A simple method for accurate endotracheal placement of an intubation tube in Guinea pigs to assess lung injury following chemical exposure.

ABSTRACT Guinea pigs are considered as the animal model of choice for toxicology and medical countermeasure studies against chemical warfare agents (CWAs) and toxic organophosphate pesticides because of the low levels of carboxylesterase compared to rats and mice. However, it is difficult to intubate guinea pigs without damaging the larynx to perform CWA inhalation experiments. We describe an easy technique of intubation of guinea pigs for accurate endotracheal placement of the intubation tube. The technique involves a speculum made by cutting the medium-size ear speculum in the midline leaving behind the intact circular connector to the otoscope. Guinea pigs were anesthetized with Telazol/meditomidine, the tongue was pulled using blunt forceps, and an otoscope attached with the specially prepared speculum was inserted gently. Insertion of the speculum raises the epiglottis and restrains the movements of vocal cord, which allows smooth insertion of the metal stylet-reinforced intubation tube. Accurate endotracheal placement of the intubation tube was achieved by measuring the length from the tracheal bifurcation to vocal cord and vocal cord to the upper front teeth. The average length of the trachea in guinea pigs (275 +/- 25 g) was 5.5 +/- 0.2 cm and the distance from the vocal cord to the front teeth was typically 3 cm. Coinciding an intubation tube marked at 6 cm with the upper front teeth accurately places the intubation tube 2.5 cm above the tracheal bifurcation. This simple method of intubation does not disturb the natural flora of the mouth and causes minimum laryngeal damage. It is rapid and reliable, and will be very valuable in inhalation exposure to chemical/biological warfare agents or toxic chemicals to assess respiratory toxicity and develop medical countermeasures.

Transcriptional induction of cholinesterase expression and protection against chemical warfare nerve agents.

We investigated whether transcriptional inducers could enhance the expression of acetylcholinesterase (AChE) in cell lines to achieve protection against organophosphate (OP) poisoning. Trichostatin A (TSA), an inhibitor of histone deacetylase that de-condenses chromatin and increases the binding of transcription factors and mRNA synthesis, induced three- to four-fold extracellular and 8-10-fold intracellular AChE expression at the optimal dose of 165-333 nM in Neuro 2A cells. Pre-treatment with TSA protected against OP exposure. Thus, transcriptional inducers, such as TSA, up-regulate AChE, which then can scavenge the OP and protect the cells from OP-induced toxicity, and are potential novel ways to treat chemical warfare nerve agent (CWNA) exposure.

Prevalence of T cell receptor zeta chain deficiency in systemic lupus erythematosus.

T cells from patients with systemic lupus erythematosus (SLE) display antigen receptor-mediated signaling aberrations associated with defective T cell receptor (TCR) zeta chain expression. We determined the prevalence of TCR zeta chain deficiency in SLE from a large cohort of unselected racially diverse patients with different levels of clinical disease activity as determined by SLE Disease Activity Index (SLEDAI). Our data show that the occurrence of TCR zeta chain deficiency is 78% in SLE patients. There was no relationship between the deficiency of TCR zeta chain and the SLEDAI scores or theapy. TCR zeta chain deficiency was also not associated with age, race or gender and persisted over a 3 year follow-up period. Thus, there is a high prevalence of TCR zeta chain deficiency in SLE patients that is independent of disease activity, and persists over time indicating an important role for TCR zeta chain deficiency in SLE pathogenesis.

Engagement of complement receptor 2 on the surface of B cells from patients with systemic lupus erythematosus contributes to the increased responsiveness to antigen stimulation.

B cells from patients with systemic lupus erythematosus (SLE) display increased responses following cross-linking of the surface antigen receptor. We explored the possibility that the increased responses are at least partially due to simultaneous cross-linking of the complement receptor 2 (CR2). To this end, we stimulated fresh B cells from SLE patients with an anti-IgD antibody conjugated to the Epstein-Barr virus gp350 protein, which binds to CR2, and recorded the free intracytoplasmic calcium response during the first 10 min. Despite the fact that SLE B cells were found to express half as many surface CR2 as normal B cells, both peak responses and the percentage of responding cells were significantly increased in the former. These observations suggest that regulatory molecules such as CR2 are involved in the increased B cell responses in SLE patients. We propose that certain immune complexes that circulate in the sera of SLE patients that have anti-surface immunoglobulin specificities and are decorated with natural ligands of CR2, such as C3d, elicit and promote B cell overactivity.

