Telmisartan provides protection against development of impaired vasodilation independently of metabolic effects in SHRSP.Z-Lepr(fa)/IzmDmcr rats with metabolic syndrome.

Metabolic syndrome is known to facilitate the development of cardiovascular disease. We have demonstrated that mesenteric arteries of SHRSP.Z-Lepr(fa)/IzmDmcr (SHRSP-fatty) rats with metabolic syndrome display an impaired vasorelaxation response mediated by nitric oxide. We examined whether the condition could be alleviated by treatment with telmisartan, an angiotensin II type 1 (AT1) receptor antagonist with PPAR-γ-activating properties and compared the results with those from pioglitazone, a PPAR-γ agonist. Telmisartan (5 mg·kg(-1)·day(-1)) or pioglitazone (2.5 mg·kg(-1)·day(-1)) was orally administered to male SHRSP-fatty rats for 8 weeks. Serum triglyceride and cholesterol levels were determined, and the oral glucose tolerance test was performed to evaluate insulin resistance. Vasodilations in response to acetylcholine and nitroprusside were determined by wire myographs under isometric tension conditions, protein expressions of soluble guanylyl cyclase in mesenteric arteries by Western blotting, and the contents of 3-nitrotyrosine in aortas by high-performance liquid chromatography with electrochemical detection. Telmisartan exerted antihypertensive effects, while pioglitazone ameliorated metabolic abnormalities in SHRSP-fatty rats. Telmisartan increased acetylcholine- and nitroprusside-induced relaxation and soluble guanylyl cyclase protein expression in mesenteric arteries and reduced 3-nitrotyrosine content in aortas. Pioglitazone displayed no such alleviating effects on vascular functions. These findings indicate that telmisartan protects against vasodilation disturbance through anti-oxidative and -nitrative stress independently of metabolic effects in SHRSP-fatty rats with metabolic syndrome.

Highly sensitive measurement of P-glycoprotein-mediated transport activity in Caco-2 cells.

We established a highly sensitive method for evaluating P-glycoprotein activity in Caco-2 cells. Using time-lapse confocal laser-scanning microscopy, we measured the change in fluorescence of residual rhodamine 123 in Caco-2 cells. Horizontal fluorescence images of rhodamine 123 in the apical and central parts of these cells were captured for 90 min. A continuous and significant decrease in the fluorescent intensity of rhodamine 123 in the apical part of the Caco-2 cells occurred during the measurement period, while the decrease in the central part was mild. The decrease in rhodamine 123 intensity in the apical part of Caco-2 cells were abolished in the presence of 10 microM digoxin, but the decrease in the central part was not. The decrease in total rhodamine 123 over whole cells was no significant influence of digoxin was observed. This residual rhodamine 123 assay for evaluating P-glycoprotein in the apical part of Caco-2 cells imaged by confocal laser scanning microscopy is more sensitive than conventional methods and will be a valuable screening tool for studying both the inhibition and induction of P-glycoprotein activity and expression. This method may also be useful for predicting P-glycoprotein-mediated alterations in the intestinal absorption of drugs.

Characterization of cardiac size and function in SHRSP.Z-Lepr(fa)/IzmDmcr rats, a new animal model of metabolic syndrome.

Cardiac structural and functional abnormalities are observed in metabolic syndrome. However, such changes have not been investigated in the SHRSP.Z-Lepr(fa)/IzmDmcr rat (SHRSP-fatty) model of metabolic syndrome. Here we compare cardiac size and hemodynamic function in these rats with their lean littermates (SHRSP) and normotensive control Wistar-Kyoto rats (WKY). In male 16-week-old SHRSP-fatty, we determined heart rate and systolic blood pressure (SBP) by tail-cuff, cardiac output (CO), subcutaneous peripheral blood flow (BF) and stroke volume (SV) by plethysmography, and systolic and diastolic functions by echocardiography. We also assessed weight and collagen type I expression by Western blot in isolated atrium and ventricle, and beat rate in isolated atrial preparation by myography. Heart rate was lower in conscious SHRSP-fatty than SHRSP, and the beat rate of isolated atria was lower in SHRSP-fatty and SHRSP than that of WKY. Atrial weight was larger in SHRSP-fatty than others. Ventricular weight of SHRSP-fatty and SHRSP was larger than WKY. There were significant inverse correlations between atrial weight and heart rate or beat rate in SHRSP-fatty. SBP, CO, BF and SV were increased in SHRSP-fatty similarly to SHRSP. Increased deceleration time and decreased E/A ratio, and preserved fractional shortening were observed in SHRSP-fatty. Expressions of collagen type I were increased in atria and ventricle of SHRSP-fatty. SHRSP-fatty with metabolic syndrome exhibit cardiac changes, including slowed heart rate, ventricular diastolic dysfunction, and fibrosis, and atrial enlargement. SHRSP-fatty may be a useful rat model to study on cardiac abnormalities in metabolic syndrome.

