Identification and characterization of ATP-dependent proton transport by rat liver multivesicular bodies

Multivesicular bodies (MVB), prelysosomal organelles in the endocytic pathway, were prepared from estrogen-treated rat livers and examined for the presence of ATP-dependent proton transport. Vesicle acidification, assessed by acridine orange fluorescence quenching, was ATP dependent (ATP much greater than GTP, UTP), was enriched 25-fold over homogenate, was abolished by pretreatment with protonophores or a nonionic detergent, exhibited a pH optimum of 7.5, was inhibited by N-ethylmaleimide (NEM) (IC50 approximately 5 microM) and N,N'-dicyclohexylcarbodiimide (IC50 approximately 5 microM), and was resistant to inhibition by vanadate, ouabain, and oligomycin. Acidification exhibited no specific cation requirement; however, maximal rates of acidification depended upon the presence of Cl- (Km approximately 20 mM). Other anions were less effective in supporting acidification (Cl- greater than Br- greater than much greater than gluconate, NO-3, SO2-4, and mannitol), and indeed NO-3 inhibited acidification even in the presence of 150 mM Cl-. The proton transport mechanism appeared to be electrogenic based on: (a) enhancement of acidification by valinomycin in the presence of K gluconate, and (b) ATP-dependent fluorescence quenching of bis(3-phenyl-5-oxoisoxasol-4-yl)pentamethine oxonol, a membrane potential-sensitive anionic dye. Furthermore, the magnitude of the pH and electrical gradients generated by the proton transport mechanism appeared to vary inversely in the presence and absence of Cl-. Finally, MVB exhibited ATPase activity that was resistant to ouabain and oligomycin, but was inhibited 32.3% by 1 mM NEM, 33.7% by 200 microM dicyclohexylcarbodiimide, and 18.7% by KNO3. In isolated MVB, therefore, the NEM-sensitive ATPase activity may represent the enzymatic equivalent of a proton pump. These studies identify and characterize an ATP-dependent electrogenic proton transport process in rat liver MVB which shares many of the properties of the proton pump described in clathrin-coated vesicles, endosomes, lysosomes, Golgi, and endoplasmic reticulum from liver and other tissues. Acidification of MVB differed somewhat from that of rat liver clathrin-coated vesicles in response to Br- and NO-3, suggesting that membrane properties of these two organelles might differ.     

Modulation of ATP-dependent Ca2+ transport in rat parotid basolateral membrane vesicles by K+ + Cl- flux

In basolateral membrane vesicles (BLMV) isolated from rat parotid glands, the initial rate of ATP-dependent Ca2+ transport, in the presence of KCl, was approx. 2-fold higher than that obtained with mannitol, sucrose or N-methyl-D-glucamine (NMDG)-gluconate. Only NH4+, Rb+, or Br- could effectively substitute for K+ or Cl-, respectively. This KCl activation was concentration dependent, with maximal response by 50 mM KCl. An inwardly directed KCl gradient up to 50 mM KCl had no effect on Ca2+ transport, while equilibration of the vesicles with KCl (greater than 100 mM) increased transport 15-20%. In presence of Cl-, 86Rb+ uptake was 2.5-fold greater than in the presence of gluconate. 0.5 mM furosemide inhibited 86Rb+ flux by approx. 60% in a Cl- medium and by approx. 20% in a gluconate medium. Furosemide also inhibited KCl activation of Ca2+ transport with half maximal inhibition either at 0.4 mM or 0.05 mM, depending on whether 45Ca2+ transport was measured with KCl (150 mM) equilibrium or KCl (150 mM) gradient. In a mannitol containing assay medium, potassium gluconate loaded vesicles had a higher (approx. 25%) rate of Ca2+ transport than mannitol loaded vesicles. Addition of valinomycin (5 microM) to potassium gluconate loaded vesicles further stimulated (approx. 30%) the Ca2+ transport rate. These results suggest that during ATP dependent Ca2+ transport in parotid BLMV, K+ can be recycled by the concerted activities of a K+ and Cl- coupled flux and a K+ conductance.     

