A perpetual switching system in pulmonary capillaries.

Of the 300 billion capillaries in the human lung, a small fraction meet normal oxygen requirements at rest, with the remainder forming a large reserve. The maximum oxygen demands of the acute stress response require that the reserve capillaries are rapidly recruited. To remain primed for emergencies, the normal cardiac output must be parceled throughout the capillary bed to maintain low opening pressures. The flow-distributing system requires complex switching. Because the pulmonary microcirculation contains contractile machinery, one hypothesis posits an active switching system. The opposing hypothesis is based on passive switching that requires no regulation. Both hypotheses were tested ex vivo in canine lung lobes. The lobes were perfused first with autologous blood, and capillary switching patterns were recorded by videomicroscopy. Next, the vasculature of the lobes was saline flushed, fixed by glutaraldehyde perfusion, flushed again, and then reperfused with the original, unfixed blood. Flow patterns through the same capillaries were recorded again. The 16-min-long videos were divided into 4-s increments. Each capillary segment was recorded as being perfused if at least one red blood cell crossed the entire segment. Otherwise it was recorded as unperfused. These binary measurements were made manually for each segment during every 4 s throughout the 16-min recordings of the fresh and fixed capillaries (>60,000 measurements). Unexpectedly, the switching patterns did not change after fixation. We conclude that the pulmonary capillaries can remain primed for emergencies without requiring regulation: no detectors, no feedback loops, and no effectors-a rare system in biology. NEW & NOTEWORTHY The fluctuating flow patterns of red blood cells within the pulmonary capillary networks have been assumed to be actively controlled within the pulmonary microcirculation. Here we show that the capillary flow switching patterns in the same network are the same whether the lungs are fresh or fixed. This unexpected observation can be successfully explained by a new model of pulmonary capillary flow based on chaos theory and fractal mathematics.

Impaired compensation to femoral artery ligation in diet-induced obese mice is primarily mediated via suppression of collateral growth by Nox2 and p47phox.

The present study was undertaken to establish the role of NADPH oxidase (Nox) in impaired vascular compensation to arterial occlusion that occurs in the presence of risk factors associated with oxidative stress. Diet-induced obese (DIO) mice characterized by multiple comorbidities including diabetes and hyperlipidemia were used as a preclinical model. Arterial occlusion was induced by distal femoral artery ligation in lean and DIO mice. Proximal collateral arteries were identified as the site of major (∼70%) vascular resistance to calf perfusion by distal arterial pressures, which decreased from ∼80 to ∼30 mmHg with ligation in both lean and DIO mice. Two weeks after ligation, significant vascular compensation occurred in lean but not DIO mice as evidenced by increased perfusion (147 ± 48% vs. 49 ± 29%) and collateral diameter (151 ± 30% vs. 44 ± 17%). Vascular mRNA expression of p22(phox), Nox2, Nox4, and p47(phox) were all increased in DIO mice. Treatment of DIO mice with either apocynin or Nox2ds-tat or with whole body ablation of either Nox2 or p47(phox) ameliorated the impairment in both collateral growth and hindlimb perfusion. Multiparametric flow cytometry analysis demonstrated elevated levels of circulating monocytes in DIO mice without impaired mobilization and demargination after femoral artery ligation. These results establish collateral resistance as the major limitation to calf perfusion in this preclinical model, demonstrate than monocyte mobilization and demarginatin is not suppressed, implicate Nox2-p47(phox) interactions in the impairment of vascular compensation to arterial occlusion in DIO mice, and suggest that selective Nox component suppression/inhibition may be effective as either primary or adjuvant therapy for claudicants.

Is the real in vivo nitric oxide concentration pico or nano molar? Influence of electrode size on unstirred layers and NO consumption.

