Subversion of human intestinal mucosa innate immunity by a Crohn's disease-associated E. coli.

Adherent-invasive Escherichia coli (AIEC), associated with Crohn's disease, are likely candidate contributory factors in the disease. However, signaling pathways involved in human intestinal mucosa innate host response to AIEC remain unknown. Here we use a 3D model of human intestinal mucosa explant culture to explore the effects of the AIEC strain LF82 on two innate immunity platforms, i.e., the inflammasome through evaluation of caspase-1 status, and NFκB signaling. We showed that LF82 bacteria enter and survive within a few intestinal epithelial cells and macrophages, without altering the mucosa overall architecture. Although 4-h infection with a Salmonella strain caused crypt disorganization, caspase-1 activation, and mature IL-18 production, LF82 bacteria were unable to activate caspase-1 and induce IL-18 production. In parallel, LF82 bacteria activated NFκB signaling in epithelial cells through IκBα phosphorylation, NFκBp65 nuclear translocation, and TNFα secretion. In addition, NFκB activation was crucial for the maintenance of epithelial homeostasis upon LF82 infection. In conclusion, here we decipher at the whole-mucosa level the mechanisms of the LF82-induced subversion of innate immunity that, by maintaining host cell integrity, ensure intracellular bacteria survival.

[Maternal virilization due to mucinous ovarian tumour: a case report]. / Tumeur ovarienne mucineuse virilisante et grossesse: à propos d'un cas.

Virilization in pregnancy is rare and mostly due to luteoma or to hyper-reactio luteinalis. We present a rare case of a virilization borderline mucinous ovarian tumour on a gravida 1 patient. The tumour was responsible for a clinical hyperandrogenism and for an increased level of testosterone. This patient was treated by ovariectomy at 31 weeks of gestation. The surgery was completed one month after delivery. There was no fetal consequence and the clinical and biological signs of virilization totally disappeared after surgery.

[Extra-uterine pelvic leiomyoma: diagnosis and practical management]. / Léiomyome pelvien extra-utérin: diagnostic et prise en charge.

OBJECTIVES: To analyze the possibilities of setting up a therapy for extra-uterine pelvic leiomyomas. METHODS: Three cases of leiomyomas of the broad ligament, of the round ligament and of the ovary, and literature review. RESULTS: Little is known about physiopathology of extra-uterine leiomyoma. The diagnosis of extra-uterine leiomyoma is based on histopathological analysis, using standard histology, and immunohistochemistry with anti-desmin and anti smooth muscle actin antibodies. The main differential diagnoses are fibroma, fibrothecoma, ovarian fibrosarcoma, and gastrointestinal stromal tumors. To define criteria of malignancy, we use Bell's classification without being sure that the uterine and extra-uterine models are comparable. So there is a risk of ignoring a low grade leiomyosarcoma. Providing therapy depends on the clinicopathologic features: the so called "parasitic leiomyoma", a tumor developed at the expense of local smooth muscle cells, metastasis of a benign metastasizing leiomyoma or leiomyomatosis peritonealis disseminata. CONCLUSION: The extra-uterine leiomyoma has no precise nosologic status and no specific criteria of benignity; thus no precise evolution can be predicted. We must be extremely careful, and the issue of the monitoring and long-term therapy of patients must come up.

Altered Calreticulin expression in human colon cancer: maintenance of Calreticulin expression is associated with mucinous differentiation.

Calreticulin is an endoplasmic reticulum luminal calcium-binding chaperone involved in various cellular functions and is a ligand for the scavenger receptor CD91. Recent studies, based on proteomic approaches on whole tissue samples containing both neoplastic and non-neoplastic cells, have shown alterations of Calreticulin expression in colon carcinomas, albeit with divergent results. The aims of this study were: 1) to assess the expression of Calreticulin and its receptor CD91 in 58 human colon adenocarcinomas, compared with paired normal mucosa, using a semi-quantitative immunohistochemical analysis, and 2) to examine associations between the tumour phenotypic features, and Calreticulin and/or CD91 expressions. Calreticulin expression was down-regulated in 51.7% human colon adenocarcinomas. Accordingly, quantitative immunoblot analysis showed that Calreticulin expression was significantly lower in human colonic cancer cell lines than in preparations of isolated human normal colonic epithelial cells. CD91 was co-expressed with Calreticulin in both normal colonic epithelial cells and pericryptic myofibroblasts. Calreticulin and CD91, that characterize the 'amateur phagocyte' function of epithelial cells, were both down-regulated in 48% of adenocarcinomas. Finally, Calreticulin expression was significantly associated with the mucinous differentiation of the tumour. Collectively, these results show that Calreticulin is likely to play a pivotal role in the differentiation of human colonic adenocarcinomas.

