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PubMed1988

The effect of DIDS on fluid reabsorption from the proximal convoluted tubule of the rat: dependence on the presence of bicarbonate.

The effects of the anion transport inhibitor DIDS (4,4'-diisothiocyano-stilbene-2,2'-disulphonate) on fluid reabsorption by the rat proximal convoluted tubule were investigated by the stationary microperfusion (split-drop) method. Addition of 10(-5) mol 1-1 DIDS to luminal and capillary perfusates in the presence of peritubular bicarbonate (25 mmol 1-1) increased the reabsorptive half-time (t 1/2) from 35.1 +/- 4.5 to 57.0 +/- 6.2 s, corresponding to a decrease in fluid reabsorption (Jv) from 1.33 +/- 0.06 to 0.89 +/- 0.09 nl min-1 mm-1. Omission of bicarbonate per se from the peritubular capillaries reduced Jv to 0.41 +/- 0.03 nl min-1 mm-1 and this reabsorptive flux was completely inhibited by the addition of 2 mmol 1-1 NaCN to both the luminal and peritubular perfusates. In the absence of bicarbonate, the addition of DIDS at either 10(-4) or 10(-5) mol 1-1 to both lumen and capillary perfusates had no significant effect on either t 1/2 or Jv. These results show that the inhibitory action of DIDS on fluid reabsorption is dependent on the presence of bicarbonate. However, the proximal tubule also reabsorbs water in the absence of bicarbonate by a mechanism that is dependent on cellular metabolism, but is insensitive to DIDS at the concentrations used in this study.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1980

Inhibitory action of DIDS on chloride transport across the amphibian cornea.

1. DIDS (4,4'-diisothiocyano-2,2'-stilbene disulphonic acid) reduced the CI Isc across the toad's (Bufo marinus) cornea. It acted on either the aqueous or tear side and these effects were additive. 2. The reduction in the Isc was equivalent to the decline in the undirectional flux of Cl from the aqueous to the tear side. The Cl flux from tear to aqueous was not changed. 3. DIDS did not change transmural Na transport. 4. The action of the diuretic bumetanide, which also inhibits Cl transport was additive to that of DIDS when both compounds were present on the aqueous though not when they were on the tear side of the cornea. 5. The results are consistent with the role of a Cl-/anion exchange mechanism in active Cl transport across the cornea. 6. A hypothesis regarding the interactions and site of action of bumetanide in relation to that of DIDS, and the Cl transport process, is proposed.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1982

Inverse effects of dansylation of red blood cell membrane on band 3 protein-mediated transport of sulphate and chloride.

1. Dansylation of the red cell membrane produces inverse effects on SO(4) (2-) and Cl(-) equilibrium exchange. The former is enhanced by several orders of magnitude (Legrum, Fasold & Passow, 1980), the latter is inhibited. Both effects are potentiated after dansylation in the presence of 2-(4-amino-3-sulphophenyl)-6-methyl-7-benzothiazol sulphonic acid (APMB), a disulphonic acid that combines non-covalently with the 4,4'-diisothiocyanate dihydrostilbene-2,2'-disulphonic acid (H(2)DIDS) binding site of the anion transport protein.2. After dansylation the maximum of the pH dependence of SO(4) (2-) exchange near pH 6.3 is replaced by a plateau. When dansylation is performed in the presence of APMB, the plateau is reached at a much higher level at around pH 7.0 and resembles that observed by Funder & Wieth (1976) for Cl(-).3. The mutual interactions between the transfer site, the H(2)DIDS binding site, and the as yet unidentified danysl chloride binding sites were studied in detail. Occupation of the H(2)DIDS binding site by the non-covalently binding agents 4,4'-dinitrostilbene-2,2'-disulphonate (DNDS), 4,4'-bis(acetamido) stilbene-2,2'-disulphonate (DAS) or APMB inhibit the enhanced SO(4) (2-) exchange across the previously dansylated membrane. The apparent K(I) value remains the same as in untreated membranes for DNDS, is reduced to 1/3 for DAS, and to 1/60 for APMB. Conversely, when dansylation is carried out while the H(2)DIDS binding site is occupied by DNDS, APMB or DAS, the enhancement of SO(4) (2-) exchange (as measured after removal of excess dansyl chloride and the additional agent) is prevented by DNDS, augmented by APMB and not affected by DAS. This suggests that the agents stabilize different conformations of the H(2)DIDS binding site that are associated with different accessibilities of the dansyl chloride binding sites.4. The SO(4) (2-) equilibrium exchange as measured at a fixed Cl(-) concentration is enhanced when the Cl(-) concentration at which dansylation is carried out is increased, indicating allosteric interactions between anion binding and the exposure of the dansyl chloride binding sites.5. The enhanced K(+) efflux from dansylated red cells is independent of the described modifications of the dansylation reaction by APMB, DAS or DNDS, demonstrating that there exists no simple correlation between the changes of anion and cation movements that are induced by dansylation.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed2002

