Contrasts in the hypo-osmoregulatory abilities of a freshwater and an anadromous population of inconnu

Juvenile freshwater and anadromous inconnu Stenodus leucichthys regulated plasma ions following a direct transfer from fresh to brackish water (10–15‰), but suffered osmoregulatory collapse with 100% mortality in 48 h when directly transferred to 25‰ salinity. Acclimation to brackish water for 2 weeks improved hypo-osmoregulatory capacity in both populations, with acclimated fish showing smaller increases in blood plasma ion concentrations, higher Na⁺-K⁺ ATPase activity, and lower mortality than non-acclimated fish following transfer to 25‰ sea water. Anadromous inconnu maintained pre-treatment plasma ion levels during acclimation, whereas these levels increased during acclimation in freshwater inconnu. Juvenile anadromous inconnu are therefore able to adapt physiologically to sea water of at least 25‰, if brackish-water acclimation is available, but freshwater inconnu have diminished saltwater tolerance, relative to the anadromous form.     

Inhibition of Calcium-Dependent ATPase from Sarcoplasmic Reticulum by a New Class of Indolizidine Alkaloids, Pumiliotoxins A, B, and 251D

Abstract: Pumiliotoxins (PTX) A, B, and 251D, members of a new class of indolizidine alkaloids isolated from the skin of poison frogs of the family Dendrobatidae, inhibit Ca²⁺-ATPase activity in sarcoplasmic reticulum vesicles from frog and rat hind-limb muscles. PTX-B and PTX-A appear to be relatively specific inhibitors of Ca²⁺-ATPase; PTX-A is much less potent than PTX-B. PTX-251D is a potent inhibitor of Ca²⁺-ATPase, and was also found to inhibit Na⁺, K⁺, and Mg²⁺-ATPases in rat brain synaptosomes. Caffeine and verapamil, two drugs known to affect calcium translocation, are very weak inhibitors of the Ca²⁺-ATPase. The K, values for inhibition of the Ca²⁺-ATPase of rat and frog sarcoplasmic reticulum by PTX-B were comparable and ranged between 22 and 36 μM. Inhibition of calcium-dependent ATPase in sarcoplasmic reticulum by pumiliotoxin-B is noncompetitive with calcium and is not readily reversible. Based on structure-activity profiles, it is concluded that inhibition of Ca²⁺-ATPase by the indolizidine alkaloids is responsible for the alkaloidelicited prolongation of twitch in intact muscle.     

Na(+)-dependent, active nucleoside transport in S49 mouse lymphoma cells and loss in AE-1 mutant deficient in facilitated nucleoside transport

S49 murine lymphoma cells were examined for expression of various nucleoside transport systems using a non-metabolized nucleoside, formycin B, as substrate. Nitrobenzylthioinosine (NBTI)-sensitive, facilitated transport was the primary nucleoside transport system of the cells. The cells also expressed very low levels of NBTI-resistant, facilitated nucleoside transport as well as of Na(+)-dependent, concentrative formycin B transport. Concentrative transport was specific for uridine and purine nucleosides, just as the concentrative nucleoside transporters of other mouse and rat cells. A nucleoside transport mutant of S49 cells, AE-1, lacked both the NBTI-sensitive, facilitated and Na(+)-dependent, concentrative formycin B transport activity, but Na(+)-dependent, concentrative transport of alpha-aminoisobutyrate was not affected.     

Effect of ABA on the Activity of Mitochondrial Membrane Bound Na+-K+ ATPase

Effect of ABA on the activity of mitochondrial membrane bound Na+-K+ATPase during isolation of mitochondria from soybean cotyledons, there was an increased activity of the mitochondrial membrane bound Na+-K+ATPase if abscisic acid (ABA) was added to the medium when soybean seedling were grown at 27 ℃ or 16℃, 40 mol/L ABA could change the turning point temperature of Arrhenius the activation energies (Ea) of Na+-K+ATPase from 36.6℃ or 22.7℃ decreased to 30. 3℃ or 17.8℃ respectively. The Km value and S0.5 value for this enzyme with ABA was higher than that without ABA. Hill coefficient (n) of this enzyme with ABA was 1.01 and without ABA was 1.89. The o2 uptake of mitochondria also increased. These results showed that the temperature of phase transition of mitochondrial membrane were decreased by ABA treatment.     

