The enhancing effects of anion channel blockers on sperm activation by bicarbonate.

We found that anion channel blockers such as phosphotungstate and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) enhanced HCO3(-)-induced activation on porcine epididymal sperm. In the presence of these compounds, HCO3- increased the motility, respiration rate and especially the cAMP content of the sperm to a greater extent than did HCO3- alone. The enhancing effects were not observed in the absence of HCO3-, but were evident when the concentration of HCO3- was low. These compounds did not significantly alter the intracellular pH and did inhibit the adenylate cyclase activity of the sperm plasma membrane. When these compounds were added to sperm homogenate with ATP, the cAMP formed was reduced compared to the control. In addition, these compounds inhibited both the SO4(2-) influx and efflux of the sperm. From these results, we conclude that the anion channel blockers tested principally inhibit the efflux of endogenous HCO3- derived from metabolic CO2, so that HCO3- accumulates intracellularly and stimulates the adenylate cyclase of the sperm.     

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^?, P_i and methanesulfonate, while it slightly increased that for cations and neutral molecules such as Na⁺, K⁺, choline and glycerol. Binding of 5μmol 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.     

Effect of staphylococcal alpha-hemolysin upon anion transport in the rabbit erythrocyte

Equilibrium exchange of SO₄^2? was measured prior to and during hemolysis in rabbit erythrocytes exposed to staphylococcal α-hemolysin. The anion-transport protein of the rabbit erythrocyte has also been identified. Equilibrium exchange of SO₄^2? was measured by both efflux and influx of ³⁵SO₄^2?. The rate of influx of SO₄^2? in rabbit erythrocytes exposed to α-hemolysin was twice that of the untreated cells. The rate of SO₄^2? efflux was unchanged by α-hemolysin. Inhibition of anion exchange with 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) did not inhibit hemolysis, therefore, the increased influx of SO₄^2? may occur through a DIDS-insensitive pathway.     

Cytosolic pH regulation in perfused rat liver: role of intracellular bicarbonate production

The contribution of metabolic bicarbonate to cytosolic pH (pH_cyto) regulation was studied on isolated perfused rat liver using phosphorus-31 NMR spectroscopy. Removal of external HCO^?₃ decreased proton efflux from 18.6±5.0 to 1.64±0.29 μmol/min per g liver wet weight (w.w.) and pH_cyto from 7.17±0.06 to 6.87±0.06. In the nominal absence of bicarbonate, inhibition of carbonic anhydrase by acetazolamide induced a further decrease of proton efflux of 0.69±0.26 μmol/min per g liver w.w. reflecting a reduction in metabolic CO₂ hydration, and hence a decrease of H⁺ and HCO^?₃ supplies. Even though 27% of the proton efflux was amiloride-sensitive under bicarbonate-free conditions, amiloride did not change pH_cyto, revealing the contribution of additional regulatory processes. Indeed, pH regulation was affected by the combined use of 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS) and amiloride since pH_cyto decreased by 0.16±0.05 and proton efflux by 0.60±0.14 μmol/min per g liver w.w. The data suggest that amiloride-sensitive or SITS-sensitive transport activities could achieve, by themselves, pH_cyto regulation. The involvement of two mechanisms, most likely Na⁺/H⁺ antiport and Na⁺:HCO^?₃ symport, was confirmed in the whole organ under intracellular and extracellular acidosis. The evidence of Na-dependent transport of HCO^?₃ in the absence of exogenous bicarbonate implies that the amount of metabolic bicarbonate is sufficient to effectively participate to pH_cyto regulation.     

A relationship between anion transport and a structural transition of the human erythrocyte membrane

Scanning microcalorimetry was employed as an aid in examining some structural features of the anion transport system in red blood cell vesicles. Two structural transitions were previously shown to be sensitive to several covalent and non-covalent inhibitors of anion transport in red cells. In this study, these transitions were selectively removed, either thermally or enzymatically, and the subsequent effect on ³⁵SO₄^2? efflux in red cell vesicles was determined. It is shown that removal of one of these transitions (B₂) has a negligible inhibitory effect on anion transport. Cytoplasmic, intermolecular disulfide linkages between band 3 dimers are known to form during the B₂ transition. The integrity of the 4,4′-diisothiocyanostilbene-2,2′-disulfonate-sensitive C transition, on the other hand, is shown to be a requirement for anion transport. The localized region of the membrane giving rise to this transition contains the transmembrane segment of band 3, as well as membrane phospholipids. The calorimetric results suggest a structure of band 3 which involves independent structural domains, and are consistent with the transmembrane segment playing a direct role in the transport process.     

