In vivo mechanisms of acetylcholine-induced vasodilation in rat sciatic nerve

We examined the importance of nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), and neurogenic activity in agonist-induced vasodilation and baseline blood flow [i.e., nerve microvascular conductance (NMVC)] in rat sciatic nerve using laser Doppler flowmetry. Agonists were acetylcholine (ACh) and 3-morpholinosydnonimine (SIN-1). Vasodilation occurring despite NO synthase (NOS) and cyclooxygenase inhibition and showing dependence on K(+) channel activity was taken as being mediated by EDHF. NOS and cyclooxygenase inhibition with N(omega)-nitro-L-arginine (L-NNA) + indomethacin (Indo) revealed two phases of ACh-induced vasodilation: an initial, transient L-NNA + Indo-resistant vasodilation, peaking at 23 +/- 6 s and lasting 145 +/- 69 s, followed by sustained L-NNA + Indo-sensitive vasodilation. L-NNA alone did not affect sustained ACh-induced vasodilation but decreased baseline NMVC by 55%. In the presence of L-NNA + Indo, the K(+) channel blocker tetraethylammonium (TEA) inhibited transient ACh-induced vasodilation by 58% and reduced baseline NMVC by 25%. SIN-1-induced vasodilation increased fourfold in the presence of L-NNA, whereas the specific guanylyl cyclase inhibitor 1H-(1, 2, 4)oxadiazolo(4,3-alpha)quinoxalin-1-one abolished it. However, in homogenates of rat sciatic nerve, SIN-1-stimulated soluble guanylyl cyclase (sGC) activity was unaffected by L-NNA. TTX affected neither SIN-1- nor ACh-induced vasodilation. In conclusion, ACh-induced vasodilation consisted of two components, the first partially mediated by EDHF and the second by a vasodilatory prostanoid + NO. Baseline NMVC was dependent on NO and EDHF. Although L-NNA enhanced SIN-1-induced vasodilation, it had no effect on sGC-activity.     

Nitric oxide exerts feedback inhibition on EDHF-induced coronary arteriolar dilation in vivo

We tested the hypothesis that nitric oxide (NO) inhibits endothelium-derived hyperpolarizing factor (EDHF)-induced vasodilation via a negative feedback pathway in the coronary microcirculation. Coronary microvascular diameters were measured using stroboscopic fluorescence microangiography. Bradykinin (BK)-induced dilation was mediated by EDHF, when NO and prostaglandin syntheses were inhibited, or by NO when EDHF and prostaglandin syntheses were blocked. Specifically, BK (20, 50, and 100 ng. kg(-1). min(-1) ic) caused dose-dependent vasodilation similarly before and after administration of N(G)-monomethyl-L-arginine (L-NMMA) (3 micromol/min ic for 10 min) and indomethacin (Indo, 10 mg/kg iv). The residual dilation to BK with L-NMMA and Indo was completely abolished by suffusion of miconazole or an isosmotic buffer containing high KCl (60 mM), suggesting that this arteriolar vasodilation is mediated by the cytochrome P-450 derivative EDHF. BK-induced dilation was reduced by 39% after inhibition of EDHF and prostaglandin synthesis, and dilation was further inhibited by combined blockade with L-NMMA to a 74% reduction in the response. This suggests an involvement for NO in the vasodilation. After dilation to BK was assessed with L-NMMA and Indo, sodium nitroprusside (SNP, 1-3 microgram. kg(-1). min(-1) ic), an exogenous NO donor, was administered in a dose to increase the diameter to the original control value. Dilation to BK was virtually abolished when administered concomitantly with SNP during L-NMMA and Indo (P < 0.01 vs. before SNP), suggesting that NO inhibits EDHF-induced dilation. SNP did not affect adenosine- or papaverine-induced arteriolar dilation in the presence of L-NMMA and Indo, demonstrating that the effect of SNP was not nonspecific. In conclusion, our data are the first in vivo evidence to suggest that NO inhibits the production and/or action of EDHF in the coronary microcirculation.     

