Modulation of macrophage procoagulant activity by arachidonic acid metabolites.

Macrophage (M phi)-mediated fibrin deposition via induction of procoagulant activity (PCA) is an important component of the host response during various infections. While endotoxin (LPS) is a well-known stimulus of PCA, the factors modulating its activity within the inflammatory microenvironment are unknown. The purpose of these studies was to determine the relative roles of two pathways of arachidonic acid metabolism, i.e., the cyclooxygenase (CO) and 5-lipoxygenase (5-LO) pathways, in modulating M phi PCA induction by LPS. Thioglycolate-elicited murine peritoneal M phi were treated with the CO inhibitor indomethacin (INDO), the 5-LO inhibitor nordihydroguaiaretic acid (NDGA), or control vehicle for 15 min prior to a 4-hr exposure to LPS (10 micrograms/ml). The ability of M phi to shorten the clotting time of plasma (i.e., PCA) was measured and clotting times were converted to PCA units via a thromboplastin standard. While CO blockade had no effect on PCA induction by LPS (without INDO 30 microM 446 +/- 131, with INDO 30 microM 546 +/- 193, mU/2 x 10(6) cells, n = 4), NDGA caused a dose-dependent inhibition (IC50 = 3 microM) without affecting cell viability (without NDGA 3 microM 446 +/- 131, with NDGA 3 microM 191 +/- 67, mU/2 x 10(6) cells, n = 6, P less than 0.05). Induction of PCA by Escherichia coli was similarly inhibited (E. coli 10(6) alone = 518 +/- 130; with NDGA 3 microM = 234 +/- 100, n = 2). Combined NDGA/INDO reduced PCA comparable to NDGA alone, ruling out the possibility that NDGA acted through generation of inhibitory prostanoids like PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of WEB 2086 on leukocyte adherence in response to hemorrhagic shock in rats

BACKGROUND: The pathogenesis of generalized microvascular injury after hemorrhagic shock is known to involve the generation of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine [PAF]). The release of PAF is manifested in several ways, including by increased vascular permeability, altered vascular reactivity, and increased leukocyte adherence to the endothelium. WEB 2086 is a PAF antagonist that has been shown experimentally to improve survival after hemorrhagic shock. The purpose of this study was to examine the efficacy of WEB 2086 in attenuating leukocyte adherence before, during, and after hemorrhagic shock. METHODS: After a control period, blood was withdrawn to reduce the mean arterial pressure to 40 mm Hg for 30 minutes in urethane-anesthetized rats. Mesenteric venules in a transilluminated segment of the small bowel were examined to quantitate leukocyte adherence using intravital microscopy. RESULTS: In sham-operated rats (control), there was minimal to no leukocyte adherence throughout the experiment. Hemorrhagic shock resulted in a significant increase in leukocyte adherence postshock during resuscitation (10.9 +/- 1.8 cells/100 microm, p < 0.01) when compared with controls. WEB 2086, when given before shock, significantly attenuated leukocyte adherence (0.1 +/- 0.08 cells/100 microm, p < 0.01) when compared with hemorrhagic shock alone. This effect of WEB 2086 on adherence could be demonstrated even when it was given during (3.5 +/- 0.9 cells/100 microm, p < 0.01) and 10 minutes into (5.8 +/- 1.1 cells/100 microm, p < 0.05) hemorrhagic shock. CONCLUSION: Our findings suggest that WEB 2086 may be of therapeutic benefit against the microvascular damage sustained after hemorrhagic shock.     

A triazolodiazepine platelet activating factor receptor antagonist (WEB 2086) reduces pulmonary dysfunction during endotoxin shock in swine

We wanted to determine the effects of WEB 2086, a platelet activating factor (PAF) antagonist, in lipopolysaccharide (LPS) shock in anesthetized pigs. In a randomized study, LPS from S. abortus equi, 2 micrograms/kg/h was given IV for six hours. Thirteen animals received LPS and WEB 2086, 10 mg/kg/h IV for 6.5 hours, beginning 30 minutes before LPS. Eleven septic controls received saline and LPS, three nonseptic controls received saline and WEB 2086, and three nonseptic controls received saline only. In six animals we investigated the effect of synthetic PAF in doses between 50 and 10,000 ng on arterial (AP) and pulmonary arterial (PAP) pressure before and during infusion of WEB 2086. The LPS-induced rise in PAP was reduced by WEB 2086 (p = 0.01) but not the decrease in AP. The LPS-induced leukopenia, hypoxia, increase in airway pressure, and release of plasminogen activator inhibitor were reduced by WEB 2086. Platelet activating factor produced an increase in PAP and a biphasic response in AP. All PAF dose response curves were shifted to the right by WEB 2086. Platelet activating factor was a pulmonary hypertensive agent and contributed to the LPS-induced respiratory alterations.     