CFTR-mediated anion secretion in parathyroid hormone-treated Caco-2 cells is associated with PKA and PI3K phosphorylation but not intracellular pH changes or Na+/K+-ATPase abundance

Parathyroid hormone (PTH) has previously been shown to enhance the transepithelial secretion of Cl^? and HCO₃^? across the intestinal epithelia including Caco-2 monolayer, but the underlying cellular mechanisms are not completely understood. Herein, we identified the major signaling pathways that possibly mediated the PTH action to its known target anion channel, i.e., cystic fibrosis transmembrane conductance regulator anion channel (CFTR). Specifically, PTH was able to induce phosphorylation of protein kinase A and phosphoinositide 3-kinase. Since the apical HCO₃^? efflux through CFTR often required the intracellular H⁺/HCO₃^? production and/or the Na⁺-dependent basolateral HCO₃^? uptake, the intracellular pH (pH_i) balance might be disturbed, especially as a consequence of increased endogenous H⁺ and HCO₃^? production. However, measurement of pH_i by a pH-sensitive dye suggested that the PTH-exposed Caco-2 cells were able to maintain normal pH despite robust HCO₃^? transport. In addition, although the plasma membrane Na⁺/K⁺-ATPase (NKA) is normally essential for basolateral HCO₃^? uptake and other transporters (e.g., NHE1), PTH did not induce insertion of new NKA molecules into the basolateral membrane as determined by membrane protein biotinylation technique. Thus, together with our previous data, we concluded that the PTH action on Caco-2 cells is dependent on PKA and PI3K with no detectable change in pH_i or NKA abundance on cell membrane.     

Intracellular signal transduction mechanisms of rat epididymal spermatozoa and their relationship to motility and metabolism

The role of intracellular signal transduction mechanisms in regulating the motility and metabolism of rat spermatozoa in undiluted caudal epididymal fluid (CEF) was examined. Samples of CEF containing immotile spermatozoa were exposed to drugs and other agents that either stimulate signal transduction pathways or mimic the action of their second messengers. Under these conditions, sperm motility in 25–30 nl of CEF was stimulated by calcium ions (Ca²⁺), N,2′ -O-dibutyryl-guanosine 3′:5′ -cyclic monophosphate (dibutryl cGMP), cyclic adenosine 3′:5′-monophosphate (cAMP), N⁶,2′-O-dibutyryladenosine 3′:5′ -cyclic monophosphate (dibutyryl cAMP), 8-bromoadenosine 3′:5′ -cyclic monophosphate (8-bromo cAMP), caffeine, theophylline and bicarbonate ions (HCO₃^?). Other agents such as magnesium ions (Mg²⁺), veratridine, phospholipase C (PLC), ionophore A23187, 1,2-dioctenoyl-sn-glycerol (DAG), phorbol 12-myristate 13-acetate, phospholipase A₂ (PLA₂), arachidonic acid, and melittin did not significantly influence motility. In the presence of radiolabelled energy substrates, untreated (immotile) spermatozoa in samples of CEF utilised D-[U-¹⁴C]glucose and [1-¹⁴C]acetate as exogenous energy sources for oxidative metabolism. No detectable ¹⁴C-lactate was produced, and none of the drugs altered the rate of glycolytic or oxidative metabolism. The findings suggest that the motility of rat caudal epididymal spermatozoa is regulated by Ca²⁺ and the guanylate cyclase and adenylate cyclase pathways, but not through the PLC and PLA₂ pathways. Also, their metabolism of exogenous substrate was uncoupled from the induction of motility, and their oxidative capacity exceeded the rate of flux of glucose-carbon through the glycolytic pathway. © 1994 Wiley-Liss, Inc.     

cAMP activates Cl−/HCO3− exchange for regulation of intracellular pH in renal epithelial cells

The role of cAMP in regulation of intracellular pH in the confluent LLC-PK₁ cells was investigated. DibutyrylcAMP and forskolin induce intracellular acidification. This acidification is inhibited by DIDS and ethacrynic acid, inhibitors of Na⁺-independent Cl^?/HCO₃^? exchange, and by removal of extracellular Cl^?. In addition, Bt₂ cAMP causes Cl^? entry into LLC-PK₁ cells. These results suggest that cAMP activates Cl^? transport, namely Na⁺-independent Cl^?/HCO₃^? exchange, which participates in pH_i regulation.     

