Inhibitors of active Ca2+ uptake by fragments of the sarcoplasmic reticulum of lobster muscle

• 1.1. Vesicles from the sarcoplasmic reticulum of lobster muscle accumulate Ca²⁺ if supplied with ATP as an energy source. A search was undertaken for inhibitors of Ca²⁺ transport.
• 2.2. p-Hydroxymercuribenzoate can completely inhibit Ca²⁺ transport and ATP hydrolysis. 2–4 Dinitrophenol inhibits uptake but not hydrolysis.
• 3.3. Sr²⁺, Ba²⁺ and Zn²⁺ inhibit uptake, perhaps by competing with Ca²⁺ for a carrier.
• 4.4. The vesicles contain acetylcholinesterase. Anticholinesterases can reduce —but not abolish—Ca²⁺ uptake. Acetylcholine has no effect on the activity of the vesicles.
• 5.5. Ca²⁺ uptake is not affected by Mn²⁺, glutamate, pilocarpine, carnosine, caffeine, strophanthidin or tetraethylammonium.
• 6.6. K⁺ is needed for maximal activity of the uptake system but not for ATP hydrolysis. Apparently K⁺ enhances the coupling between the energy supply and the carrier mechanism.

     

Behaviour and haemolymphatic ionic composition of the intertidal crab Chasmagnathus granulata dana, 1851 (Crustacea: Decapoda) during emersion

• 1.1. Behavioural observations and haemolymphatic measurements of Na⁺ K⁺ and Ca⁺ were performed in Chasmagnalhus granulata during emersion.
• 2.2. Activity levels were found to be higher during voluntary emersion periods than when the animals were submerged. A lt₅₀ of 39.45 hr was observed when no access to water was allowed.
• 3.3. The Na⁺ and K⁺ and Ca⁺ levels increased during aerial exposure. The Na⁺ and K⁺ levels were restored prior the end of the experimental period. Mechanisms for such regulation are therefore discussed. The Ca²⁺ levels, remaining high during emersion, are probably a result of acid-base balance adjustments.

     

Influence of deionized water supplemented or not with different ions on prolactin cell activity and osmotic regulation in the goldfish

• 1.1. Goldfish were kept in deionized water (DW), DW + Na⁺ (0.35 mM), DW + K⁺ (0.05 mM), DW + Ca²⁺ (2mM) and DW + Mg²⁺ (0.2 mM). In Ca-free environments, prolactin cells appear unaffected. Stimulated calcium-sensitive cells (pars intermedia) may elaborate a hypercalcemic factor.
• 2.2. Fecal excretion, reduced in all groups, remains noticeable in DW + Ca²⁺
• 3.3. Ionic losses, very low in all groups, are minimal in DW. Supplementation with K⁺ increases Na⁺ loss.
• 4.4. Plasma Na⁺ Ca²⁺, and osmolarity decrease in DW, and still more in DW + K⁺. Ca²⁺' and Mg²⁺ partly suppress hyponatremia.
• 5.5. In goldfish kept in DW and subsequently in DW + Ca²⁺, calcemia increases.

     

High-affinity Ca2+-Mg2+-ATPase in kidney of euryhaline Gillichthys mirabilis: kinetics,subcellular distribution and effects of salinity

• 1.1. Two components of Ca²⁺-Mg²⁺-ATPase are observed in kidneys of G. mirabilis. The high-affinity component has a K_0.5Ca of 0.23μM; the low-affinity activity K_0.5Ca is 90–110μM. The high-affinity activity requires Mg²⁺, displays Michaelis-Menten kinetics, has peak activity at 1.2 μM Ca²⁺, and is insensitive to ouabain and Na⁺ azide.
• 2.2. In subcellular fractions, the high-affinity component segregates with Na⁺-K⁺-ATPase and is localized predominantly in BLM. The low-affinity component is broadly distributed among membranous organelles, including brush border, and may be equivalent to alkaline phosphatase.
• 3.3. Specific activity of the high-affinity Ca²⁺-Mg²⁺-ATPase is modestly increased following adaptation of fish to FW, but total renal high-affinity activity is greatest in the hypertrophied kidneys of FW-adapted fish and is least in kidneys of fish adapted to 200% SW.
• 4.4. High-affinity Ca²⁺-Mg²⁺-ATPase may be associated with active Ca²⁺ transport or with regulation of intracellular Ca²⁺ concentration of tubular cells.

