NO-mediated activation of KATP channels contributes to cutaneous thermal hyperemia in young adults.

Local skin heating to 42°C causes cutaneous thermal hyperemia largely via nitric oxide (NO) synthase (NOS)-related mechanisms. We assessed the hypothesis that ATP-sensitive K+ (KATP) channels interact with NOS to mediate cutaneous thermal hyperemia. In 13 young adults (6 women, 7 men), cutaneous vascular conductance (CVC) was measured at four intradermal microdialysis sites that were continuously perfused with 1) lactated Ringer solution (control), 2) 5 mM glibenclamide (KATP channel blocker), 3) 20 mM NG-nitro-l-arginine methyl ester (NOS inhibitor), or 4) a combination of KATP channel blocker and NOS inhibitor. Local skin heating to 42°C was administered at all four treatment sites to elicit cutaneous thermal hyperemia. Thirty minutes after the local heating, 1.25 mM pinacidil (KATP channel opener) and subsequently 25 mM sodium nitroprusside (NO donor) were administered to three of the four sites (each 25-30 min). The local heating-induced prolonged elevation in CVC was attenuated by glibenclamide (19%), but the transient initial peak was not. However, glibenclamide had no effect on the prolonged elevation in CVC in the presence of NOS inhibition. Pinacidil caused an elevation in CVC, but this response was abolished at the glibenclamide-treated skin site, demonstrating its effectiveness as a KATP channel blocker. The pinacidil-induced increase in CVC was unaffected by NOS inhibition, whereas the increase in CVC elicited by sodium nitroprusside was partly (15%) inhibited by glibenclamide. In summary, we showed an interactive effect of KATP channels and NOS for the plateau of cutaneous thermal hyperemia. This interplay may reflect a vascular smooth muscle cell KATP channel activation by NO.

Abnormal neurovascular coupling as a cause of excess cerebral vasodilation in familial migraine.

AIMS: Acute migraine attack in familial hemiplegic migraine type 2 (FHM2) patients is characterized by sequential hypo- and hyperperfusion. FHM2 is associated with mutations in the Na, K-ATPase α2 isoform. Heterozygous mice bearing one of these mutations (α2+/G301R mice) were shown to have elevated cerebrovascular tone and, thus, hypoperfusion that might lead to elevated concentrations of local metabolites. We hypothesize that these α2+/G301R mice also have increased cerebrovascular hyperaemic responses to these local metabolites leading to hyperperfusion in the affected part of the brain. METHODS AND RESULTS: Neurovascular coupling was compared in α2+/G301R and matching wild-type (WT) mice using Laser Speckle Contrast Imaging. In brain slices, parenchymal arteriole diameter and intracellular calcium changes in neuronal tissue, astrocytic endfeet, and smooth muscle cells in response to neuronal excitation were assessed. Wall tension and smooth muscle membrane potential were measured in isolated middle cerebral arteries. Quantitative polymerase chain reaction, western blot, and immunohistochemistry were used to assess the molecular background underlying the functional changes. Whisker stimulation induced larger increase in blood perfusion, i.e. hyperaemic response, of the somatosensory cortex of α2+/G301R than WT mice. Neuronal excitation was associated with larger parenchymal arteriole dilation in brain slices from α2+/G301R than WT mice. These hyperaemic responses in vivo and ex vivo were inhibited by BaCl2, suggesting involvement of inward-rectifying K+ channels (Kir). Relaxation to elevated bath K+ was larger in arteries from α2+/G301R compared to WT mice. This difference was endothelium-dependent. Endothelial Kir2.1 channel expression was higher in arteries from α2+/G301R mice. No sex difference in functional responses and Kir2.1 expression was found. CONCLUSION: This study suggests that an abnormally high cerebrovascular hyperaemic response in α2+/G301R mice is a result of increased endothelial Kir2.1 channel expression. This may be initiated by vasospasm-induced accumulation of local metabolites and underlie the hyperperfusion seen in FHM2 patients during migraine attack.

Effect of Soluble Epoxide Hydrolase on the Modulation of Coronary Reactive Hyperemia: Role of Oxylipins and PPARγ.