Effect of vanadate on ATP-induced increase in intracellular calcium ion levels in human umbilical vein endothelial cells.

We investigated the effect of ammonium vanadate (vanadate) on ATP-induced increases in intracellular calcium ion level ([Ca(2+)](i)) of human umbilical vein endothelial cells (HUVEC) by fluorescence confocal microscopic imaging using the Ca(2+)-sensitive probe Calcium Green 1/AM. The ATP analogue 2-methylthio-ATP (2meS-ATP), at 10 microM, significantly increased the [Ca(2+)](i) of HUVEC, and this was abolished by 1 microM thapsigargin (a calcium pump inhibitor), whereas extracellular free calcium had no effect. Vanadate at 10 microM also significantly increased the [Ca(2+)](i) of HUVEC, which was abolished by 1 microM thapsigargin. However, vanadate at 1 microM did not exert such a significant effect. We thus examined the influence of < or =1 microM vanadate for 24 h on 2meS-ATP-induced increase in [Ca(2+)](i). Vanadate significantly reduced the action of 2meS-ATP at 1 microM but not at 0.1 microM. Endogenously released ATP is known to induce various actions on endothelial cells. The present results suggest that vanadate exerts a regulatory influence on the function of vascular endothelial cells.

Effects of nicorandil on sympathetic neurotransmission in rat caudal artery.

1. We examined the effects of nicorandil, an ATP-sensitive potassium (K(ATP)) channel opener and nitric oxide donor, on the release of noradrenaline from vascular sympathetic nerves. This effect was compared to the effect on vascular smooth muscle. 2. Caudal artery preparations from Wistar rats were electrically stimulated (1 Hz, 0.5-ms duration) and noradrenaline release in the artery was detected using an high-pressure liquid chromatography-electrochemical detection technique. The pharmacological properties of the prejunctional effect of nicorandil were determined using the nonselective K(ATP) channel blocker glibenclamide, the pancreatic beta-cell and brain-type K(ATP) channel blocker tolbutamide, and the smooth muscle-type K(ATP) channel blocker PNU-37883A. 3. Nicorandil inhibited the electrical stimulation-evoked noradrenaline release in a concentration-dependent manner. This inhibitory effect was abolished by 1 micromol/L glibenclamide and 10 micromol/L tolbutamide, but was not affected by 10 micromol/L PNU-37883A or 0.3 micromol/L ODQ. Nicorandil did not affect the noradrenaline transporter uptake 1 in the adrenergic nerve terminals. 4. Nicorandil produced a relaxation response in a concentration-dependent manner in the caudal artery pre-contracted with 0.3 micromol/L noradrenaline. This relaxation response was significantly diminished in the presence of 1 micromol/L glibenclamide, 10 micromol/L PNU-37883A and 0.3 micromol/L ODQ but not by 10 micromol/L tolbutamide. 5. These findings suggest that nicorandil inhibits noradrenaline release via the K(ATP) channels of sympathetic nerves. These channels may be pharmacologically different from those of vascular smooth muscle.

Coronary vascular dysfunction promoted by oxidative-nitrative stress in SHRSP.Z-Lepr(fa) /IzmDmcr rats with metabolic syndrome.