Localization and characterization of transport-related elements in the plasma membrane of turtle bladder epithelial cells

A mixed membrane preparation obtained from turtle bladder epithelial cells contains (Na⁺ + K⁺)-ATPase, adenylate cyclase and protein kinase, which interact with ouabain, norepinephrine and cyclic AMP, respectively. When such a preparation is obtained from bladders which had been preexposed to serosal fluids containing the tritiated form of 4,4′-diisothiocyano-2,2′-disulfonic stilbene, the subsequently isolated membrane proteins are enriched in tritium as well as in the afore-mentioned enzymes, none of which is inhibited. Freeflow electrophoresis separates the mixed membrane preparation into two distinguishable groups: one, construed as apical membranes, is enriched in norepinephrine-sensitive adenylate cyclase and cyclic AMP-sensitive protein kinase; the other, construed as basal-lateral membranes, is enriched in ouabain-sensitive ATPase and 4,4′-diisothiocyano-2,2′-disulfonic stilbene-binding proteins.The physiological counterparts of these enzymatically defined membrane markers are the mucosal sidedness of the transport effects of norepinephrine and cyclic AMP derivatives and the serosal sidedness of the transport effects of ouabain and disulfonic stilbenes in the intact turtle bladder. The discreteness and ion selectivity of each membrane-bound, transport-related element are discussed in relation to the corresponding characteristics of each transport process in vivo; the possibility of regulation of anion transport by adenylate cyclase-protein kinase system is also discussed.     

A comparison of the inhibitory potency of reversibly acting inhibitors of anion transport on chloride and sulfate movements across the human red cell membrane

The effects of a variety of chemically diverse, reversibly acting inhibitors have been measured on both Cl^? and SO₄^2? equilibrium exchange across the human red cell membrane. The measurements were carried out under the same conditions (pH 6.3, 8°C) and in the same medium for both the Cl^? and SO₂⁴ tracer fluxes. Under these conditions the rate constant for Cl^?-Cl^? exchange is about 20 000 times larger than that for SO₄^2?-SO₄^2? exchange. Despite this large difference in the rates of transport of the two anions, eight different reversibly acting inhibitors have virtually the same effect on the Cl^? and SO₄^2? transport. The proteolytic enzyme papain also has the same inhibitory effect on both the Cl^? and SO₄^2? self-exchange. In addition, the slowly penetrating disulfonate 2-(4′-aminophenyl)-6-methylbenzenethiazol-3′,7-disulfonic acid (APMB) is 5-fold more effective from the outer than from the inner membrane surface in inhibiting both Cl^? and SO₄^2? self-exchange. We interpret these results as evidence that the rapidly penetrating monovalent anion Cl^? and the slowly penetrating divalent anion SO₄^2? are transported by the same system.     

Effect of 3-isobutyl-1-methylxanthine on HCO3− transport in turtle bladder: Evidence for electrogenic HCO3− secretion

Ouabain-treated turtle bladders bathed on both surfaces by identical HCO₃^?/CO₂-containing, Cl^?-free Na⁺ media exhibit a short-circuit current (I_sc) and transepithelial potential (p.d.) serosa electronegative to mucosa. Addition of 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cyclic nucleotide phosphodiesterase, rapidly reverses the direction of the I_sc and p.d.. The IBMX-induced reversal of I_sc and p.d. is (1) dependent on the presence of HCO₃^? (and CO₂) in the serosal bathing fluid, (2) independent of Na⁺ and other ions in the bathing medium, (3) decreased by inhibitors of carbonic anhydrase or oxidative metabolism, (4) increased by the serosal addition of cyclic AMP or the disulfonic stilbene, SITS. The results constitute evidence that the reversed I_sc elicited by IBMX represents electrogenic secretion of HCO₃^?.     

Anion channels in the sea urchin sperm plasma membrane

Ionic fluxes in sea urchin sperm plasma membrane regulate cell motility and the acrosome reaction (AR). Although cationic channels mediate some of the ionic movements, little is known about anion channels in these cells. The fusion of sperm plasma membranes into lipid bilayers allowed identification of a 150 pS anion channel. This anion channel was enriched from detergent-solubilized sperm plasma membranes using a wheat germ agglutinin Sepharose column. Vesicles formed from this preparation were fused into black lipid membranes (BLM), yielding single channel anion-selective activity with the properties of those found in the sperm membranes. The following anion selectivity sequence was found: NO₃^? > CNS^? > Br^? > CI^?. This anion channel has a high open probability at the holding potentials tested, it is partially blocked by 4,4′-diisothiocyano-2,2′ -stilbendisulfonic acid (DIDS), and it often displays substates. The sperm AR was also inhibited by DIDS. © 1993 Wiley-Liss, Inc.     