Coronary reactive hyperemia (CRH) is a physiological response to ischemic insult that prevents the potential harm associated with an interruption of blood supply. The relationship between the pharmacologic inhibition of soluble epoxide hydrolase (sEH) and CRH response to a brief ischemia is not known. sEH is involved in the main catabolic pathway of epoxyeicosatrienoic acids (EETs), which are converted into dihydroxyeicosatrienoic acids (DHETs). EETs protect against ischemia/reperfusion injury and have numerous beneficial physiological effects. We hypothesized that inhibition of sEH by t-AUCB enhances CRH in isolated mouse hearts through changing the oxylipin profiles, including an increase in EETs/DHETs ratio. Compared to controls, t-AUCB-treated mice had increased CRH, including repayment volume (RV), repayment duration, and repayment/debt ratio (p < 0.05). Treatment with t-AUCB significantly changed oxylipin profiles, including an increase in EET/DHET ratio, increase in EpOME/DiHOME ratio, increase in the levels of HODEs, decrease in the levels of mid-chain HETEs, and decrease in prostanoids (p < 0.05). Treatment with MS-PPOH (CYP epoxygenase inhibitor) reduced CRH, including RV (p < 0.05). Involvement of PPARγ in the modulation of CRH was demonstrated using a PPARγ-antagonist (T0070907) and a PPARγ-agonist (rosiglitazone). T0070907 reduced CRH (p < 0.05), whereas rosiglitazone enhanced CRH (p < 0.05) in isolated mouse hearts compared to the non-treated. These data demonstrate that sEH inhibition enhances, whereas CYP epoxygenases-inhibition attenuates CRH, PPARγ mediate CRH downstream of the CYP epoxygenases-EET pathway, and the changes in oxylipin profiles associated with sEH-inhibition collectively contributed to the enhanced CRH.

Purinergic glio-endothelial coupling during neuronal activity: role of P2Y1 receptors and eNOS in functional hyperemia in the mouse somatosensory cortex.

Impairment of moment-to-moment adjustment of cerebral blood flow (CBF) via neurovascular coupling is thought to play a critical role in the genesis of cognitive impairment associated with aging and pathological conditions associated with accelerated cerebromicrovascular aging (e.g., hypertension, obesity). Although previous studies demonstrate that endothelial dysfunction plays a critical role in neurovascular uncoupling in these conditions, the role of endothelial NO mediation in neurovascular coupling responses is not well understood. To establish the link between endothelial function and functional hyperemia, neurovascular coupling responses were studied in mutant mice overexpressing or deficient in endothelial NO synthase (eNOS), and the role of P2Y1 receptors in purinergic glioendothelial coupling was assessed. We found that genetic depletion of eNOS (eNOS(-/-)) and pharmacological inhibition of NO synthesis significantly decreased the CBF responses in the somatosensory cortex evoked by whisker stimulation and by administration of ATP. Overexpression of eNOS enhanced NO mediation of functional hyperemia. In control mice, the selective and potent P2Y1 receptor antagonist MRS2179 attenuated both whisker stimulation-induced and ATP-mediated CBF responses, whereas, in eNOS(-/-) mice, the inhibitory effects of MRS2179 were blunted. Collectively, our findings provide additional evidence for purinergic glio-endothelial coupling during neuronal activity, highlighting the role of ATP-mediated activation of eNOS via P2Y1 receptors in functional hyperemia.

Targeting Adenosine Monophosphate-Activated Protein Kinase by Metformin Adjusts Post-Ischemic Hyperemia and Extracellular Neuronal Discharge in Transient Global Cerebral Ischemia.

OBJECTIVE: I/R and its subsequent reactive hyperemia results in different adverse effects such as brain edema and BBB disruption. AMPK activation has been perceived as one of the target factors for I/R treatment. We investigated the effect of Met (an AMPK activator) on some physiological parameters including vascular responses, hyperemia, BBB disruption, and electrophysiological activity following tGCI. METHODS: Rats were pretreated with Met for two weeks and CC was administered half an hour before tGCI. Brain vascular responses, hyperemia, BBB disruption, and electrophysiological activity were evaluated following the ischemia. RESULTS: Met attenuated BBB disruption and reactive hyperemia in tGCI rats compared with the untreated I/R rats (p < 0.001). Met administration along with CC in the ischemic rats reversed the beneficial effects of Met on BBB disruption and reactive hyperemia (p < 0.001). Electrophysiological records indicated that Met increased spike rates in the ischemic rats comparing with I/R rats (p < 0.001), whereas, CC administration blocked the beneficial effects of Met on the neuronal discharges (p < 0.05). CONCLUSION: We established a regulatory role for AMPK in vascular and electrophysiological responses to tGCI. Studies are ongoing to determine if activation of AMPK in the reperfusion period would offer similar protection.

Regulatory mechanism of the gastric hyperemic response following barrier disruption: roles of cyclooxygenase-1, the prostaglandin E2/EP1 receptor and sensory neurons.