Increased pulmonary responses to acute ozone exposure in obese db/db mice

Epidemiological studies indicate the incidence of asthma is increased in obese and overweight humans. Responses to ozone (O(3)), an asthma trigger, are increased in obese (ob/ob) mice lacking the satiety hormone leptin. The long form of leptin receptor (Ob-R(b)) is required for satiety; mice lacking this receptor (db/db mice) are also substantially obese. Here, wild-type (WT) and db/db mice were exposed to air or O(3) (2 ppm) for 3 h. Airway responsiveness, measured by the forced oscillation technique, was greater in db/db than WT mice after air exposure. O(3)-induced increases in pulmonary resistance and airway responsiveness were also greater in db/db mice. BALF eotaxin, IL-6, KC, and MIP-2 increased 4 h after O(3) exposure and subsided by 24 h, whereas protein and neutrophils continued to increase through 24 h. For each outcome, the effect of O(3) was significantly greater in db/db than WT mice. Previously published results obtained in ob/ob mice were similar except for O(3)-induced neutrophils and MIP-2, which were not different from WT mice. O(3) also induced pulmonary IL-1beta and TNF-alpha mRNA expression in db/db but not ob/ob mice. Leptin was increased in serum of db/db mice, and pulmonary mRNA expression of short form of leptin receptor (Ob-R(a)) was similar in db/db and WT mice. These data confirm obese mice have innate airway hyperresponsiveness and increased pulmonary responses to O(3). Differences between ob/ob mice, which lack leptin, and db/db mice, which lack Ob-R(b) but not Ob-R(a), suggest leptin, acting through Ob-R(a), can modify some pulmonary responses to O(3).     

In eNOS knockout mice skeletal muscle arteriolar dilation to acetylcholine is mediated by EDHF

The mechanisms that account for acetylcholine (ACh)-induced responses of skeletal muscle arterioles of mice lacking endothelial nitric oxide (NO) synthase (eNOS-KO) were investigated. Isolated, cannulated, and pressurized arterioles of gracilis muscle from male eNOS-KO (74.1 +/- 2.3 microm) and wild-type (WT, 87.2 +/- 2.1 microm) mice developed spontaneous tone accounting for 63 and 61% of their passive diameter (116.8 +/- 3.4 vs. 143.2 +/- 2.8 microm, respectively) and dilated dose-dependently to ACh (10(-9)-10(-7) M). These dilations were significantly smaller in vessels of eNOS-KO compared with WT mice (29.2 +/- 2.0 microm vs. 46.3 +/- 2.1 microm, at maximum concentration) but responses to the NO donor, sodium nitrite (NaNO(2), 10(-6)-3 x 10(-5) M), were comparable in the vessels of the two strains. N(G)-nitro-L-arginine (L-NNA, 10(-4) M), an inhibitor of eNOS, inhibited ACh-induced dilations by 60-90% in arterioles of WT mice but did not affect responses in those of eNOS-KO mice. In arterioles of eNOS-KO mice, dilations to ACh were not affected by indomethacin but were essentially abolished by inhibitors of cytochrome P-450, clotrimazole (CTZ, 2 x 10(-6) M) or miconazole (MCZ, 2 x 10(-6) M), as well as by either high K(+) (40 mM) or iberiotoxin [10(-7) M, a blocker of Ca(2+)-dependent K(+) channels (K(Ca) channels)]. On the other hand, in WT arterioles CTZ or MCZ inhibited ACh-induced dilations only by approximately 10% and only in the presence of L-NNA. These results indicate that in arterioles of eNOS-KO mice, endothelium-derived hyperpolarizing factor (EDHF), synthesized via cytochrome P-450, accounts entirely for the mediation of ACh-induced dilation via an increase in K(Ca)-channel activity. In contrast, in arterioles of WT mice, endothelium-derived NO predominantly mediates ACh-induced dilation in which participation of EDHF becomes apparent only after inhibition of NO synthesis.     

Endothelium-derived relaxing factors in the kidney of spontaneously hypertensive rats

Acetylcholine (ACh)-induced vasodilation is mainly due to endothelium-derived nitric oxide (EDNO) and hyperpolarizing factor (EDHF). To explore the mechanisms underlying attenuated endothelium-dependent vasodilation in hypertensive arteries, we measured the EDNO released from isolated kidneys of spontaneously hypertensive rats (SHR) using a sensitive chemiluminescence assay system of NO. ACh-induced renal vasodilation was significantly smaller in SHR than in the normotensive control, Wistar-Kyoto rats (WKY). However, ACh-induced NO release did not differ between SHR and WKY (10⁻⁷ M: SHR +37 ± 2 [SE] vs. WKY +32 ± 4 fmol/min/g kidney). Perfusion with a 20 mEq/L high-K⁺ buffer, which is reported to inhibit action of EDHF, significantly reduced ACh-induced vasorelaxation in WKY but not in SHR, resulting in identical renal perfusion pressure in SHR and wKY under these conditions. These results indicate that attenuated ACh-induced vasorelaxation in the SHR kidney may be attributed to a decrease in EDHF rather than that in EDNO.     