Platelet-activating factor antagonists do not attenuate delayed posttraumatic cerebral edema in rats

Platelet-activating factor (PAF) receptor antagonists reportedly improve early postischemic neurological recovery and cerebral blood flow in selected experimental models. Their effects on posttraumatic cerebral edema have, however, not been examined. In a rat model of right hemispheric percussive cerebral trauma, we examined the effects of two PAF receptor antagonists on posttraumatic edema formation. Two groups of rats received either BN 52021 (n = 14) or WEB 2086 (n = 11), 10 mg/kg i.v. at 15 min posttrauma. Two other groups treated with the BN 52021 (n = 17) and WEB 2086 (n = 10) vehicles served as controls. Hemispheric percent brain water was determined at 24 h. Edema occurred in all groups. Neither PAF receptor antagonist significantly reduced right hemispheric percent brain water (81.08 +/- 0.25 and 81.04 +/- 0.15 in Bn 52021 and WEB 2086-treated rats, respectively, versus 81.31 +/- 0.23 and 81.14 +/- 0.17% brain water in BN 52021 vehicle and WEB 2086 vehicle-treated rats). Mortality was not statistically different between groups. These data do not support a major role for PAF in the development of posttraumatic cerebral edema.     

Receptor-mediated increase in cytosolic calcium in LLC-PK1 cells by platelet activating factor and thromboxane A2.

Several studies indicate an important role of platelet activating factor (PAF) and thromboxane A2 (TXA2) in glomerular pathophysiology. However, the potential role of PAF or TXA2 in renal tubular pathophysiology has received little attention, and the presence of functional receptors for these autacoids in renal tubular epithelium has not been previously studied. We examined the effects of PAF and the TXA2 analogue, ONO11113, on the cytosolic free calcium concentration [( Ca2+]i) in cultured LLC-PK1 cell line using a fluorescent probe, fura-2. In these cells, the addition of PAF or ONO11113 caused a significant increment in [Ca2+]i in a dose-dependent manner: both agonists (10(-7) M) increased [Ca2+]i from 148 +/- 16 to 288 +/- 39 nM and from 130 +/- 8 to 240 +/- 18 nM, with the values of EC50 for PAF and ONO11113 being 17 +/- 4 and 17 +/- 2 nM, respectively. These effects were both rapid and transient, returning to baseline in two minutes. The effect of PAF was selectively blocked by PAF receptor antagonist BN50730, but not by TXA2 receptor antagonist L657925. Similarly ONO11113 response was abolished by L657925, but not by BN50730. PAF- or ONO11113-challenged cells did not respond to a second addition of the same agent and showed heterologous desensitization to the other agonist. The initial peaks of [Ca2+]i as well as the sustained elevations in [Ca2+]i induced by PAF or ONO11113 were reduced following the chelation of extracellular Ca2+ by 10 mM ethylene glycol-bis(beta-aminomethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of a platelet activating factor antagonist, WEB 2086, on allergen induced asthmatic responses.

BACKGROUND--Platelet activating factor (PAF) has been implicated in the pathogenesis of airway hyperresponsiveness in asthma. The purpose of this study was to evaluate the effects of a selective PAF antagonist (WEB 2086), given in doses known to antagonise the effects of inhaled PAF in human subjects, on allergen induced early and late asthmatic responses and on airway hyperresponsiveness. METHODS--Eight atopic, mildly asthmatic subjects were studied during a screening period and two treatment periods. During the screening period subjects inhaled an allergen to which they were known to be sensitised and the response was measured as the fall in the forced expired volume in one second (FEV1) to show the presence of early (0-1 h) and late (3-7 h) asthmatic responses. On another day the subjects inhaled allergen diluent. During the treatment periods subjects inhaled allergen after one week's pretreatment with WEB 2086 (100 mg three times a day) or placebo administered in a randomised, double blind, crossover fashion. Histamine airway responsiveness was measured 24 hours before and 24 hours after allergen and the results were expressed as the provocative concentration causing a 20% fall in FEV1 (PC20). RESULTS--The maximal early asthmatic response after allergen with placebo treatment was 18.4% (SE 4.4%) and with WEB 2086 18.9% (4.4%). The maximal late response with placebo treatment was 21.7% (5.3%) and with WEB 2086 21.2% (3.0%). The log difference (before and after allergen) in histamine PC20 was 0.35 (0.06) after placebo treatment and 0.30 (0.1) after WEB 2086. CONCLUSIONS--These results indicate that one week of treatment with an orally administered PAF antagonist (WEB 2086) does not attenuate allergen induced early or late responses or airway hyperresponsiveness.     