We herein reviewed the mechanism underlying the gastric hyperemic response following barrier disruption, with a focus on cyclooxygenase (COX) isozymes, prostaglandin (PG) E2, and capsaicin-sensitive afferent neurons. Mucosal damage was induced by exposing the stomach to 20 mM taurocholate (TC) with 50 mM HCl. The TC treatment disrupted surface epithelial cells, and then increased acid back-diffusion and mucosal blood flow (GMBF) in the stomachs of rats or wild-type mice. This hyperemic response in the rat stomach was inhibited by indomethacin without affecting acid back-diffusion, which resulted in the aggravation of lesions. The effect of indomethacin was mimicked by loxoprofen and the selective COX-1 inhibitor, SC-560, but not by the selective COX-2 inhibitor, celecoxib. The GMBF responses induced by TC were similarly observed in the stomachs of wild-type mice and EP3 receptor knockout mice, but not in mice lacking the EP1 receptor or pretreated with an EP1 antagonist. The increase in the GMBF response associated with acid back-diffusion after the TC treatment was also inhibited by the chemical ablation of capsaicin-sensitive afferent neurons, but not capsazepine, a TRPV1 antagonist. Thus, endogenous PGE2 produced by COX-1 plays a role in the gastric hyperemic response following barrier disruption of the stomach by interacting with capsaicin-sensitive afferent neurons, mainly through EP1 receptors, and facilitating the GMBF response to acid back-diffusion. These findings have also contributed to a deeper understanding of mucosal defensive mechanisms following barrier disruption and the development of new strategies for the treatment of gastrointestinal diseases.

Reactive hyperemia occurs via activation of inwardly rectifying potassium channels and Na+/K+-ATPase in humans.

RATIONALE: Reactive hyperemia (RH) in the forearm circulation is an important marker of cardiovascular health, yet the underlying vasodilator signaling pathways are controversial and thus remain unclear. OBJECTIVE: We hypothesized that RH occurs via activation of inwardly rectifying potassium (KIR) channels and Na(+)/K(+)-ATPase and is largely independent of the combined production of the endothelial autocoids nitric oxide (NO) and prostaglandins in young healthy humans. METHODS AND RESULTS: In 24 (23±1 years) subjects, we performed RH trials by measuring forearm blood flow (FBF; venous occlusion plethysmography) after 5 minutes of arterial occlusion. In protocol 1, we studied 2 groups of 8 subjects and assessed RH in the following conditions. For group 1, we studied control (saline), KIR channel inhibition (BaCl2), combined inhibition of KIR channels and Na(+)/K(+)-ATPase (BaCl2 and ouabain, respectively), and combined inhibition of KIR channels, Na(+)/K(+)-ATPase, NO, and prostaglandins (BaCl2, ouabain, L-NMMA [N(G)-monomethyl-L-arginine] and ketorolac, respectively). Group 2 received ouabain rather than BaCl2 in the second trial. In protocol 2 (n=8), the following 3 RH trials were performed: control; L-NMMA plus ketorolac; and L-NMMA plus ketorolac plus BaCl2 plus ouabain. All infusions were intra-arterial (brachial). Compared with control, BaCl2 significantly reduced peak FBF (-50±6%; P<0.05), whereas ouabain and L-NMMA plus ketorolac did not. Total FBF (area under the curve) was attenuated by BaCl2 (-61±3%) and ouabain (-44±12%) alone, and this effect was enhanced when combined (-87±4%), nearly abolishing RH. L-NMMA plus ketorolac did not impact total RH FBF before or after administration of BaCl2 plus ouabain. CONCLUSIONS: Activation of KIR channels is the primary determinant of peak RH, whereas activation of both KIR channels and Na(+)/K(+)-ATPase explains nearly all of the total (AUC) RH in humans.

Effect of human 15-lipoxygenase-1 metabolites on vascular function in mouse mesenteric arteries and hearts.