[Interest of selective lymphadenectomy in patients with vulvar cancer]. / Intérêt de la technique de lymphadénectomie sélective dans les cancers de vulve.

Vulvar cancer represents approximately 4% of all gynecologic malignancies and the most important prognosis factor in this cancer is the status of the regional lymph nodes. The radical inguinal lymphadenectomy, associated or not with radiotherapy, is accompanied by high morbidity, which can affect 50% of the patients. The sentinel node detection appears now to be feasible in patients with vulvar carcinoma, in order to reduce the morbidity of inguinal lymphadenectomy. But contrary to breast cancer, the learning curve is not easy to obtain because of the low number of cases. That is why we have described the procedure of selective lymphadenectomy. The aim of this technique is to remove the blue and/or marked inguinal lymph node and any other palpable lymph node, without a real radical inguinal lymphadenectomy. Thus, since November 2003, 4 procedures have been performed in total. With the lymphoscintigraphy, we identified 17 marked lymph node and we finally obtained 28 lymph nodes after surgery, with only one metastatic lymph node. There was no complication after our procedure. Selective lymphadenectomy appears to be a new procedure which may reduce the morbidity of usual inguinal lymphadenectomy.

Methods for detecting internalized, FM 1-43 stained particles in epithelial cells and monolayers.

The membrane dye FM 1-43 has frequently been used to quantify exocytosis in neurons. In epithelia, intense lateral intracellular space staining and fluctuations in baseline labeling produced inconsistent results. Membrane retrieved in the presence of FM 1-43 retains the dye, however, and cells that undergo compensatory endocytosis during and following evoked exocytosis contain punctate, fluorescent particles after washout of external stain. As an alternative measure of trafficking, we quantified the fluorescent puncta retained after dye washout and tested our method on both coverslip-grown cell clusters and filter-grown intact monolayers. Images for analysis were acquired using serial sectioning with either epifluorescence or confocal microscopy. Tests with an intestinal goblet cell line that exhibits basal and ATP-stimulated granule trafficking confirmed that 1), the algorithm identified the same number of internalized particles with either epifluorescence or confocal microscopy acquired images; 2), low density clusters exhibited significantly more internalized particles per cell than either filter-grown monolayers or high density clusters; 3), ATP stimulation significantly increased the number of internalized particles in all preparations; and 4), the number of particles internalized was comparable to capacitance measurements of exocytosis. This method provides a single technique for quantifying membrane trafficking in both monolayers and unpolarized cells.

Phosphohistone H3 labelling for histoprognostic grading of breast adenocarcinomas and computer-assisted determination of mitotic index.

BACKGROUND: Microscopic evaluation of mitotic figures is a routine procedure in the assessment of the histoprognostic grade of tumours. Nevertheless, their count may be fraught with difficulties. As histone H3 phosphorylation at serine 10 is closely linked to chromosomal condensation, a new monoclonal antibody directed to phosphorylated histone H3 (PPH3) was recently proposed to detect mitotic cells. AIM: To test the reliability of this antibody in detecting and counting mitotic figures in sections of breast adenocarcinomas, because of the importance of mitotic count in histoprognostic grading. METHODS: The pattern of PPH3 staining in formalin-fixed paraffin wax-embedded tissues, including normal tissues and a series of 39 breast adenocarcinomas, was examined. A new computer-assisted method was also developed for determining the mitotic index. RESULTS AND CONCLUSIONS: In all tissues tested, PPH3-labelled mitotic figures were easily detected, allowing a rapid identification of the area of highest mitotic activity. In breast carcinomas, a strong correlation was observed between PPH3-stained and haematoxylin and eosin-stained mitotic counts (r = 0.86, p<0.0001). Counting of prophase nuclei that coexpress cyclin B1, a marker of the G2/M phase, was possible by PPH3 staining; its accuracy led us to reconsider the tumour grade in three cases. Finally, an automatic computer-assisted method was designed for assessing mitotic index with confocal microscopy and image-analysis software.

