was not abolished by the use of a nitric oxide synthase (NOS) inhibitor, whereas it was abolished by the use of cyclooxygenase (COX), thromboxane synthase (TXS), and thromboxane-prostanoid (TP) receptor antagonist. Further, the difference in UDP-induced contraction was not abolished by the use of a cPLA2 inhibitor, whereas it was abolished by the use of an iPLA2 inhibitor. UDP increased TXA2 release in both groups, and its level was similar in both groups. Moreover, the release of PGE2, PGF2α, and PGI2 was similar among the groups. Under NOS inhibition, TP receptor agonist-induced contraction increased in the AGE-BSA-treated group (vs. control group). In conclusion, the increase in UDP-induced carotid arterial contraction by AGE-BSA can be attributed to an increase in the COX/TXS/TP receptor pathway, particularly, TP receptor signaling.

Direct Impairment of the Endothelial Function by Acute Indoxyl Sulfate through Declined Nitric Oxide and Not Endothelium-Derived Hyperpolarizing Factor or Vasodilator Prostaglandins in the Rat Superior Mesenteric Artery.

Upon stimulation, endothelial cells release various factors to regulate the vascular tone. In particular, vasorelaxing factors, called endothelium-derived relaxing factors (EDRFs), are altered in the production and/or release, as well as their signaling every vessel and under pathophysiological states, including cardiovascular, kidney, and metabolic diseases. Although indoxyl sulfate is known as a protein-bound uremic toxin and circulating levels are elevated in the impaired kidney functions, direct impact on the vascular function, especially EDRF's signaling, remains unclear. In this study, we hypothesize that acute exposure to indoxyl sulfate could alter vascular relaxation in the rat superior mesenteric artery. Accordingly, we measured acetylcholine (ACh)-induced endothelium-dependent relaxation in the absence and presence of several inhibitors to divide into each EDRF, including nitric oxide (NO), vasodilator prostaglandins (PGs), and endothelium-derived hyperpolarizing factor (EDHF). Indoxyl sulfate reduced the sensitivity to ACh but not sodium nitroprusside. Under cyclooxygenase (COX) inhibition or inhibitions of COX plus source of EDHF, such as small (SKCa)- and intermediate (IKCa)-conductance calcium-activated K+ channels, the decreased sensitivity to ACh in indoxyl sulfate exposed vessel was still preserved. However, under inhibition of NO synthase (NOS) or inhibitions of NOS and COX, the difference of sensitivity to ACh between vehicle and indoxyl sulfate was eliminated. These findings indicated that acute exposure of indoxyl sulfate in the rat superior mesenteric artery specifically explicitly impaired NO signaling but not EDHF or vasodilator PGs.

Effect of Equol on Vasocontractions in Rat Carotid Arteries Treated with High Insulin.

Previous research has indicated that high insulin affects vascular function. Equol is an active metabolite of daidzein, an isoflavone produced from soy by intestinal microbial flora, with beneficial effects on the vascular system. This study investigated whether equol was beneficial for vascular function under high insulin conditions. Using organ culture techniques, rat carotid arteries were treated for 23 ± 1 h with a vehicle, high insulin (100 nM), or equol (100 µM) plus high insulin (100 nM). Vascular isometric forces were measured by the organ bath technique. In each endothelium-intact ring, the contractions induced by high-K+, noradrenaline, or by serotonin (5-HT) were similar for the vehicle, insulin, and equol + insulin treatments. Contractions induced by a selective 5-HT2A receptor agonist (TCB2) increased with insulin treatment (vs. vehicle), but less so with equol + insulin. Under basal conditions, a selective 5-HT2B receptor agonist (BW723C86) did not induce contraction; following precontraction by a thromboxane analog, it induced contraction but not relaxation. These responses were similar across the three treatments. Acetylcholine-induced relaxations were also similar for the three treatments. In the endothelium-denuded preparations, 5-HT-induced contraction was augmented with insulin treatment (vs. vehicle) but less so by equol + insulin treatment. These differences in 5-HT-induced contractions were eliminated by iberiotoxin, a large-conductance calcium-activated K+ channel (BKCa) inhibitor. These results suggest that equol exerts a preventive effect on the enhancement of 5-HT-induced contraction by high insulin (possibly mediated by the 5-HT2A receptor), and that these effects may be attributed to the activation of BKCa channels in vascular smooth muscle.

