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S Fujii

Publications and source records attributed to S Fujii.

At least 19 recordsLinked to original sources
PubMed1992

Adenosine A2 receptor antagonist facilitates the reversal of long-term potentiation (depotentiation) of evoked postsynaptic potentials but inhibits that of population spikes in hippocampal CA1 neurons.

The effects of the adenosine A2 receptor antagonist CP-66713 on the reversal of long-term potentiation (LTP) were studied in CA1 neurons of guinea pig hippocampal slices. Reduction of LTP (depotentiation, DP) was effected by delivering a train of low-frequency afferent stimuli (depotentiation stimulation, DPS) which was given 20 min after tetanus (100 Hz, 100 pulses). DPS (1 Hz, 1000 pulses) was applied during perfusion of CP-66713 (10 microM). In the presence of CP-66713, DPS reduced the potentiated component of the slope of the field EPSP (S-EPSP) and the amplitude of the population spike (A-PS) by 101.7% and 19.1% of the potentiated amount, respectively (mean, n = 8). The reduction of LTP of the S-EPSP was significantly larger than the control (68.5% (mean, n = 6)) while that of the A-PS was significantly smaller than the control (80.1% (mean, n = 6)). Based on these results, we conclude that the inhibition of DP in the EPSP, the facilitation of DP in the PS, and the consequent attenuation in the EPSP-PS relationship follow from activation of adenosine A2 receptors.

Animals↗
PubMed1992

Tissue factor potentiates the factor VIIa-catalyzed hydrolysis of an ester substrate.

We designed a simple and sensitive method to assay the activity of the factor VIIa-tissue factor complex, using as a substrate N alpha-benzyloxycarbonyl-L-arginine p-nitrobenzyl ester (Z-Arg-ONb) (Zur, M., and Nemerson, Y. (1978) J. Biol. Chem. 253, 2203-2209). The principle was to measure the amount of p-nitrobenzyl alcohol released during ester hydrolysis using reversed-phase high performance liquid chromatography. Z-Arg-ONb had a broad specificity for plasma serine proteases and factor IXa. Using this method, we examined the effect of tissue factor on the esterase activity of factor VIIa under various conditions. We found that tissue factor greatly potentiates the factor VIIa-catalyzed hydrolysis of Z-Arg-ONb. Phospholipids were not required for the factor VIIa-catalyzed hydrolysis of Z-Arg-ONb, even in the presence of tissue factor. The Km value of factor VIIa alone toward the ester substrate was six times higher than that of a VIIa-tissue factor complex (3.2 versus 0.54 mM), whereas the kcat value was 12 times lower than that of the VIIa-tissue factor complex (14.3 versus 173 s-1). Thus, tissue factor apparently affects the catalytic site of factor VIIa and enhances hydrolysis of the ester substrate. This enhancing effect of tissue factor disappeared on removal of the gamma-carboxyglutamic acid domain from factor VIIa, whereas the esterase activity in the absence of tissue factor was not affected by this modification. The gamma-carboxyglutamic acid domain is probably required as a potent determinant for interactions with tissue factor, even in the absence of phospholipids in the reaction mixture.

Amino Acid Sequence↗
PubMed1992

Expression and growth-promoting effect of adult T-cell leukemia-derived factor. A human thioredoxin homologue in hepatocellular carcinoma.

Adult T-cell leukemia-derived factor (ADF), originally defined as an interleukin-2 receptor inducer, is a human thioredoxin homologue. ADF is detected in many malignant tissues and has a growth-promoting effect on transformed cells. In this study, ADF expression was examined immunohistochemically in human liver cell lines and liver tissues, and its growth-promoting effect was tested on human hepatoma cells. On three liver cell line--PLC/PRF/5, HepG2, and Chang liver cells--ADF stained positively and also was detected by immunoblotting. ADF had strong staining in the fetal liver (n = 8), although it was faint in the normal adult liver (n = 6). In hepatocellular carcinoma (n = 25), ADF expression generally was enhanced and was very strong in 52% (13 of 25) of the cases, although it was moderate in cases of chronic hepatitis or cirrhosis. ADF augmented the growth of PLC/PRF/5 cells and showed an additive effect with epidermal growth factor. These results indicate possible involvement of ADF in cell activation and growth of hepatocytes, as is the case with lymphocytes.

