Role of erythrocyte-released ATP in the regulation of microvascular oxygen supply in skeletal muscle.

In a 1914 book entitled The Respiratory Function of the Blood, Joseph Barcroft stated that 'the cell takes what it needs and leaves the rest'. He postulated that there must be both a 'call for oxygen' and a 'mechanism by which the call elicits a response...' In the past century, intensive investigation has provided significant insights into the haemodynamic and biophysical mechanisms involved in supplying oxygen to skeletal muscle. However, the identification of the mechanism by which tissue oxygen needs are sensed and the affector responsible for altering the upstream vasculature to enable the need to be appropriately met has been a challenge. In 1995, Ellsworth et al. proposed that the oxygen-carrying erythrocyte, by virtue of its capacity to release the vasoactive mediator ATP in response to a decrease in oxygen saturation, could serve both roles. Several in vitro and in situ studies have established that exposure of erythrocytes to reduced oxygen tension induces the release of ATP which does result in a conducted arteriolar vasodilation with a sufficiently rapid time course to make the mechanism physiologically relevant. The components of the signalling pathway for the controlled release of ATP from erythrocytes in response to exposure to low oxygen tension have been determined. In addition, the implications of defective ATP release on human pathological conditions have been explored. This review provides a perspective on oxygen supply and the role that such a mechanism plays in meeting the oxygen needs of skeletal muscle.

Simvastatin and GGTI-2133, a geranylgeranyl transferase inhibitor, increase erythrocyte deformability but reduce low O(2) tension-induced ATP release.

Statin drugs inhibit 3-hydroxy-3-methylglutaryl CoA reductase, which reduces the synthesis of both cholesterol and isoprenoids (geranylgeranyl pyrophosphate and farnesyl pyrophosphate), with the latter being lipid molecules responsible for the posttranslational modification of small GTP-binding proteins such as Rho. Effects of statins, independent of lowering blood cholesterol levels, are thought to occur by inhibition of Rho/Rho kinase. The Rho kinase inhibitor Y-27632 has been reported to increase both erythrocyte deformability and low O2 tension-induced ATP release. Here, we tested the hypothesis that by inhibiting Rho/Rho kinase, simvastatin would increase both erythrocyte deformability and low O2 tension-induced ATP release. Male Sprague-Dawley rats were divided into two groups, control or simvastatin treated [simvastatin-supplemented chow (0.02%)], for 4 wk. Simvastatin treatment increased rat erythrocyte deformability compared with controls (n = 6, P < 0.05). However, erythrocytes of simvastatin-treated rats (n = 9, P < 0.05) exhibited impaired low O2 tension-induced ATP release. Similarly, the geranylgeranyl transferase inhibitor GGTI-2133 (10 µM) also increased deformability and impaired low O2 tension-induced ATP release in healthy human erythrocytes (P < 0.05). Interestingly, ATP release in response to mastoparan 7 (n = 7, P < 0.05), which directly activates Gi, and isoproterenol (n = 5, P < 0.05), which signals through Gs, was not altered by incubation with GGTI-2133. These results suggest that although statins increase erythrocyte deformability, likely by inhibiting geranylgeranylation, the finding that both statins and a geranylgeranyl transferase inhibitor attenuated low O2 tension-induced ATP release demonstrates that factors in addition to erythrocyte deformability are critical for ATP release in response to this physiological stimulus.

The Rho kinase inhibitor Y-27632 increases erythrocyte deformability and low oxygen tension-induced ATP release.

