Combined Inhibition Of Nitric Oxide Research Articles

Effects of progesterone treatment on endothelium-dependent coronary relaxation in ovariectomized rats

AimAlthough progesterone (P4) has a beneficial effect on the cardiovascular system, P4 actions on the coronary bed have not yet been fully elucidated. This study evaluated the effect of progesterone treatment on endothelium-dependent coronary vascular reactivity in Wistar rats. Main methodsEight-week-old adult rats were divided into Sham, Ovariectomized (OVX), Ovariectomized and progesterone treated (OVX P4). The OVX P4 group received daily doses of progesterone (2 mg/kg/day). Vascular reactivity was assessed by a modified Langendorff technique. The intensity of eNOS, Akt, and gp91phox protein expression was quantified by Western blotting. Superoxide anion (O2●-) and hydrogen peroxide (H2O2) production was measured by dihydroethidium and 2′,7′-dichlorofluorescein, respectively. Key findingsTreatment with P4 was able to prevent the reduction in baseline coronary perfusion pressure induced by ovariectomy. We observed that endothelium-dependent coronary vasodilation was reduced in the OVX group and potentiated in the OVX P4 group. Following the inhibition of the nitric oxide (NO) pathway, the bradykinin-induced relaxing response was potentiated in the OVX P4 group. With regard to the combined inhibition of NO and prostanoids pathways, the OVX P4 group showed a greater relaxing response, similar to what was found upon individual inhibition of NO. After the combined inhibition of NO, prostanoids and epoxyeicosatrienoic acids' pathways, the vasodilatory response induced by BK was abolished in all groups. SignificanceTreatment with P4 prevented oxidative stress induced by ovariectomy. These results suggest that progesterone has a beneficial action on the coronary vascular bed.

Inhibition of Na+/K+‐ATPase and KIR channels abolishes hypoxic hyperaemia in resting but not contracting skeletal muscle of humans

Increasing blood flow (hyperaemia) to exercising muscle helps match oxygen delivery and metabolic demand. During exercise in hypoxia, there is a compensatory increase in muscle hyperaemia that maintains oxygen delivery and tissue oxygen consumption. Nitric oxide (NO) and prostaglandins (PGs) contribute to around half of the augmented hyperaemia during hypoxic exercise, although the contributors to the remaining response are unknown. In the present study, inhibiting NO, PGs, Na+ /K+ -ATPase and inwardly rectifying potassium (KIR ) channels did not blunt augmented hyperaemia during hypoxic exercise beyond previous observations with NO/PG block alone. Furthermore, although inhibition of only Na+ /K+ -ATPase and KIR channels abolished hyperaemia during hypoxia at rest, it had no effect on augmented hyperaemia during hypoxic exercise. This is the first study in humans to demonstrate that Na+ /K+ -ATPase and KIR channel activation is required for augmented muscle hyperaemia during hypoxia at rest but not during hypoxic exercise, thus providing new insight into vascular control. Exercise hyperaemia in hypoxia is augmented relative to the same exercise intensity in normoxia. During moderate-intensity handgrip exercise, endothelium-derived nitric oxide (NO) and vasodilating prostaglandins (PGs) contribute to ∼50% of the augmented forearm blood flow (FBF) response to hypoxic exercise (HypEx), although the mechanism(s) underlying the remaining response are unclear. We hypothesized that combined inhibition of NO, PGs, Na+ /K+ -ATPase and inwardly rectifying potassium (KIR ) channels would abolish the augmented hyperaemic response in HypEx. In healthy young adults, FBF responses were measured (Doppler ultrasound) and forearm vascular conductance was calculated during 5min of rhythmic handgrip exercise at 20% maximum voluntary contraction under regional sympathoadrenal inhibition in normoxia and isocapnic HypEx (O2 saturation ∼80%). Compared to control, combined inhibition of NO, PGs, Na+ /K+ -ATPase and KIR channels (l-NMMA+ketorolac+ouabain+BaCl2; Protocol 1; n=10) blunted the compensatory increase in FBF during HypEx by ∼50% (29±6mLmin-1 vs. 62±8mLmin-1 , respectively, P<0.05). By contrast, ouabain+BaCl2 alone (Protocol 2; n=10) did not affect this augmented hyperaemic response (50±11mLmin-1 vs. 60±13mLmin-1 , respectively, P>0.05). However, the blocked condition in both protocols abolished the hyperaemic response to hypoxia at rest (P<0.05). We conclude that activation of Na+ /K+ -ATPase and KIR channels is involved in the hyperaemic response to hypoxia at rest, although it does not contribute to the augmented exercise hyperaemia during hypoxia in humans.