Carcinoma, Hepatocellular↗
PubMed1992

Biosynthesis of blood group I and i antigens in rat tissues. Identification of a novel beta 1-6-N-acetylglucosaminyltransferase.

The beta-galactoside beta 1-6- and beta 1-3-N-acetylglucosaminyltransferases (beta 1-6GnT and beta 1-3GnT) that synthesize blood group I and i antigens were identified in rat tissues, using pyridylaminated lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-PA) as an acceptor. The products of the transferase reactions were separated on high performance liquid chromatography. The products of the transferase reactions were identified by 1H NMR as (formula; see text) and GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-PA. The product for beta 1-6GnT was also identified by methylation analysis. Kinetic experiments were carried out using rat serum for beta 1-3GnT and partially purified enzyme from rat intestine for beta 1-6GnT. beta 1-3GnT has a pH optimum of 7.5 and requires Mn2+ for optimal activity. beta 1-6GnT has a pH optimum of 7.0 and does not require Mn2+. Studies on the substrate specificity of each enzyme indicated that the preferred substrate for beta 1-3GnT had the general structure Gal beta 1-4GlcNAc-OR and, for beta 1-6GnT, Gal beta 1-4GlcNAc beta 1-3Gal-OR where R = sugar. This is the first demonstration that the beta 1-6GnT acts on an internal galactose of lacto-N-neotetraose and paragloboside, and the enzyme appears to be a novel enzyme in terms of substrate specificity.

Animals↗
PubMed1992

Determinants of induction of increased synthesis of plasminogen activator inhibitor type-1 in human endothelial cells by t-PA.

Tissue-type plasminogen activator (t-PA) induces synthesis of a rapidly acting inhibitor, plasminogen activator inhibitor type-1 (PAI-1) in human umbilical vein endothelial cells (HUVEC) in culture. In vivo, an analogous process may induce negative feedback on the fibrinolytic system. To define specific determinants in the t-PA molecule contributing to the induction, PAI-1 synthesis was characterized in 35S-methionine labeled HUVEC in response to several molecular variants of t-PA. Catalytically active variants devoid of several specific structural domains in the A-chain retained the capacity to form complexes with PAI-1 and to induce increased concentrations of total PAI-1 (free and complexed) in conditioned media without depleting PAI-1 from the extracellular matrix. Surprisingly, a mutant t-PA with markedly reduced catalytic activity reflecting replacement of the active site serine with threonine (S478T) formed complexes with PAI-1 and induced increased PAI-1 synthesis as well. However, in contrast to wild-type t-PA and A-chain variants, it did not release 35S-methionine labeled PAI-1 from the extracellular matrix. Thus, its effects appeared to reflect increased secretion exclusively. Our results suggest that induction of PAI-1 synthesis in HUVEC by t-PA depends on its protease domain but that an active site serine is not a requirement.

Binding Sites↗
PubMed1992

Synergistic induction of plasminogen activator inhibitor type-1 in HEP G2 cells by thrombin and transforming growth factor-beta.

Plasminogen activator inhibitor type-1 (PAI-1) is a physiologic modulator of the fibrinolytic system. Its activity in plasma increases in diverse thrombotic states. The large synthetic capacity of the liver make it a source of potentially large amounts of PAI-1. Because thrombin activity increases in association with thrombotic disorders and because specific binding sites for thrombin have been identified on hepatocytes, we characterized the effect of thrombin on hepatocyte PAI-1 production. Incubation of Hep G2 cells with human alpha-thrombin resulted in a dose- and time-dependent increase in the concentration of PAI-1 in conditioned media. This effect was inhibited completely by hirudin and by antithrombin III. Steady-state levels of both the 3.2-kb and 2.2-kb forms of PAI-1 mRNA increased after stimulation of the cells with thrombin, indicating that thrombin influences PAI-1 expression in Hep G2 cells at the pretranslational level. Incubation of Hep G2 cells with alpha-thrombin and either platelet lysates or purified transforming growth factor-beta (TGF-beta), both previously shown to augment hepatocyte PAI-1 expression, resulted in a synergistic increase in the concentration of PAI-1 in conditioned media. PAI-1 mRNA appeared to be synergistically increased as well. Thus, thrombin increases expression of both PAI-1 protein and mRNA in Hep G2 cells and exerts synergistic effects with TGF-beta. These results underscore the potential importance of inhibition of thrombin under conditions in which thrombolysis is induced pharmacologically.