     

Erythrocyte membrane anion-sensitive Mg2+-ATPase—Identity with monovalent cation sensitive Ca2+-Mg2+-ATPase

• 1.1. Activation of Mg²⁺-ATPase of rabbit and guinea-pig erythrocyte membrane by bicarbonate or chloride could be completely abolished by ethylene-glycol-bis-(β-aminoethylether)-N,N'-tetraacetic acid. The anion stimulation was actually an activation of contaminating Ca²⁺ -stimulated Mg²⁺-ATPase by monovalent cations associated with the anions.
• 2.2. Guinea-pig red cell Ca²⁺-Mg²⁺-ATPase could be activated by both sodium and potassium while the rabbit enzyme was sensitive only to sodium. The concentrations of monovalent cations for half-maximal stimulation of Ca²⁺-Mg²⁺-ATPase are: k_na+ = 40.8 mM, k_k+ = 12.2 mM (guinea-pig); K_Na+ = 13.3mM (rabbit).
• 3.3. Potassium enhanced activation of rabbit erythrocyte membrane Ca²⁺-Mg²⁺-ATPase by red cell Ca²⁺-Mg²⁺-ATPase activator protein. With the guinea pig enzyme, neither sodium nor potassium enhanced activator stimulation of Ca²⁺-Mg²⁺-ATPase.
• 4.4. Ca²⁺-Mg²⁺-ATPase of aged rabbit erythrocyte membrane responded to sodium but not to activator protein.
• 5.5. Triton X-100 solubilized rabbit erythrocyte membrane Ca²⁺-Mg²⁺-ATPase has an apparent molecular weight of 371,000. It did not respond to the activator.
• 6.6. One major and three minor proteins, visualized by SDS-polyacrylamide gel electrophoresis, were extracted from rabbit erythrocyte membrane by 50 μM chlorpromazine.

     

Calcium-dependent potassium conductance in the photoresponse of a nudibranch mollusk

• 1.1. The response to light of Hermissenda photoreceptors when recorded intracellularly without interference from synaptic and action potentials consisted of three phases: an early depolarization (ED) followed by hyperpolarization (dip) and subsequent depolarization (tail).
• 2.2. The ED and the dip were associated with increased membrane conductance while decreased membrane conductance was involved with the tail.
• 3.3. The dip reversal potential was − 82.1 ± 5.3 mV and its amplitude varied inversely with the log of [K⁺].
• 4.4. Perfusing with agents which block K⁺ current like 4AP, Quinine, Quinidine or injection of TEA eliminated the dip and its associated increased membrane conductance, thus further supporting the role of K⁺ conductance in producing the dip.
• 5.5. The dip was enhanced by increased [Ca²⁺]_o, reduced by decreased [Ca²⁺]_o and abolished together with its associated increased membrane conductance when perfused with either D600, Cd²⁺, Mg²⁺, Mn²⁺, or Co²⁺, which block transmembrane Ca²⁺ current.
• 6.6. The dip and its associated increased membrane conductance were abolished by intracellular injection of EGTA and enhanced by perfusion with Ruthenium red.
• 7.7. Intracellular injection of Ca²⁺ mimicked the dip: membrane conductance was increased and the cell hyperpolarized.
• 8.8. These results indicate that the increase in intracellular [Ca²⁺] is primarily responsible for the light-induced increase of K⁺ conductance during the dip. The possible source of the Ca²⁺ is, at least in part, extracellular due to activation of an inward Ca²⁺ current.