1. Metabolic syndrome is an independent risk factor for cardiovascular disease. SHRSP.Z-Lepr(fa) /IzmDmcr (SHRSP fatty) rat, established as a new rat model of metabolic syndrome, spontaneously develops obesity, severe hypertension and shows hypertriglyceridaemia, hypercholesterolaemia and abnormal glucose tolerance. Using SHRSP fatty rats, we examined whether or not oxidative stress was correlated with vascular dysfunction in small and large calibre coronary arteries in ex vivo beating hearts, isolated mesenteric arteries and aortas in comparison with normal rats, Wistar-Kyoto rats (WKY). Vasodilation of coronary arteries was determined by microangiography of the Langendorff heart. 2. Compared with WKY, acetylcholine (ACh) and sodium nitroprusside (SNP)-induced relaxations were impaired in the coronary arteries of SHRSP fatty rats. The mesenteric arteries and aorta of SHRSP fatty rats showed impaired relaxation responses to ACh and SNP, decreased 3',5'-monophosphate (cGMP) production, and reduced soluble guanylyl cyclase protein expression. Superoxide release, angiotensin II and 3-nitrotyrosine contents were increased. 3. SHRSP fatty rats were orally administered olmesartan, an angiotensin II receptor type 1 (AT(1) ) antagonist, and amlodipine, a calcium channel blocker, at doses of 5 and 8mg/kg per day, respectively, for 8weeks. Both olmesartan and amlodipine reduced blood pressure, but only olmesartan prevented the development of abnormal vascular and biochemical parameters in the SHRSP fatty rats. 4. The results showed that in the SHRSP fatty rats, the impaired nitric oxide- and cGMP-mediated relaxation of vascular smooth muscle cells were linked to AT(1) receptor-induced oxidative-nitrative stress, which occurred concurrently with severe hypertension and metabolic abnormalities in vivo.

Possible participation of chloride ion channels in ATP release from cancer cells in suspension.

1. Cancer cells must detach from the primary focus to initiate the process of metastasis. Previously, we demonstrated that intracellular Ca(2+) levels are increased in endothelial cells in the presence of cancer cells and that ATP derived from these cells causes this increase. The present study clarifies the mechanism of ATP release from cancer cells by investigating the effects of Cl(-) channel inhibitors and other drugs on ATP release from human fibrosarcoma cells (HT-1080 cells). 2. Levels of extracellular ATP and its metabolites were measured using high-performance liquid chromatography (HPLC) with fluorescent detection. 3. Significantly more extracellular ATP was released by suspended than by adherent HT-1080 cells. The Cl(-) channel inhibitors 5-nitro-2-(3-phenylpropylamino) benzoic acid (100 micromol/L), gadolinium (100 micromol/L) and niflumic acid (100 micromol/L) all significantly inhibited ATP release from HT-1080 cells (1 x 10(3) /mL) to 39.7 +/- 6.5, 28.5 +/- 2.5 and 82.5 +/- 4.1% of control, respectively. 4. Neither of the p-glycoprotein inhibitors (i.e. 50 micromol/L quinidine and 90 micromol/L verapamil) had any effect on ATP release from HT-1080 cells. The gap junction hemichannel inhibitor Gap26 (300 micromol/L) slightly, but significantly, decreased ATP release by approximately 20%. The gap junction inhibitor 18-alpha-glycyrrhetinic acid (10 micromol/L) tended to inhibit ATP release from HT-1080 cells, but the difference did not reach statistical significance. 5. These findings indicate that Cl(-) channels play the most important role in ATP release from detached cancer cells and that gap junction hemichannels are also associated with ATP release.

Effect of P2 receptor on the intracellular calcium increase by cancer cells in human umbilical vein endothelial cells.

One of the important functions of vascular endothelial cells is as a barrier between blood and vascular tissue. This led us to speculate that cancer cells affect endothelial cells during metastasis. In the present study, we investigated the influence of human fibrosarcoma cells (HT-1080) on human umbilical vein endothelial cells (HUVEC), particularly intracellular calcium ion levels ([Ca2+]i), which are known to be an important intracellular signal transduction factor. HUVEC were treated with a fluorescent marker, and the fluorescence intensity of [Ca2+]i was then measured by phase contrast microscopic imaging. Extracellular adenosine triphosphate (ATP) release was measured using the chemiluminescence of luciferin-luciferase and a photon counting imaging system. HT-1080 (5x10(4) cells per dish) was found to increase [Ca2+]i in HUVEC. This [Ca2+]i rise was significantly reduced by U-73122 (phospholipase C inhibitor, 1 microM) and thapsigargin (calcium pump inhibitor, 1 microM). Interestingly, the [Ca2+]i rise in HUVEC was also significantly reduced by pyridoxalphosphare-6-azophenyl-2', 4'-disulfonic acid, a P2Y receptor antagonist (100 microM) and apyrase, a nucleotidase inhibitor (2 U/ml). In addition, we observed ATP release from HT-1080. These results suggest that [Ca2+]i in HUVEC was increased through the phospholipase C-IP3 pathway via ATP release from cancer cells. We previously reported that extracellular ATP increased [Ca2+]i and enhanced macromolecular permeability via the P2Y receptor. In tumor metastasis, cancer cells may exploit these regulatory mechanisms in the endothelial cell layer.

