Elevated thromboxane levels in the rat during endotoxic shock: protective effects of imidazole, 13-azaprostanoic acid, or essential fatty acid deficiency.

The potential deleterious role of the proaggregatory vasoconstrictor, thromboxane A(2), in endotoxic shock was investigated in rats. Plasma thromboxane A(2) was determined by radioimmunoassay of its stable metabolite thromboxane B(2). After intravenous administration of Salmonella enteritidis endotoxin (20 mg/kg), plasma thromboxane B(2) levels increased from nondetectable levels (<375 pg/ml) in normal control rats to 2,054+/-524 pg/ml (n = 8), within 30 min to 2,071+/-429 at 60 min, and decreased to 1,119+/-319 pg/ml, at 120 min. Plasma levels of prostaglandin E also increased from 146+/-33 pg/ml in normal controls (n = 5) to 2,161+/-606 pg/ml 30 min after endotoxin (n = 5). In contrast to shocked controls, rats pretreated with imidazole, a thromboxane synthetase inhibitor, or essential fatty acid-deficient rats, which are deficient in arachidonate and its metabolites, did not exhibit significant elevations in plasma levels of thromboxane B(2). Imidazole did not however inhibit endotoxin-induced elevations in plasma prostaglandin E. Essential fatty acid deficiency significantly reduced mortality to lethal endotoxic shock. This refractoriness could be duplicated in normal rats pretreated with the fatty acid cyclo-oxygenase inhibitor, indomethacin (10 mg/kg), intravenously 30 min before endotoxin injection. Imidazole (30 mg/kg) administered intraperitoneally 1 h before or intravenously 30 min before endotoxin, also significantly (P < 0.01) reduced mortality from lethal endotoxin shock to 40% compared to a control mortality of 95% at 24 h. Likewise pretreatment with 13-azaprostanoic acid (30 mg/kg), a thromboxane antagonist, reduced mortality from endotoxic shock at 24 h from 100% in control rats to only 50% (P < 0.01). The results suggest that endotoxin induces increased synthesis of thromboxane A(2) that may contribute to the pathogenesis of endotoxic shock.

Altered responses to modulators of guanine nucleotide binding protein activity in endotoxin tolerance.

The effects of cholera toxin or pertussis toxin and nonhydrolyzable GTP analogs on Salmonella enteritidis endotoxin stimulation of iTxB2 and i6-keto-PGF1 alpha synthesis in control and endotoxin tolerant rat peritoneal macrophages were determined. Pretreatment with pertussis toxin alone had no effect on basal macrophage iTxB2 or i6-keto-PGF1 alpha production, but pertussis toxin (0.1, 1.0 and 10 ng/ml) significantly inhibited endotoxin-stimulated iTxB2 and i6-keto-PGF1 alpha synthesis. Pretreatment with cholera toxin, which did not affect basal iTxB2 or i6-keto-PGF1 alpha synthesis, significantly enhanced endotoxin-induced synthesis of iTxB2 and i6-keto-PGF1 alpha. The effects of pertussis and cholera toxin with or without endotoxin were significantly (P less than 0.05) less in macrophages from endotoxin tolerant rats compared to control macrophages. GTP[gamma-S] (100 microM) significantly increased iTxB2 synthesis and significantly augmented endotoxin-stimulated iTxB2 synthesis in control macrophages (P less than 0.05). However, in macrophages from endotoxin tolerant rats the effect of GTP[gamma-S] on iTxB2 synthesis was significantly less (P less than 0.05) compared to control macrophages. Collectively, these data suggest that: (1) guanine nucleotide binding regulatory proteins mediate endotoxin-stimulated arachidonic acid metabolism in rat peritoneal macrophages; and (2) endotoxin tolerance induces alterations in guanine nucleotide binding protein activity.

Arachidonic acid turnover in peritoneal macrophages is altered in endotoxin-tolerant rats.