Antithrombin III↗
PubMed1992

Expression of adult T-cell leukaemia-derived factor, a human thioredoxin homologue, in the human ovary throughout the menstrual cycle.

An immunohistochemical study of the expression of adult T-cell leukaemia-derived factor (ADF), a human thioredoxin homologue, was performed in the normal human ovary throughout the menstrual cycle. Primordial follicles were negative for ADF. Both granulosa cells and theca interna cells at the stages of preantral and antral follicles contained ADF. The staining intensity of these cells was very strong in the preovulatory dominant follicle. After ovulation, both granulo-lutein and theca-lutein cells were positive for ADF. During pregnancy, the theca-lutein cells revealed very intense ADF staining. The theca interna cells of the atretic follicles showed ADF staining, while the granulosa cells of such follicles did not. These results suggest that ADF localizes in the ovarian steroidogenic cells which have the binding sites of either luteinizing hormone or follicle-stimulating hormone, and that ADF expression is closely associated with the activity of the ovarian steroidogenic cells.

Adult↗
PubMed1992

Expression of c-erbB-2 protein and epidermal growth factor receptor in normal tissues of the female genital tract and in the placenta.

The c-erbB-2 (HER-2/neu) protein is a membrane glycoprotein growth factor receptor that has molecular homology with the epidermal growth factor receptor (EGFR). To investigate the relationship between the expression of c-erbB-2 protein and EGFR in the tissues of the human female genital tract and in the placenta, we examined the immunohistochemical reactivity of monoclonal antibodies against both of these proteins. In the müllerian-derived genital tract, epithelial cells of the fallopian tube, endometrium, and endocervix showed reactivity for c-erbB-2 protein, whereas reactivity for EGFR was distributed mainly in the stromal cells throughout the menstrual cycle and during pregnancy. In addition, the staining intensity for EGFR in the endometrial stroma increased with its decidualization. In the exocervical squamous epithelium, basal cells were c-erbB-2 protein-negative and EGFR-positive, but the more differentiated squamous cells of the intermediate layer were c-erbB-2 protein-positive and EGFR-negative. In the placental tissues, cytotrophoblasts and syncytiotrophoblasts of the chorionic villi were c-erbB-2 protein-negative and EGFR-positive. In contrast, intermediate trophoblasts in the extravillous space were c-erbB-2 protein-positive and EGFR-negative. Thus, there is an inverse relationship between the expression of c-erbB-2 protein and EGFR in the tissues of the female genital tract and in the placenta. This suggests that there may be a regulatory mechanism(s) for the expression of both proteins that is associated with the differentiation and/or function of cells in the female genital tract and the placenta.

ErbB Receptors↗
PubMed1992

Immunohistochemical localization of c-erbB-2 protein and epidermal growth factor receptor in normal surface epithelium, surface inclusion cysts, and common epithelial tumours of the ovary.

The c-erbB-2 (HER-2/neu) protein is a membrane glycoprotein growth factor receptor showing molecular homology with the epidermal growth factor receptor (EGFR). We examined the immunohistochemical reactivity of monoclonal antibodies against both of these proteins in normal surface epithelium, surface inclusion cysts, and common epithelial tumours of the ovary. The ovarian tumours were classified as benign (16), borderline malignant (2), and malignant (19). Normal surface ovarian epithelium was weakly positive for both c-erbB-2 protein and EGFR. In surface inclusion cysts, however, the epithelial cells lining the lumen exhibited stronger staining for c-erbB-2 protein, but no staining for EGFR. All 16 benign ovarian tumours and the 2 borderline malignant ovarian tumours were positive for c-erbB-2 protein and negative for EGFR. Of the ovarian carcinomas, 13 of the 19 (68.4%) were positive for c-erbB-2 protein and negative for EGFR, while 4 showed positivity for both c-erbB-2 protein and EGFR. Two cases were negative for both proteins. Expression of both c-erbB-2 protein and EGFR was found in endometrioid carcinoma with squamous differentiation and in clinically advanced poorly differentiated serous carcinomas. Expression of c-erbB-2 protein appears to be increased and that of EGFR is reduced in the early stage of epithelial ovarian oncogenesis. The expression of EGFR with c-erbB-2 protein in ovarian carcinoma is related both to histological differentiation and/or advanced clinical stage.

Adolescent↗
PubMed1992

Glucagon-glucose (GG) test for the estimation of the insulin reserve in diabetes.