Glomerular macrophages produce reactive oxygen species in experimental glomerulonephritis

The production of reactive oxygen species by intraglomerular macrophages was assessed in a macrophage dependent model of diffuse proliferative glomerulonephritis in rabbits. Glomerular macrophages were obtained from isolated nephritic glomeruli by short term (60 min) culture. Control macrophage populations were simultaneously obtained from peripheral blood (blood monocytes) and lung lavage fluid (alveolar macrophages). Superoxide anion (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH.) production was assessed. Glomerular macrophage production of O2- (48.9 +/- 5.5 nmol/hr/10(6) cells), H2O2 (4.4 +/- 2.5 nmol/hr/10(6) cells) and OH. (57.8 +/- 4.7 U/hr/10(6) cells) was significantly greater than the production of reactive oxygen species seen with control monocyte populations: alveolar macrophages, O2- 9.8 +/- 2.0 nmol/hr/10(6) cells; H2O2 0.6 +/- 0.3 nmol/hr/10(6) cells; OH. 11.0 +/- 1.8 U/hr/10(6) cells; blood monocytes, O2- 8.6 +/- 1.4 nmol/hr/10(6) cells; OH. 9.9 +/- 1.2 U/hr/10(6) cells, (all P less than 0.05 cf. glom macs). Hydrogen peroxide production by blood monocytes (1.6 +/- 0.9 nmol/hr/10(6) cells) was less than glomerular macrophages, however this difference was not statistically significant. The enhanced production of reactive oxygen species by glomerular macrophages in this macrophage dependent model of glomerulonephritis suggests that these mononuclear cells are locally activated within the glomerulus following recruitment from the circulation. Reactive oxygen species production by glomerular macrophages may contribute to their ability to induce glomerular basement membrane injury in this disease.     

The effects of cyclosporine and cyclosporine metabolites in experimental small intestinal transplantation

Cyclosporine metabolites (CM) were compared with cyclosporine for their in vitro and in vivo immunosuppressive, nephrotoxic, and hepatotoxic effects using (A) in vitro mixed lymphocyte induction of monocyte/macrophage procoagulant activity (PCA), an accurate marker of allograft rejection; (B) in vitro toxic effects on renal cells in culture; and (C) a unidirectional rejection model of rat small intestinal transplantation (SIT). CM were composed of OL1, OL17, OL18, and two additional peaks C and H, (peak C: mass = 1235, 15.3% of total CM, peak H: mass = 1205, 6.3% of total CM). In vitro, CM fully suppressed the one-way mixed lymphocyte culture-induced PCA from 52.5 +/- 8.2 mU/10(6) PBM to the basal level 22.3 +/- 6.6 mU/10(6) PBM (P less than 0.01), which was comparable to CsA (21.3 +/- 5.5 mU/10(6) PBM). Lewis rats that had received Lewis-Brown Norway F1 hybrid intestinal allografts when treated with CM, demonstrated near-normal histology with minimal signs of rejection as compared with the fulminant clinical and histological rejection observed in the control (untreated and Cremaphor/NaCl treated) animals. PCA was markedly elevated in the control animals, 278 +/- 172 (untreated) and 160 +/- 98 mU/10(6) PBM (Cremaphor/normal saline treated), whereas CsA-treated allogeneic transplants expressed only basal levels of PCA (14.0 +/- 4 mU/10(6) PBM) (P less than 0.01), associated with normal histology. CM-treated animals expressed PCA levels of 27.0 +/- 10 mU/10(6) PBM, which was significantly different from both control and CsA-treated animals (P less than 0.01). In contrast to CsA-treated animals, CM-treated allogeneic transplants demonstrated no apparent renal or hepatic toxicity, as measured by blood urea nitrogen (25.3 +/- 9.5 vs. 10.0 +/- 5.3 mg/dl), alkaline phosphatase (160.7 +/- 29.3 vs. 100.3 +/- 19.5 U/L), and aspartate transaminase (96.7 +/- 23.7 vs. 61.7 +/- 11.7 U/L) (P less than 0.01). Similarly, in contrast to CsA, CM had minimal or no toxicity in renal epithelial and mesangial cells in culture, as measured by minimal or no inhibition of DNA, RNA, and protein synthesis. These results suggest that CM have potent immunosuppressive properties with no apparent nephrotoxicity and hepatotoxicity in vitro and in vivo.     

