Adrenomedullin-induced dilation of human placental arteries is modulated by an endothelium-derived constricting factor

Adrenomedullin is synthesized and secreted by fetoplacental tissues. Given that the placenta lacks autonomic innervation, we proposed that adrenomedullin acts locally to control blood flow in the placental vasculature through a balance of dilatory and constrictive pathways. Placental stem villous arteries (200 microm) from normotensive human pregnancies were dissected and mounted on a wire myograph. The vessels were preconstricted with the thromboxane A(2) mimetic U46619 (EC(80) concentration), and exposed to cumulative concentrations of adrenomedullin (1 x 10(-9) to 3 x 10(-7) mol/L). Adrenomedullin caused concentration-dependent vasorelaxation which, in endothelium-intact vessels, was attenuated in the presence of the nitric oxide synthase inhibitor L-NMMA. This suggested that the vasodilation was mediated, at least in part, through nitric oxide. However, removal of the endothelium did not similarly alter the response. Nor did L-NMMA have any effect in endothelium-denuded vessels. We hypothesized that adrenomedullin must induce release of both endothelium-derived relaxing (nitric oxide) and constricting factors. When we blocked the two major pathways through which adrenomedullin is known to induce vasodilation, by incubating the vessels with L-NMMA (nitric oxide synthase inhibitor) and Rp-cAMPS (cAMP-dependent protein kinase inhibitor), adrenomedullin induced concentration-dependent vasoconstriction. This was not mediated through endothelin, since addition of the non-specific endothelin receptor antagonist PD142893 failed to alter the response to adrenomedullin. We conclude that, in addition to increasing endothelial nitric oxide biosynthesis in placental stem villous arteries, adrenomedullin induces release of an endothelium-derived constricting factor.     

Role of nitric oxide in methacholine-induced sweating and vasodilation in human skin.

The purpose of this study was to determine whether the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) demonstrates significant muscarinic-receptor antagonism during methacholine (MCh)-stimulated sweating in human forearm skin. Three intradermal microdialysis probes were placed in the skin of eight healthy adults (4 men and 4 women). MCh in the range of 0.033-243 mM in nine steps was perfused through a microdialysis probe with and without the presence of the nitric oxide synthase inhibitor L-NAME (10 mM) or the L-arginine analog NG-monomethyl-L-arginine (L-NMMA; 10 mM). Local sweat rate (sweat rate) and skin blood flow (laser-Doppler velocimetry) were measured directly over each microdialysis probe. We observed similar resting sweat rates at MCh only, MCh and L-NAME, and MCh and L-NMMA sites averaging 0.175 +/- 0.029, 0.186 +/- 0.034, and 0.139 +/- 0.027 mg x min(-1) x cm(-2), respectively. Peak sweat rate (0.46 +/- 0.11, 0.56 +/- 0.16, and 0.53 +/- 0.16. mg x min(-1) x cm(-2)) was also similar among all three sites. MCh produced a sigmoid-shape dose-response curve and 50% of the maximal attainable response (0.42 +/- 0.14 mM for MCh only) was shifted rightward shift in the presence of L-NAME or L-NMMA (2.88 +/- 0.79 and 3.91 +/- 1.14 mM, respectively; P < 0.05). These results indicate that nitric oxide acts to augment MCh-stimulated sweat gland function in human skin. In addition, L-NAME consistently blunted the MCh-induced vasodilation, whereas L-NMMA did not. These data support the hypothesis that muscarinic-induced dilation in cutaneous blood vessels is not mediated by nitric oxide production and that the role of L-NAME in attenuating acetylcholine-induced vasodilation may be due to its potential to act as a muscarinic-receptor antagonist.     

Clonidine-induced coronary vasodilatation in isolated guinea pig heart is not mediated by endothelial alpha2 adrenoceptors.

