Vanadate inhibition of ATP-dependent H+ transport in membrane vesicles from turtle bladder epithelial cells

The ATP-dependent proton transport into vesicles of a mixed membrane fraction obtained from turtle bladder epithelial cells consists of at least two kinetically defined moieties: one, which is maximally inhibited by 25% with nanomolar levels of vanadate, but not inhibited at all with equimolar levels of N-ethylmaleimide, and another, which is maximally inhibited by 70% with micromolar levels of N-ethylmaleimide and by 25% with equimolar levels of vanadate. In contrast to the transport function, the associated enzymatic function (the ouabain-resistant ATPase activity) in these membranes, not inhibited by nanomolar levels of vanadate or N-ethylmaleimide, is maximally inhibited by 40% with micromolar levels of vanadate and by 13% with equimolar levels of N-ethylmaleimide. Independent of these kinetic differences between the enzyme and the transport functions, membranes containing the N-ethylmaleimide-sensitive proton transport function are electrophoretically separable from those containing the vanadate-sensitive transport function. For example, the kinetically defined, vanadate-sensitive proton transport function is recovered exclusively and kinetically identified in one of four electrophoretic membrane fractions, EF-II; while the N-ethylmaleimide-sensitive function is recovered in EF-III as well as in EF-II. Membranes of EF-IV, maximally enriched in ouabain-resistant ATPase activity, possess no proton transport function at all, even in the absence of N-ethylmaleimide or vanadate. Additional data under in vivo as well as under in vitro conditions are required to prove that the vanadate-sensitive proton transport in these vesicles is an in vitro manifestation of the mechanism responsible for generating the vanadate-sensitive luminal acidification process under in vivo conditions in the intact turtle bladder.     

Metabolic pathways coupled to H+ transport in turtle urinary bladder

Summary Active H⁺ transport in the turtle urinary bladder is mediated by an ATPase. Although the source of ATP is usually mitochondrial oxidative phosphorylation, it is possible because of intracellular compartmentalization or cellular heterogeneity that one metabolic pathway exclusively provides ATP to the pump. To examine this we performed several types of experiments. In one, the coupling between the rate of transport and the rate of oxidation of¹⁴C-labeled substrates was studied. We found that there was coupling between H⁺ transport and glucose, butyrate, oleate, and -OH-butyrate oxidation. In another set of experiments we depleted turtle bladders of their endogenous substrates and tested the effect of a number of substrates on the rate of transport. We found that glucose, pyruvate, lactate, actetate, butyrate and -OH butyrate all stimulated H⁺ transport. In a third set of experiments we found no coupling between H⁺ transport and lactate production. Finally, we found that reduction of H⁺ transport by mucosal acidification resulted in an increase in epithelial cell ATP concentrations and a decrease in ADP levels.These results suggest that the H⁺ pump receives its ATP from carbohydrate and fatty acid oxidation. The changes in ATP and ADP levels provide an initial explanation for the coupling of H⁺ transport to the rate of cellular oxidative metabolism.     

Active H+ transport in the turtle urinary bladder. Coupling of transport to glucose oxidation

The turtle urinary bladder acidifies the contents of its lumen by actively transporting protons. H+ secretion by the isolated bladder was measured simultaneously with the rate of 14CO2 evolution from [14C]glucose. The application of an adverse pH gradient resulted in a decline in the rate of H+ secretion (JH) and in the rate of glucose oxidation (JCO2). The changes in JH and JCO2 were linear functions of the pH difference across the membrane. Hence, JH and JCO2 were linearly related to each other. The slope, deltaJH/deltaJCO2 was found to be similar in half-bladders from the same animal but was seen to vary widely in a population of turtles. To investigate the effect of pH gradients on deltaJH/deltaJCO2, two experiments were performed in each of 14 hemibladders. In one, JH and JCO2 were altered by changing the luminal pH. In the other, they were altered by changing the ambient pCO2 while the luminal pH was kept constant. The average slope, deltaJH/deltaJCO2, in the presence of pH gradients was 14.45 eq-mol-1. In the absence of gradients in the same hemibladders it was 14.72, delta = 0.27 +/- 1.46. The results show that H+ transport is organized in such a way that leaks to protons in parallel to the pump are negligible. Analysis of the transport system by use of the Essig-Caplan linear irreversible thermodynamic formalism shows that the system is tightly coupled. The degree of coupling, q, given by that analysis was measured and found to be at or very near the maximum theoretical value.     

