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)

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1 The effects of pretreatment with the thromboxane synthetase inhibitor UK 37248 (dazoxiben) administered 30 min before intravenous endotoxin (S. enteriditis) in the rat was investigated. 2 Plasma prostaglandins and thromboxanes were determined via radioimmunoassay. Endotoxaemia was associated with significant elevations above control values (<200pg/ml) in plasma thromboxane B2 (TXB2), prostaglandin E (PGE) and 6-keto-prostaglandin Fla (6-keto-PGFia). Within 30 min after endotoxin administration plasma immunoreactive (i) iTXB2 was 875±90pg/ml (n=9), iPGE was 1670±271 (n=9) and i6-keto-PGFia was 1191±209 pg/ml (n= 10). By 4 h plasma iTXB2 was 1743±328 pg/ml (n=5), iPGE was 2589±494pg/ml (n=9) and i6-keto-PGFia was 4251±984pg/ml (n=10). UK 37248 pretreatment resulted in a significant (P<0.001) decrease in plasma iTXB2 at 30min and 4h to 193±28pg/ml (n=5) and 421±57pg/ml (n=5), respectively. Unexpectedly UK 37248 also significantly decreased plasma i6-keto PGFia at 30 min and 4 h to 360±75 pg/ml (n= 10) (P<0.005) and 1920±513 pg/ml (n= 10) (P<0.05), respectively. iPGE plasma levels were not significantly changed in the UK 37248-pretreated rats 30 min (2210±370 pg/ml (n=9) or 4 h 3529± 1093 pg/ml (n= 13) after endotoxin compared to the vehicle-treated rats. 3 UK 37248 significantly (P<0.05) reduced the endotoxin mortality rate at 24 h from 69% (n= 13) to 30% (n= 13). UK 37248 also reduced splanchnic infarction from 90% (n = 20) to 6% (n = 16). 4 UK 37248 significantly improved the endotoxin-induced thrombocytopaenia, disseminated intravascular coagulation, hypoglycaemia and lysosomal labilization. 5 We conclude that UK 37248 provides significant beneficial effects in experimental endotoxic shock in the rat.

Ca2+ is thought to play a role in the enhancement of water permeability of toad urinary bladder epithelial cells by antidiuretic hormone (ADH) or theophylline. This study examined the effects of ADH and theophylline on intracellular free Ca2+ ([Ca2+]i) and total cellular exchangeable Ca2+ in isolated toad bladder epithelial cells. ADH or theophylline enhanced water permeability maximally by 15-25 min after a 4-min lag. 45Ca2+ efflux, a probe for total cellular exchangeable (plasma membrane plus intracellular) Ca2+, was enhanced by ADH within 2 min and returned to control by 8 min. Chlortetracycline fluorescence, a probe for intracellular Ca2+ only, was not affected, suggesting that ADH released only plasma membrane-bound Ca2+. Theophylline enhanced 45Ca2+ efflux and decreased chlortetracycline fluorescence, suggesting release of Ca2+ from intracellular sources. Both agents decreased [Ca2+]i as assessed by quin-2 fluorescence with a time course similar to the enhancement in water permeability. The results suggest that the changes in membrane-bound Ca2+ and [Ca2+]i induced by ADH and theophylline may play a role in the enhanced permeability to water in response to these agents.

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