Functional role of endothelial alpha(2)-adrenoceptor in coronary circulation remains unclear. Clonidine, an agonist of alpha(2)-adrenoceptors, was reported to induce coronary vasodilatation via stimulation of endothelial alpha(2)-adrenoceptors or coronary vasoconstriction involving vascular smooth muscle alpha(2)-adrenoceptors. Moreover, H(2) receptor-dependent responses to clonidine were described. Here, we reassess the contribution of endothelial alpha(2)-adrenoceptor and H(2) receptors to coronary flow and contractility responses induced by clonidine in the isolated guinea pig heart. We found that clonidine (10(-9) - 10(-6) M) produced concentration-dependent coronary vasoconstriction without a significant change in contractility. This response was inhibited by the alpha(1)/alpha(2)-adrenoceptor antagonist - phentolamine (10(-5) M) and the selective alpha(2)-adrenoceptor antagonist yohimbine (10(-6) M), but it was not changed by the selective alpha(1)-adrenoceptor antagonist prazosin (10(-6) M). In the presence of nitric oxide synthase inhibitor, L-NAME (10(-4) M) the clonidine-induced vasoconstriction was potentiated. Clonidine at high concentrations of 10(-5) - 3 x 10(-5) M produced coronary vasodilatation, and an increase in myocardial contractility. These responses were abolished by a selective H(2)-receptor antagonist, ranitidine (10(-5) M), but not by phentolamine (10(-5) M). We conclude that in the isolated guinea pig heart, clonidine-induced vasoconstriction is mediated by activation of smooth muscle alpha(2)-adrenoceptors whereas clonidine-induced coronary vasodilatation is mediated by activation of vascular H(2) histaminergic receptors. Accordingly, endothelial alpha(2)-adrenoceptors does not seem to play a major role in coronary flow response induced by clonidine.     

Combined NO and PG inhibition augments alpha-adrenergic vasoconstriction in contracting human skeletal muscle

Sympathetic alpha-adrenergic vasoconstrictor responses are blunted in the vascular beds of contracting muscle (functional sympatholysis). We tested the hypothesis that combined inhibition of nitric oxide (NO) and prostaglandins (PGs) restores sympathetic vasoconstriction in contracting human muscle. We measured forearm blood flow via Doppler ultrasound and calculated the reduction in forearm vascular conductance in response to alpha-adrenergic receptor stimulation during rhythmic handgrip exercise (6.4 kg) and during a control nonexercise vasodilator condition (using intra-arterial adenosine) before and after combined local inhibition of NO synthase (NOS; via N(G)-nitro-L-arginine methyl ester) and cyclooxygenase (via ketorolac) in healthy men. Before combined inhibition of NO and PGs, the forearm vasoconstrictor responses to intra-arterial tyramine (which evoked endogenous noradrenaline release), phenylephrine (a selective alpha1-agonist), and clonidine (an alpha2-agonist) were significantly blunted during exercise compared with adenosine treatment. After combined inhibition of NO and PGs, the vasoconstrictor responses to all alpha-adrenergic receptor stimuli were augmented by approximately 10% in contracting muscle (P <0.05), whereas the responses to phenylephrine and clonidine were also augmented by approximately 10% during passive vasodilation in resting muscle (P <0.05). In six additional subjects, PG inhibition alone did not alter the vasoconstrictor responses in resting or contracting muscles. Thus in light of our previous findings, it appears that inhibition of either NO or PGs alone does not affect functional sympatholysis in healthy humans. However, the results from the present study indicate that combined inhibition of NO and PGs augments alpha-adrenergic vasoconstriction in contracting muscle but does not completely restore the vasoconstrictor responses compared with those observed during passive vasodilation in resting muscle.     

Nitric oxide regulates retinal vascular tone in humans

The purpose of the present study was to investigate the contribution of basal nitric oxide (NO) on retinal vascular tone in humans. In addition, we set out to elucidate the role of NO in flicker-induced retinal vasodilation in humans. Twelve healthy young subjects were studied in a three-way crossover design. Subjects received an intravenous infusion of either placebo or NG-monomethyl-L-arginine (L-NMMA; 3 or 6 mg/kg over 5 min), an inhibitor of NO synthase. Thereafter, diffuse luminance flicker was consecutively performed for 16, 32, and 64 s at a frequency of 8 Hz. The effect of L-NMMA on retinal arterial and venous diameter was assessed under resting conditions and during the hyperemic flicker response. Retinal vessel diameter was measured with a Zeiss retinal vessel analyzer. L-NMMA significantly reduced arterial diameter (3 mg/kg: -2%; 6 mg/kg: -4%, P < 0.001) and venous diameter (3 mg/kg: -5%; 6 mg/kg: -8%, P < 0.001). After placebo infusion, flicker induced a significant increase in retinal vessel diameter (P < 0.001). At a flicker duration of 64 s, arterial diameter increased by 4% and venous diameter increased by 3%. L-NMMA did not abolish these hyperemic responses but blunted venous vasodilation (P = 0.017) and arterial vasodilation (P = 0.02) in response to flicker stimulation. Our data indicate that NO contributes to basal retinal vascular tone in humans. In addition, NO appears to play a role in flicker-induced vasodilation of the human retinal vasculature.     