Disturbances in nitric oxide/cyclic guanosine monophosphate system in SHR/NDmcr-cp rats, a model of metabolic syndrome.

Metabolic syndrome is a cluster of metabolic abnormalities, including hypertension, hyperlipidemia, hyperinsulinemia, glucose intolerance and obesity. In such lifestyle-related diseases, impairment of nitric oxide (NO) production or bioactivity has been reported to lead to the development of atherogenic vascular diseases. Therefore, in the present study we investigated changes in the NO/cyclic guanosine monophosphate (cGMP) system in aortas of SHR/NDmcr-cp (cp/cp) rats (SHR-cp), a model of the metabolic syndrome. In aortas of SHR-cp, endothelium-dependent relaxations induced by acetylcholine and endothelium-independent relaxations induced by sodium nitroprusside were significantly impaired in comparison with Wistar-Kyoto rats. Furthermore, protein levels of soluble guanylyl cyclase and cGMP levels induced by sodium nitroprusside were significantly decreased. In contrast, protein levels of endothelium NO synthase and cGMP levels induced by acetylcholine were significantly increased, and plasma NO2 plus NO3 levels were also increased. The levels of lipid peroxide in plasma and the contents of 3-nitrotyrosine, a biomarker of peroxynitrite, in aortas were markedly increased. These findings indicate that in the aortas of SHR-cp, NO production from the endothelium is augmented, although the NO-induced relaxation response is impaired. Enhanced NO production may be a compensatory response to a variety of factors, including increases in oxidative stress.

ATP participates in the regulation of microvessel permeability.

We demonstrated previously that stimulation of the P2Y receptor enhanced the macromolecular permeability of cultured endothelial cell monolayers via the paracellular pathway. To determine whether the P2Y receptor participates in the regulation of permeability in intact microvessels, we have examined the effects of exogenous and endogenous ATP on the permeation of the surface tissue of perfused rat tail caudal artery using a fluorescein isothiocyanate-dextran (FD-4; MW 4400; 1.0 mg mL(-1)). The permeation of FD-4 was assessed by a confocal fluorescence imaging system. We found that 2-methylthioadenosine 5'-triphosphate, a P2Y receptor agonist, enhanced the fluorescence intensity of FD-4 in the surface of the rat caudal artery tissue and that it was inhibited by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, a P2 receptor antagonist. Also, noradrenaline, a sympathetic neurotransmitter, and bradykinin, an inflammatory autacoid, enhanced the fluorescence intensity of FD-4 in the surface tissue of the rat caudal artery. The enhancement by noradrenaline was significantly inhibited by the P2 receptor antagonist. In addition, noradrenaline and bradykinin caused the release of ATP, ADP, AMP and adenosine from the endothelium of the rat caudal artery. These results indicated that the exogenous and endogenous ATP increased the macromolecular permeability of blood capillaries via the P2Y receptor. Such purinergic regulation of endothelial permeability may function in physiological and pathophysiological conditions.

Myosin light chain kinase and Rho-kinase participate in P2Y receptor-mediated acceleration of permeability through the endothelial cell layer.

We have shown that P2Y receptor stimulation accelerates macromolecular permeation through the endothelial cell layer. To elucidate the mechanism of this acceleration, we examined the effects of ML-9, a myosin light chain kinase inhibitor, and Y-27632, a Rho-kinase inhibitor, on fluorescein isothiocyanate dextran (FD-4) permeation across the human umbilical vein endothelial cell monolayer. FD-4 permeation was analysed by high-performance liquid chromatography fluorescence detection. A P2Y receptor agonist, 2meS-ATP, enhanced the permeability of FD-4, which was inhibited by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a P2Y-receptor antagonist. The 2meS-ATP-induced increase in the permeability of FD-4 was significantly inhibited by ML-9. Also, Y-27632 prevented the 2meS-ATP-induced increase in the permeability of FD-4. Neither ML-9 nor Y-27632 influenced the spontaneous permeation of FD-4. These results suggest that phosphorylation of the myosin light chain may play an important role in the purinergic regulation of macromolecular permeation through the vascular endothelium.

