Uridine Adenosine Tetraphosphate Research Articles

Biphasic effects of single-dose intravenous injection of uridine adenosine tetraphosphate on blood pressure in mice

PurposeTo explore the effects of a single dose of uridine adenosine tetraphosphate (Up4A) administered through the tail vein, on the blood pressure of mice.MethodsThe mice were separated into three groups: the Up4A group, the norepinephrine (NA) group, and the α, β-methylene adenosine triphosphate (α, β-meATP) group. Each group of mice were injected drugs through the tail vein at 1, 3, 10, and 30 nmol/kg doses in an ascending order. Additionally, six mice were injected Up4A through the tail vein at 20, 40, 60, and 80 nmol/kg doses in an ascending order. The administration intervals for each dose were 20 min.ResultsMice in these groups experienced a rapid increase in blood pressure, reaching its peak within 10 s after drug administration. It took approximately 120 s for the blood pressure to return to baseline levels after the administration of the drugs in both the NA and α, β-meATP groups. After higher doses of Up4A were administered to the mice, their blood pressure exhibited biphasic changes. Initially, blood pressure of the mice rapidly dropped to a minimum within 10 s, then rose rapidly to a peak within 30 s. Subsequently, it gradually declined, taking around 10 min to return to the levels before the drug administration.ConclusionCompared to NA and α, β-meATP, Up4A, which contains purine and pyrimidine components, displayed a weaker blood pressure-elevating potency. Through its corresponding structure, Up4A exerted vasodilatory and vasoconstrictive effects throughout the entire experiment resulting in biphasic changes in blood pressure.

Indoxyl sulfate decreases uridine adenosine tetraphosphate-induced contraction in rat renal artery.

The protein-bound uremic toxin indoxyl sulfate has negative effects on a variety of physiological activities including vascular function. Uridine adenosine tetraphosphate (Up4A), a new dinucleotide molecule affects vascular function including induction of vasocontraction, and aberrant responsiveness to Up4A is evident in arteries from disorders such as hypertension and diabetes. The link between indoxyl sulfate and the Up4A-mediated response is, however, unknown. We used Wistar rat's renal arteries to see if indoxyl sulfate will affect Up4A-mediated vascular contraction. In renal arteries of indoxyl sulfate, the contractile response generated by Up4A was dramatically reduced compared to the non-treated control group. Indoxyl sulfate increased endothelin-1-induced contraction but had no effect on phenylephrine, thromboxane analog, or isotonic K+-induced renal arterial contractions. UTP, ATP, UDP, and ADP-produced contractions were reduced by indoxyl sulfate. CH223191, an aryl hydrocarbon receptor (AhR) antagonist, did not reverse Up4A, and UTP contraction decreases caused by indoxyl sulfate. The ectonucleotidase inhibitor ARL67156 prevents indoxyl sulfate from reducing Up4A- and UTP-mediated contractions. In conclusion, we discovered for the first time that indoxyl sulfate inhibits Up4A-mediated contraction in the renal artery, possibly through activating ectonucleotidase but not AhR. Indoxyl sulfate is thought to play a function in the pathophysiology of purinergic signaling.

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.

Activation of adenosine A2A but not A2B receptors is involved in uridine adenosine tetraphosphate-induced porcine coronary smooth muscle relaxation

Activation of both adenosine A2A and A2B receptors (A2BR) contributes to coronary vasodilation. We previously demonstrated that uridine adenosine tetraphosphate (Up4A) is a novel vasodilator in the porcine coronary microcirculation, acting mainly on A2AR in smooth muscle cells (SMC). We further investigated whether activation of A2BR is involved in Up4A-mediated coronary SMC relaxation. Both A2AR and A2BR may stimulate H2O2 production leading to activation of KATP channels in SMCs, we also studied the involvement of H2O2 and KATP channels in Up4A-mediated effect. Coronary small arteries dissected from the apex of porcine hearts were mounted on wire myograph for Up4A concentration responses. Up4A-induced coronary SMC relaxation was attenuated by A2AR but not A2BR antagonism or non-selective P2R antagonism, despite greater endogenous A2BR expression vs. A2AR in both coronary small arteries and primary cultured coronary SMCs. Moreover, Up4A-induced coronary SMC relaxation was blunted by H2O2 catabolism. This effect was not altered by KATP channel blockade. Combination of H2O2 catabolism and A2AR antagonism attenuated Up4A-induced coronary SMC relaxation to the similar extent as A2AR antagonism alone. Collectively, Up4A-induced porcine coronary SMC relaxation is mediated by activation of A2AR-H2O2 pathway. This process does not involve A2BR, P2R or KATP channels.