Regional heterogeneity in acetylcholine-induced relaxation in rat vascular bed: role of calcium-activated K+ channels

Ca+ -activated K+ -channels (KCa) regulate vasomotor tone via smooth muscle hyperpolarization and relaxation. The relative contribution of the endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation differs depending on vessel type and size. It is unknown whether these KCa channels are differentially distributed along the same vascular bed and hence have different roles in mediating the EDHF response. We therefore assessed the role of small- (SKCa), intermediate- (IKCa), and large-conductance (BKCa) channels in mediating acetylcholine-induced relaxations in both first- and fourth-order side branches of the rat superior mesenteric artery (MA1 and MA4, respectively). Two-millimeter segments of each MA were mounted in the wire myograph, incubated with Nomega-nitro-L-arginine methyl ester (L-NAME, 100 micromol/l) and indomethacin (10 micromol/l), and precontracted with phenylephrine (10 micromol/l). Cumulative concentration-response curves to ACh (0.001-10 micromol/l) were performed in the absence or presence of selective KCa channel antagonists. Apamin almost completely abolished these relaxations in MA4 but only partially blocked relaxations in MA1. The selective IKCa channel blocker 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) caused a significantly greater inhibition of the ACh-induced relaxation in MA4 compared with MA1. Iberiotoxin had no inhibitory effect in MA4 but blunted relaxation in MA1. Relative mRNA expression levels of SKCa (rSK1, rSK3, and rSK4 = rIK1) were significantly higher in MA4 compared with MA1. BKCa (rBKalpha1 and rBKbeta1) genes were similar in both MA1 and MA4. Our data demonstrate regional heterogeneity in SKCa and IKCa function and gene expression and stress the importance of these channels in smaller resistance-sized arteries, where the role of EDHF is more pronounced.     

Receptor-activating peptides for PAR-1 and PAR-2 relax rat gastric artery via multiple mechanisms

Receptor-activating peptides for protease-activated receptors (PARs) 1 or 2 enhance gastric mucosal blood flow (GMBF) and protect against gastric mucosal injury in rats. We thus examined and characterized the effects of PAR-1 and PAR-2 agonists on the isometric tension in isolated rat gastric artery. The agonists for PAR-2 or PAR-1 produced vasodilation in the endothelium-intact arterial rings, which was abolished by removal of the endothelium. The mechanisms underlying the PAR-2- and PAR-1-mediated relaxation involved NO, endothelium-derived hyperpolarizing factor (EDHF) and prostanoids, to distinct extent, as evaluated by use of inhibitors of NO synthase, cyclo-oxygenase and Ca2+-activated K+ channels. The EDHF-dependent relaxation responses were significantly attenuated by gap junction inhibitors. These findings demonstrate that endothelial PAR-1 and PAR-2, upon activation, dilate the gastric artery via NO and prostanoid formation and also EDHF mechanisms including gap junctions, which would enhance GMBF.     

Responses of cerebral arterioles to ADP: eNOS-dependent and eNOS-independent mechanisms

ADP mediates platelet-induced relaxation of blood vessels and may function as an important intercellular signaling molecule in the brain. We used pharmacological and genetic approaches to examine mechanisms that mediate responses of cerebral arterioles to ADP, including the role of endothelial nitric oxide synthase (eNOS). We examined responses of cerebral arterioles (control diameter approximately 30 microm) in anesthetized wild-type (WT, eNOS+/+) and eNOS-deficient (eNOS-/-) mice using a cranial window. In WT mice, local application of ADP produced vasodilation that was not altered by indomethacin but was reduced by approximately 50% by NG-nitro-L-arginine (L-NNA) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (inhibitors of NOS and soluble guanylate cyclase, respectively). In eNOS-/- mice, responses to ADP were largely preserved, and a significant component of the response was resistant to L-NNA (a finding similar to that in WT mice treated with L-NNA). In the absence of L-NNA, responses to ADP were markedly reduced by charybdotoxin plus apamin [inhibitors of Ca2+-dependent K+ channels and responses mediated by endothelium-derived hyperpolarizing factor (EDHF)] in both WT and eNOS-/- mice. Thus pharmacological and genetic evidence suggests that a significant portion of the response to ADP in cerebral microvessels is mediated by a mechanism independent of eNOS. The eNOS-independent mechanism is functional in the absence of inhibited eNOS and most likely is mediated by an EDHF.     