Coupling between H+ transport and anaerobic glycolysis in turtle urinary bladder: Effects of inhibitors of H+ ATPase

Summary The coupling between H⁺ transport (J _H) and anaerobic glycolysis was examinedin vitro in an anaerobic preparation of turtle urinary bladder.J _H was measured as the short-circuit current after Na⁺ transport was abolished with ouabain and by pH stat titration. The media were gassed with N₂ and 1% CO₂ (PO₂<0.5 mm Hg) and contained 10mm glucose. Under these conditions,J _H was not inhibited by 3mm serosal (S) cyanide or by 0.1mm mucosal (M) dinitrophenol. Control anerobic lactate production (J _lac) of 47 bladders was plotted as a function of simultaneously measuredJ _H. The slope ofJ _lac onJ _H was 0.58±0.12 with an intercept forJ _lac atJ _H=0 of 0.55 mol/hr. Values for J _lac/J _H were determined in groups of individual bladders whenJ _H was inhibited by an opposing pH gradient (0.55±0.16), by acetazolamide (0.58±0.19) and by dicyclohexylcarbodiimide, DCCD (0.58±0.14). The constancy of J _lac/J _H indicates a high degree of coupling betweenJ _H andJ _lac. Since the anaerobic metabolism of glucose produces one ATP for each lactate formed, the J _lac/J _H values can be used to estimate the stoichiometry of H⁺ translocation. The movement of slightly less than 2 H⁺ ions is coupled to the hydrolysis of one ATP. During anaerobiosis (absence of mitochondrial ATPase function) the acidification pump was not inhibited byM addition of oligomycin but was inhibited byM addition of DCCD and Dio-9, inhibitors of H⁺ flow in the proteolipid portion of H⁺-translocating ATPases. DCCD inhibited anaerobicJ _H without change in J _lac/J _H or basalJ _lac and, therefore, acted primarily on the H⁺ pump.S addition of vanadate also inhibitedJ _H, but the inhibition was associated with an increase inJ _lac. The site of this apparent uncoupling remains to be defined. The acidification pump of the luminal cell membrane of the turtle bladder has H⁺-ATPase characteristics that differ from mitochondrial ATPase in that H⁺ transport is oligomycin-resistant and vanadate-sensitive. As judged from the flows of H⁺ and lactate, the H⁺/ATP stoichiometry of the pump is about 2.     

Calmodulin-sensitive ATP-dependent Ca2+ transport across adipocyte plasma membranes

An ATP-dependent transport system which is active at concentrations of free Ca2+ in the submicromolar range has been identified in adipocyte plasma membranes. The system appears to represent the functional component of the high affinity insulin-sensitive calcium-stimulated, magnesium-dependent adenosine triphosphatase preveiously described in the same preparation (Pershadsingh, H. A., and McDonald, J. M. (1979) Nature 281, 495-497). This ATP-dependent Ca2+ transport pump was stimulated approximately 3-fold by the Ca2+-dependent regulatory protein, calmodulin. This effect was confined to the plasma membrane since a similar effect was undetectable in the fraction enriched in endoplasmic reticulum. Calmodulin stimulation was dose-dependent but saturable with half-maximal activation occurring at 0.72 microgram/ml (43 nM). Calmodulin appeared to stimulate the system primarily by decreasing the apparent half-maximal saturation constant for free Ca2+ from 0.20 +/- 0.04 microM to 0.07 +/- 0.01 microM (n = 3). The Hill coefficient increased from 1.6 +/- 0.2 to 3.2 +/- 0.6 (n = 3), thus showing an increased positive cooperativity which allows the pump to be activated by an exceedingly narrow Ca2+ threshold in the presence of calmodulin. The calmodulin stimulation of the plasma membrane Ca2+ extrusion pump in adipocytes, working in opposition to metabolic signals which increase cytoplasmic Ca2+, could constitute a self-regulating negative feedback device for maintaining a low steady state level of intracellular Ca2+. This feedback system may be of critical importance in regulation of cellular metabolism by insulin.     