Primed neutrophils injure rat lung through a platelet-activating factor-dependent mechanism.

Bacterial lipopolysaccharide (LPS) promotes transient lung neutrophil sequestration. These LPS-primed neutrophils, when stimulated by an N-formyl peptide (FNLP), promote lung injury. We hypothesized that LPS-primed, FNLP-stimulated neutrophils promote lung injury through a platelet-activating factor (PAF)-dependent mechanism. Rats were pretreated with either saline or WEB2170, a PAF receptor antagonist (10 mg/kg po). One hour after pretreatment, rats were administered intraperitoneal LPS (salmonella typhimurium lipopolysaccharide, 500 micrograms/kg) followed 6 hr later by intravenous FNLP (250 micrograms/kg infused over 30 min). Two hours after the initiation of FNLP infusion, rats were sacrificed and assays were performed to measure: (1) lung neutrophil sequestration with myeloperoxidase (MPO) activity; (2) circulating neutrophil activation with nitroblue tetrazolium (NBT) staining, and (3) lung microvascular leak with 125I-albumin flux. We found that lung myeloperoxidase, circulating neutrophil NBT staining, and lung 125I-albumin flux were increased (P less than 0.05) in saline-pretreated LPS/FNLP rats, relative to control. While lung MPO remained increased (P less than 0.05) in WEB2170-pretreated LPS/FNLP rats, circulating neutrophil NBT and lung 125I-albumin flux were decreased (P less than 0.05) relative to those in saline-pretreated rats. We conclude that PAF mediates LPS/FNLP-induced neutrophil activation and lung injury, but is independent from lung neutrophil sequestration. Thus, lung neutrophil sequestration does not inevitably produce lung injury. Rather, neutrophils can accumulate in the lung without causing lung injury if neutrophil activation can be blocked.     

Vaccinia virus-induced inhibition of nitric oxide production

BACKGROUND: The role of nitric oxide (NO) in the host defense against viruses has not been well defined. Several studies have implicated NO as responsible for the destruction of a variety of viruses. However, others have reported that certain viruses can impair the ability of macrophages to produce NO. This study was initiated to determine the ability of macrophages to produce NO in response to vaccinia virus infection. METHODS: RAW 264.7 murine macrophages in minimum essential medium were exposed to virus-containing supernatants for 1 h before stimulation with Escherichia coli lipopolysaccharide (LPS, 0.001 and 1.0 microg/ml). After further 24-h incubations, nitrite concentration, cell viability, and inducible nitric oxide synthase (iNOS) were quantitated. RESULTS: The viral preparation alone did not stimulate nitric oxide synthesis (measured as nitrite) by macrophages. However, macrophages exposed to 0.001 and 1.0 microg/ml LPS produced 7.7 +/- 0.6 and 16.6 +/- 0.8 nmole/1.1 x 10(6) cells/24-h nitrite, respectively. Production of nitrite caused cell death. Macrophages incubated with vaccinia virus prior to exposure to LPS resulted in a dose-dependent decrease in nitrite production. An 80% inhibition of nitrite was noted when macrophages were exposed to vaccinia virus (m.o.i. 10(-4)) plus LPS (1.0 microg/ml) (P < 0.05). Further study showed that this inhibition was not associated with changes in cell viability or substrate availability, but was associated with a marked reduction in iNOS protein. When the virus was inactivated with UV-irradiation, the same incubation caused a 46% inhibition of nitrite production (P < 0.05 vs active virus). However, this effect occurred without altering the quantity of iNOS protein. CONCLUSION: These results indicate that active vaccinia virus inhibits the ability of stimulated macrophages to produce NO by hindering iNOS protein expression. Because live viral particles were not entirely required for this inhibition, it is possible that by products of viral infection, such as soluble viral proteins, may also be responsible for this effect.     

Thiopental Alters Contraction, Intracellular Ca2+, and pH in Rat Ventricular Myocytes

Background: Myocardial contractility is regulated by intracellular concentration of free Ca2+ ([Ca2+]i) and myofilament Ca2+ sensitivity. The objective of this study was to elucidate the direct effects of thiopental on cardiac excitation-contraction coupling using individual, field-stimulated ventricular myocytes.

Methods: Freshly isolated rat ventricular myocytes were loaded with the Ca2+ indicator, fura-2, and placed on the stage of an inverted fluorescence microscope in a temperature-regulated bath. [Ca2+]i (340/380 ratio) and myocyte shortening (video-edge detection) were monitored simultaneously in individual cells field-stimulated at 0.3 Hz. Amplitude and timing of myocyte shortening and [Ca2+]i were compared before and after addition of thiopental. Intracellular pH was measured with the pH indicator, BCECF (500/440 ratio). Real-time uptake of Ca2+ into isolated sarcoplasmic reticulum vesicles was measured using fura-2 free acid in the extravesicular compartment. One hundred thirty-two cells were studied.