Nitric oxide-cGMP-mediated vasoconstriction and effects of acetylcholine in the branchial circulation of the eel

Information about the presence and effects of nitric oxide (NO) in fish vasculature is scant and contradictory. We have studied the NO/cGMP system in the branchial circulation of the teleost Anguilla anguilla using a branchial basket preparation under basal conditions and cholinergic stimulation. The effects of endogenous and exogenous NO were tested with L-arginine, the nitric oxide synthase (NOS) substrate, and the NO donors 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside (SNP), respectively. L-arginine (from 10(-11) to 10(-6) M) and the NO donors (starting from 10(-14) M) caused dose-dependent vasoconstriction. Conversely, in the ACh-pre-contracted preparations both donors elicited vasodilation. SIN-1-induced vasoconstriction was due to NO generation: it was increased by superoxide dismutase (SOD) and blocked by NO scavenger hemoglobin. Pre-treatment with sGC inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) inhibited the effects of SIN-1 and SNP. The stable cGMP analogue 8-bromo-guanosine 3',5'-cyclic monophosphate (8-Br cGMP) induced dose-dependent vasoconstriction. Unexpectedly, three NOS inhibitors, N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-monomethyl-L-arginine (L-NMMA), L-N(5)-(1-iminoethyl) ornithine (L-NIO), caused mild vasoconstriction. ACh caused vasoconstriction, but at pico- and nanomolar concentrations it caused mild but significant vasodilation in 40% of the preparations. Both responses, blocked by atropine and pirenzepine, required an intact endothelium. The ACh-induced vasoconstriction was substantially independent of a NO-cGMP mechanism.     

Blockade of nitric oxide formation impairs adrenergic-induced ACTH and corticosterone secretion.

It has been suggested that adrenergic agents might modulate the L-arginine-NO pathway. Sympathomimetic agonists enhance the basal release of NO, and noradrenaline increases the synthesis of nitric oxide synthase (NOS) in the medial basal hypothalamus in vitro. In the present study possible involvement of NO in central stimulation of the hypothalamic-pituitary-adrenal (HPA) axis by adrenergic agents was investigated in conscious rats. The nitric oxide synthase blocker N(omega)-nitro-L-arginine methyl ester (L-NAME 2 and 10 microg) was administered intracerebroventricularly (i.c.v.) 15 min before the adrenergic agonist given by the same route; 1 h later the rats were decapitated. Plasma levels of ACTH and corticosterone were measured. L-NAME significantly diminished the ACTH and corticosterone response to phenylephrine (30 microg), an alpha1-adrenergic receptor agonist. These hormone responses to clonidine (10 microg), an alpha2-receptor agonist, were dose-dependently suppressed or totally abolished by L-NAME. A significant rise in the ACTH and corticosterone secretion induced by isoprenaline (10 microg), a beta-adrenergic receptor agonist, was only moderately diminished by pretreatment with L-NAME. These results indicate that NOS is considerably involved in central stimulation of the HPA axis by alpha1- and alpha2-adrenergic receptor agonists, and that NO mediates the stimulatory action of these agonists on ACTH and corticosterone secretion. The stimulation induced by beta-adrenergic receptors is only moderately affected by endogenous NO.     

No alpha-adrenoreceptor-induced C-fiber activation in healthy human skin.

In healthy volunteers, flare responses induced by norepinephrine (NE) iontophoresis have been observed. However, as NE iontophoresis is a combined electrical and chemical stimulus axon, reflexes cannot be directly linked to pharmocological activity of NE. Different concentrations of NE, clonidine (CL), and phenylephrine (PE) (NE: 10(-10)-10(-3) M; CL and PE: 10(-8)-10(-3) M) were applied via intradermal microdialysis fibers into the skin of healthy volunteers. Simultaneously, skin blood flow was visualized by laser-Doppler imaging scans and quantified in a vasoconstriction skin area directly above the membranes to control drug effects and in expected axon reflex vasodilation areas that were 0.75 cm apart. NE, PE, and CL caused dose-dependent vasoconstriction. However, neither in the presumed axon reflex areas (quantitative analysis) nor on laser-Doppler imaging pictures (qualitative analysis) were any vasodilation observed. Even at concentrations causing maximum vasoconstriction (10(-3) M for any drug), no vasodilation was induced. Our results indicate that, in healthy human skin, exogenously supplied alpha-adrenoreceptor agonists alone do not activate nociceptors sufficiently to induce axon reflex flare.     

