Ethanol and its non-oxidative metabolites profoundly inhibit CFTR function in pancreatic epithelial cells which is prevented by ATP supplementation.

Excessive alcohol consumption is a major cause of acute pancreatitis, but the mechanism involved is not well understood. Recent investigations suggest that pancreatic ductal epithelial cells (PDECs) help defend the pancreas from noxious agents such as alcohol. Because the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel plays a major role in PDEC physiology and mutated CFTR is often associated with pancreatitis, we tested the hypothesis that ethanol affects CFTR to impair ductal function. Electrophysiological studies on native PDECs showed that ethanol (10 and 100 mM) increased basal, but reversibly blocked, forskolin-stimulated CFTR currents. The inhibitory effect of ethanol was mimicked by its non-oxidative metabolites, palmitoleic acid ethyl ester (POAEE) and palmitoleic acid (POA), but not by the oxidative metabolite, acetaldehyde. Ethanol, POAEE and POA markedly reduced intracellular ATP (ATPi) which was linked to CFTR inhibition since the inhibitory effects were almost completely abolished if ATPi depletion was prevented. We propose that ethanol causes functional damage of CFTR through an ATPi-dependent mechanism, which compromises ductal fluid secretion and likely contributes to the pathogenesis of acute pancreatitis. We suggest that the maintenance of ATPi may represent a therapeutic option in the treatment of the disease.

Gray matter textural heterogeneity as a potential in-vivo biomarker of fine structural abnormalities in Asperger syndrome.

Brain imaging studies contribute to the neurobiological understanding of Autism Spectrum Conditions (ASC). Herein, we tested the prediction that distributed neurodevelopmental abnormalities in brain development impact on the homogeneity of brain tissue measured using texture analysis (TA; a morphological method for surface pattern characterization). TA was applied to structural magnetic resonance brain scans of 54 adult participants (24 with Asperger syndrome (AS) and 30 controls). Measures of mean gray-level intensity, entropy and uniformity were extracted from gray matter images at fine, medium and coarse textures. Comparisons between AS and controls identified higher entropy and lower uniformity across textures in the AS group. Data reduction of texture parameters revealed three orthogonal principal components. These were used as regressors-of-interest in a voxel-based morphometry analysis that explored the relationship between surface texture variations and regional gray matter volume. Across the AS but not control group, measures of entropy and uniformity were related to the volume of the caudate nuclei, whereas mean gray-level was related to the size of the cerebellar vermis. Similar to neuropathological studies, our study provides evidence for distributed abnormalities in the structural integrity of gray matter in adults with ASC, in particular within corticostriatal and corticocerebellar networks. Additionally, this in-vivo technique may be more sensitive to fine microstructural organization than other more traditional magnetic resonance approaches and serves as a future testable biomarker in AS and other neurodevelopmental disorders.

Prefrontal activity in Huntington's disease reflects cognitive and neuropsychiatric disturbances: the IMAGE-HD study.

Functional integrity of prefrontal cortico-striatal circuits underlying executive functioning may be compromised by basal ganglia degeneration during Huntington's disease (HD). This study investigated challenged inhibitory attentional control with a shifting response-set (SRS) task whilst assessing neural response via functional magnetic resonance imaging (fMRI) in 35 healthy controls, 35 matched pre-symptomatic (pre-HD) and 30 symptomatic (symp-HD) participants. A ≥70% performance accuracy threshold allowed confident identification of neural activity associated with SRS performance in a sub-set of 33 healthy controls, 32 pre-HD and 20 symp-HD participants. SRS activated dorsolateral prefrontal and dorsal anterior cingulate cortices, premotor, parietal, and basal ganglia regions and deactivated subgenual anterior cingulate cortex. Symp-HD participants showed greater prefrontal functional responses relative to controls and pre-HD, including larger activations and larger deactivations in response to cognitive challenge, consistent with compensatory neural recruitment. We then investigated associations between prefrontal BOLD responses, SRS performance accuracy and neuropsychiatric disturbance in all participants, including those below SRS performance accuracy threshold. We observed that reduced prefrontal responsivity in symp-HD was associated with reduced accuracy in SRS performance, and with increased neuropsychiatric disturbance within domains including executive dysfunction, pathological impulses, disinhibition, and depression. These findings demonstrate prefrontal response during inhibitory attentional control usefully characterises cognitive and neuropsychiatric status in symp-HD. The functional integrity of compensatory prefrontal responses may provide a useful marker for treatments which aim to sustain cognitive function and delay executive and neuropsychiatric disturbance.