Inhibition of anion permeability of sarcoplasmic reticulum vesicles by stilbene derivatives and the identification of an inhibitor-binding protein

The permeability of sarcoplasmic reticulum vesicles to sulfate ions was inhibited by diisothiocyano-1,2-diphenylethane-2,2′-disulfonic acid (H₂DIDS), which is a potent inhibitor of anion permeability in red blood cell membrane. The amount of H₂DIDS bound to the vesicles was determined by using [³H]-H₂DIDS. Apparent half inhibition of sulfate permeation was observed on the binding of 2.5 μmol/g protein. SDS-polyacrylamide gel electrophoresis of the vesicles treated with [³H]H₂DIDS showed that about 10% of the total bound H₂DIDS corresponds to a 100 000-dalton protein, but the remaining 90% to non-protein components. The content of the H₂DIDS-binding protein was about 0.5 μmol/g protein. These results suggest that the H₂DIDS-binding protein is different from the calcium pump protein and is possibly an anion transport system similar to band 3 in red blood cell membrane.     

The ascidian sperm reaction: evidence for Cl- and HCO3- involvement in acid release

Ascidia callosa sperm are triggered to undergo initiation of the sperm reaction (mitochondrial swelling) by increasing the pH or lowering the Na⁺ concentration of the medium. The optimal [Na⁺] for acid release is 20 mM with excellent correlation between acid release and initiation of morphological changes. Increasing the [K⁺] to around 20 mM inhibits acid release when applied up to 1 min after triggering the sperm but with less inhibition at 2 and 4 min, suggesting that K⁺ inhibits initiation of acid release rather than acid release itself. Acid release and the sperm reaction can also be triggered by Cl^?-free (NO^?₃ or glutamate substituted) seawater (SW). Cl^? efflux accompanies H⁺ efflux with twice as many Cl^? being released as H⁺. Both H⁺ and Cl^? release in Cl^?-free SW are dependent upon CO₂ being present in HCO^?₃-free medium, suggesting that H⁺ efflux is in part Cl^? and HCO^?₃-mediated. However, the chloride channel blocking agent SITS has no effect on H⁺ release and augments Cl^? release. Acid release results in a substantial increase in internal pH as determined by partitioning of 9-amino acridine. We envision acid release from ascidian sperm as involving two systems, the Na⁺-dependent acidification system of unreacted sperm and the Cl^?- and HCO^?₃-mediated H⁺ release at activation. The mechanism controlling acid release would then involve inactivation of the internal acidification process and activation of the chloride-bicarbonate-mediated alkalinization process.     

Identification of an anion channel protein from electric organ of Narke japonica

The anion permeability of membrane vesicles prepared from the electric organ of $\text{Narke japonica}$ was inhibited by the addition of 4,4′-diisothiocyano-stilbene-2,2′-disulfonic acid (DIDS). The permeability was measured by measuring changes in the scattered-light intensity caused by the osmotic volume change of vesicles; and also by the efflux measurement of ions from the vesicles using radioisotopes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of membrane vesicles treated with dihydro analog of DIDS ([³H]H₂DIDS) showed that the H₂DIDS binding protein has a molecular weight of 180,000, and exists in membrane vesicles as a dimer formed by a disulfide bond between monomers of molecular weight 90,000.     

Stimulatory effect of PACAP on gastroduodenal bicarbonate secretion in rats

The effects of pituitary adenylate cyclase activating polypeptides (PACAPs) on gastroduodenal HCO₃⁻ secretion were investigated in anesthetized rats and compared with those of vasoactive intestinal polypeptide (VIP). Under urethane anesthesia, a rat stomach mounted in an ex vivo chamber (in the absence of acid secretion) or a rat proximal duodenal loop was perfused with saline, and the HCO₃⁻ secretion was measured at pH 7.0 using a pH-stat method and by adding 10 mM HCl. Intravenous injection of PACAP-27 stimulated HCO₃⁻ secretion in a dose-dependent manner in the duodenum but not in the stomach; at 8 nmol/kg PACAP-27 increased the HCO₃⁻ secretion to maximal values of four times greater than basal levels, although this peptide had no effect on duodenal HCO₃⁻ secretion after intracisternal administration (1 nmol/rat). PGE₂ (300 μg/kg, iv) significantly increased HCO₃⁻ secretion in both the stomach and the duodenum. The potency of duodenal HCO₃⁻ secretory action was in the following order; PACAP-27 > PACAP-38 = VIP, and that of PACAP-27 was about 100-fold greater than that of PGE₂. The duodenal HCO₃⁻ secretory action of PACAP-27 as well as PGE₂ was markedly potentiated by prior administration of isobutylmethyl xanthine (10 mg/kg, sc), the inhibitor of phosphodiesterase. Folskolin (250 μg/kg, iv), the stimulator of adenylate cyclase, also increased HCO₃⁻ secretion in the duodenum but not in the stomach. These results suggest that: 1) PACAPs are potent stimulators of HCO₃⁻ secretion in the duodenum but not in the stomach; 2) this action is mediated by cAMP through stimulation of adenylate cyclase; 3) cAMP is a mediator in duodenal but not gastric HCO₃⁻ secretion; and 4) PACAPs may be involved in the peripheral regulation of duodenal HCO₃⁻ secretion.     

