Preparation and characterization of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and related stilbene disulfonates

4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and other 4,4'-stilbene-2,2'-disulfonate derivatives used as reagents in histochemistry and physiology have been prepared in their E isomeric form, and rearranged to the Z isomers by irradiation with visible light. Infrared, and 1H and 13C nuclear magnetic resonance spectra were recorded for these compounds, and used to establish the chemical structures. In particular, it was shown that the E-isomer of SITS decomposed in aqueous solution by hydrolysis of both the acetamido and isocyano groups yielding a diamine; disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) also decomposed in solution, while disodium 4,4'-dinitrostilbene-2,2'-sulfonate (DNDS) rearranged from the E-isomer to the Z-isomer when solutions were kept unprotected from light. These results indicate that benchworkers should not be surprised when commercial samples of such stilbenes contain large amounts of various types of impurities.     

The use of triplet-sensitized photochromism phenomenon for the study of dynamic interaction of membrane proteins

Possibility of registration of protein interactions in the membranes was demonstrated. The membrane preparation of Na+, K+ ATPase was used in the investigations. The Na+, K+ ATPase was bound with 4-acetoamido-4'-isothiocyanatostilbene-2,2' disullfonic acid (SITS) and erythrosinisothiocyanate (ERITC). The label/Na+,K+ATPase (M/M) ratio was equal to 1:1 for SITS and changed from 1:1 to 1:5 for ERITC. The cis-trans isomerization of SITS was initiated by triplet-triplet energy transfer from light excited ERITC to SITS. The kinetics of isomerization was recorded by the SITS fluorescence measurements. The rate constant of triplet-triplet energy transfer (kT) from ERITC to cis isomer of SITS, (3 divided by 7) X 10(3) M-1 s-1 was determined at 25 degrees C. The kT value of the energy transfer between loose molecules of erythrosine and SITS in buffer solution equaled to 7 X 10(7) M-1 s-1. This drop of kT in the membrane at 10(4) reflected the decrease in the frequency of label collisions caused by the increase in the media viscosity and steric hindrances.     

Permeation of the erythrocyte stroma by superoxide radical.

Superoxide anion, generated by xanthine oxidase within vesicles formed from washed erythrocyte ghosts, crosses the vesicle membrane to reduce cytochrome c in the medium (Lynch, R. E., and Fridovich, I. (1978) J. Biol. Chem, 253, 1838-1845). To determine whether O2- could travel through the membrane in the "channel" for the exchange of stable anions, the effects of two specific inhibitors of anion exchange, 4-acetamido-4'-isothiocyano-2,2' disulfonic acid stilbene (SITS) and 4,4'-diisothiocyano-2,2' disulfonic acid stilbene (DIDS), on the escape of O2- from vesicles were studied. The reduction of external cytochrome c, caused by O2- produced by the enzymic turnover of internal xanthine oxidase, was 85 to 90% inhibited by SITS and DIDS. If SITS impeded the egress of O2- from vesicles, it should enhance the internal effects of O2- and antagonize the inhibition of these effects by external superoxide dismutase. External superoxide dismutase inhibited the lysis of vesicles containing xanthine oxidase. SITS (60 micron) partially reversed this inhibition. It appears that O2- can cross the membrane of the erythrocyte in the anion channel.     

Disulfonic stilbene derivatives open the Ca2+ release channel of sarcoplasmic reticulum

Ca2+ channels of isolated sarcoplasmic reticulum were incorporated into a planar lipid bilayer and their pharmacological properties were studied. The results show that the channel is a Ca2+-induced Ca2+ release channel like that observed in skinned muscle fibers and isolated vesicles. (i) The open channel probability was increased by the addition of micromolar amounts of Ca2+ to the cis (myoplasmic) side and further increased by millimolar ATP. (ii) The channel was closed by millimolar Mg2+ and micromolar ruthenium red. We found that two disulfonic stilbene derivatives, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), when added to the cis side open the channel and lock it irreversibly at open without changing the single channel conductance. Ca2+ efflux from SR vesicles was also enhanced by SITS and DIDS, as monitored by a tracer assay. Further, Ag+ activated the channel transiently. These results suggest that certain amino and SH residues play important roles in gating the Ca2+ channel.     