Feedback inhibition in the inner plexiform layer underlies the surround-mediated responses of AII amacrine cells in the mammalian retina.

Intracellular recordings were made from narrow-field, bistratified AII amacrine cells in the isolated, superfused retina-eyecup of the rabbit. Pharmacological agents were applied to neurons to dissect the synaptic pathways subserving AII cells so as to determine the circuitry generating their off-surround responses. Application of the GABA antagonists, picrotoxin, bicuculline and 1,2,5,6-tetrahydropyridine-4-yl methylphosphinic acid (TPMPA) all increased the on-centre responses of AII amacrine cells, but attenuated the off-surround activity. At equal concentrations, picrotoxin was approximately twice as effective as bicuculline or TPMPA in modifying the response activity of AII amacrine cells. These results indicate that the mechanism underlying surround inhibition of AII amacrine cells includes activation of both GABA(A) and GABA(C) receptors in an approximately equal ratio. Application of the GABA antagonists also increased the size of on-centre receptive fields of AII amacrine cells. Again, picrotoxin was most effective, producing, on average, a 54 % increase in the size of the receptive field, whereas bicuculline and TPMPA produced comparable 34 and 33 % increases, respectfully. Application of the voltage-gated sodium channel blocker TTX produced effects on AII amacrine cells qualitatively similar to those of the GABA blockers. Intracellular application of the chloride channel blocker 4,4'-dinitro-stilbene-2,2'-disulphonic acid (DNDS) abolished the direct effects of GABA on AII amacrine cells. Moreover, DNDS increased the amplitude of both the on-centre and off-surround responses. The failure of DNDS to block the off-surround activity indicates that it is not mediated by direct GABAergic inhibition. Taken together, our results suggest that surround receptive fields of AII amacrine cells are generated indirectly by the GABAergic, reciprocal feedback synapses from S1/S2 amacrine cells to the axon terminals of rod bipolar cells.

Animals↗
PubMed2005

Modulation of human cytochrome P450 1B1 expression by 2,4,3',5'-tetramethoxystilbene.

We have previously shown that 2,4,3',5'-tetramethoxystilbene (TMS), a synthetic trans-stilbene analog, is one of the most potently selective inhibitors of recombinant human cytochrome P450 1B1 (CYP1B1) in vitro. In the present studies, the effects of TMS on CYP1B1 expression were investigated in human cancer cells. TMS significantly inhibited CYP1-mediated 7-ethoxyresorufin O-deethylation activity in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced MCF-7 cells or lung microsomes of Sprague-Dawley rats treated with 7,12-dimethylbenz[a]anthracene. TCDD-stimulated CYP1B1 protein and mRNA expression was significantly suppressed by TMS in a concentration-dependent manner in MCF-7, MCF-10A, and HL-60 cells. Whereas TMS down-regulated TCDD-induced CYP1B1 gene expression, the levels of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator mRNA expression were not changed by TMS treatment. In human cancer cells, TMS induced apoptotic cell death, and the cytotoxic effects of TMS were significant when the cells were incubated with TCDD. CYP1B1 was able to convert TMS to a metabolite(s) when incubated with NADPH. Metabolic activation of TMS by CYP1B1 induced by TCDD may mediate cellular toxicity of TMS in human cancer cells because the sensitivity to TMS in MCF-7 cells treated with TCDD was more significant than in HL-60 cells treated with TCDD. Taken together, our results indicate that TMS acts as a strong modulator of CYP1B1 gene expression as well as a potent selective inhibitor in vitro. The ability of TMS to induce apoptotic cell death in tumor cells, as well as CYP1B1 inhibition, may contribute to its usefulness for cancer chemoprevention.