Type 2 diabetes impairs insulin receptor substrate-2-mediated phosphatidylinositol 3-kinase activity in primary macrophages to induce a state of cytokine resistance to IL-4 in association with overexpression of suppressor of cytokine signaling-3

Chronic elevation of proinflammatory markers in type 2 diabetes (T2D) is well defined, but the role of anti-inflammatory cytokines in T2D is less clear. In this study, we report that normal IL-4-dependent elaboration of IL-1 receptor antagonist (IL-1RA) requires IRS-2-mediated PI3K activity in primary macrophages. We also show that macrophages isolated from obese/diabetic db/db mice have impaired IRS-2-mediated PI3K activity and constitutively overexpress suppressor of cytokine signaling (SOCS)-3, which impairs an important IL-4 anti-inflammatory function. Peritoneal proinflammatory cytokine levels were examined in diabese (db/db) mice, and IL-6 was found to be nearly 7-fold higher than in nondiabese (db/+) control mice. Resident peritoneal macrophages were isolated from db/db mice and were found to constitutively overexpress IL-6 and were unable to elaborate IL-1RA in response to IL-4-like db/+ mouse macrophages. Inhibition of PI3K with wortmannin or blockage of IRS-2/PI3K complex formation with a cell permeable IRS-2-derived tyrosine phosphopeptide inhibited IL-4-dependent IL-1RA production in db/+ macrophages. Examination of IL-4 signaling in db/db macrophages revealed that IL-4-dependent IRS-2/PI3K complex formation and IRS-2 tyrosine phosphorylation was reduced compared with db/+ macrophages. SOCS-3/IL-4 receptor complexes, however, were increased in db/db mouse macrophages compared with db/+ mice macrophages as was db/db mouse macrophage SOCS-3 expression. These results indicate that in the db/db mouse model of T2D, macrophage expression of SOCS-3 is increased, and impaired IL-4-dependent IRS-2/PI3K formation induces a state of IL-4 resistance that disrupts IL-4-dependent production of IL-1RA.     

Cytochrome P-450 metabolites but not NO,PGI2, and H2O2 contribute to ACh-induced hyperpolarization of pressurized canine coronary microvessels

The endothelium-dependent hyperpolarization of cells has a crucial role in regulating vascular tone, especially in microvessels. Nitric oxide (NO) and prostacyclin (PGI2), in addition to endothelium-derived hyperpolarizing factor (EDHF), have been reported to hyperpolarize vascular smooth muscle in several organs. Studies have reported the hyperpolarizing effects of these factors are increased by a stretch in large coronary arteries. EDHF has not yet been identified and cytochrome P-450 metabolites and H2O2 are candidates for EDHF. With the use of the membrane potential-sensitive fluorescent dye bis-(1,3-dibutylbarbituric acid)trimethione oxonol [DiBAC4(3)], we examined whether NO, PGI2, cytochrome P-450 metabolites, and H2O2 contribute to ACh-induced hyperpolarization in pressurized coronary microvessels. Canine coronary arterial microvessels (60-356 mum internal diameter) were cannulated and pressurized at 60 cmH2O in a vessel chamber perfused with physiological salt solution containing DiBAC4(3). Fluorescence intensity and diameter were measured on a computer. There was a linear correlation between changes in the fluorescence intensity and membrane potential. ACh significantly decreased the fluorescence intensity (hyperpolarization) of the microvessels without any inhibitors. Endothelial damage caused by air perfusion abolished the ACh-induced decrease in fluorescence intensity. The inhibitors of NO synthase and cyclooxygenase did not affect the ACh-induced decreases in the fluorescence intensity. The addition of 17-octadecynoic acid, a cytochrome P-450 monooxygenase inhibitor, to those inhibitors significantly attenuated the ACh-induced decreases in fluorescence intensity, whereas catalase, an enzyme that dismutates H2O2 to form water and oxygen, did not. Furthermore, catalase did not affect the vasodilation produced by ACh. These results indicate that NO and PGI2 do not contribute to the ACh-induced hyperpolarization and that the cytochrome P-450 metabolites but not H2O2 are involved in EDHF-mediated hyperpolarization in canine coronary arterial microvessels.     