Defective FcgammaRIIb1 signaling contributes to enhanced calcium response in B cells from patients with systemic lupus erythematosus.

B lymphocytes from patients with systemic lupus erythematosus (SLE) display enhanced B cell antigen receptor (BCR)-mediated early signal transduction events, including increased fluxes of intracytoplasmic calcium ([Ca(2+)](i)). Because crosslinking of FcgammaRIIb1 (CD32) in normal B cells suppresses the BCR-initiated signal transduction process, we investigated whether the increased BCR-initiated [Ca(2+)](i) response in SLE B cells is the consequence of decreased FcgammaRIIb1-mediated suppression. To this end, we used flow cytometry to study the [Ca(2+)](i) responses of indo-1-loaded negatively gated B cells stimulated with F(ab')(2) fragments or whole IgG anti-human micro Ab. We found that the ratio of F(ab')(2) to whole anti-micro Ab [Ca(2+)](i) response was significantly lower in SLE B cells compared to B cells from patients with other systemic rheumatic diseases or normal individuals (P < 0.01). Because the surface expressions of FcgammaRIIb1 and surface IgM were similar in B cells from SLE patients and disease and normal controls, these data indicate a decrease in FcgammaRIIb-mediated suppression in SLE B cells. In addition, the whole IgG anti-micro Ab but not its F(ab')(2) fragment caused increased redistribution of SH2 domain-containing inositol 5'phosphatase in SLE compared to normal and disease control B cells. In conclusion, deficient FcgammaRIIb1-mediated suppression contributes to the augmented [Ca(2+)](i) responses of human SLE B cells.

High dose of dexamethasone upregulates TCR/CD3-induced calcium response independent of TCR zeta chain expression in human T lymphocytes.

Glucocorticoids are very potent anti-inflammatory and immunosuppressive agents that modulate cellular immune responses, although, the molecular mechanisms that impart their complex effects have not been completely defined. We have previously demonstrated that dexamethasone (Dex), a synthetic glucocorticoid, biphasically modulates the expression of TCR (T cell receptor) zeta chain in human T cells. At 10 nM, it induced the expression of TCR zeta chain whereas at 100 nM, it inhibited its expression. In parallel to the upregulation of TCR zeta chain, the TCR/CD3-mediated [Ca(2+)](i) response was enhanced in 10 nM Dex-treated cells. However, at 100 nM, Dex treatment enhanced TCR/CD3-mediated [Ca(2+)](i) response without the induction of TCR zeta chain expression. Because the classical transcriptional model of glucocorticoid action cannot account for the effects of high dose of Dex, here we studied alternative mechanisms of action. We show that, increased and more sustained TCR/CD3-mediated [Ca(2+)](i) response was also observed in 100 nM Dex-treated cells in the presence of actinomycin D or cycloheximide suggesting that cellular transcription and/or de novo protein synthesis are not required for the induction. The TCR/CD3-mediated hyper [Ca(2+)](i) response in 100 nM Dex-treated cells was readily reversible by short-term culture in steroid-free medium. RU-486, a competitive antagonist of Dex, inhibited the increase in [Ca(2+)](i) response suggesting that the effect of Dex is mediated through the glucocorticoid receptor. Although the lipid-raft association of the TCR zeta chain was not significantly increased, high-dose of Dex increased the amount of ubiquitinated form of the TCR zeta chain in the cell membrane along with increased levels of actin. Fluorescence microscopy showed that high-dose of Dex alters the distribution of the TCR zeta chain and form more distinct clusters upon TCR/CD3 stimulation. These results suggest that high dose of Dex perturbs the membrane distribution of TCR zeta chain leading to more functional signaling clusters that result in increased TCR/CD3-mediated [Ca(2+)](i) response independent of TCR zeta chain expression.

Identification of differentially expressed genes in human memory (CD45RO+) CD4+ T lymphocytes.

The precise nature and development of the memory (CD45RO+) CD4+ T lymphocytes remain unknown. In this study, we analyzed differential gene expression of human memory CD4+ T lymphocytes in relation to their naive counterparts. A suppression subtractive hybridization technique was used to isolate and clone differentially expressed genes in the memory subset with respect to the naive subset. We screened approximately 300 clones by dot blot analysis and sequenced 23 differentially expressed clones. GenBank sequence homology search showed that these clones included genes for transcription factors, enzymes and immunomodulatory molecules. Differential expression of a subset of these genes was further confirmed by RT-PCR and densitometric analysis revealed that they were expressed five to eightfold more in memory than naive CD4+ T lymphocytes. Collectively, these results suggest that multiple genes with different functions contribute to the development of immunological memory in human T lymphocytes.