Automated differentiation of pre-diagnosis Huntington's disease from healthy control individuals based on quadratic discriminant analysis of the basal ganglia: the IMAGE-HD study.

We investigated two measures of neural integrity, T1-weighted volumetric measures and diffusion tensor imaging (DTI), and explored their combined potential to differentiate pre-diagnosis Huntington's disease (pre-HD) individuals from healthy controls. We applied quadratic discriminant analysis (QDA) to discriminate pre-HD individuals from controls and we utilised feature selection and dimension reduction to increase the robustness of the discrimination method. Thirty six symptomatic HD (symp-HD), 35 pre-HD, and 36 control individuals participated as part of the IMAGE-HD study and underwent T1-weighted MRI, and DTI using a Siemens 3 Tesla scanner. Volume and DTI measures [mean diffusivity (MD) and fractional anisotropy (FA)] were calculated for each group within five regions of interest (ROI; caudate, putamen, pallidum, accumbens and thalamus). QDA was then performed in a stepwise manner to differentiate pre-HD individuals from controls, based initially on unimodal analysis of motor or neurocognitive measures, or on volume, MD or FA measures from within the caudate, pallidum and putamen. We then tested for potential improvements to this model, by examining multi-modal MRI classifications (volume, FA and MD), and also included motor and neurocognitive measures, and additional brain regions (i.e., accumbens and thalamus). Volume, MD and FA differed across the three groups, with pre-HD characterised by significant volumetric reductions and increased FA within caudate, putamen and pallidum, relative to controls. The QDA results demonstrated that the differentiation of pre-HD from controls was highly accurate when both volumetric and diffusion data sets from basal ganglia (BG) regions were used. The highest discriminative accuracy however was achieved in a multi-modality approach and when including all available measures: motor and neurocognitive scores and multi-modal MRI measures from the BG, accumbens and thalamus. Our QDA findings provide evidence that combined multi-modal imaging measures can accurately classify individuals up to 15 years prior to onset when therapeutic intervention is likely to have maximal effects in slowing the trajectory of disease development.

Autism attenuates sex differences in brain structure: a combined voxel-based morphometry and diffusion tensor imaging study.

BACKGROUND AND PURPOSE: It has been proposed that autism spectrums condition may represent a form of extreme male brain (EMB), a notion supported by psychometric, behavioral, and endocrine evidence. Yet, limited data are presently available evaluating this hypothesis in terms of neuroanatomy. Here, we investigated sex-related anatomic features in adults with AS, a "pure" form of autism not involving major developmental delay. MATERIALS AND METHODS: Males and females with AS and healthy controls (n = 28 and 30, respectively) were recruited. Structural MR imaging was performed to measure overall gray and white matter volume and to assess regional effects by means of VBM. DTI was used to investigate the integrity of the main white matter tracts. RESULTS: Significant interactions were found between sex and diagnosis in total white matter volume, regional gray matter volume in the right parietal operculum, and fractional anisotropy (FA) in the body of the CC, cingulum, and CR. Post hoc comparisons indicated that the typical sexual dimorphism found in controls, whereby males have larger FA and total white matter volume, was absent or attenuated in participants with AS. CONCLUSIONS: Our results point to a fundamental role of the factors that underlie sex-specific brain differentiation in the etiology of autism.

The neural basis of illusory gustatory sensations: two rare cases of lexical-gustatory synaesthesia.

Lexical-gustatory synaesthesia is a rare phenomenon in which the individual experiences flavour sensations when they read, hear, or imagine words. In this study, we provide insight into the neural basis of this form of synaesthesia using functional neuroimaging. Words known to evoke pleasant, neutral, and unpleasant synaesthetic tastes and synaesthetically tasteless words were presented to two lexical-gustatory synaesthetes, during fMRI scanning. Ten non-synaesthetic participants were also scanned on the same list of words. The synaesthetic brain displayed a different pattern of activity to words when compared to the non-synaesthetes, with insula activation related to viewing words that elicited tastes that have an associated emotional valence (i.e., pleasant or unpleasant tastes). The subjective intensity of the synaesthesia was correlated with activity in the medial parietal lobes (precuneus/retrosplenial cortex), which are implicated in polymodal imagery and self-directed thought. This region has also previously been activated in studies of lexical-colour synaesthesia, suggesting its role may not be limited to the type of synaesthesia explored here.