Characterization of a H+-ATPase in rat brain synaptic vesicles. Coupling to L-glutamate transport

Synaptic vesicles contain a H+-ATPase that generates a proton electrochemical gradient (delta mu H+) required for the uptake of neurotransmitters into the organelles. In this study, the synaptic vesicle H+-ATPase was examined for structural and functional similarities with other identified ATPases that generate a delta mu H+ across membranes. The synaptic vesicle H+-ATPase displayed immunological similarity with the 115-, 72-, and 39-kDa subunits of a vacuolar-type H+-ATPase purified from chromaffin granules. Functionally, the ATP-dependent H+ pumping across synaptic vesicles and ATP hydrolysis were sensitive to the sulfhydryl-modifying reagents, N-ethylmaleimide and 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, at concentrations known to affect vacuolar-type H+-ATPases. In addition, as with vacuolar-type H+-ATPases, the presence of NO3-, SO4(2-), or F- inhibited the generation of a delta mu H+, but addition of vanadate or oligomycin had no effect. The delta mu H+ is a function of the pH gradient (delta pH) and membrane potential (delta psi sv) across the synaptic vesicle. Acidification (delta pH) of the synaptic vesicle interior was enhanced in the presence of permeant anions, such as Cl-, or the K+ ionophore, valinomycin. In the absence of permeant anions, the H+-ATPase generated a delta psi sv that effected the transport of L-glutamate into the synaptic vesicles. Dissipation of delta psi sv by incubation with increased external Cl- or nigericin resulted in the abolition of glutamate uptake, despite the continued maintenance of a delta mu H+ across the synaptic vesicle as a substantial delta pH. The results suggest that the synaptic vesicle H+-ATPase is of a vacuolar type and energizes the uptake of anionic glutamate by virtue of the delta psi sv component of the delta mu H+ it generates.     

The lysosomal H+ pump: 8-azido-ATP inhibition and the role of chloride in H+ transport

Lysosomes (tritosomes) were purified from the livers of rats injected with Triton WR 1339. The lysosomes developed an Mg2+-ATP-dependent pH gradient as measured by Acridine orange accumulation. H+ transport was supported by chloride, but not sulfate, and was independent of the cation used. H+ transport and Mg2+-stimulated ATPase was inhibited by diethylstilbesterol (K0.5 = 2 microM). N-Ethylmaleimide inhibited H+ transport (K0.5 = 30 microM). At low concentrations of N-ethylmaleimide, ATP partially protected H+ transport from inhibition with N-ethylmaleimide. Photolysis with 8-azido-ATP inhibited H+ transport and Mg2+-stimulated ATPase activity. Under these same conditions, 8-azido-[alpha-32P]ATP reacted with a number of polypeptides of the intact lysosome and lysosomal membranes. Pump-dependent potentials were measured using the fluorescent potential-sensitive dye, DiSC3(5) (3,3'-dipropylthiocarbocyanine) and ATP-dependent potential generation was inhibited by diethylstilbesterol. Chloride, but not sulfate reduced the magnitude of the ATP-dependent membrane potential, as measured using merocyanine 540. The chloride conductance, independent of ATP, was of sufficient magnitude to generate a H+ gradient driven by external chloride in the presence of tetrachlorosalicylanilide. In Cl- free media, ATP-dependent H+ transport was restored to control levels by outwardly directed K+ gradients in the presence of valinomycin. The role of cell Cl- is to provide the necessary conductance for supporting lysosomal acidification by the electrogenic proton pump.     

An ATP-driven Cl- pump in the brain

EDTA-treated microsomes prepared from rat brain mainly consisted of sealed membrane vesicles 200-500 nm in diameter and were rich in both Cl- -ATPase and Na+,K+-ATPase activities. Such Cl- -ATPase-rich membrane vesicles accumulated Cl- in an ATP-dependent and osmotically reactive manner in the presence of 1 nM ouabain. The Cl- uptake was maximally stimulated by ATP with a Km value of 1.5 mM; GTP, ITP, and UTP partially stimulated Cl- uptake, but CTP, beta, gamma-methylene ATP, ADP, and AMP did not. The ATP-dependent Cl- uptake was accelerated by an increase in the medium Cl- concentration with a Km value of 7.4 mM. Such stimulation of Cl- uptake by ATP was dependent on the pH of the medium, with an optimal pH of 7.4, and also on the temperature of the medium, with an optimal range of 37-42 degrees C. Ethacrynic acid dose dependently inhibited the ATP-dependent Cl- uptake with a concentration for half-maximal inhibition at 57 microM. N-ethylmaleimide (0.1 mM) completely inhibited and sodium vanadate (1 mM) partially inhibited the ATP-dependent Cl- uptake. The membrane vesicles did not accumulate H+ in the Cl- uptake assay medium. The ATP-dependent Cl- uptake profile agreed with that of Cl- -ATPase activity reported previously (Inagaki, C., Tanaka, T., Hara, M., and Ishiko, J. (1985) Biochem. Pharmacol. 34, 1705-1712), and this strongly supports the idea that Cl- -ATPase in the brain actively transports Cl-.     