Generation and biochemical analysis of human effector CD4 T cells: alterations in tyrosine phosphorylation and loss of CD3zeta expression.

Human effector T cells have been difficult to isolate and characterize due to their phenotypic and functional similarity to the memory subset. In this study, a biochemical approach was used to analyze human effector CD4 T cells generated in vitro by activation with anti-CD3 and autologous monocytes for 3 to 5 days. The resultant effector cells expressed the appropriate activation/differentiation markers and secreted high levels of interferon gamma (IFN-gamma) when restimulated. Biochemically, effector CD4 T cells exhibited increases in total intracellular tyrosine phosphorylation and effector-associated phosphorylated species. Paradoxically, these alterations in tyrosine phosphorylation were concomitant with greatly reduced expression of CD3zeta and CD3epsilon signaling subunits coincident with a reduction in surface T-cell receptor (TCR) expression. Because loss of CD3zeta has also been detected in T cells isolated ex vivo from individuals with cancer, chronic viral infection, and autoimmune diseases, the requirements and kinetics of CD3zeta down-regulation were examined. The loss of CD3zeta expression persisted throughout the course of effector T-cell differentiation, was reversible on removal from the activating stimulus, and was modulated by activation conditions. These biochemical changes occurred in effector T cells generated from naive or memory CD4 T-cell precursors and distinguished effector from memory T cells. The results suggest that human effector T-cell differentiation is accompanied by alterations in the TCR signal transduction and that loss of CD3zeta expression may be a feature of chronic T-cell activation and effector generation in vivo. (Blood. 2001;97:3851-3859)

T cell signaling abnormalities in systemic lupus erythematosus are associated with increased mutations/polymorphisms and splice variants of T cell receptor zeta chain messenger RNA.

OBJECTIVE: T cells from patients with systemic lupus erythematosus (SLE) display antigen receptor-mediated signaling aberrations associated with defective T cell receptor (TCR) zeta chain protein and messenger RNA (mRNA) expression. This study was undertaken to explore the possibility that coding-region mutations/polymorphisms of the TCR zeta chain could account for its decreased expression and altered signaling in SLE T cells. METHODS: TCR zeta chain mRNA from 48 SLE patients, 18 disease controls, and 21 healthy volunteers was reverse transcribed, amplified by polymerase chain reaction, and cloned, and complementary DNA (cDNA) was sequenced. DNA sequences from multiple clones were analyzed for silent single-nucleotide polymorphisms, mutations, and splice variations, to promote the identification of heterozygosity. RESULTS: DNA sequence analysis revealed several widely distributed missense mutations and silent polymorphisms in the coding region of the TCR zeta chain, which were more frequent in SLE patients than in patients with other rheumatic diseases or healthy controls (P < 0.0001). Several of the missense mutations were located in the 3 immunoreceptor tyrosine activation motifs or the GTP binding domain, and this could lead to functional alterations in the TCR zeta chain. A splice variant of the TCR zeta chain with a codon CAG (glutamine) insertion between exons IV and V was found in half of the SLE and control samples. Two larger spliced isoforms of the TCR zeta chain, with an insertion of 145 bases and 93 bases between exons I and II, were found only in SLE T cells. We also identified various alternatively spliced forms of the TCR zeta chain resulting from the deletion of individual exons II, VI, or VII, or a combined deletion of exons V and VI; VI and VII; II, III, and IV; or V, VI, and VII in SLE T cells. The frequency of the deletion splice variants was significantly higher in SLE than in control samples (P = 0.004). These variations were observed in cDNA and may not reflect the status of the genomic DNA. CONCLUSION: These findings demonstrate that heterogeneous mutations/polymorphisms and alternative splicing of TCR zeta chain cDNA are more frequent in SLE T cells than in T cells from non-SLE subjects and may underlie the molecular basis of known T cell signaling abnormalities in this disease.

Fc epsilon receptor type I gamma chain replaces the deficient T cell receptor zeta chain in T cells of patients with systemic lupus erythematosus.