     

Bioelectrical potentials across the mantle epithelium of Achatina fulica

• 1.1. The shell side of the mantle of Achatina fulica is several millivolts positive to the blood side in vitro.
• 2.2. The electrical potential does not depend on Na⁺, Ca²⁺, Mg²⁺, K⁺ or HCO₃⁻ but requires the presence of chloride on the shell side.
• 3.3. The potential difference and short-circuit current ranged from 3.0 to 30.0 mV and 15.0 to 75 μA/cm² with averages at 10m V and 50 μA/cm² respectively.
• 4.4. The electrical gradient is reduced by 2,4-dinitrophenol, thiocyanate and furosemide but not by ouabain, CO₂ or acetozolamide.
• 5.5. It is suggested that the nature and mechanism of electrogenesis in Achatina parallels that of the Helix mantle.

     

Effects of Na+ and/or K+ on the Mg2+-dependent ATPase activities in shrimp (Macrobrachium amazonicum) gill homogenates

• 1.1. Homogenates of gills from the freshwater shrimp M. amazonicum exhibit the following ATPase activities: (i) a basal, Mg²⁺-dependent ATPase; (ii) an ouabain-sensitive, Na⁺ + K⁺-stimulated ATPase; (iii) an ouabain-insensitive, Na⁺-stimulated ATPase; and (iv) an ouabain-insensitive, K⁺-stimulated ATPase.
• 2.2. K⁺ suppresses the Na⁺-stimulated ATPase activity in a mixed-type kind of inhibition, whereas Na⁺ does not exert any noticeable effect on the K⁺-stimulated ATPase activity.
• 3.3. The Na⁺- and the K⁺-stimulated ATPase activities are totally inhibited by 5 mM ethacrynic acid in the incubation medium.
• 4.4. The Na⁺- and the K⁺-stimulated ATPase activities are not expressions of the activation of a Ca-ATPase.
• 5.5. The possible localization and roles of the described ATPases within the gill epithelium are briefly discussed and evaluated.

     

Na+ regulation and (Na++ K+)ATPase activity in the euryhaline fiddler crab Uca minax (Le conte)

• 1.1. Specific activity and kinetic characteristics of the (Na⁺ + K⁺)ATPase have been investigated in the gill epithelium of the hyper-hypoosmoregulator crab Uca minax.
• 2.2. (Na⁺ +K⁺)ATPase activity is shown to be at least three times higher in the posterior gills.
• 3.3. The kinetic study supports the hypothesis of the existence of two different (Na⁺ + K⁺)ATPases: the enzyme activity in the posterior gills could be involved in the transepithelial transport of Na⁺ while the activity of the anterior gills could be responsible for the intracellular regulation of Na⁺ and K⁺.
• 4.4. Significant and specific changes in (Na⁺ +K⁺)ATPase activity occur upon acclimation to media of various salinities.

     

Ionic dependence and vanadate sensitivity of (Na+, K+)-ATPase isolated from branchial sac of ascidians

• 1.1. Ion dependence and vanadium-induced inhibition on branchial sac ATPase in five species of ascidian Phlebobranchiata (vanadium-accumulating) and Stolidobranchiata (iron-accumulating) were studied.
• 2.2. The ATPase was obtained from the microsomal fraction, which was prepared from each ascidian branchial sac.
• 3.3. The ATPase was dependent on Mg²⁺ and activated by exogenous Na⁺ + K⁺.
• 4.4. Ouabain inhibited the ATPase activity in vitro, 10 μM to 100 μM vanadate, in vitro, suppressed the (Na⁺, K⁺)-ATPase.

     

Crayfish retinular cells: Influence of extracellular sodium and calcium upon receptor potential

• 1.1. In crayfish, light stimulation of the retinular cells induces a depolarizing receptor potential.
• 2.2. Experiments were designed to determine the role of Na⁺ and Ca²⁺ on receptor potential during dark And light states.
• 3.3. Depolarization depends on Na⁺ and Ca²⁺ availability to the retinular cell.
• 4.4. Repolarization velocity and response duration depend on extracellular Ca²⁺ availability.
• 5.5. Light adaptation increases receptor potential dependence on calcium and sodium ions.
• 6.6. We analyse these results with respect to other invertebrate photoreceptors.

     

Mg2+-dependent (Na+ + K+)- and Na+-ATPases in the kidneys of the gilthead bream (Sparus auratus L.)