Antibiotic therapy reduces nitrosative stress and programmed cell death in the rabbit foetal lung.

The correlation of clinical and epidemiological data suggests that intrauterine infection/inflammation can promote foetal lung injury. The aim of this study was: 1) to characterise the early inflammatory response elicited in infected foetal lungs, in terms of nitric oxide-derived oxidative stress and programmed cell death; and 2) to investigate the effects of antibiotic therapy on these parameters. A previously described rabbit experimental model of materno-foetal infection was used. Animals were divided into three groups: controls; Escherichia coli infected (12 h); and E. Coli infected (12 h) and treated (24 h gentamicin+ceftriaxone). Foetal lungs were examined in terms of histology, nitric oxide synthase (NOS) activity, immunohistochemical detection of 3-nitrotyrosine, and detection of apoptotic cells by the TUNEL assay and Hoechst staining. In the infected group, a moderate inflammatory response was observed, associated with a significant increase in inducible NOS activity, the formation of 3-nitrotyrosine residues in epithelial and immune cells, the down-regulation of constitutive NOS activity and clusters of apoptotic cells, as compared with the control group. Early antibiotic therapy, initiated at 12 h post-inoculation, elicited a significant decrease in the infection-induced nitrosative stress. Levels of 3-nitrotyrosine and of apoptotic cells were decreased in the infected-and-treated group compared with the infected group, mainly by the re-expression of constitutive NOS and of the basal level of inducible NOS. Altogether, these findings indicate that early antibiotic therapy can curb the inflammatory reaction and help avert antenatal lung injury, which is known to be involved in the onset of bronchopulmonary dysplasia.

Loss of NOS1 expression in high-grade renal cell carcinoma associated with a shift of NO signalling.

In normal human kidney, NOS1 and soluble guanylate cyclase (sGC) are expressed in tubular epithelial cells, suggesting a physiological autocrine NO signalling pathway. Therefore, we investigated both NOS1 and sGC expressions in benign and malignant renal tumours. In addition, we examined the pattern of protein tyrosine nitration in normal and tumour tissue. NOS1 expression and activity were found to be downregulated, correlating with the tumour grade, as shown by immunohistochemistry, quantitative RT-PCR analysis, and histochemical detection of the NADPH-diaphorase activity of nitric oxide synthases (NOS). These results show that the autocrine NO signalling pathway is maintained in benign tumours and lost in malignant tumours. In contrast, sGC expression was maintained in renal tumours whatever the tumour type, a finding showing that tumour cells remain sensitive to the bioregulatory role of exogeneous NO(*). Finally, the staining pattern of protein tyrosine nitration, assessed by immunohistochemistry, parallelled that of NOS1 expression in normal renal parenchyma and benign tumours, supporting the concept that protein nitration was accounted for by NOS1 activity. In contrast, in malignant tumours, protein tyrosine nitration was accounted for by the production of reactive nitrogen oxide species by the inflammatory infiltrate. Altogether, these findings argue for a pattern of NO signalling similar in normal kidney and benign renal tumours, whereas it is completely different in malignant renal tumours.

Niflumic acid inhibits ATP-stimulated exocytosis in a mucin-secreting epithelial cell line.

ATP is an efficacious secretagogue for mucin and chloride in the epithelial cell line HT29-Cl.16E. Mucin release has been measured as [3H]glucosamine-labeled product in extracellular medium and as single-cell membrane capacitance increases indicative of exocytosis-related increases in membrane area. The calcium-activated chloride channel blocker niflumic acid, also reported to modulate secretion, was used to probe for divergence in the purinergic signaling of mucin exocytosis and channel activation. With the use of whole cell patch clamping, ATP stimulated a transient capacitance increase of 15 +/- 4%. Inclusion of niflumic acid significantly reduced the ATP-stimulated capacitance change to 3 +/- 1%, although normalized peak currents were not significantly different. Ratiometric imaging was used to assess intracellular calcium (Cai2+) dynamics during stimulation. In the presence of niflumic acid, the ATP-stimulated peak change in Cai2+ was unaffected, but the initial response and overall time to Cai2+ peak were significantly affected. Excluding external calcium before ATP stimulation or including the capacitative calcium entry blocker LaCl3 during stimulation muted the initial calcium transient similar to that observed with niflumic acid and significantly reduced peak capacitance change, suggesting that a substantial portion of the ATP-stimulated mucin exocytosis in HT29-Cl.16E depends on a rapid, brief calcium influx through the plasma membrane. Niflumic acid interferes with this influx independent of a chloride channel blockade effect.