Altered Purinergic Receptor Sensitivity in Type 2 Diabetes-Associated Endothelial Dysfunction and Up₄A-Mediated Vascular Contraction.

Purinergic signaling may be altered in diabetes accounting for endothelial dysfunction. Uridine adenosine tetraphosphate (Up4A), a novel dinucleotide substance, regulates vascular function via both purinergic P1 and P2 receptors (PR). Up4A enhances vascular contraction in isolated arteries of diabetic rats likely through P2R. However, the precise involvement of PRs in endothelial dysfunction and the vasoconstrictor response to Up4A in diabetes has not been fully elucidated. We tested whether inhibition of PRs improved endothelial function and attenuated Up4A-mediated vascular contraction using both aortas and mesenteric arteries of type 2 diabetic (T2D) Goto Kakizaki (GK) rats vs. control Wistar (WT) rats. Endothelium-dependent (EDR) but not endothelium-independent relaxation was significantly impaired in both aortas and mesenteric arteries from GK vs. WT rats. Non-selective inhibition of P1R or P2R significantly improved EDR in aortas but not mesenteric arteries from GK rats. Inhibition of A1R, P2X7R, or P2Y6R significantly improved EDR in aortas. Vasoconstrictor response to Up4A was enhanced in aortas but not mesenteric arteries of GK vs. WT rats via involvement of A1R and P2X7R but not P2Y6R. Depletion of major endothelial component nitric oxide enhanced Up4A-induced aortic contraction to a similar extent between WT and GK rats. No significant differences in protein levels of A1R, P2X7R, and P2Y6R in aortas from GK and WT rats were observed. These data suggest that altered PR sensitivity accounts for endothelial dysfunction in aortas in diabetes. Modulating PRs may represent a potential therapy for improving endothelial function.

Uridine Adenosine Tetraphosphate-Induced Coronary Relaxation Is Blunted in Swine With Pressure Overload: A Role for Vasoconstrictor Prostanoids

Plasma levels of the vasoactive substance uridine adenosine tetraphosphate (Up4A) are elevated in hypertensive patients and Up4A-induced vascular contraction is exacerbated in various arteries isolated from hypertensive animals, suggesting a potential role of Up4A in development of hypertension. We previously demonstrated that Up4A produced potent and partially endothelium-dependent relaxation in the porcine coronary microvasculature. Since pressure-overload is accompanied by structural abnormalities in the coronary microvasculature as well as by endothelial dysfunction, we hypothesized that pressure-overload blunts the coronary vasodilator response to Up4A, and that the involvement of purinergic receptors and endothelium-derived factors is altered. The effects of Up4A were investigated using wire-myography in isolated coronary small arteries from Sham-operated swine and swine with prolonged (8 weeks) pressure overload of the left ventricle induced by aortic banding (AoB). Expression of purinergic receptors and endothelium-derived factors was assessed in isolated coronary small arteries using real-time PCR. Up4A (10-9 to 10-5 M) failed to produce contraction in isolated coronary small arteries from either Sham or AoB swine, but produced relaxation in preconstricted arteries, which was significantly blunted in AoB compared to Sham. Blockade of purinergic P1, and P2 receptors attenuated Up4A-induced coronary relaxation more, while the effect of P2X1-blockade was similar and the effects of A2A- and P2Y1-blockade were reduced in AoB as compared to Sham. mRNA expression of neither A1, A2, A3, nor P2X1, P2X7, P2Y1, P2Y2, nor P2Y6-receptors was altered in AoB as compared to Sham, while P2Y12 expression was higher in AoB. eNOS inhibition attenuated Up4A-induced coronary relaxation in both Sham and AoB. Additional blockade of cyclooxygenase enhanced Up4A-induced coronary relaxation in AoB but not Sham swine, suggesting the involvement of vasoconstrictor prostanoids. In endothelium-denuded coronary small arteries from normal swine, thromboxane synthase (TxS) inhibition enhanced relaxation to Up4A compared to endothelium-intact arteries, to a similar extent as P2Y12 inhibition, while the combination inhibition of P2Y12 and TxS had no additional effect. In conclusion, Up4A-induced coronary relaxation is blunted in swine with AoB, which appears to be due to the production of a vasoconstrictor prostanoid, likely thromboxane A2.