OBJECTIVE: There is a debate if the [NO] required to influence vascular smooth muscle is below 50 nM or much higher. Electrodes with 30 µm and larger diameter report [NO] below 50 nM, whereas those with diameters of <10-12 µm report hundreds of nM. This study examined how size of electrodes influenced [NO] measurement due to NO consumption and unstirred layer issues. METHODS: Electrodes were 2 mm disk, 30 µm × 2 mm carbon fiber, and single 7 µm diameter carbon fiber within open tip microelectrode, and exposed 7 µm carbon fiber of ~15 µm to 2 mm length. RESULTS: All electrodes demonstrated linear calibrations with sufficient stirring. As stirring slowed, 30 µm and 2 mm electrodes reported much lower [NO] due to unstirred layers and high NO consumption. The three 7 µm microelectrodes had minor stirring issues. With limited stirring with NO present, 7 µm open tip microelectrodes advanced toward 30 µm and 2 mm electrodes experienced dramatically decreased current within 10-50 µm of the larger electrodes due to high NO consumption. None of the 7 µm microelectrodes interacted. CONCLUSIONS: The data indicate large electrodes underestimate [NO] due to excessive NO consumption under conditions where unstirred layers are unavoidable and true microelectrodes are required for valid measurements.

Rapid and slow nitric oxide responses during conducted vasodilation in the in vivo intestine and brain cortex microvasculatures.

Conduction of arteriolar vasodilation is initiated by activation of nitric oxide (NO) mechanisms, but dependent on conduction of hyperpolarization. Most studies have used brief (<1 second) activation of the initial vasodilation to evaluate the fast conduction processes. However, most arteriolar mechanisms involving NO production persist for minutes. In this study, fast and slower components of arteriolar conduction in the in vivo rat brain and small intestine were compared using three-minute stimulation of NO-dependent vasodilation and measurement of [NO] at the distal sites. Within 10-15 seconds, both vasculatures had a rapidly conducted vasodilation and dilation at distance had a fast but small [NO] component. A slower but larger distal vasodilation occurred after 60-90 seconds in the intestine, but not the brain, and was associated with a substantial increase in [NO]. This slowly developed dilation appeared to be caused by flow mediated responses of larger arterioles as smaller arterioles dilated to lower downstream resistance. These results indicate while the intestinal and cerebral arterioles have a fast conducted vasodilation system, the intestinal arterioles also have a slower but larger dilation of major arterioles that is NO related and dependent on the conduction of vasodilation between small arterioles.

Nitric oxide formation by lymphatic bulb and valves is a major regulatory component of lymphatic pumping.

Microscopic lymphatics produce nitric oxide (NO) during contraction as flow shear activates the endothelial cells. The valve leaflets and bulbous valve housing contain a large amount of endothelial nitric oxide synthase (eNOS) due both to many endothelial cells and increased expression of eNOS. Direct NO measurements indicate the valve area has a 30-50% higher NO concentration ([NO]) than tubular regions although both regions generate equivalent relative increases in [NO] with each contraction. We hypothesize that 1) the greater eNOS and [NO] of the bulb region would have greater effects to lower pumping activity of the overall lymphatic than occurs in tubular regions and 2), the elevated [NO] in the bulb region may be because of high NO production in the valve leaflets that diffuses to the wall of the bulb. Measurement of [NO] with a micropipette inside the lymphatic bulb revealed the valve leaflets generate ~50% larger [NO] than the bulb wall in the in vivo rat mesenteric lymphatics. The valves add NO to the lymph that quickly diffuses to the bulb wall. Bradykinin locally released iontophoretically from a micropipette on both bulbs and tubes increased the [NO] in a dose-dependent manner up to ~50%, demonstrating agonist activation of the NO pathway. However, pumping output determined by contraction frequency and stroke volume decreased much more for the bulb than tubular areas in response to the bradykinin. In effect, NO generation by the bulb area and its valves limits the pumped flow of the total lymphatic by lowering frequency and stroke volume of individual contractions.

Abnormal nitric oxide production in aged rat mesenteric arteries is mediated by NAD(P)H oxidase-derived peroxide.