Systemic alpha-adrenergic and nitric oxide inhibition on basal limb blood flow: effects of endurance training in middle-aged and older adults

Endurance training improves endothelium-dependent vasodilation, yet it does not increase basal blood flow in the legs. We determined the effects of a 3-mo aerobic exercise intervention on basal leg blood flow and alpha-adrenergic vasoconstriction and nitric oxide (NO) release in seven apparently healthy middle-aged and older adults (60 +/- 3 yr). Basal femoral artery blood flow (via Doppler ultrasound) (pretraining: 354 +/- 29; posttraining: 335 +/- 34 ml/min) and vascular conductance did not change significantly with the exercise training. Before the exercise intervention, femoral artery blood flow increased 32 +/- 16% with systemic alpha-adrenergic blockade (with phentolamine) (P < 0.05), and the addition of nitric oxide synthase (NOS) inhibition using N(G)-monomethyl-L-arginine (L-NMMA) did not affect femoral artery blood flow. After training was completed, femoral artery blood flow increased 47 +/- 7% with alpha-adrenergic blockade (P < 0.01) and then decreased 18 +/- 7% with the subsequent administration of L-NMMA (P < 0.05). Leg vascular conductance showed a greater alpha-adrenergic blockade-induced vasodilation (+1.7 +/- 0.5 to +3.0 +/- 0.5 units, P < 0.05) as well as NOS inhibition-induced vasoconstriction (-0.8 +/- 0.4 to -2.7 +/- 0.7 units, P < 0.05) after the exercise intervention. Resting plasma norepinephrine concentration significantly increased after the training. These results suggest that regular aerobic exercise training enhances NO bioavailability in middle-aged and older adults and that basal limb blood flow does not change with exercise training because of the contrasting influences of sympathetic nervous system activity and endothelium-derived vasodilation on the vasculature.     

Both endothelium and afferent nerve endings play a role in acetylcholine-induced renal vasodilation

We investigated the nature and signaling pathways of endothelium- and sensory-nerve ending-derived substances involved in acetylcholine-induced vasodilation in rat isolated perfused kidney. Endothelial denudation by Triton X-100 (0.2%, 0.1 ml) or depletion of afferent nerve endings by capsaicin (10(-6) mol/l) attenuated acetylcholine-induced vasodilation. When these two agents were administered together, the response to acetylcholine was completely inhibited. CGRP1 receptor blocker CGRP 8-37 (10(-7) mol/l) and adenosine A(2) receptor antagonist ZM 241 385 (10(-7) mol/l) inhibited acetylcholine-induced dilation. When indomethacin (10(-5) mol/l), a cyclooxygenase inhibitor, l-NOARG (10(-4) mol/l), a nitric oxide (NO) synthase inhibitor, and potassium chloride (30 mmol/l), to test EDHF response, were perfused simultaneously, the inhibition was greater than that was observed with each agent alone. Guanylate cyclase inhibitor ODQ (10(-5) mol/l) or protein kinase A inhibitor KT 5720 (5x10(-7) mol/l) inhibited acetylcholine-induced dilation. Gap junction uncoupler 18alpha-glycyrrhetinic acid (10(-4) mol/l) caused an uncontrollable increase in basal perfusion pressure making it impossible to test against acetylcholine-induced dilation. Our data suggest that NO, prostanoids, EDHF, and CGRP released from vascular endothelium and afferent nerve endings participate in acetylcholine-induced vasodilation and their signal transduction molecules include protein kinase A and guanylate cyclase.     