Results: Field stimulation increased [Ca2+]i from 85 +/- 10 nM to 355 +/- 22 nM (mean +/- SEM). Myocytes shortened by 10% of resting cell length (127 +/- 5 [micro sign]m). Times to peak [Ca2+]i and shortening were 139 +/- 6 and 173 +/- 7 msec, respectively. Times to 50% recovery for [Ca2+]i and shortening were 296 +/- 6 and 290 +/- 6 ms, respectively. Addition of thiopental (30-1,000 [micro sign]M) resulted in dose-dependent decreases in peak [Ca2+]i and myocyte shortening. Thiopental altered time to peak and time to 50% recovery for [Ca2+]i and myocyte shortening and inhibited the rate of uptake of Ca2+ into isolated sarcoplasmic reticulum vesicles. Thiopental did not, however, alter the amount of Ca2+ released in response to caffeine in sarcoplasmic reticulum vesicles or intact cells. Thiopental (100 [micro sign]M) increased intracellular pH and caused an upward shift in the dose-response curve to extracellular Ca2+ for shortening, with no concomitant effect on peak [Ca2+]i. These effects were abolished by ethylisopropyl amiloride, an inhibitor of Na+ -H+ exchange.     

Monocyte/macrophage procoagulant activity as a measure of immune responsiveness in Lewis and brown Norway inbred rats. Discordance with lymphocyte proliferative assays

In vitro lymphocyte proliferative assays were performed using Lewis (Lew) and Brown Norway (BN) rats, and compared to induction of monocyte/macrophage procoagulant activity (PCA) in a mixed lymphocyte culture and by endotoxin (LPS) (E. Coli 0111:B4). Splenic mononuclear cells from Lew rats had significantly greater mitogen-induced proliferation to concanavalin A (P = .002) and phytohemagglutinin (P = 0.007). The Lew cells also showed greater allogeneically induced proliferation by BN cells in a one-way MLC in comparison to the reciprocal BN proliferative response (P less than 0.04). PCA induction in peripheral blood mononuclear cells (PBM) by allogeneic stimulation in MLC or total content PCA by LPS did not vary significantly between the 2 strains (P greater than 0.5). Induction of PCA by LPS was rapid, with a moderate rise over basal activity at 3 hr and maximal activity at 6 hr. Two-way allogeneic induction of PCA in PBM from BN and Lew rats resulted in PCA elevation by 3 hr, which became maximal at 18 hr. One-way MLC with Lew or BN cells as responders resulted in moderate increases in PCA by 3-6 hr, with equivalent maximal activities recorded at 18 hr. Viable PCA accounted for 26-32% of total content PCA in both Lew and BN rats. Maximal allogeneic PCA induction by MLC was 14-18% of PCA induced by LPS and required a longer incubation for its expression. Our results indicate that in vitro PCA expression by Lew and BN PBM following allogeneic or endotoxin stimulation shows little interstrain variability in comparison to lymphocyte proliferative responses. Thus PCA appears to more closely reflect the observed in vivo responses of these strains to allogeneic challenge.     

On the mechanisms of impaired phagocytosis in phosphate depletion.

Phosphate depletion (PD) impairs the phagocytic ability of polymorphonuclear leukocytes (PMNL). This derangement has been attributed to the low ATP content of PMNL in PD. The mechanisms responsible for the low ATP content are not well defined. Phosphorus deficiency, per se, and/or other cellular metabolic consequences of PD such as a rise in cytosolic calcium ([Ca2+]i) could be responsible. Indeed, PD is associated with a rise in [Ca2+]i in other cells, and such an event may inhibit mitochondrial ATP production. It is also not evident whether the impaired phagocytosis in PD is due to low ATP content and/or a rise in the [Ca2+]i of PMNL. The study presented here examined levels of [Ca2+]i, ATP content, and the phagocytic ability of PMNL from PD and pair-weighed (PW) rats and evaluated the potential beneficial effect of treatment with verapamil (V), which may prevent a rise in [Ca2+]i and the consequent effects on ATP content and the phagocytosis of PMNL. The resting levels of [Ca2+]i of PMNL from PD rats (148 +/- 3.9 nM) were significantly (P less than 0.01) higher, and the ATP contents (4.8 +/- 0.2 nmol/5 x 10(6) PMNL) were significantly (P less than 0.01) lower than in PW (111 +/- 2.8 nM and 9.3 +/- 0.3 nmol/5 x 10(6) PMNL), PW-V (114 +/- 2.2 nM and 9.3 +/- 0.28 nmol/5 x 10(6) PMNL), and PD-V (112 +/- 1.8 nM and 6.6 +/- 0.19 nmol/5 x 10(6) PMNL) animals. Despite the normal [Ca2+]i in the PMNL of PD-V rats, their ATP contents were still significantly (P less than 0.01) lower than those of PW or PW-V rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet-activating factor and bacteremia-induced pulmonary hypertension

