Contribution of nitric oxide and prostaglandins to reactive hyperemia in the human forearm

Engelke, Keith A., John R. Halliwill, David N. Proctor, NikiM. Dietz, and Michael J. Joyner. Contribution of nitric oxide andprostaglandins to reactive hyperemia in the human forearm. J. Appl. Physiol. 81(4):1807-1814, 1996.We investigated the separate and combinedcontributions of nitric oxide (NO) and vasodilating prostaglandins asmediators of reactive hyperemia in the human forearm. Forearm bloodflow (FBF) was measured with venous occlusion plethysmography after 5 min of ischemia. In one protocol (n = 12), measurements were made before and after intra-arterialadministration of the NO synthase inhibitorN^G-monomethyl-L-arginine(L-NMMA) to one forearm. In aseparate protocol (n = 7),measurements were made before and after systemic administration of thecyclooxygenase inhibitor ibuprofen and again afterL-NMMA.L-NMMA reduced baseline FBF atrest (2.7 ± 0.4 to 1.6 ± 0.2 ml ^· 100 ml^{1 ·} min¹;P < 0.05) and had a modesteffect on peak forearm vascular conductance and flow (forearm vascularconductance = 31.1 ± 3.1 vs. 25.7 ± 2.5 ml ^· min^{1 ·} 100 mlforearm^{1 ·} 100 mmHg of perfusionpressure^{1 ·} min¹,P < 0.05; FBF = 26.6 ± 2.9 vs.22.8 ± 2.6 ml ^· 100 ml^{1 ·} min¹,P = 0.055). Total excessflow above baseline during reactive hyperemia was unaffected byL-NMMA (14.3 ± 3.0 vs. 13.1 ± 2.3 ml/100 ml; P < 0.05).Ibuprofen did not change FBF at rest, reduced peak FBF from 27.6 ± 1.9 to 20.3 ± 2.7 ml ^· 100 ml^{1 ·} min¹(P < 0.05), but had no effect ontotal excess flow above baseline. Infusion ofL-NMMA after ibuprofen reducedFBF at rest by 40%, had no effect on peak flow, but reduced totalexcess flow above baseline from 12.0 ± 2.5 to 7.6 ± 1.3 ml/100ml (P < 0.05). These datademonstrate that NO synthase inhibition has a modest effect on peakvasodilation during reactive hyperemia but plays a minimal role later.Prostaglandins appear to be important determinants of peak flow. Theeffects of NO synthase inhibition during reactive hyperemia may also bepotentiated by concurrent cyclooxygenase inhibition.

     

Effect of luteal phase elevation in core temperature on forearm blood flow during exercise

Kolka, Margaret A., and Lou A. Stephenson. Effect ofluteal phase elevation in core temperature on forearm blood flow duringexercise. J. Appl. Physiol. 82(4):1079-1083, 1997.Forearm blood flow (FBF) as an index of skinblood flow in the forearm was measured in five healthy women by venousocclusion plethysmography during leg exercise at 80% peak aerobicpower and ambient temperature of 35°C (relative humidity 22%;dew-point temperature 10°C). Resting esophagealtemperature (T_es) was 0.3 ± 0.1°C higher in the midluteal than in the early follicular phase ofthe menstrual cycle (P < 0.05).Resting FBF was not different between menstrual cycle phases. TheT_es threshold for onset of skinvasodilation was higher (37.4 ± 0.2°C) in midluteal than inearly follicular phase (37.0 ± 0.1°C; P < 0.05). The slope of the FBF toT_es relationship was not different between menstrual cycle phases (14.0 ± 4.2 ml · 100 ml¹ · min¹ · °C¹for early follicular and 16.3 ± 3.2 ml · 100 ml¹ · min¹ · °C¹for midluteal phase). Plateau FBF was higher during exercise inmidluteal (14.6 ± 2.2 ml · 100 ml¹ · min¹ · °C¹)compared with early follicular phase (10.9 ± 2.4 ml · 100 ml¹ · min¹ · °C¹;P < 0.05). The attenuation of theincrease in FBF to T_es occurred when T_es was 0.6°C higher andat higher FBF in midluteal than in early follicular experiments(P < 0.05). In summary, the FBF response is different during exercise in the two menstrual cycle phasesstudied. After the attenuation of the increase in FBF and whileT_es was still increasing, thegreater FBF in the midluteal phase may have been due to the effects ofincreased endogenous reproductive endocrines on the cutaneousvasculature.