Dysfunction of purinergic regulation of sympathetic neurotransmission in SHR/NDmcr-cp (SHR-cp) rat.

1. The effect of 2-chloroadenosine (2CA), a P1 receptor agonist and beta,gamma-methylene ATP (betagamma mATP), a P2 receptor agonist, on the overflow of endogenous noradrenaline (NE) and the contractile response were examined in the electrically field-stimulated (EFS) (1 Hz) caudal artery obtained from Wistar-Kyoto (WKY) and SHR/NDmcr-cp (SHR-cp) rats. 2. Both 2CA and betagamma mATP reduced the EFS-evoked release of NE from the arteries of WKY. Also, 2CA significantly reduced the EFS-evoked contractile response in WKY, while it had no effect at all in SHR-cp. Betagamma mATP significantly reduced the EFS-evoked contractile response in both WKY and SHR-cp. Both 2CA and betagamma mATP did not affect the contractile response induced by NE at 1 micromol/L. 3. These results indicate that in the caudal arteries of SHR-cp, the P2 agonist but not the P1 agonist is functional in the prejunctional inhibitory regulation of adrenergic neurotransmission. This P1 dysfunction may play a role in the sympathetic hyperinnervation in metabolic syndrome.

A novel method using confocal laser scanning microscopy for sensitive measurement of P-glycoprotein-mediated transport activity in Caco-2 cells.

OBJECTIVES: The aim of this study was to use time-lapse confocal laser scanning microscopy to establish a more sensitive and specific method for evaluating P-glycoprotein activity in Caco-2 cells. METHODS: The change in the fluorescence of residual rhodamine 123 at the apical and central regions of Caco-2 cells was measured in the presence of digoxin or St John's wort by using time-lapse confocal laser scanning microscopy. The data were compared with measurements made using conventional techniques, a fluorescence microplate reader and a fluorescence microscope. KEY FINDINGS: The percentage decrease of rhodamine 123 caused by 10 µm digoxin or 0.1 µg/ml St John's wort was significantly larger in the apical region of the Caco-2 cell than in the central region or in the whole cell. The digoxin-induced inhibition in the apical region as measured by time-lapse confocal laser scanning microscopy was greater than that measured in the whole cell by a microplate reader or a fluorescence microscope. CONCLUSIONS: The assay of residual rhodamine 123 in the apical region of Caco-2 cells by confocal laser scanning microscopy was more sensitive than the conventional methods using a microplate reader or fluorescence microscopy. It will be a valuable screening tool for studying both the inhibition and induction of P-glycoprotein activity.

Effects of ATP on the intracellular calcium level in the osteoblastic TBR31-2 cell line.

We investigated the effects of extracellular ATP on TBR31-2 cells established from the bone marrow of transgenic mice harboring the temperature-sensitive simian virus (SV) 40 T-antigen gene. These cells showed the capacity to differentiate toward osteoblasts and could be enhanced by bone morphogenetic protein (BMP)-2, an inducer of osteoblasts. The intracellular calcium ion level ([Ca(2+)](i)) in differentiating TBR31-2 cells was measured by fluorescence confocal microscopic imaging using the Ca(2+)-sensitive probe, Calcium Green 1/AM. P2 receptor agonists, such as ATP (1 microM), uridine 5'-triphosphate (1 microM), and ADP (1 microM), significantly increased the [Ca(2+)](i) of TBR31-2 cells in 2-d and 5-d cultures, but a potent P2X receptor agonist, alpha,beta-methylene ATP (10 microM), did not increase [Ca(2+)](i). The increase in [Ca(2+)](i) induced by ATP in the 2-d culture tended to be higher than in the 5-d culture. The increase in [Ca(2+)](i) of both cultures was inhibited by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, a P2 receptor antagonist. However, in an external Ca(2+)-free condition ATP-induced increase in [Ca(2+)](i) was unchanged at either stage. U73122, phospholipase C inhibitor and Thapsigargin, a calcium-pump inhibitor, significantly inhibited the increase in [Ca(2+)](i) at both stages. Reverse transcription-polymerase chain reaction analysis showed that the expression of P2Y receptor mRNA was higher in the 2-d culture than in the 5-d culture. These results indicate that ATP induces the increase in [Ca(2+)](i) from the calcium store through activating P2Y receptors in TBR31-2 cells and that the 2-d culture can respond to ATP more than the 5-d culture due to the higher expression of P2Y receptors. This suggests that the physiological role of ATP in osteoblasts is altered during differentiation.