Peritoneal macrophages from endotoxin-tolerant rats have been found to exhibit depressed metabolism of arachidonic acid (AA) to prostaglandins and thromboxane in response to endotoxin. The effect of endotoxin tolerance on AA turnover in peritoneal macrophages was investigated by measuring [14C]AA incorporation and release from membrane phospholipids. Endotoxin tolerance did not affect the amount of [14C]AA incorporated into macrophages (30 min-24 h). However, the temporal incorporation of [14C]AA into individual phospholipid pools (15 min-24 h) was altered. In endotoxin-tolerant macrophages, [14C]AA incorporation into phosphatidylcholine (PC) (2, 4, 24 h) and phosphatidylethanolamine (PE) (8 h) was increased, while the incorporation into phosphatidylserine (PS) (2-24 h) was reduced (P less than 0.005) compared to control macrophages. There was no change in [14C]AA incorporation into phosphatidylinositol (PI). Following 2 or 24 h of incorporation of [14C]AA, macrophages were incubated (3 h) with endotoxin (50 micrograms/ml) or A23187 (1 microM), and [14C]AA release was measured. Endotoxin-tolerant macrophages released decreased (P less than 0.05) amounts of [14C]AA in response to both endotoxin and the calcium ionophore A23187 compared to controls. Control macrophages in response to endotoxin released [14C]AA from PC, PI and PE. In contrast, tolerant cells released [14C]AA only from PC (P less than 0.05). A23187 released [14C]AA from all four pools in the control cells, but only from PC and PE in the tolerant cells. These data demonstrate that endotoxin tolerance alters the uptake and release of AA from specific macrophage phospholipid pools. These results suggest that changes in AA turnover and/or storage are associated with endotoxin tolerance.

Macrophage eicosanoid formation is stimulated by platelet arachidonic acid and prostaglandin endoperoxide transfer.

The present study was designed to determine whether platelets transfer arachidonic acid or prostaglandin endoperoxide intermediates to macrophages which may be further metabolized into cyclooxygenase products. Adherent peritoneal macrophages were prepared from rats fed either a control diet or an essential fatty acid-deficient diet, and incubated with a suspension of washed rat platelets. Macrophage cyclooxygenase metabolism was inhibited by aspirin. In the presence of a thromboxane synthetase inhibitor, 7-(1-imidazolyl)heptanoic acid, immunoreactive 6-ketoprostaglandin F1 alpha formation was significantly increased 3-fold. Since this increase was greater (P less than 0.01) than that seen with either 7-(1-imidazolyl)heptanoic acid-treated platelets or aspirin-treated macrophages alone, these results indicate that shunting of endoperoxide from platelets to macrophages may have occurred. In further experiments, macrophages from essential fatty acid-deficient rats were substituted for normal macrophages. Essential fatty acid-deficient macrophages, depleted of arachidonic acid, produced only 2% of the amount of eicosanoids compared to macrophages from control rats. When platelets were exposed to aspirin, stimulated with thrombin, and added to essential fatty acid-deficient macrophages, significantly more immunoreactive 6-ketoprostaglandin F1 alpha was formed than in the absence of platelets. This increased macrophage immunoreactive 6-ketoprostaglandin F1 alpha synthesis, therefore, must have occurred from platelet-derived arachidonic acid. These data indicate that in vitro, in the presence of an inhibition of thromboxane synthetase, prostaglandin endoperoxides, as well as arachidonic acid, may be transferred between these two cell types.

Endotoxin-induced arachidonic acid metabolism requires de novo protein synthesis and protein kinase C activation.

The mechanisms whereby bacterial endotoxins stimulate arachidonic acid metabolism in macrophages are uncertain. Both protein kinase C activation and de novo protein synthesis occur in macrophages in response to endotoxin. In this study we evaluated the time course and role of protein kinase C and de novo protein synthesis in endotoxin stimulated arachidonic acid metabolism in resident rat peritoneal macrophages. Thromboxane (TX) B2 was measured as the representative arachidonic acid metabolite synthesized in response to Salmonella enteritidis endotoxin, calcium ionophore A23187, or phorbol 12-myristate 13-acetate (PMA). The effect of inhibition of protein kinase C by 1-(5-isoquinolinsulfonyl)-2-methylpiperazine dihydrochloride (H-7) and staurosporine on endotoxin- and A23187-induced TXB2 synthesis was examined. The potential roles of transcriptional and translational events in endotoxin- and A23187-stimulated TXB2 synthesis were determined by utilizing the transcriptional inhibitors camptothecin (10 microM) or actinomycin D (0.08 microM), and the translational inhibitor cycloheximide (0.1 microM). Whereas, A23187 stimulated maximal TXB2 synthesis within 15 min, endotoxin showed a more prolonged time course with a 12-fold increase in TXB2 synthesis above basal levels after 3 h (P less than 0.05). PMA induced an approx. 8-fold increase above basal TXB2 levels that was blocked by inhibition of transcription with actinomycin D. H-7 (10 microM to 50 microM) inhibited endotoxin- and A23187-stimulated eicosanoid synthesis. Staurosporine (0.2 microM) produced a selective 66% inhibition of endotoxin, but not A23187-stimulated TXB2 synthesis. Endotoxin-induced TXB2 production was significantly (P less than 0.05) inhibited by staurosporine, camptothecin, actinomycin D or cycloheximide at intervals from 30 min prior to, through 60 min after endotoxin stimulation. These studies suggest a role for protein kinase C activation and de novo protein synthesis in endotoxin signal transduction events leading to increased macrophage arachidonic acid metabolism. These intracellular events are essential in sustaining the prolonged inflammatory response to endotoxin.