Acute Exposure to Indoxyl Sulfate Impairs Endothelium-Dependent Vasorelaxation in Rat Aorta.

Gut microbiota are emerging as potential contributors to the regulation of host homeostasis. Dysbiosis of the gut microbiota associated with increased intestinal permeability facilitates the passage of endotoxins and other microbial products, including indoxyl sulfate in the circulation. Although an emerging body of evidence has suggested that indoxyl sulfate is a key substance for the development of chronic kidney disease, few studies have investigated the direct association of indoxyl sulfate with vascular function. We hypothesized that indoxyl sulfate adversely affects vascular function. Aortas isolated from male Wistar rat were examined in the presence or absence of indoxyl sulfate to assess the vascular function, including vasorelaxation and vasocontraction. Indoxyl sulfate (vs. vehicle) (1) decreased vasorelaxation induced by acetylcholine (ACh) but not by sodium nitroprusside; (2) had no significant alterations of noradrenaline-induced vasocontraction in the absence and presence of endothelium; (3) decreased adenylyl cyclase activator (forskolin)-induced vasorelaxation, while such a difference was eliminated by endothelial denudation; and (4) decreased vasorelaxations induced by calcium ionophore (A23187) and transient receptor potential vanilloid 4 agonist (GSK1016790A). The indoxyl sulfate-induced decrease in the vasorelaxations induced by ACh and A23187 increased by cell-permeant superoxide dismutase or by organic anion transporter inhibitor. However, apocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, had no effects on vasorelaxations induced by ACh, A23187, forskolin, and GSK1016790A in the presence of indoxyl sulfate. These results suggest that indoxyl sulfate directly affects the vascular function, particularly, endothelium-dependent vasorelaxation, and this effect may be attributable to increased oxidative stress after cell transportion via organic anion transporter, and such increased oxidative stress may not be attributable to activation of NADPH oxidase activation.

Alteration of Vascular Responsiveness to Uridine Adenosine Tetraphosphate in Aortas Isolated from Male Diabetic Otsuka Long-Evans Tokushima Fatty Rats: The Involvement of Prostanoids.

We investigated whether responsiveness to dinucleotide uridine adenosine tetraphosphate (Up4A) was altered in aortas from type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats compared with those from age-matched control Long-Evans Tokushima Otsuka (LETO) rats at the chronic stage of disease. In OLETF aortas, we observed the following: (1) Up4A-induced contractions were lower than those in the LETO aortas under basal conditions, (2) slight relaxation occurred due to Up4A, but this was not observed in phenylephrine-precontracted LETO aortas, (3) acetylcholine-induced relaxation was reduced (vs. LETO), and (4) prostanoid release (prostaglandin (PG)F2α, thromboxane (Tx)A2 metabolite, and PGE2) due to Up4A was decreased (vs. LETO). Endothelial denudation suppressed Up4A-induced contractions in the LETO group, but increased the contractions in the OLETF group. Under nitric oxide synthase (NOS) inhibition, Up4A induced contractions in phenylephrine-precontracted aortas; this effect was greater in the LETO group (vs. the OLETF group). The relaxation response induced by Up4A was unmasked by cyclooxygenase inhibitors, especially in the LETO group, but this effect was abolished by NOS inhibition. These results suggest that the relaxant component of the Up4A-mediated response was masked by prostanoids in the LETO aortas and that the LETO and OLETF rats presented different contributions of the endothelium to the response.