Characterization of the porcine constitutive androstane receptor (CAR) and its splice variants.

Porcine constitutive androstane receptor (CAR; NR1I3) was cloned and compared for homology and activity with mouse and human CAR (mCAR, hCAR). Porcine CAR (pgCAR) was 86% and 75% homologous to hCAR at the nucleotide and protein levels. Five alternatively spliced variants of pgCAR were identified, each of which generated a truncated protein product. Real-time polymerase chain reaction (PCR) analyses showed that these variants were present in pig liver cDNA samples from 4.61% to 9.20% of total pgCAR. pgCAR and hCAR responded similarly to more ligands than did hCAR and mCAR. The known hCAR agonist (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) activated pgCAR, while the murine agonist 1,4 bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) had no effect. 5beta-dihydrotestosterone was identified as a novel inverse agonist of both pgCAR and hCAR. pgCAR splice variant 2 (SV2) had a dose-dependent dominant negative effect on the activity of wild-type pgCAR in dual luciferase assays. SV2 had no effect against pgPXR (pregnane X receptor) or pgFXR (farnesoid X receptor) activity when using PXR- or FXR-specific reporters.

Effects of bile acids on pancreatic ductal bicarbonate secretion in guinea pig.

BACKGROUND AND AIMS: Acute pancreatitis is associated with significant morbidity and mortality. Bile reflux into the pancreas is a common cause of acute pancreatitis and, although the bile can reach both acinar and ductal cells, most research to date has focused on the acinar cells. The aim of the present study was to investigate the effects of bile acids on HCO(3)(-) secretion from the ductal epithelium. METHODS: Isolated guinea pig intralobular/interlobular pancreatic ducts were microperfused and the effects of unconjugated chenodeoxycholate (CDC) and conjugated glycochenodeoxycholate (GCDC) on intracellular calcium concentration ([Ca(2+)](i)) and pH (pH(i)) were measured using fluorescent dyes. Changes of pH(i) were used to calculate the rates of acid/base transport across the duct cell membranes. RESULTS: Luminal administration of a low dose of CDC (0.1 mM) stimulated ductal HCO(3)(-) secretion, which was blocked by luminal H(2)DIDS (dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). In contrast, both luminal and basolateral administration of a high dose of CDC (1 mM) strongly inhibited HCO(3)(-) secretion. Both CDC and GCDC elevated [Ca(2+)](i), and this effect was blocked by BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid), caffeine, xestospongin C and the phospholipase C inhibitor U73122. BAPTA-AM also inhibited the stimulatory effect of low doses of CDC on HCO(3)(-) secretion, but did not modulate the inhibitory effect of high doses of CDC. CONCLUSIONS: It is concluded that the HCO(3)(-) secretion stimulated by low concentrations of bile acids acts to protect the pancreas against toxic bile, whereas inhibition of HCO(3)(-) secretion by high concentrations of bile acids may contribute to the progression of acute pancreatitis.

Exploring the assessors' and nurses' experience of formal assessment of clinical competency in the administration of blood components.

Blood transfusion, clinical competency, assessment, evaluation The change in nurse education from apprenticeship training to the higher education setting, has raised concerns about the lack of practical skills newly qualified nurses have on registration. Every practitioner must be able to administer blood components safely however, the Serious Hazards of Transfusion (SHOT) scheme have consistently demonstrated that 'wrong blood' incidents are the major cause of morbidity and mortality related to transfusion in the United Kingdom. As a result the SHOT working group have recommended that all practitioners should have their clinical competency formally assessed. This paper describes the development and evaluation of a tool for assessing clinical competency for staff involved in transfusing blood. The evaluation used a triangulated approach of phenomenology and survey. The tool was piloted in two different clinical settings by four registered nurses who each assessed two nurses. Individual semi-structured interviews were conducted to collate the nurses' and the assessors' experience of the process. The study participants were of the opinion that assessing clinical competency using a criterion-referenced tool gave practitioners the opportunity to relate theory to practice, promote best practice and encourage adherence to hospital transfusion policies. Formal assessment of clinical competency is therefore, a vehicle that could be used to promote safe transfusion practice, ensuring the safety of patients is paramount.