Accumulation of iron in the rabbit erythroid cell as affected by ouabain, sodium and potassium ions, and temperature.

Reticulocytosis was induced in rabbits with phenylhydrazine. The accumulation of a small part of (59)Fe in blood cells of these animals was inhibited by ouabain and related to changes in extracellular sodium and potassium concentrations. Sodium increases movement from the cell surface into the cell, whereas potassium and ouabain decrease this movement. (59)Fe movement was found to be temperature-dependent. Thus, the Na-K ATPase system appears to be important in the movement of iron from the cell membrane (stroma) to the cell interior, but influences only a small part of the total iron transport.

Studies on the beneficial effects of aspirin in endotoxic shock. Relationship to inhibition of arachidonic acid metabolism.

Endotoxic shock is associated with increased metabolism of arachidonic acid into thromboxanes and prostaglandins. This study assessed the effects of varied doses of aspirin, an inhibitor of arachidonic acid metabolism, on Salmonella enteritidis endotoxin-induced mortality, plasma levels of arachidonate metabolites, and other pathophysiologic sequelae in Long-Evans rats. Aspirin in doses of 3.75, 15, and 30 mg/kg given 30 minutes prior to endotoxin challenge significantly (p less than 0.01) improved 24-hour survival rates from 11 percent to approximately 65 percent, but 100 mg/kg afforded no protection. Pretreatment with aspirin (15 or 100 mg/kg) 30 minutes prior to endotoxin also significantly (p less than 0.001) decreased the endotoxin-induced elevations in plasma levels of immunoreactive thromboxane B2, a stable metabolite of thromboxane A2, and immunoreactive 6-keto prostaglandin F1 alpha, a stable metabolite of prostacyclin. Aspirin doses of 15 or 100 mg/kg given 24 hours prior to challenge with endotoxin significantly improved 24-hour survival rates from 17 percent to 42 percent (p less than 0.01) and 44 percent (p less than 0.005), respectively. Pretreatment with an aspirin dose of 15 mg/kg 24 hours prior to challenge with endotoxin significantly (p less than 0.05) inhibited thrombin-induced immunoreactive thromboxane B2 synthesis in platelet-rich plasma (in vitro) and endotoxin-induced immunoreactive 6-keto-prostaglandin F1 alpha and immunoreactive thromboxane B2 synthesis by rat peritoneal macrophages. Although 24-hour pretreatment with aspirin (15 or 100 mg/kg) significantly (p less than 0.001) reduced endotoxin-induced elevations in immunoreactive thromboxane B2, only the 100 mg/kg dose significantly lowered plasma levels of immunoreactive 6-keto prostaglandin F1 alpha. These observations are consistent with the notion that the beneficial effects of aspirin seen in experimental endotoxic shock may be mediated, in part, via inhibition of arachidonic acid metabolism.

Kinin effects on chloride secretion do not require eicosanoid synthesis.

The actions of bradykinin on colonic epithelia from essential fatty acid-deficient (EFAD) rats has been examined. Electrogenic chloride secretion as short circuit current (SCC) and release of immunoreactive prostaglandin E2 (iPGE2) and i 6-keto PGF1 alpha have been measured. Resting release of prostanoids was significantly less in EFAD than in control tissues. Bradykinin, in a maximally effective concentration, produced no increase in prostanoid release in EFAD tissues in contrast to controls, while the SCC response was 55% of that in controls. In EFAD tissues the SCC response to bradykinin was the same whether or not the cyclooxygenase inhibitor piroxicam was present. EFAD tissues were not more sensitive to prostaglandins than control tissues. We conclude that while prostaglandin release contributes to the totality of the response to bradykinin, the latter's effect on electrogenic chloride secretion does not require the obligatory production of arachidonic acid metabolites.