Previous work in our laboratory showed increased basal periarterial nitric oxide (NO) and H2O2 concentrations in the spontaneously hypertensive rat, characterized by oxidant stress, as well as impaired flow-mediated NO production that was corrected by a reduction of periarterial H2O2. Aging is also associated with an increase in vascular reactive oxygen species and results in abnormal vascular function. The current study was designed to assess the role of H2O2 in regulating NO production during vascular aging. In vivo, real-time NO and H2O2 concentrations were measured by microelectrodes in mesenteric arteries of retired breeder (aged; 8-12 mo) and young (2 to 3 mo) Wistar-Kyoto rats under conditions of altered flow. The results in aged rats revealed elevated basal NO (1,611+/-286 vs. 793+/-112 nM, P<0.05) and H2O2 concentrations (16+/-2 vs. 9+/-1 microM, P<0.05) and a flow-mediated increase in H2O2 but not NO production. Pretreatment of aged rats with the antioxidant apocynin lowered both basal H2O2 (8+/-1 microM) and NO (760+/-102 nM) to young levels and restored flow-mediated NO production. Similar results were obtained with the NAD(P)H oxidase inhibitor gp91ds-tat. In addition, acute incubation with topical polyethylene-glycolated catalase lowered the baseline NO concentration and restored flow-mediated NO production. Taken together, the data indicate that elevated baseline and suppressed flow-mediated NO production in aged Wistar-Kyoto rats are mediated by NAD(P)H oxidase-derived H2O2.

Transfer of nitric oxide by blood from upstream to downstream resistance vessels causes microvascular dilation.

The discovery that hemoglobin, albumin, and glutathione carry and release nitric oxide (NO) may have consequences for movement of NO by blood within microvessels. We hypothesize that NO in plasma or bound to proteins likely survives to downstream locations. To confirm this hypothesis, there must be a finite NO concentration ([NO]) in arteriolar blood, and upstream resistance vessels must be able to increase the vessel wall [NO] of downstream arterioles. Arteriolar blood NO was measured with NO-sensitive microelectrodes, and vessel wall [NO] was consistently 25-40% higher than blood [NO]. Localized suppression of NO production in large arterioles over 500-1,000 microm with L-nitroarginine reduced the [NO] approximately 40%, indicating as much as 60% of the wall NO was from blood transfer. Flow in mesenteric arteries was elevated by occlusion of adjacent arteries to induce a flow-mediated increase in arterial NO production. Both arterial wall and downstream arteriolar [NO] increased and the arterioles dilated as the blood [NO] was increased. To study receptor-mediated NO generation, bradykinin was locally applied to upstream large arterioles and NO measured there and in downstream arterioles. At both sites, [NO] increased and both sets of vessels dilated. When isoproterenol was applied to the upstream vessels, they dilated, but neither the [NO] or diameter downstream arterioles increased. These observations indicate that NO can move in blood from upstream to downstream resistance vessels. This mechanism allows larger vessels that generate large [NO] to influence vascular tone in downstream vessels in response to both flow and receptor stimuli.

Phasic contractions of rat mesenteric lymphatics increase basal and phasic nitric oxide generation in vivo.

Multiple investigators have shown interdependence of lymphatic contractions on nitric oxide (NO) activity by pharmacological and traumatic suppression of endothelial NO synthase (eNOS). We demonstrated that lymphatic diastolic relaxation is particularly sensitive to NO from the lymphatic endothelium. The predicted mechanism is shear forces produced by the lymph flow during phasic pumping, activating eNOS in the lymphatic endothelium to produce NO. We measured [NO] during phasic contractions using microelectrodes on in situ mesenteric lymphatics in anesthetized rats under basal conditions and with an intravenous saline bolus (0.5 ml/100 g) or infusion (0.5 ml x 100 g(-1) x h(-1)). Under basal conditions, [NO] measured on the tubular portions of the lymphatics was approximately 200-250 nM, slightly higher than in the adjacent adipocyte microvasculature, whereas [NO] measured on the lymphatic bulb surface was approximately 400 nM. Immunohistochemistry of eNOS in isolated lympathics indicated a much greater expression in the lymph valves and surrounding bulb area than in the tubular regions. During phasic lymphatic contractions, the valve and tubular [NO] increased with each contraction, and during intravenous saline infusion, [NO] increased in proportion to the contraction frequency and, presumably, lymph flow. The partial blockade of eNOS over approximately 1 cm length with N(omega)-nitro-L-arginine methyl ester lowered the [NO]. These in vivo data document for the first time that both valvular and tubular lymphatic segments increase NO generation during each phasic contraction and that [NO] summated with increased contraction frequency. The combined data predict regional variations in eNOS and [NO] in the tubular and valve areas, plus the summated NO responses dependent on contraction frequency provide for a complex relaxation mechanism involving NO.