     

NO does not mediate inhibitory neural responses in sheep airway and bronchial vascular smooth muscle

Endogenous nitric oxide (NO) influences acetylcholine-inducedbronchovascular dilation in sheep and is a mediator of the airway smooth muscle inhibitory nonadrenergic, noncholinergic neural responsein several species. This study was designed to determine the importanceof NO as a neurally derived modulator of ovine airway and bronchialvascular smooth muscle. We measured the response of pulmonaryresistance (RL) and bronchialblood flow (br) to vagal stimulationin 14 anesthetized, ventilated, open-chest sheep duringthe following conditions: 1)control; 2) infusion of the -agonist phenylephrine to reduce baseline br bythe same amount as would be produced by infusion ofN-nitro-L-arginine(L-NNA), a NO synthaseinhibitor; 3) infusion ofL-NNA(10² M); and4) after administration of atropine(1.5 mg/kg). The results showed that vagal stimulation produced anincrease in RL andbr in periods 1, 2, and 3 (P < 0.01) that was not affected byL-NNA. Afteratropine was administered, there was no increase inbr or RL. Invitro experiments on trachealis smooth muscle contracted with carbachol showed no effect ofL-NNA on neural relaxation butshowed a complete blockade with propranolol(P < 0.01). In conclusion, thevagally induced airway smooth muscle contraction and bronchial vasculardilation are not influenced by NO, and the sheep's trachealis muscle,unlike that in several other species, does not have inhibitorynonadrenergic, noncholinergic innervation.

     

Bioactive nitric oxide concentration does not increase during reactive hyperemia in human skin.

This study examined whether nitric oxide (NO) is involved in the cutaneous response to reactive hyperemia (RH) in the human forearm. We enrolled seven healthy volunteers. NO concentrations were monitored using a NO selective amperometric electrode (ISO-NOP200, World Precision Instruments) inserted into the skin of the forearm. Laser-Doppler flowmetry (Moor Instruments) was used for monitoring skin blood flow (SkBF) at the same site. SkBF and NO levels were monitored and recorded continuously throughout the experiment. An intradermal microdialysis probe was inserted adjacent to the NO electrode for drug delivery. Data collection began 140 min after the NO electrodes and microdialysis probes were inserted. RH was achieved by the inflation of a blood pressure cuff to 25 mmHg above systolic pressure for 7 min after which the pressure in the cuff was abruptly released. Acetylcholine (ACh) was given by microdialysis probe at the end of RH study to verify the ability of the electrode system to detect changes in the NO concentration. SkBF and NO data before RH and immediately, 2, 5, 7, and 10 min after cuff deflation were used for analysis. SkBF increased immediately after release of the occlusion (P < 0.0001) and remained elevated for 2 min. No significant NO changes occurred with the increases in LDF. ACh induced increases in both SkBF and NO (P < 0.000 and P < 0.037, respectively). We conclude that RH increases SkBF by mechanisms that do not require a measurable increase in NO concentrations.     

Nonselective cation conductance activated by muscarinic and purinergic receptors in rat spiral ganglion neurons

The present studycharacterizes the ionic conductances activated by acetylcholine (ACh)and ATP, two candidate neuromodulators, in isolated spiral ganglionneurons (SGNs). Brief application (1 s) of ACh evoked in adose-dependent manner (EC₅₀ = 4.1 µM) a reversibleinward current with a long latency (average 1.3 s), at holdingpotential (V_h) = 50 mV. This current wasreversibly blocked by atropine and mimicked by muscarine. Applicationof ATP also evoked a reversible inward current atV_h = 50 mV, but the current showed twocomponents. A fast component with a short latency was largely reducedwhen N-methyl-D-glucamine (NMDG) replaced extracellular sodium, implying a P2X-like ionotropic conductance. Thesecond component had a longer latency (average 1.1 s) and waspresumably activated by metabotropic P2Y-like receptors. The secondcomponent of ATP-evoked current shared similar characteristics with theresponses evoked by ACh: the current reversed near 0 mV, displayedinward rectification, could be carried by NMDG, and was insensitive toextracellular and intracellular calcium. This ACh-/ATP-evokedconductance was reversibly inhibited by preapplication of ionomycin.These results suggest that muscarinic receptors and purinergicmetabotropic receptors activate a similar large nonselective cationconductance via a common intracellular pathway in SGNs, a candidatemechanism to regulate neuronal excitability of SGNs.