Human submucosal neurones regulate intestinal epithelial cell proliferation: evidence from a novel co-culture model.

The role of the human enteric nervous system (ENS) in the control of the intestinal epithelium organization and proliferation is unknown. To address this issue, we developed a novel co-culture model, consisting of human submucosa containing the submucosal plexus and a human colonic epithelial monolayer. After 3 days in basal conditions (i.e. in absence of neuronal activation) epithelium disorganization and proliferation occurred. In contrast, electrical activation of submucosal neurones maintained monolayer organization and decreased cell proliferation. These effects were blocked by tetrodotoxin and a vasoactive intestinal peptide (VIP) receptor antagonist, and reproduced by VIP. In conclusion, our study suggests that the human ENS is involved in the control of epithelial cell proliferation.

Overexpression of the CD155 gene in human colorectal carcinoma.

BACKGROUND AND AIMS: The Tage4 gene (tumour associated glycoprotein E4) is overexpressed in rat colon tumours and Min mouse intestinal adenomas. The rat Tage4 protein has approximately 40% identity with human CD155, a member of the immunoglobulin superfamily coding for a transmembrane protein capable of serving as an entry receptor for poliovirus, porcine pseudorabies virus, and bovine herpesvirus 1. Analysis of the rat Tage4 gene has revealed structural and functional similarities with the human CD155 gene. We therefore investigated expression of the CD155 gene in human colorectal carcinomas. METHODS: Overall CD155 expression was assessed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical analysis using tissue specimens from patients with colorectal adenomas and adenocarcinomas. We also used a qualitative RT-PCR assay to determine relative expression of different splicing variants in each sample. RESULTS: mRNA levels of CD155 were increased in six of six colorectal cancer tissues compared with the tumour free colon mucosa. Immunohistochemical analysis revealed an increased level of CD155 protein in 12 of 12 samples. The qualitative RT-PCR assay revealed that relative expression of the different CD155 variant transcripts was similar in the different normal and cancer samples tested, indicating that this overexpression is not associated with a particular mRNA variant generated by alternative splicing of the CD155 gene. CONCLUSION: We have shown for the first time that the CD155 gene is overexpressed in colorectal carcinoma and that this overexpression begins at an early stage in tumorigenesis and continues to late stages.

[Protective effect of an apoptosis inhibitor in a new model of hepatitis induced by interleukin-4 in the rat]. / Effet protecteur d'un peptide inhibiteur de l'apoptose dans un nouveau modèle d'hépatite induite par l'interleukine-4 chez le rat.

OBJECTIVES: Interleukin-4 is a cytokine with pleiotropic effects on many cells. The effects of its expression on the liver remain unclear. To obtain organ-localized cytokine expression and analyze its effect on the liver, recombinant adenovirus with coding sequences of interleukin-4 were transduced to rat livers. METHODS: Adenovirus with coding sequences of rat interleukin-4 were injected into the portal vein of Wistar rats. Microscopic examination of the liver was performed. The effects of interleukin-4 were confirmed in vitro on primary cultured rat hepatocytes. The same analysis was performed after intraperitoneal injection of l'YVADcmk, an inhibitor of the interleukin 1 converting enzyme. RESULTS: Interleukin-4 expression due to the recombinant adenovirus produced dose-related, potentially lethal, severe hepatitis. This hepatitis was characterized by a leucocyte infiltrate mainly composed of eosinophilic polymorphonuclear and mast cells with numerous apoptotic hepatocytes. Intraperitoneal injection of YVADcmk decreased hepatocyte apoptosis and biological hepatitis and prevented death. CONCLUSION: These results suggested that YVADcmk might be used in fulminant hepatitis in which apoptosis is predominant.

The in vitro manipulation of carbohydrate metabolism: a new strategy for deciphering the cellular defence mechanisms against nitric oxide attack.