Anion regulation of active transport of protons across the membrane of the synaptic vesicles of the brain

The dependence of active transport of H+ on the presence of anions in synaptic vesicle membranes from rat brain was studied. The H+ transport was measured by monitoring the acidification of the vesicles with a permeant weak base-acridine orange. The fluorescence changes in the latter were proportional to the magnitude of artificially imposed pH gradients (delta pH). The ATP-dependent generation of delta pH was completely dependent on the presence of a permeant anion, was maximal at 150 mM Cl- and was inhibited, when the medium osmolarity was further increased by sucrose or KCl. At 150 mM only Br-, similar to Cl-, behaved as permeant anions, whereas I- was effective only at low (5-20 mM) concentrations. The anions--SCN-, ClO4-, HSO3- and I-(10-20 mM) as well as 4-acetamido-4'-isothiocyanatostilbene-2.2'-disulfonate (K0.5 = 14 microM) blocked the ATP-dependent generation of delta pH observed in the presence of Cl-, while other anions tested (F-, phosphate, bicarbonate, some organic anions) were virtually without effect and did not support the H+ transport. The dependence of the rate and extent of H+ accumulation on Cl- concentration was sigmoidal with a Hill coefficient of 2.8 and a Km value of 85-90 mM. The effects of anions point to the presence in the membrane of synaptic vesicles of an anion (chloride) channel whose conductance can regulate the H+ transport by switching it from an electrogenic to an electroneutral (coupled entry of H+ and Cl-) mode of operation.     

Modulation of coated vesicle chloride channel activity and acidification by reversible protein kinase A-dependent phosphorylation.

We have previously shown that activity of a Cl- channel is required for acidification of clathrin-coated vesicles by the coated vesicle (H+)-ATPase (Arai, H., Pink, S. and Forgac, M. (1989) Biochemistry 28, 3075-3082). We demonstrate that activity of the coated vesicle Cl- channel is modulated by phosphorylation. Cl- conductance was measured in a reconstituted preparation of coated vesicle membrane proteins using the Cl(-)-sensitive fluorescence probe, 6-methoxy-N-(3-sulfopropyl)quinolinium. Treatment of coated vesicle membranes with alkaline phosphatase resulted in a 25 +/- 5% decrease in Cl- channel activity. A parallel decrease in ATP-dependent acidification of coated vesicles was also observed. The decrease in Cl- conductance and ATP-dependent acidification was reversed by treatment with protein kinase A and MgATP; the alkaline phosphatase inhibitor, sodium orthovanadate, blocked the inhibition of acidification. These results indicate that Cl- conductance in coated vesicles is modulated by a protein kinase A-dependent phosphorylation and that this modulation in turn affects ATP-dependent acidification.     

Effects of Monovalent Ions on the Transport of Noradrenaline Across the Plasma Membrane of Neuronal Cells (PC-12 Cells)

The dependence on Na+, K+, and Cl- of uptake and accumulation of [3H]noradrenaline was studied in plasma membrane vesicles isolated from PC-12 pheochromocytoma cells. Plasma membrane vesicles accumulated [3H]noradrenaline when an inward-directed gradient for Na+ and an outward-directed gradient for K+ were imposed across the vesicle membrane. Under these conditions, initial rates of uptake of [3H]noradrenaline were saturable (Km = 0.14 microM) and inhibited by a series of substrates and inhibitors of "uptake". The IC50 values were positively correlated with those for inhibition of uptake into intact PC-12 cells. Uptake and accumulation of [3H]noradrenaline in plasma membrane vesicles were absolutely dependent on external Na+ and Cl-; they were dependent on an inwardly directed gradient for Na+ but less dependent on an inwardly directed gradient for Cl-. Internal K+ strongly enhanced uptake and accumulation of [3H]noradrenaline. Rb+, but not Li+, had the capacity to replace internal K+. Two explanations are proposed for this effect of internal K+: (a) creation of a K+ diffusion potential (inside negative) provides a driving force for inward transport, and/or (b) K+ increases the turnover rate by formation of a highly mobile potassium-carrier complex. A hypothetical scheme for the transport of noradrenaline is presented.     