A plant lipid and the platelet-activating factor stimulate ATP-dependent H+ transport in isolated plant membrane vesicles

A plant lipid was isolated from zucchini (Cucurbita pepo L.) membranes and from soybean (Glycine max [L.] Merr) phospholipids by thinlayer chromatography and further purified by high-performance liquid chromatography. This plant lipid was chromatographically very similar to the platelet-activating factor, an ether phospho-lipid with hormone-like properties found in mammals. Both the plant lipid and the platelet-activating factor stimulated ATP-dependent H⁺ transport in isolated membrane vesicles from zucchini hypocotyls.Abbreviations HPLC high-performance liquid chromatography - PAF platelet-activating factor     

ATP-dependent H+ transport in synaptosomal membrane vesicles of the rat brain

H+ transport into synaptosomal membrane vesicles of the rat brain was stimulated by ATP and to a lesser extent by GTP, but not by ITP, CTP, UTP, ADP, AMP or beta, gamma-methylene ATP. ATP at concentrations up to 200 mM concentration-dependently stimulated the rate of H+ transport with a Km value of 0.6 mM, but at higher concentrations of this nucleotide the rate decreased. Other nucleotides such as CTP, UTP, GTP and AMP, or products of ATP hydrolysis i.e. ADP and Pi also reduced the ATP-stimulated H+ transport. The inhibition by GTP and ADP was not affected by the ATP concentration. These findings suggest that plasma membranes of nerve endings transport H+ from inside to outside of the cells utilizing energy from ATP hydrolysis, and that this transport is regulated by the intracellular concentration of nucleotides and Pi on sites other than those involved in substrate binding.     

Effects of valinomycin on vanadate-sensitive and vanadate-resistant H+ transport in vesicles from turtle bladder epithelium: evidence for a K+/H+ exchanger

The vanadate-sensitive component of the ATP-dependent H+ gradient formed in isolated vesicles from a urinary epithelium was abolished by valinomycin omission. This suggests that vanadate-sensitive H+ transport has an absolute requirement for intravesicular K+ and that the transport may be due to a K+/H+ exchanger. Sensitivity to the inhibitor SCH28080 supports this conclusion. On the other hand, valinomycin affects the initial velocity of vanadate-resistant transport without altering its maximum gradient. This is consistent with the development of a membrane potential consequent to electrogenic uniport H+ transport.     

Calmodulin-induced activation of ATP-dependent Ca2+ transport in plasma membranes of the myometrium

Calmodulin activates the ATP-dependent transport of Ca2+. The V0 value for this reaction in the absence of calmodulin is 0.82, that in the presence of 10(-7) M calmodulin is 5 times as high, i. e. 4.5 nmol 45Ca2+/mg protein/min. The Vmax value in the absence of calmodulin is 2.07, that with the activator is 4.33 nmol 45Ca2+/mg protein/min. The corresponding Km values are 0.75 X 10(-6) M and 0.66 X 10(-7) M, respectively, i. e., the affinity of the Ca-pump for Ca2+ increases. The half-maximum Ca-binding activity of calmodulin measured with a help of the fluorescent probe, N-phenyl-1-naphthylamine (PNA), is observed at 5 X 10(-7) M Ca2+. Mg2+ (3 mM) decreases 10-fold the Ca-binding affinity. No significant effect of ATP on the Ca-binding properties of calmodulin was found; the Hill coefficient is suggestive of a positive cooperativity of this reaction. A comparison of dependences of the calmodulin-stimulated component of ATP-dependent transport of Ca2+ in myometrium plasma membranes and of the Ca-binding activity of calmodulin measured with a help of PNA suggests that the effect of calmodulin on the affinity of the Ca-pump for Ca2+ can also be realized when some (but not all) Ca-binding sites in the calmodulin molecule are saturated with Ca2+.     