Low oxygen (O(2)) tension and mechanical deformation are stimuli for ATP release from erythrocytes. It has been shown previously that rabbit erythrocytes made less deformable with diamide, a thiol cross-linking agent, release less ATP in response to low O(2) tension, suggesting a link between these two stimuli. In nonerythroid cells, activation of the Rho/Rho kinase signaling pathway has been reported to decrease cell deformability by altering Rho kinase-dependent cytoskeleton-protein interactions. We investigated the hypothesis that the Rho kinase inhibitor Y-27632 would increase erythrocyte deformability and thereby increase low O(2) tension-induced ATP release from erythrocytes. Here we show that Y-27632 (1 µM) increases erythrocyte deformability (5%) and increases low O(2) tension-induced ATP release (203%) from healthy human erythrocytes. In addition, we found that, when erythrocytes were made less deformable by incubation with diamide (100 µM), Y-27632 restored both deformability and low O(2) tension-induced ATP release to levels similar to those measured in the absence of diamide. These findings suggest that the Rho kinase inhibitor Y-27632 is able to reverse the diamide-induced decrease in erythrocyte deformability and rescue low O(2) tension-induced ATP release. These results further support a link between erythrocyte deformability and ATP release in response to low O(2) tension.

Erythrocytes as controllers of perfusion distribution in the microvasculature of skeletal muscle.

In 1929, August Krogh identified the matching of oxygen (O(2)) supply with demand in skeletal muscle as a fundamental physiological process. In the intervening decades, much research has been focused on elucidating the mechanisms by which this important process occurs. For any control system to be effective, there must be a means by which the need is determined and a mechanism by which that information is coupled to an appropriate response. The focus of this review was to highlight current research in support of the hypothesis that the mobile erythrocyte, when exposed to reduced O(2) tension, releases ATP in a controlled manner. This ATP interacts with purinergic receptors on the endothelium producing both local and conducted vasodilation enabling the erythrocyte to distribute perfusion to precisely match O(2) delivery with need in skeletal muscle. If this is an important mechanism for normal physiological control of microvascular perfusion, defects in this process would be anticipated to have pathophysiological consequences. Individuals with either type 2 diabetes (DM2) or pre-diabetes have microvascular dysfunction that contributes to morbidity and mortality. DM2 erythrocytes and erythrocytes incubated with insulin at levels similar to those seen in pre-diabetes fail to release ATP in response to reduced O(2) tension. Knowledge of the components of the signal transduction pathway for low O(2) -induced ATP release suggest novel therapeutic approaches to ameliorating this defect. Although the erythrocyte may be but one component of the complex O(2) delivery process, it appears to play an important role in distributing oxygen within the microvasculature.

Timing of fledging is influenced by glucocorticoid physiology in Laysan Albatross chicks.

Fledging is a major life transition for birds, when juveniles move from the safety of a nest into an environment where they must find food and avoid predators. The timing of fledging within a season can have significant effects on future survival and breeding success. Proximate triggers of fledging are unknown: though wing development is likely a primary factor, other physiological changes, such as elevated plasma corticosterone (CORT), may affect fledging behavior. Laysan Albatross (Phoebastria immutabilis) chicks have an extended post-hatching period during which they reach 150% of adult mass. However, approaching fledging, chicks fast for days to weeks and lose mass while still putting energy into feather growth. We evaluated chick morphology and physiology to elucidate proximate triggers of fledging. As in some other species, CORT increased as chicks fasted and lost body mass. At the same time, corticosteroid binding globulin (CBG) declined, thus amplifying free CORT prior to fledging. Once chicks reached a morphological threshold, free CORT levels predicted how long they stayed at the colony: chicks with higher free CORT fledged sooner. To perturb the relationship between body condition, endocrine physiology, and fledging behavior, we supplementally fed chicks for the month before fledging. Fed birds had a slower decrease in body mass, slower decrease in CBG, slower increase in free CORT, and stayed at the colony longer after reaching a morphological threshold. Our study suggests that as chicks lose mass, free CORT acts as a signal of energetic or nutritional state to adjust the timing of fledging.

Plasma-binding globulins and acute stress response.

Within studies of acute stress physiology an increase in glucocorticoid secretion is thought to be the primary mediator of tissue response to stress. Corticosteroid-binding globulin may regulate tissue availability of steroids, but has not been considered a dynamic component of the acute stress response. Here, we examined CBG level over the common 60-minute time frame in an acute capture and handling protocol to investigate whether CBG capacity is dynamic or static over short stressors. Using a comparative approach, we measured CBG response to capture and handling stress in nine species of birds, representing five orders and nine families. CBG capacity significantly declined within 30 - 60 minutes of capture in five of the nine species examined. This decline may serve to significantly increase the level of corticosterone reaching tissues during acute stress.