Loss of EGF binding and cation transport response during differentiation of mouse neuroblastoma cells

Mouse neuroblastoma cells (clone N1E-115) differentiate in culture upon withdrawal of serum growth factors and acquire the characteristics of neurons. We have shown tht exponentially growing N1E-115 cells possess functional epidermal growth factor (EGF) receptors but that the capacity for binding EGF and for stimulation of DNA synthesis is lost as the cells differentiate. Furthermore, in exponentially growing cells, EGF induces a rapid increase in amiloride-sensitive Na+ influx, followed by stimulation of the (Na+-K+)ATPase, indicating that activation of the Na+/H+ exchange mechanism in N1E-115 cells [1] may be induced by EGF. The ionic response is also lost during differentiation, but we have shown that the stimulation of both Na+ and K+ influx is directly proportional to the number of occupied receptors in all cells whether exponentially growing or differentiating, thus only indirectly dependent on the external EGF concentration. The linearity of the relationships indicates that there is no rate-limiting step between EGF binding and the ionic response. Our data would suggest that as neuroblastoma cells differentiate and acquire neuronal properties, their ability to respond to mitogens, both biologically and in the activation of cation transport processes, progressively decreases owing to the loss of the appropriate receptors.     

ATP dependent primary active transport of xenobiotic-glutathione conjugates by human erythrocyte membrane

We have demonstrated the presence of a dinitrophenyl glutathione (Dnp-SG) stimulated ATPase in human erythrocyte membranes. This ATPase mediates the active transport of glutathione — xenobiotic conjugate such as Dnp-SG from erythrocytes into the plasma. It is suggested that this transport system is distinct from the system which actively transports oxidized glutathione from the erythrocytes.     

Fine structure and function of the gut epithelium of pike eel larvae

The development of the alimentary canal of pike eel, Muraenesox cinereus , that were hatched and reared in the laboratory, was examined. The larvae did not feed on zooplankton or phytoplankton and survived for 10 days. Histological and histochemical investigations showed that the alimentary canal was morphologically and functionally differentiated in 3 days after hatching and before exhaustion of the yolk. The foregut was not functional in absorption. The midgut absorptive cell was characterized by a well-developed lamellar membranous structure in the basal portion of the cytoplasm. Na¹-K¹ ATPase activity was demonstrated on the lamellar membrane of the structure. It suggests that water and solute transport is quite active in the midgut epithelium. Orally administered HRP (horseradish peroxidase) was ingested and intracellularly digested in the hindgut absorptive cells. Some of the HRP was transported, without complete digestion, into tissue spaces under the epithelium. Evidence suggests that pike eel larvae possibly utilize dissolved organic matter, including soluble macromolecules, as their nutrition sources.     

Effects of bicarbonate on fluid and electrolyte transport by guinea pig and rabbit gallbladder: Stimulation of absorption

Summary The effect of bicarbonate (HCO₃) on fluid absorption by guinea pig gallbladder was investigatedin vitro. Stimulation of fluid absorption was concentration dependent resulting in a fourfold increase in transport over the range 1 to 50mm. Phosphate, Tris, glycodiazine and glutamine buffers failed to substitutte for HCO₃ in stimulating absorption. Unidirectional²²Na fluxes were measured across short-circuited sheets of guinea pig and rabbit gallbladders mounted in Ussing-type chambers. In both species the net Na flux was unaffected by serosal HCO₃ alone but was stimulated by addition of HCO₃ to the mucosal bathing solution. Transepithelial electrical potential difference in rabbit gallbladder was about 1.4 mV (lumen positive) when HCO₃ was present in the mucosal or in both compartments. This fell to 0.2 mV under HCO₃-free conditions or when HCO₃ was present only in the serosal solution. The respective values for guinea pig gallbladder were –1.6 and –0.6 mV (lumen negative). HCO₃ stimulation of Na absorption by guinea pig gallbladder was abolished by increasing the bathing pH from 7.4 to 7.8, an effect resulting mainly from a reduction inJ _mis ^Na . Tris buffer (25mm) inhibited HCO₃-dependent fluid absorption in this species completely at pH 8.5 and partially at 7.5. These results indicate that HCO₃ stimulates gallbladder transport in both species by an action from the mucosal side. This effect cannot be attributed to simple buffering of H⁺ but may be explained by the participation of HCO₃ in the maintenance of intracellular H⁺ for a Na/H-exchange.     