Lipoxygenases regulate vascular function by metabolizing arachidonic acid (AA) to dilator eicosanoids. Previously, we showed that endothelium-targeted adenoviral vector-mediated gene transfer of the human 15-lipoxygenase-1 (h15-LO-1) enhances arterial relaxation through the production of vasodilatory hydroxyepoxyeicosatrienoic acid (HEETA) and trihydroxyeicosatrienoic acid (THETA) metabolites. To further define this function, a transgenic (Tg) mouse line that overexpresses h15-LO-1 was studied. Western blot, immunohistochemistry and RT-PCR results confirmed expression of 15-LO-1 transgene in tissues, especially high quantity in coronary arterial wall, of Tg mice. Reverse-phase HPLC analysis of [(14)C]-AA metabolites in heart tissues revealed enhanced 15-HETE synthesis in Tg vs. WT mice. Among the 15-LO-1 metabolites, 15-HETE, erythro-13-H-14,15-EETA, and 11(R),12(S),15(S)-THETA relaxed the mouse mesenteric arteries to the greatest extent. The presence of h15-LO-1 increased acetylcholine- and AA-mediated relaxation in mesenteric arteries of Tg mice compared to WT mice. 15-LO-1 was most abundant in the heart; therefore, we used the Langendorff heart model to test the hypothesis that elevated 15-LO-1 levels would increase coronary flow following a short ischemia episode. Both peak flow and excess flow of reperfused hearts were significantly elevated in hearts from Tg compared to WT mice being 2.03 and 3.22 times greater, respectively. These results indicate that h15-LO-1-derived metabolites are highly vasoactive and may play a critical role in regulating coronary blood flow.

Sympathetic activation increases NO release from eNOS but neither eNOS nor nNOS play an essential role in exercise hyperemia in the human forearm.

Nitric oxide (NO) release from endothelial NO synthase (eNOS) and/or neuronal NO synthase (nNOS) could be modulated by sympathetic nerve activity and contribute to increased blood flow after exercise. We examined the effects of brachial-arterial infusion of the nNOS selective inhibitor S-methyl-l-thiocitrulline (SMTC) and the nonselective NOS inhibitor N(G)-monomethyl-l-arginine (l-NMMA) on forearm arm blood flow at rest, during sympathetic activation by lower body negative pressure, and during lower body negative pressure immediately after handgrip exercise. Reduction in forearm blood flow by lower body negative pressure during infusion of SMTC was not significantly different from that during vehicle (-28.5 ± 4.02 vs. -34.1 ± 2.96%, respectively; P = 0.32; n = 8). However, l-NMMA augmented the reduction in forearm blood flow by lower body negative pressure (-44.2 ± 3.53 vs. -23.4 ± 5.71%; n = 8; P < 0.01). When lower body negative pressure was continued after handgrip exercise, there was no significant effect of either l-NMMA or SMTC on forearm blood flow immediately after low-intensity exercise (P = 0.91 and P = 0.44 for l-NMMA vs. saline and SMTC vs. saline, respectively; each n = 10) or high-intensity exercise (P = 0.46 and P = 0.68 for l-NMMA vs. saline and SMTC vs. saline, respectively; each n = 10). These results suggest that sympathetic activation increases NO release from eNOS, attenuating vasoconstriction. Dysfunction of eNOS could augment vasoconstrictor and blood pressure responses to sympathetic activation. However, neither eNOS nor nNOS plays an essential role in postexercise hyperaemia, even in the presence of increased sympathetic activation.

Control of microvascular PO2 kinetics following onset of muscle contractions: role for AMPK.

The microvascular partial pressure of oxygen (Pmv(o(2))) kinetics following the onset of exercise reflects the relationship between muscle O(2) delivery and uptake (Vo(2)). Although AMP-activated protein kinase (AMPK) is known as a regulator of mitochondria and nitric oxide metabolism, it is unclear whether the dynamic balance of O(2) delivery and Vo(2) at exercise onset is dependent on AMPK activation level. We used transgenic mice with muscle-specific AMPK dominant-negative (AMPK-DN) to investigate a role for skeletal muscle AMPK on Pmv(o(2)) kinetics following onset of muscle contractions. Phosphorescence quenching techniques were used to measure Pmv(o(2)) at rest and across the transition to twitch (1 Hz) and tetanic (100 Hz, 3-5 V, 4-ms pulse duration, stimulus duration of 100 ms every 1 s for 1 min) contractions in gastrocnemius muscles (each group n = 6) of AMPK-DN mice and wild-type littermates (WT) under isoflurane anesthesia with 100% inspired O(2) to avoid hypoxemia. Baseline Pmv(o(2)) before contractions was not different between groups (P > 0.05). Both muscle contraction conditions exhibited a delay followed by an exponential decrease in Pmv(o(2)). However, compared with WT, AMPK-DN demonstrated 1) prolongation of the time delay before Pmv(o(2)) began to decline (1 Hz: WT, 3.2 ± 0.5 s; AMPK-DN, 6.5 ± 0.4 s; 100 Hz: WT, 4.4 ± 1.0 s; AMPK-DN, 6.5 ± 1.4 s; P < 0.05), 2) a faster response time (i.e., time constant; 1 Hz: WT, 19.4 ± 3.9 s; AMPK-DN, 12.4 ± 2.6 s; 100 Hz: WT, 15.1 ± 2.2 s; AMPK-DN, 9.0 ± 1.7 s; P < 0.05). These findings are consistent with the presence of substantial mitochondrial and microvascular dysfunction in AMPK-DN mice, which likely slows O(2) consumption kinetics (i.e., oxidative phosphorylation response) and impairs the hyperemic response at the onset of contractions thereby sowing the seeds for exercise intolerance.