Partial purification and characterization of an anion-activated ATPase from radish microsomes

Previous investigation showed two distinct ATP-dependent proton-transporting systems in microsomal vesicle from radish seedlings, one inhibited by vanadate and one inhibited by NO-3. On the bases of the effects of these inhibitors we could discriminate two distinct ATPase activities in the same material. The NO-3 sensitive activity was separated from the vanadate-sensitive activity and partially purified by a single-step chromatographic method, which lead to approx 35-fold purification from the microsomes and to a specific activity of 2.3 mumol Pi X min-1 X mg protein-1, at 30 degrees C. The partially purified activity was specific for ATP, some activity being observed toward GTP, and even less toward CTP, UTP and ITP. No significant Pi hydrolysis was found with ADP, AMP, p-nitrophenylphosphate and glucose 6-phosphate. ADP but not AMP was inhibiting in the presence of ATP. The activity was dependent on divalent cations in the order of preference: Mg2+ greater than Mn2+ greater than Co2+ greater than Ca2+ greater than Zn2+. The activity was unaffected by monovalent cations, strongly activated by Cl-, inhibited by 90% by 50 mM NO-3, virtually unaffected by oligomycin and NaN3. At least 90% of the activity was abolished in the presence of each: 10 microM N,N'-dicyclohexylcarbodiimide, 10 microM erythrosin B, 10 mu mersalyl, 100 microM trimethyltin, 100 microM diethylstilbestrol, 100 microM N-ethylmaleimide. No inhibition has been found in the presence of Ca2+, at a concentration blocking the vanadate-sensitive activity. Nigericin, gramicidin and carbonylcyanide p-trifluoromethoxyphenylhydrazone stimulated the activity of this preparation after it was incubated in the presence of sonicated phospholipids, suggesting the capacity of the ATPase to function as a H+-transporting system. All characteristics mentioned were closely similar to those described in the vacuolar ATPases.     

ATP-dependent and DCCD-insensitive Cl- uptake by membrane vesicles from the rat brain plasma membrane fractions

ATP-dependent Cl- uptake by membrane vesicles from the rat brain plasma membrane fractions was not affected by the addition of 40 mM of K+, Na+ or HCO3- to the assay medium. Na+ and K+ did not alter the uptake even in the presence of a K+ ionophore, valinomycin (10 microM), or a H+/K+ exchanger, nigericin (10 microM), whereas in the presence of both of these ionophores, K+, but not Na+, reduced the Cl- uptake. Inhibitors of proton pump activity, N,N'-dicyclohexylcarbodiimide (1 mM) and 5-(N,N-hexamethylene)amiloride (40 microM), however, did not affect the Cl- uptake. These findings suggest the presence of a primary Cl- transport system probably associated with passive H+ flux in the brain plasma membranes.     

Characterization of an electrogenic ATP and chloride-dependent proton translocating pump from rat renal medulla

To study acidification mechanisms in the distal nephron, microsomes were prepared from rat renal medulla by differential centrifugation. Microsomes were enriched in the enzyme marker gamma-glutamyl transferase and contained an ATP-dependent proton pump, as evidenced by ATP-dependent, 3,3',4',5-tetrachlorosalicylanilide-reversible quenching of acridine orange fluorescence. Acidification was vanadate-insensitive, but was completely inhibited by micromolar N-ethylmaleimide. Maximal acidification was achieved in the presence of halide (Cl-, Br-) only and was not attainable with potassium-valinomycin diffusion potentials without halide ion. Microsomal ATPase activity was neither chloride- nor N-ethylmaleimide-sensitive. A chloride conductance was observed only with vesicles which had undergone ATP-dependent acidification. An ATP-dependent, N-ethylmaleimide-inhibitable, 3,3',4',5-tetrachlorosalicylanilide-reversible, and chloride-attenuated quench of bis(1,3-dibutylbarbituric acid-(5] pentamethinoxonol fluorescence was seen, consistent with net transfer of positive charge into the vesicles. Nonetheless, positive intravesicular potentials increased the ATP-dependent initial acidification rate, perhaps by increasing availability of chloride ion to the transport site. Our results are consistent with an electrogenic, ATP-dependent proton pump regulated by a voltage-sensitive chloride site.     