The role of G protein beta subunits in the release of ATP from human erythrocytes.

Previously, we demonstrated that adenosine triphosphate (ATP) is released from human erythrocytes in response to mechanical deformation and that this release requires activation of a signal-transduction pathway involving adenylyl cyclase and the heterotrimeric G protein, Gs. Here we investigate the role of heterotrimeric G proteins of the Gi subtype in the release of ATP from human erythrocytes. In addition, we determined the profile of heterotrimeric G protein beta subunits present in these erythrocyte membranes. The activity of Gi was stimulated by incubation of erythrocytes (20% hematocrit) with mastoparin (10 microM). ATP release was measured using the luciferin/luciferase assay. Heterotrimeric G protein beta subunits present in erythrocyte membranes were resolved using gel electrophoresis and subunit specific antibodies. Incubation of human erythrocytes with mastoparan (an activator of Gi/o) resulted in a 4.1 +/- 0.6-fold increase in ATP present in the medium (P<0.01). Human erythrocyte membranes stain positively for beta subunit types 1, 2, 3 and 4, all of which been reported to activate of some isoforms of adenylyl cyclase. Activation of the heterotrimeric G protein, Gi, results in ATP release from erythrocytes. This effect is may be related to the activity of beta subunits associated with this G protein in the human erythrocyte.

Participation of cAMP in a signal-transduction pathway relating erythrocyte deformation to ATP release.

Previously, we reported that red blood cells (RBCs) of rabbits and humans release ATP in response to mechanical deformation and that this release of ATP requires the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). It was reported that cAMP, acting through a cAMP-dependent protein kinase, PKA, is an activator of CFTR. Here we investigate the hypothesis that cAMP stimulates ATP release from RBCs. Incubation of human and rabbit RBCs with the direct activator of adenylyl cyclase, forskolin (10 or 100 microM), with IBMX (100 microM), resulted in ATP release and increases in intracellular cAMP. In addition, epinephrine (1 microM), a receptor-mediated activator of adenylyl cyclase, stimulated ATP release from rabbit RBCs. Moreover, incubation of human and rabbit RBCs with an active cAMP analog [adenosine 3'5'-cyclic monophosphorothioate Sp-isomer (Sp-cAMP, 100 microM)] resulted in ATP release. In contrast, forskolin and Sp-cAMP were without effect on dog RBCs, cells known not to release ATP in response to deformation. When rabbit RBCs were incubated with the inactive cAMP analog and inhibitor of PKA activity, adenosine 3',5'-cyclic monophosphorothioate Rp-isomer (100 microM), deformation-induced ATP release was attenuated. These results are consistent with the hypothesis that adenylyl cyclase and cAMP are components of a signal-transduction pathway relating RBC deformation to ATP release from human and rabbit RBCs.

Receptor-mediated activation of the heterotrimeric G-protein Gs results in ATP release from erythrocytes.

BACKGROUND: Adenosine triphosphate (ATP), released from the erythrocyte in response to mechanical deformation, decreased oxygen tension or reduced pH, has been suggested to be an important determinant of vascular resistance in several vascular beds. Mechanical deformation-induced ATP release from rabbit and human erythrocytes was reported to require the activity of the cystic fibrosis transmembrane conductance regulator (CFTR), suggesting that a signal transduction pathway involving CFTR mediates ATP release from erythrocytes. Here we investigate the hypothesis that the heterotrimeric G-protein Gs is also involved in this signal transduction pathway. MATERIALS AND METHODS: The heterotrimeric G-protein Gs was identified in rabbit and human erythrocyte membranes, using gel electrophoresis. The concentration of ATP released into a suspension of erythrocytes, incubated with iloprost or epinephrine, was measured using the luciferin/luciferase assay. RESULTS: The 45 kDa form of the heterotrimeric G-protein Gs was identified in rabbit and human erythrocyte membranes. Incubation of rabbit erythrocytes with iloprost (n=18) or epinephrine (n=6) increased the ATP concentration by 106+/-16% and 156+/-54%, respectively. Epinephrine-induced changes in ATP concentrations were prevented by pretreatment with propranolol. CONCLUSIONS: The heterotrimeric G-protein Gs is present in erythrocyte membranes. Receptor-mediated activation of Gs results in ATP release. These results are consistent with the hypothesis that Gs is a component of a signal transduction pathway for ATP release from erythrocytes.