BACKGROUND: Acute lung injury is a common complication of gram-negative sepsis. Pulmonary hypertension and increased lung vascular permeability are central features of lung injury following experimental bacteremia. Platelet-activating factor is a prominent proinflammatory mediator during bacterial sepsis. Our previous studies have demonstrated that exogenous administration of platelet-activating factor (PAF) induces pulmonary edema without causing pulmonary hypertension. Interestingly, inhibition of PAF activity during Escherichia coli bacteremia prevents the development of both pulmonary hypertension and pulmonary edema. These data suggest that PAF contributes to pulmonary hypertension during sepsis, but that this is unlikely to be a direct vascular effect of PAF. The goal of the present study was to investigate the mechanism by which acute E. coli bacteremia induces pulmonary injury and to define the role that PAF plays in this injury. We hypothesized that the effects of PAF on pulmonary hypertension during bacteremia are due to the effects of PAF on other vascular mediators. Several studies suggest that PAF induces the expression of endothelin-1 (ET), a potent peptide vasoconstrictor. Further, our previous studies have implicated ET as a central mediator of systemic vasoconstriction during bacteremia. We therefore sought to assess whether ET is modulated by PAF. E. coli has also been demonstrated to increase endothelial production of nitric oxide (NO), which contributes to maintenance of basal vascular tone in the pulmonary circulation. We hypothesized that PAF might increase pulmonary vascular resistance during bacteremia by activating neutrophils, increasing expression of ET, and decreasing the tonic release of NO. Furthermore, we hypothesized that hypoxic vasoconstriction did not contribute to pulmonary vasoconstriction during the first 120 min of E. coli bacteremia. METHODS: Pulmonary artery pressure (PAP), blood pressure (BP), heart rate (HR), and arterial blood gases (ABG) were measured in anesthetized spontaneously breathing adult male Sprague-Dawley rats. E. coli (10(9) CFU/100 g body wt) was injected at t = 0, and hemodynamic data were obtained at 10-min intervals and ABG data at 30-min intervals for a total of 120 min. Sham animals were treated equally but received normal saline in place of E. coli. In treatment groups, a 2.5 mg/kg dose of WEB 2086, a PAF receptor antagonist, was administered intravenously 15 min prior to the onset of sepsis or sham sepsis. The groups were (1) intravenous E. coli (n = 5); (2) intravenous WEB 2086 pretreatment + intravenous E. coli (n = 5); (3) intravenous WEB 2086 alone (n = 5); and (4) intravenous normal saline (n = 6). Nitric oxide metabolites (NOx) and ET concentrations were assayed from arterial serum samples obtained at the end of the protocol. Lung tissue was harvested for measurement of myeloperoxidase (MPO) activity and pulmonary histology. RESULTS: E. coli bacteremia increased HR, PAP, and respiratory rate early during sepsis (within 20 min), while hypoxemia, hypotension, and hemoconcentration were not manifest until the second hour. Pretreatment with WEB 2086 completely abrogated all of these changes. E. coli bacteremia increased the activity of serum ET, lung MPO, and neutrophil sequestration in the lung parenchyma via a PAF-dependent mechanism. However, the mechanism of increased production of NO appears to be PAF independent. CONCLUSIONS: These data support the hypothesis that E. coli bacteremia rapidly induces pulmonary hypertension stimulated by PAF and mediated at least in part by endothelin-1 and neutrophil activation and sequestration in the lung. Microvascular injury with leak is also mediated by PAF during E. coli bacteremia, but the time course of resultant hypoxemia and hemoconcentration is slower than that of pulmonary hypertension. The contribution of hypoxic vasoconstriction in exacerbating pulmonary hypertension in gram-negative sepsis is probably a late     