This study was aimed at examining the effects of manipulating the carbohydrate source of the culture medium on the cellular sensitivity of epithelial cells to an oxidative attack. Our rationale was that substituting galactose for glucose in culture media would remove the protection afforded by glucose utilization in two major metabolic pathways, i.e. anaerobic glycolysis and/or the pentose phosphate pathway (PPP), which builds up cellular reducing power. Indeed, we show that the polarized human colonic epithelial cell line HT29-Cl.16E was sensitive to the deleterious effects of the NO donor PAPANONOate [3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine] only in galactose-containing medium. In such medium NO attack led to cytotoxic and apoptotic cell death, associated with formation of derivatives of NO auto-oxidation (collectively termed NOx) and peroxynitrite, leading to intracellular GSH depletion and nitrotyrosine formation. The addition of 2-deoxyglucose, a non-glycolytic substrate, to galactose-fed cells protected HT29-Cl. 16E cells from NO attack and maintained control GSH levels through its metabolic utilization in the PPP, as shown by (14)CO(2) production from 2-deoxy[1-(14)C]glucose. Therefore, increasing the availability of reducing equivalents without interfering with energy metabolism is able to prevent NO-induced cell injury. Finally, this background provides the conceptual framework for establishing nutritional manipulation of cellular metabolic pathways that could provide new means for (i) deciphering the mechanisms of cell injury by reactive nitrogen species and reactive oxygen species at the whole-cell level and (ii) establishing the hierarchy of intracellular defence mechanisms against these attacks.

Interleukin 1 and interleukin 1beta converting enzyme (caspase 1) expression in the human colonic epithelial barrier. Caspase 1 downregulation in colon cancer.

BACKGROUND: Interleukin (IL) 1beta converting enzyme (now known as caspase 1) is able to process pro-IL-1beta into its active form and is involved in proapoptotic signalling. AIMS: To characterise IL-1 and caspase 1 expression in human colonic epithelial cells. METHODS: Intracellular IL-1 content (IL-1alpha and IL-1beta) was measured by ELISA in freshly isolated human normal colonocytes. Caspase 1 expression was determined both at the mRNA level using in situ hybridisation and reverse transcription polymerase chain reaction, and at the protein level by immunoblotting experiments using antibodies specific for the proform of caspase 1 and for its cleavage forms. RESULTS: Low amounts of IL-1beta were found in nearly all preparations (92%), and IL-1alpha was detected in only 45% of human colonocyte preparations. The normal colonic epithelium strongly expressed caspase 1, both at the mRNA level and at the protein level in its latent form. In contrast, caspase 1 was not expressed in colon cancer (primary colonic adenocarcinomas and cancer cell lines). CONCLUSIONS: The demonstration that the human colonic epithelial barrier is able to express caspase 1 and its substrate IL-1beta reinforces the concept that, under certain conditions, the epithelium could trigger an inflammatory reaction. In addition, the finding that caspase 1 was downregulated in colonic adenocarcinomas supports the concept that disrupted apoptosis pathways may be involved in tumour formation and/or may confer resistance to treatment.

Detection of translocation t(11;14)(q13;q32) in mantle cell lymphoma by fluorescence in situ hybridization.

To assess an unequivocal diagnosis of mantle cell lymphoma (MCL), we have developed a fluorescence in situ hybridization (FISH) assay, enabling the demonstration of t(11;14)(q13;q32) directly on pathological samples. We have first selected CCND1 and IGH probes encompassing the breakpoint regions on both chromosomes. Then, we have defined experimental conditions enabling us to obtain bright clear-cut signals in all of the samples, independently of the initial fixation conditions. We have analyzed single-cell suspensions from 26 formalin-fixed, paraffin-embedded MCL samples with this set of probes. In all cases, we have found a fusion signal (ie, a t(11;14)(q13;q32) translocation) in 14% to 99% of cells (median, 87%). So far, IGH-CCND1 fusions have been detected in all of the 51 MCL patients that we have analyzed by FISH (either on paraffin-embedded tumor samples or on peripheral blood samples). Regarding the low sensitivity of other techniques used to diagnose t(11;14)(q13;q32) (ie, 70% to 75% for cytogenetics and 50% to 60% for polymerase chain reaction), our FISH assay is by far the most sensitive technique. Moreover, because of the quality of the fluorescent signals and the rapidity of the experiment, this technique is widely applicable, even in routine cytogenetics or pathology laboratories. As MCL patients are usually refractory to standard therapy, an unambiguous diagnosis is needed to propose adapted therapeutic strategies, and this highly sensitive assay may be of great value for accurate diagnosis in difficult cases.