A sensitive technique for the determination of anion exchange activities in brush-border membrane vesicles. Evidence for two exchangers with different affinities for HCO3- and SITS in rat intestinal epithelium

A large percentage (up to 70%) of 36Cl- influx in brush-border membrane vesicles from rat small intestine under equilibrium exchange conditions was found to be mediated by SITS-inhibitable anion exchange. This Cl-/anion exchange could be measured 10-15-times more sensitive by determining the uptake of trace amounts of 125I- driven by a large Cl- gradient (in greater than out) generated by passing the vesicles through an anion-exchange column. Voltage clamping of the vesicle membrane with K+ and valinomycin did not effect the chloride driven 125I- uptake, showing that the 'overshooting' I- uptake was not mediated by an electrical diffusion potential, as might be generated by the Cl- gradient in the presence of a chloride channel. The Cl-/anion exchange was further characterized in brush-border membrane vesicles from both rat ileum and jejunum by studying the inhibitory action of various anions on the Cl- driven I- uptake. NO3-, Cl-, SCN- and formate at 2 mM could inhibit Cl-/I- exchange for more than 80%. The ileal brush-border membrane vesicles displayed a clear heterogeneity with respect to the inhibitory action of SO2-(4), SITS and HCO-3 on Cl-/I- exchange. Approximately 30% of the Cl-/I- exchange was insensitive to SO2-(4) and showed a relatively low sensitivity to SITS (IC50 = 1 mM) but could be inhibited for 80% by 2 mM HCO-3. Presumably this component represents Cl-/OH- or Cl-/HCO-3 exchange. The residual 70% showed a high sensitivity to SO2-(4) (IC50 = 0.5 mM) and SITS (IC50 = 2.5 microM) but was less sensitive to HCO-3. This part of the exchange activity showed inhibition characteristics very similar to the Cl-/I- exchange in the jejunal vesicles. The latter process was also inhibited for 80% by 2 mM oxalate. As discussed in this paper both exchangers may be involved in the electroneutral transport of NaCl across the apical membrane of the small intestinal villus cell.     

Inhibition by local anesthetics and barbiturates of the active transport of H+ in the synaptic vesicle membranes of the brain

The effects of local anesthetics and barbiturates on the ATP-dependent H+ transport in synaptic vesicle membranes from rat brain were studied using a fluorescent probe, acridine orange. Local anesthetics depressed the active H+ transport with the following order of potencies: tetracaine trimecaine lidocaine procaine. Respective IC50 values were 0.07, 0.28, 0.46 and 0.60 mM. The local anesthetics also disrupted the endogenous pH gradient seen in the absence of ATP. Barbiturates inhibited the active H+ transport showing IC50 values in the range of 2-5 mM except for benzobarbital and barbital characterized by IC50 values of 0.5 and 20 mM, respectively. The order of potencies was benzobarbital hexobarbital amobarbital pentobarbital phenobarbital barbital. The endogenous pH gradient was not affected by the barbiturates. The results show that local anesthetics disrupt the H+ transport by acting as permeable weak bases (uncouplers) whereas barbiturates are likely to block and anion channel which maintains electroneutrality of the H+ transport in the membrane of synaptic vesicles.     