Inhibition by orthovanadate of ATP-dependent Ca2+ transport in microsomes isolated from rat liver

A technique employing sucrose-density centrifugation for the enrichment of rat liver microsomes and rat liver plasma membranes in separate subcellular fractions is described. The fractions are enriched in glucose 6-phosphatase and 5'-nucleotidase, respectively, and are free of cytochrome oxidase activity. Vanadate-sensitive Ca2+ transport activity (half-maximal inhibition at approximately 10 microM vanadate, corresponding to approximately 12 nmol/mg of protein) was detected in only that fraction enriched in microsomal membranes. Inhibition by vanadate of ATP-dependent Ca2+ transport is noncompetitive with respect to added Ca2+ but competitive with respect to added ATP. Because it inhibits ATP-dependent Ca2+ transport in rat liver microsomes but not in rat liver plasma membranes, vanadate becomes a useful tool to distinguish in vitro between these two transport systems.     

Modulation of H+ transport mechanisms by interleukin-1 in isolated bovine articular chondrocytes.

The proinflammatory cytokine interleukin-1 (IL-1) promotes the degradation of articular cartilage by inhibiting matrix synthesis and stimulating degradative enzyme activity. Generation of nitric oxide (NO) in response to IL-1 is implicated in these actions. The catabolic actions of IL-1 can be inhibited by manoeuvres which are predicted to dissipate H+ gradients across the chondrocyte plasma membrane. In the present study, the effects of IL-1 on H+ extrusion from bovine articular chondrocytes were investigated. pH was measured using the H+-sensitive fluorescent dye BCECF. Cells were acidified by ammonium rebound and the contribution of the Na+-H+ exchanger (NHE) and of the vacuolar H+-ATPase to acid extrusion was characterised by ion substitution and inhibitor studies. Overnight (18 h) exposure to IL-1 stimulated acid extrusion in a dose-dependent fashion. This effect represented stimulation of both NHE and the ATPase. Characterisation of the timecourse of this response indicated that, while stimulation of acid extrusion was rapid, effects on the ATPase were only apparent after greater than 8h incubation with the cytokine. In keeping with this observation, the protein synthesis inhibitor cycloheximide abolished the stimulatory effect of IL-1 on ATPase-mediated extrusion. The upregulation of ATPase activity by IL-1 was inhibited by the NOS inhibitor L-NAME and by the NO scavenger PTIO. In cells which had not been exposed to IL-1, treatment with the NO donor SNAP also stimulated acid extrusion by the ATPase. In contrast, NHE activity was not altered by any of these compounds. Taken together, these results imply that IL-1 can stimulate acid extrusion in chondrocytes and that this reflects rapid upregulation of NHE with slower induction of H+-ATPase activity which requires elevated levels of NO. While ATPase induction involves protein synthesis, this process may not constitute synthesis of ATPase proteins per se, but rather of some associated regulatory process.     

Serosal Na/Ca exchange and H+ and Na+ transport by the turtle and toad bladders

Summary A Na/Ca exchange system has been described in the plasma membrane of several tissues and seems to regulate the concentration of calcium in cytosol. Replacement of extracellular Na by sucrose increases calcium uptake into and decreases calcium efflux from the cell, leading to an increase in cytosolic calcium. The effect of an increase in cytosolic calcium mediated by the Na/Ca exchange system on H⁺ and Na transport in the turtle and toad bladder was investigated by replacing serosal Na isosmotically by sucrose or choline. Replacement of serosal by sucrose was associated with a significant inhibition of H⁺ secretion or Na transport which was reversible by addition of NaCl. Replacement of mucosal Na by sucrose failed to alter H⁺ secretion. Removal of serosal Na was associated with a significant increase in⁴⁵Ca uptake which could be blocked by pretreatment with lanthanum chloride. Pretreatment with lanthanum chloride blunted the inhibitory effect of replacement of serosal Na by sucrose on H⁺ and Na transport, thus suggesting that the increase in calcium uptake and the inhibition of transport are causally related. Under anaerobic conditions the rate of H⁺ or Na transport are linked to the rate of lactate production. The inhibition of Na or H⁺ transport by removal of serosal Na was accompanied by a proportional decrease in lactate production, thus suggesting that an increase in cytosolic calcium does not inhibit transport by uncoupling glycolysis from transport. Replacement of serosal Na by sucrose did not alter the force of the H⁺ or Na pump but led to an increase in resistance of the active pathway of H⁺ and Na transport. The inhibition of Na transport by replacement of serosal Na with sucrose could be reversed by addition of amphotericin B, an agent which increases luminal permeability to Na, thus suggesting that decreased Na entry across the apical membrane is the mechanism responsible for the inhibition of Na transport. The results of the present studies strongly suggest that an increase in cytosolic calcium through the serosal Na/Ca exchange system inhibits H⁺ and Na transport in the turtle and toad bladder probably by increasing the resistance of the luminal membrane.     