Cutaneous constitutive nitric oxide synthase activation in postural tachycardia syndrome with splanchnic hyperemia.

Models of microgravity are linked to excessive constitutive nitric oxide (NO) synthase (NOS), splanchnic vasodilation, and orthostatic intolerance. Normal-flow postural tachycardia syndrome (POTS) is a form of chronic orthostatic intolerance associated with splanchnic hyperemia. To test the hypothesis that there is excessive constitutive NOS in POTS, we determined whether cutaneous microvascular neuronal NO and endothelial NO are increased. We performed two sets of experiments in POTS and control subjects aged 21.4 ± 2 yr. We used laser-Doppler flowmetry to measure the cutaneous response to local heating as an indicator of bioavailable neuronal NO. To test for bioavailable endothelial NO, we infused intradermal acetylcholine through intradermal microdialysis catheters and used the selective neuronal NOS inhibitor l-N(ω)-nitroarginine-2,4-L-diamino-butyric amide (N(ω), 10 mM), the selective inducible NOS inhibitor aminoguanidine (10 mM), the nonspecific NOS inhibitor nitro-l-arginine (NLA, 10 mM), or Ringer solution. The acetylcholine dose response and the NO-dependent plateau of the local heating response were increased in POTS compared with those in control subjects. The local heating plateau was significantly higher, 98 ± 1%maximum cutaneous vascular conductance (%CVC(max)) in POTS compared with 88 ± 2%CVC(max) in control subjects but decreased to the same level with N(ω) (46 ± 5%CVC(max) in POTS compared with 49 ± 4%CVC(max) in control) or with NLA (45 ± 3%CVC(max) in POTS compared with 47 ± 4%CVC(max) in control). Only NLA blunted the acetylcholine dose response, indicating that NO produced by endothelial NOS was released by acetylcholine. Aminoguanidine was without effect. This is consistent with increased endothelial and neuronal NOS activity in normal-flow POTS.

Role of endothelial nitric oxide synthase in the development of portal hypertension in the carbon tetrachloride-induced liver fibrosis model.

Portal hypertension (PHT) is a complication of liver cirrhosis and directly increases mortality and morbidity by increasing the propensity of venous hemorrhage. There are two main underlying causations for PHT, increased hepatic resistance and systemic hyperdynamic circulation. Both are related to localized aberrations in endothelial nitric oxide synthase (eNOS) function and NO biosynthesis. This study investigates the importance of eNOS and systemic hyperdynamic-associated hyperemia to better understand the pathophysiology of PHT. Wild-type and eNOS(-/-) mice were given the hepatotoxin CCl(4) for 4-12 wk. Hepatic fibrosis was determined histologically following collagen staining. Portal venous pressure, hepatic resistance, and hyperemia were determined by measuring splenic pulp pressure (SPP), hepatic portal-venous perfusion pressure (HPVPP), abdominal aortic flow (Qao), and portal venous flow (Qpv). Hepatic fibrosis developed equally in wild-type and eNOS(-/-) CCl(4)-exposed mice. SPP, Qao, and Qpv increased rapidly in wild-type CCl(4)-exposed mice, but HPVPP did not. In eNOS(-/-) CCl(4) mice, Qao was not increased, SPP was partially increased, and HPVPP and Qpv were increased nonsignificantly. We concluded that the systemic hyperemia component of hyperdynamic circulation is eNOS dependent and precedes increased changes in hepatic resistance. Alternative mechanisms, possibly involving cyclooxygenase, may contribute. eNOS maintains normal hepatic resistance following CCl(4)-induced fibrosis. Consequently, increased portal pressure following chronic CCl(4) exposure is linked to hyperdynamic circulation in wild-type mice and increased hepatic resistance in eNOS(-/-) mice.

Role of Cu,Zn-SOD in the synthesis of endogenous vasodilator hydrogen peroxide during reactive hyperemia in mouse mesenteric microcirculation in vivo.