     

Forearm training attenuates sympathetic responses to prolonged rhythmic forearm exercise

Sinoway, Lawrence, Jeffrey Shenberger, Gretchen Leaman,Robert Zelis, Kristen Gray, Robert Baily, and Urs Leuenberger. Forearm training attenuates sympathetic responses to prolonged rhythmic forearm exercise. J. Appl.Physiol. 81(4): 1778-1784, 1996.We previouslydemonstrated that nonfatiguing rhythmic forearm exercise at 25%maximal voluntary contraction (12 2-s contractions/min) evokessympathoexcitation without significant engagement ofmetabolite-sensitive muscle afferents (B. A. Batman, J. C. Hardy, U. A. Leuenberger, M. B. Smith, Q. X. Yang, and L. I. Sinoway.J. Appl. Physiol. 76: 1077-1081,1994). This is in contrast to the sympathetic nervous system responsesobserved during fatiguing static forearm exercise wheremetabolite-sensitive afferents are the key determinants of sympatheticactivation. In this report we examined whether forearm exercisetraining would attenuate sympathetic nervous system responses torhythmic forearm exercise. We measured heart rate, mean arterial bloodpressure (MAP), muscle sympathetic nerve activity (microneurography),plasma norepinephrine (NE), and NE spillover and clearance (tritiatedNE kinetics) during nonfatiguing rhythmic forearm exercise before andafter a 4-wk unilateral forearm training paradigm. Training had noeffect on forearm mass, maximal voluntary contraction, or heart ratebut did attenuate the increase in MAP (increase in MAP: from 15.2 ± 1.8 before training to 11.4 ± 1.4 mmHg after training;P < 0.017), muscle sympathetic nerve activity (increase in bursts: from 10.8 ± 1.4 before training to6.2 ± 1.1 bursts/min after training;P < 0.030), and the NE spillover(increase in arterial spillover: from 1.3 ± 0.2 before training to0.6 ± 0.2 nmol ^· min^{1 ·} m²after training, P < 0.014; increasein venous spillover: from 2.0 ± 0.6 beforetraining to 1.0 ± 0.5 nmol ^· min^{1 ·} m²after training, P < 0.037) seen inresponse to exercise performed by the trained forearm. Thus forearmtraining reduces sympathetic responses during a nonfatiguing rhythmichandgrip paradigm that does not engage muscle metaboreceptors. Wespeculate that this effect is due to a conditioning-inducedreduction in mechanically sensitive muscle afferentdischarge.

     

Inhibition of nitric oxide synthase slows heart rate recovery from cholinergic activation

The role ofnitric oxide (NO) in the cholinergic regulation of heart rate(HR) recovery from an aspect of simulated exercise wasinvestigated in atria isolated from guinea pig to test the hypothesisthat NO may be involved in the cholinergic antagonism of the positivechronotropic response to adrenergic stimulation. Inhibition of NOsynthesis withN^G-monomethyl-L-arginine(L-NMMA, 100 µM) significantlyslowed the time course of the reduction in HR without affecting themagnitude of the response elicited by bath-applied ACh (100 nM) orvagal nerve stimulation (2 Hz). The half-times(t_1/2) of responses were 3.99 ± 0.41 s in control vs. 7.49 ± 0.68 s inL-NMMA(P < 0.05). This was dependent onprior adrenergic stimulation (norepinephrine, 1 µM). The effect ofL-NMMA was reversed byL-arginine (1 mM; t_1/2 4.62 ± 0.39 s). The calcium-channelantagonist nifedipine (0.2 µM) also slowed the kinetics of thereduction in HR caused by vagal nerve stimulation. However, thet_1/2 for the reduction in HR with antagonists (2 mM Cs⁺ and 1 µM ZD-7288) of thehyperpolarization-activated current were significantlyfaster compared with control. There was no additional effect ofL-NMMA orL-NMMA+L-arginineon vagal stimulation in groups treated with nifedipine,Cs⁺, or ZD-7288. Weconclude that NO contributes to the cholinergic antagonism of thepositive cardiac chronotropic effects of adrenergic stimulation byaccelerating the HR response to vagal stimulation. This may involve aninterplay between two pacemaking currents (L-type calcium channelcurrent and hyperpolarization-activated current). Whether NO modulatesthe vagal control of HR recovery from actual exercise remains to bedetermined.