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.

Divergent coronary flow responses to uridine adenosine tetraphosphate in atherosclerotic ApoE knockout mice

Uridine adenosine tetraphosphate (Up4A) exerts potent relaxation in porcine coronary arteries that is reduced following myocardial infarction, suggesting a crucial role for Up4A in the regulation of coronary flow (CF) in cardiovascular disorders. We evaluated the vasoactive effects of Up4A on CF in atherosclerosis using ApoE knockout (KO) mice ex vivo and in vivo. Functional studies were conducted in isolated mouse hearts using the Langendorff technique. Immunofluorescence was performed to assess purinergic P2X1 receptor (P2X1R) expression in isolated mouse coronary arteries. In vivo effects of Up4A on coronary blood flow (CBF) were assessed using ultrasound. Infusion of Up4A (10−9–10−5 M) into isolated mouse hearts resulted in a concentration-dependent reduction in CF in WT and ApoE KO mice to a similar extent; this effect was exacerbated in ApoE KO mice fed a high-fat diet (HFD). The P2X1R antagonist MRS2159 restored Up4A-mediated decreases in CF more so in ApoE KO + HFD than ApoE KO mice. The smooth muscle to endothelial cell ratio of coronary P2X1R expression was greater in ApoE KO + HFD than ApoE KO or WT mice, suggesting a net vasoconstrictor potential of P2X1R in ApoE KO + HFD mice. In contrast, Up4A (1.6 mg/kg) increased CBF to a similar extent among the three groups. In conclusion, Up4A decreases CF more in ApoE KO + HFD mice, likely through a net upregulation of vasoconstrictor P2X1R. In contrast, Up4A increases CBF in vivo regardless of the atherosclerotic model.

Loss of nitric oxide-mediated inhibition of purine neurotransmitter release in the colon in the absence of interstitial cells of Cajal.

Regulation of colonic motility depends on the integrity of enteric inhibitory neurotransmission mediated by nitric oxide (NO), purine neurotransmitters, and neuropeptides. Intramuscular interstitial cells of Cajal (ICC-IM) and platelet-derived growth factor receptor-α-positive (PDGFRα+) cells are involved in generating responses to NO and purine neurotransmitters, respectively. Previous studies have suggested a decreased nitrergic and increased purinergic neurotransmission in KitW/KitW-v (W/Wv ) mice that display lesions in ICC-IM along the gastrointestinal tract. However, contributions of NO to these phenotypes have not been evaluated. We used small-chamber superfusion assays and HPLC to measure the spontaneous and electrical field stimulation (EFS)-evoked release of nicotinamide adenine dinucleotide (NAD+)/ADP-ribose, uridine adenosine tetraphosphate (Up4A), adenosine 5'-triphosphate (ATP), and metabolites from the tunica muscularis of human, monkey, and murine colons and circular muscle of monkey colon, and we tested drugs that modulate NO levels or blocked NO receptors. NO inhibited EFS-evoked release of purines in the colon via presynaptic neuromodulation. Colons from W/Wv, Nos1-/- , and Prkg1-/- mice displayed augmented neural release of purines that was likely due to altered nitrergic neuromodulation. Colons from W/Wv mice demonstrated decreased nitrergic and increased purinergic relaxations in response to nerve stimulation. W/Wv mouse colons demonstrated reduced Nos1 expression and reduced NO release. Our results suggest that enhanced purinergic neurotransmission may compensate for the loss of nitrergic neurotransmission in muscles with partial loss of ICC. The interactions between nitrergic and purinergic neurotransmission in the colon provide novel insight into the role of neurotransmitters and effector cells in the neural regulation of gastrointestinal motility.NEW & NOTEWORTHY This is the first study investigating the role of nitric oxide (NO) and intramuscular interstitial cells of Cajal (ICC-IM) in modulating neural release of purines in colon. We found that NO inhibited release of purines in human, monkey, and murine colons and that colons from KitW/KitW-v (W/Wv ) mice, which present with partial loss of ICC-IM, demonstrated augmented neural release of purines. Interactions between nitrergic and purinergic neurotransmission may affect motility in disease conditions with ICC-IM deficiencies.