Stilbene disulfonic acids inhibit synexin-mediated membrane aggregation and fusion

Stilbene disulfonic acids inhibit surfactant secretion from lung epithelial type II cells by an undefined mechanism, and inhibit CD4 mediated cell-cell fusion. We have previously shown that lung synexin promotes in vitro fusion of lamellar bodies and plasma membranes, an obligatory process for surfactant secretion. This study investigates the effect of stilbene disulfonic acids, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), and 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMDS), on synexin-mediated liposome aggregation and fusion. Structurally, these three stilbene compounds differ in the number of isothiocyano groups present (DIDS = 2, SITS = 1, and AMDS = 0). At 10 μg synexin/ml, DIDS and SITS inhibited synexin-mediated liposome aggregation with an EC₅₀ of 3.5 μM and 148 μM, respectively. In comparison, AMDS was least inhibitory (EC₅₀ > 1 mM). Thus, the inhibitory potency (DIDS > SITS > AMDS) was partly dependent upon the number of isothiocyano groups. The EC₅₀ was also dependent on synexin concentration. Stilbene disulfonic acids were also inhibitory for arachidonic acid-enhanced synexin-mediated liposome fusion. The EC₅₀ for DIDS and SITS for fusion were similar to that for liposome aggregation. Ca²⁺-induced synexin polymerization, measured by 90° light scattering, was increased by DIDS, suggesting binding of stilbene disulfonic acids to synexin. The binding of DIDS to synexin was dependent on the molar ratio of synexin to DIDS. These results indicate that stilbene disulfonic acids interact directly with synexin to inhibit membrane aggregation and fusion. Our results suggest that such inhibition of synexin activity may contribute towards inhibition of surfactant secretion by DIDS, and support a physiological role for synexin in lung surfactant secretion.     

Studies on the interaction of matrix-bound inhibitor with Band 3, the anion transport protein of human erythrocyte membranes

The effect of temperature and chemical modification on the interaction of the human erythrocyte Band 3 protein (the anion transport protein) with 4-acetamido-4'-isothiocyanostilbene 2,2'-disulfonate (SITS; Ki = 10 microM)-Affi-Gel 102 resin was studied. Band 3 binds to the affinity resin in two states; weakly bound, which is eluted by 1 mM 4-benzamido-4'-aminostilbene 2,2'-disulfonate (BADS; Ki = 2 microM), and strongly bound, which is eluted only under denaturing conditions by 1% lithium dodecyl sulfate (LDS). At 4 degrees C, most of band 3 was present initially in the weakly bound form and very little in the strongly bound form. With longer incubations at 4 degrees C, the weakly bound form was slowly converted to the strongly bound form. At 37 degrees C, most of Band 3 was rapidly converted to the strongly bound form, with some Band 3 still remaining in the weakly bound form. Band 3 dimers, labelled with 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS) in one monomer, did bind to immobilized SITS but did not become tightly bound upon incubation at 37 degrees C. Since the covalent attachment of DIDS to one monomer prevented the adjacent monomer from becoming tightly bound to immobilized SITS ligand, this observation suggests that the inhibitor-binding sites of the two adjacent monomers must be interacting with each other. When the inhibitor site of Band 3 was selectively modified by citrate in the presence of 1-ethyl-3-(3-azonia-4,4-dimethylpentyl)carbodiimide (EAC), Band 3 bound to the resin was more easily eluted by BADS, suggesting reduced affinity for immobilized SITS. However, citrate-modified Band 3 did become tightly bound upon incubation at 37 degrees C.     