In vitro pharmacological actions of sinomenine on the smooth muscle and the endothelial cell activity in rat aorta

Vasodilating actions of sinomenine were examined using rat aorta ring strips. Sinomenine (0.1 to 100 microM) dilated norepinephrine (NE, 5 microM)-induced vasoconstriction in a concentration-dependent manner reaching 68.8+/-5.1% (n=6, P<0.01) at a concentration of 100 microM. Sinomenine (100 microM) also attenuated KCl (60 mM) and phorbol 12, 13-dibutyrate (PDB, a protein kinase C, PK-C, activator, 300 nM)-induced vasoconstriction by 86.9+/-8.5% (n=6, P<0.01) and 49.9+/-9.8% (n=6, P<0.01), respectively. Pretreatment with nicardipine (a Ca2+ channel antagonist), staurosporine (a PK-C inhibitor), NG-monomethyl-L-arginine acetate (L-NMMA, a nitric oxide, NO, synthesis inhibitor), and indomethacin (a cyclooxygenase inhibitor) were carried out. Nicardipine (0.1 microM) led to a significant decrease in the vasodilating potential of sinomenine (at 100 microM, 68.8+/-5.1% vs. 35.5+/-6.9%; n=5, P<0.001). Pretreatment with staurosporine (30 nM) reduced sinomenine-associated vasodilation from 68.8+/-5.1% to 49.5+/-7.7% (n=5, P<0.001), and L-NMMA (100 microM) and indomethacin (10 microM), to 25.3+/-2.3% (n=5, P<0.001) and to 37.1+/-9.3% (n=5, P<0.001), respectively. The responses were almost similar to the results without endothelium. Therefore, these results indicate that sinomenine causes the vasorelaxation by the mechanisms involved with the inhibitions of Ca2+ channel and PK-C activity, and also with the activations of NO and prostaglandin (PG) I2 syntheses in endothelium.     

Mechanisms of vasoactive intestinal peptide-mediated vasodilation in human skin.

Vasoactive intestinal peptide (VIP) is known to induce histamine release in human skin and to include a nitric oxide (NO)-dependent dilation in several other vascular beds. However, the relative contribution of histamine and NO to VIP-mediated vasodilation in human skin is unknown. Forty-three subjects volunteered to participate in two studies designed to examine the mechanism of VIP-mediated vasodilation in human skin. Study 1 examined the contribution of NO in the skin blood flow response to eight doses of VIP ranging from 25 to 800 pmol. In addition, study 1 examined a specific role for NO in VIP-mediated dilation. Study 2 examined the relative contribution of NO and histamine to VIP-mediated dilation via H1 and H2 histamine receptors. Infusions were administered to skin sites via intradermal microdialysis. Red blood cell flux was measured by using laser-Doppler flowmetry (LDF), and cutaneous vascular conductance (CVC; LDF/mean arterial pressure) was calculated and normalized to maximal vasodilation. VIP-mediated vasodilation includes a NO-dependent component at doses above 100 pmol, where NO synthase inhibition significantly attenuates CVC (P < 0.05). Inhibition of H1 receptors attenuates the rise in CVC to exogenous VIP (P < 0.05); however, combined H1-receptor inhibition and NO synthase inhibition further reduced VIP-mediated vasodilation compared with either H1 inhibition or NO synthase inhibition alone (P < 0.05). In contrast to H1-receptor inhibition, H2-receptor inhibition did not affect vasodilation to exogenous VIP. Thus, in human skin, VIP-mediated vasodilation includes a NO-dependent component that could not be explained by H1- and H2-receptor activation.     

Control of cardiovascular function in the icefish Chionodraco hamatus: involvement of serotonin and nitric oxide

The involvement of nitric oxide (NO) in the branchial circulation and cardiac performance of the Antarctic hemoglobinless icefish Chionodraco hamatus was investigated using isolated and perfused head and working heart preparations. In the branchial vasculature under basal (i.e. unstimulated conditions), the nitric oxide synthase (NOS) inhibitor L-NIO (L-N(5)-(1-iminoethyl) ornithine, 10(-5) and 10(-4) M), caused a marked vasoconstriction (20%), indicating a basal nitrergic vasodilator tone, while the dose-response curve of the NO donor SIN-1 (3-morpholinosydnonimine) showed a dose-dependent vasodilator effect. Acetylcholine induced a dose-dependent branchial vasoconstriction mediated by muscarinic receptors, since the effects were abolished by pre-treatment with atropine (10(-4) M). Serotonin (5-HT) induced a dose-dependent branchial methysergide-sensitive vasoconstriction which was abolished by pre-treatment with L-NIO, indicating a NO-dependent mechanism. On the isolated heart, the NOS inhibitor L-NMMA (N(G)-monomethyl-L-arginine) 10(-4) M produced a small, but significant decrease of heart rate and, as a consequence, a decrease of power output, while the NO donor sodium nitroprusside (SNP) 10(-4) M elicited increases of stroke volume, stroke work and power output, suggesting an exogenous NO-dependent positive inotropism. Exposure of the bulbus arteriosus to L-NMMA was without any appreciable effect. In contrast, SNP produced a notable relaxation of the bulbus wall with a marked increase of its stiffness, as indicated by the increase of systolic and diastolic dP/dt max (23 and 104%, respectively).     