Impaired release of ATP from red blood cells of humans with primary pulmonary hypertension.

Previously, we reported that in the isolated perfused rabbit lung, red blood cells (RBCs) obtained from either rabbits or healthy humans were a required component of the perfusate to unmask evidence of nitric oxide (NO) participation in regulation of the pulmonary circulation. In addition, we found that mechanical deformation of rabbit and healthy human RBCs released ATP, a known agonist for enhanced NO synthesis. In contrast, RBCs obtained from patients with cystic fibrosis (CF) did not release ATP in response to mechanical deformation. The coexistence of airway disease and alveolar hypoxia in patients with CF precluded the drawing of conclusions relating a defect in RBC ATP release with the pulmonary hypertension associated with CF. Airway disease and alveolar hypoxia are not, however, features of primary pulmonary hypertension (PPH), a human condition of unknown etiology. We postulated that a defect in NO generation might contribute to the increased pulmonary vascular resistance in PPH, and as a first step, we hypothesized that RBCs obtained from patients with PPH would not release ATP. In contrast to RBCs of healthy humans, when RBCs of PPH patients were passed through filters (average pore size 12, 8, or 5 microm), ATP was not released and the RBCs exhibited reduced deformability. Moreover, when incubated with the active cAMP analogue, Sp-cAMP (100 microM), an activator of the CF transmembrane conductance regulator, ATP was not released. These results demonstrate that RBCs obtained from patients with PPH fail to release ATP whether the stimulus is mechanical or pharmacological. Thus, failure of RBCs to release ATP in patients with PPH might be a major pathogenetic factor that accounts for the heretofore unknown etiology of their pulmonary hypertension.

Red blood cell regulation of microvascular tone through adenosine triphosphate.

The matching of blood flow with metabolic need requires a mechanism for sensing the needs of the tissue and communicating that need to the arterioles, the ultimate controllers of tissue perfusion. Despite significant strides in our understanding of blood flow regulation, the identity of the O(2) sensor has remained elusive. Recently, the red blood cell, the Hb-containing O(2) carrier, has been implicated as a potential O(2) sensor and contributor to this vascular control by virtue of its concomitant carriage of millimolar amounts of ATP, which it is able to release when exposed to a low-O(2) environment. To evaluate this possibility, we exposed perfused cerebral arterioles to low extraluminal O(2) in the absence and presence of red blood cells or 6% dextran and determined both vessel diameter and ATP in the vessel effluent. Only when the vessels were perfused with red blood cells did the vessels dilate in response to low extraluminal O(2). In addition, this response was accompanied by a significant increase in vessel effluent ATP. These findings support the hypothesis that the red blood cell itself serves a role in determining O(2) supply to tissue.

Deformation-induced ATP release from red blood cells requires CFTR activity.

Recently, it was reported that rabbit and human red blood cells (RBCs) release ATP in response to mechanical deformation. Here we investigate the hypothesis that the activity of the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP binding cassette, is required for deformation-induced ATP release from RBCs. Incubation of rabbit RBCs with either of two inhibitors of CFTR activity, glibenclamide (10 microM) or niflumic acid (20 microM), resulted in inhibition of deformation-induced ATP release. To demonstrate the contribution of CFTR to deformation-induced ATP release from human RBCs, cells from healthy humans, patients with cystic fibrosis (CF), or patients with chronic obstructive lung disease (COPD) unrelated to CF were studied. RBCs of healthy humans and COPD patients released ATP in response to mechanical deformation. In contrast, deformation of RBCs from patients with CF did not result in ATP release. We conclude that deformation-induced ATP release from rabbit and human RBCs requires CFTR activity, suggesting a previously unrecognized role for CFTR in the regulation of vascular resistance.