OBJECTIVE: T cells from the majority of patients with systemic lupus erythematosus (SLE) express significantly lower levels of T cell receptor zeta chain, a critical signaling molecule. However, TCR/CD3 triggering of SLE T cells shows increased phosphorylation of downstream signaling intermediates and increased [Ca2+]i response, suggesting the presence of alternative signaling mechanisms. We investigated whether Fcepsilon receptor type I gamma chain (FcepsilonRIgamma) could substitute for TCR zeta chain and contribute to T cell signaling in SLE. METHODS: T cells were purified from the peripheral blood of 21 patients with SLE and 5 healthy volunteers. The expression of FcepsilonRIgamma was investigated using immunoblotting, reverse transcriptase-polymerase chain reaction, and flow cytometry methods. Involvement of the FcepsilonRIgamma in T cell signaling was studied by immunoprecipitation and/or immunoblotting after TCR/CD3 stimulation. RESULTS: Western blotting and densitometric analysis showed that the expression of FcepsilonRIgamma in SLE T cells was 4.3-fold higher than in normal T cells (P < 0.001). Flow cytometric analyses of T lymphocyte subsets revealed that the proportions of FcepsilonRIgamma+,CD3+, FcepsilonRIgamma+,CD4+, and FcepsilonRIgamma+, CD8+ cells were significantly greater in SLE patients than in healthy controls (P < 0.001). Immunoprecipitation of SLE T cell lysates with an anti-FcepsilonRIgamma antibody showed that FcepsilonRIgamma associates with the tyrosine kinase Syk and the CD3epsilon chain, suggesting that FcepsilonRIgamma is functionally involved in TCR signaling. CONCLUSION: These results demonstrate that the FcepsilonRIgamma chain is expressed at high levels in a large proportion of SLE T cells. The increased expression of FcepsilonRIgamma chain in SLE T cells may account in part for the aberrant antigen receptor-initiated signaling and contribute to the diverse cellular abnormalities found in this disease.

Dexamethasone modulates TCR zeta chain expression and antigen receptor-mediated early signaling events in human T lymphocytes.

Dexamethasone is a potent anti-inflammatory and immunosupressive agent that has complex, yet incompletely defined, effects on the immune response. Here, we explored the effect of dexamethasone on the expression of TCR zeta chain and TCR/CD3-induced early signaling events in human T lymphocytes. Immunoblotting studies using TCR zeta chain specific mAb showed a dose-dependent biphasic effect of dexamethasone on TCR zeta chain expression, that is, it was increased when cells were incubated with 10 nM, whereas the expression was decreased when incubated with 100 nM dexamethasone. The dose-dependent biphasic effect of dexamethsone on the TCR zeta chain expression was also revealed by FACS analysis of permeabilized cells. Time course studies showed that upregulation of the TCR zeta chain at 10 nM dexamethasone reached maximum levels at 24 h and remained elevated up to 48 h. Other subunits of the TCR/CD3 complex were minimally affected under these conditions. The increased expression of the TCR zeta chain following treatment with 10 nM dexamethasone correlated with increased anti-CD3 antibody-induced tyrosine phosphorylation of the TCR zeta chain and downstream signaling intermediate ZAP-70 and PLC gamma with faster kinetics. Similarly, the induction of TCR zeta chain expression at 10 nM dexamethasone correlated with increased and more sustained TCR/CD3-mediated [Ca(2+)](i) response. Reporter gene assays using TCR zeta chain promoter-driven luciferase gene constructs in Jurkat cells showed that treatment with 10 nM dexamethasone increased TCR zeta chain promoter activity and that the region between -160 and +58 was responsible for the observed effect. These results suggest that dexamethasone primarily acts at the transcriptional level and differentially modulates TCR zeta chain expression and antigen receptor-mediated early signaling events in human peripheral T lymphocytes.

Polymorphisms/mutations of TCR-zeta-chain promoter and 3' untranslated region and selective expression of TCR zeta-chain with an alternatively spliced 3' untranslated region in patients with systemic lupus erythematosus.