Magnesium permeability of sarcoplasmic reticulum. Mg2+ is not countertransported during ATP-dependent Ca2+ uptake by sarcoplasmic reticulum

Magnesium transport across sarcoplasmic reticulum (SR) vesicles was investigated in reaction mixtures of various composition using antipyrylazo III or arsenazo I to monitor extravesicular free Mg2+. The half-time of passive Mg2+ efflux from Mg2+-loaded SR was 100 s in 100 mM KCl, 150 S in 100 mM K gluconate, and 370 S in either 100 mM Tris methanesulfonate or 200 mM sucrose solutions. The concentration and time course of Mg2+ released into the medium was also measured during ATP-dependent Ca2+ uptake by SR. In reaction mixtures containing up to 3 mM Mg2+, small changes in free magnesium of 10 microM or less were accurately detected without interference from changes in free Ca2+ of up to 100 microM. Three experimental protocols were used to determine whether the increase of free [Mg2+] in the medium after an addition of ATP was due to Mg2+ dissociated from ATP following ATP hydrolysis or to Mg2+ translocation from inside to outside of the vesicles. 1) In the presence of ATP-regenerating systems which maintained constant ATP to ADP ratios and normal rates of active Ca2+ uptake, the increase of Mg2+ in the medium was negligible. 2) Mg2+ released during ATP-dependent Ca2+ uptake by SR was similar to that observed during ATP hydrolysis catalyzed by apyrase, in the absence of SR. 3) In SR lysed with Triton X-100 such that Ca2+ transport was uncoupled from ATPase activity, the rate and amount of Mg2+ release was greater than that observed during ATP-dependent Ca2+ uptake by intact vesicles. Taken together, the results indicate that passive fluxes of Mg2+ across SR membranes are 10 times faster than those of Ca2+ and that Mg2+ is not counter-transported during active Ca2+ accumulation by SR even in reaction mixtures containing minimal concentrations of membrane permeable ions that could be rapidly exchanged or cotransported with Ca2+ (e.g. K+ or Cl-).     

ATP-dependent Ca2+ transport in the rat parotid basolateral plasma membrane is regulated by calmodulin

Calmodulin regulation of ATP-dependent Ca2+ transport activity was assessed in inverted basolateral plasma membrane vesicles (BLMV) isolated from rat parotid glands. The initial rate of Ca2+ transport in media containing 100 nM Ca2+ was stimulated by approximately 60% at maximal concentrations (300 nM) of exogenously added calmodulin (CAM). Half-maximal activation was obtained at 50 and 175 nM CAM in KCl and mannitol containing assay media, respectively. In the KCl medium, addition of 300 nM CAM increased the affinity of the BLMV Ca2+ transport activity for Ca2+ from approximately 70 nM, in the absence of added CAM, to approximately 50 nM. Vmax was consistently increased by approximately 20% under these conditions. When BLMV were treated with ethylene glycol bis(beta-aminoethylether) N,N'-tetraacetic acid (EGTA) (200 microM), the affinity of the transporter for Ca2+ decreased by 50% to approximately 150 nM, with no change in Vmax. When CAM was added to the EGTA-treated membranes, Ca2+ transport activity was comparable to that obtained when CAM was added directly to control, untreated BLMV. The CAM antagonists, trifluoperazine (TFP), W-7, and calmidazolium, inhibited Ca2+ transport in the presence of CAM. Half-maximal inhibition of transport was achieved by 12 microM TFP and 20 microM W-7. Calmidazolium (1 microM) inhibited Ca2+ transport by 75%. The inhibitory effects on ATP-dependent Ca2+ transport exerted by these agents were not due to an increase in the passive permeability of the membranes to Ca2+. Furthermore, in the absence of added CAM, the inhibitory effects of these agents on initial Ca2+ transport rate was decreased. The data presented suggest that the Ca2+-dependent interaction of CAM with the ATP-dependent Ca2+ transporter in rat parotid BLMV modifies the kinetic properties of this Ca2+ transporting mechanism.     

Biochemical characterization of the Na+/K+/Cl- co-transport in chick cardiac cells

Cultured chick cardiac cells possess a Na+K+Cl-co-transport system that is inhibited by the "loop diuretics" benzmetanide (IC50 = 0.3 microM), bumetanide (IC50 = 0.6 microM), piretanide (IC50 = 1.5 microM) and furosemide (IC50 = 5 microM). The K0.5 values for Cl- and Na+ activation of the bumetanide-sensitive 86Rb+ uptake are 59 mM and 40mM respectively. Bumetanide also inhibits a 22Na+ uptake component that is suppressed when external Cl- or K+ are substituted by impermeant ions. The ratio of bumetanide-sensitive 86Rb+ to 22Na+ uptake is close to 1. The cardiac Na+/K+/Cl- cotransport is a major uptake pathway for Na+ and K+. It accounts for 50% of the initial rate of 86Rb+ uptake and 17% of the initial rate of 22Na+ uptake by chick cardiac cells. It is activated two-fold by an hyperosmotic shock produced with 200 mM mannitol.     