We have recently demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor and that endothelial Cu/Zn-superoxide dismutase (SOD) plays an important role in the synthesis of endogenous H2O2 in both animals and humans. We examined whether SOD plays a role in the synthesis of endogenous H2O2 during in vivo reactive hyperemia (RH), an important regulatory mechanism. Mesenteric arterioles from wild-type and Cu,Zn-SOD(-/-) mice were continuously observed by a pencil-type charge-coupled device (CCD) intravital microscope during RH (reperfusion after 20 and 60 s of mesenteric artery occlusion) in the cyclooxygenase blockade under the following four conditions: control, catalase alone, N(G)-monomethyl-L-arginine (L-NMMA) alone, and L-NMMA + catalase. Vasodilatation during RH was significantly decreased by catalase or L-NMMA alone and was almost completely inhibited by L-NMMA + catalase in wild-type mice, whereas it was inhibited by L-NMMA and L-NMMA + catalase in the Cu,Zn-SOD(-/-) mice. RH-induced increase in blood flow after L-NMMA was significantly increased in the wild-type mice, whereas it was significantly reduced in the Cu,Zn-SOD(-/-) mice. In mesenteric arterioles of the Cu,Zn-SOD(-/-) mice, Tempol, an SOD mimetic, significantly increased the ACh-induced vasodilatation, and the enhancing effect of Tempol was decreased by catalase. Vascular H(2)O(2) production by fluorescent microscopy in mesenteric arterioles after RH was significantly increased in response to ACh in wild-type mice but markedly impaired in Cu,Zn-SOD(-/-) mice. Endothelial Cu,Zn-SOD plays an important role in the synthesis of endogenous H(2)O(2) that contributes to RH in mouse mesenteric smaller arterioles.

Systemic nitric oxide synthase inhibition improves coronary flow reserve to adenosine in patients with significant stenoses.

We studied the impact of systemic infusion of the nitric oxide synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (L-NMMA) on coronary flow reserve (CFR) in patients with coronary artery disease (CAD). We have previously demonstrated that CFR to adenosine was significantly increased after systemic infusion of L-NMMA in normal volunteers but not in recently transplanted denervated hearts. At baseline, myocardial blood flow (MBF; ml x min(-1) x g(-1)) was measured at rest and during intravenous administration of adenosine (140 microg x kg(-1) x min(-1)) in 10 controls (47 +/- 5 yr) and 10 CAD patients (58 +/- 8 yr; P < 0.01 vs. controls) using positron emission tomography and (15)O-labeled water. Both MBF measurements were repeated during intravenous infusion of 10 mg/kg L-NMMA. CFR was calculated as the ratio of MBF during adenosine to MBF at rest. CFR was significantly higher in healthy volunteers than in CAD patients and increased significantly after L-NMMA in controls (4.00 +/- 1.10 to 6.15 +/- 1.35; P < 0.0001) and in patients, both in territories subtended by stenotic coronary arteries (>70% luminal diameter; 2.06 +/- 1.13 to 3.21 +/- 1.07; P < 0.01) and in remote segments (3.20 +/- 1.23 to 3.92 +/- 1.62; P < 0.05). In conclusion, CFR can be significantly increased in CAD by a systemic infusion of L-NMMA. Similarly to our previous findings in normal volunteers, this suggests that adenosine-induced hyperemia in CAD patients is constrained by a mechanism that can be relieved by systemic NOS inhibition with L-NMMA.

Cyclooxygenase and nitric oxide synthase dependence of cutaneous reactive hyperemia in humans.

We tested the hypothesis that cyclooxygenases (COXs) or COX products inhibit nitric oxide (NO) synthesis and thereby mask potential effects of NO on reactive hyperemia in the cutaneous circulation. We performed laser-Doppler flowmetry (LDF) with intradermal microdialysis in 12 healthy volunteers aged 19-25 yr. LDF was expressed as the percent cutaneous vascular conduction (%CVC) or as the maximum %CVC (%CVC(max)) where CVC is LDF/mean arterial pressure. We tested the effects of the nonisoform-specific NO synthase inhibitor nitro-L-arginine (NLA, 10 mM), the nonspecific COX inhibitor ketorolac (Keto, 10 mM), combined NLA + Keto, and NLA + sodium nitroprusside (SNP, 28 mM) on baseline and reactive hyperemia flow parameters. We also examined the effects of isoproterenol, a beta-adrenergic agonist that causes prostaglandin-independent vasodilation to correct for the increase in baseline flow caused by Keto. When delivered directly into the intradermal space, Keto greatly augments all aspects of the laser-Doppler flow response to reactive hyperemia: peak reactive hyperemic flow increased from 41 +/- 5 to 77 +/- 7%CVC(max), time to peak flow increased from 17 +/- 3 to 56 +/- 24 s, the area under the reactive hyperemic curve increased from 1,417 +/- 326 to 3,376 +/- 876%CVC(max).s, and the time constant for the decay of peak flow increased from 100 +/- 23 to 821 +/- 311 s. NLA greatly attenuates the Keto response despite exerting no effects on baseline LDF or on reactive hyperemia when given alone. Low-dose NLA + SNP duplicates the Keto response. Isoproterenol increased baseline and peak reactive flow. These results suggest that COX inhibition unmasks NO dependence of reactive hyperemia in human cutaneous circulation.