Identification of the Cl− transport site of human red blood cells by a kinetic analysis of the inhibitory effects of a chemical probe

H₂DIDS, the dihydro analog of DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid) can interact covalently with membrane sites, resulting in an irreversible inhibition of anion exchange. At low temperatures (0°C) and for relatively short times, however, its interaction is largely reversible, so that a kinetic analysis of the nature of its inhibitory effect on Cl^? self exchange can be performed. The effects of variations in the chloride concentration on the inhibitory potency of H₂DIDS are consistent with the concept that Cl^? and H₂DIDS compete for the transport site of the anion exchange system. The value of K_i for H₂DIDS is 0.046 μM, indicating that H₂DIDS has a higher affinity for the transport system than any other inhibitor so far examined. If, as seems probable, the covalent labelling of H₂DIDS occurs at the same site as the reversible binding, H₂DIDS can be used as a covalent label for the transport site. The specific localization of H₂DIDS in the band-3 protein thus indicates that this protein participates directly in anion exchange.     

Role of CFTR and anion exchanger in bicarbonate fluxes in C127 cell lines

C127 cell lines transfected with wtCFTR, ΔF508CFTR or vector were employed to determine HCO⁻₃ fluxes in the presence or absence of functional CFTR, using the pH-sensitive dye BCECF. Both cytosolic alkalinization and acidification were due to activity of anion exchanger and were similar in the three cell lines, indicating that expression of CFTR did not influence anion exchanger activity. In C127wt cells only, cAMP elevating agents significantly stimulated HCO⁻₃ fluxes, insensitive to the inhibitor of anion exchanger 4,4′-diisothiocyanate dihydrostilbene-2,2′-disulfonic acid, suggesting that activated CFTR directly mediates both HCO⁻₃ influx and efflux and therefore can contribute to intracellular and extracellular pH regulation.     

Effects of 4-acetamido-4′-isothiocyano-2,2′-disulfonic stilbene on ion transport in turtle bladders

The disulfonic stilbene (4-acetamido-4′-isothiocyano-2,2′-disulfonic stilbene) is found to be more potent than acetazolamide as an anion transport inhibitor in the turtle bladder, but less potent than acetazolamide as a carbonic anhydrase inhibitor. The anion-dependent (HCO₃^-−, Cl⁻) moeity of the short-circuiting current is eliminated by 4-acetamido-4′-isothiocyano-2,2′-disulfonic stibene, but only after its addition to the serosal bathing fluid. Whereas 4-acetmido-4′-isothiocyano-2,2′-disulfonic stilbene has no effect om Na⁺transport across the bladder, it is more potent than ouabain as an inhibitor of microsomal (Na⁺+K⁺)-ATPase of both turtle bladder and eel electric organ.     

ACTH-induced refractoriness in cultured adrenal cell line (Y1).

Treatment of cultured mouse adrenal cells Y₁ with ACTH induced cell refractoriness to further hormonal stimulation. When ACTH was added to the cells every 2 hours the first addition increased the levels of 20αOH-progesterone and cAMP secreted into the medium. Upon the second and third additions of ACTH the levels of 20αOH-progesterone and cAMP secreted were greatly diminished and upon the fourth addition of ACTH were absent. Prolonged incubation (14 hours) with different concentrations of ACTH (5 × 10^?11 M to 10^?6 M) induced a dose-related steroidogenic refractoriness to further ACTH stimulation, 10^?8 M ACTH inducing complete refractoriness. The number of ACTH binding sites of cell particles prepared from desensitized cells was similar to that of the control but ACTH failed to stimulate the adenylate cyclase of desensitized cells, whereas the enzyme responded fully to NaF and Gpp(NH)p. The cAMP phosphodiesterase activity was similar in both desensitized and control cells. In addition the steroidogenic response to dibutyryl cAMP of desensitized cells was abolished. Thus, ACTH-induced adrenal cell desensitization seems to be related to at least two phenomena : a defect in the “coupling” between the hormone-receptor sites and the adenylate cyclase and an alteration of certain steps beyond cAMP formation.