Active sodium transport by the isolated toad bladder

Studies were made of the active ion transport by the isolated urinary bladder of the European toad, Bufo bufo, and the large American toad, Bufo marinus. The urinary bladder of the toad is a thin membrane consisting of a single layer of mucosal cells supported on a small amount of connective tissue. The bladder exhibits a characteristic transmembrane potential with the serosal surface electrically positive to the mucosal surface. Active sodium transport was demonstrated by the isolated bladder under both aerobic and anaerobic conditions. Aerobically the mean net sodium flux across the bladder wall measured with radioactive isotopes, Na²⁴ and Na²², just equalled the simultaneous short-circuit current in 42 periods each of 1 hour's duration. The electrical phenomenon exhibited by the isolated membrane was thus quantitatively accounted for solely by active transport of sodium. Anaerobically the mean net sodium flux was found to be slightly less than the short-circuit current in 21 periods of observation. The cause of this discrepancy is not known. The short-circuit current of the isolated toad bladder was regularly stimulated with pure oxytocin and vasopressin when applied to the serosal surface under aerobic and anaerobic conditions. Adrenaline failed to stimulate the short-circuit current of the toad bladder.     

ATP-dependent Na+ transport in cardiac sarcolemmal vesicles

Although the enzyme (Na+ + K+)-ATPase has been extensively characterized, few studies of its major role, ATP-dependent Na+ pumping, have been reported in vesicular preparations. This is because it is extremely difficult to determine fluxes of isotopic Na+ accurately in most isolated membrane systems. Using highly purified cardiac sarcolemmal vesicles, we have developed a new technique to detect relative rates of ATP-dependent Na+ transport sensitively. This technique relies on the presence of Na+-Ca2+ exchange and ATP-driven Na+ pump activities on the same inside-out sarcolemmal vesicles. ATP-dependent Na+ uptake is monitored by a subsequent Nai+-dependent Ca2+ uptake reaction (Na+-Ca2+ exchange) using 45Ca2+. We present evidence that the Na+-Ca2+ exchange will be linearly related to the prior active Na+ uptake. Although this method is indirect, it is much more sensitive than a direct approach using Na+ isotopes. Applying this method, we measure cardiac ATP-dependent Na+ transport and (Na+ + K+)-ATPase activities in identical ionic media. We find that the (Na+ + K+)-ATPase and the Na+ pump have identical dependencies on both Na+ and ATP. The dependence on [Na+] is sigmoidal, with a Hill coefficient of 2.8. Na+ pumping is half-maximal at [Na+] = 9 mM. The Km for ATP is 0.21 mM. ADP competitively inhibits ATP-dependent Na+ pumping. This approach should allow other new investigations on ATP-dependent Na+ transport across cardiac sarcolemma.     

ATP-dependent CA2+ transport in endoplasmic reticulum isolated from roots ofLepidium sativum L.