Preserved reflex cutaneous vasodilation in cystic fibrosis does not include an enhanced nitric oxide-dependent mechanism.

In humans, vasoactive intestinal peptide (VIP) may play a role in reflex cutaneous vasodilation during body heating. We tested the hypothesis that the nitric oxide (NO)-dependent contribution to active vasodilation is enhanced in the skin of subjects with cystic fibrosis (CF), compensating for sparse levels of VIP. In 2 parallel protocols, microdialysis fibers were placed in the skin of 11 subjects with CF and 12 controls. Lactated Ringer was perfused at one microdialysis site and NG-nitro-L-arginine methyl ester (2.7 mg/ml) was perfused at a second microdialysis site. Skin blood flow was monitored over each site with laser-Doppler flowmetry. In protocol 1, local skin temperature was increased 0.5 degrees C every 5 s to 42 degrees C, and then it maintained at 42 degrees C for approximately 45 min. In protocol 2, subjects wore a tube-lined suit perfused with water at 50 degrees C, sufficient to increase oral temperature (Tor) 0.8 degrees C. Cutaneous vascular conductance (CVC) was calculated (flux/mean arterial pressure) and scaled as percent maximal CVC (sodium nitroprusside; 8.3 mg/ml). Vasodilation to local heating was similar between groups. The change (Delta%CVCmax) in CVC with NO synthase inhibition on the peak (9+/-3 vs. 12+/-5%CVCmax; P=0.6) and the plateau (45+/-3 vs. 35+/-5%CVCmax; P=0.1) phase of the skin blood flow response to local heating was similar in CF subjects and controls, respectively. Reflex cutaneous vasodilation increased CVC in CF subjects (58+/-4%CVCmax) and controls (53+/-4%CVCmax; P=0.37) and NO synthase inhibition attenuated CVC in subjects with CF (37+/-6%CVCmax) and controls (35+/-5%CVCmax; P=0.8) to a similar degree. Thus the preservation of cutaneous active vasodilation in subjects with CF is not associated with an enhanced NO-dependent vasodilation.     

Role of nitric oxide in exercise sympatholysis.

The production of nitric oxide is the putative mechanism for the attenuation of sympathetic vasoconstriction (sympatholysis) in working muscles during exercise. We hypothesized that nitric oxide synthase blockade would eliminate the reduction in alpha-adrenergic-receptor responsiveness in exercising skeletal muscle. Ten mongrel dogs were instrumented chronically with flow probes on the external iliac arteries of both hindlimbs and a catheter in one femoral artery. The selective alpha(1)-adrenergic agonist (phenylephrine) or the selective alpha(2)-adrenergic agonist (clonidine) was infused as a bolus into the femoral artery catheter at rest and during mild and heavy exercise. Before nitric oxide synthase inhibition with N(G)-nitro-l-arginine methyl ester (l-NAME), intra-arterial infusions of phenylephrine elicited reductions in vascular conductance of -91 +/- 3, -80 +/- 5, and -75 +/- 6% (means +/- SE) at rest, 3 miles/h, and 6 miles/h and 10% grade, respectively. Intra-arterial clonidine reduced vascular conductance by -65 +/- 6, -39 +/- 4, and -30 +/- 3%. After l-NAME, intra-arterial infusions of phenylephrine elicited reductions in vascular conductance of -85 +/- 5, -85 +/- 5, and -84 +/- 5%, whereas clonidine reduced vascular conductance by -67 +/- 5, -45 +/- 3, and -35 +/- 3%, at rest, 3 miles/h, and 6 miles/h and 10% grade. alpha(1)-Adrenergic-receptor responsiveness was attenuated during heavy exercise. In contrast, alpha(2)-adrenergic-receptor responsiveness was attenuated even at a mild exercise intensity. Whereas the inhibition of nitric oxide production eliminated the exercise-induced attenuation of alpha(1)-adrenergic-receptor responsiveness, the attenuation of alpha(2)-adrenergic-receptor responsiveness was unaffected. These results suggest that the mechanism of exercise sympatholysis is not entirely mediated by the production of nitric oxide.     