Stilbene derivatives inhibit the activity of the inner mitochondrial membrane chloride channels

Ion channels selective for chloride ions are present in all biological membranes, where they regulate the cell volume or membrane potential. Various chloride channels from mitochondrial membranes have been described in recent years. The aim of our study was to characterize the effect of stilbene derivatives on single-chloride channel activity in the inner mitochondrial membrane. The measurements were performed after the reconstitution into a planar lipid bilayer of the inner mitochondrial membranes from rat skeletal muscle (SMM), rat brain (BM) and heart (HM) mitochondria. After incorporation in a symmetric 450/450 mM KCl solution (cis/trans), the chloride channels were recorded with a mean conductance of 155 ± 5 pS (rat skeletal muscle) and 120 ± 16 pS (rat brain). The conductances of the chloride channels from the rat heart mitochondria in 250/50 mM KCl (cis/trans) gradient solutions were within the 70–130 pS range. The chloride channels were inhibited by these two stilbene derivatives: 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS). The skeletal muscle mitochondrial chloride channel was blocked after the addition of 1 mM DIDS or SITS, whereas the brain mitochondrial channel was blocked by 300 μM DIDS or SITS. The chloride channel from the rat heart mitochondria was inhibited by 50–100 μM DIDS. The inhibitory effect of DIDS was irreversible. Our results confirm the presence of chloride channels sensitive to stilbene derivatives in the inner mitochondrial membrane from rat skeletal muscle, brain and heart cells.     

Effect of disulfonic stilbene anion-channel blockers on the guinea-pig myocardium

The role of anions in the maintenance of tension in electrically driven left atria isolated from guinea pigs has been examined. The disulfonic stilbene anion-channel blockers SITS (4-acetamido-4'-isothiocyanostilbene 2'-disulfonate) and DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate) decreased the contractile force developed in a time- and concentration-dependent manner. As in the red cell anion channel, DIDS was more potent than SITS, but the maximal inhibition of tension produced by N-(4-azido-2-nitrophenyl)-2-aminoethyl sulfonate (NAP-taurine) was considerably lower than the near maximal inhibition produced by SITS and DIDS. The inhibition by SITS and DIDS was irreversible, suggesting a covalent interaction, and could not be overcome by increasing the calcium concentration or the frequency of stimulation. Consistent with a requirement for chloride anion, substitution of chloride and bicarbonate by the impermeant anion gluconate did not support contraction, while only partial tension was maintained with the lipophilic anions acetate and thiocyanate. Incubation of atria with 400 microM SITS blocked both 36Cl and 45Ca uptake to a similar extent, whereas the efflux of both these ions was not affected by incubation of the atria with SITS. The blockade by disulfonic stilbene anion-channel blockers of the contraction of the guinea pig myocardium may result from impairment of excitation-contraction coupling.     

Purification of bicarbonate-sensitive sperm adenylylcyclase by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid-affinity chromatography.

Anion transport inhibitors, such as SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid) and heparin, inhibit reversibly the bicarbonate-sensitive adenylylcyclase of porcine sperm plasma membrane. In the light of this, SITS- and heparin-affinity chromatographies were applied in order to purify sperm adenylylcyclase. SITS-Affi-Gel 102 binds proteins extracted from the porcine cauda epididymal sperm plasma membrane by Lubrol-PX, more selectively than heparin-agarose. However, recovery of adenylylcyclase activity is higher when heparin-agarose is used. The hormone-sensitive liver adenylylcyclase, which is less sensitive to bicarbonate than sperm enzyme, has less affinity for these affinity resins than sperm enzyme. Adenylylcyclase can be purified to apparent homogeneity on two-dimensional gel electrophoresis (isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis) from the Lubrol-PX extract of the purified sperm plasma membrane by using SITS-affinity chromatography at the first step of the purification followed by preparative isoelectric focusing and gel filtration. The molecular weight and pI of the purified enzyme are 46,300 and 6.9, respectively. The purified enzyme activity is highly dependent on Mn2+. Bicarbonate activates even the purified enzyme both by decreasing Km and by increasing Vmax.     