Chronic production of peroxynitrite in the vascular wall impairs vasorelaxation function in SHR/NDmcr-cp rats, an animal model of metabolic syndrome.

We have previously reported that peroxynitrite is involved in dysfunction of nitric oxide (NO)-mediated vasorelaxation in SHR/NDmcr-cp rats (SHR-cp), which display typical symptoms of metabolic syndrome. This study investigated whether peroxynitrite is actually generated in the vascular wall with angiotensin II-induced NADPH-oxidase activation, thus contributing to the dysfunction. In isolated mesenteric arteries of male 18-week-old SHR-cp, relaxations in response to acetylcholine and sodium nitroprusside were impaired compared with that in Wistar-Kyoto rats. This impaired relaxation was not restored by treatment with apocynin, an NADPH-oxidase inhibitor. Protein expression of endothelial NO synthase increased while that of soluble guanylyl cyclase (sGC) decreased in the artery. We observed increased production of superoxide anions and peroxynitrite from the artery and their inhibition by apocynin, and also increased contents of nitrotyrosine, a biomarker of peroxynitrite, in mesenteric arteries and angiotensin II in aortas. Long-term (8 weeks) administration of telmisartan, an angiotensin II type 1-receptor antagonist, prevented the impaired vasorelaxation, decreased sGC expression and increased nitrotyrosine content in mesenteric arteries. These findings suggest that in the vascular wall of SHR-cp, peroxynitrite is continually produced by the reaction of NO with NADPH oxidase-derived superoxide via angiotensin II and gradually denatures sGC protein, leading to vasorelaxation dysfunction.

Long-term feeding of Ginkgo biloba extract impairs peripheral circulation and hepatic function in aged spontaneously hypertensive rats.

We previously demonstrated that Ginkgo biloba extract (GBE) produces an anti-hypertensive effect via enhanced vasodilation responses in young spontaneously hypertensive rats (SHR) and hepatic hypertrophy occurs with increased cytochrome P450 (CYP) mRNA expression in young rats. In the present study, to clarify whether these actions of GBE are observed in older rats, we investigated cardiovascular functions and hepatic CYP protein expressions in aged SHR fed a control diet or a diet containing 0.5% GBE for 4 weeks. In aged SHR, GBE feeding significantly increased liver weight per 100 g body weight without changing the body weight. Furthermore, significant increases in alanine aminotransferase level in serum and marked increase in CYP2B protein expression in the liver were observed in aged SHR fed GBE. On the other hand, GBE feeding did not affect blood pressure, but significantly reduced heart rate and blood flow velocity in tail arteries of aged SHR. Furthermore, GBE feeding did not affect contractile response to phenylephrine, relaxation responses to not only sodium nitroprusside but also acetylcholine, and protein levels of endothelium nitric oxide synthase and soluble guanylate cyclase in aortas of aged SHR. These results suggested that long-term GBE feeding impairs peripheral circulation due to bradycardia and hepatic function in aged SHR. Thus, in the elderly population with hypertension, the use of GBE may need to be assessed for effects on heart rate and liver function.

Impaired effect of salt loading on nitric oxide-mediated relaxation in aortas from stroke-prone spontaneously hypertensive rats.

1. The present study was designed to characterize the effects of salt on vasorelaxation via the nitric oxide (NO)/cGMP pathway in stroke-prone spontaneously hypertensive rats (SHRSP), which are highly salt sensitive. 2. Male 8-week-old SHRSP were given 1% NaCl solution as drinking water for 4 weeks, whereas control animals were given water only. 3. In aortic rings from salt-loaded SHRSP, relaxations in response to acetylcholine and sodium nitroprusside were significantly impaired compared with those in the control. In the presence of zaprinast, a cGMP-specific cyclic nucleotide phosphodiesterase (PDE)-5 inhibitor, the cGMP levels induced by these drugs were significantly reduced by salt loading, but remained unchanged in the absence of zaprinast. The protein levels of endothelial NO synthase, soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase (PKG) remained unchanged with salt loading, but those of PDE-5 decreased significantly and those of phosphorylated PKG tended to decrease, although the change was not statistically significant. Salt loading significantly impaired the relaxation in response to 8-bromo-cGMP. 4. These results indicate that, in aortas from SHRSP, salt loading causes impairment of relaxation in response to NO, which may be due to a decrease in cGMP production by sGC and impairment of the relaxation pathway downstream of cGMP, which, in turn, probably causes a decrease in PKG activity. Reduced PDE-5 protein expression may act, in part, as a compensatory response to impairment of cGMP-mediated relaxation.