Contracting human skeletal muscle maintains the ability to blunt α1‐adrenergic vasoconstriction during KIR channel and Na+/K+‐ATPase inhibition

During exercise there is a balance between vasoactive factors that facilitate increases in blood flow and oxygen delivery to the active tissue and the sympathetic nervous system, which acts to limit muscle blood flow for the purpose of blood pressure regulation. Functional sympatholysis describes the ability of contracting skeletal muscle to blunt the stimulus for vasoconstriction, yet the underlying signalling of this response in humans is not well understood. We tested the hypothesis that activation of inwardly rectifying potassium channels and the sodium-potassium ATPase pump, two potential vasodilator pathways within blood vessels, contributes to the ability to blunt α1 -adrenergic vasoconstriction. Our results show preserved blunting of α1 -adrenergic vasconstriction despite blockade of these vasoactive factors. Understanding this complex phenomenon is important as it is impaired in a variety of clinical populations. Sympathetic vasoconstriction in contracting skeletal muscle is blunted relative to that which occurs in resting tissue; however, the mechanisms underlying this 'functional sympatholysis' remain unclear in humans. We tested the hypothesis that α1 -adrenergic vasoconstriction is augmented during exercise following inhibition of inwardly rectifying potassium (KIR ) channels and Na(+) /K(+) -ATPase (BaCl2 +ouabain). In young healthy humans, we measured forearm blood flow (Doppler ultrasound) and calculated forearm vascular conductance (FVC) at rest, during steady-state stimulus conditions (pre-phenylephrine), and after 2min of phenylephrine (PE; an α1 -adrenoceptor agonist) infusion via brachial artery catheter in response to two different stimuli: moderate (15% maximal voluntary contraction) rhythmic handgrip exercise or adenosine infusion. In Protocol1 (n=11 subjects) a total of six trials were performed in three conditions: control (saline), combined enzymatic inhibition of nitric oxide (NO) and prostaglandin (PG) synthesis (l-NMMA+ketorolac) and combined inhibition of NO, PGs, KIR channels and Na(+) /K(+) -ATPase (l-NMMA+ketorolac+BaCl2 +ouabain). In Protocol2 (n=6) a total of four trials were performed in two conditions: control (saline), and combined KIR channel and Na(+) /K(+) -ATPase inhibition. All trials occurred after local β-adrenoceptor blockade (propranolol). PE-mediated vasoconstriction was calculated (%ΔFVC) in each condition. Contrary to our hypothesis, despite attenuated exercise hyperaemia of ∼30%, inhibition of KIR channels and Na(+) /K(+) -ATPase, combined with inhibition of NO and PGs (Protocol1) or alone (Protocol2) did not enhance α1 -mediated vasoconstriction during exercise (Protocol1: -27±3%; P=0.2vs. control, P=0.4vs. l-NMMA+ketorolac; Protocol2: -21±7%; P=0.9vs. control). Thus, contracting human skeletal muscle maintains the ability to blunt α1 -adrenergic vasoconstriction during combined KIR channel and Na(+) /K(+) -ATPase inhibition.

Evidence for a functional vasodilatatory role for hydrogen sulphide in the human cutaneous microvasculature.

Hydrogen sulphide (H2 S) is vasoprotective, attenuates inflammation and modulates blood pressure in animal models; however, its specific mechanistic role in the human vasculature remains unclear. In the present study, we report the novel finding that the enzymes responsible for endogenous H2 S production, cystathionine-γ-lyase and 3-mercaptopyruvate sulphurtransferase, are expressed in the human cutaneous circulation. Functionally, we show that H2 S-induced cutaneous vasodilatation is mediated, in part, by tetraethylammonium-sensitive calcium-dependent potassium channels and not by ATP-sensitive potassium channels. In addition, nitric oxide and cyclo-oxygenase-derived byproducts are required for full expression of exogenous H2 S-mediated cutaneous vasodilatation. Future investigations of the potential role for H2 S with respect to modulating vascular function in humans may have important clinical implications for understanding the mechanisms underlying vascular dysfunction characteristic of multiple cardiovascular pathologies. The present study aimed to identify the presence of cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulphurtransferase (3-MST), which endogenously produce hydrogen sulphide (H2 S), and to functionally examine the mechanisms of H2 S-induced vasodilatation in the human cutaneous microcirculation. CSE and 3-MST were quantified in forearm skin samples from 5 healthy adults (24±3years) using western blot analysis. For functional studies, microdialysis fibres were placed in the forearm skin of 12 healthy adults (25±3years) for graded infusions (0.01-100mm) of sodiumsulphide (Na2 S) and sodium hydrogen sulphide (NaHS). To define the mechanisms mediating H2 S-induced vasodilatation, microdialysis fibres were perfused with Ringer solution (control), a ATP-sensitive potassium channel (KATP ) inhibitor, an intermediate calcium-dependent potassium channel (KCa ) inhibitor, a non-specific KCa channel inhibitor or triple blockade. To determine the interaction of H2 S-mediated vasodilatation with nitric oxide (NO) and cyclo-oxygenase (COX) signalling pathways, microdialysis fibres were perfused with Ringer solution (control), a non-specific NO synthase inhibitor, a non-selective COX inhibitor or combined inhibition during perfusion of increasing doses of Na2 S. CSE and 3-MST were expressed in all skin samples. Na2 S and NaHS elicited dose-dependent vasodilatation. Non-specific KCa channel inhibition and triple blockade blunted Na2 S-induced vasodilatation (P<0.05), whereas KATP and intermediate KCa channel inhibition had no effect (P>0.05). Separate and combined inhibition of NO and COX attenuated H2 S-induced vasodilatation (all P<0.05). CSE and 3-MST are expressed in the human microvasculature. Exogenous H2 S elicits cutaneous vasodilatation mediated by KCa channels and has a functional interaction with both NO and COX vasodilatatory signalling pathways.