     

Effect of concentration on albumin diffusion in lung interstitium

The transport of macromolecules through the lung interstitiumdepends on both bulk transport of fluid and diffusion. In the presentstudy, we studied the diffusion of albumin. Isolated rabbit lungs wereinflated with silicon rubber via airways and blood vessels, and twochambers were bonded to the sides of a 0.5-cm-thick slab that encloseda vessel with an intersititial cuff. One chamber was filled with eitheralbumin solution (2 or 5 g/dl) containing tracer¹²⁵I-albumin or with tracer¹²⁵I-albumin alone; the other wasfilled with Ringer solution. Unbound ¹²⁵I was removed from the tracerby dialysis before use. The chamber with Ringer solution was placed inthe well of a NaI(Tl) scintillation detector. Diffusion oftracer through the interstitium was measured continuously for 60 h.Tracer mass (M) showed a time(t) delay followed by an increase toa steady-state flow(dM/dtconstant). Albumin diffusion coefficient(D) was given byL²/(6T),where T was the time intercept of thesteady-stateM-t line at zero M, andL was interstitial length.Interstitial cuff thickness-to-vessel radius ratio(Th₀/R)was estimated by using Fick's law for steady-state diffusion. BothD andTh₀/Rwere independent of albumin concentration.D averaged 6.6 × 10⁷cm²/s, similar to the freeD for albumin. Values ofTh₀/Raveraged 0.047 ± 0.024 (SD), near the values measuredhistologically. Thus pulmonary interstitial constituents offered norestriction to the diffusion of albumin.

     

cGMP-mediated Ca2+ release from IP3-insensitive Ca2+ stores in smooth muscle

Recent studies on the role of nitric oxide (NO) ingastrointestinal smooth muscle have raised the possibility thatNO-stimulated cGMP could, in the absence of cGMP-dependent proteinkinase (PKG) activity, act as aCa²⁺-mobilizing messenger[K. S. Murthy, K.-M. Zhang, J.-G. Jin, J. T. Grider, and G. M. Makhlouf. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28):G660-G671, 1993]. This notion was examined indispersed gastric smooth muscle cells with 8-bromo-cGMP (8-BrcGMP) andwith NO and vasoactive intestinal peptide (VIP), which stimulate endogenous cGMP. In muscle cells treated with cAMP-dependent protein kinase (PKA) and PKG inhibitors (H-89 and KT-5823), 8-BrcGMP (10 µM),NO (1 µM), and VIP (1 µM) stimulated⁴⁵Ca²⁺release (21 ± 3 to 30 ± 1% decrease in⁴⁵Ca²⁺cell content); Ca²⁺ releasestimulated by 8-BrcGMP was concentration dependent with anEC₅₀ of 0.4 ± 0.1 µM and athreshold of 10 nM. 8-BrcGMP and NO increased cytosolic freeCa²⁺ concentration([Ca²⁺]_i)and induced contraction; both responses were abolished after Ca²⁺ stores were depleted withthapsigargin. With VIP, which normally increases[Ca²⁺]_iby stimulating Ca²⁺ influx,treatment with PKA and PKG inhibitors caused a further increase in[Ca²⁺]_ithat reverted to control levels in cells pretreated with thapsigargin. Neither Ca²⁺ release norcontraction induced by cGMP and NO in permeabilized muscle cells wasaffected by heparin or ruthenium red.Ca²⁺ release induced by maximallyeffective concentrations of cGMP and inositol 1,4,5-trisphosphate(IP₃) was additive, independent of which agent was applied first. We conclude that, in the absence ofPKA and PKG activity, cGMP stimulatesCa²⁺ release from anIP₃-insensitive store and that itseffect is additive to that of IP₃.