Effects of a pyridine derivative thromboxane synthetase inhibitor and its inactive isomer in endotoxic shock in the rat.

1 We investigated the effects of a pyridine derivative thromboxane synthetase inhibitor and its inactive ortho isomer on arachidonic acid metabolism and pathophysiological sequelae of endotoxic shock. In vehicle-treated rats, 30 min after intravenous S. enteritidis endotoxin (15 mg/kg), plasma iTxB2 (the stable metabolite of thromboxane A2) increased from non-detectable levels (less than 100 pg/ml) to 763 +/- 250 pg/ml (n = 10). Plasma i6-keto-PGF1 alpha (the stable metabolite of prostacyclin, PGI2) increased to 1850 +/- 426 pg/ml, (n = 9) and plasma iPGE increased to 2350 = 560 (n = 5). Pretreatment with the pyridine active (PA) meta isomer (30 mg/kg i.p.) significantly (P less than 0.05) suppressed iTxB2 to 390 +/- 31 pg/ml (n = 10) although 6-keto-PGF1 alpha levels (1294 +/- 358 pg/ml, n = 5) and plasma iPGE (2847 +/- 1103 pg/ml, n = 5) were not significantly different from the shocked control values. In contrast, pretreatment with, the pyridine inactive (PI) ortho isomer did not significantly affect endotoxin-induced iTxB2 (1431 +/- 194 pg/ml, n = 5) or i6-keto-PGF1 alpha synthesis (628 +/- 266 pg/ml, n = 5). 2 Pretreatment of rats with the Tx synthetase inhibitor, PA, significantly enhanced (P less than 0.05) survival and prevented splanchnic infarction relative to both endotoxin shocked control rats and those pretreated with the PI isomer. 3 Significantly reduced lysosomal labilization, hepatocellular dysfunction and elevations in serum fibrin/fibrinogen degradation products were seen only in groups pretreated with the PA isomer. 4 The beneficial effects of the latter compound in endotoxic shock thus appear to be due to inhibition of Tx synthesis, since its ortho isomer did not inhibit TxA2 synthesis nor did it protect against endotoxic shock.

Role of thromboxane, prostaglandins and leukotrienes in endotoxic and septic shock.

Intravenous bolus endotoxin elicits a marked but transient increase in plasma TxB2 and 6-keto-PGF1 alpha in a large number of species. A smaller, delayed and more prolonged increase in TxB2 and 6-keto-PGF1 alpha are reported in animals with septic shock, i.e., those with fecal peritonitis or cecal ligation. Thromboxane synthetase inhibitors or antagonists attenuate endotoxin-induced acute cardiopulmonary changes, the delayed increase in serum lysosomal enzymes, fibrin/fibrinogen degradation products and the thrombocytopenia in a number of species. While these drugs increase survival of rats or mice following endotoxin they do not alter survival of rats in septic shock. These results support the hypothesis that TxA2 exerts a pathophysiologic effect in shock following bolus endotoxin. In contrast, nonsteroidal antiinflammatory drugs (NSAID) and dietary essential fatty acid deficiency increase survival of rats subjected to endotoxin shock, and survival time in models of septic shock. These results also suggest that some other cyclooxygenase product(s) is involved in septic shock due to fecal peritonitis or cecal ligation. Preliminary experimental studies indicate salutary effects of leukotriene inhibitors and antagonists in endotoxin shock and in models of acute pulmonary injury. Clinical studies have demonstrated elevated plasma TxB2 and 6-keo-PGF1 alpha concentrations in patients with septic shock, and elevated LTD4 in pulmonary edema fluid of patients with the adult respiratory distress syndrome. In view of these clinical and experimental results, clinical trials of NSAID and/or leukotriene inhibitors/antagonists should be considered.

Essential fatty acid-deficient rats are resistant to oleic acid-induced pulmonary injury.