Contribution of vascular hyperpolarization to augmented skeletal muscle hyperemia during hypoxic exercise in humans (1079.20)

Exercise hyperemia during hypoxia is augmented relative to normoxia. At moderate exercise intensities, nitric oxide (NO) and vasodilating prostaglandins (PGs) explain ~50% of this response, yet the remaining signaling mechanisms remain unclear. We hypothesized that vascular hyperpolarization via activation of inwardly rectifying potassium (KIR) channels and Na+/K+‐ATPase accounts for the remaining ~50% of the augmented hyperemia. In 10 young adults, we measured forearm blood flow (FBF; Doppler ultrasound) during 5 min of rhythmic handgrip exercise at 20% maximal voluntary contraction in normoxia (NormEx) and isocapnic hypoxia (HypEx; O2 saturation 80%) before and after local combined inhibition of NO synthase (L‐NMMA), cyclooxygenase (ketorolac), KIR channels (barium chloride) and Na+/K+‐ATPase (ouabain). All trials were performed during local α‐ and β‐adrenoceptor blockade to isolate local vasodilation. In control conditions, FBF during HypEx was augmented vs. NormEx by 62±8 ml/min (P&lt;0.05). In the blocked condition, this compensatory increase in FBF was only 29±6 ml/min (47±10% decrease), but O2 extraction was increased to maintain forearm VO2. These findings are similar to our previous observations with combined inhibition of NO and PGs alone, suggesting that vascular hyperpolarization via these pathways does not contribute to the augmented hyperemic response during hypoxic exercise.Grant Funding Source: Supported by NIH award HL‐095573 (FAD)

Activation of KIR channels and Na+/K+‐ATPase is not obligatory to blunt sympathetic vasoconstriction in contracting human skeletal muscle (1170.6)

Activation of inwardly‐rectifying potassium (KIR) channels and Na+/K+‐ATPase regulates muscle blood flow during mild exercise; whether these pathways are involved in the blunting of sympathetic α‐adrenergic vasoconstriction in active muscle is unknown. We tested the hypothesis that α1‐adrenergic vasoconstriction (phenylephrine; PE) is augmented during exercise after inhibition of KIR channels and Na+/K+‐ATPase. Young adults (n=11) were instrumented with a brachial artery catheter (drug infusions and blood pressure) and forearm blood flow (Doppler ultrasound) was measured and vascular conductance (FVC) calculated at rest, during minute 4 of steady‐state hyperemic conditions (moderate handgrip exercise or adenosine; ADO), and after 2 min of PE in these conditions: control (saline), combined inhibition of nitric oxide (NO; L‐NMMA) and prostaglandins (PGs; ketorolac) and combined inhibition of NO, PGs, KIR channels and Na+/K+‐ATPase (L‐NMMA+ketorolac+BaCl2+ouabain). In control, exercise blunted PE‐mediated vasoconstriction (%ΔFVC) compared to ADO (‐21±3% vs ‐46±6%; P&lt;0.05) and this was unchanged with L‐NMMA+ketorolac (‐24±3% vs ‐47±5%). The addition of BaCl2+ouabain had no effect on vasoconstriction during exercise (‐27±3%), indicating that activation of KIR channels and Na+/K+‐ATPase is not obligatory to blunt sympathetic vasoconstriction in contracting human muscle.Support HL102720

Mechanisms of rapid vasodilation after a brief contraction in human skeletal muscle

A monophasic increase in skeletal muscle blood flow is observed after a brief single forearm contraction in humans, yet the underlying vascular signaling pathways remain largely undetermined. Evidence from experimental animals indicates an obligatory role of vasodilation via K⁺-mediated smooth muscle hyperpolarization, and human data suggest little to no independent role for nitric oxide (NO) or vasodilating prostaglandins (PGs). We tested the hypothesis that K⁺-mediated vascular hyperpolarization underlies the rapid vasodilation in humans and that combined inhibition of NO and PGs would have a minimal effect on this response. We measured forearm blood flow (Doppler ultrasound) and calculated vascular conductance 10 s before and for 30 s after a single 1-s dynamic forearm contraction at 10%, 20%, and 40% maximum voluntary contraction in 16 young adults. To inhibit K⁺-mediated vasodilation, BaCl₂ and ouabain were infused intra-arterially to inhibit inwardly rectifying K⁺ channels and Na⁺-K⁺-ATPase, respectively. Combined enzymatic inhibition of NO and PG synthesis occurred via NG-monomethyl-L-arginine (L-NMMA; NO synthase) and ketorolac (cyclooxygenase), respectively. In protocol 1 (n = 8), BaCl₂ + ouabain reduced peak vasodilation (range: 30-45%, P < 0.05) and total postcontraction vasodilation (area under the curve, ~55-75% from control) at all intensities. Contrary to our hypothesis, L-NMMA + ketorolac had a further impact (peak: ~60% and area under the curve: ~80% from control). In protocol 2 (n = 8), the order of inhibitors was reversed, and the findings were remarkably similar. We conclude that K⁺-mediated hyperpolarization and NO and PGs, in combination, significantly contribute to contraction-induced rapid vasodilation and that inhibition of these signaling pathways nearly abolishes this phenomenon in humans.