5,6-Epoxyeicosatrienoic acid reduces increases in pulmonary vascular resistance in the dog.

We recently reported that canine pulmonary microsomes metabolize arachidonic acid to all four regioisomeric epoxyeicosatrienoic acids (EET). 5,6-EET dilates blood vessels in several nonpulmonary vascular beds, often in a cyclooxygenase-dependent manner. The present study was designed to determine whether 5,6-EET can decrease pulmonary vascular resistance (PVR) in the intact pulmonary circulation. In isolated canine lungs perfused with physiological salt solution, a constant infusion of U-46619 (3.28 +/- 0.99 nmol/min) increased PVR 62.1 +/- 4.5%. Administration of 5,6-EET (10(-5) M) into the perfusate reduced the U-46619-mediated increase in PVR by 23.6 +/- 6.1%. These effects of U-46619 and 5,6-EET were limited to changes in resistance solely in the pulmonary venous segment. In contrast, venous as well as arterial segmental resistances were increased in 5-hydroxytryptamine (5-HT)-treated lungs. However, in the latter instance, 5,6-EET reduced arterial but not venous segmental resistance. 5,6-EET increased pulmonary PGI2 synthesis from 70.5 +/- 18.4 to 675.9 +/- 125.4 ng/min. In the presence of indomethacin (10(-4) M), 5,6-EET did not increase PGI2 synthesis nor did it decrease U-46619- or 5-HT-mediated increases in PVR. In canine intrapulmonary vessels, 5,6-EET decreased active tension in veins contracted with U-46619. 5,6-EET decreased active tension in arteries but not veins contracted with 5-HT, consistent with results in the perfused lungs. These results demonstrate that 5, 6-EET is a vasodilator in the intact pulmonary circulation. Its dilator activity depends on the constrictor agent present, the segmental resistance, and cyclooxygenase activity.

Biosynthesis of sulfidopeptide leukotrienes via the transfer of leukotriene A4 from polymorphonuclear cells to bovine retinal pericytes.

Administration of exogenous sulfidopeptide leukotrienes (LTs) is associated with enhanced microvascular permeability. In addition, endogenous LTs have been implicated as participants in permeability (nonhydrostatic) edema formation. The source of LTs for interaction with the microvasculature is, however, unknown. We hypothesized that pericytes contribute to vascular LT synthesis. Under basal conditions and after incubation with either the calcium ionophore, A23187 (0-1 microM), or arachidonic acid (20 microM), bovine retinal pericytes (BRPs) did not produce significant amounts of sulfidopeptide LTs. In contrast, in the presence of polymorphonuclear leukocytes (PMNs), which can synthesize LTA4, but not sulfidopeptide leukotrienes, incubation of BRPs with A23187 resulted in dose-dependent increases in LTC4/D4/E4 production (peak: 35.4 +/- 5 pg/microg protein; n = 12). Similarly, BRPs, incubated with exogenous, authentic LTA4 (10 microM), synthesized sulfidopeptide LTs (peak: 18.9 +/- 5 pg/microg protein, n = 3). Preincubation (30 min) of BRPs with PMNs and the lipoxygenase inhibitor, esculetin (1 x 10(-)4 M; n = 12), reduced peak A23187-induced production of LTs by 63.9 +/- 7%. Finally, Northern blot analysis revealed mRNA for 5-lipoxygenase to be present in human and bovine PMNs, but not in BRPs. These results suggest that pericytes produce sulfidopeptide LTs only when provided with LTA4 from an external source such as the PMN. Interactions between pericytes and PMNs may lead to the production of sulfidopeptide LTs, which, in turn, could alter microvascular permeability.

Nitric oxide opposes phorbol ester-induced increases in pulmonary microvascular permeability in dogs.