     

Relationship between paxillin and myosin phosphorylation during muscarinic stimulation of smooth muscle

The tyrosinephosphorylation of paxillin increases in association with forcedevelopment during tracheal smooth muscle contraction, suggesting thatpaxillin plays a role in the contractile activation of smooth muscle[Z. L. Wang, F. M. Pavalko, and S. J. Gunst. Am.J. Physiol. 271 (CellPhysiol. 40): C1594-C1602, 1996]. We compared the Ca²⁺ sensitivity ofthe tyrosine phosphorylation of paxillin and myosin light chain (MLC)phosphorylation in tracheal muscle and evaluated whether MLCphosphorylation is necessary to induce paxillin phosphorylation. Ca²⁺-depleted muscle strips werestimulated with10⁷-10⁴M acetylcholine (ACh) in 0, 0.05, 0.1, or 0.5 mM extracellular Ca²⁺. In the absence ofextracellular Ca²⁺,10⁴ M ACh induced a maximalincrease in paxillin phosphorylation without increasing MLCphosphorylation or force. Increases in extracellularCa²⁺ concentration did not furtherincrease paxillin phosphorylation. However, during stimulation with10⁶ M ACh, paxillinphosphorylation increased with increases in extracellular Ca²⁺ concentration. We concludethat the tyrosine phosphorylation of paxillin can be stimulated bysignaling pathways that do not depend onCa²⁺ mobilization and that theactivation of contractile proteins is not required to elicit paxillinphosphorylation.

     

Reflex responses to regional venous pooling during lower body negative pressure in humans

Halliwill, John R., Lori A. Lawler, Tamara J. Eickhoff,Michael J. Joyner, and Sharon L. Mulvagh. Reflex responses toregional venous pooling during lower body negative pressure in humans.J. Appl. Physiol. 84(2): 454-458, 1998.Lower body negative pressure is frequently used to simulateorthostasis. Prior data suggest that venous pooling in abdominal orpelvic regions may have major hemodynamic consequences. Therefore, we developed a simple paradigm for assessing regional contributions tovenous pooling during lower body negative pressure. Sixteen healthy menand women underwent graded lower body negative pressure protocols to 60 mmHg while wearing medical antishock trousers to prevent venous poolingunder three randomized conditions:1) no trouser inflation (control),2) only the trouser legs inflated, and 3) the trouser legs andabdominopelvic region inflated. Without trouser inflation, heart rateincreased 28 ± 4 beats/min, mean arterial pressure fell 3 ± 2 mmHg, and forearm vascular resistance increased 51 ± 9 units at 60 mmHg lower body negative pressure. With inflation of eitherthe trouser legs or the trouser legs and abdominopelvic region, heartrate and mean arterial pressure did not change during lower bodynegative pressure. By contrast, although the forearm vasoconstrictorresponse to lower body negative pressure was attenuated by inflation ofthe trouser legs (forearm vascular resistance 33 ± 10 units,P < 0.05 vs. control), attenuation was greater with the inflation of the trouser legs and abdominopelvic region (forearm vascular resistance 16 ± 5 units,P < 0.05 vs. control and trouserlegs-only inflation). Thus the hemodynamic consequences of pooling inthe abdominal and pelvic regions during lower body negative pressureappear to be less than in the legs in healthy individuals.

     

Cellular Responses to Mechanical Stress: Invited Review: Effects of flow on vascular endothelial intracellular calcium signaling of rat aortas ex vivo

To study the effects of flow on in situendothelial intracellular calcium concentration([Ca²⁺]_i) signaling, rat aortic rings wereloaded with fura 2, mounted on a tissue flow chamber, and divided intocontrol and flow-pretreated groups. The latter was perfused with bufferat a shear stress of 50 dyns/cm² for 1 h. Endothelial[Ca²⁺]_i responses to ACh or shear stresseswere determined by ratio image analysis. Moreover, ACh-induced[Ca²⁺]_i elevation responses were measured ina calcium-free buffer, or in the presence of SKF-96365, to elucidatethe role of calcium influx in the flow effects. Our results showed that1) ACh increased endothelial[Ca²⁺]_i in a dose-dependent manner, and theseresponses were incremented by flow-pretreatment; 2) thedifferences in ACh-induced [Ca²⁺]_i elevationbetween control and flow-pretreated groups were abolished by SKF-96365or by Ca²⁺-free buffer; and 3) in the presenceof 10⁵ M ATP, shear stress induced dose-dependent[Ca²⁺]_i elevation responses that were notaltered by flow-pretreatment. In conclusion, flow-pretreatment augmentsthe ACh-induced endothelial calcium influx in rat aortas ex vivo.