The residual B-cell function was examined by means of the plasma C-peptide response 6 min after a combined injection of glucagon and glucose (GG test) or conventional glucagon test (G test) in four insulin-dependent diabetic patients (IDDM group), in 18 diabetic patients treated with insulin (Insulin group), 31 treated with oral hypoglycemic agents (SU group) and 27 treated with diet only (Diet group) and in 22 borderline cases. By GG test, 6-min C-peptide values of the IDDM group were 0.27 +/- 0.05 nM (n = 4) and were significantly lower than those of the Insulin group (0.89 +/- 0.09 nM, n = 12), the SU group (1.42 +/- 0.10 nM, n = 13), the Diet group (2.47 +/- 0.22 nM, n = 11) and the borderline cases (3.38 +/- 0.22 nM, n = 11). Patients with a 6-min C-peptide concentration below 0.75 nM by GG test appeared to be insulin-requiring patients. In the G test, plasma C-peptide concentrations at 6 min were 0.35 +/- 0.08 nM in the IDDM group (n = 2), 0.72 +/- 0.20 nM in the Insulin group (n = 7), 1.08 +/- 0.09 nM in the SU group (n = 20), 1.40 +/- 0.19 nM in the Diet group (n = 17) and 2.05 +/- 0.21 nM in the borderline cases (n = 12). Some of the Diet group patients showed extremely low C-peptide responses. When comparing the GG test and G test in individual cases, a greater C-peptide response was seen with the GG test in all cases except for IDDM patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

"Plasminogen steal" and clot lysis.

Although initially developed to reduce the risk of bleeding, second-generation (clot-selective) thrombolytic agents have been found to induce more prompt and frequent recanalization than do nonselective, first-generation agents. To determine whether they do so in part by preserving clot-associated plasminogen, human whole blood clots formed in Chandler tubes were studied. Addition of suprapharmacologic concentrations of recombinant tissue-type plasminogen activator (rt-PA) to the media bathing mature clots led to a paradoxic impairment of clot lysis and a concomitant concentration-dependent depletion of clot-associated plasminogen (Western blot analysis). In contrast, supplementation of the plasma with plasminogen (0.27 mg/ml) led to significant conservation of both plasma and clot-associated plasminogen (p less than or equal to 0.05, n = 4), and prevented the diminution of clot lysis (p less than or equal to 0.05; n = 4). Fibrinogen degradation products did not account for the attenuation of lysis with the highest concentrations of rt-PA. In concentrations equivalent to those that were induced by the highest concentrations of rt-PA evaluated, fibrinogen degradation products potentiated rather than inhibited lysis (p less than or equal to 0.05, n = 4), probably by stimulating rt-PA activity directly. When preformed clots were incubated with plasminogen-depleted plasma plus 1,000 ng/ml rt-PA, the plasminogen content in residual clot declined (9.36 +/- 0.46 versus 12.39 +/- 0.69 ng/mg clot found in nondepleted plasma; p less than or equal to 0.05; n = 6). Furthermore, clot lysis was attenuated completely.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Coagulation↗
PubMed1992

3 alpha-hydroxysteroid dehydrogenase activity catalyzed by purified pig adrenal 20 alpha-hydroxysteroid dehydrogenase.

In earlier studies, two distinct molecules, 20 alpha-HSD-I and 20 alpha-HSD-II, responsible for 20 alpha-HSD activity of pig adrenal cytosol were purified to homogeneity and characterized [S. Nakajin et al., J. Steroid Biochem. 33 (1989) 1181-1189]. We report here that the purified 20 alpha-HSD-I, which mainly catalyzes the reduction of 17 alpha-hydroxyprogesterone to 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one, catalyzes 3 alpha-hydroxysteroid oxidoreductase activity for 5 alpha (or 5 beta)-androstanes (C19), 5 alpha (or 5 beta)-pregnanes (C21) in the presence of NADPH as the preferred cofactor. The purified enzyme has a preference for the 5 alpha (or 5 beta)-androstane substrates rather than 5 alpha (or 5 beta)-pregnane substrates, and the 5 beta-isomers rather than 5 alpha-isomers, respectively. Kinetic constants in the reduction for 5 alpha-androstanedione (Km; 3.3 microM, Vmax; 69.7 nmol/min/mg) and 5 beta-androstanedione (Km; 7.7 microM, Vmax; 135.7 nmol/min/mg) were demonstrated for comparison with those for 17 alpha-hydroxyprogesterone (Km; 26.2 microM, Vmax; 1.3 nmol/min/mg) which is a substrate for 20 alpha-HSD activity. Regarding oxidation, the apparent Km and Vmax values for 3 alpha-hydroxy-5 alpha-androstan-17-one were 1.7 microM and 43.2 nmol/min/mg, and 1.2 microM and 32.1 nmol/min/mg for 3 alpha-hydroxy-5 beta-androstan-17-one, respectively. 20 alpha-HSD activity in the reduction of 17 alpha-hydroxyprogesterone catalyzed by the purified enzyme was inhibited competitively by addition of 5 alpha-DHT with a Ki value of 2.0 microM. Furthermore, 17 alpha-hydroxyprogesterone inhibited competitively 3 alpha-HSD activity with a Ki value of 150 microM.