ATP‐mediated vasodilatation occurs via activation of inwardly rectifying potassium channels in humans

Circulating ATP possesses unique vasomotor properties in humans and has been hypothesized to play a role in vascular control under a variety of physiological conditions. However, the primary downstream signalling mechanisms underlying ATP-mediated vasodilatation remain unclear. The purpose of the present experiment was to determine whether ATP-mediated vasodilatation is independent of nitric oxide (NO) and prostaglandin (PG) synthesis and occurs primarily via the activation of Na(+)/K(+)-ATPase and inwardly rectifying potassium (K(IR)) channels in humans. In all protocols, young healthy adults were studied and forearm vascular conductance (FVC) was calculated from forearm blood flow (measured via venous occlusion plethysmography) and intra-arterial blood pressure to quantify local vasodilatation. Vasodilator responses (%FVC) during intra-arterial ATP infusions were unchanged following combined inhibition of NO and PGs (n = 8; P > 0.05) whereas the responses to KCl were greater (P < 0.05). Combined infusion of ouabain (to inhibit Na(+)/K(+)-ATPase) and barium chloride (BaCl(2); to inhibit K(IR) channels) abolished KCl-mediated vasodilatation (n = 6; %FVC = 134 ± 13 vs. 4 ± 5%; P < 0.05), demonstrating effective blockade of direct vascular hyperpolarization. The vasodilator responses to three different doses of ATP were inhibited on average 56 ± 5% (n = 16) following combined ouabain plus BaCl(2) infusion. In follow-up studies, BaCl(2) alone inhibited the vasodilator responses to ATP on average 51 ± 3% (n = 6), which was not different than that observed for combined ouabain plus BaCl(2) administration. Our novel results indicate that the primary mechanism of ATP-mediated vasodilatation is vascular hyperpolarization via activation of K(IR) channels. These observations translate in vitro findings to humans in vivo and may help explain the unique vasomotor properties of intravascular ATP in the human circulation.

ATP‐mediated vasodilation occurs via vascular hyperpolarization in humans

ATP possesses unique vasomotor properties and has been hypothesized to contribute to the control of vascular tone under a variety of physiological conditions; however, in humans, the underlying vasomotor signaling mechanisms remain unknown. We sought to determine whether ATP‐mediated vasodilation occurs via vascular hyperpolarization in young healthy humans. In Protocol 1 (n=8), progressive doses of ATP and potassium chloride (KCl) were infused via brachial artery catheter. Forearm vascular conductance (FVC) was calculated from forearm blood flow (venous occlusion plethysmography) and intra‐arterial blood pressure to quantify local vasodilation. Vasodilatory responses (%ΔFVC) to ATP infusions were unchanged following combined inhibition of nitric oxide (NO) and prostaglandins (PGs; P&gt;0.05) whereas the response to KCl was significantly greater (P&lt;0.05). In Protocol 2 (n=21), infusion of both ouabain (to inhibit Na+/K+ ATPase) and barium chloride (BaCl2; to inhibit KIR channels) abolished KCl‐mediated vasodilation (%ΔFVC= 134±13 vs 4±5%; P&lt;0.05) indicating effective inhibition of vasodilation mediated via hyperpolarization. On average, forearm vasodilation to 3 doses of ATP was inhibited ~60% following combined ouabain+BaCl2 infusion. Our novel results indicate that the majority of vasodilation in response to ATP occurs via vascular hyperpolarization in humans. Supported by HL102720

Augmented skeletal muscle hyperaemia during hypoxic exercise in humans is blunted by combined inhibition of nitric oxide and vasodilating prostaglandins

Exercise hyperaemia in hypoxia is augmented relative to the same level of exercise in normoxia. At moderate exercise intensities, the mechanism(s) underlying this augmented response are currently unclear. We tested the hypothesis that endothelium-derived nitric oxide (NO) and vasodilating prostaglandins (PGs) contribute to the augmented muscle blood flow during hypoxic exercise relative to normoxia. In 10 young healthy adults, we measured forearm blood flow (FBF; Doppler ultrasound) and calculated the vascular conductance (FVC) responses during 5 min of rhythmic handgrip exercise at 20% maximal voluntary contraction in normoxia (NormEx) and isocapnic hypoxia (HypEx; O2 saturation ∼85%) before and after local intra-brachial combined blockade of NO synthase (NOS; via N(G)-monomethyl-L-arginine: L-NMMA) and cyclooxygenase (COX; via ketorolac). All trials were performed during local α- and β-adrenoceptor blockade to eliminate sympathoadrenal influences on vascular tone and thus isolate local vasodilatation. Arterial and deep venous blood gases were measured and oxygen consumption (VO2) was calculated. In control (saline) conditions, FBF after 5 min of exercise in hypoxia was greater than in normoxia (345 ± 21 ml min(−1) vs. 297 ± 18 ml min(−1); P < 0.05). After NO–PG block, the compensatory increase in FBF during hypoxic exercise was blunted ∼50% and thus was reduced compared with control hypoxic exercise (312 ± 19 ml min(−1); P < 0.05), but this was not the case in normoxia (289 ± 15 ml min(−1); P = 0.33). The lower FBF during hypoxic exercise was associated with a compensatory increase in O2 extraction, and thus VO2 was maintained at normal control levels (P = 0.64–0.99). We conclude that under the experimental conditions employed, NO and PGs have little role in normoxic exercise hyperaemia whereas combined NO–PG inhibition reduces hypoxic exercise hyperaemia and abolishes hypoxic vasodilatation at rest. Additionally, VO2 of the tissue was maintained in hypoxic conditions at rest and during exercise, despite attenuated oxygen delivery following NO–PG blockade, due to an increase in O2 extraction at the level of the muscle.