Because leukotrienes and prostaglandins are inflammatory mediators derived from arachidonic acid, their potential role in oleic acid-induced lung injury was evaluated in control and in essential fatty acid-deficient (EFAD) rats depleted of arachidonic acid substrate. In control rats, oleic acid (0.06 ml/kg iv) increased the pulmonary permeability index (measured by scintigraphy) from -10 +/- 13 x 10(-6) s-1 to 217 +/- 20 x 10(-6) s-1 and 118 +/- 13 x 10(-6) s-1 at 5 and 50 min (P less than 0.05), respectively. It also caused arterial hypoxemia at 30 min (P less than 0.05). Compared with saline controls, oleic acid increased bronchoalveolar lavage fluid levels of immunoreactive (i) LTC4/D4, iLTB4, (P less than 0.01), and 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) (P less than 0.05). In EFAD rats, oleic acid failed to significantly increase the lung permeability index at 5 and 50 min. In contrast to control rats, oleic acid failed to cause hypoxemia in the EFAD rats. Bronchoalveolar lavage levels of iLTB4 and i6-keto-PGF1 alpha after oleic acid in EFAD rats were lower compared with oleic acid controls, whereas iLTC4/D4 in the oleic acid EFAD group was not decreased. Treatment with intraperitoneal ethyl arachidonate (400 mg over 2 wk) reversed the resistance of EFAD rats such that the pulmonary edema (P less than 0.05) was evident after oleic acid. This latter group also manifested a significant (P less than 0.05) rise in the bronchoalveolar lavage levels of iLTB4 and i6-keto-PGF1 alpha. These results suggest that arachidonic acid metabolites contribute to oleic acid-induced pulmonary permeability.

Endotoxin tolerance is associated with altered GTP-binding protein function.

Previous studies have suggested that guanine nucleotide regulatory (G) proteins modulate endotoxin-stimulated peritoneal macrophage arachidonic acid (AA) metabolism. Endotoxin-stimulated metabolism of AA by peritoneal macrophages is decreased in endotoxin tolerance (Rogers et al. Prostaglandins 31: 639-650, 1986). These observations led to a study of G protein function and AA metabolism by peritoneal macrophages in endotoxin tolerance. Endotoxin tolerance was induced by the administration of sublethal doses of endotoxin. AA metabolism was assessed by measurement of thromboxane B2 (TxB2), a cyclooxygenase metabolite. NaF (5 mM), an activator of G proteins, significantly stimulated TxB2 synthesis in control macrophages from 7.7 +/- 0.2 to 19.1 +/- 0.6 (SE) ng/ml (P less than 0.05) at 2 h and was partially inhibited by pertussis toxin, suggesting a G protein-dependent mechanism. Salmonella enteritidis endotoxin (50 micrograms/ml) stimulated a similar increase in TxB2 levels (23 +/- 0.4 ng/ml, P less than 0.05). In contrast to control macrophages, macrophages from endotoxin-tolerant rats stimulated with either NaF or S. enteritidis endotoxin had TxB2 levels that were only 30 and 2% of the respective stimulated control cells. Basal guanosine-triphosphatase (GTPase) activity (33 +/- 6 pmol.mg-1.min-1) in endotoxin-tolerant macrophage membranes was significantly lower (P less than 0.05) than control basal activity (158 +/- 5 pmol.mg-1.min-1). This suppression of macrophage GTPase activity was apparent 48 h after the first in vivo sublethal endotoxin injection (100 micrograms/kg ip). The reduced GTPase activity paralleled in vitro cellular hyporesponsiveness to endotoxin-stimulated TxB2 production.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalase pretreatment attenuates oleic acid-induced edema in isolated rabbit lung.

Because reactive O2 metabolites have been demonstrated to be potent mediators of vascular dysfunction and are synthesized by lung tissue, their involvement as mediators of oleic acid (OA)-induced pulmonary edema in the isolated Krebs-perfused rabbit lung was assessed. Injection of OA (0.1 ml) into the pulmonary artery after vehicle pretreatment induced marked increases in lung weight [50.4 +/- 13.9 vs. 4.2 +/- 2.0 (SE) g 45 min after OA or vehicle, respectively, P less than 0.05], an index of pulmonary edema, and airway pressure. OA also caused a significant though minimal increase in pulmonary arterial pressure. Pretreatment with catalase (1,000 U/ml), a scavenger of H2O2, significantly (P less than 0.05, Friedman's) attenuated the increases in lung weight (50.4 +/- 13.9 vs. 15.1 +/- 4.9 g), airway pressure, and pulmonary arterial pressure. In contrast to catalase, pretreatment with Cu-tryptophan (40 microM), a lipid-soluble scavenger of superoxide, provided no protective effect by itself, nor was there any potentiation of protection when combined with catalase. Further evidence implicating O2 metabolites in OA-induced edema was obtained by electron paramagnetic resonance (EPR) spectroscopy of perfusate samples to which the spin trap, sodium 3,5-dibromo-4-nitrosobenzenesulfonate (10 mM), was added. Analysis of these samples revealed the presence of free radicals after OA. Pretreatment with catalase (1,000 U/ml) and superoxide dismutase (250 U/ml) attenuated the EPR signal, indicating that proximal formation of O2 free radicals was in part responsible for the signal. These results suggest that reactive O2 metabolites are mediators of OA-induced pulmonary edema in the isolated perfused rabbit lung.