Leukocyte activation in ischemia-reperfusion injury of skeletal muscle

Polymorphonuclear leukocyte (PMN) participation in the pathophysiology of the reperfusion injury following skeletal muscle ischemia has become recognized. We measured the activation of PMNs as evidenced by production of superoxide anion (O2-) in the isolated canine gracilis muscle preparation of ischemia-reperfusion injury. PMNs were isolated from the gracilis muscle venous effluent and central venous blood after 6 hr of bilateral gracilis ischemia and 1 hr of reperfusion in five dogs. Baseline samples were obtained prior to ischemia from the central venous circulation. Liberation of O2- from PMNs and from PMNs stimulated by opsonized zymosan was determined by measuring ferricytochrome reduction. Results are expressed as nanomoles of O2- produced/2 x 10(6) PMN +/- SEM. O2- production by unstimulated cells was increased from 0.33 +/- 0.15 nmole in the baseline samples to 0.96 +/- 0.08 nmole in the central venous sample (P less than 0.01). With stimulation by zymosan, production increased from 10.3 +/- 1.4 nmole in the baseline samples to 15.2 +/- 1.1 nmole in the central venous sample (P less than 0.05) and to 15.5 +/- 0.9 nmole in the gracilis venous sample (P less than 0.01). These increases in superoxide production were not seen in the three sham-operated animals. Mean infarct size determined by planimetry was 55%. O2- produced by PMNs from central venous blood correlated with infarct size (r = 0.934, P = 0.02). These data imply that PMNs are activated by muscular ischemia, and the degree of activation is directly related to the extent of the muscle infarction.     

Glomerular interleukin 1 production is dependent on macrophage infiltration in anti-GBM glomerulonephritis

Both macrophages and glomerular mesangial cells have the potential to synthesize interleukin 1 (IL-1), however, their respective contributions to IL-1 production in anti-GBM glomerulonephritis (GN) are unknown. To address this problem, IL-1 production by glomeruli from rabbits with macrophage-associated anti-GBM GN (passive autologous anti-GBM GN [PAGBMGN]) and macrophage independent (heterologous phase) anti-GBM GN was studied. Macrophage-infiltrated nephritic glomeruli produced IL-1 bioactivity which was inhibitable by an anti-IL-1 antibody, and had a molecular weight consistent with rabbit IL-1. Glomerular IL-1 production in PAGBMGN was markedly augmented (1.43 +/- 0.79 U/10(3) glomeruli [gloms]/24 hr) compared to normal glomeruli (0.13 +/- 0.06 U/10(3) gloms/24 hr, P less than 0.05) or glomeruli from rabbits with macrophage independent GN (0.11 +/- 0.07 U/10(3) gloms/24 hr, P less than 0.05). IL-1 production by glomeruli from leukocyte depleted rabbits with PAGBMGN (0.16 +/- 0.07 U/10(3) gloms/24 hr) was not significantly elevated compared to normal glomeruli. Glomerular macrophages from rabbits with PAGBMGN produced more IL-1 (3.62 +/- 1.63 U/10(3) cells/24 hr) than blood monocytes (0.51 +/- 0.30 U/10(3) cells/24 hr) or alveolar macrophages (0.24 +/- 0.12 U/10(3) cells/24 hr) from the same animals. These results show that in experimental anti-GBM GN where injury is macrophage dependent, IL-1 production is also macrophage dependent and infiltrating glomerular macrophages are the major source of IL-1. Further, as glomerular IL-1 production was not significantly augmented in GN in the absence of macrophages, glomerular deposition of immunoglobulin and complement alone do not stimulate significant IL-1 production by intrinsic glomerular cells in experimental anti-GBM GN.     

Effects of Halothane on Sarcoplasmic Reticulum Calcium Release Channels in Porcine Airway Smooth Muscle Cells

Background: Volatile anesthetics relax airway smooth muscle (ASM) by altering intracellular Ca2+ concentration ([Ca2+]i). The authors hypothesized that relaxation is produced by decreasing sarcoplasmic reticulum Ca2+ content via increased Ca2+ "leak" through both inositol trisphosphate (IP3) and ryanodine receptor channels.

Methods: Enzymatically dissociated porcine ASM cells were exposed to acetylcholine in the presence or absence of 2 minimum alveolar concentration (MAC) halothane, and IP3 levels were measured using radioimmunoreceptor assay. Other cells were loaded with the Ca2+ indicator fluo-3 and imaged using real-time confocal microscopy.

