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.

Analysis of the interactions of Nrf-2, PMF-1, and CSN-7 with the 5'-flanking sequence of the mouse 4E-BP1 gene.

Nuclear factor erythroid 2-related factor 2 (Nrf-2) binds to a specific polyamine responsive element (PRE) in the promoter region of the spermidine-spermine acetyltransferase (SSAT) gene, a key component of the polyamine catabolic pathway. Regulation of SSAT gene transcription requires the additional interaction of Nrf-2 with polyamine modulated factor 1 (PMF-1). Likewise, transcription of the eukaryotic initiation factor 4E binding protein 1 (4E-BP1) gene is regulated in a polyamine-dependent manner, but the actual mechanism has not previously been determined. Analysis of the 5'-flanking sequence of the murine 4E-BP1 gene indicated the presence of several potential PRE sites, which might be involved in regulating its transcription. Our goal in this research was to determine potential interactions between Nrf-2, PMF-1, the human homologue of the Arabidopsis signalosome complex (CSN-7), and these potential PRE sites. Four PCR fragments containing regions with considerable homology (78%) to the human PRE were generated from the 5'-flanking sequence of the mouse 4E-BP1 gene and the fragments were used in electrophoretic gel mobility shift and supershift assays. Purified Nrf-2 interacted with all four of these fragments, and similar gel shifts were observed with both cytoplasmic and nuclear fractions of NIH-3T3 cells. However, polyamine depletion with difluoromethylornithine (DFMO) eliminated the gel shift. Supershift assays indicated that the shift was due to the binding of Nrf-2, and the binding was competitive with a known Nrf-2 binding sequence. Purified PMF-1 did not bind any of the PCR fragments alone, but when added with Nrf-2, decreased the magnitude of the gel shift for one of the fragments (PRE located at -2060 relative to the transcription start site). CSN-7 did not interact with the sequences, nor did it inhibit protein/DNA interaction. These data indicate a possible mechanism by which polyamines enhance the binding of a Nrf-2/PMF-1 complex to the 5'-flanking region of the 4E-BP1 gene. Since polyamines increase expression of the 4E-BP1 gene, it seems likely that formation of this complex is involved in its transcriptional regulation.

Polyamines regulate eukaryotic initiation factor 4E-binding protein 1 gene transcription.

Difluoromethylornithine-induced polyamine depletion produced a significant fall in the rate of 4E-BP1 gene transcription in IEC-6 cells, without a change in stability of the 4E-BP1 message. The effect was reversed by the addition of exogenous putrescine. Decreased 4E-BP1 gene transcription produced a concomitant fall in steady-state concentration of the 4E-BP1 protein. Segments of the 4E-BP1 gene 5' flanking sequence were inserted into a GFP reporter construct. While all the segments containing the first 500 nucleotides 5' to exon 1 were capable of driving GFP expression, two regions (between -2465 and -1965, and between -896 and 511) did so in a polyamine-dependent manner. Steady-state concentration of ornithine decarboxylase (ODC), the first enzyme in the polyamine biosynthetic pathway, was increased in response to polyamine depletion. These data provide a mechanism by which polyamines affect transcription of the 4E-BP1 gene, which in turn affect translation of ODC and perhaps other cap-dependent proteins.

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.

Role of cyclooxygenase-2 in oleic acid-induced acute lung injury.

Eicosanoid production appears to be important to both edemagenesis and the pattern of pulmonary perfusion in experimental acute lung injury (ALI). We hypothesized that these effects could be mediated by the inducible form of cyclooxygenase (COX-2). We used positron emission tomography to evaluate the pulmonary perfusion pattern in dogs given oleic acid (OA) only (n = 6), the novel COX-2 inhibitor SC-236 50 min before OA (n = 3), and SC-236 given 20 min before endotoxin (Etx), followed by OA given 30 min after Etx (n = 5). Thromboxane B(2) (TXB(2)) and prostacyclin (6-keto prostaglandin F(1alpha); 6-keto PGF(1alpha)) metabolite concentrations in plasma and lung tissue were measured in these groups and in another group given Etx + OA (n = 4). Inhibition of COX-2 before administration of OA alone or before administration of Etx and OA did not have any significant effect on plasma or lung tissue concentrations of TXB(2). However, inhibition of COX-2 prior to Etx and OA significantly reduced the plasma and lung tissue concentrations of 6-keto PGF(1alpha) as compared with those in the group given only Etx + OA. Moreover, SC-236 prevented the expected loss of perfusion redistribution associated with Etx + OA only. The effect of endotoxin on pulmonary perfusion in ALI is therefore the result of a COX-2-mediated increase in prostacyclin production in lung tissue.