Periadventitial adipose tissue impairs coronary endothelial function via PKC-beta-dependent phosphorylation of nitric oxide synthase.

Endogenous periadventitial adipose-derived factors have been shown to contribute to coronary vascular regulation by impairing endothelial function through a direct inhibition of endothelial nitric oxide synthase (eNOS). However, our understanding of the underlying mechanisms remains uncertain. Accordingly, this study was designed to test the hypothesis that periadventitial adipose tissue releases agents that attenuate coronary endothelial nitric oxide production via a protein kinase C (PKC)-beta-dependent mechanism. Isometric tension studies were conducted on isolated canine circumflex coronary arteries with and without natural amounts of periadventitial adipose tissue. Adipose tissue significantly diminished coronary endothelial-dependent vasodilation and nitric oxide production in response to bradykinin and acetylcholine. The selective inhibition of endothelial PKC-beta with ruboxistaurin (1 microM) abolished the adipose-induced impairment of bradykinin-mediated coronary vasodilation and the endothelial production of nitric oxide. Western blot analysis revealed a significant increase in eNOS phosphorylation at the inhibitory residue Thr(495) in arteries exposed to periadventitial adipose tissue. This site-specific phosphorylation of eNOS was prevented by the inhibition of PKC-beta. These data demonstrate that periadventitial adipose-derived factors impair coronary endothelial nitric oxide production via a PKC-beta-dependent, site-specific phosphorylation of eNOS at Thr(495).

Independent regulation of periarteriolar and perivenular nitric oxide mechanisms in the in vivo hamster cheek pouch microvasculature.

OBJECTIVE: We tested the hypothesis that differential stimulation of nitric oxide (NO) production can be induced in pre- and postcapillary segments of the microcirculation in the hamster cheek pouch. MATERIALS AND METHODS: We applied acetylcholine (ACh) or platelet-activating factor (PAF) topically and measured perivascular NO concentration ([NO]) with NO-sensitive microelectrodes in arterioles and venules of the hamster cheek pouch. We also measured NO in cultured coronary endothelial cells (CVEC) after ACh or PAF. RESULTS: ACh increased periarteriolar [NO] significantly in a dose-dependent manner. ACh at 1 microM increased [NO] from 438.1+/-43.4 nM at baseline to 647.9+/-66.3 nM, while 10 microM of ACh increased [NO] from baseline to 1,035.0+/-59.2 nM (P<0.05). Neither 1 nor 10 microM of ACh changed perivenular [NO] in the hamster cheek pouch. PAF, at 100 nM, increased perivenular [NO] from 326.6+/-50.8 to 622.8+/-41.5 nM. Importantly, 100 nM of PAF did not increase periarteriolar [NO]. PAF increased [NO] from 3.6+/-2.1 to 455.5+/-19.9 in CVEC, while ACh had no effect. CONCLUSIONS: We conclude that NO production can be stimulated in a differential manner in pre- and postcapillary segments in the hamster cheek pouch. ACh selectively stimulates the production of NO only in arterioles, while PAF stimulates the production of NO only in venules.

NAD(P)H oxidase-derived peroxide mediates elevated basal and impaired flow-induced NO production in SHR mesenteric arteries in vivo.