     

Peroxynitrite and nitric oxide differ in their effects on pig coronary artery smooth muscle

Peroxynitrite generated in arteries fromsuperoxide and nitric oxide (NO) may damage their function. Here, wecompare the effects of peroxynitrite and peroxynitrite/NO-generatingagents SIN-1 (3-morpholinosydnonimine hydrochloride), SNAP(S-nitroso-N-acetyl-penicillamine), SNP (sodiumnitroprusside), and NONOate (spermine NONOate) on pig coronary artery.Deendothelialized artery rings were pretreated with these agents andthen washed before examining their contractility. Pretreatment with allagents (200 µM) results in a decrease in the force of contraction inresponse to the sarco(endo)plasmic Ca²⁺ (SERCA) pumpinhibitor cyclopiazonic acid (CPA): SNAP > NONOate  peroxynitrite  SIN-1 > SNP. Pretreatment with SNAP,NONOate, or SIN-1 also inhibits the force of contraction produced with 30 mM KCl, with SNAP being the most potent. Including catalase plussuperoxide dismutase (SOD) during the preincubation has no effect. Including an NO scavenger[2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] or a guanylate cyclase inhibitor(1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) partially protects against SNAP. Pretreatment of cultured cells with peroxynitrite, but not with SNAP, inhibits the Ca²⁺transients produced in response to CPA. Pretreating isolated membranevesicles with peroxynitrite inhibits the Ca²⁺ uptake due tothe SERCA pump, with all the other agents being less effective. Thusperoxynitrite and NO both inhibit the CPA-induced contractions indeendothelialized artery rings, peroxynitrite by damage to the SERCApump and NO possibly by a step downstream from the increase incytosolic Ca²⁺.

     

Mechanosensitive tyrosine phosphorylation of paxillin and focal adhesion kinase in tracheal smooth muscle

We investigatedthe role of the integrin-associated proteins focal adhesion kinase(FAK) and paxillin as mediators of mechanosensitive signal transductionin tracheal smooth muscle. In muscle strips contracted isometricallywith ACh, we observed higher levels of tyrosine phosphorylation of FAKand paxillin at the optimal muscle length(L_o) than atshorter muscle lengths of 0.5 or 0.75 L_o. Paxillinphosphorylation was also length sensitive in muscles activated byK⁺ depolarization and adjustedrapidly to changes in muscle length imposed after contractileactivation by either ACh or K⁺depolarization. Ca²⁺ depletion didnot affect the length sensitivity of paxillin and FAK phosphorylationin muscles activated with ACh, indicating that the mechanotransductionprocess can be mediated by aCa²⁺-independent pathway. SinceCa²⁺-depleted muscles do notgenerate significant active tension, this suggests that themechanotransduction mechanism is sensitive to muscle length rather thantension. We conclude that FAK and paxillin participate in anintegrin-mediated mechanotransduction process in tracheal smoothmuscle. We propose that this pathway may initiate alterations in smoothmuscle cell structure and contractility via the remodeling of actinfilaments and/or via the mechanosensitive regulation ofsignaling molecules involved in contractile protein activation.