Purinergic and cholinergic agonists induce exocytosis from the same granule pool in HT29-Cl.16E monolayers.

Several secretagogues induce mucin secretion in epithelial monolayers, as determined by measuring released granule contents. To assess whether different agonists act on the same granule pool, capacitance changes in intact monolayers of the goblet cell line HT29-Cl.16E were measured by a novel impedance method. Apical ATP (purinergic agonist) and basolateral carbachol (cholinergic agonist) induce rapid exocytosis with maximal capacitance changes within 3 min. The maximal levels of exocytosis that can be induced by optimal concentrations of either agonist are the same and produce a 30-40% increase in total monolayer capacitance. When ATP and carbachol are applied simultaneously, the magnitude of exocytosis is unchanged from the single-secretagogue level. The recovery of capacitance to baseline (endocytosis) is significantly faster after ATP stimulation than after carbachol stimulation. When ATP and carbachol are applied sequentially at doses that give maximal exocytosis, the magnitude of the capacitance increase produced by the second secretagogue is less than or equal to that of the capacitance decrease during the recovery period. Together, these data suggest that purinergic and cholinergic agonists act on the same granule pool.

Control of growth and differentiation of normal human epithelial cells through the manipulation of reactive nitrogen species.

In this work, we addressed the issue of whether exogenous NO and ONOO- (peroxynitrite) are able to alter growth, viability and/or differentiation of normal epithelial cells using cultured normal human keratinocytes as a model. 3-Morpholino-sydnonimine (SIN-1), a donor of both NO and O2(-)., leading to the production of ONOO-, dose-dependently inhibited growth of human keratinocytes without loss of viability. This inhibitory effect was lowered when SIN-1 was transformed into a pure NO donor by scavenging O2(-). with superoxide dismutase/catalase. Finally, scavenging NO release from SIN-1 with carboxy-1H-imidazol-1-yloxy,2-(4-carboxyp henyl)-4,5-dihydro-4,4,5,5 -tetramethyl-3-oxide (PTIO) resulted in a loss of the inhibitory effect of SIN-1. Together these findings suggest that both ONOO- and NO exert a growth inhibitory effect on human keratinocytes without cytotoxicity. Further support for this conclusion came from the treatment of human keratinocytes with the NO. donor propanamine 3-(2-hydroxy-2-nitroso-1-propyl hydrazino) or with authentic peroxynitrite. Moreover, only SIN-1 or peroxynitrite, and not NO, was able to trigger the expression of markers of terminal differentiation in human keratinocytes. From a physiological perspective, the ability of peroxynitrite, a known genotoxic and potentially carcinogenic agent, to direct proliferating keratinocytes towards terminal differentiation may be crucial to protect the genomic stability of this barrier epithelium.

Purinergic agonists, but not cAMP, stimulate coupled granule fusion and Cl- conductance in HT29-Cl.16E.

Cl- conductance and capacitance were simultaneously measured in the mucin-secreting cell line, HT29-Cl.16E (Cl.16E), and its sister cell line, HT29-Cl.19A (Cl.19A), which lacks mucin granules. Purinergic stimulation by extracellular ATP transiently increased Cl- conductance in both cell lines with similar peak increases of 0.92 and 1.00 nS/pF in Cl.16E and Cl.19A cells, respectively (baseline of 0.08 nS/pF). Cell capacitance increased only in Cl.16E cells (17% above baseline of 22 pF in Cl.16E and 1% above baseline of 18 pF in Cl.19A cells). Wortmannin inhibited the purinergically activated Cl- conductance and capacitance increases in Cl.16E by 50 and 80%, respectively, but had no effects in Cl.19A cells. In Cl.16E cells, adenosine 3',5'-cyclic monophosphate (cAMP) signaling increased Cl- conductance from 0.08 to 0.52 nS/pF without changing capacitance. Cl- secretion in Cl.16E monolayers was additive in response to supramaximal stimulation of purinergic receptors and adenylyl cyclase, even though granule fusion is nine times greater with purinergic than adenylyl cyclase stimulation. In conclusion, 1) wortmannin does not directly inhibit activation of Cl- conductance, 2) at least 50% of purinergically activated Cl- conductance in Cl.16E is associated with granule fusion, and 3) cAMP-activated Cl- conductance is not associated with granules.