Electrogenic behavior of the human red cell Ca2+ pump revealed by disulfonic stilbenes

A systematic study was made of the action of 4-acet-amido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) on active Ca2+ transport of human erythrocytes. Pumping activity was estimated in inside-out vesicles (IOV's) by means of Ca2+-selective electrodes or use of tracer 45Ca2+. The stilbenes exhibited an approximately equal inhibitory potency and their action could be overcome by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) at low but not at high stilbene concentrations. In the absence of DIDS, Ca2+ transport was not affected upon addition of valinomycin, but it was appreciably reduced when vesicles were preincubated with low DIDS concentrations. Such an effect was strictly dependent on the external K+ concentration and it was abolished when valinomycin was added together with FCCP. Similar results were obtained using IOV's prepared from intact cells which had been previously exposed to the stilbene. The findings clearly demonstrate the presence in human red cells of a partially electrogenic Ca2+ pump, exchanging one Ca2+ ion for one proton.     

The role of anion channels in the mechanism of acrosome reaction in bull spermatozoa.

The involvement of anion channels in the mechanism of the acrosome reaction (AR) was investigated. The AR was induced by Ca2+ or by addition of the Ca2+ ionophore A23187. The occurrence of AR was determined by following the release of acrosin from the cells. In order to investigate the role of anion channels in the AR, several anion-channel inhibitors were tested, mainly DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). Other blockers, like SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid), furosemide, probenecid and pyridoxal 5-phosphate, were also tested. We found that DIDS binds covalently to sperm plasma membrane in a time- and concentration-dependent manner. Maximal binding occurs after 2 h with 0.3 mM DIDS. DIDS and SITS inhibit AR in a concentration-dependent manner. The IC50 of DIDS and SITS in the presence of A23187 is 0.15 and 0.22 mM, respectively. Tributyltin chloride (TBTC), an Cl-/OH- exchanger, partially overcomes DIDS inhibition of the AR. HCO3- is required for a maximal acrosin release and Ca(2+)-uptake, in the presence or absence of A23187. It is known that HCO3- activates adenylate cyclase and therefore, increases the intracellular level of cAMP. The inhibition of the AR by DIDS decreases from 95 to 50% when (dibutyryl cyclic AMP (dbcAMP) was added, i.e., HCO3- is no longer required while elevating the level of cAMP in an alternative way. Moreover, we show that the stimulatory effect of HCO3- on Ca(2+)-uptake is completely inhibited by DIDS. We conclude that DIDS inhibits AR by blocking anion channels, including those that transport HCO3- into the cell.     

Inhibition of anion permeability of sarcoplasmic reticulum vesicles by 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonate

The permeabilities of sarcoplasmic reticulum vesicle membrane for various ions and neutral molecules were measured by following the change in light scattering intensity due to the osmotic volume change of the vesicles. 4-Acetoamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS), which is a potent inhibitor for the anion permeability of red blood cells membrane, inhibited the permeability of sarcoplasmic reticulum for anions such as Cl-, Pi and methanesulfonate, while it slightly increased that for cations and neutral molecules such as Na+, K+, choline and glycerol. Binding of 5 mumol SITS/g protein was necessary for the inhibition of anion permeability. These results suggest the existence of a similar anion transport system in sarcoplasmic reticulum membrane as revealed in red blood cell membrane.     

Voltage-dependent chloride conductance of the squid axon membrane and its blockade by some disulfonic stilbene derivatives

When giant axons of squid, Sepioteuthis, were bathed in a 100 mM Ca-salt solution containing tetrodotoxin (TTX) and internally perfused with a solution of 100 mM tetraethylammonium-salt (TEA-salt) or tetramethylammonium-salt (TMA-salt), the membrane potential was found to become sensitive to anions, especially Cl-. Membrane currents recorded from those axons showed practically no time-dependent properties, but they had a strong voltage-dependent characteristic, i.e., outward rectification. Cl- had a strong effect upon the voltage-dependent membrane currents. The nonlinear property of the currents was almost completely suppressed by some disulfonic stilbene derivatives applied intracellularly, such as 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which are blockers of chloride transport. On the basis of these experimental results, it is concluded that a voltage-dependent chloride-permeable channel exists in the squid axon membrane. The chloride permeability (PCl) is a function of voltage, and its value at the resting membrane (Em = -60 mV) is calculated, using the Goldman-Hodgkin-Katz equation, to be 3.0 X 10(-7) cm/s.     