A vast majority of systemic lupus erythematosus (SLE) patients display decreased expression of TCR zeta-chain mRNA, a critical signaling molecule implicated in the selection of the TCR repertoire and in the prevention of autoimmunity. To identify the molecular mechanisms involved in the downregulation of TCR zeta-chain transcripts in SLE T cells, we investigated the possibility of polymorphisms/mutations in the promoter and the 3' untranslated region. PCR, cloning and sequence analysis of the promoter region from the genomic DNA showed significantly higher number of polymorphisms in SLE T cells compared to non-SLE control subjects (P = 0.044). Promoter sequence was also analysed from granulocytes to delineate the possibility of somatic mutations in activated SLE T cells. Promoter polymorphisms were significantly higher in granulocytes of SLE patients compared to non-SLE controls (P = 0.048), suggesting that these polymorphisms were of genomic origin. Nucleotide analysis of the promoter sequence revealed a -76T insertion compared to the published sequence, in all of the SLE samples and controls. RT-PCR analysis of the TCR zeta-chain 3' untranslated region showed a 344 bp product in addition to the expected 906 bp product. Cloning and sequence analysis of the 344 bp product indicated that it is an alternatively spliced form with both splicing donor and acceptor sites, resulting in deletion of nucleotides 672-1233 of TCR zeta-chain mRNA. Unlike the nomal TCR zeta-chain, the expression of TCR zeta-chain with the alternatively spliced 344 bp 3' untranslated region was higher in SLE T cells compared to non-SLE controls. The number of mutations/polymorphisms in the 906 bp TCR zeta-chain 3' untranslated region were significantly higher in SLE T cells compared to non-SLE subjects (P = 0.032). Frequent mutations/polymorphisms and aberrant splicing of the downstream 3' untranslated region may affect the stability and/or transport of TCR zeta-chain mRNA, leading to its downregulation in SLE T cells.

Immune cell signaling in lupus.

The fate of the lymphocyte is determined by integration of signals delivered after the binding of antigen to the surface antigen receptor, signals delivered by cytokines that bind to their surface receptors, and signals initiated after the engagement of other surface receptors, known as costimulatory molecules. The summation of this input determines whether the immune cell will become stimulated, ignore the signal (anergy), or die (apoptosis). Antigen-receptor signaling events are abnormal in lupus lymphocytes, manifested by increased calcium responses and hyperphosphorylation of several cytosolic protein substrates. Further down, at the gene transcription level, the activity of the nuclear factor kappaB is decreased. These events are underwritten by defective T cell receptor zeta chain expression, overexpression of the gamma chain of the Fc(epsilon)RI that functions as an alternate of zeta chain, and decreased p65 -Rel A protein that is responsible for the inducible NFkappaB activity. Accumulated research data have enabled us to begin deciphering the molecular basis of the abnormal lupus lymphocyte and may lead to the development of new medicinal treatments for lupus.

Heat stress downregulates TCR zeta chain expression in human T lymphocytes.

After heat treatment, human T lymphocytes downregulate the T-cell receptor (TCR)/CD3-mediated [Ca(2+)](i) response and production of inositol triphosphate. Here we demonstrate that heat treatment of T lymphocytes at sublethal temperature decreases the expression of TCR zeta chain, which plays a critical role in the regulation of TCR/CD3-mediated signal transduction. Downregulation of TCR zeta chain in heat-treated T cells was observed at 8 h and reached a maximum at 16 h. Under these conditions, the expression of CD3 epsilon or TCR alphabeta chains was minimally affected. Consistent with the decrease in TCR zeta chain, a reduction in the level of TCR/CD3 induced tyrosine phosphorylation of several cellular protein substrates, and a delay in the kinetics of peak tyrosine phosphorylation was observed in heat-treated T cells. Interestingly, analysis of the TCR zeta chain content in the detergent-insoluble membrane fraction showed that heat treatment induces translocation of soluble TCR zeta chain to the cell membranes. In addition, the mRNA level of TCR zeta chain was reduced in heat-treated T cells. Correlative with the downregulation of TCR zeta chain mRNA, the level of the TCR zeta chain transcription factor Elf-1 was also reduced in heat-treated cells. We conclude that heat stress causes a decrease in the level of TCR zeta chain by increasing its association with the membranes and decreasing the transcription of the TCR zeta gene. Decreased expression of the TCR zeta chain is apparently responsible for the decreased TCR/CD3 responses of T cells.

Inhibition of the cytotoxicity of protein toxins by a novel plant metabolite, mansonone-D.