Interaction of t-Butylbicyclophosphorothionate with γ-Aminobutyric Acid-Gated Chloride Channels in Cultured Cerebral Neurons

The role of t-butylbicyclophosphorothionate (TBPS) as an antagonist of gamma-aminobutyric acid (GABA) was studied with primary cultures of neurons from the chick embryo cerebrum. The addition of GABA stimulated the uptake of 36Cl- by neurons and the dose dependence of this effect followed hyperbolic kinetics with a K0.5 = 1.3 microM for GABA. TBPS proved to be a potent inhibitor of GABA-dependent Cl- uptake (IC50 = 0.30 microM). Analysis of the kinetics of this process revealed that TBPS is a noncompetitive inhibitor (Ki = 0.15 microM) with respect to GABA. Scatchard analysis of direct binding of [35S]TBPS to membranes isolated from neuronal cultures gave curvilinear plots. These could be resolved by nonlinear regression methods into two components with KD values of 3.1 nM and 270 nM. The TBPS binding constant for this lower affinity site agreed well with the IC50 and Ki values for inhibition of Cl- flux, suggesting that this site is physiologically relevant to GABA antagonism. GABA was a noncompetitive displacer of [35S]TBPS binding to the lower affinity site. The Ki value for this displacement by GABA (1.7 microM) was comparable to the value for GABA enhancement of Cl- flux. The binding of [35S]TBPS to its low-affinity site on neuronal membranes was ninefold higher in the presence of Cl- than with gluconate, an impermeant anion. The rank order for anion stimulation of [35S]TBPS binding was Br- greater than or equal to SCN- greater than Cl- greater than or equal to NO3- greater than I- greater than F- greater than gluconate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation Characteristics of Brain Clathrin-Coated Vesicle Endogenous Proteins

Clathrin-coated vesicles purified from bovine brain express protein kinase activity on two principal endogenous vesicle-associated substrates: a 50,000-Mr polypeptide (pp50) and clathrin-associated protein2 (CAP2; the faster-migrating clathrin light chain). Various exogenous substrates, e.g., casein, phosvitin, histone II, and histone III, also are phosphorylated. The pp50 protein kinase activity of clathrin-coated vesicles is not modulated by Ca2+, calmodulin, phosphatidylserine, or cyclic AMP. On the other hand, phosphorylation of the other endogenous substrates requires certain activators, including histone, polylysine, polyarginine, or polyethylenimine. Phosphate incorporation into pp50 was sensitive to divalent cations that inhibit sulfhydryl-dependent enzymes in the following order of potency: Zn2+ greater than Hg2+ greater than Cd2+, Cu2+, and Pb2+. Phosphate incorporation into CAP2 with polylysine present was insensitive to divalent cations. The alkylating agents dithiodinitrobenzene, phenacyl bromide, and N-ethylmaleimide inhibited phosphate incorporation into pp50 up to 90% without affecting incorporation into the other substrates. Vanadium pentoxide inhibited phosphorylation of CAP2 but had a minimal effect on pp50. CAP2 kinase activity was separated from the coated vesicle membrane and from dis-assembled clathrin triskelions, coeluting with the assembly polypeptide complex on a Sepharose 4B column. It retained phosphorylation properties similar to those of intact vesicles. These data imply that clathrin-coated vesicle kinases are elements of the coat proteins and may be involved in the assembly/disassembly of clathrin triskelions or interactions of coated vesicles with other cellular components.     