Sendai virus-mediated CFTR gene transfer to the airway epithelium.

The potential for gene therapy to be an effective treatment for cystic fibrosis has been hampered by the limited gene transfer efficiency of current vectors. We have shown that recombinant Sendai virus (SeV) is highly efficient in mediating gene transfer to differentiated airway epithelial cells, because of its capacity to overcome the intra- and extracellular barriers known to limit gene delivery. Here, we have identified a novel method to allow the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA sequence to be inserted within SeV (SeV-CFTR). Following in vitro transduction with SeV-CFTR, a chloride-selective current was observed using whole-cell and single-channel patch-clamp techniques. SeV-CFTR administration to the nasal epithelium of cystic fibrosis (CF) mice (Cftr(G551D) and Cftr(tm1Unc)TgN(FABPCFTR)#Jaw mice) led to partial correction of the CF chloride transport defect. In addition, when compared to a SeV control vector, a higher degree of inflammation and epithelial damage was found in the nasal epithelium of mice treated with SeV-CFTR. Second-generation transmission-incompetent F-deleted SeV-CFTR led to similar correction of the CF chloride transport defect in vivo as first-generation transmission-competent vectors. Further modifications to the vector or the host may make it easier to translate these studies into clinical trials of cystic fibrosis.

Extracellular zinc stimulates a calcium-activated chloride conductance through mobilisation of intracellular calcium in renal inner medullary collecting duct cells.

We have used the perforated patch clamp and fura-2 fluorescence techniques to study the effect of extracellular Zn(2+) on whole-cell Ca(2+)-activated Cl(-) currents (I (CLCA)) in mouse inner medullary collecting duct cells (mIMCD-3). I (CLCA) was spontaneously active in 74% of cells under basal conditions and displayed time and voltage-independent kinetics and an outwardly rectifying current/voltage relationship (I/V). Addition of zinc chloride (10-400 microM) to the bathing solution resulted in a dose-dependent increase in I (CLCA) with little change in Cl(-) selectivity or biophysical characteristics, whereas gadolinium chloride (30 microM) and lanthanum chloride (100 microM) had no significant effect on the whole-cell current. Using fura-2-loaded mIMCD-3 cells, extracellular Zn(2+) (400 microM) stimulated an increase in intracellular Ca(2+) to an elevated plateau. The Zn(2+)-stimulated [Ca(2+)](i) increase was inhibited by thapsigargin (200 nM), the IP(3) receptor antagonist 2-aminoethoxydiphenyl borate (10 microM) and removal of bath Ca(2+). Pre-exposure to Zn(2+) (400 microM) markedly attenuated the ATP (100 microM)-stimulated [Ca(2+)](i) increase. These data are consistent with the hypothesis that extracellular Zn(2+) stimulates an increase in [Ca(2+)](i) by a release of calcium from thapsigargin/IP(3) sensitive stores. A possible physiological role for a divalent metal ion receptor, distinct from the extracellular Ca(2+)-sensing receptor, in IMCD cells is discussed.

Using stable isotope analysis with telemetry or mark-recapture data to identify fish movement and foraging.

Information about animal movements has often been inferred from stable isotope analysis (SIA), but is dependent on animals assimilating site-specific isotopic signatures via diet. This potential weakness in ecological interpretation can be overcome by using other investigative tools that provide precise information about individual movement patterns. In this paper, we demonstrate the value of combining SIA with telemetry or mark-recapture data from trapping, electrofishing and remote detection of individuals to study the movement and feeding ecology of fishes in different habitats. In a fjord lake system in Newfoundland, Canada, juvenile Atlantic salmon delayed downstream migration (smolts) or actively moved into a large lake (parr) where they foraged for periods reflecting different life history strategies. In the Miramichi River (New Brunswick, Canada), SIA provided evidence of distinct foraging habitats (tributary versus large river). By tracking fish implanted with passive integrated transponder (PIT) tags, we distinguished between movements related to foraging versus seeking cool water refugia during high temperature events. Finally, site fidelity and limited mobility of slimy sculpin, a small benthic fish, was established where delta13C in muscle tissue showed a progressive enrichment downstream and where a median displacement of <10 m was estimated for sculpin tagged with PIT tags. Technological improvements have permitted non-destructive tissue sampling of wild fishes for SIA, and the tagging and remote detection of animals smaller than was previously possible. These advancements and the combination of investigative tools promise new insights into animal ecology.