Peroxynitrite is Involved in the dysfunction of vasorelaxation in SHR/NDmcr-cp rats, spontaneously hypertensive obese rats.

SHR/NDmcr-cp (SHR-cp) rats display typical symptoms and features of the metabolic syndrome. We previously reported that endothelium-dependent relaxation decreases in the thoracic aortas of SHR-cp rats, despite increased nitric oxide (NO) production from the endothelium. In the present study, to search for the reasons for this contradiction, we investigated whether vascular abnormality could be reduced by treatment of SHR-cp rats with antihypertensive drugs; a calcium channel blocker (amlodipine), an alpha 2 and imidazoline receptor agonist (moxonidine), and an angiotensin II type 1 (AT1) receptor antagonist (telmisartan). Telmisartan but not amlodipine and moxonidine ameliorated the impairment of relaxation in response to acetylcholine and the increased protein expression of endothelium NO synthase in thoracic aortas. All three drugs significantly lowered the blood pressure. Telmisartan decreased the serum levels of lipid peroxide and 8-hydroxy-2'-deoxyguanosine, oxidative stress markers, and also the aortic levels of the protein expression of gp91, a component of NADPH oxidase, and 3-nitrotyrosine, a biomarker of peroxynitrite. These findings suggest that NADPH oxidase-derived superoxide, probably produced due to stimulation of AT1 receptors, reacts with NO to form peroxynitrite, and consequently decreases active NO, leading to attenuation of endothelium-dependent relaxation. Angiotensin receptor antagonists may be effective for preventing endothelial dysfunction in metabolic syndrome.

P2Y receptor-mediated Ca(2+) signaling increases human vascular endothelial cell permeability.

We investigated the effects of P2-receptor agonists on cell size, intracellular calcium levels ([Ca(2+)](i)), and permeation of FITC-labeled dextran (FD-4) as well as the relationship between these effects in human umbilical vein endothelial cells (HUVEC). FD-4 concentration, cell size, and [Ca(2+)](i) were analyzed by HPLC with fluorescence, phase contrast microscopic imaging, and fluorescent confocal microscopic imaging, respectively. The P2Y(1)-receptor agonists 2-methylthio ATP (2meS-ATP) and ADP decreased cell size and increased [Ca(2+)](i) in HUVEC. The P2Y(2)-receptor agonist UTP increased [Ca(2+)](i), but did not influence cell size. The P2X-receptor agonist alpha,beta-methylene ATP did not induce either response. The decrease in size and increase in [Ca(2+)](i) by 2meS-ATP were blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, P2Y(1)-antagonist), thapsigargin (Ca(2+)-pump inhibitor), and U73122 (phospholipase C inhibitor). Furthermore, 2meS-ATP (P2Y(1)-receptor agonist) enhanced permeation of FD-4 through the endothelial cell monolayer. The 2meS-ATP-induced enhancement of the permeation was also prevented by PPADS, thapsigargin, and U73122. These results indicate that activation of P2Y receptors induces a decrease in cell size, an increase in [Ca(2+)](i), and may participate in facilitating macromolecular permeability in HUVEC.

P2Y receptor-mediated enhancement of permeation requires Ca2+ signalling in vascular endothelial cells.

1. We investigated the effects of 2-methylthioATP (2meS-ATP; a P2Y receptor agonist) on the permeation of fluorescein isothiocyanate (FITC)-labelled dextran, transendothelial electrical resistance (TEER) and intracellular calcium levels ([Ca2+]i) in cultured endothelial cells isolated from the rat caudal artery. 2. The cellular transport of FITC-labelled dextran was enhanced and TEER of the endothelial monolayer was reduced by 2meS-ATP. Both these effects were prevented by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, a P2Y receptor antagonist, which also inhibited the increase in [Ca2+]i induced by 2meS-ATP in endothelial cells. 3. The increase in [Ca2+]i induced by 2meS-ATP was inhibited by thapsigargin (a Ca2+ pump inhibitor) and by U-73122 (a phospholipase C inhibitor). 4. These findings suggested that activation of the P2Y receptor enhances the passage of material in the endothelium, which is associated with Ca2+ signalling in endothelial cells.