Altered purinergic signaling in uridine adenosine tetraphosphate-induced coronary relaxation in swine with metabolic derangement

We previously demonstrated that uridine adenosine tetraphosphate (Up4A) induces potent and partially endothelium-dependent relaxation in the healthy porcine coronary microvasculature. We subsequently showed that Up4A-induced porcine coronary relaxation was impaired via downregulation of P1 receptors after myocardial infarction. In view of the deleterious effect of metabolic derangement on vascular function, we hypothesized that the coronary vasodilator response to Up4A is impaired in metabolic derangement, and that the involvement of purinergic receptor subtypes and endothelium-derived vasoactive factors (EDVFs) is altered. Coronary small arteries, dissected from the apex of healthy swine and swine 6 months after induction of diabetes with streptozotocin and fed a high-fat diet, were mounted on wire myographs. Up4A (10−9–10−5 M)-induced coronary relaxation was maintained in swine with metabolic derangement compared to normal swine, despite impaired endothelium-dependent relaxation to bradykinin and despite blunted P2X7 receptor and NO-mediated vasodilator influences of Up4A. Moreover, a thromboxane-mediated vasoconstrictor influence was unmasked. In contrast, an increased Up4A-mediated vasodilator influence via P2Y1 receptors was observed, while, in response to Up4A, cytochrome P450 2C9 switched from producing vasoconstrictor to vasodilator metabolites in swine with metabolic derangement. Coronary vascular expression of A2A and P2X7 receptors as well as eNOS, as assessed with real-time PCR, was reduced in swine with metabolic derangement. In conclusion, although the overall coronary vasodilator response to Up4A was maintained in swine with metabolic derangement, the involvement of purinergic receptor subtypes and EDVF was markedly altered, revealing compensatory mechanisms among signaling pathways in Up4A-mediated coronary vasomotor influence in the early phase of metabolic derangement. Future studies are warranted to investigate the effects of severe metabolic derangement on coronary responses to Up4A.

Impaired Aortic Contractility to Uridine Adenosine Tetraphosphate in Angiotensin II-Induced Hypertensive Mice: Receptor Desensitization?

We previously showed that uridine adenosine tetraphosphate (Up4A)-mediated aortic contraction is partly mediated through purinergic P2X1 receptors (P2X1R). It has been reported that the plasma level of Up4A is elevated in hypertensive patients, implying a potential role for Up4A-P2X1R signaling in hypertension. This study investigated the vasoactive effect of Up4A in aortas isolated from angiotensin (Ang) II-infused (21 days) hypertensive mice. Blood pressure was measured by tail cuff plethysmography. Aortas were isolated for isometric tension measurements, and protein expression was analyzed by western blot. Mean and systolic arterial pressures were elevated by ~50% in Ang II-infused mice. Protein levels of both AT1R and P2X1R were upregulated in Ang II-infused aortas. Surprisingly, Up4A (10-9-10-5 M)-induced concentration-dependent contraction was significantly impaired in Ang II-infused mice. Studies in control mice revealed that both P2X1R (MRS2159) and AT1R (losartan) antagonists significantly attenuated Up4A-induced aortic contraction. In addition, desensitization of AT1R by prior Ang II (100 nM) exposure had no effect on Up4A-induced aortic contraction. However, subsequent serial exposure responses to Up4A-induced aortic contraction were markedly reduced, suggesting a desensitization of purinergic receptors. This desensitization was further confirmed in control mice by prior exposure of aortas to the P2X1R desensitizer α, β-methylene ATP (10 μM). Despite upregulation of AT1R and P2X1R in hypertension, Up4A-mediated aortic contraction was impaired in Ang II-infused mice, likely through the desensitization of P2X1R but not AT1R. This implies that vascular P2X1R activity, rather than plasma Up4A level, may determine the role of Up4A in hypertension.