Antisense oligonucleotides against cytochrome P450 2C8 attenuate EDHF-mediated Ca(2+) changes and dilation in isolated resistance arteries.

Using a novel vessel culture technique in combination with antisense oligonucleotide transfection, we tested whether the endothelium-derived hyperpolarizing factor (EDHF) is a cytochrome P450 (CYP)-related compound. Isolated resistance arteries from hamster gracilis muscle (n=19) were perfused and exposed to antisense (As), sense (S), or scrambled (Scr) oligonucleotides against the coding region of CYP2C8/9, an isoform expressed in endothelial cells. Thereafter, NO- and prostaglandin-independent, EDHF-mediated vascular responses associated with hyperpolarization [i.e., decrease in smooth muscle calcium (Fura 2) and vasodilation] were studied after the application of acetylcholine (ACh). These EDHF-mediated responses were markedly attenuated (by 70%) by As- but not by S- or Scr-oligonucleotide treatment. However, the responses to norepinephrine (0.3 micromol/l), the NO donor sodium nitroprusside (1 micromol/l), and the K(Ca) channel activator NS1619 (100 micromol/l) were unaltered. As treatment, which specifically targeted the endothelial layer (as assessed by confocal microscopy), had no inhibitory effect on increases in endothelial calcium to ACh. It is concluded that a CYP2C8/9-related isoform functions as an EDHF synthase in hamster resistance arteries and that a product of this enzyme is an EDHF, or at least an integral part of the signaling cascade leading to EDHF-mediated responses.-Bolz, S.-S., Fisslthaler, B., Pieperhoff, S., de Wit, C., Fleming, I., Busse, R., Pohl, U. Antisense oligonucleotides against cytochrome P450 2C8 attenuate EDHF-mediated Ca(2+) changes and dilation in isolated resistance arteries.     

Selected Contribution: Aging impairs nitric oxide and prostacyclin mediation of endothelium-dependent dilation in soleus feed arteries.

We tested the hypothesis that endothelium-dependent dilation in soleus muscle feed arteries (SFA) is impaired by aging due to attenuated nitric oxide (NO)-mediated vasodilation. SFA were isolated from young (4 mo) and old (24 mo) male Fischer 344 rats and cannulated with two glass micropipettes for examination of endothelium-dependent [flow or acetylcholine (ACh)] and endothelium-independent [sodium nitroprusside (SNP)] vasodilator function. Flow- and ACh-induced dilation was significantly attenuated by age, whereas dilation to SNP was not compromised. To determine the mechanism(s) by which aging affected dilator responses to flow and ACh, dilation was assessed in the presence of Nomega-nitro-L-arginine (L-NNA; to inhibit NO synthase), indomethacin (Indo; to inhibit cyclooxygenase), and L-NNA + Indo. In the presence of L-NNA, Indo, or L-NNA + Indo, flow-induced dilation was inhibited in young SFA, resulting in a response to flow that was no longer greater than old SFA. In the presence of L-NNA or Indo, ACh-induced dilation was not significantly inhibited in young or old SFA; however, double blockade with L-NNA + Indo inhibited ACh-induced dilation in young SFA such that the response to ACh was no longer greater than old SFA. Collectively, these data indicate that aging impairs vasodilator responses in SFA by attenuating NO- and prostacyclin-mediated, endothelium-dependent, dilation.     

Hydrogen peroxide acts as an EDHF in the piglet pial vasculature in response to bradykinin

We investigated the mechanism of EDHF-mediated dilation to bradykinin (BK) in piglet pial arteries. Topically applied BK (3 micromol/l) induced vasodilation (62 +/- 12%) after the administration of N(omega)-nitro-L-arginine methyl ester (L-NAME) and indomethacin, which was inhibited by endothelial impairment or by the BK(2) receptor antagonist HOE-140 (0.3 micromol/l). Western blotting showed the presence of BK(2) receptors in brain cortex and pial vascular tissue samples. The cytochrome P-450 antagonist miconazole (20 micromol/l) and the lipoxygenase inhibitors baicalein (10 micromol/l) and cinnamyl-3,4-dyhydroxy-alpha-cyanocinnamate (1 micromol/l) failed to reduce the BK-induced dilation. However, the H(2)O(2) scavenger catalase (400 U/ml) abolished the response (from 54 +/- 11 to 0 +/- 2 microm; P < 0.01). The ATP-dependent K(+) (K(ATP)) channel inhibitor glibenclamide (10 micromol/l) had a similar effect as well (from 54 +/- 11 to 16 +/- 5 microm; P < 0.05). Coapplication of the Ca(2+)-dependent K(+) channel inhibitors charybdotoxin (0.1 micromol/l) and apamin (0.5 micromol/l) failed to reduce the response. We conclude that H(2)O(2) mediates the non-nitric oxide-, non-prostanoid-dependent vasorelaxation to BK in the piglet pial vasculature. The response is mediated via BK(2) receptors and the opening of K(ATP) channels.     