An in vitro model for the study of phagocytosis of damaged hepatocytes by rat Kupffer cells.

AIMS/BACKGROUND: One function of Kupffer cells is the phagocytosis of nonviable hepatocytes. Our aims were to develop a model for phagocytosis of damaged hepatocytes by rat Kupffer cells in vitro, and to characterise prostaglandin E2 (PGE2), prostacyclin (PGI), and tumour necrosis factor-alpha (TNF) production in this model. METHODS: Kupffer cells were incubated alone or with damaged hepatocytes for up to 18 h, then washed and cultured for up to 66 h. To compare mediator responses produced during inert particle phagocytosis, Kupffer cells were also incubated with latex beads. RESULTS: Phagocytic uptake of hepatocyte debris was confirmed in at least 50% of Kupffer cells. A dissociation between TNF and PGI responses was found for both latex beads and damaged hepatocytes, such that a TNF secretory response was not triggered by either stimulus whereas PGI production was increased for both. Although phagocytosis of beads increased PGE2 production, phagocytosis of hepatocytes did not. CONCLUSIONS: Phagocytosis of damaged hepatocytes by Kupffer cells results in the production of PGI but not PGE2 or TNF.

Response to inhaled nitric oxide in acute lung injury depends on distribution of pulmonary blood flow prior to its administration.

Responses to inhaled nitric oxide (iNO) in acute lung injury (ALI), as evidenced by improvements in oxygenation, are variable. We hypothesized that the effect of iNO may be related to the pre-iNO distribution of pulmonary blood flow (PBF). In the present study we evaluated the effect of iNO on PBF in normal healthy dogs and in a canine model of ALI induced by oleic acid (OA). In Group "OA only" (n = 5), ALI was induced by central venous injection of 0.08 ml/kg OA. In Group "E+OA" (n = 5), hypoxic pulmonary vasoconstriction after ALI was blocked with low-dose endotoxin (15 microg/kg of Escherichia coli endotoxin) administered 30 min before giving the same dose of OA. Measurements of regional PBF and lung water concentration (LWC) using positron emission tomography (PET) and H215O were performed before and after OA or placebo, and then again at concentrations of 10, 40, and 0 ppm iNO. One hundred twenty minutes after OA injury, PaO2/FIO2 fell significantly in Group OA only, from 567 +/- 32 to 437 +/- 67 mm Hg. In these animals, PBF redistributed from the dorsal edematous regions of the lungs to the nondependent zones, thus partially preserving normal ventilation/ perfusion relationships. As in the normal animals, in Group OA only, iNO did not significantly change either PBF or oxygenation. In Group E+OA, the administration of low-dose endotoxin eliminated perfusion redistribution from the dorsal edematous lung regions. As a result, PaO2/FIO2 fell from 558 +/- 70 to 119 +/- 53 mm Hg, a decrease that was significantly greater than that in Group OA only. In Group E+OA, administration of iNO restored perfusion redistribution to a similar level as in Group OA only, which was associated with a significant improvement in PaO2/FIO2, from 119 +/- 53 to 251 +/- 159 (10 ppm iNO), and 259 +/- 165 mm Hg (40 ppm iNO). We conclude that the effect of iNO on oxygenation after ALI depends on the pre-iNO perfusion pattern, which may help explain the variable response to iNO often observed in patients with acute respiratory distress syndrome.

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.

Involvement of cytochrome P-450 enzyme activity in the control of microvascular permeability in canine lung.

Products of cytochrome P-450 enzymes may play a role in capacitative Ca2+ entry in endothelial cells, which can promote a rise in vascular permeability. Thapsigargin (150 nM) stimulated capacitative Ca2+ entry and increased the capillary filtration coefficient (Kf,c) in isolated normal canine lung lobes. Pretreatment of the lobes with cytochrome P-450 inhibitors clotrimazole (10 microM) or 17-octadecynoic acid (5 microM) abolished the thapsigargin-induced increases in Kf,c. Because clotrimazole also blocks Ca2+-activated K+ channels, the K+-channel blocker tetraethylammonium (10 mM) was used to ensure that permeability was not influenced by this mechanism. Tetraethylammonium did not affect thapsigargin-induced permeability. The effects of the cytochrome P-450 arachidonic acid metabolite 5,6-epoxyeicosatrienoic acid (EET) were also investigated in lobes taken from control dogs and dogs with pacing-induced heart failure (paced at 245 beats/min for 4 wk). 5,6-EET (10 microM) significantly increased Kf,c in lobes from the control but not from the paced animals. We conclude that cytochrome P-450 metabolites are involved in mediating microvascular permeability in normal canine lungs, but an absence of 5,6-EET after heart failure does not explain the resistance of lungs from these animals to permeability changes.

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.