AB309. SPR-36 Loss of nitric oxide-mediated inhibition of purine neurotransmitter release in the colon in the absence of interstitial cells of Cajal

ObjectiveRegulation of colonic motility depends upon the integrity of enteric inhibitory neurotransmission mediated by nitric oxide (NO), multiple purine neurotransmitters, and neuropeptides. Intramuscular interstitial cells of Cajal (ICC-IM) and platelet-derived growth factor receptor α-positive (PDGFRα+) cells are involved in generating responses to NO and purine neurotransmitters, respectively. Previous studies have reported reduced nitrergic neurotransmission and upregulation of purinergic signaling in gastrointestinal muscles from KitW/KitW-v (W/Wv) mice that display viable lesions in ICC-IM along the GI tract, including the colon. However, contributions of NO to these phenotypes have not been evaluated. The present study was undertaken to determine whether neural release of purines is altered in colons from W/Wv mice and to investigate the role of NO in these mechanisms.MethodsWe utilized small-chamber superfusion assays and high-performance liquid chromatography with fluorescence detection (HPLC-FLD) to measure the spontaneous and electrical field stimulation (EFS)-evoked release of nicotinamide adenine dinucleotide (NAD+)/ADP-ribose, uridine adenosine tetraphosphate (Up4A), adenosine 5'-triphosphate (ATP), and metabolites from the tunica muscularis of human and murine colons, and we tested drugs that modulate NO levels or blocked NO receptors.ResultsNO inhibited EFS-evoked release of NAD+/ADP-ribose, Up4A, and ATP in the colon via presynaptic neuromodulation. Colons from W/Wv, Nos1−/−, and Prkg1−/− mice displayed augmented neural release of purines that was likely due to altered nitrergic neuromodulation. Colons from W/Wv mice demonstrated decreased nitrergic and increased purinergic relaxations in response to nerve stimulation. Extracellular purine metabolism was not altered in W/Wv colons. W/Wv mouse colons also demonstrated reduced Nos1 expression and reduced NO release, suggesting that NO available for prejunctional inhibition of purine release is likely reduced in these tissues.ConclusionsNO mediates inhibition of purine release that may act to prevent “over-inhibition” of colonic muscles during increased motor neuron activation. Enhanced purinergic neurotransmission may compensate for the loss of nitrergic neurotransmission in muscles with partial loss of ICC. The interactions between nitrergic and purinergic neurotransmission in the colon provides novel insight into the role of neurotransmitters and effector cells in the neural regulation of gastrointestinal motility.Funding Source(s)a grant from the National Institutes of Health, USA, DK 41315

Uridine adenosine tetraphosphate acts as a proangiogenic factor in vitro through purinergic P2Y receptors.

Uridine adenosine tetraphosphate (Up4A), a dinucleotide, exerts vascular influence via purinergic receptors (PR). We investigated the effects of Up4A on angiogenesis and the putative PR involved. Tubule formation assay was performed in a three-dimensional system, in which human endothelial cells were cocultured with pericytes with various Up4A concentrations for 5 days. Expression of PR subtypes and angiogenic factors was assessed in human endothelial cells with and without P2Y6R antagonist. No difference in initial tubule formation was detected between Up4A stimulation and control conditions at day 2 In contrast, a significant increase in vascular density in response to Up4A was observed at day 5 Up4A at an optimal concentration of 5 μM promoted total tubule length, number of tubules, and number of junctions, all of which were inhibited by the P2Y6R antagonist MRS2578. Higher concentrations of Up4A (10 μM) had no effects on angiogenesis parameters. Up4A increased mRNA level of P2YRs (P2Y2R, P2Y4R, and P2Y6R) but not P2XR (P2X4R and P2X7R) or P1R (A2AR and A2BR), while Up4A upregulated VEGFA and ANGPT1, but not VEGFR2, ANGPT2, Tie1, and Tie2. In addition, Up4A increased VEGFA protein levels. Transcriptional upregulation of P2YRs by Up4A was inhibited by MRS2578. In conclusion, Up4A is functionally capable of promoting tubule formation in an in vitro coculture system, which is likely mediated by pyrimidine-favored P2YRs but not P2XRs or P1Rs, and involves upregulation of angiogenic factors.

Impaired Vascular Contractility to Uridine Adenosine Tetraphosphate (Up4A) in Angiotensin (Ang) II‐Induced Hypertensive Mice: The Receptor Desensitization?