Angiotensin II and nitric oxide in neural control of intrarenal blood flow

We investigated the roles of the renin-angiotensin system and the significance of interactions between angiotensin II and nitric oxide, in responses of regional kidney perfusion to electrical renal nerve stimulation (RNS) in pentobarbital sodium-anesthetized rabbits. Under control conditions, RNS (0.5-8 Hz) reduced total renal blood flow (RBF; -89 +/- 3% at 8 Hz) and cortical perfusion (CBF; -90 +/- 2% at 8 Hz) more than medullary perfusion (MBF; -55 +/- 5% at 8 Hz). Angiotensin II type 1 (AT(1))-receptor antagonism (candesartan) blunted RNS-induced reductions in RBF (P = 0.03), CBF (P = 0.007), and MBF (P = 0.04), particularly at 4 and 8 Hz. Nitric oxide synthase inhibition with N(G)-nitro-L-arginine (L-NNA) enhanced RBF (P = 0.003), CBF (P = 0.001), and MBF (P = 0.03) responses to RNS, particularly at frequencies of 2 Hz and less. After candesartan pretreatment, L-NNA significantly enhanced RNS-induced reductions in RBF (P = 0.04) and CBF (P = 0.007) but not MBF (P = 0.66). Renal arterial infusion of angiotensin II (5 ng.kg(-1).min(-1)) selectively enhanced responses of MBF to RNS in L-NNA-pretreated but not in vehicle-pretreated rabbits. In contrast, greater doses of angiotensin II (5-15 ng.kg(-1).min(-1)) blunted responses of MBF to RNS in rabbits with intact nitric oxide synthase. These results suggest that endogenous angiotensin II enhances, whereas nitric oxide blunts, neurally mediated vasoconstriction in the renal cortical and medullary circulations. In the renal medulla, but not the cortex, angiotensin II also appears to be able to blunt neurally mediated vasoconstriction.     

L-NG-monomethyl arginine inhibits the vasodilating effects of low dose of endothelin-3 on rat mesenteric arteries

We have reported that low doses of endothelin-3 (ET-3) elicited continuous vasodilation of rat mesenteric arteries, which is possibly related to endothelium-derived relaxing factor (EDRF). In order to clarify whether or not the vasodilating effects of ET-3 are associated with EDRF, we examined the effects of L-NG-monomethyl arginine (L-NMMA), an analog of L-arginine, on low-dose ET-3 induced vasodilation of rat mesente-Hc arteries. Infusion of 50 microM L-NMMA inhibited the vasodilation induced by 10(-13) M ET-3 and rather elicited an increase in perfusion pressure, which itself was decreased by infusion of 150 microM L-arginine. In the presence of 50 microM L-NMMA, 10(-13) M ET-3 did not elicit any vasodilation of the mesenteric arteries preconstricted with NE, in which 150 microM L-arginine, but not D-arginine, caused considerable vasodilation. These data suggest that the vasodilating effects of low doses of ET-3 are associated with EDRF as an endothelium-derived nitric oxide.     

Nitric oxide and attenuated reflex cutaneous vasodilation in aged skin

Thermoregulatory cutaneous vasodilation is diminished in the elderly. The goal of this study was to test the hypothesis that a reduction in nitric oxide (NO)-dependent mechanisms contributes to the attenuated reflex cutaneous vasodilation in older subjects. Seven young (23 +/- 2 yr) and seven older (71 +/- 6 yr) men were instrumented with two microdialysis fibers in the forearm skin. One site served as control (Ringer infusion), and the second site was perfused with 10 mM N(G)-nitro-l-arginine methyl ester to inhibit NO synthase (NOS) throughout the protocol. Water-perfused suits were used to raise core temperature 1.0 degrees C. Red blood cell (RBC) flux was measured with laser-Doppler flowmetry over each microdialysis fiber. Cutaneous vascular conductance (CVC) was calculated as RBC flux per mean arterial pressure, with values expressed as a percentage of maximal vasodilation (infusion of 28 mM sodium nitroprusside). NOS inhibition reduced CVC from 75 +/- 6% maximal CVC (CVC(max)) to 53 +/- 3% CVC(max) in the young subjects and from 64 +/- 5% CVC(max) to 29 +/- 2% CVC(max) in the older subjects with a 1.0 degrees C rise in core temperature. Thus the relative NO-dependent portion of cutaneous active vasodilation (AVD) accounted for approximately 23% of vasodilation in the young subjects and 60% of the vasodilation in the older subjects at this level of hyperthermia (P < 0.001). In summary, NO-mediated pathways contributed more to the total vasodilatory response of the older subjects at high core temperatures. This suggests that attenuated cutaneous vasodilation with age may be due to a reduction in, or decreased vascular responsiveness to, the unknown neurotransmitter(s) mediating AVD.     