Results: Halothane increased IP3 concentrations in the presence and absence of acetylcholine. Inhibition of phospholipase C blunted the IP3 response to halothane. Exposure to 2 MAC halothane induced a transient [Ca2+]i response, suggesting depletion of sarcoplasmic reticulum Ca2+. Exposure to 20 [mu]m Xestospongin D, a cell-permeant IP3 receptor antagonist, resulted in a 45 +/- 13% decrease in the [Ca2+]i response to halothane compared with halothane exposure alone. In permeabilized cells, Xestospongin D or 0.5 mg/ml heparin decreased the [Ca2+]i response to halothane by 65 +/- 13% and 68 +/- 22%, respectively, compared with halothane alone. In both intact and permeabilized cells, 20 [mu]m ryanodine blunted the [Ca2+]i response to halothane by 32 +/- 13% and 39 +/- 21%, respectively, compared with halothane alone. Simultaneous exposure to Xestospongin D and ryanodine completely inhibited the [Ca2+]i response to halothane.     

Effects of advanced glycation end products on cytosolic Ca2+ signaling of cultured human mesangial cells.

Advanced glycation end product (AGE) accumulation in a high glucose (HG) environment is thought to mediate some of the vascular complications of diabetes. Transmembrane signaling of contractile cells is generally inhibited by HG, with implications for systemic and target organ hemodynamics. In the kidney, glomerular mesangial cells grown in HG media are hyporesponsive to the effects of vasoconstrictor agents, possibly explaining the hyperfiltration and increased capillary pressure that eventually lead to diabetic glomerulopathy. To verify whether AGE binding to specific mesangial receptors could mediate these effects of HG, cultured human mesangial cells (HMC) were exposed to in vitro glycated bovine serum albumin (BSA) for 60 min at 37 degrees C before measurement of cytosolic Ca2+ ([Ca2+]i) by microfluorometric techniques in monolayers or single cells. AGE-BSA (2 mg/ml) reduced Ca2+ release from intracellular stores by 1 microM angiotensin II from peak [Ca2+]i levels of 843+/-117 to 390+/-50 nM in monolayers and from 689+/-68 to 291+/-36 nM in individual cells (P < 0.05). Nonglycated BSA and BSA exposed to 250 mM glucose-6-phosphate for 30 d in the presence of 250 mM aminoguanidine (AMGD), an inhibitor of nonenzymatic glycation, had no effect on the angiotensin II-induced [Ca2+]i spike (peak 766+/-104 and 647+/-87 nM, monolayers/ single cells, respectively, P = NS). AGE also inhibited store-operated Ca2+ influx through plasma membrane channels, assessed by addition of 1 to 10 mM extracellular Ca2+ to cells previously held in Ca2(+)-free media (control 339+/- 46/593 +/- 51, +AGE-BSA 236 +/- 25/390 +/- 56, +AMGD 483+/-55/ 374+/-64 nM [Ca2+]i, monolayers/single cells at 10 mM Ca2+, respectively; +AGE-BSA, P < 0.05 versus control). Contrary to HG, AGE-BSA did not translocate protein kinase C isoforms alpha, zeta, and delta to the plasma membrane. Culture of HMC in HG supplemented with 1 mM AMGD prevented downregulation of [Ca2+]i signaling. These data suggest that glycated macromolecules or matrix components may inhibit transmembrane Ca2+ signaling of glomerular cells through binding to a specific AGE receptor, thus mediating some of the known functional effects of HG on the kidney.     

Is macrophage-induced microthrombosis during endotoxemia dependent on prostaglandin synthesis?

Steroids and nonsteroidal anti-inflammatory drugs (NSAID), in addition to inhibiting prostaglandin synthesis, have recently been shown to block production of procoagulant activity (PCA) by alveolar macrophages (A-M phi). This inhibition may ameliorate one deleterious effect of endotoxin (LPS), since LPS-induced PCA may contribute to the diffuse microvascular thrombosis (DIC) and subsequent organ failure seen in endotoxemia. However, the results of treatment of endotoxemia with steroids of NSAID are variable or short-lived. To help elucidate the basis for these results, the effect of these agents on production of PCA by another M phi population, the hepatic M phi (H-M phi PCA is functionally discrete from A-M phi PCA) was evaluated. Four agents, (hydrocortisone, indomethacin, ibuprofen and acetylsalicylic acid (ASA)) used to both block production of prostaglandins and ameliorate the deleterious effects of LPS in vivo were tested on the production of rabbit M phi PCA in vitro in homogeneous cultures. PCA was measured by a standardized one-step coagulation assay. Treatment with LPS (1 to 10 g/ml) produced maximal stimulation of PCA production with a peak at 8 hr with PCA levels 20- to 30+-fold higher than controls. The effect of pretreatment (T-10 min) of M phi cultures with the various inhibitors on PCA production at 8 hr post-LPS was evaluated, (N = 6 to 10 experiments).(ABSTRACT TRUNCATED AT 250 WORDS)