We previously showed (Zhou et. al., Vascular Pharmacol. 73:78–85, 2015) that Up4A‐mediated vascular contraction is partly through purinergic P2X1 receptors (P2X1R). Since the plasma level of Up4A is elevated in hypertensives, which implies a crucial role for Up4A‐P2X1R signalling in hypertension. We investigated the vasoactive effect of Up4A in Ang II‐infused mice. Mean arterial blood pressure (MAP) was measured daily in saline or Ang II‐infused mice. Aortas were isolated for isometric tension measurement. Protein expression of Ang II type 1 receptor (AT1) and P2X1R was assessed by Western blot. MAP was elevated by ~50% in 3‐week Ang II‐infused mice indicating hypertension. Protein levels of both AT1 and P2X1R were upregulated in Ang II‐infused aortas. Surprisingly, Up4A (10−9–10−5 M)‐induced concentration‐dependent contraction in saline‐infused aortas was significantly impaired in Ang II group. Both P2X1R antagonist MRS2159 and AT1 antagonist losartan attenuated Up4A‐induced aortic contraction in WT. However, Up4A‐induced aortic contraction with AT1 desensitization by the prior Ang II (100 nM) stimulation in WT was similar to that without the Ang II stimulation, while the second Up4A‐induced aortic contraction was markedly reduced as compared to the first exposure in the same preparation, suggesting a desensitization of P2R. This desensitization was further confirmed by P2X1R desensitizer α, β‐methylene ATP (10 μM). In conclusion, despite the upregulation of AT1 and P2X1R in hypertension, Up4A‐mediated aortic contraction was impaired in Ang II‐infused mice, which was likely through the desensitization of P2X1R but not AT1. This study implies that vascular P2R activity rather than plasma Up4A level may determine the role of Up4A in hypertension.Support or Funding InformationSupported by HL02339, HL094447 from NIH and MEPSCKL201301 from Sichuan Medical University

Diabetes and Age-Related Differences in Vascular Function of Renal Artery: Possible Involvement of Endoplasmic Reticulum Stress.

To study the time-course relationship between vascular functions and endoplasmic reticulum (ER) stress in type 2 diabetes, we investigated vascular function and associated protein expression, including cyclo-oxygenase (COX), ER stress, and apoptotic markers, in renal arteries (RA) from type 2 diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats at the young adult (4 months old) and aged (18 months old) stages. In the RA of aged OLETF (vs. young OLETF), we found: (1) Increased contractions induced by uridine adenosine tetraphosphate (Up4A) and phenylephrine, (2) decreased relaxation and increased contraction induced by acetylcholine (ACh) at lower and higher concentrations, respectively, and (3) increased expression of COX-1 and C/EBP-homologous protein (CHOP, a pro-apoptotic protein). In aged rats, the expression of COX-1, COX-2, PDI (an ER protein disulfide isomerase), Bax (a proapoptotic marker), and CHOP were increased in RA from OLETF rats (vs. age-matched control Long-Evans Tokushima Otsuka [LETO] rats). Up-regulation of PDI and Bax were seen in the RA from young OLETF (vs. young LETO) rats. No age-related alterations were apparent in the above changes in RA from LETO rats, excluding ACh-induced contraction. Short-term treatment with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA, 100 mg/kg per day, intraperitoneally for 1 week) to OLETF rats at the chronic stage of the disease (12 months old) could suppress renal arterial contractions induced by Up4A and ACh. These results suggest that a long-term duration of disease may be important for the development of vascular dysfunction rather than aging per se. The early regulation of ER stress may be important against the development of diabetes-associated vascular dysfunction.

血管内皮由来収縮因子として発見されたウリジンアデノシンテトラフォスフェート（Up<sub>4</sub>A）の血管機能に対する役割

血管内皮細胞は，血液と接する血管内腔に存在し様々な因子を放出することから，最大の内分泌器官と考えられている．2005年に新規血管内皮由来収縮因子として報告されたウリジンアデノシンテトラフォスフェート（Up4A）は，その構造体にプリンとピリミジンを含む非常にユニークな物質である．これまで，様々なモデル動物標本を用いた検討からUp4Aは収縮反応や弛緩反応を誘発するといった血管緊張性を調節することが明らかとなったが，病態下におけるUp4Aの作用に関しては全く不明であった．我々は，生活習慣病の主翼である高血圧と糖尿病に着目し，これらのモデル動物を用いて，病態下でUp4Aの収縮力が血管部位によって異なることや，血管平滑筋におけるUp4Aの収縮機序の一端を明らかとした．また，Up4Aは，血管緊張性調節のみならず，平滑筋の増殖や遊走，炎症，石灰化にも関与することが報告され病態形成への役割も明らかになりつつある．今後，この新たな血管作動性物質であるUp4Aの研究がさらに進むことにより，生理的および病態生理的な役割が明確になることが望まれる．