     

Influence of group B streptococci on piglet pulmonary artery response to bradykinin

To study whether a sepsis-induced increase indes-Arg⁹-bradykinin(des-Arg⁹-BK) and bradykinin (BK)B₁-receptor activity participatesin the observed increase in pulmonary vascular resistance in neonatal group B streptococcal sepsis (GBS), isometric force bioassays ofpulmonary artery (PA) rings were studied, after 4-h exposure to eitherKrebs or GBS, by using the following protocols:1) BK dose-response curve,2) vascular response to BK withN^G-nitro-L-arginine methyl ester(L-NAME), and3) response todes-Arg⁹-BK (BK metabolite andB₁ agonist). PA rings exposed toBK resulted in contraction in the GBS group and a decrease in restingtension in the Control group (P = 0.034) at a concentration of10⁵ M. GBS-treated PA ringscontracted more to des-Arg⁹-BKthan did Controls (P < 0.001). BK(10⁶ M) relaxedpreconstricted PA rings incubated in GBS less than BK relaxed Controls(P < 0.001), and preincubation withL-NAME decreased relaxation inboth. These results suggest that GBS decreased endothelium-dependent BKrelaxation and increased contractile response todes-Arg⁹-BK. We speculate thatthis occurs secondary to upregulation of B₁ receptors reflected byB₁-agonist-mediated PA contraction.

     

Failure of prostaglandins to modulate the time course of blood flow during dynamic forearm exercise in humans

Shoemaker, J. K., H. L. Naylor, Z. I. Pozeg, and R. L. Hughson. Failure of prostaglandins to modulate the time course ofblood flow during dynamic forearm exercise in humans.J. Appl. Physiol. 81(4):1516-1521, 1996.The time course and magnitude of increases inbrachial artery mean blood velocity (MBV; pulsed Doppler), diameter(D; echo Doppler), mean perfusionpressure (MPP; Finapres), shear rate ( = 8 ^· MBV/D), andforearm blood flow (FBF = MBV ^· r²)were assessed to investigate the effect that prostaglandins (PGs) haveon the hyperemic response on going from rest to rhythmic exercise inhumans. While supine, eight healthy men performed 5 min of dynamichandgrip exercise by alternately raising and lowering a 4.4-kg weight(~10% maximal voluntary contraction) with a work-to-rest cycle of1:1 (s/s). When the exercise was performed with the arm positionedbelow the heart, the rate of increase in MBV and wasfaster compared with the same exercise performed above the heart.Ibuprofen (Ibu; 1,200 mg/day, to reduce PG-induced vasodilation) andplacebo were administered orally for 2 days before two separate testingsessions in a double-blind manner. Resting heart rate was reduced inIbu (52 ± 3 beats/min) compared with placebo (57 ± 3 beats/min)(P < 0.05) without change to MPP.With placebo, D increased in both armpositions from ~4.3 mm at rest to ~4.5 mm at 5 min of exercise(P < 0.05). This response was notaltered with Ibu (P > 0.05). Ibudid not alter the time course of MBV or forearm blood flow(P > 0.05) in either arm position. The was significantly greater in Ibu vs. placebo at 30 and 40 s of above the heart exercise and for all time points after 25 sof below the heart exercise (P < 0.05). Because PG inhibition altered the time course of at the brachial artery, but not FBF, it was concludedthat PGs are not essential in regulating the blood flow responses todynamic exercise in humans.

     

Effects of nitric oxide synthase inhibition on cutaneous vasodilation during body heating in humans

We sought toexamine further the potential role of nitric oxide (NO) in the neurallymediated cutaneous vasodilation in nonacral skin during body heating inhumans. Six subjects were heated with a water-perfused suit whilecutaneous blood flow was measured by using laser-Doppler flowmetersplaced on both forearms. The NO synthase inhibitorN^G-monomethyl-L-arginine(L-NMMA) was given selectivelyto one forearm via a brachial artery catheter after marked cutaneousvasodilation had been established. During body heating, oraltemperature increased by 1.1 ± 0.1°C while heart rate increasedby 30 ± 6 beats/min. Mean arterial pressure stayed constant at 84 ± 2 mmHg. In the experimental forearm, cutaneous vascularconductance (CVC; laser-Doppler) decreased to 86 ± 5% of the peakresponse to heating (P < 0.05 vs.pre-L-NMMA values) afterL-NMMA infusion. In somesubjects, L-NMMA caused CVC tofall by ~30%; in others, it had little impact on the cutaneouscirculation. CVC in the control arm showed a similar increase withheating, then stayed constant whileL-NMMA was given to thecontralateral side. These results demonstrate that NO contributesmodestly, but not consistently, to cutaneous vasodilation during bodyheating in humans. They also indicate that NO is not the only factorresponsible for the dilation.