In addition to its effects on vascular tone, nitric oxide (NO) has been suggested to function as a participant in fluid homeostasis affecting interactions between the endothelium and circulating inflammatory cells. The role of NO in the increased microvascular permeability of acute lung injury, however, remains controversial. We investigated the hypothesis that NO opposes increases in pulmonary vascular permeability after phorbol myristate acetate administration, i.e., in a model of neutrophil-dependent acute lung injury. In anesthetized dogs, phorbol myristate acetate (10 microg/kg, i.v.) had no effect on pulmonary arterial pressure (Ppa) or extravascular lung water. After pretreatment with the NO synthesis inhibitor, NG-nitro-L-arginine methyl ester (10 mg/kg, i.v. ; 5 mg/kg/hr), an identical dose of phorbol myristate acetate resulted in a 20 +/- 8 mm Hg (P < .01) increase in pulmonary arterial pressure and a 186 +/- 86% (P < .01) increase in extravascular lung water. To determine if the pulmonary edema was related to increases in microvascular pressure or to changes in the microvascular permeability coefficient, experiments were performed in isolated blood-perfused dog lungs. The addition of phorbol myristate acetate (4.2 x 10(-8) M) to the perfusate was without effect on microvascular pressure or pulmonary capillary filtration coefficient. However, after NG-nitro-L-arginine methyl ester (100 microM), phorbol myristate acetate resulted in increases in both microvascular pressure and permeability coefficient that were prevented by pretreatment with L-arginine (1 mM). These data support the hypothesis that endogenous NO opposes increases in pulmonary vascular permeability as well as microvascular pressure in this neutrophil-dependent model of acute lung injury resulting in preservation of the endothelial barrier to the passage of water and solutes and prevention of the formation of pulmonary edema.

Vascular endothelial growth factor mRNA in pericytes is upregulated by phorbol myristate acetate.

Increased microvascular permeability, which occurs in conditions such as the adult respiratory distress syndrome and diabetes mellitus, is related to physicochemical alterations in the microvascular barrier. We postulate that, in part, capillary pericytes affect microvascular permeability via production of a vasoactive cytokine, viz, vascular endothelial growth factor (VEGF), also known as vascular permeability factor. The goal of the present study was to evaluate the effects of phorbol myristate acetate (PMA), a substance known to produce nonhydrostatic pulmonary edema in intact animals, on VEGF gene expression in pericyte cultures. Microvascular pericytes were isolated from bovine retinas using magnetic microspheres coated with 3G5 monoclonal antibody. Pericyte identity was confirmed both morphologically and by immunostaining for alpha-smooth muscle actin and 3G5 ganglioside. The cultured pericytes were stimulated with N(omega)-nitro-L-arginine methyl ester (L-NAME, 1 x 10(-4) mmol/L), angiotensin II (1 x 10(-6) mmol/L), and PMA (5 x 10(-8) mmol/L), selected because of their ability to upregulate VEGF mRNA expressions in other cell types. Northern blot analysis was performed using [32P]dCTP labeled human VEGF cDNA (Genentech). Lane-loading differences were normalized using mouse GAPDH control cDNA probe. VEGF mRNA expression was upregulated by PMA (10(-9) to 10(-6) mol/L) in a dose-dependent manner, whereas neither L-NAME nor angiotensin II affected VEGF mRNA expression in pericytes. These results support the hypothesis that pericytes increase permeability of the endothelial barrier through increased VEGF production.

ATP: the red blood cell link to NO and local control of the pulmonary circulation.

Recently, we reported that rabbit red blood cells (RBCs) were required for the expression of nitric oxide (NO) activity on pulmonary vascular resistance (PVR) in rabbit lungs. Here, we investigate the hypothesis that RBCs participate in the regulation of PVR via release of ATP in response to mechanical deformation that, in turn, evokes vascular NO synthesis. We found that rabbit and human RBCs, but not dog RBCs, release ATP in response to mechanical deformation. To determine the contribution of this ATP to NO synthesis and PVR, we compared the effects of human and dog RBCs on pressure-flow relationships in isolated rabbit lungs. In the presence of human RBCs, NG-nitro-L-arginine methyl ester (100 microM) produced a shift in the pressure-flow relationship consistent with a reduction in vascular caliber. NG-nitro-L-arginine methyl ester had no effect in lungs perfused with dog RBCs. These results suggest a unique mechanism for the control of PVR in rabbits and humans whereby release of ATP by RBCs in response to mechanical deformation leads to stimulation of NO synthesis that, in turn, modulates the PVR.