Anticarcinogenic Agents↗
PubMed2003

Induction of multidrug resistance protein 3 in rat liver is associated with altered vectorial excretion of acetaminophen metabolites.

Treatment with the microsomal enzyme inducer trans-stilbene oxide (TSO) can decrease biliary excretion of acetaminophen-glucuronide (AA-GLUC) and increase efflux of AA-GLUC into blood. The hepatic canalicular multidrug resistance protein (Mrp) 2 and sinusoidal protein Mrp3 transport AA-GLUC conjugates into bile and blood, respectively. Thus, TSO-induced alterations in the vectorial excretion of AA-GLUC may occur via increased hepatic Mrp3 levels. The goal of this study was to determine whether TSO, diallyl sulfide (DAS), and oltipraz (OLT) treatments can up-regulate Mrp3 protein expression, and whether treatment with DAS and OLT can correspondingly increase hepatovascular efflux of AA metabolites. Rats were administered phenobarbital, TSO, DAS, OLT, or vehicle for 4 days. Interestingly, all of the chemicals increased the plasma concentration and urinary excretion of AA-GLUC and decreased its biliary excretion. In control animals, approximately 77% and 23% of AA-GLUC was excreted into bile or urine, respectively, whereas with inducer-pretreated animals, <32% of AA-GLUC was excreted into bile and >68% was excreted into urine. Correspondingly, all of the compounds increased hepatic Mrp3 mRNA levels by 13- to 37-fold and protein levels by 2- to 6-fold, respectively. In conclusion, these studies correlate increased Mrp3 protein levels in liver with increased hepatovascular excretion of AA-GLUC and suggest that induction of Mrp3 affects the route of drug excretion.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2006

Potentiation of a survival signal in the ischemic heart by resveratrol through p38 mitogen-activated protein kinase/mitogen- and stress-activated protein kinase 1/cAMP response element-binding protein signaling.

Resveratrol (3,4',5-trihydroxy-trans-stilbene), a naturally occurring polyphenolic compound found abundantly in grape skins and red wines, has been found to pharmacologically precondition the heart against ischemia reperfusion injury through the potentiation of a survival signal involving cAMP response element-binding protein-dependent phosphatidylinositol 3-kinase-Akt-BclII pathway. The present study was designed to determine whether, similar to ischemic preconditioning, resveratrol uses mitogen-activated protein kinases (MAPKs) as upstream signaling targets. The isolated rat hearts were preperfused for 15 min with Krebs-Henseleit bicarbonate buffer in the absence (control) or presence of extracellular signal-regulated kinase (ERK) 1/2 inhibitor 2'-amino-3'-methoxyflavone (PD98059), p38 MAPK inhibitor 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole (SB-202190), mitogen- and stress-activated protein kinase 1 (MSK-1) inhibitor N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89), protein kinase A inhibitor (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3fg: 3',2',1'-kl]-pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester (KT5720), resveratrol only, resveratrol plus PD98059, resveratrol plus SB-202190, resveratrol plus H89, or resveratrol plus KT5720. Consistent with previous reports, resveratrol provided cardioprotection as evidenced by its ability to improve postischemic ventricular function, reduction of myocardial infarct size, and cardiomyocyte apoptosis. The cardioprotection afforded by resveratrol was partially abolished with PD98059 or SB-202190, suggesting that ERK1/2 and p38 MAPK play roles in resveratrol-mediated preconditioning. An MSK-1 inhibitor, H89, abolished resveratrol-mediated preconditioning, indicating MSK-1 to be the downstream target molecule for both ERK1/2 and p38 MAPK. KT5720 had no effect on resveratrol-mediated cardioprotection. Corroborating these results, Western blot analysis revealed phosphorylation of ERK1/2, p38 MAPK, MAPK-activated protein (MAPKAP) kinase 2, and MSK-1 with resveratrol and inhibition of phosphorylation with corresponding inhibitors. These results showed for the first time that resveratrol triggers an MAPK signaling pathway involving ERK1/2 and p38 MAPK, the former using MSK-1 as the downstream target and the latter, using both MAPKAP kinase 2 and MSK-1 as downstream targets.