Anion Channel Blockers Inhibit the Acrosome Reaction of Echinoderm Sperm

Two types of anion channel blockers, SITS (4-acetamide-4'-isothiocyanostilbene-2,2'-disulfonic acid) and DIDS (4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid), inhibited jelly-induced acrosorne reaction in starfish and sea urchin. In starfish sperm, both of the blockers reversibly inhibited the formation of acrosomal process but they had no effect on either the acrosomal exocytosis or acid release from the sperm. Complete acrosome reaction occurred even in Cl⁻- and SO₄²⁻-free artificial seawater whereas HCO₃⁻was required for the acrosomal exocytosis. Importance of anion transport in acrosome reaction is discussed.     

Ion channels on synaptic vesicle membranes studied by planar lipid bilayer method.

An anion selective channel and three types of cation selective channels were found in planar lipid bilayers incorporating synaptic vesicles from rat brains. In asymmetric KCl solutions (cis: 300 mM/trans: 150 mM), the anion selective channel showed a single-channel conductance of 94 pS and was inactivated by negative voltages and by 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonic acid disodium salt (SITS). In the same solution, single-channel conductances of three types of cation selective channels were 250 pS (Type 1), 248 pS (Type 2), and 213 pS (Type 3), respectively. These channels resembled one another in single-channel conductances but were different in gating behaviors. Type 1 channel, which was most frequently observed, had a remarkable subconducting state (175 pS). Type 2 channel had a flickering state that increased as the potential became more positive, and a long inactive state that increased as the potentials were more negative. Type 3 channel, which was also sensitive to the potentials, had the open-channel probability increased as the potential became more positive.     

Modulation and block of the plasma membrane anion channel of guard cells by stilbene derivatives

An anion channel in the plasma membrane of guard cells (GCAC1) provides a regulatory element for the voltage-dependent release of anions during stomatal closure (Keller et al. 1989) as well as excitability (Hedrich et al. 1990). Recognition sites for plant growth hormones on the extracellular surface of GCAC1 further indicate that this channel may also serve as a transduction element in hormone signaling (Marten et al. 1991 a). Stilbene derivatives were used to study the inhibitor-structure channel-function relationship of GCAC1. We have analyzed the activity, voltage-gate and kinetics of this channel as affected by stilbenes. The stilbene derivatives SITS and DNDS caused a shift in activation potential and a decrease in the peak current amplitude. Channel block through the action of DIDS, on the other hand, was not accompanied by a shift in voltage-dependence. Differences in the dose-dependence of the two effects give clues to the presence of channel sites responsible for gate-shifting and block. The ability to inhibit anion currents (K_d) increased in the sequence: SITS (4 µM) < DNDS (0.5 µM) < DIDS (0.2 µM). All inhibitors reversibly blocked the anion channel from the extracellular side. Channel block on the level of single anion-channels is characterized by a reduction of long open-transitions into flickering bursts and a decrease in channel amplitude.Abbreviations DIDS 4,4-Diisothiocyanostilbene-2,2-disulfonic acid - SITS 4-Acetamido-4-isothiocyanostilbene-2,2-disulfonic acid - DNDS 4,4-dinitrostilbene-2,2-disulfonic acid - NPPB 5-Nitro-2-(3-phenylpropylamio)benzoic acid - IAA-94 [(6,7-Dichloro2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5y1)oxy] acetic acid - A-9-C Anthracene-9-carboxylic acid - TEA Tetraethylammonium     