Inhibition of cytochrome P-450 attenuates hypoxemia of acute lung injury in dogs.

The intravenous administration of ethchlorvynol (ECV), in dogs, resulted in an acute lung injury (ALI) characterized by a 200 +/- 80% increase in venous admixture and a 142 +/- 30% increase in extravascular lung water (EVLW). Pretreatment with the cytochrome P-450 inhibitor 8-methoxypsoralen prevented the ECV-induced increase in venous admixture but not the increased EVLW. These findings parallel those reported for cyclooxygenase inhibition in ECV-induced ALI and suggest that an arachidonic acid (AA) metabolite of pulmonary cytochrome P-450 activity may mediate the increase in venous admixture of ALI. We demonstrate that canine pulmonary microsomes metabolize [1-(14)C]AA to a variety of products, including the cytochrome P-450 metabolites 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid (EET). In prostaglandin F2 alpha-contracted, isolated pulmonary venous rings, 5,6-EET induced relaxation in a concentration-dependent manner. This action of 5,6-EET was prevented by indomethacin (10(-5) M). These results suggest that may serve as the cyclooxygenase-dependent endogenous pulmonary vasodilator responsible for the increase in venous admixture of ECV-induced ALI.

Hypotheses regarding the role of pericytes in regulating movement of fluid, nutrients, and hormones across the microcirculatory endothelial barrier.

A decade ago, we initiated studies to define relationship(s) between products of 5-lipoxygenase-mediated arachidonic acid metabolism and altered microvascular permeability. Patients with permeability (nonhydrostatic) pulmonary edema (adult respiratory distress syndrome) and intact animal models of permeability edema, produced with agents that required neutrophils (phorbol myristate acetate) and those that did not (ethchlorvynol), invariably revealed the presence of leukotrienes; in contrast, leukotrienes were not detected in cases of hydrostatic pulmonary edema. In isolated perfused canine lung, we identified increases in microvascular permeability coefficients in response to the injurious agent. Permeability coefficients were not increased when injurious agents were given in the presence of 5-lipoxygenase inhibitors. To define further the relationships between leukotriene generation and edema formation, we postulated that leukotrienes effected contraction of capillary pericytes, thereby increasing pore size of endothelial intercellular junctions and enhancing movement across the microvascular barrier. We isolated pericytes from bovine retinas, identified them morphologically and by staining characteristics, and, in preliminary experiments, found that they do not possess the 5-lipoxygenase enzyme; however, when cocultured with neutrophils, which possess 5-lipoxygenase but cannot synthesize sulfidopeptide leukotrienes because of their lack of glutathione S-transferase, sulfidopeptide leukotriene synthesis ensued. In view of the anatomic position of pericytes, evidence that they participate in endothelial transport, their ability to contract, and evidence of cell-to-cell communication, we propose that pericytes control the movement of fluid, solutes, hormones, and small and large molecules across the microvascular endothelium.

Effect of L-NAME on pressure-flow relationships in isolated rabbit lungs: role of red blood cells.

Nitric oxide (NO) is produced by and relaxes pulmonary arteries and veins; however, a role for NO as a participant in the control of pulmonary vascular resistance (PVR) remains to be defined. Here we investigated the hypothesis that for NO to serve as a determinant of PVR in the rabbit requires the presence of blood. In isolated blood-perfused rabbit lungs, NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) increased PVR and the slope of the pressure-flow relationship. These effects of L-NAME were prevented by pretreatment with L-arginine. In contrast, in lungs perfused with a physiological salt solution, L-NAME had no effect on PVR or the pressure-flow relationship. The addition of washed red blood cells (RBCs) to physiological salt solution, but not the addition of plasma and platelets, restored the response to L-NAME. This effect of RBCs was not reproduced by increasing perfusate viscosity with dextran. These results suggest that, in the rabbit lung, NO is a determinant of PVR in the presence of blood. Moreover, that aspect of blood that permits the generation of NO appears to be related to the RBC and not to perfusate viscosity.