Effects of N-ethylmaleimide on ouabain-insensitive cation fluxes in human red cell ghosts

In red cells of several species, the sulfhydryl reagent N-ethylmaleimide activates a Cl- -dependent, ouabain-resistant K+ transport pathway. Here we report our attempts to demonstrate ouabain-resistant Cl- -dependent K+ fluxes stimulated by N-ethylmaleimide in resealed human red cell ghosts using Rb+ as a K+ analogue. In contrast to intact cells, the rate constants of the base level Rb+ efflux in ghosts were similar in NaNO3 and NaCl (okRb = 0.535 +/- 0.079 h-1 and 0.534 +/- 0.085 h-1, respectively), while 1 mM N-ethylmaleimide stimulated Rb+ efflux strongly in NaNO3 (okRb = 14.26 +/- 1.32 h-1) and moderately in NaCl (okRb = 2.73 +/- 0.54 h-1). This effect was dependent on the presence of internal ATP. Stimulation of Rb+ efflux was observed in the presence of greater than or equal to 0.2 mM N-ethylmaleimide and increased at pH values approaching 8.0, consistent with titration of SH groups. N-Ethylmaleimide-stimulated Rb+ efflux was approx. 50% inhibited by 100 microM quinine sulfate whereas 1 microM bumetanide had no effect. In NaCl the N-ethylmaleimide-stimulated efflux saturated with initial internal ghost Rb+ concentration, but rates increased linearly in NaNO3. Replacement of external Na+ with glucamine or choline decreased the N-ethylmaleimide-stimulated Rb+ efflux, suggesting a role for external Na+. N-Ethylmaleimide-stimulated Rb+ efflux was greater in buffers with lipophilic anions such as SCN- or NO3- than in solutions with Cl- or acetate. However, the cation selectivity of the pathway studied was low, as Li+ efflux was also stimulated by N-ethylmaleimide. We conclude that the effect of N-ethylmaleimide on ouabain-resistant cation effluxes of human red cell ghosts is very different from the selective action of N-ethylmaleimide on Rb+ influxes in intact red cells.     

Characterisation of the effects of anthranilic and (indanyloxy) acetic acid derivatives on chloride transport in membrane vesicles.

The effects of the Cl- channel blockers, NPPB, IAA94/95 and a number of related compounds on 36Cl- transport in membrane vesicles from bovine kidney cortex and rabbit ileum mucosa brush borders have been studied. These vesicles have been previously shown to be enriched in Cl- channel and Cl-/anion cotransport activity, respectively. Chloride transport was assayed in both types of vesicles by measuring the uptake of 36Cl- in response to an outwardly-directed Cl- concentration gradient. In kidney microsomes, a large proportion of the observed 36Cl- uptake was mediated by an electrogenic uniport and could be substantially reduced by clamping the membrane potential at zero mV using K+ and valinomycin. Chloride uptake was inhibited by both NPPB and IAA94/95 with apparent IC50 values of around 10 microM under optimal conditions (i.e., 4 min uptake at 4 degrees C). Under other conditions (e.g., 10 min uptake at 25 degrees C), where uptake had reached a steady-state level, much higher concentrations of inhibitor were required to cause inhibition. Therefore, previous differences in the reported potency of these compounds may, in part, have been due to the conditions under which Cl- uptake was measured. In addition, both NPPB and, to a lesser extent, IAA94/95 were found to have other effects on the vesicles, in that, when added at a concentration of 100 microM, they induced a leakage of pre-accumulated 36Cl-. This was probably caused by either dissipation of membrane potential or damage to the vesicle membranes. The sulphonic acid derivatives of NPPB and IAA94/95 (NPPB-S and ISA94/95, respectively) blocked 36Cl- uptake with around the same potency as NPPB and IAA94/95, but did not cause any non-specific Cl- leakage, when added at concentrations up to 100 microM. Inhibition of 36Cl- uptake by all four compounds was almost completely reversible. However, when vesicles were incubated with the inhibitors in the presence of an outward Cl- concentration gradient, or if vesicles were freeze/thawed in the presence of the compounds, inhibition could be only partially reversed. In rabbit brush border membrane vesicles, 36Cl- uptake was not reduced when the vesicles were voltage clamped using valinomycin and K+, and was therefore probably mediated by Cl-/Cl- exchange. However, despite the lack of effect of valinomycin, 36Cl- uptake was inhibited by both NPPB (approx. 80% inhibition at 100 microM) and, to a lesser extent, by IAA94/95 (approx. 30% inhibition at 100 microM).(ABSTRACT TRUNCATED AT 400 WORDS)