Endoplasmic reticulum membranes were isolated from roots of garden cress (Lepidium sativum L. cv Krause) using differential and discontinuous sucrose gradient centrifugation. The endoplasmic reticulum fraction was 80% rough endoplasmic reticulum oriented with the cytoplasmic surface directed outward and contaminated with 12% unidentified smooth membranes and 8% mitochondria. Marker enzyme analysis showed that the activity for endoplasmic reticulum was enriched 2.4-fold over total membrane activity while no other organelle activity showed an enrichment. All evidence indicated that the fraction was composed of highly enriched endoplasmic reticulum membranes. Ca²⁺ uptake activity was measured using the filter technique described by Gross and Marmé (1978). The results of these experiments showed an ATP-dependent, oxalate-stimulated Ca²⁺ uptake into vesicles of the endoplasmic reticulum fraction. The majority of the transport activity was microsomal since specific inhibitors of mitochondrial Ca²⁺ transport (ruthenium red, LaCl₃ and oligomycin) inhibited the activity by only 25%. Sodium azide showed no inhibition. The transport was likely directly coupled to ATP hydrolysis since there was no inhibition with carbonylcyanidem-chlorophenylhydrazone. The transport activity was specific for ATP showing only 36% and 29% of the activity with inosine diphosphate and guanosine 5′-triphosphate, respectively. The results indicate a Ca²⁺ transport function located on the endoplasmic reciculum of garden cress roots.     

Control of active proton transport in turtle urinary bladder by cell pH

The rate of active H+ secretion (JH) across the luminal cell membrane of the turtle bladder decreases linearly with the chemical (delta pH) or electrical potential gradient (delta psi) against which secretion occurs. To examine the control of JH from the cell side of the pump, acid-base changes were imposed on the cellular compartment by increasing serosal[HCO3-] at constant PCO2 or by varying PCO2 at constant [HCO3-]. When serosal [HCO3-] was increased from 0 to 60 mM, cell [H+] decreased, as estimated by the 5,5-dimethyloxazoladine-2,4- dione method. JH was a saturable function of cell [H+], with an apparent Km of 25 nM. When PCO2 was varied between 1 and 20% at various serosal Km of 25 nM. When PCO2 was varied between 1 and 20% at various serosal [HCO3-], the PCO2 required to reach a maximal JH increased with [HCO3-] so that JH was a function of cell [H+] rather than of cell [HCO3-] or CO2. The proton pump was controlled asymmetrically with respect to the pH component of the electrochemical potential for protons, microH. On the cell side of the pump, a delta pH of < 1 U was required to vary JH between maximal and zero values, whereas on the luminal side a delta pH of 3 U was required. Cell [H+] regulates JH by determining the availability of H+ to the pump in a relationship resembling Michaelis-Menten kinetics. Increasing luminal [H+] generates an energy barrier at a luminal pH near 4.4 that equals the free energy (per H+ translocated) of the metabolic driving reaction.     

Evidence that ATP-dependent Ca2+ transport in rat parotid microsomal membranes requires charge compensation.

ATP-dependent Ca2+ transport was investigated in a rat parotid microsomal-membrane preparation enriched in endoplasmic reticulum. Ca2+ uptake, in KCl medium, was rapid, linear with time up to 20 s, and unaffected by the mitochondrial inhibitors NaN3 and oligomycin. This Ca2+ uptake followed Michaelis-Menten kinetics, and was of high affinity (Km approximately 38 nM) and high capacity (approximately 30 nmol/min per mg of protein). In the presence of oxalate, Ca2+ uptake continued to increase for at least 5 min, reaching an intravesicular accumulation approx. 10 times higher than without oxalate. Ca2+ uptake was dependent on univalent cations in the order K+ = Na+ greater than trimethylammonium+ greater than mannitol and univalent anions in the order Cl- greater than acetate- greater than Br- = gluconate- = NO3- greater than SCN-. Ca2+ uptake was not elevated if membranes were incubated in the presence of a lipophilic anion (NO3-) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Ca2+ transport was altered by changes in the K+-diffusion potential of the membranes. A relatively negative K+-diffusion potential increased the initial rate of Ca2+ accumulation, whereas a relatively positive potential decreased Ca2+ accumulation. In the presence of an outwardly directed K+ gradient, nigericin had no effect on Ca2+ uptake. In aggregate, these studies suggest that the ATP-dependent Ca2+-transport mechanism present in rat parotid microsomal membranes exhibits an electrogenic Ca2+ flux which requires the movement of other ions for charge compensation.     