Constrictor prostanoids and uridine adenosine tetraphosphate: vascular mediators and therapeutic targets in hypertension and diabetes.

Vascular dysfunction plays a pivotal role in the development of systemic complications associated with arterial hypertension and diabetes. The endothelium, or more specifically, various factors derived from endothelial cells tightly regulate vascular function, including vascular tone. In physiological conditions, there is a balance between endothelium-derived factors, that is, relaxing factors (endothelium-derived relaxing factors; EDRFs) and contracting factors (endothelium-derived contracting factors; EDCFs), which mediate vascular homeostasis. However, in disease states, such as diabetes and arterial hypertension, there is an imbalance between EDRF and EDCF, with a reduction of EDRF signalling and an increase of EDCF signalling. Among EDCFs, COX-derived vasoconstrictor prostanoids play an important role in the development of vascular dysfunction associated with hypertension and diabetes. Moreover, uridine adenosine tetraphosphate (Up4 A), identified as an EDCF in 2005, also modulates vascular function. However, the role of Up4 A in hypertension- and diabetes-associated vascular dysfunction is unclear. In the present review, we focused on experimental and clinical evidence that implicate these two EDCFs (vasoconstrictor prostanoids and Up4 A) in vascular dysfunction associated with hypertension and diabetes.

Mechanisms underlying uridine adenosine tetraphosphate-induced vascular contraction in mouse aorta: Role of thromboxane and purinergic receptors

Uridine adenosine tetraphosphate (Up4A), a novel endothelium-derived vasoactive agent, is proposed to play a role in cardiovascular disorders and induces aortic contraction through activation of cyclooxygenases (COXs). We and others demonstrated that activation of A1 or A3 adenosine receptors (ARs) results in vascular contraction via thromboxane (TX) A2 production. However, the mechanisms of Up4A-induced vascular contraction in mouse aorta are not understood. We hypothesize that Up4A-induced aortic contraction is through COX-derived TXA2 production, which requires activation of A1 and/or A3AR. Concentration responses to Up4A were conducted in isolated aorta. The TXB2 production, a metabolite of TXA2, was also measured. Up4A (10−9-10−5M) produced a concentration-dependent contraction >70%, which was markedly attenuated by COX and COX1 but not by COX2 inhibition. Notably, Up4A-induced aortic contraction was blunted by both TX synthase inhibitor ozagrel and TXA2 receptor (TP) antagonist SQ29548. Surprisingly, A3AR deletion had no effect on Up4A-induced contraction. Moreover, A1AR deletion or antagonism as well as A1/A3AR deletion potentiated Up4A-induced aortic contraction, suggesting a vasodilator influence of A1AR. In contrast, non-selective purinergic P2 receptor antagonist PPADS significantly blunted Up4A-induced aortic contraction to a similar extent as selective P2X1R antagonist MRS2159, the latter of which was further reduced by addition of ozagrel. Endothelial denudation almost fully attenuated Up4A-induced contraction. Furthermore, Up4A (3μM) increased TXB2 formation, which was inhibited by either MRS2159 or ozagrel. In conclusion, Up4A-induced aortic contraction depends on activation of TX synthase and TP, which partially requires the activation of P2X1R but not A1 or A3AR through an endothelium-dependent mechanism.