Animals↗
PubMed2006

Specific plasma membrane binding sites for polyphenols, including resveratrol, in the rat brain.

Using [3H]resveratrol (3,5,4'-trihydroxy-trans-stilbene) as radioligand, we investigated the possible existence of specific polyphenol binding sites at the level of the cellular plasma membrane in rat brain. Specific [3H]resveratrol binding sites were found to be enriched in the plasma membrane pellet with lower levels in the nuclear and cell debris fraction. Specific [3H]resveratrol binding to the plasma membrane fraction was sensitive to trypsin digestion and protein denaturation but not to DNase and RNase treatment. Saturation binding experiments revealed that specific [3H]resveratrol recognized a single class of sites with an apparent affinity (KD) of 220+/-45 nM and a maximal capacity (Bmax) of 1060+/-120 fmol/mg protein. Various polyphenols and resveratrol derivatives competed against specific [3H]resveratrol binding in rat brain plasma membrane homogenates with the tea catechin gallates (epigallocatechin gallate and epicatechin gallate) displaying the highest affinities (Ki=25-45 nM) followed by resveratrol (Ki=102 nM). Quantitative autoradiographic studies revealed that specific [3H]resveratrol binding sites are broadly distributed in the rat brain, with highest levels of labeling seen in the choroid plexus and subfornical organ. Finally, the potency of various polyphenols and resveratrol analogs in protecting hippocampal cells against beta-amyloid-induced toxicity correlated well (r=0.74) with their apparent affinity in the [3H]resveratrol binding assay. Taken together, these results suggest that the neuroprotective action of various polyphenols and resveratrol analogs could be mediated by the activation of common "receptor" binding sites particularly enriched at the level of the cellular plasma membrane in the rat brain.

Animals↗
PubMed2006

Desoxyrhaponticin (3,5-dihydroxy-4'-methoxystilbene 3-O-beta-D-glucoside) inhibits glucose uptake in the intestine and kidney: In vitro and in vivo studies.

Rhubarb extracts have been reported to improve oral glucose tolerance in diabetic animals. In the present study we have investigated the antidiabetic actions of desoxyrhaponticin, a major stilbene in rhubarb, as a glucose uptake inhibitor. Desoxyrhaponticin was demonstrated to inhibit glucose uptake in rabbit intestinal membrane vesicles as well as in rat everted gut sleeves, with IC50 values of 148.3 and 30.9 microM, respectively. Kinetics studies revealed that desoxyrhaponticin is a competitive inhibitor of glucose uptake in both systems. Moreover, desoxyrhaponticin could reduce glucose uptake in the intestinal membrane vesicles of both normal and diabetic rats. In addition, glucose uptake in the renal membrane vesicles of both normal and diabetic rats was reduced by desoxyrhaponticin. Under the inhibition of desoxyrhaponticin, uptake of glucose in both the intestinal and renal membrane vesicles of the normal rats was no different from that of the diabetic rats. The IC50 values of the uptake inhibition in the renal membrane vesicles of normal and diabetic rats were 118.8 and 115.7 microM, respectively. In a type 2 diabetic animal model in which rats have been treated with streptozotocin at the neonatal stage, postprandial hyperglycemia was significantly suppressed by oral administration of this compound (300 mg/kg b.wt.). These results suggest that desoxyrhaponticin is an agent that is potentially effective in controlling postprandial hyperglycemia in diabetes. The in vivo antidiabetic action of this compound can be explained, in part at least, by inhibition of glucose transport in the small intestine and inhibition of glucose reabsorption in the kidney.