NAD(P)H oxidase modulates angiogenesis and the development of portosystemic collaterals and splanchnic hyperaemia in portal hypertensive rats.

BACKGROUND: Recent studies have shown the presence of vascular endothelial growth factor (VEGF)-dependent splanchnic angiogenesis in experimental models of portal hypertension, and the role of such neovascularisation on the development of both portosystemic collaterals and hyperdynamic splanchnic circulation. However, the mechanisms modulating angiogenesis in portal hypertension are unknown. Experimental evidence indicates that NAD(P)H oxidase is required for VEGF-induced angiogenesis. Interestingly, we have recently shown that splanchnic NAD(P)H oxidase activity is significantly increased in portal hypertensive rats. Therefore, it could be possible that activated NAD(P)H oxidases modulate angiogenesis in portal hypertension. AIM: To determine the effects of chronic NAD(P)H oxidase inhibition on angiogenesis and splanchnic haemodynamics in portal hypertensive rats. METHODS: Partial portal vein-ligated and sham-operated rats were treated with the NAD(P)H oxidase inhibitor apocynin, or with vehicle for 5 days. Then, the expression of angiogenesis markers (western blotting), the formation of portosystemic collaterals (radioactive microspheres) and the production of superoxide anion (lucigenin-enhanced chemiluminescence) were determined. Mean arterial pressure, portal pressure, and superior mesenteric arterial blood flow and resistance were also measured. RESULTS: In portal hypertensive rats, NAD(P)H oxidase blockade significantly decreased portosystemic collateral formation, and superior mesenteric arterial flow. It also reduced the splanchnic expression of VEGF, VEGF receptor-2 and CD31, and attenuated the increased production of superoxide, compared with vehicle. CONCLUSIONS: NAD(P)H oxidase plays an important role in experimental portal hypertension, modulating splanchnic angiogenesis, the formation of portosystemic collaterals and the development of splanchnic hyperdynamic circulation. These results suggest that NAD(P)H oxidase may represent a new target in the treatment of portal hypertension.

AMPD1 gene polymorphism and the vasodilatory response to ischemia.

Peripheral vasculature resistance can play an important role in affecting blood pressure and the development of cardiovascular disease. A better understanding of the genes that encode vasodilators, such as adenosine, will provide insight into the mechanisms underlying cardiovascular disease. We tested whether the adenosine monophosphate deaminase-1 (AMPD1) C34T gene polymorphism was associated with the vasodilatory response to ischemia in Caucasian females aged 18-35 years. Blood samples (n = 58) were analyzed for the C34T variant and resulted in the following genotype groups: CC (n = 45) and CT (n = 13). Mean blood pressure (MBP), heart rate, and forearm blood flow (FBF) measured by venous occlusion plethysmography were measured at baseline and at 1 (peak FBF), 2 and 3 min of vasodilation during reactive hyperemia following 5 min of arm ischemia. To control for interindividual variability in baseline FBF and forearm vascular resistance (FVR) the percent change in FBF and FVR were calculated for each min. The percent decrease in FVR was significantly greater in the CT compared to the CC genotype group (-40+/-4% vs. -24+/-3%, P = 0.01) during the 2nd min of reactive hyperemia. The percent increase in FBF tended to be greater in the CT compared to the CC genotype group (+69+/-9% vs. +42+/-9%, P = 0.07) during the 2nd min of reactive hyperemia after adjustment for percent body fat. Consistent with previous findings of increased production of adenosine during exercise in individuals carrying a T allele, our findings suggest that the AMPD1 C34T polymorphism is associated with vasodilatory response to ischemia in the peripheral vasculature because individuals with the T allele had a greater vasodilatory response to ischemia.

F0F1 ATP synthase activity is differently modulated by coronary reactive hyperemia before and after ischemic preconditioning in the goat.