Mechanisms of Uridine Adenosine Tetraphosphate (Up4A)‐induced Contraction in Mice Aorta: Role of Thromboxane (TXA2) and Purinergic Receptors (P2R)

Up4A, a novel endothelium‐derived vasoconstrictor, is proposed to play a role in cardiovascular disorders and induces aortic contraction through cyclooxygenases (COX). We demonstrated earlier that activation of A3 adenosine receptors (AR) results in aortic contraction via TXA2 production. However, the mechanisms of Up4A‐induced contraction are not understood. We hypothesize that Up4A‐induced contraction is via A3AR‐mediated TXA2 production. Concentration responses to Up4A were conducted in aorta from WT and A3AR KO. Up4A (10‐9‐10‐5 M) produced a dose‐dependent contraction (~80%), which was attenuated by non‐selective COX (indomethacin, 10 μM) and selective COX1 (SC560, 10 nM) but not by COX2 inhibition (NS398, 1 μM). Notably, Up4A‐induced contraction was blunted by TXA2 synthase inhibitor ozagrel (10 μM) and TXA2 receptor antagonist SQ29548 (1 μM). Non‐selective AR antagonist 8PST (100 μM) nor A3AR deletion affected Up4A‐induced contraction. However, both non‐selective P2R antagonist PPADS (10 μM) and P2X1R antagonist MRS2159 (30 μM) significantly attenuated Up4A‐induced contraction (~46% and 40%), respectively. Combination of ozagrel with MRS2159 further decreased this effect (~20%). Endothelial denudation almost fully reduced Up4A‐induced contraction. Furthermore, TXA2 production increased in vessels treated with Up4A (3 μM) (26.6±0.5 vs. 83.4±0.8 pg/mg), which was inhibited by either MRS2159 (49.0±0.7 pg/mg) or Ozagrel (42.4±0.5 pg/mg). In conclusion, Up4A‐induced contraction is via TXA2 production, which partially requires the activation of P2X1R but not A3AR through an endothelium‐dependent mechanism (Supported by HL 027339, HL 094447 and MEPSCKL201301).

MP31-12 TAMSULOSIN IMPROVES MEMORY FUNCTION BY ACTIVATING NMDA RECEPTORS IN RAT HIPPOCAMPUS

INTRODUCTION AND OBJECTIVES: Prostate smooth muscle contraction may be critical for pathophysiology and therapy of lower urinary tract symptoms (LUTS) suggestive of benign prostate hyperplasia. Although application of a1-blockers represents the goldstandard for medical treatment, their effects are limited to max. 50%. The reasons are unknown, while only little is known about contributions of nonadrenergic mediators to prostate contraction. In vivo, different mediators probably do not occur separately, but rather act simultaneously on smooth muscle cells. Here, we compared different contractile mediators with norepinephrine, and adressed their cooperative effects in contraction of human prostate smooth muscle. METHODS: Prostate tissues were obtained from radical prostatectomy (n1⁄472 patients). Contractility of prostate strips was assessed in an organ bath. Receptor expression was detected by Western blot analysis and fluorescence staining. RESULTS: Maximum endothelin-1 (ET-1)(0.1-3 mM), endothelin-2 (ET-2)(0.1-3 mM), and norepinephrine (NE)(0.1-100 mM) induced contractions were of similar magnitude (ET-1 116 23% of KCl-induced contraction; ET-2 86 17%; NE 117 18%). Maximum contractions by the thromboxane analogue, U46619 (10 mM) amounted on average around 50% of NE-induced contraction (63 9% of KCl). ET-1or ET-2-induced contractions were not affected by the a1-blocker tamsulosin (300 nM), while NE-induced contraction was virtually completely abolished and U46619-induced contraction was slightly inhibited. Dopamine (0.1-300 mM) and serotonin (0.1-300 mM) induced only very slight contractions (22 4% and 9 2% of KCl) of prostate strips, which may be of minor or local relevance. Carbachol and uridine adenosine tetraphosphate did not induce contractions. After maximumNE-induced precontraction, ET-1 did not further increase tension. In contrast, contractile effects of ET-1 (3 mM) and U46619 (10 mM) were additive, so that tension after combined application of ET-1 and U46619 (172 25% of KCl) was higher than maximumNE(100 mM) induced tension (p<0.05). Endothelin receptor A and B were detectable by Western blot in all samples. Double fluorescence staining suggests expression in stromal smooth muscle cells. CONCLUSIONS: NE-induced contraction of human prostate smooth muscle can be exceeded by cooperative actions of endothelins and thromboxane. These contractions may even occur under treatment with a1-blockers. Such contributions of non-adrenergic mediators might explain the limited efficacy of a1-blockers.