Combined inhibition of nitric oxide and vasodilating prostaglandins abolishes forearm vasodilatation to systemic hypoxia in healthy humans

We tested the hypothesis that nitric oxide (NO) and vasodilating prostaglandins (PGs) contribute independently to hypoxic vasodilatation, and that combined inhibition would reveal a synergistic role for these two pathways in the regulation of peripheral vascular tone. In 20 healthy adults, we measured forearm blood flow (Doppler ultrasound) and calculated forearm vascular conductance (FVC) responses to steady-state (SS) isocapnic hypoxia (O₂ saturation ~85%). All trials were performed during local α- and β-adrenoceptor blockade (via a brachial artery catheter) to eliminate sympathoadrenal influences on vascular tone and thus isolate local vasodilatory mechanisms. The individual and combined effects of NO synthase (NOS) and cyclooxygenase (COX) inhibition were determined by quantifying the vasodilatation from rest to SS hypoxia, as well as by quantifying how each inhibitor reduced vascular tone during hypoxia. Three hypoxia trials were performed in each subject. In group 1 (n = 10), trial 1, 5 min of SS hypoxia increased FVC from baseline (21 ± 3%; P < 0.05). Infusion of N(G)-nitro-L-arginine methyl ester (L-NAME) for 5 min to inhibit NOS during continuous SS hypoxia reduced FVC by -33 ± 3% (P < 0.05). In Trial 2 with continuous NOS inhibition, the increase in FVC from baseline to SS hypoxia was similar to control conditions (20 ± 3%), and infusion of ketorolac for 5 min to inhibit COX during continuous SS hypoxia reduced FVC by -15 ± 3% (P < 0.05). In Trial 3 with combined NOS and COX inhibition, the increase in FVC from baseline to SS hypoxia was abolished (~3%; NS vs. zero). In group 2 (n = 10), the order of NOS and COX inhibition was reversed. In trial 1, five minutes of SS hypoxia increased FVC from baseline (by 24 ± 5%; P < 0.05), and infusion of ketorolac during SS hypoxia had minimal impact on FVC (-4 ± 3%; NS). In Trial 2 with continuous COX inhibition, the increase in FVC from baseline to SS hypoxia was similar to control conditions (27 ± 4%), and infusion of L-NAME during continuous SS hypoxia reduced FVC by -36 ± 7% (P < 0.05). In Trial 3 with combined NOS and COX inhibition, the increase in FVC from baseline to SS hypoxia was abolished (~3%; NS vs. zero). Our collective findings indicate that (1) neither NO nor PGs are obligatory to observe the normal local vasodilatory response from rest to SS hypoxia; (2) NO regulates vascular tone during hypoxia independent of the COX pathway, whereas PGs only regulate vascular tone during hypoxia when NOS is inhibited; and (3) combined inhibition of NO and PGs abolishes local hypoxic vasodilatation (from rest to SS hypoxia) in the forearm circulation of healthy humans during systemic hypoxia.

Influence of Combined Nitric Oxide and Prostaglandin Inhibition on Contraction‐induced Rapid Vasodilation

A monophasic increase in skeletal muscle blood flow is observed following a brief single forearm contraction in young healthy adults. The factors facilitating this rapid vasodilatory response are unclear. We tested the hypothesis that nitric oxide (NO) and prostaglandins (PGs) contribute to contraction-induced rapid vasodilation in humans. We measured forearm blood flow (Doppler ultrasound) and calculated vascular conductance 10s prior to, and for 30s after a single 1-s dynamic forearm contraction at mild and moderate exercise intensity. Trials were performed in eight young subjects before and after combined inhibition of NO and PG synthesis. For both workloads, the immediate and peak dilatory responses (cardiac cycles 1 – 4 P>0.05) were not influenced by combined inhibition, however the sustained dilation was blunted (cardiac cycles 5 – 23; P<0.05). The total hyperemic response to a single contraction was significantly attenuated at both mild and moderate contraction intensity (~70%). The present study indicates that NO and vasodilating PGs contribute to the latter sustained portion of the contraction-induced rapid vasodilatory response. Our collective findings suggest that multiple vasoactive signals are likely acting in concert, and have a specific role in coordinating the magnitude and temporal pattern of contraction-induced rapid vasodilation. Supported by HL-095573

Contribution of adenosine to compensatory dilation in hypoperfused contracting human muscles is independent of nitric oxide