Nitric oxide (NO) and reactive oxygen species (ROS) have fundamentally important roles in the regulation of vascular tone and remodeling. Although arterial disease and endothelial dysfunction alter NO and ROS levels to impact vasodilation and vascular structure, direct measurements of these reactive species under in vivo conditions with flow alterations are unavailable. In this study, in vivo measurements of NO and H2O2 were made on mesenteric arteries to determine whether antioxidant therapies could restore normal NO production in spontaneously hypertensive rats (SHR). Flow was altered from approximately 50-200% of control in anesthetized Wistar-Kyoto rats (WKY) and SHR by selective placement of microvascular clamps on adjacent arteries while NO and H2O2 were directly measured with microelectrodes. Relative to WKY, SHR had significantly increased baseline NO and H2O2 concentrations (2,572 +/- 241 vs. 1,059 +/- 160 nM, P < 0.01; and 26 +/- 7 vs. 7 +/- 1 microM, P < 0.05, respectively). With flow elevation, H2O2 but not NO increased in SHR; NO but not H2O2 was elevated in WKY. Apocynin and polyethylene-glycolated catalase decreased baseline SHR NO and H2O2 to WKY levels and restored flow-mediated NO production. Suppression of NAD(P)H oxidase with gp91ds-tat decreased SHR H2O2 to WKY levels. Addition of topical H2O2 to increase peroxide to the basal concentration measured in SHR elevated WKY NO to levels observed in SHR. The results support the hypothesis that increased vascular peroxide in SHR is primarily derived from NAD(P)H oxidase and increases NO concentration to levels that cannot be further elevated with increased flow. Short-term and even acute administration of antioxidants are able to restore normal flow-mediated NO signaling in young SHR.

Endogenous adipose-derived factors diminish coronary endothelial function via inhibition of nitric oxide synthase.

Adipocytokines may be the molecular link between obesity and vascular disease. However, the effects of these factors on coronary vascular function have not been discerned. Accordingly, the goal of this investigation was to delineate the mechanisms by which endogenous adipose-derived factors affect coronary vascular endothelial function. Both isolated canine coronary arteries and coronary blood flow in anesthetized dogs were studied with and without exposure to adipose tissue. Infusion of adipose-conditioned buffer directly into the coronary circulation did not change baseline hemodynamics; however, endothelial-dependent vasodilation to bradykinin was impaired both in vitro and in vivo. Coronary vasodilation to sodium nitroprusside was unaltered by adipose tissue. Oxygen radical formation did not cause the impairment because quantified dihydroethidium staining was decreased by adipose tissue and neither a superoxide dismutase mimetic nor catalase improved endothelial function. Inhibition of nitric oxide (NO) synthase with L-NAME diminished bradykinin-mediated relaxations and eliminated the subsequent vascular effects of adipose tissue. In vitro measurement of NO demonstrated that adipose tissue exposure quickly lowered baseline NO and abolished bradykinin-induced NO production. The results indicate that adipose tissue releases factor(s) that selectively impair endothelial-dependent dilation via inhibition of NO synthase-mediated NO production.

Cerebral microvascular nNOS responds to lowered oxygen tension through a bumetanide-sensitive cotransporter and sodium-calcium exchanger.

Na(+) cotransporters have a substantial role in neuronal damage during brain hypoxia. We proposed these cotransporters have beneficial roles in oxygen-sensing mechanisms that increase periarteriolar nitric oxide (NO) concentration ([NO]) during mild to moderate oxygen deprivation. Our prior studies have shown that cerebral neuronal NO synthase (nNOS) is essential for [NO] responses to decreased oxygen tension and that endothelial NO synthase (eNOS) is of little consequence. In this study, we explored the mechanisms of three specific cotransporters known to play a role in the hypoxic state: KB-R7943 for blockade of the Na(+)/Ca(2+) exchanger, bumetanide for the Na(+)-K(+)-2Cl(-) cotransporter, and amiloride for Na(+)/H(+) cotransporters. In vivo measurements of arteriolar diameter and [NO] at normal and locally reduced oxygen tension in the rat parietal cortex provided the functional analysis. As previously found for intestinal arterioles, bumetanide-sensitive cotransporters are primarily responsible for sensing reduced oxygen because the increased [NO] and dilation were suppressed. The Na(+)/Ca(2+) exchanger facilitated increased NO formation because blockade also suppressed [NO] and dilatory responses to decreased oxygen. Amiloride-sensitive Na(+)/H(+) cotransporters did not significantly contribute to the microvascular regulation. To confirm that nNOS rather than eNOS was primarily responsible for NO generation, eNOS was suppressed with the fusion protein cavtratin for the caveolae domain of eNOS. Although the resting [NO] decreased and arterioles constricted as eNOS was suppressed, most of the increased NO and dilatory response to oxygen were preserved because nNOS was functional. Therefore, nNOS activation secondary to Na(+)-K(+)-2Cl(-) cotransporter and Na(+)/Ca(2+) exchanger functions are key to cerebral vascular oxygen responses.