20-Hydroxysteroid Dehydrogenases↗
PubMed1992

Altered expression of E-cadherin in gastric cancer tissues and carcinomatous fluid.

Expression of E-cadherin in 21 patients with various histological types of gastric carcinomas was studied by immunoperoxidase staining. Intercellular boundaries of almost all cancer cells in well and moderately differentiated adenocarcinomas stained as deeply for E-cadherin as normal gastric mucosa. However, singly infiltrating cells of those histological types were poorly stained. In poorly differentiated adenocarcinomas, cancer cells forming clusters stained lightly and those infiltrating singly stained even less. In signet ring cell carcinomas, hardly any staining was observed. In each histological type, the staining patterns and intensity at different layers of the gastric wall, were essentially the same. Cancer cells from carcinomatous ascites of gastric adenocarcinomas and pancreatic adenocarcinomas, and those from pleural effusion of lung adenocarcinomas were also studied by immunofluorescence staining. Of 11 specimens, ten were negative and only one from a lung adenocarcinomas was positively stained. By phase-contrast microscopic observations, none of these cancer cells including those from the lung adenocarcinomas, formed obvious cell-cell contacts. Cell aggregation assays confirmed the above results. The molecular weight of E-cadherin of cancer cells of lung adenocarcinomas was less than intact E-cadherin as revealed by Western blot analysis. These results suggest that depressed expression and/or impaired function of E-cadherin in cancer cells, facilitates their liberation from primary sites to infiltrate freely into tissue or fluid.

Adenocarcinoma↗
PubMed1992

Cranio-nasal median splitting for radical resection of craniopharyngioma.

A new surgical approach for radical resection of craniopharyngioma is presented. This approach (cranio-nasal median splitting) involves craniotomy in the centre of the frontal bone, removal of the median portion of the supraorbital bar that incorporates the nasal bone, and detachment of the medial canthal ligaments. The frontal lobes, the cribriform plates, the planum sphenoidale, and the upper nasal cavities are split in the midline. The extraventricular surface of the hypothalamus, the pituitary stalk, and the posterior portion of the Willis' arterial ring are well visualized through the midline infrachiasmatic route. The intraventricular surface of the hypothalamus is also visible in the same operative field through the lamina terminalis and/or the anterior portion of the corpus callosum. This excellent visualization is quite helpful for minimizing operative injury to the hypothalamus and the pituitary stalk whichever surface of the third ventricular floor the tumour is situated upon. Three cases of craniopharyngioma operated upon by this approach are presented. Discussions are focused not only on the indication, but on the advantages and disadvantages of this approach. The surgical techniques for reconstruction of the cranial base are also described, together with some precautions that should be taken to prevent possible postoperative complications.

Adult↗
PubMed1992

Dysregulation of adult T-cell leukemia-derived factor (ADF)/thioredoxin in HIV infection: loss of ADF high-producer cells in lymphoid tissues of AIDS patients.

