Cyclothiazide potentiates agonist responses at human AMPA/kainate receptors expressed in oocytes.

Cloned human AMPA/kainate subunits were functionally expressed in Xenopus oocytes. Cyclothiazide potentiated kainate-evoked currents by 682 +/- 122% (mean +/- S.E.M., n = 5), 1396 +/- 55% (n = 4), and 690 +/- 40% (n = 14) in oocytes expressing GluR1, GluR2, and GluR1 + GluR2 receptors, respectively. AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate)-induced currents were also potentiated by cyclothiazide. GYKI 52466 (1-(4-amino-phenyl)-4-methyl-7,8-methylendioxyl-5H-2,3-benzod++ + iazepine hydrochloride) attenuated cyclothiazide potentiation in all cases. Thus, modulatory sites for cyclothiazide and GYKI 52466 exist on individual human AMPA/kainate receptor subunits. Additionally, kainate appears to act as a desensitizing partial agonist at human GluR1 and GluR2 receptor subunits.

Transcriptional inhibition of endotoxin-induced monokine synthesis following heat shock in murine peritoneal macrophages.

The role of heat shock proteins (HSP) during the inflammatory response has been controversial. The effect of heat shock (HS) on the synthesis of the monokines tumor necrosis factor (TNF) and interleukin 1 (IL-1) by endotoxin-stimulated thioglycollate-elicited peritoneal macrophages was investigated. HS was deemed to have affected macrophages if a 70 kD HSP appeared on SDS gels; identity of this protein as the highly conserved HSP70 was then confirmed by immunoprecipitation. Maximal increases in HSP70 were apparent 2-5 h after HS at 45 degrees C for 12 min. Synthesis of HSP70 was no longer detected 24 h post HS. A reciprocal relationship between HSP70 and TNF was apparent in kinetic studies. TNF was not detected in culture supernatants if macrophages were endotoxin-stimulated 2 to 6 h after HS; however, the same stimulation 24 h later induced significant TNF secretion. RNA analysis of HS and non-HS macrophage cultures demonstrated a 60-fold reduction in TNF message in the HS macrophages 1 h after endotoxin stimulation. TNF mRNA levels remained depressed at 6 h while the HSP70 message had increased 30-fold. The ability of HS macrophages to ingest antibody-coated erythrocytes was not significantly affected following heat treatment. Macrophage response to HS can be said to inhibit transcription of inducible monokines while retaining other macrophage functions.

Differential expression of interleukin-1 and tumor necrosis factor in murine septic shock models.

Murine shock models have employed bolus endotoxin as well as cecal ligation and puncture (CLP) in an attempt to understand the pathophysiologic changes associated with human septic shock. Injection of endotoxin results in a rapid but transient rise in tumor necrosis factor (TNF), with maximal levels between 1 and 2 hr followed by an increase in serum interleukin-1 (IL-1) by 3 hr which remains elevated at 24 hr. CLP animals in contrast do not demonstrate an elevation in serum TNF, and the increase in IL-1 is less significant relative to the endotoxin model. Whereas both models demonstrate comparable increases in serum amyloid A protein and result in host death between 24 and 48 hr, these models respond differently to therapeutic modalities. Steroids and an anti-TNF monoclonal antibody administered prophylactically are effective at preventing death in the endotoxin model and yet had no beneficial effect in antibiotic-treated CLP animals. Experiments with adrenalectomized mice suggest that the absence of serum TNF in the CLP model is in part due to the modulatory effects of endogenous corticosteroids.

Endotoxin-macrophage interaction: post-translational regulation of tumor necrosis factor expression.

Thioglycollate-elicited murine peritoneal macrophages produce significant quantities of TNF 2 to 4 h after induction with bacterial endotoxin, LPS. However, macrophages exposed to a second LPS stimulus are refractory and the amount of TNF detected in these supernatants is reduced 10- to 50-fold. The acquisition of the refractory state in vitro or in vivo requires the continued presence of LPS for a minimum of 6 to 8 h, is optimal by 20 h, and is reversible. Refractory macrophages incubated for an additional 48 h in the absence of LPS produce significant quantities of TNF after reexposure to endotoxin. Although LPS refractory macrophages do not release TNF in response to a secondary endotoxin challenge, riboprobe ribonuclease protection assays demonstrated amplification of TNF message, suggesting that post-transcriptional events are involved in the regulation of TNF production in endotoxin refractory macrophages. Immunoprecipitation studies revealed the accumulation of the 26-kDa TNF precursor in lysates of refractory macrophages, thus demonstrating a post-translational regulatory process. Although LPS refractory macrophages do not release TNF in response to a second LPS stimulus, ingestion of zymosan by these cells results in the release of significant quantities of TNF. Furthermore, LPS-refractory macrophages do not demonstrate a reduction in other effector functions including Fc-mediated erythrophagocytosis. Therefore, the LPS refractory state is a metabolically dependent post-translational regulatory process, which requires continuous LPS exposure, is specific in which macrophage effector functions are inhibited, and is reversible with further incubation or by non-LPS-related stimuli.