We have studied the effect of several structurally related mansonones on the cytotoxicity of plant and bacterial toxins in Vero and BER-40, a brefeldin A-resistant mutant of Vero cells. Mansonone-D (MD), a sesquiterpenoid ortho-naphthoquinone, inhibited the cytotoxicity of ricin, modeccin, Pseudomonas toxin, and diphtheria toxin in Vero cells to different extents. The inhibition of ricin cytotoxicity was dose dependent and reversed upon removal of the drug. Protection of ricin cytotoxicity was also observed in the presence of cycloheximide, indicating that de novo protein synthesis is not required for the protective effect. Although MD inhibited the degradation and excretion of ricin, the binding and internalization of ricin was not affected. In contrast, MD strongly reduced the specific binding of diphtheria toxin in Vero cells. Fluorescence microscopic studies show that MD treatment dramatically alters the morphology of the Golgi apparatus in Vero cells. The kinetic studies reveal that the protection of ricin cytotoxicity is the consequence of decreased toxin translocation to the cytosol in MD-treated cells. The reactive ortho-quinone moiety of MD is important for the protective effect as thespesone, a para-naphthoquinone with a heterocyclic ring structure identical to that of MD, did not inhibit the cytotoxicity of toxins. Thespone, a dehydromansonone-D, lacking two hydrogens from the heterocyclic dihydrofuran ring of MD, inhibited the cytotoxicity of ricin, but was albeit less potent than MD. Neither mansonone-E nor mansonone-H with reactive ortho-quinone moiety, but with a different heterocyclic structure, had any effect on the cytotoxicity of ricin indicating that the protective effect of MD is specifically related to the overall structure of the metabolite.

Ilimaquinone inhibits the cytotoxicities of ricin, diphtheria toxin, and other protein toxins in Vero cells.

Ilimaquinone (IQ), a metabolite from sea sponges, has been shown to cause the breakdown of Golgi membranes into small vesicular structure and to inhibit protein transport without eliciting the retrograde transport of the Golgi enzymes to the endoplasmic reticulum [P. A. Takizawa, J. K. Yucel, B. Viet, D. J. Faulkner, T. Deerinck, G. Soto, M. Ellismann, and V. Malhotra, Cell (1993) 73, 1079-1090]. We have found that incubation of Vero cells with IQ inhibited the cytotoxicity of ricin in a dose-dependent manner. The inhibition was reversed upon the removal of IQ. Neither binding and internalization of 125I-ricin nor the translocation of ricin to the cytosol was affected by IQ. However, IQ significantly inhibited the recycling and degradation of internalized 125I-ricin. Preincubation with IQ also prevented the enhancement of ricin cytotoxicity by NH4Cl or nigericin. The inhibition of ricin cytotoxicity by IQ was observed in the presence of cycloheximide, indicating that de novo protein synthesis is not required for IQ-mediated protection of Vero cells from ricin cytotoxicity. In contrast to perinuclear distribution of TRITC-labeled ricin in Vero cells, TRITC-ricin appeared in numerous small vesicles dispersed throughout the cytoplasm in IQ-treated Vero cells. Double labeling with C6-NBD-ceramide and TRITC-labeled ricin showed that these ricin-containing vesicles were distinct from the IQ-induced breakdown product of the Golgi membranes. Like brefeldin A (BFA), IQ inhibited the cytotoxicities of abrin, modeccin, Pseudomonas toxin, and Shiga-like toxin in Vero cells. Unlike BFA, IQ also inhibited the cytotoxicity of diphtheria toxin (DT). Inhibition of DT cytotoxicity was the consequence of a decreased specific binding of the toxin in the IQ-treated cells.

Involvement of the Golgi region in the intracellular trafficking of cholera toxin.

The intracellular pathway following receptor-mediated endocytosis of cholera toxin was studied using brefeldin A (BFA), which inhibited protein secretion and induced dramatic morphological changes in the Golgi region. In both mouse Y1 adrenal cells and CHO cells, BFA at 1 micrograms/ml caused a 80-90% inhibition of the cholera toxin (CT)-induced elevation of intracellular cAMP. The inhibition of the cytotoxicity of CT by BFA was also observed in a rounding assay of Y1 adrenal cells. The inhibition of CT cytotoxicity by BFA was dose dependent, with the ID50 value similar to the LD50 of BFA in Y1 adrenal cells. Binding and internalization of [125I]-labeled cholera toxin in Y1 adrenal cells was not affected by BFA. Unlike the BFA-sensitive cell lines such as Y1 adrenal and CHO cells, BFA at 1 micrograms/ml did not inhibit the cytotoxicity of CT in PtK1 cells, of which the Golgi structure was BFA-resistant. These results strongly suggest that a BFA-sensitive Golgi is required for the protection of CT cytotoxicity by BFA. In contrast, elevation of the intracellular cAMP by forskolin, which acts directly on the plasma membrane adenylate cyclase, was not affected by BFA. These observations indicate that the intoxication of target cells by CT requires an intact Golgi region for its intracellular trafficking and/or processing. In this respect, CT shares a common intracellular pathway with ricin, Pseudomonas toxin, and modeccin, even though their structures and modes of action are very different.