     

Nitric oxide and cutaneous active vasodilation during heat stress in humans

Whether nitric oxide (NO) is involved incutaneous active vasodilation during hyperthermia in humans is unclear.We tested for a role of NO in this process during heat stress(water-perfused suits) in seven healthy subjects. Two forearm siteswere instrumented with intradermal microdialysis probes. One site wasperfused with the NO synthase inhibitorN^G-nitro-L-argininemethyl ester (L-NAME)dissolved in Ringer solution to abolish NO production. The other sitewas perfused with Ringer solution only. At those sites, skin blood flow(laser-Doppler flowmetry) and sweat rate were simultaneously andcontinuously monitored. Cutaneous vascular conductance, calculated fromlaser-Doppler flowmetry and mean arterial pressure, was normalized tomaximal levels as achieved by perfusion with the NO donor nitroprusside through the microdialysis probes. Under normothermic conditions, L-NAME did not significantlyreduce cutaneous vascular conductance. During hyperthermia, with skintemperature held at 38-38.5°C, internal temperature rose from36.66 ± 0.10 to 37.34 ± 0.06°C (P < 0.01). Cutaneous vascularconductance at untreated sites increased from 12 ± 2 to 44 ± 5% of maximum, but only rose from 13 ± 2 to 30 ± 5% ofmaximum at L-NAME-treated sites(P < 0.05 between sites) during heatstress. L-NAME had no effect onsweat rate (P > 0.05). Thuscutaneous active vasodilation requires functional NO synthase toachieve full expression.

     

Nitric oxide regulates oxygen uptake and hydrogen peroxide release by the isolated beating rat heart

Isolated rat heart perfused with 1.5-7.5µM NO solutions or bradykinin, which activates endothelial NOsynthase, showed a dose-dependent decrease in myocardial O₂uptake from 3.2 ± 0.3 to 1.6 ± 0.1 (7.5 µM NO, n = 18,P < 0.05) and to 1.2 ± 0.1 µM O₂ · min¹ · gtissue¹ (10 µM bradykinin, n = 10,P < 0.05). Perfused NO concentrations correlated with aninduced release of hydrogen peroxide (H₂O₂) inthe effluent (r = 0.99, P < 0.01). NO markedlydecreased the O₂ uptake of isolated rat heart mitochondria(50% inhibition at 0.4 µM NO, r = 0.99,P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b andcytochrome c, which accounts for the effects in O₂uptake and H₂O₂ release. Most NO was bound tomyoglobin; this fact is consistent with NO steady-state concentrationsof 0.1-0.3 µM, which affect mitochondria. In the intact heart,finely adjusted NO concentrations regulate mitochondrial O₂uptake and superoxide anion production (reflected byH₂O₂), which in turn contributes to thephysiological clearance of NO through peroxynitrite formation.

     

Effects of NO donors and synthase agonists on endothelial cell uptake of L-Arg and superoxide production

It is commonly believed thatthe activity of NO synthase (NOS) solely controls NO production fromits substrates, L-Arg and O₂. The Michaelis-Menten constant(K_m) of NOS forL-Arg is in the micromolarrange; cellular levels of L-Argare much higher. However, evidence strongly suggests that cellularsupply of L-Arg may becomelimiting and lead to reduced NO and increased superoxide anion(O₂·) formation, promotingcardiovascular dysfunction. Uptake ofL-Arg into cells occursprimarily (~85%) through the actions of aNa⁺-independent, carrier-mediatedtransporter (system y⁺). We haveexamined the effects of NOS agonists (substance P, bradykinin, and ACh)and NO donors(S-nitroso-N-acetyl-penicillamine and dipropylenetriamine NONOate) on transport ofL-Arg into bovine aorticendothelial cells (BAEC). Our results demonstrate that NOS agonistsincrease y⁺ transporter activity.A rapidly acting NO donor initially increases L-Arg uptake; however, afterlonger exposure, L-Arg uptake is suppressed. Exposure of BAEC withoutL-Arg to substance P and aCa²⁺ ionophore (A-23187) increasedO₂· formation, which was blockedwith concurrent presence ofL-Arg or the NOS antagonistN-nitro-L-arginine methyl ester.We conclude that factors including NO itself controly⁺ transport function and theproduction of NO and O₂·.