The amplitude of coronary reactive hyperemia (CRH), elicited by 15 s of ischemia, is reduced in hearts subjected to 5 min of ischemic preconditioning (IP). F0F1 ATP synthase activity and ATP concentration are also altered by IP. We hypothesized that F0F1 ATP synthase is differently modulated by the inhibitor protein IF(1) during CRH elicited before (CRHnp) and after (CRHprec) IP. Hemodynamic parameters were recorded in 10 anesthetized goats. Myocardial biopsies were obtained before IP (Cnp), during CRHnp, 4 and 6 min after the onset of CRHnp, after IP (Cprec), during CRHprec, and 4 min after CRHprec. F0F1 ATP synthase activity, ATP concentration, and ATP-to-ADP ratio (ATP/ADP) were determined. Compared with CRHnp, IP blunted CRHprec. F0F1 ATP synthase activity transiently increased during CRHnp, decreased 4 min after CRHnp, and returned to control 2 min later; it was lower after IP (Cprec) and did not change during and after CRHprec. All these changes in activity were modulated by IF1. During CRHnp, ATP concentration and ATP/ADP were reduced compared with Cnp and began to rise 6 min thereafter. During Cprec, both parameters were transiently reduced but increased during and after CRHprec. Hence, during CRHnp, F0F1 ATP synthase activity transiently increases and then decreases significantly. The short-lasting inhibition of the enzyme may explain why a few seconds of occlusion do not induce IP. After IP, F0F1 ATP synthase activity is blunted, and it is not affected by a subsequent 15 s of occlusion, which induces a blunted CRHprec. These results suggest that postischemic long-lasting inhibition of F0F1 ATP synthase activity may be a feature of the preconditioned heart. The increase in ATP concentration after preconditioning is in agreement with previous reports of reduced ATP hydrolysis by cytoplasmic ATPases.

Nitric oxide synthase in healthy and inflamed human dental pulp.

Nitric oxide synthase (NOS) plays a significant role in the pathogenesis of pulpitis. In this study, we hypothesized the existence of endothelial (eNOS) and inducible (iNOS) enzyme isoforms in human dental pulp. Extracted third molar pulps were divided into groups based on clinical diagnosis: healthy, hyperemic, and irreversible pulpitis. We have localized the eNOS and iNOS by immunohistochemistry and have tested their mRNA expression by RT-PCR and protein levels by Western blots. eNOS is present in the endothelial cells and odontoblasts of the healthy pulp, but an elevation of eNOS mRNA and protein levels with a concomitant dilation of vessels was characteristic under pathological conditions. Healthy pulp tissue failed to exhibit any iNOS; however, acute inflammation enhanced the mRNA and protein levels of iNOS, mainly in the leukocytes. There are differences in localization and expression between eNOS and iNOS in healthy and inflamed dental pulp.

Ammonia-induced heme oxygenase-1 expression in cultured rat astrocytes and rat brain in vivo.

Ammonia is a key factor in the pathogenesis of hepatic encephalopathy (HE), which is a major complication in acute and chronic liver failure and other hyperammonemic states. The molecular mechanisms underlying ammonia neurotoxicity and the functional consequences of ammonia on gene expression in astrocytes are incompletely understood. Using cDNA array hybridization technique we identified ammonia as a trigger of heme oxygenase-1 (HO-1) mRNA levels in cultured rat astrocytes. As shown by Northern and Western blot analysis, HO-1 mRNA levels were upregulated by ammonia (0.1-5 mmol/L) after 24 h and protein expression after 72 h in astrocytes. These ammonia effects on HO-1 are probably triggered to a minor extent by ammonia-induced glutamine synthesis or by astrocyte swelling, because HO-1 expression was not inhibited by the glutamine synthetase inhibitor methionine sulfoximine (which abrogated ammonia-induced cell swelling in cultured astrocytes), and ammonia-induced HO-1 expression could only partly be mimicked by hypoosmotic astrocyte swelling. Hypoosmotic (205 mOsm/L) exposure of astrocytes led even to a decrease in HO-1 mRNA levels within 4 h, whereas hyperosmotic (405 mOsm/L) exposure increased HO-1 mRNA expression. After 24 h, hypoosmolarity slightly raised HO-1 mRNA expression. Taurine and melatonin diminished ammonia-induced HO-1 mRNA or protein expression, whereas other antioxidants (dimethylthiourea, butylated hydroxytoluene, N-acetylcysteine, and reduced glutathione) increased HO-1 mRNA levels under ammonia-free conditions. An in vivo relevance is suggested by the finding that increased HO-1 expression occurs in the brain cortex from acutely ammonia-intoxicated rats. It is concluded that ammonia-induced HO-1 expression may contribute to cerebral hyperemia in hyperammonic states.