We previously demonstrated that nitric oxide (NO) contributes to compensatory vasodilation in the contracting human forearm subjected to acute hypoperfusion. We examined the potential role of an adenosine-NO interaction to this response in 17 male subjects (25 ± 2 yr). In separate protocols subjects performed rhythmic forearm exercise (20% of maximum) while hypoperfusion was evoked by balloon inflation in the brachial artery above the elbow. Each trial included exercise before inflation, exercise with inflation, and exercise after deflation (3 min each). Forearm blood flow (FBF; ultrasound) and local [brachial artery catheter pressure (BAP)] and systemic [mean arterial pressure (MAP); Finometer] arterial pressure were measured. In protocol 1 (n = 10), exercise was repeated during nitric oxide synthase inhibition [N(G)-monomethyl-L-arginine (L-NMMA)] alone and during L-NMMA-aminophylline (adenosine receptor blockade) administration. In protocol 2, exercise was repeated during aminophylline alone and during aminophylline-L-NMMA. Forearm vascular conductance (FVC; ml·min(-1)·100 mmHg(-1)) was calculated from blood flow (ml/min) and BAP (mmHg). Percent recovery in FVC during inflation was calculated as (steady-state inflation + exercise value - nadir)/[steady-state exercise (control) value - nadir]. In protocol 1, percent recovery in FVC was 108 ± 8% during the control (no drug) trial. Percent recovery in FVC was attenuated with inhibition of NO formation alone (78 ± 9%; P < 0.01 vs. control) and was attenuated further with combined inhibition of NO and adenosine (58 ± 9%; P < 0.01 vs. L-NMMA). In protocol 2, percent recovery was reduced with adenosine receptor blockade (74 ± 11% vs. 113 ± 6%, P < 0.01) compared with control drug trials. Percent recovery in FVC was attenuated further with combined inhibition of adenosine and NO (48 ± 11%; P < 0.05 vs. aminophylline). Our data indicate that adenosine contributes to compensatory vasodilation in an NO-independent manner during exercise with acute hypoperfusion.

Blockade Of Prostaglandins And Nitric Oxide In-vivo Reduces State 3 Mitochondrial Respiration In Human Skeletal Muscle

Combined inhibition of nitric oxide (NO) and prostaglandins (PG) reduces skeletal muscle blood flow and oxygen delivery at rest and during exercise. It has recently been found that combined inhibition of NO and PG blockade reduces muscle oxygen uptake during exercise (Mortensen et al. 2007) suggesting a partial inhibition of aerobic metabolism or improved muscle efficiency of oxidative phosphorylation or actin-myosin cycling. PURPOSE: The purpose of this study was to examine in humans the independent and combined effect of NO and PG blockade with L-NMMA and Indomethacin (Indo) on mitochondrial respiration in muscle from biopsies of the vastus lateralis (VL) following knee extension (KE) exercise. METHODS: Mitochondrial respiration was measured ex-vivo by high resolution respirometry in saponin-permeabilized fibers following 6 min KE in control (CON, n=8), arterial infusion of LNMMA (n=4) and Indo (n=4) randomized in order, followed by combined inhibition of NO and PG (L-NMMA + Indo, n=8). RESULTS. ADP-stimulated state 3 respiration with substrates for complex I (glutamate, malate) was reduced 50% by Indo compared to CON. State 3 O2 flux with convergent electron input through both complex I and II with addition of succinate was equally reduced with Indo (20%) and L-NMMA + Indo (15%) compared to CON and LNMMA alone. Uncoupled O2 flux determined by titration of the ionophore FCCP was not increased above state 3 respiration with Indo suggesting tight coupling to oxidative phosphorylation. Graded titrations of L-NMMA into the respirometer in CON muscle reduced state 3 respiration by 12% only at a supra-physiological dose, while titration of Indo caused a linear reduction at all doses within and above the physiological range. CONCLUSION: The results indicate that inhibition of PG by indomethacin inhibits state 3 mitochondrial respiration primarily at complex I of the respiratory chain. This effect may in part explain the in-vivo reduction in muscle O2 uptake during exercise with double blockade of NO and PG, since blockade of NO by L-NMMA counteracts the inhibitory effect of Indo.

Evidence Mounts That Nitrite Contributes to Hypoxic Vasodilation in the Human Circulation

Hypoxic vasodilation is a conserved physiological response to hypoxia that matches blood flow and oxygen delivery to tissue metabolic demand. This fundamental physiological process has been characterized for >100 years since the initial description by Roy and Brown1 in 1880. Hypoxic vasodilation requires a sensor mechanism that can detect a divergence in the normal relationship between delivered blood oxygen and tissue oxygen consumption.2 This hypoxic sensor mechanism must be coupled to the feedback generation of vasodilatory effectors that increase blood flow to maintain adequate tissue oxygenation. In short, hypoxic vasodilation requires hypoxia and/or pH sensing coupled to the release of a vasodilating signal. In mammalian species, the set point for hypoxic vasodilation occurs as the hemoglobin desaturates from 60% to 40%, around a partial pressure of oxygen ranging from 40 to 20 mm Hg.3 Despite the fact that the hypoxic vasodilation response was discovered almost 150 years ago, the identities of the oxygen sensor mechanism and the specific feedback vasodilator effectors remain uncertain. Although a number of mediators have been considered, including adenosine, nitric oxide (NO), ATP-sensitive potassium (KATP) channels, endothelium-derived hyperpolarizing factor (candidates include CO, H2O2, or ONOO−), and prostacyclin,2,4 the specific blockade of many of these pathways fails to completely inhibit hypoxic vasodilation.2 Article p 670 The present study published by Maher and colleagues5 in this issue of Circulation provides compelling evidence in normal human volunteers that the circulating anion salt nitrite (NO2−) may be an effector of hypoxic vasodilation. They report that nitrite potently vasodilates the venous forearm circulation under resting conditions and potently vasodilates the arteriolar circulation during experimental hypoxia, achieved by systemic breathing of 11% oxygen. Their studies indicate that the vasodilatory effect of nitrite is maximal in the deoxygenated venous …