Effects of dinitrophenol and oligomycin on the coupling between anaerobic metabolism and anaerobic sodium transport by the isolated turtle bladder

Dinitrophenol (1 x 10^-5 M) has been found to inhibit anaerobic sodium transport by the isolated urinary bladder of the fresh water turtle. Concurrently, anaerobic glycolysis was stimulated markedly. However, tissue ATP levels diminished only modestly, remaining at approximately 75% of values observed under anaerobic conditions without DNP. The utilization of glucose (from endogenous glycogen) corresponded closely to that predicted from the molar quantities of lactate formed. Thus the glycolytic pathway was completed in the presence of DNP and if ATP were synthesized normally during glycolysis, synthesis should have been increased. On the other hand, the decrease in Na transport should have decreased ATP utilization. Oligomycin did not block sodium transport either aerobically or anaerobically, but ATP concentrations did decrease. When anaerobic glycolysis was blocked by iodoacetate, pyruvate did not sustain sodium transport thus suggesting that no electron acceptors were available in the system. Two explanations are entertained for the anaerobic effect of DNP: (a) Stimulation by DNP of plasma membrane as well as mitochondrial ATPase activity; (b) inhibition of a high energy intermediate derived from glycolytic ATP or from glycolysis per se. The arguments relevant to each possibility are presented in the text. Although definitive resolution is not possible, we believe that the data favor the hypothesis that there was a high energy intermediate in the anaerobic system and that this intermediate, rather than ATP, served as the immediate source of energy for the sodium pump.     

Proton transport and membrane shuttling in turtle bladder epithelium

Summary Proton secretion in the urinary bladder of the freshwater turtle is mediated by proton pumps located in the apical membrane of carbonic-anhydrase (CA)-rich cells. It has been proposed that the rate of proton transport is regulated by endocytotic and exocytotic fusion processes which alter the apical membrane area, and hence number of exposed pumps. Three techniques were used to study this process. Analyses of transepithelial impedance provided estimates of transport-associated changes in net membrane area, as well as other electrical parameters. Electron microscopy allowed visualization of the endocytotic vesicles thought to be involved in the process. Finally, uptake of a florescent fluid-phase markerprovided measurements of the rates of endocytosis. We report the following: (i) endocytotic and exocytotic processes occur primarily in the CA-rich cells; (ii) inhibition of proton transport resulting from 0.5mm acetazolamide (AZ) results in a decrease in the apical membrane area of approximately 0.47 cm²/cm² tissue; (iii) the apical membrane specific conductance of the CA-rich cells is approximately 220 S/F, and possibly represents a Cl^– conductance that may function in counter-ion flow; (iv) the decline in transport following AZ is not directly proportional to the decline in apical membrane area, suggesting that changes in pump kinetics are also involved in the regulation of transport; (v) the CA-rich cells exhibit a high rate of constitutive pinocytosis, and hence membrane shuttling, which appears to be independent of the rate of transport; (vi) AZ induces a transient increase in the rates of endocytosis and shuttling; and (vii) the transport-associated changes in apical membrane area may reflect an effect of AZ on a regulated endocytotic pathway which is distinct from the pinocytotic process.     

ATP-dependent Ca2+ transport in vesicles isolated from the bile canalicular region of the hepatocyte plasma membrane

Three plasma membrane subfractions have been isolated and characterized from rat liver cells. The high affinity Ca2+-stimulated ATPase is highly enriched in the bile canalicular subfraction. Taking into account cross-contamination by the blood sinusoidal and lateral membranes it is suggested that the high-affinity Ca2+-ATPase is located exclusively in this fraction. The high-affinity Ca2+-ATPase is coupled to Ca2+ transport, is calmodulin-insensitive, sensitive to vanadate under appropriate experimental conditions and is strongly inhibited by La3+. In the presence of Ca2+ and ATP the ATPase forms a phosphorylated intermediate of molecular mass about 200 kDa.