Animals↗
PubMed2005

Resveratrol inhibits angiotensin II- and epidermal growth factor-mediated Akt activation: role of Gab1 and Shp2.

trans-Resveratrol (RV), a polyphenolic stilbene derivative found in grape skin and other food products, has been proposed to exert beneficial effects in cardiovascular disease. Our group has shown previously that RV inhibits angiotensin II (Ang II)-induced Akt activation and, consequently, vascular smooth muscle cell (VSMC) hypertrophy. In this work, to identify the molecular target of RV, we investigated the impact of RV on early signaling cascades in rat aortic VSMCs triggered by Ang II and epidermal growth factor (EGF). We show that RV does not influence Ang II-mediated transactivation of EGF-receptor but potently inhibits EGF-induced phosphorylation of Akt kinase, suggesting that RV acts downstream of EGF-receptor transactivation in VSMCs. Recent evidence indicates that the adapter molecule Gab1, together with the protein tyrosine phosphatase Shp2, is critically involved in regulating the strength and duration of phosphatidylinositol-3-kinase (PI3K) and Akt activation upon EGF stimulation in fibroblasts. Our results show that stimulation of VSMCs with EGF as well as Ang II leads to a rapid tyrosine phosphorylation of Gab1 and its association with the p85 subunit of PI3K. RV attenuates these processes. Experiments performed in Shp2-deficient fibroblasts revealed that RV does not inhibit EGF-stimulated Akt activation in these cells, suggesting that Shp2 is necessary for the inhibitory effect of RV on the PI3K/Akt pathway. Furthermore, RV treatment activates Shp2. We therefore propose that RV blocks Akt activation in Ang II- and EGF-stimulated VSMCs by activating Shp2, thus preventing interaction between Gab1 and PI3K that is necessary for further signal transduction.

3T3 Cells↗
PubMed2003

Resveratrol increases serine15-phosphorylated but transcriptionally impaired p53 and induces a reversible DNA replication block in serum-activated vascular smooth muscle cells.

Resveratrol (RV), a polyphenolic stilbene derivative, has been proposed to exert a plethora of beneficial cardiovascular effects. Of these, in particular, inhibition of vascular smooth muscle cell (VSMC) proliferation shows great promise for preventing cardiovascular disease. In the present study, we show that RV leads to a reversible arrest in early S phase of the VSMC cycle, accompanied by an accumulation of hyperphosphorylated retinoblastoma protein. In contrast to studies with other cell systems, RV decreases cellular levels of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1). This is of particular interest because phosphorylated p53 protein (serine(15)) is strongly enhanced by this substance. We further found that RV only slightly inhibits phosphorylation of Erk 1/2, protein kinase B/Akt, and p70(S6) kinase upon serum stimulation. Thus, inhibition of these kinases is not likely to contribute to the cell cycle effect of RV. Importantly, the observed S phase arrest is not linked to an increase in apoptotic cell death: there was no detectable increase in apoptotic nuclei and in levels of the proapoptotic protein Bax. This is the first study elucidating the molecular pathways mediating the antiproliferative properties of RV in VSMCs.

Animals↗
PubMed1998

Design of organic molecules with large two-photon absorption cross sections.