Effect of the LTD4 receptor antagonist LY203647 on endotoxic shock sequelae in the rat.

This study investigated the effect of a novel LTD4 receptor antagonist LY203647 on Salmonella enteritidis endotoxin-induced shock sequelae in anesthetized rats. LY203647 (30 mg/kg i.v.) or vehicle was given 10 min prior to endotoxin (10 mg/kg i.v.) or its vehicle, and the hematocrit, mean arterial pressure and circulating leukocyte counts were determined. LY203647 significantly inhibited endotoxin-induced hemoconcentration up to 90 min post-endotoxin (46.7 +/- 1.3 vs. 51.9 +/- 2.4% at 30 min post-endotoxin, 45.9 +/- 1.1 vs. 53.1 +/- 1.4% at 90 min post-endotoxin, N = 8-9, P less than 0.05). The endotoxin-induced decreases in mean arterial pressure were also attenuated by LY203647, -29 +/- 5 vs. -56 +/- 9 mm Hg at 60 min post-endotoxin and -42 +/- 4 vs. -60 +/- 9 mm Hg at 90 min post-endotoxin (N = 9-10, P less than 0.05). LY203647 also attenuated endotoxin-induced decreases in leukocyte count in arterial blood. A study of differential counts in circulating leukocytes (N = 3) showed that endotoxin induced complete disappearance in circulating neutrophils. The circulating lymphocyte count was decreased by 30 +/- 10 and 41 +/- 1% at 15 and 30 min post-endotoxin, respectively. LY203647 inhibited endotoxin-induced lymphopenia (P less than 0.05) but failed to alter endotoxin-induced neutropenia. These data suggest that LTD4 may play an important role in mediating hemoconcentration, hypotensive and lymphocytopenic sequelae of endotoxin shock.

Transforming growth factor beta 1 alters rat peritoneal macrophage mediator production and improves survival during endotoxic shock.

Transforming Growth Factor beta 1 (TGF-beta 1) is a potent anti-inflammatory cytokine involved in wound healing. Because of its anti-inflammatory actions, the effects of TGF-beta 1 were studied in vitro on rat macrophage inflammatory mediator production and in vivo on Salmonella enteritidis endotoxin (LPS) induced lethality. TGF-beta 1's effect on stimulated rat peritoneal macrophage (MO) production of prostacyclin and nitric oxide were assessed via measurement of their stable metabolites, 6-keto-PGF-1 alpha and nitrite, respectively. TGF-beta 1 (10 ng/ml) pretreatment (3 hours) resulted in significant reductions (p < 0.05) of LPS (50 micrograms/ml) stimulated, lipid A (1 and 10 micrograms/ml) stimulated, arachidonic acid (16 microM) stimulated, and Ca++ ionophore (10 microM) stimulated MO 6-keto-PGF 1 alpha production. These data suggest that TGF-beta 1 inhibits LPS stimulated MO 6-keto-PGF-1 alpha production by acting at, or distally to, phospholipase A2, possibly at the level of cyclooxygenase in the arachidonic acid cascade. In a similar study, TGF-beta 1 pretreatment led to a significant (p < 0.05) reduction of LPS stimulated MO nitrite production. In subsequent studies, the effects of TGF-beta 1 were studied in vivo on rats challenged with lethal doses of LPS. Rats pretreated with TGF-beta 1 (250 ng/rat i.v.) exhibited a significant increase (p < 0.01, n = 15 rats/group) in mean survival time compared to control rats. The increased survival time of TGF-beta 1 pretreated rats with LPS shock may be due, in part, to altered production of MO mediators.