Intrusion of fluids into nanogrooves: how geometry determines the shape of the gas-liquid interface.

We study the shape of gas-liquid interfaces forming inside rectangular nanogrooves (i.e., slit-pores capped on one end). On account of purely repulsive fluid-substrate interactions the confining walls are dry (i.e., wet by vapor) and a liquid-vapor interface intrudes into the nanogrooves to a distance determined by the pressure (i.e., chemical potential). By means of Monte Carlo simulations in the grand-canonical ensemble (GCEMC) we obtain the density rho(z) along the midline (x = 0) of the nanogroove for various geometries (i.e., depths D and widths L) of the nanogroove. We analyze the density profiles with the aid of an analytic expression which we obtain through a transfer-matrix treatment of a one-dimensional effective interface Hamiltonian. Besides geometrical parameters such as D and L , the resulting analytic expression depends on temperature T , densities of coexisting gas and liquid phases in the bulk rho g,l(x) and the interfacial tension gamma. The latter three quantities are determined in independent molecular dynamics simulations of planar gas-liquid interfaces. Our results indicate that the analytic formula provides an excellent representation of rho(z) as long as L is sufficiently small. At larger L the meniscus of the intruding liquid flattens. Under these conditions the transfer-matrix analysis is no longer adequate and the agreement between GCEMC data and the analytic treatment is less satisfactory.

Extracellular arginine rapidly dilates in vivo intestinal arteries and arterioles through a nitric oxide mechanism.

OBJECTIVE: Arginine used for nitric oxide formation can be from intracellular stores or transported into cells. The study evaluated the rapidity, and primary site of NO and vascular resistance responses to arginine at near physiological concentrations (100-400 microM). METHODS: Arginine was applied to a single arteriole through a micropipette to determine the fastest possible responses. For vascular blood flow and [NO] responses, arginine was added to the bathing media. RESULTS: Dilation of single arterioles to arginine began in 10-15 seconds and application over the entire vasculature increased [NO] in approximately 60-90 seconds, and flow increased within 120-300 seconds. Resting periarteriolar [NO] for arterioles was 493.6 +/- 30.5 nM and increased to 696.1 +/- 68.2 and 820.1 +/- 110.5 nM at 200 and 400 microM L-arginine. The blood flow increased 50% at 400-1200 microM L-arginine. The reduced arterial resistance during topical arginine was significantly greater than microvascular resistance at 100 and 200 microM arginine. All responses were blocked by L-NAME. CONCLUSIONS: This study demonstrated arterial resistance responses are as or more responsive to arginine induced NO formation as arterioles at near physiological concentrations of arginine. The vascular NO and resistance responses occurred rapidly at L-arginine concentrations at and below 400 microM, which predict arginine transport processes were involved.

Nitric oxide production by mouse renal tubules can be increased by a sodium-dependent mechanism.