A strategy for the design of molecules with large two-photon absorption cross sections, delta, was developed, on the basis of the concept that symmetric charge transfer, from the ends of a conjugated system to the middle, or vice versa, upon excitation is correlated to enhanced values of delta. Synthesized bis(styryl)benzene derivatives with donor-pi-donor, donor-acceptor-donor, and acceptor-donor-acceptor structural motifs exhibit exceptionally large values of delta, up to about 400 times that of trans-stilbene. Quantum chemical calculations performed on these molecules indicate that substantial symmetric charge redistribution occurs upon excitation and provide delta values in good agreement with experimental values. The combination of large delta and high fluorescence quantum yield or triplet yield exhibited by molecules developed here offers potential for unprecedented brightness in two-photon fluorescent imaging or enhanced photosensitivity in two-photon sensitization, respectively.

Aniline Compounds↗
PubMed2006

Production of resveratrol in recombinant microorganisms.

Resveratrol production in Saccharomyces cerevisiae was compared to that in Escherichia coli. In both systems, 4-coumarate:coenzyme A ligase from tobacco and stilbene synthase from grapes were expressed. When p-coumaric acid was used as the precursor, resveratrol accumulations in the culture medium were observed to be comparable in E. coli (16 mg/liter) and yeast (6 mg/liter).

Acyltransferases↗
PubMed1984

Oxalate and chloride absorption by the rabbit colon: sensitivity to metabolic and anion transport inhibitors.

The effects of transport inhibitors on the movements of oxalate and chloride across the isolated short circuited rabbit colon were studied. Net oxalate absorption was shown in this species and was shown to be an energy dependent process as indicated by its sensitivity to 2-4 dinitrophenol (DNP) 10(-4)M. Mucosal addition of 4-acetamido-4-isothiocyano-2,-stilbene-2,2'-disulfonic acid (SITS) 10(-4)M abolished the net flux of both oxalate and chloride. Acetazolamide (8 mM) in bicarbonate free buffer significantly reduced the mucosal to serosal flux of both anions. These results suggest that in rabbit colon, oxalate and chloride share a common transport pathway and implicate the chloride bicarbonate exchange system. This study also confirms that chloride absorption by the short circuited rabbit colon is an electrically silent process and presents evidence that suggests that chloride absorption is mediated by a chloride bicarbonate exchange system located in the apical membrane of absorbing colonic epithelial cells.

2,4-Dinitrophenol↗
PubMed1983

Nonadditive effects of combined in vivo hepatic microsomal enzyme inducers in the Wistar rat.

Compounds that are known to increase the hepatic microsomal cytochrome P-450 dependent monooxygenases were administered to adult female rats, alone or in combination, to determine whether their effects on certain substrate oxidations were additive. 3-Methylcholanthrene (3-MC) and pregnenolone-16 alpha-carbonitrile (PCN), known to induce different forms of cytochrome P-450, when administered together increased benzo[a]pyrene oxidation to the same level as observed following 3-MC treatment alone. Phenobarbital (Pb) and PCN when administered concomitantly increased benzo[a]pyrene, amino-pyrine, and ethylmorphine metabolism to the same extent as seen following PCN administration alone. Both compounds are known to induce different forms of cytochrome P-450. Nonadditive effects were also observed with Pb and spironolactone, as well as with Pb and trans-stilbene oxide. Treatment of adult male rats with either PCN or 3-MC resulted in significantly smaller increases in benzo[a]pyrene oxidation than observed in adult female rats. These results suggest that oxidative metabolism in hepatic microsomes is not the sum of activities of a number of cytochrome P-450s, but may represent the activity of a single predominant hemeprotein. In addition, it appears that the oxidation of substrate by a particular cytochrome P-450, in intact microsomes, is greatly influenced by the presence of another form.

Animals↗
PubMed2002

Functional and molecular evidence for Na(+)-HCO cotransporter in porcine vas deferens epithelia.