Maturation of membrane function: transport of amino acid by rat erythroid cells.

The membrane changes which occur during cellular maturation of erythroid cells have been investigated. The transport of alpha-aminoisobutyric acid, alanine, and N-methylated-alpha-aminoisobutyric acid have been studied in the erythroblastic leukemic cell, the reticulocyte, and the erythrocyte of the Long-Evans rat. The dependence of amino acid transport on extracellular sodium concentration was investigated. Erythrocytes were found to transport these amino acids only by Na-independent systems. The steady state distribution ratio was less than 1. Reticulocytes were found to transport alpha-aminoisobutyric acid and alanine by Na-dependent systems, but only small amounts of N-methylated-alpha-aminoisobutyric acid. Small amounts of these amino acids were transported by Na-independent systems. The steady state distribution ratio was greater than one for Na-dependent transport. The erythroblastic leukemia cell, a model immature erythroid cell, showed marked Na-dependence (greater than 90%) for alpha-aminoisobutyric acid and alanine transport, and greater than 80% for the Na-dependent transport of N-methyl-alpha-aminoisobutyric acid. The steady state distribution ratio for the Na-dependent transport was greater than 4. In the erythroblastic leukemic cell, at least three Na-dependent systems are present: one includes alanine and alpha-aminoisobutyric acid, but excludes N-methyl-alpha-aminoisobutyric acid; one is for alpha-aminoisobutyric acid, alanine and also N-methyl-alpha-aminoisobutyric acid; and one is for N-methyl-alpha-aminoisobutyric acid alone. In the reticulocyte, the number of Na-dependent systems are reduced to two: one for alpha-aminoisobutyric acid and alanine; one for N-methyl-alpha-aminoisobutyric acid. In the erythrocytes, no Na-dependent transport was found. Therefore, maturation of the blast cell to the mature erythrocyte is characterized by a systematic loss in the specificity and number of transport system for amino acids.

Amino acid transport in thymic- and spleen-derived lymphocytes.

We have examined the transport of amino acids by the sodium-dependent "A" and "ASC" system in thymic- and splenic-derived lymphocytes from the Long-Evans rat. Lymphocytes derived from the thymus transport amino acids by both the "A" and "ASC" systems, whereas lymphocytes from the spleen transport amino acids by the "ASC" system only. Thymic lymphocytes are capable of establishing a steady state distribution ratio of 7.9 for 2-aminoisobutyric acid, but splenic lymphocytes can attain only 3.5. The steady state distribution ratio of alanine was the same in both cell types. Sodium-independent transport is also different in splenic and thymic lymphocytes. But both cells move amino acids by a Na+-independent system for mediated exchange-diffusion. The studies show that lymphocytes derived from the spleen and thymus transport amino acids differently, and that the "T" lymphocytes from the spleen have membrane transport systems different from "T" lymphocytes from the thymus.

A kinetic analysis of the release of acidic amino acids from rat cortical synaptosomes following pre-loading with [14C]glutamic acid.

The kinetics of the release of acidic amino acids have been studied in rat cortical synaptosomes. After pre-loading for 30 minutes in labeled glutamate, labeled glutamate and aspartate appear to be totally releasable. However, extra-synaptosomal Ca does not facilitate release. When elevated [K]0 was used to depolarize, release was unaffected by removal of Ca from the incubation medium. When veratridine was used as a depolarizing agent, presence of Ca in the incubation medium inhibited release. In all solutions, semi-log plots of synaptosomal label content as a function of time were non-linear, which is incompatible with release from a single compartment. Previous studies of the effect of membrane potential on transport led to the development of a carrier model which should participate in depolarization induced release (19). Under the conditions used in the present studies, this carrier should be saturated. When the data were fitted to a two compartment model, with release from compartment A linear with compartment size and release from compartment B via a saturated carrier, an excellent fit was obtained. Under control conditions, about 90% of the labeled amino acid is in compartment B and about 70% of the total release is from this compartment. Rate of release is greatly accelerated in depolarizing solutions. Under depolarizing conditions, there is a large shift of labeled amino acid from compartment B to compartment A and release from compartment A predominates. Analysis of the results under the several depolarizing conditions used shows that the present results are consistent with the predictions of the carrier model which has been developed from previous studies of the Na and membrane potential dependence of glutamate transport.