In vivo role of heme oxygenase in ischemic coronary vasodilation

The heart constitutively expresses heme oxygenase (HO)-2, which catabolizes heme-containing proteins to produce biliverdin and carbon monoxide (CO). The heart also contains many possible substrates for HO-2 such as heme groups of myoglobin and cytochrome P-450s, which potentially could be metabolized into CO. As a result of observations that CO activates guanylyl cyclase and induces vascular relaxation and that HO appears to confer protection from ischemic injury, we hypothesized that the HO-CO pathway is involved in ischemic vasodilation in the coronary microcirculation. Responses of epicardial coronary arterioles to ischemia (perfusion pressure approximately 40 mmHg; flow velocity decreased by approximately 50%; dL/dt reduced by approximately 60%) were measured using stroboscopic fluorescence microangiography in 34 open-chest anesthetized dogs. Ischemia caused vasodilation of coronary arterioles by 36 +/- 6%. Administration of N(G)-monomethyl-L-arginine (L-NMMA, 3 micromol.kg(-1).min(-1) intracoronary), indomethacin (10 mg/kg iv), and K(+) (60 mM, epicardial suffusion) to prevent the actions of nitric oxide, prostaglandins, and hyperpolarizing factors, respectively, partially inhibited dilation during ischemia (36 +/- 6 vs. 15 +/- 4%; P < 0.05). The residual vasodilation during ischemia after antagonist administration was inhibited by tin mesoporphyrin IX (SnMP, 10 mg/kg iv), which is an inhibitor of HO (15 +/- 4 vs. 7 +/- 2%; P < 0.05 vs. before SnMP). The guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (10(-5) M, epicardial suffusion) also inhibited vasodilation during ischemia in the presence of L-NMMA with indomethacin and KCl. Moreover, administration of heme-L-arginate, which is a substrate for HO, produced dilation after ischemia but not after control conditions. We conclude that during myocardial ischemia, HO-2 activation can produce cGMP-mediated vasodilation presumably via the production of CO. This vasodilatory pathway appears to play a backup role and is activated only when other mechanisms of vasodilation during ischemia are exhausted.     

NO modulates norepinephrine release in human skeletal muscle: implications for neural preconditioning

The purpose of this study was to estimate muscle interstitial norepinephrine (NE) levels during exercise and to determine whether nitric oxide (NO) modulates NE release in the skeletal muscle in humans. We measured interstitial dialysate concentrations of NE with two microdialysis probes inserted into the forearm. Probes were perfused with saline and the NO synthesis inhibitor N(G)-monomethyl-L-arginine (L-NMMA), respectively. Dialysate samples were collected during two sequential 20-min intense dynamic handgrip periods, preceded by 40-min baseline periods. On a different day, forearm ischemia was performed instead of the first exercise period. Exercise increased dialysate NE from 172 +/- 42 to 270 +/- 45 pg/ml (83% increase, P < 0.02, n = 6). Probes perfused with L-NMMA had a 136 +/- 39% greater dialysate NE compared with probes perfused with saline (225 +/- 25 vs. 125 +/- 25 pg/ml, P < 0.001, n = 9). The exercise-induced increase in NE (125 +/- 52%) was attenuated if preceded by exercise (34 +/- 34%) or ischemia (40 +/- 36%; P = 0.06, n = 6), suggesting a neural preconditioning effect. This attenuation was not observed in probes perfused with L-NMMA. We propose that NO modulates NE release in skeletal muscle, that ischemic exercise increases muscle interstitial NE, and that this increase can be attenuated by a preconditioning effect mediated in part by NO.