Short-term increases in intraluminal pressure reverse age-related decrements in endothelium-dependent dilation in soleus muscle feed arteries.

We tested the hypothesis that short-term increases in intraluminal pressure improve endothelium-dependent dilation and increase endothelial nitric oxide (NO) synthase (eNOS) expression in senescent soleus muscle feed arteries (SFA). SFA isolated from young (4 mo) and old (24 mo) Fischer 344 rats were cannulated and pressurized at 90 (p90) or 130 (p130) cmH(2)O for 4 h. At the end of the 4-h protocol, pressure in p130 SFA was lowered to 90 cmH(2)O for examination of endothelium-dependent (flow- or ACh-induced) vasodilation. Flow- and ACh-induced dilations were blunted in old p90 SFA relative to young p90 SFA. Pretreatment with increased pressure (p130) improved flow- and ACh-induced dilations in old SFA, such that vasodilator responses were similar to those in young SFA. In the presence of N(omega)-nitro-l-arginine (l-NNA) or l-NNA + indomethacin (Indo), flow-induced dilation was inhibited in old p130 SFA, such that the response was not greater than the response in old p90 SFA. In old p130 SFA, ACh-induced dilation was inhibited by l-NNA + Indo (not l-NNA alone). In a separate experiment, SFA were pressurized at 70, 90, 110, or 130 cmH(2)O for 4 h, and eNOS mRNA and protein content were assessed. Increased pressure induced eNOS mRNA expression in young (not old) SFA. eNOS protein content was not altered in young or old SFA. These results indicate that short-term increases in intraluminal pressure improve endothelium-dependent dilation in senescent SFA, in part by enhancing NO bioavailability; however, the beneficial effect was not associated with increased eNOS expression.     

miR-200s Contribute to Interleukin-6 (IL-6)-induced Insulin Resistance in Hepatocytes

By influencing the activity of the PI3K/AKT pathway, IL-6 acts as an important regulator of hepatic insulin resistance. miR-200s have been shown to control growth by regulating PI3K, but the role of miR-200s in the development of hepatic insulin resistance remains unclear. The present study showed that elevated serum concentration of IL-6 is associated with decreased levels of miR-200s, impaired activation of the AKT/glycogen synthase kinase (GSK) pathway, and reduced glycogenesis that occurred in the livers of db/db mice. As shown in the murine NCTC 1469 hepatocytes and the primary hepatocytes treated with 10 ng/ml IL-6 for 24 h and in 12-week-old male C57BL/6J mice injected with 16 μg/ml IL-6 by pumps for 7 days, IL-6 administration induced insulin resistance through down-regulation of miR-200s. Moreover, IL-6 treatment inhibited the phosphorylation of AKT and GSK and decreased the glycogenesis. The effects of IL-6 could be diminished by suppression of FOG2 expression. We concluded that IL-6 treatment may impair the activities of the PI3K/AKT/GSK pathway and inhibit the synthesis of glycogen, perhaps via down-regulating miR-200s while augmenting FOG2 expression.     

Peroxynitrite reduces the endothelium-derived hyperpolarizing factor component of coronary flow-mediated dilation in PECAM-1-knockout mice