• 1.1. The (Na⁺ + K⁺)- and Na⁺-ATPases, both present in kidney microsomes of Sparus auratus L., have different activities and optimal assay conditions as, in the first of the two stocks of fish used (A), the spec. act. of the former is 51.7 μmol P_i mg prot⁻¹ hr⁻¹ at pH 7.5, 100 mM Na⁺, 10 mM K⁺, 17.5 mM Mg²⁺, 7.5 mM ATP and that of the latter is 6.5 μmol P_i mg prot⁻¹ hr⁻¹ at pH 6.5, 40 mM Na⁺, 4.0 mM Mg²⁺, 2.5 mM ATP.
• 2.2. Ouabain and vanadate specifically inhibit the (Na⁺ + K⁺)-ATPase but not the Na⁺-ATPase that is preferentially inhibited by ethacrynic acid.
• 3.3. While the (Na⁺ + K⁺)-ATPase is strictly specific for ATP and Na⁺, Na⁺-ATPase can be activated by various monovalent cations and, apart from ATP, hydrolyses CTP, though less efficiently.
• 4.4. The second stock B, subjected to higher salinity than A, shows an acidic shifted Na⁺-ATPase optimal pH, opposed to the stability of that of the (Na⁺ + K⁺)-ATPase, a decreased (Na⁺ + K⁺)-ATPase and a strikingly depressed Na⁺-ATPase.
• 5.5. The results are compared with literature data and discussed on the basis of the presumptive different roles as well as functional prevalence in various salinities of the two ATPases.

     

Calcium uptake in the gorgonian Leptogorgia virgulata. The effects of atpase inhibitors

• 1.1. Longitudinally split or completely regenerated branch tips from Leplogorgia virgulata show no differences in calcium uptake between control and ouabain treatments. This indicates that there is no ouabain sensitive Na⁺, K⁺-ATPase involved in calcium uptake.
• 2.2. The tissue fractions of both regenerated and split branch tips show, at certain times, higher calcium uptake than control fractions. In the spicule fractions of these tips calcium uptake decreases in vandate treated specimens.
• 3.3. Pulse-chase experiments show an initial rapid release of calcium from the tips into surrounding seawater.
• 4.4. The results may suggest the presence of outwardly directed calcium pumps on the basal/lateral and apical plasma membranes of the epithelial cells. Outwardly directed calcium pumps may also be envisaged on the cell membranes of scleroblasts. In addition, pumps may move calcium into specific organelles of the scleroblasts en route to the spicule forming vacuoles.
• 5.5. These pumps are likely to be Ca²⁺-ATPase.

     

The effect of mercury and aluminum on sodium-potassium-Mg2+ dependent-adenosine triphosphatase activity of Electrophorus electricus (L.) electrocyte

• 1.1. Na⁺,K⁺-ATPase, which mediates the active transport of Na⁺ and K⁺ across the plasma membrane, is found in equivalent amounts in both plasma membranes of the electrocyte, the anterior, non-innervated (fraction P₂) and the posterior, innervated (fraction P₃) obtained by differential centrifu gation of Electrophorus electricus (L.) electric organ.
• 2.2. The kinetic effects of Hg²⁺ and A1³⁺, described as neurotoxic metals, on the Na⁺,K⁺-ATPase activity of the two membrane fractions (P₂ and P₃) were analysed with respect to Na⁺ and K⁺ ions, after the I₅₀ estimation of each metal.
• 3.3. Mercury is a potent Na⁺,K⁺-ATPase inhibitor in the nanomolar range. In all cases, it behaved as a mixed partial hyperbolic inhibitor.
• 4.4. Aluminum was shown to be a poor enzyme inhibitor. Changing the K⁺ concentration, it behaved as a mixed linear inhibitor (P₂ fraction) and as a non-essential mixed activator (fraction P₃). Aluminum behaved as a partial hyperbolic inhibitor for both P₂ and P₃ fractions with respect to Na⁺ concentration.
• 5.5. The observation of the variable kinetic behaviour of P₂ and P₃ led us to attribute these differences to the Na⁺,K⁺-ATPase electrocyte isoenzymes which occur in different proportions in these fractions (Gomes-Quintana et al., 1992 Comp. biochem. Physiol.103B/3 623–628).