Kinetics and regulation of a Ca2+-activated Cl- conductance in mouse renal inner medullary collecting duct cells.

Using the whole cell patch-clamp technique, a Ca2+-activated Cl- conductance (CaCC) was transiently activated by extracellular ATP (100 microM) in primary cultures of mouse inner medullary collecting duct (IMCD) cells and in the mouse IMCD-K2 cell line. ATP also transiently increased intracellular Ca2+ concentration ([Ca2+]i) from 100 nM to peak values of approximately 750 nM in mIMCD-K2 cells, with a time course similar to the ATP-induced activation and decay of the CaCC. Removal of extracellular Ca2+ had no major effect on the peak Cl- conductance or the increase in [Ca2+]i induced by ATP, suggesting that Ca2+ released from intracellular stores directly activates the CaCC. In mIMCD-K2 cells, a rectifying time- and voltage-dependent current was observed when [Ca2+]i was fixed via the patch pipette to between 100 and 500 nM. Maximal activation occurred at approximately 1 microM [Ca2+]i, with currents losing any kinetics and displaying a linear current-voltage relationship. From Ca2+-dose-response curves, an EC50 value of approximately 650 nM at -80 mV was obtained, suggesting that under physiological conditions the CaCC would be near fully activated by mucosal nucleotides. Noise analysis of whole cell currents in mIMCD-K2 cells suggests a single-channel conductance of 6-8 pS and a density of approximately 5,000 channels/cell. In conclusion, the CaCC in mouse IMCD cells is a low-conductance, nucleotide-sensitive Cl- channel, whose activity is tightly coupled to changes in [Ca2+]i over the normal physiological range.

Investigation of the effects of extracts of Manilkara zapota (L.) P. van Royen on blood pressure and heart rate of rats

The objective of the study was to investigate the hypotensive effect produced by leaf extracts of Manilkara zapota. Methanol extracts of Manilkara zapota leaves were prepared using a soxhlet apparatus. The methanol was removed with a rotor evaporator. Sprague-Dawley rats were anaesthetized with urethane (1.2 mg/kb) and doses of 0.63, 1.25, 2.5, 5.0, 10.0 and 20.0 mg/kg body weight of extract were administered intavenously. Saline (0.9 percent) was given as a control. The effects on blood pressure and heart rate were recorded using a Pressure transducer (Spectramed model 23XL) coupled to a Grass polygraph (model 79E). The plant extract showed a dose-related hypotensive activity and no significant change in heart rate (P.0.05). Toxicity was observed with doses greater than 20mg/kg body weight. These results indicate that Manilkara zapota leaf tea used in folklore medicine to treat hypertension does indeed show various degrees of hypotensive activity when tested in lab animals. However, larger doses were toxic. It is therefore necessary to assess the beneficial as well as the adverse effects of this herb before usage by the hypertensive patient

Steady-state visually evoked potential topography during processing of emotional valence in healthy subjects.

The International Affective Picture System (IAPS) is increasingly used in brain imaging studies to examine emotional processes. This task allows valence and arousal content to be systematically investigated; however, previous studies have generally failed to select images that vary in one dimension as well as hold constant the variability on the other dimension. In addition, no studies have investigated the temporal structure associated with the conscious, ongoing processing of emotional stimuli following systematic selection of IAPS images. The aim of the present study was therefore to use steady-state probe topography (SSPT) to examine the steady-state visually evoked potentials (SSVEPs) associated with the processing of pleasant and unpleasant images low in arousal content. Seventy-five IAPS images, categorized as unpleasant, neutral, or pleasant, were presented to 16 healthy subjects while brain activity was recorded from 64 scalp sites. Analysis subtracted the activity associated with the presentation of neutral images from the activity associated with the presentation of pleasant as well as unpleasant images. Results demonstrate that both pleasant and unpleasant valence is associated with transient, widespread, and bilateral frontal SSVEP latency reductions. Unpleasant images were also associated with a transient bilateral anterior frontal amplitude decrease. Latency reductions are interpreted as increases in neural information processing speed, while amplitude reductions are interpreted in the current paper as analogous to an event-related desynchronisation commonly associated with the alpha bandwidth. These key findings support previous literature in terms of there being substantial overlap in frontal neural circuitry when the brain processes pleasant and unpleasant valence relative to neutral valence.