Extracellular protons regulate the extracellular cation selectivity of the sodium pump

The effects of 0.3-10 nM extracellular protons (pH 9.5-8.0) on ouabain-sensitive rubidium influx were determined in 4,4'-diisocyanostilbene-2, 2'-disulfonate (DIDS)-treated human and rat erythrocytes. This treatment clamps the intracellular H. We found that rubidium binds much better to the protonated pump than the unprotonated pump; 13-fold better in rat and 34-fold better in human erythrocytes. This clearly shows that protons are not competing with rubidium in this proton concentration range. Bretylium and tetrapropylammonium also bind much better to the protonated pump than the unprotonated pump in human erythrocytes and in this sense they are potassium-like ions. In contrast, guanidinium and sodium bind about equally well to protonated and unprotonated pump in human red cells. In rat red cells, protons actually make sodium bind less well (about sevenfold). Thus, protons have substantially different effects on the binding of rubidium and sodium. The effect of protons on ouabain binding in rat red cells was intermediate between the effects of protons on rubidium binding and on sodium binding. Remarkably, all four cationic inhibitors (bretylium, guanidinium, sodium, and tetrapropylammonium) had similar apparent inhibitory constants for the unprotonated pump ( approximately 5-10 mM). The K(d) for proton binding to the human pump, with the empty transport site facing extracellularly is 13 nM, whereas the extracellular transport site loaded with sodium is 9.5 nM, and with rubidium is 0.38 nM. In rat red cells there is also a substantial difference in the K(d) for proton binding to the sodium-loaded pump (14.5 nM) and the rubidium-loaded pump (0.158 nM). These data suggest that important rearrangements occur at the extracellular pump surface as the pump moves between conformations in which the outward facing transport site has sodium bound, is empty, or has rubidium bound and that guanidinium is sodium-like and bretylium and tetrapropylammonium are rubidium-like.     

Active calcium transport via coupling between the enzymatic and the ionophoric sites of Ca2+ + Mg2+-ATPase

The 20K dalton fragment of Ca²⁺ + Mg²⁺-ATPase obtained from the tryptically digested sarcoplasmic reticulum has been further purified using Bio-Gel P-100. This removed low-molecular-weight UV-absorbing and positive Lowry-reacting contaminants. The ionophoric activity of the 20K fragment in both oxidized cholesterol and phosphatidylcholine: cholesterol membranes is unaltered by this further purification. The 20K selectivity sequence in phosphatidylcholine: cholesterol membranes is Ba²⁺ > Ca²⁺ > Sr²⁺ > Mn²⁺ Mg²⁺. Digestion of intact sarcoplasmic reticulum vesicles with trypsin, which results in the dissection of the hydrolytic site (30K) from the ionophoric site (20K), is shown to disrupt energy transduction between ATP hydrolysis and calcium transport. This further implicates the 20K dalton fragment as a calcium transport site. These data and previous evidence are discussed in terms of a proposed model for the ATPase molecular structure and the mechanism of cation transport in sarcoplasmic reticulum.     

Potassium transport in Escherichia coli: sodium is not a substrate of the potassium uptake system TrkA

Abstract In Escherichia coli cells depleted of both sodium and potassium, the potassium uptake system TrkA mediated a slow, electrogenic uptake of potassium. Electroneutrality was maintained by the extrusion of protons. Internal, but not external sodium stimulated potassium uptake. This extra uptake was coupled to a stoichiometric extrusion of sodium. Triethanolamine also stimulated potassium uptake, presumably by increasing the cytoplasmic buffer capacity. These results are taken to mean that sodium is not a substrate of the TrkA system, but stimulates TrkA activity by facilitating the reentry of protons through the sodium-proton antiporter, and thereby preventing a prohibitive increase in cytoplasmic pH.     

Inhibition of hexose transport and abrogation of pH homeostasis in the intraerythrocytic malaria parasite by an O-3-hexose derivative

An O-3-hexose derivative, shown previously to inhibit a malaria parasite hexose transporter expressed in Xenopus oocytes as well as to suppress the multiplication of parasites, both in vitro and in vivo, was shown here to block the uptake of hexose sugars into isolated blood-stage parasites. This led to a decline in ATP levels and the loss of intracellular pH control. The results are consistent with those obtained with the cloned transporter. They support the notion that the transporter mediates uptake of glucose into the intraerythrocytic parasite and provide further support for the view that it is a suitable antimalarial drug target.     