Synergistic hemodynamic effects of low-dose endotoxin and acute lung injury.

We evaluated the effects of low-dose endotoxin (15 microg/kg) on the pulmonary and systemic responses to oleic acid (OA)-induced acute lung injury in dogs. Animals given endotoxin alone (n = 5) showed a modest decrease in arterial blood pressure, but no effects on pulmonary hemodynamics, blood gases, cardiac output, or lung water accumulation. Animals (n = 6) given only OA (0.08 ml/kg) showed the expected development of mild-moderate pulmonary hypertension, a comparable reduction in arterial blood pressure, hypoxemia, increased lung water concentration, and an altered intrapulmonary perfusion pattern, as assessed by positron emission tomography. Animals (n = 7) given the same dose of endotoxin, followed 30 min later by the same dose of OA, developed a similar increase in lung water concentration as the group given OA alone, but failed to develop pulmonary hypertension or to redistribute pulmonary blood flow away from the edematous lung regions. In addition, arterial blood pressure fell significantly more than in the other groups. These responses were associated with a 30-fold increase in circulating prostacyclin (assayed as 6-keto prostaglandin F1 alpha [PGF1alpha]). The effects on systemic blood pressure, intrapulmonary blood flow redistribution, and eicosanoid production were eliminated by pretreating (n = 5) animals with meclofenamate (2 mg/kg). The results are consistent with a "priming" effect of low-dose endotoxin on the pulmonary endothelium, with exaggerated prostacyclin production in response to a subsequent lung injury. This interaction leads to altered intrapulmonary hemodynamics that exacerbate the development of hypoxemia, and to significant decreases in systemic blood pressure. To the extent that the lung is the most likely source of the increased prostacyclin production, the synergistic effects of low-dose endotoxin and lung injury may produce a kind of "lung shock."

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.

The adenovirus E3-14.7K protein and the E3-10.4K/14.5K complex of proteins, which independently inhibit tumor necrosis factor (TNF)-induced apoptosis, also independently inhibit TNF-induced release of arachidonic acid.

Tumor necrosis factor (TNF) is an inflammatory cytokine that inhibits the replication of many viruses in cultured cells. We have reported that adenovirus (Ad) infection of TNF-resistant mouse cells renders them susceptible to lysis by TNF and that two sets of proteins encoded by the E3 transcription unit block TNF cytolysis. The E3 protein sets are named E3-14.7K (14,700 kDa) and E3-10.4K/14.5K (a complex of two proteins of 10,400 and 14,500 kDa). TNF activation of the 85-kDa cytosolic phospholipase A2 (cPLA2) is thought to be essential for TNF cytolysis (i.e.,TNF-induced apoptosis). Here we provide evidence that cPLA2 is important in the response of Ad-infected cells to TNF and that the mechanism by which E3-14.7K and E3-10.4K/14.5K inhibit TNF cytolysis is by inhibiting TNF activation of cPLA2. cPLA2 cleaves arachidonic acid (AA) specifically from membrane phospholipids; therefore, cPLA2 activity was measured by the release of 3H-AA from cells prelabeled with 3H-AA. Uninfected cells or cells infected with wild-type Ad were not lysed and did not release 3H-AA in response to TNF. In contrast, TNF treatment induced cytolysis and 3H-AA release in uninfected cells sensitized to TNF by treatment with cycloheximide and also in infected cells sensitized to TNF by expression of E1A. In C127 cells, in which either E3-14.7K or E3-10.4K/14.5K inhibits TNF cytolysis, either set of proteins inhibited TNF-induced release of 3H-AA. In C3HA cells, in which E3-14.7K but not E3-10.4K/14.5K prevents TNF cytolysis, E3-14.7K but not E3-10.4K/14.5K prevented TNF-induced release of 3H-AA. When five virus mutants with lesions in E3-14.7K were examined, there was a perfect correlation between a mutant's ability to inhibit both TNF-induced cytolysis and release of 3H-AA. E3-14.7K expressed in two stably transfected C127 cell lines prevented both TNF-cycloheximide-induced cytolysis and release of 3H-AA. The E3 proteins also prevented TNF-induced cytolysis and release of 3H-AA in mouse L929 cells, which are spontaneously sensitive to TNF. TNF cytolysis was blocked by dexamethasone, an inhibitor of PLA2 activity, and by nordihydroquaiaretic acid, which inhibits the metabolism of AA to the leukotrienes. Indomethacin, which blocks the formation of prostaglandins from AA, did not inhibit TNF cytolysis. The leukotrienes and prostaglandins are amplifiers of the inflammatory response. We propose that E3-14.7K and E3-10.4K/14.5K function independently in Ad infection to inhibit both cytolysis and inflammation induced by TNF.