Renal tubules process large amounts of NaCl that other investigators indicate increases tubular generation of nitric oxide. We questioned whether medullary or superficial cortical tubules would have the greater increase in nitric oxide concentration, [NO], when stressed by sodium and if the sodium/calcium exchanger was involved. Sodium stress in proximal tubules is due to the large amount of sodium absorbed and medullary tubules exist in a hypertonic sodium environment. To sodium stress the tissue, mouse kidney slices were exposed to monensin to allow passive entry of sodium ions from isotonic media and in separate studies, 400 and 600 mOsm NaCl was used. [NO] was measured with microelectrodes. Monensin (10 microM) caused a sustained increase in medullary and cortical [NO] to approximately 180% of control and 400 mOsm NaCl caused a similar initial increase in [NO] that then subsided. 600 mOsm NaCl caused a more sustained increase in [NO] of >250% of control. L-NAME strongly attenuated the increased [NO] during sodium stress. The increase in [NO] during NaCl elevation was due to sodium ions because mannitol hyperosmolarity caused approximately 20% of the increase in [NO]. Entry of sodium during NaCl hyperosmolarity was through bumetanide sensitive channels because the drug suppressed increased [NO]. Blockade of the sodium/calcium ion exchanger strongly suppressed the increased [NO] during monensin, to increase sodium entry into cells, and the elevated NaCl concentration. The data support a sodium-NO linkage that increased NO signaling in proportion to sodium stress by cortical tubules and was highly dependent upon sodium-calcium exchange.

Cerebral microvascular dilation during hypotension and decreased oxygen tension: a role for nNOS.

Endothelial (eNOS) and neuronal nitric oxide synthase (nNOS) are implicated as important contributors to cerebral vascular regulation through nitric oxide (NO). However, direct in vivo measurements of NO in the brain have not been used to dissect their relative roles, particularly as related to oxygenation of brain tissue. We found that, in vivo, rat cerebral arterioles had increased NO concentration ([NO]) and diameter at reduced periarteriolar oxygen tension (Po(2)) when either bath oxygen tension or arterial pressure was decreased. Using these protocols with highly selective blockade of nNOS, we tested the hypothesis that brain tissue nNOS could donate NO to the arterioles at rest and during periods of reduced perivascular oxygen tension, such as during hypotension or reduced local availability of oxygen. The decline in periarteriolar Po(2) by bath manipulation increased [NO] and vessel diameter comparable with responses at similarly decreased Po(2) during hypotension. To determine whether the nNOS provided much of the vascular wall NO, nNOS was locally suppressed with the highly selective inhibitor N-(4S)-(4-amino-5-[aminoethyl]aminopentyl)-N'-nitroguanidine. After blockade, resting [NO], Po(2), and diameters decreased, and the increase in [NO] during reduced Po(2) or hypotension was completely absent. However, flow-mediated dilation during occlusion of a collateral arteriole did remain intact after nNOS blockade and the vessel wall [NO] increased to approximately 80% of normal. Therefore, nNOS predominantly increased NO during decreased periarteriolar oxygen tension, such as that during hypotension, but eNOS was the dominant source of NO for flow shear mechanisms.

Transport of extracellular l-arginine via cationic amino acid transporter is required during in vivo endothelial nitric oxide production.

In cultured endothelial cells, 70-95% of extracellular l-arginine uptake has been attributed to the cationic amino acid transporter-1 protein (CAT-1). We tested the hypothesis that extracellular l-arginine entry into endothelial cells via CAT-1 plays a crucial role in endothelial nitric oxide (NO) production during in vivo conditions. Using l-lysine, the preferred amino acid transported by CAT-1, we competitively inhibited extracellular l-arginine transport into endothelial cells during conditions of NaCl hyperosmolarity, low oxygen, and flow increase. Our prior studies indicate that each of these perturbations causes NO-dependent vasodilation. The perivascular NO concentration ([NO]) and blood flow were determined in the in vivo rat intestinal microvasculature. Suppression of extracellular l-arginine transport significantly and strongly inhibited increases in vascular [NO] and intestinal blood flow during NaCl hyperosmolarity, lowered oxygen tension, and increased flow. These results suggest that l-arginine from the extracellular space is accumulated by CAT-1. When CAT-1-mediated transport of extracellular l-arginine into endothelial cells was suppressed, the endothelial cell NO response to a wide range of physiological stimuli was strongly depressed.