Platelet endothelial cell adhesion molecule 1 (PECAM-1) is capable of transducing signals in endothelial cells exposed to shear; however, the biological consequences of this signal transduction are unknown. Because shear stress elicits flow-mediated dilation (FMD), we examined whether steady-state FMD in mouse coronary arteries (MCAs) is affected in the PECAM-1 knockout (KO) mouse. MCAs were isolated from wild-type (WT) or KO mice and prepared for videomicroscopy, histofluorescence, Western blotting, and immunohistochemistry. FMD was examined in the absence and presence of N(omega)-nitro-l-arginine methyl ester (l-NAME) and l-NAME+indomethacin (INDO). FMD was reduced in KO relative to WT MCAs, but the l-NAME-inhibitable portion of FMD was similar between the two. The INDO-sensitive component of FMD was diminished in KO MCAs. In contrast, the residual component of dilation, presumably because of endothelium-derived hyperpolarizing factor (EDHF), was abolished in KO MCAs. Histofluorescence showed relatively more superoxide (O2-.; oxy-ethidium fluorescence) and peroxide production (dihydrochlorofluorescene fluoresecence) in KO MCAs at rest. Flow augmented O2-. and peroxide production in WT MCAs but had little effect on KO MCAs. Enhanced nitric oxide generation was observed in arteries from KO mice, accompanied with increased eNOS S1177 phosphorylation. In vessels from KO mice, treatment with ebselen decreased peroxynitrite (ONOO-) formation and improved the reduced FMD, largely due to restoration of the presumed EDHF component. These results suggest that PECAM-1 is necessary for normal FMD in the mouse coronary circulation. In the absence of this adhesion and signaling molecule, ONOO- production is increased concomitant with a reduction in both the EDHF and INDO-sensitive components of FMD.     

Adiponectin abates diabetes-induced endothelial dysfunction by suppressing oxidative stress, adhesion molecules, and inflammation in type 2 diabetic mice

Adiponectin (APN) can confer protection against metabolism-related illnesses in organs such as fat, the liver, and skeletal muscle. However, it is unclear whether APN improves endothelial-dependent nitric oxide-mediated vasodilation in type 2 diabetes and, if so, by what mechanism. We tested whether exogenous APN delivery improves endothelial function in type 2 diabetic mice and explored the mechanisms underlying the observed improvement. To test the hypothesis, we injected adenovirus APN (Ad-APN) or adenovirus β-galactosidase (Ad-βgal; control virus) via the tail vein in control (m Lepr(db)) and diabetic (Lepr(db); db/db) mice and studied vascular function of the aorta ex vivo. Ad-APN improved endothelial-dependent vasodilation in db/db mice compared with Ad-βgal, whereas Ad-APN had no further improvement on endothelial function in control mice. This improvement was completely inhibited by a nitric oxide synthase inhibitor (N(G)-nitro-l-arginine methyl ester). Serum triglyceride and total cholesterol levels were increased in db/db mice, and Ad-APN significantly reduced triglyceride levels but not total cholesterol levels. Immunoblot results showed that interferon-γ, gp91(phox), and nitrotyrosine were markedly increased in the aorta of db/db mice. Ad-APN treatment decreased the expression of these proteins. In addition, mRNA expression of TNF-α, IL-6, and ICAM-1 was elevated in db/db mice, and Ad-APN treatment decreased these expressions in the aorta. Our findings suggest that APN may contribute to an increase in nitric oxide bioavailability by decreasing superoxide production as well as by inhibiting inflammation and adhesion molecules in the aorta in type 2 diabetic mice.     

Aging associated with mild dyslipidemia reveals that COX-2 preserves dilation despite endothelial dysfunction

The endothelial function declines with age, and dyslipidemia (DL) has been shown to hasten this process by favoring the generation of reactive oxygen species (ROS). Cyclooxygenase-2 (COX-2) can be induced by ROS, but its contribution to the regulation of the endothelial function is unknown. Since COX-2 inhibitors may be deleterious to the cardiovascular system, we hypothesized that DL leads to ROS-dependent endothelial damage and a protective upregulation of COX-2. Dilations to acetylcholine (ACh) of renal arteries isolated from 3-, 6-, and 12-mo-old wild-type (WT) and DL mice expressing the human ApoB-100 were recorded with or without COX inhibitors and the antioxidant N-acetyl-l-cystein (NAC). Nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) were inhibited using N(omega)-nitro-l-arginine (l-NNA) and a depolarizing solution, respectively. In WT mice, the dilation to ACh declined at 12 mo but was insensitive to COX-1/2 inhibition alone or with NAC. DL led to an early endothelial dysfunction at 6 mo, normalized, however, by NAC. At 12 mo, vascular sensitivity to ACh was further reduced by DL. At this age, selective COX-2 inhibition reduced the dilation, whereas addition of NAC improved it. In 3- and 6-mo-old WT mice, l-NNA significantly reduced the dilation, whereas it limited the dilation only in 3-mo-old DL mice. EDHF-dependent dilation remains identical in both groups. These data suggest that COX-2 activity confers endothelium-dependent vasodilatory function in aged DL mice in the face of a pro-oxidative environment. Upregulation of this pathway compensates for the early loss of the contribution of NO in DL mice.     