Effect of scavengers of active oxygen species and pretreatment with acetyl-salicylic acid on the injury to cultured endothelial cells by thrombin-stimulated platelets

Summary Thrombin-stimulated human platelets adhere to and injure cultured human endothelial cells. We hypothesize that generation of active oxygen species by the stimulated platelets are involved in the injury. To confirm this, catalase [final concentration (8.25 μg/ml)], superoxide dismutase (SOD) (10 μg/ml), ofd-mannitol (9 mg/ml) were added to the cell culture medium before the experiments. Platelet suspension (200.000/μl) and thrombin (4 U/ml) were added and the culture dishes shaken for 15 min at room temperature. In separate experiments the endothelial cells were pretreated with acetylsalicylic acid (0.05, 0.1, or 0.5 mM) to test whether the arachidonic acid metabolism of the endothelial cells is involved in the injury process. In preliminary experiments we were able to confirm that platelets, when stimulated by thrombin, produce chemiluminescence which was suppressed by mannitol but not by catalase or SOD. The degree of injury to cultured endotheial cells by thrombin-stimulated platelets, as measured by release of⁵¹Cr from prelabeled endothelial cells, was reduced significantly with the presence of mannitol, but only moderately when catalase or SOD had been added. Morphometric quantification based on scanning electron micrographs of the endothelial cells after exposure to thrombin-stimulated platelets in the presence of catalase or mannitol showed a reduced number of injured cells. Pretreatment of the endothelial cells with acetylsalicylic acid did not cause any significant change in the degree of endothelial cell injury as measured by the⁵¹Cr release. It is concluded that active oxygen species, in particular hydroxyl radicals, may be generated during thrombin stimulation of platelets and cause injury to the endothelial cells. This work was supported by the Norwegian Research Council for Science and the Humanities and the Norwegian Council for Cardiovascular Diseases. We express our gratitude for their grants.     

Barium modifies the concentration dependence of active potassium transport by insect midgut

Summary The rate of active K⁺ transport by the isolated lepidopteran midgut shows a rectangular hyperbolic relation to [K⁺] over the range 20 to 70mm K⁺ in the absence of any divalent cation. Addition of Ba⁺⁺ to the hemolymph (K⁺ uptake) side introduces a linear component to the concentration dependence, such that active K^– transport is decreased at [K⁺] of 55mm or less, but increased transiently at higher [K⁺]. As [Ba⁺⁺] is increased over the range 2 to 8mm the linear component increases and the saturating component decreases; in 8mm Ba⁺⁺ the concentration dependence is dominated by the linear component. The effect of Ba⁺⁺ cannot easily be accounted for by simple competition with K⁺ for basal membrane uptake sites. Similar effects might be exercised by other alkali earth cations, since the concentration dependence of active K⁺ transport possesses a substantial linear component in solutions containing 5mm Ca⁺⁺ and 5mm Mg⁺⁺ (the alkali earth metal concentrations of standard lepidopteran saline).     

Neurochemical characterization of the release and uptake of dopamine in ventral tegmental area and serotonin in substantia nigra of the mouse

In the present report, fast-scan cyclic voltammetry was used to identify the monoamines that were released by electrical stimulation in mouse brain slices containing ventral tegmental area (VTA), substantia nigra (SN) -pars compacta (SNc) and -pars reticulata (SNr). We showed that voltammograms obtained in mouse VTA were consistent with detection of a catecholamine, while those in both subregions of the SN were consistent with detection of an indolamine, based on the reduction peak potentials. We used pharmacological blockade and genetic deletion of monoamine transporters to further confirm the identity of released monoamines in mouse midbrain and to assess the control of monoamines by their transporters in each brain region. Inhibition of dopamine and norepinephrine transporters by nomifensine (1 and 10 microm) decreased uptake rates in the VTA, but did not change uptake rates in either subregion of the SN. Serotonin transporter inhibition by fluoxetine (10 microm) decreased uptake rates in the SNc and SNr, but was without effect in the VTA. Selective inhibition of the norepinephrine transporter by desipramine (10 microm) had no effect in any brain region. Using dopamine transporter- and serotonin transporter-knockout mice, we found decreased uptake rates in VTA and SN subregions, respectively. Peak signals recorded in each midbrain region were pulse number dependent and exhibited limited frequency dependence. Thus, dopamine is predominately detected by voltammetry in mouse VTA, while serotonin is predominately detected in mouse SNc and SNr. Furthermore, active uptake occurs in these areas and can be altered only by specific uptake inhibitors, suggesting a lack of heterologous uptake. In addition, somatodendritic dopamine release in VTA was not mediated by monoamine transporters. This work offers an initial characterization of voltammetric signals in the midbrain of the mouse and provides insight into the regulation of monoamine neurotransmission in these areas.     