     

A comparison of the effect of alternating current electronarcosis,rectified current electronarcosis and chemical anaesthesia on the blood physiology of the freshwater bream Oreochromis mossambicus (peters)—III. The effect on the plasma electrolytes Ca2+, Na+ and K+ and on the osmotic pressure of the blood

• 1.1. The effects of alternating current electronarcosis, rectified current electronarcosis and chemical anaesthesia (benzocaine hydrochloride) on plasma electrolytes and on the osmotic pressure of the blood of the freshwater bream Oreochromis mossambicus were evaluated.
• 2.2. Plasma Ca²⁺, Na⁺ and K⁺ concentrations and the osmotic pressure of the blood were monitored over a period of 7 days.
• 3.3. The results showed that the different electrolytes respond differently to the different techniques.
• 4.4. Chemical anaesthesia exhibited the least effects on the parameters studied.

     

Effect of Li+ and Ba2+ on the electrocyte membrane-bound (Na+ + K+)-ATPase

• 1.1. Membrane-bound (Na⁺ + K⁺)-ATPase activity from the non-innervated and innervated faces of Electrophorus electricus (L.) electric organ, obtained by differential centrifugation, was measured using AChE as an enzyme marker for membranes derived from the post-synaptic area (fraction P₃) of the electrolyte.
• 2.2. The effect of Li⁺ and Ba²⁺ on (Na⁺ + K⁺)-ATPase activity of the two membrane fractions (P₂ and P₃) was analysed with respect to K⁺ and Mg²⁺ ions, after the I₅₀ estimation.
• 3.3. The kinetics of the reactions with these cations were investigated showing that Li⁺ inhibits P₂ uncompetitively and for P₃ presented a mixed type inhibition.
• 4.4. Ba²⁺ behaved as an hyperbolic mixed type inhibitor for P₂ and a linear mixed type inhibitor for P₃ fraction.

     

Ouabain sensitive Na+/K+-transporting ATPase from the brain of Poekilocerus bufonius

• 1.1. The properties of Na⁺/K⁺-transporting ATPase in microsomal fractions from the nervous tissue of the grasshopper, Poekilocerus bufonius were investigated.
• 2.2. Two components of ATPase activity are present.
• 3.3. Inclusion of 1 mM ouabain in the incubation media reduced the activity of total and Na⁺/K⁺-ATPase by 57 and 79%, respectively.
• 4.4. The maximum velocity (V_max) was decreased by the addition of 1 mM ouabain, whereas the apparent K_m value was not affected indicating a non-competitive type of inhibition.
• 5.5. The calculated value of the pI₅₀ was 6.4 (I₅₀ = 3.98 × 10⁻⁷M) for ouabain inhibition of the enzyme showing great sensitivity to the cardiac glycoside ouabain.
• 6.6. The present results show that the physicochemical properties of Na⁺/K⁺-transporting ATPase from the brain of P. bufonius are essentially the same as for the enzyme prepared from the excretory system of the insect which has been previously investigated.
• 7.7. Dissimilarities were also observed between these tissues in the way that the enzyme from the brain was sensitive to ouabain inhibition with a non-competitive type rather than a ouabain-resistance and a competitive type of inhibition for the enzyme from the excretory system.
• 8.8. These dissimilarities are probably due to different isoenzyme patterns available in the same insect.

     

Osmolality and electrolyte concentration of hemolymph and the problem of ion and volume regulation of cells in higher insects

• 1.1. The study was carried out on 22 species of insects from 5 orders. The osmolality of their hemolymph varied from 319 to 421 mOsm/kg H₂O, concentration of Na⁺ 4.6 to 118 mM/l, K⁺ 6.3 to 73mM/l, Ca²⁺ 3.6 to 12.9 mM/l, Mg²⁺ 2.3 to 76 mM/l. The most abundant cation in the hemolymph of insects from higher orders is either K⁺ or Mg²⁺.
• 2.2. In the muscles of lower and higher insects K⁺ is usually within 80–120 mM/kg wet wt.
• 3.3. Most Ca²⁺ and Mg²⁺ in hemolymph is bound with protein and low molecular anions, concentration of free Ca²⁺ is 0.9-2.1mM/l Mg²⁺ 3.7–8.0 mM/l.
• 4.4. It is concluded that, in insects, potassium hemolymph, cell volume regulation and accumulation of ions in the cell, are ensured by an increased osmolality of hemolymph due to a high percentage contribution of low molecular organic substances which are retained in the hemolymph due to the absence of filtration apparatus in the Malpighian tubules.