Combined inhibition of NO, prostaglandins and EDHFs inhibits ATP‐induced vasodilation in the human leg

Adenosine-5′-triphosphate (ATP) can bind to P2y-purinergicreceptors and induce vasodilation in skeletal muscle, but the precise mechanisms by which this occurs remain unclear. To investigate whether ATP induced vasodilation is mediated by the action of nitric oxide (NO), prostaglandins and/or endothelial-derived hyperpolarizing factors (EDHFs), we measured leg hemodynamics before and during 5 min of intra-arterial infusion of ATP (0.41±0.04μmol min−1;mean±SEM) in 8 healthy, male subjects (24±2 years, 76±3 kg) with and without co-infusion of indomethacin (INDO; 621±17 μg min−1), NG-mono-methyl-L-arginine (L-NMMA; 12.4±0.3 mg min−1), tetraethylammonium chloride (TEA; 12.4±0.3 mg min−1) or INDO + L-NMMA + TEA. Basal leg blood flow (LBF) was 0.46±0.06 l min−1, but was lower with either L-NMMA (0.32±0.03) or all three inhibitors (0.28±0.06)(P<0.05), whereas infusion of either TEA or INDO had no effect on basal flow. ATP infusion increased LBF to 2.4±0.2 l min−1, but with co-infusion of either INDO or L-NMMA alone, or all three inhibitors combined, it was 13±8, 22±8 and 39±5 % lower, respectively, than with ATP infusion alone. Infusion of TEA did not alter the ATP induced hyperemia. The lower LBF with ATP combined with INDO, L-NMMA and triple blockade was associated with a 8–12% higher blood pressure and a 15–45 % lower vascular conductance, while heart rate and leg VO2 remained similar. These results indicate that both NO and prostaglandins play an important role in ATP mediated vasodilatation, whereas EDHFs appear to be less important. Supported by Lundbeck Foundation

Combined Treatment With a Nonselective Nitric Oxide Synthase Inhibitor (L-NMMA) and Indomethacin Increases Ductus Constriction in Extremely Premature Newborns

Studies in premature animals suggest that 1) prolonged tight constriction of the ductus arteriosus is necessary for permanent anatomic closure and 2) endogenous nitric oxide (NO) and prostaglandins both play a role in ductus patency. We hypothesized that combination therapy with an NO synthase (NOS) inhibitor [N(G)-monomethyl-L-arginine (L-NMMA)] and indomethacin would produce tighter ductus constriction than indomethacin alone. Therefore, we conducted a phase I and II study of combined treatment with indomethacin and L-NMMA in newborns born at <28 weeks' gestation who had persistent ductus flow by Doppler after an initial three-dose prophylactic indomethacin course (0.2, 0.1, 0.1 mg/kg/24 h). Twelve infants were treated with the combined treatment protocol [three additional indomethacin doses (0.1 mg/kg/24 h) plus a 72-hour L-NMMA infusion]. Thirty-eight newborns received three additional indomethacin doses (without L-NMMA) and served as a comparison group. Ninety-two percent (11/12) of the combined treatment group had tight ductus constriction with elimination of Doppler flow. In contrast, only 42% (16/38) of the comparison group had a similar degree of constriction. L-NMMA infusions were limited in dose and duration by acute side effects. Doses of 10-20 mg/kg/h increased serum creatinine and systemic blood pressure. At 5 mg/kg/h, serum creatinine was stable but systemic hypertension still limited L-NMMA dose. We conclude that combined inhibition of NO and prostaglandin synthesis increased the degree of ductus constriction in newborns born at <28 weeks' gestation. However, the combined administration of L-NMMA and indomethacin was limited by acute side effects in this treatment protocol.

Impact of combined NO and PG blockade on rapid vasodilation in a forearm mild-to-moderate exercise transition in humans.