Modification of K conductance of the squid axon membrane by SITS

The effects of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) on the K conductance, gK, were studied in internally perfused giant axons from squid, Doryteuthis. SITS at 3-200 microM was applied intracellularly by adding the reagent to the internal perfusion fluid. Three remarkable changes in gK were noted: there was a slowing of the opening and closing rates of the K channel in the whole voltage region; K channels modified with SITS started to open at voltages below -100 mV, and thus 30% of total K channels were open at the level of normal resting potential (approximately -60 mV) after the maximal drug effect was attained (less than 30 microM); there was a disappearance of gK inactivation that became distinct at relatively high temperature (greater than 8 degrees C). These drug effects depended solely on the drug concentration, not on factors such as repetitive alterations of the membrane potential, and the changes in gK were almost irreversible. Another disulfonic stilbene derivative, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), had similar effects on gK, but the effects were approximately 1.5 times stronger. These changes in gK were somewhat similar to alterations in gNa produced by an application of veratridine, batrachotoxin, and grayanotoxin, which are known as Na channel openers.     

Inactivation of the Na,K-ATPase by modification of Lys-501 with 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS).

The sodium pump or Na,K-ATPase, maintains the Na+ and K+ gradients across eukaryotic cell membranes at the expense of ATP. Incubation of purified canine renal Na,K-ATPase with 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) inhibited the ATPase activity. Both the labeling of the protein and the loss of ATPase activity were prevented by co-incubation with ADP (acting as an ATP analog) or KCl. Only the alpha-subunit was labeled by SITS. The alpha-subunit from the inhibited enzyme was extensively digested with trypsin, and SITS-labeled peptides were purified by reverse-phase HPLC and sequenced. The amino acid sequence determined, His-Leu-Leu-Val-Met-X-Gly-Ala-Pro-Glu, indicated that SITS modifies Lys-501 (X) on the alpha-subunit of Na,K-ATPase.     

SITS Blockade Induces Multiple Subconductance States in a Large Conductance Chloride Channel

The effect of the chloride channel blocker 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS) on the gating and amplitude of an endothelial chloride channel was explored using the outside-out configuration of the patch-clamp technique. Under control conditions the channel displayed two main gating modes: shut and fully open. Transitions to equally spaced subconductance states were rarely observed (less than 10 events/minute). At low concentrations (<45 μm), SITS increased the number of transitions to the three subconductance states in a concentration-dependent manner, while reducing the number of transitions to the fully open state. This effect was maintained after removing SITS from the bath solution, suggesting that the modifications in the channel induced by SITS were irreversible. All four conducting states had similar current-voltage relationships. At higher concentrations (>45 μm), SITS reduced the amplitude of all conducting states (three subconductances and fully open). This effect was fully reversible upon SITS removal from the bath solution. A half-inhibitory concentration (IC₅₀) of 55.6 ± 2.7 μm (+60 mV) and 66.7 ± 2.2 (−60 mV) was obtained from the fitting to a Langmuir function. All these results are compatible with the existence of two SITS binding sites in the chloride channel: one of high affinity responsible for the increment in the number of transitions to subconductance states, and one low affinity binding site involved in the reduction of the amplitude of all conducting states. Received: 29 October 1998/Revised: 16 February 1999     

Properties of chloride transport in barnacle muscle fibers.

Unidirectional chloride-36 fluxes were measured in internally dialyzed barnacle giant muscle fibers. About 50--60% of the Cl efflux was irreversibly blocked by the amino-group reactive agent, 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (SITS), when it was applied either intra- or extracellularly. Similarly, Cl influx was also blocked by SITS. No significant effect on [Cl]i of SITS was noted in intact muscle fibers. However, the rate of net Cl efflux from muscle fibers which were Cl-loaded by overnight storage at 6 degrees C could be slowed by SITS treatment. Two classes of anions were defined based upon their effects on Cl efflux. Methanesulfonate and nitrate inhibited Cl efflux either when they replaced external chloride or when they were added to a constant external chloride concentration. The other group of anions (propionate, formate) stimulated both Cl efflux and influx and such stimulation could be blocked by SITS. Propionate influx was not nearly as large as the stimulated Cl efflux and was unaffected by SITS. Neither the effects of SITS nor those of the anion substitutes could be simply accounted for by changes in the membrane resting potential or conductance. These results suggest a mediated transport system for chloride across the barnacle sarcolemma.