     

Osmoregulation in the brook trout,Salvelinus fontinalis—II. Effects of size,age and photoperiod on seawater survival and ionic regulation

• 1.1. Brook trout (Salvelinus fontinalis) of a single genetic stock, and hatched at the same time, were raised under two photoperiod and two feeding regimes to obtain fish of the same age but with different sizes and photoperiod experiences. In 11 experiments over 1.5 firs, fish were gradually exposed to 32 ppt seawater for 20 days to investigate the ontogeny of salinity tolerance.
• 2.2. Daily changes in plasma osmolarity, [Na⁺], [Cl⁻], [K⁺], [Mg²⁺], thyroxine, hematocrit and gill Na⁺,K⁺-ATPase during adaptation to 10, 20 and 32 ppt were examined in one experiment.
• 3.3. Size was the primary determinant of seawater survival (r² = 0.77) the effect of size on seawater survival slowed after fish reached a fork length of 14 cm. The effect of age on seawater survival (r² = 0.65) was through its covariance with size.
• 4.4. Photoperiod affected seawater survival only through its influence on the timing of male maturation, which decreased salinity tolerance.
• 5.5. Regulation of plasma osmolarity, [Na⁺], [Cl⁻], [K²⁺], [Mg²⁺] and hematocrit in sea water increased linearly with size over the entire range of sizes (6–32 em).
• 6.6. Gill Na⁺,K⁺-ATPase activity after 20 days in seawater decreased with increasing size of brook trout, possibly reflecting decreased demand for active ion transport in larger fish.
• 7.7. Plasma thyroxine concentrations declined in seawater, but no definitive role of this hormone in seawater adaptation was found.
• 8.8. Size dependent survival and osmoregulatory ability of brook trout is compared to other salmonids and a conceptual model is developed.

     

Ca2+ - and Mg2+-dependent ATPase activities in the deoxycholate-treated rat heart sarcolemma

• 1.1. Isolated rat heart sarcolemma was treated with different concentrations of an ionic detergent, deoxycholate (DOC) and ATP hydrolysis in the presence of Ca²⁺ or Mg²⁺ was determined.
• 2.2. Both Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were decreased in the DOC-treated membranes; however, the depression of Mg²⁺-dependent ATPase activity was greater than that of Ca²⁺-dependent ATPase.
• 3.3. The differential changes in Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were apparent when incubations with DOC were carried out for different time intervals and at different temperatures.
• 4.4. In DOC-treated preparations, the K_m value for Ca²⁺-dependent ATPase was decreased whereas that for Mg²⁺-dependent ATPase was increased. The half maximal velocities of the Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase enzyme reactions in the treated preparations were obtained at a DOC: membrane protein ratio of 3.0 and 0.6, respectively.
• 5.5. In the DOC-treated membranes exhibiting the half maximal velocities of enzyme reactions, the K_i value for Ca²⁺-dependent ATPase was drastically reduced but remained unchanged for Mg²⁺-dependent ATPase.
• 6.6. The DOC treatment was associated with a loss of protein as well as phospholipids and resulted in changes in the ultrastructural integrity of the membrane.
• 7.7. Varying degrees of decreases in the activities of sarcolemmal adenylate cyclase. (Na⁻-K⁺)-ATPase. 5'-nucleotidase and calcium binding were seen upon DOC treatment.
• 8.8. The extent of reduction in Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were also different when the membrane was treated with a non-ionic detergent, Lubrol PX.
• 9.9. These data suggest that Ca²⁺-dependent ATPase in heart sarcolemma is more resistant than Mg²⁺-dependent ATPase to detergent treatments and further indicate some differences in the properties of these enzymes.