Adult T-cell leukemia (ATL)-derived factor (ADF) is a multifunctional protein homologous to thioredoxin (TRX) with co-cytokine and thiol-dependent reducing activities. ADF/thioredoxin production is enhanced in T cells transformed by HTLV-I. We have examined the effect of HIV-1 infection on ADF/TRX expression using specific antibody against ADF/TRX. Lymph nodes from 5 AIDS and 1 AIDS-related complex (ARC) patients were examined. As a control, 8 HIV noninfected lymph nodes, including 3 cases with hyperplasia, were also examined. Immunohistopathological studies using normal HIV noninfected lymph nodes showed that ADF/TRX high-producer (ADFh) cells were macrophages and cells with dendritic morphology in the paracortical area. Abundant ADFh cells were observed in HIV noninfected hyperplastic lymph nodes. The number of ADFh cells was low in hyperplastic lymph nodes from an ARC patient. All of the lymph nodes of 5 AIDS cases were atrophic and the number of ADFh cells were extremely low. To verify these histochemical studies, we examined the effect of in vitro HIV infection on ADF/TRX expression in HTLV-I (+) T-cell lines. Western blot analysis showed that a reduction of ADF/TRX in HIV-1-infected SKT-1B and MT-2 cells, and the reduction inversely correlated with p24 antigen level. On the basis of the above in vivo and in vitro findings, we imply that the levels of ADF/TRX were down-regulated by HIV-1 infection and that the down-regulation may play a role for pathophysiology of HIV-infected individuals.

AIDS-Related Complex↗
PubMed1992

Resonance Raman study on complexes of medium-chain acyl-CoA dehydrogenase.

Resonance Raman (RR) spectra of the complex of pig kidney medium-chain acyl-CoA dehydrogenase with acetoacetyl-CoA and of the purple complex formed upon the addition of octanoyl-CoA to the dehydrogenase were obtained. RR spectra were also measured for the complexes prepared by using isotopically labeled compounds, i.e., [3-13C]-, [1,3-13C]-, and [2,4-13C2]acetoacetyl-CoA; [1-13C]octanoyl-CoA; the dehydrogenase reconstituted with [4a-13C]- and [4,10a-13C2]FAD. Both bands of oxidized flavin and acetoacetyl-CoA were resonance-enhanced in the 632.8 nm excited spectra of the acetoacetyl-CoA complex; this confirms that the broad long-wavelength absorption band is a charge-transfer absorption band between oxidized flavin and acetoacetyl-CoA. The 1,622 cm-1 band was assigned to the C(3)=O stretching mode coupling with the C(2)-H bending mode of the enolate form of acetoacetyl-CoA and the bands at 1,483 and 1,119 cm-1 were assigned to bands associated with the C(2)=C(1)-O- moiety. Both bands of fully reduced flavin and the substrate were resonance-enhanced in the 632.8 nm excited spectra of the purple complex. As the enzyme is already reduced, the substrate must be oxidized to octenoyl-CoA; the complex is a charge-transfer complex between the reduced enzyme and octenoyl-CoA. The low frequency value of the 1,577 cm-1 band, which is associated with the C(2)-C(1)=O moiety of the octenoyl-CoA, suggests that the enzyme-bound octenoyl-CoA has an appreciable contribution of C(2)=C(1)-O-.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyl Coenzyme A↗
PubMed1992

Direct evidence for a basolateral membrane Cl- conductance in toad retinal pigment epithelium.

There is now evidence that a Cl- conductance on the basal membrane of the retinal pigment epithelium (RPE) is involved in the generation of both the fast oscillation and the light peak of the direct-current electroretinogram as well as being critical for transepithelial fluid and salt movement. In the present study, we characterized the basolateral membrane Cl- conductance of an in vitro preparation of toad RPE-choroid using conventional and Cl(-)-selective microelectrodes. Under control conditions, the potential across the apical (Vap) and basal (Vba) membranes averaged -60 +/- 2 and -45 +/- 2 mV, respectively (n = 40). Intracellular Cl- activity (aiCl = 20 +/- 1 mM) was distributed above equilibrium across both membranes, consistent with active accumulation of Cl-. A sixfold decrease in Cl- in the basal bath depolarized Vba by 12 +/- 1 mV (n = 17) and increased the apparent basal membrane resistance. By sequential measurement of aiCl and subepithelial Cl- activity during a step decrease in basal Cl-, we constructed the change in Cl- equilibrium potential (ECl) across the basal membrane. Estimation of the change in basal membrane electromotive force during the change in ECl gave an average value for the Cl- transference number (TCl) of 0.45. Further evidence for a Cl- conductance was obtained by measuring changes in aiCl induced by transepithelial current. Depolarizing Vba elevated aiCl, whereas hyperpolarizing Vba had the opposite effect, consistent with conductive Cl- movement across the basal membrane. Both the amplitude of the Cl- diffusion potential and the current-induced changes in aiCl were reduced by basal perfusion with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (250-500 microM), a blocker of Cl- channels in some epithelia.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