Lack of inhibition of vasopressin-stimulated NA+K+ATPase by atrial natriuretic factor in the rat renal medullary thick ascending limb of Henle's loop

The anti-diuretic hormone, arginine vasopressin (AVP) stimulates the activity of Na+K+ATPase in the rat renal medullary thick ascending limb of Henle's loop (mTAL). Atrial natriuretic factor (ANF) has been suggested to exert a tubular effect on the mammalian nephron, perhaps in part, by interacting with other hormones. In the present study, we investigated the effect of rat ANF with and without AVP upon mTAL Na+K+ATPase activity using cytochemical methods. ANF alone failed to inhibit or stimulate Na+K+ATPase activity in mTAL at any of the concentrations tested (10 nmol-0.1 pmol l-1). Unlike the rat hypothalamic digitalis-like factor, ANF (10 nmol-10 fmol l-1) did not inhibit Na+K+ATPase activity after stimulation with AVP (1 fmol l-1) for either 4 or 10 min. The results suggest that ANF does not exert an effect on mTAL, either alone or in conjunction with AVP.     

An electron microscopic study of the transport of peroxidases in the endothelium of mouse aorta

Summary Aortic endothelium presents a continuous barrier to diffusion of macromolecules. The cell margins overlap for long distances and there are multiple points of contact between the cell membranes at which the intercellular cleft is reduced to 30–40 Å or less, and free diffusion of lanthanum is impeded at some points of apposition. Macromolecular transport through the endothelium of mouse aorta was studied with the help of horseradish peroxidase (HRP) and bovine milk lactoperoxidase. Following injection of 0.25–0.5 mg of HRP no tracer was detected in the intercellular clefts even though it was seen in plasmalemmal vesicles and subendothelial space. However, when 5 mg of HRP was injected in either 0.05 or 0.5 ml of saline, transport of the enzyme occurred through both the intercellular clefts and via the plasmalemmal vesicles. On the other hand, lactoperoxidase of m.w. 82000 was transported through the plasmalemmal vesicles only. The findings were discussed with reference to the transport of serum lipoproteins and it was suggested that low and high density lipoproteins would be transported via the plasmalemmal vesicles.The excellent technical help of Miss R. Ben-Moshe and Mrs. A. Mandeles is gratefully acknowledged. This study was supported in part by a grant from the Myra Kurland Heart Fund, Chicago, Ill., and by a grant 06-101-1 of the National Institute of Health, United States Public Health Service.     

The response of electron transport mediated by active NADPH dehydrogenase complexes to heat stress in the cyanobacterium Synechocystis 6803

The electron-transport machinery in photosynthetic membranes is known to be very sensitive to heat. In this study, the rate of electron transport (ETR) driven by photosystem I (PSI) and photosystem II (PSII) during heat stress in the wild-type Synechocystis sp. strain PCC 6803 (WT) and its ndh gene inactiva-tion mutants △ndhB (M55) and △ndhD1/ndhD2 (D1/D2) was simultaneously assessed by using the novel Dual-PAM-100 measuring system. The rate of electron transport driven by the photosystems (ETRPSs) in the WT, M55, and D1/D2 cells incubated at 30℃ and at 55℃ for 10 min was compared. Incubation at 55 ℃ for 10 min significantly inhibited PSII-driven ETR (ETRPSII) in the WT, M55 and D1/D2 cells, and the ex-tent of inhibition in both the M55 and D1/D2 cells was greater than that in the WT cells. Further, PSI-driven ETR (ETRPSI) was stimulated in both the WT and D1/D2 cells, and this rate was increased to a greater extent in the D1/D2 than in the WT cells. However, ETRPSI was considerably inhibited in the M55 cells. Analysis of the effect of heat stress on ETRPSs with regard to the alterations in the 2 active NDH-1 complexes in the WT, M55, and D1/D2 cells indicated that the active NDH-1 supercomplex and medi-umcomplex are essential for alleviating the heat-induced inhibition of ETRPSII and for accelerating the heat-induced stimulation of ETRPSI, respectively. Further, it is believed that these effects are most likely brought about by the electron transport mediated by each of these 2 active NDH-1 complexes.     

Stimulation of the Na+/K+ Pump Activity During Electrogenic Uptake of Acidic Amino Acid Transmitters by Rat Brain Synaptosomes

Addition of D-aspartate, a substrate for the high-affinity transport of acidic amino acid transmitters, to suspensions of rat brain synaptosomes increased the rate of O2 consumption, uptake of 86Rb, and transport of 2-[3H]deoxyglucose. Stimulation of all three processes was abolished in the presence of ouabain. D-Aspartate had no effect on respiration in the medium in which NaCl was replaced by choline chloride. The ratio of the ouabain-sensitive increase in 86Rb uptake to that in O2 consumption was 12 to 1, which gives a calculated 86Rb(K+)/ATP of 2. It is concluded that electrogenic, high-affinity transport of sodium-D-aspartate into synaptosomes stimulates the activity of the Na+/K+ pump through an increase in [Na+]i.