We tested the hypothesis that nitric oxide (NO) and prostaglandins (PGs) contribute to the rapid vasodilation that accompanies a transition from mild to moderate exercise. Nine healthy volunteers (2 women and 7 men) lay supine with forearm at heart level. Subjects were instrumented for continuous brachial artery infusion of saline (control condition) or combined infusion of N(G)-nitro-L-arginine methyl ester (L-NAME) and ketorolac (drug condition) to inhibit NO synthase and cyclooxygenase, respectively. A step increase from 5 min of steady-state mild (5.4 kg) rhythmic, dynamic forearm handgrip exercise (1 s of contraction followed by 2 s of relaxation) to moderate (10.9 kg) exercise for 30 s was performed. Steady-state forearm blood flow (FBF; Doppler ultrasound) and forearm vascular conductance (FVC) were attenuated in drug compared with saline (control) treatment: FBF = 196.8 +/- 30.8 vs. 281.4 +/- 34.3 ml/min and FVC = 179.3 +/- 29.4 vs. 277.8 +/- 34.8 ml.min(-1).100 mmHg(-1) (both P < 0.01). FBF and FVC increased from steady state after release of the initial contraction at the higher workload in saline and drug conditions: DeltaFBF = 72.4 +/- 8.7 and 52.9 +/- 7.8 ml/min, respectively, and DeltaFVC = 66.3 +/- 7.3 and 44.1 +/- 7.0 ml.min(-1).100 mmHg(-1), respectively (all P < 0.05). The percent DeltaFBF and DeltaFVC were not different during saline infusion or combined inhibition of NO and PGs: DeltaFBF = 27.2 +/- 3.1 and 28.1 +/- 3.8%, respectively (P = 0.78) and DeltaFVC = 25.7 +/- 3.2 and 26.0 +/- 4.0%, respectively (P = 0.94). The data suggest that NO and vasodilatory PGs are not obligatory for rapid vasodilation at the onset of a step increase from mild- to moderate-intensity forearm exercise. Additional vasodilatory mechanisms not dependent on NO and PG release contribute to the immediate and early increase in blood flow in an exercise-to-exercise transition.

Increased epoxyeicosatrienoic acid formation in the rat kidney during liver cirrhosis.

Vascular complications during liver cirrhosis are often severe, particularly in the kidney. These complications are the result of complex and poorly understood interactions between the injured liver and other organs such as the lungs, heart, and kidney. The purpose of this study was to investigate the alterations to renal hemodynamics during cirrhosis, focusing on the actions of epoxyeicosatrienoic acids (EET), known to be potent regulators of renal hemodynamics. Cirrhosis was induced in rats by common bile duct ligation (CBDL), and they were compared with sham rats. Experiments were conducted 4 wk after either the sham or CBDL surgery. Vasoreactivity was assessed in isolated perfused kidneys. cPLA(2) expression and cytochrome P450 (CYP450) expression were measured using Western blot. cPLA(2) enzymatic activity was measured by radioenzymatic assay. EET production was measured using rpHPLC analysis. The major findings were that kidneys from CBDL rats had significantly greater acetylcholine-induced vasodilation that was partially blocked by nitric oxide (NO) and prostaglandin inhibition and fully blocked by the combined inhibition of NO, prostaglandins, and CYP450 metabolites. Expression and activity of cPLA(2) in CBDL kidneys was increased, providing arachidonic acid substrate to the CYP450 enzymes. Finally, expression and activity of CYP450 enzymes was elevated in CBDL kidneys, resulting in significantly greater production of the vasodilating 11,12-EET and 14,15-EET. While it is well documented that renal vasoconstriction leading to impaired renal function occurs during cirrhosis, our data clearly demonstrate that endogenous production of EET is increased in cirrhotic kidneys. This may be a homeostatic response to preserve renal perfusion.

Epoxygenase Metabolites Contribute to Nitric Oxide-Independent Afferent Arteriolar Vasodilation in Response to Bradykinin

In the kidney, epoxyeicosatrienoic acids (EETs) have been suggested to be endothelium-derived hyperpolarizing factors (EDHFs). The aim of the present study was to determine the contribution of EETs to the preglomerular vasodilation elicited by bradykinin. Sprague-Dawley rats were studied utilizing an in vitro perfused juxtamedullary nephron preparation. The afferent arteriolar diameter was determined and the diameter averaged 19 ± 1 µm (n = 26) at a renal perfusion pressure of 100 mm Hg. Addition of 1, 10 and 100 nM bradykinin to the perfusate dose-dependently increased afferent arteriolar diameter by 5 ± 1, 12 ± 2 and 17 ± 2%, respectively. The nitric oxide inhibitor N^ω-nitro-L-arginine reduced bradykinin-induced afferent arteriolar vasodilation by 50%, and the diameter increased by 9 ± 2% in response to 100 nM bradykinin. Epoxygenase inhibitors N-methylsulphonyl-6-(2-propargyloxyphenyl)hexanamide or miconazole greatly attenuated the nitric oxide-independent component of the vasodilation elicited by bradykinin. Cyclooxygenase (COX) inhibition attenuated the nitric oxide-independent vasodilation elicited by 1 nM bradykinin but did not significantly affect the vascular response to 100 nM bradykinin. Combined inhibition of nitric oxide, COX and epoxygenase pathways completely abolished bradykinin-mediated afferent arteriolar vasodilation. In additional studies, renal microvessels were isolated and incubated with bradykinin and samples were analyzed by NICI/GC/MS. Under control conditions, renal microvascular EET levels averaged 49 ± 9 pg/mg/20 min (n = 7). In the presence of bradykinin, EET levels were significantly higher and averaged 81 ± 11 pg/mg/20 min (n = 7). These data support the concept that EETs are EDHFs and contribute to the nitric oxide-independent afferent arteriolar vasodilation elicited by bradykinin.