This study focused on the role of sodium-bicarbonate cotransporter (NBC1) in cAMP-stimulated ion transport in porcine vas deferens epithelium. Ion substitution experiments in modified Ussing chambers revealed that cAMP-mediated stimulation was dependent on the presence of Na(+), HCO, and Cl(-) for a full response. HCO-dependent current was unaffected by acetazolamide, bumetanide, or amiloride but was inhibited by basolateral 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Na(+)-driven, HCO-dependent, stilbene-inhibitable anion flux was observed across the basolateral membrane of selectively permeabilized monolayers. Results of radiotracer flux studies suggest a 4,4'-dinitrostilbene-2,2'-disulfonate-sensitive stoichiometry of 2 base equivalents per Na(+). Antibodies raised against rat kidney NBC epitopes (rkNBC; amino acids 338-391 and 928-1035) identified a single band of ~145 kDa. RT-PCR detected NBC1 message in porcine vas deferens epithelia. These results demonstrate that vas deferens epithelial cells possess the proteins necessary for the vectoral transport of HCO and that these mechanisms are maintained in primary culture. Taken together, the results indicate that vas deferens epithelia play an active role in male fertility and have implications for our understanding of the relationship between cystic fibrosis and congenital bilateral absence of the vas deferens.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

Kinetics of reversible DIDS inhibition of chloride self exchange in human erythrocytes.

The capnophorin (band 3)-mediated chloride self exchange flux in intact erythrocytes and in resealed erythrocyte ghosts was determined at pH 7.3 by measuring the unidirectional efflux of 36Cl-. The time-dependent irreversible inactivation of the anion transport system by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) was measured as the relative change of the unidirectional 36Cl efflux rate. The rate of covalent DIDS binding under conditions of excess DIDS in solution that ensure a complete irreversible inhibition followed an exponential time course with a rate coefficient Kcov (min-1). The Arrhenius activation enthalpy of Kcov was constant, 114 kJ/mol, at 0-38 degrees C. At 38 and 0 degrees C, Kcov was 0.5 min-1 [half time (T1/2) = ln2/Kcov = 1.3 min] and 0.004 min-1 (T1/2 = 178 min), respectively. The slow irreversible DIDS binding to the anion transport system at 0 degrees C allows a determination of the kinetics of the reversible DIDS reaction. The pseudo first-order rate constant for binding, kon, was 3.5 X 10(5) (M.s)-1. The apparent dissociation constant, KD, determined from the steady-state binding to the erythrocyte membrane was 3.1 X 10(-8) M at an equal internal and external Cl- concentration of 165 mM (0 degrees C). The value of KD shows that DIDS is the most efficient reversible inhibitor among the stilbene derivatives so far studied. Maximum reversible inhibition by DIDS was obtained by binding of a minimum of approximately 10(6) molecules/cell membrane. The number is similar to that obtained from studies of irreversible DIDS binding.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Histamine-induced chloride channels in apical membrane of isolated rabbit parietal cells.

The electrical properties of the apical membrane of isolated rabbit parietal cells were studied using the patch-clamp technique. The apical membrane of the parietal cells plated on Matrigel and maintained in culture conditions was identified by lectin-binding studies. Cell-attached and excised inside-out patches from 10(-4) M cimetidine-treated parietal cells infrequently contained Cl- channels (9% of the patches). A single class of voltage-dependent outwardly rectifying Cl- channels with 24 +/- 1-pS conductance was observed in 75% of the patches from cells stimulated (acid secreting) by 10(-4) M histamine. Other anions passed through these channels with a permeability sequence of I- (1.2) greater than Br- (1.1) greater than or equal to Cl- (1.0) greater than NO3- (0.7) greater than SO4(2-) (0.1), but there was a very low permeability for Na+ or K+ (PCl-/PNa+ or PCl-/PK+ greater than 5). In inside-out patch configurations the Cl- channel was insensitive to Ba2+ and stilbene derivatives but was inhibited by diphenylamine-2-carboxylic acid in a manner characteristic of a reversible open-channel blocker. It is concluded that H2-receptor agonist stimulation of acid secretion by rabbit parietal cells activates Cl- channels in the apical cell membrane.

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