Neuronal nitric oxide synthase-derived hydrogen peroxide is a major endothelium-dependent relaxing factor

Endothelium-dependent vasorelaxation in large vessels is mainly attributed to Nomega-nitro-L-arginine methyl ester (L-NAME)-sensitive endothelial nitric oxide (NO) synthase (eNOS)-derived NO production. Endothelium-derived hyperpolarizing factor (EDHF) is the component of endothelium-dependent relaxations that resists full blockade of NO synthases (NOS) and cyclooxygenases. H2O2 has been proposed as an EDHF in resistance vessels. In this work we propose that in mice aorta neuronal (n)NOS-derived H2O2 accounts for a large proportion of endothelium-dependent ACh-induced relaxation. In mice aorta rings, ACh-induced relaxation was inhibited by L-NAME and Nomega-nitro-L-arginine (L-NNA), two nonselective inhibitors of NOS, and attenuated by selective inhibition of nNOS with L-ArgNO2-L-Dbu-NH2 2TFA (L-ArgNO2-L-Dbu) and 1-(2-trifluoromethylphehyl)imidazole (TRIM). The relaxation induced by ACh was associated with enhanced H2O2 production in endothelial cells that was prevented by the addition of L-NAME, L-NNA, L-ArgNO2-L-Dbu, TRIM, and removal of the endothelium. The addition of catalase, an enzyme that degrades H2O2, reduced ACh-dependent relaxation and abolished ACh-induced H2O2 production. RT-PCR experiments showed the presence of mRNA for eNOS and nNOS but not inducible NOS in mice aorta. The constitutive expression of nNOS was confirmed by Western blot analysis in endothelium-containing vessels but not in endothelium-denuded vessels. Immunohistochemistry data confirmed the localization of nNOS in the vascular endothelium. Antisense knockdown of nNOS decreased both ACh-dependent relaxation and ACh-induced H2O2 production. Antisense knockdown of eNOS decreased ACh-induced relaxation but not H2O2 production. Residual relaxation in eNOS knockdown mouse aorta was further inhibited by the selective inhibition of nNOS with L-ArgNO2-L-Dbu. In conclusion, these results show that nNOS is constitutively expressed in the endothelium of mouse aorta and that nNOS-derived H2O2 is a major endothelium-dependent relaxing factor. Hence, in the mouse aorta, the effects of nonselective NOS inhibitors cannot be solely ascribed to NO release and action without considering the coparticipation of H2O2 in mediating vasodilatation.     

Implication of adenosine A2A receptors in hypotension-induced vasodilation and cerebral blood flow autoregulation in rat pial arteries

This study aimed to evaluate the role for adenosine A2A receptors in the autoregulatory vasodilation to hypotension in relation with cerebral blood flow (CBF) autoregulation in rat pial arteries. Changes in pial artery diameters were observed directly through a closed cranial window. Vasodilation induced by adenosine was markedly suppressed by ZM 241385 (1 micromol/l, A2A antagonist) and alloxazine (1 micromol/l, A2B antagonist), but not by 8-cyclopentyltheophylline (CPT, 1 micromol/l, A1 antagonist). CGS-21680-induced vasodilation was more strongly inhibited by ZM 241385 (25.3-fold; P<0.05) than by alloxazine. In contrast, 5'-N-ethylcarboxamido-adenosine (NECA)-induced vasodilation was more prominently suppressed by alloxazine (12.0-fold; P<0.001) than by ZM 241385. The autoregulatory vasodilation in response to acute hypotension of the pial arteries was significantly suppressed by ZM 241385, but not by CPT and alloxazine. Consistent with this finding, the lower limit of CBF autoregulation significantly shifted to a higher blood pressure by 1 micromol/l of ZM 241385 (53.0+/-3.9 mm Hg to 69.2+/-2.9 mm Hg, P<0.01) and 10 micromol/l of glibenclamide (54.7+/-6.5 mm Hg to 77.9+/-4.2 mm Hg, P<0.001), but not by CPT and alloxazine. Thus, it is suggested that adenosine-induced vasodilation of the rat pial artery is mediated via activation of adenosine A2A and A2B receptors, but not by A1 subtype, and activation of adenosine A2A receptor preferentially contributes to the autoregulatory vasodilation via activation of ATP-sensitive K+ channels in response to hypotension and maintenance of CBF autoregulation.