Correction of delF508-CFTR activity with benzo(c)quinolizinium compounds through facilitation of its processing in cystic fibrosis airway cells.

A number of genetic diseases, including cystic fibrosis, have been identified as disorders of protein trafficking associated with retention of mutant protein within the endoplasmic reticulum. In the presence of the benzo(c)quinolizinium drugs, MPB-07 and its congener MPB-91, we show the activation of cystic fibrosis transmembrane conductance regulator (CFTR) delF508 channels in IB3-1 human cells, which express endogenous levels of delF508-CFTR. These drugs were without effect on the Ca(2+)-activated Cl- transport, whereas the swelling-activated Cl- transport was found altered in MPB-treated cells. Immunoprecipitation and in vitro phosphorylation shows a 20% increase of the band C form of delF508 after MPB treatment. We then investigated the effect of these drugs on the extent of mislocalisation of delF508-CFTR in native airway cells from cystic fibrosis patients. We first showed that delF508 CFTR was characteristically restricted to an endoplasmic reticulum location in approximately 80% of untreated cells from CF patients homozygous for the delF508-CFTR mutation. By contrast, 60-70% of cells from non-CF patients showed wild-type CFTR in an apical location. MPB-07 treatment caused dramatic relocation of delF508-CFTR to the apical region such that the majority of delF508/delF508 CF cells showed a similar CFTR location to that of wild-type. MPB-07 had no apparent effect on the distribution of wild-type CFTR, the apical membrane protein CD59 or the ER membrane Ca(2+),Mg-ATPase. We also showed a similar pharmacological effect in nasal cells freshly isolated from a delF508/G551D CF patient. The results demonstrate selective redirection of a mutant membrane protein using cell-permeant small molecules of the benzo(c)quinolizinium family and provide a major advance towards development of a targetted drug treatment for cystic fibrosis and other disorders of protein trafficking.

The voltage-dependent Cl(-) channel ClC-5 and plasma membrane Cl(-) conductances of mouse renal collecting duct cells (mIMCD-3).

1. We have tested the hypothesis that the voltage-dependent Cl(-) channel, ClC-5 functions as a plasma membrane Cl(-) conductance in renal inner medullary collecting duct cells. 2. Full-length mouse kidney ClC-5 (mClC-5) was cloned and transiently expressed in CHO-K1 cells. Fast whole-cell patch-clamp recordings confirmed that mClC-5 expression produces a voltage-dependent, strongly outwardly rectifying Cl(-) conductance that was unaffected by external DIDS. 3. Slow whole-cell recordings, using nystatin-perforated patches from transfected CHO-K1 cells, also produced voltage-dependent Cl(-) currents consistent with ClC-5 expression. However, under this recording configuration an endogenous DIDS-sensitive Ca(2+)-activated Cl(-) conductance was also evident, which appeared to be activated by green fluorescent protein (GFP) transfection. 4. A mClC-5-GFP fusion protein was transiently expressed in CHO-K1 cells; confocal laser scanning microscopy (CLSM) showed localization at the plasma membrane, consistent with patch-clamp experiments. 5. Endogenous expression of mClC-5 was demonstrated in mouse renal collecting duct cells (mIMCD-3) by RT-PCR and by immunocytochemistry. 6. Using slow whole-cell current recordings, mIMCD-3 cells displayed three biophysically distinct Cl(-)-selective currents, which were all inhibited by DIDS. However, no cells exhibited whole-cell currents that had mClC-5 characteristics. 7. Transient transfection of mIMCD-3 cells with antisense mClC-5 had no effect on the endogenous Cl(-) conductances. Transient transfection with sense mClC-5 failed to induce the Cl(-) conductance seen in CHO-K1 cells but stimulated levels of the endogenous Ca(2+)-activated Cl(-) conductance 24 h post-transfection. 8. Confocal laser scanning microscopy of mIMCD-3 cells transfected with mClC-5-GFP showed that the protein was absent from the plasma membrane and was instead localized to acidic endosomal compartments. 9. These data discount a major role for ClC-5 as a plasma membrane Cl(-) conductance in mIMCD-3 cells but suggest a role in endosomal function.