A Cl-/HCO-3-ATPase in the gills of Carassius auratus. Its inhibition by thiocyanate.

An ATPase is demonstrated in plasma membrane fractions of goldfish gills. This enzyme is stimulated by Cl- and HCO-3, inhibited by SCN-. Biochemical characterization shows that HCO-3 stimulation (Km = 2.5 mequiv./l) is specifically inhibited in a competitive fashion by SCN- (Ki = 0.25 mequiv./l). This residual Mg2+-dependent activity is weakly affected by SCN-. In the microsomal fraction chloride stimulation of the enzyme occurs in the presence of HCO-3 (Km for chloride = 1 mequiv/l); no stimulation is observed in the absence of HCO-3. Thiocyanate exhibits a mixed type of inhibition (Ki = 0.06 mequiv./l) towards the Cl- stimulation of the enzyme. Bicarbonate-dependent ATPase from the mitochondrial fraction is stimulated by Cl-, but this enzyme has a relatively weak affinity for this substrate (Km = 14 mequiv./l).

Characterization of valine transport in sea urchin eggs.

In unfertilized eggs, the mechanism of valine uptake can be summarized as follows. It is saturable over the external concentration of valine and insensitive to the presence of external sodium, depletion of cellular energy supplies and intracellular acidosis. The activation energy for the transport reaction (16.3 kcal/mol) is within the range of values reported for active transport of small molecules. In fertilized eggs, the total rate of valine uptake can be divided into two components: (i) a Na+-insensitive uptake which accounts for about 7% of total absorption as shown by studies in Na+-free medium seems to possess the same characteristics as in unfertilized eggs, (ii) a Na+-dependent transport of valine which constitutes the main entry is formed about 5 min after fertilization. It follows Michaelis-Menten kinetics characterized by 15-fold increase in Vmax with no change in Km. These two mechanisms have characteristics in common, such as their insensitivity to metabolic energy supply, their energy of activation and their ability to concentrate valine. The relationship between the establishment of the Na+-dependent valine uptake and the ionic events triggered by fertilization is discussed.

Calcium pools in sea urchin eggs: roles of endoplasmic reticulum and mitochondria in relation to fertilization.

A preparation of sea urchin eggs permeabilized with digitonin (40 microM for 2.5 min) was used to study the kinetic characteristics of the two cellular compartments suspected to play a key role in cellular calcium transfer during fertilization: an ATP-dependent Ca2+ pool (Km = 0.47 microM; Vm = 0.48 nmol/min.mg protein) probably located in the endoplasmic reticulum and a mitochondrial Ca2+ pool (Km = 1.50 microM; Vm = 0.12 nmol/min.mg protein). Fertilization triggered a decrease in the rate of ATP dependent uptake by the non-mitochondrial pool (Km = 0.59 microM; Vm = 0.15 nmol/min.mg protein) while it transiently increased the Ca2+ uptake into mitochondria (2 min post-fertilization: Km = 2.20 microM; Vm = 0.40 nmol/min.mg protein). Microanalysis studies performed on quickly frozen, freeze substituted and embedded eggs showed a transient Ca2+ enrichment of mitochondria soon after fertilization thus suggesting that mitochondria behave as a Ca2+ sink at fertilization. Results are discussed in relation to the role of endoplasmic reticulum and mitochondria in handling free calcium during the early period following sea urchin egg fertilization.

Cl- -HCO3- dependent ATPase in gills of freshwater and seawater adapted eels (Anguilla anguilla L.).

The gills of both seawater and freshwater adapted eels have an ATPase activity which is stimulated by anions in the presence of Mg2+. Plasma membranes were distinguished from mitochondrial membranes with specific enzyme markers, the membrane fractions separated on a discontinuous sucrose gradient, and the ATPase activity of the plasma membranes studied. Activation by the anions of Cl- or HCO3- followed Michaelis-Menten kinetics and was competitively inhibited by SCN-. The Cl- and HCO3- activation characteristics were determined: no differences between the plasma membrane ATPase activities of freshwater and seawater-adapted fishes were observed. Maximal activity measurements after solubilization of the enzymes by Triton X 100 confirmed these findings. The function of a membrane anion-dependent ATPase in the brachial epithelium of euryhaline fish is discussed.

Ryanodine Activates Sea Urchin Eggs: (sea urchin egg/activation/ryanodine/inositol phosphate/heparin).

We have studied the effect on sea urchin eggs of ryanodine, a plant alkaloid that causes muscle contraction by opening calcium channels in the sarcoplasmic reticulum terminal cisternae. Ryanodine, although it is less effective that IP3 , produces full or partial activation in 62% of injected sea urchin eggs. In addition ryanodine inhibits in a dose dependant manner 45 Ca pumping in the isolated egg cortex or in eggs permeabilized with digitonin. Efflux experiments show that in fact ryanodine as IP3 stimulates the release of calcium sequestered intracellularly. We further show that these effects of ryanodine are inhibited by Mg++ , ruthenium red and heparin. Our results suggest that ryanodine-sensitive intracellular calcium channels exist in the sea urchin egg.

Bovine pancreatic trypsin inhibitor and homologous polypeptide inhibitors in nephron cells.

Bovine pancreatic trypsin inhibitor (BPTI, aprotinin) is a fifty-eight amino acid polypeptide, which is present together with related molecular isoforms in various bovine organs. In the present study these protease inhibitors were isolated from bovine kidney by affinity chromatography on immobilized trypsin and a subsequent FPLC step. Due to their electrophoretic, structural, and inhibitory properties, the inhibitors were strictly similar to the polypeptides identified previously in other bovine organs. Immunohistochemical experiments showed a widespread localization of these polypeptides in nephron epithelial cells (proximal and distal tubules, loop of Henle, collecting tubules).

alpha-Bungarotoxin-acetylcholine receptors in the chick ciliary ganglion: effects of deafferentation and axotomy.

alpha-Bungarotoxin (alpha-BuTX) binds in a saturable and practically irreversible fashion to membrane-associated receptors in the ciliary ganglion of the adult chick. The binding of toxin to receptors is competitively inhibited by nicotinic cholinergic ligands, and for these properties the receptors are regarded as acetylcholine receptors of the nicotinic type (alpha-buTX-AChRs). The rate constant of association (K1) and dissociation (K-1) of the toxin-receptor reaction has been estimated to be K1 = 7.4 x 104 M(-1) sec(-1) and K-1 = 9.6 X 10(-6) sec(-1), respectively. Light autoradiography shows that most, if not all, the receptors are related to surface membrane, probably to synaptic areas of both choroid and ciliary neurons. The choroid neurons contain more receptors than the ciliary ones. A single chick ciliary ganglion binds specifically 47 fmole of alpha-BuTX in situ corresponding to 2.83 x 1010 alpha-BuTX-AChRs/ganglion. No changes in number and distribution of the toxin receptors occur following preganglionic denervation. Conversely, postganglionic axotomy causes a rapid disappearance of the receptors in situ. Since binding experiments in vitro revealed a partial, instead of a total, loss of the receptors, it is suggested that the disappearance of the receptors in situ includes both a partial loss of the original receptors and the masking of the residual ones.

HgCl2-induced cell injury. Differential effects on membrane-located transport systems in unfertilized and fertilized sea urchin eggs.

The inhibitory potency of HgCl2 on amino acid and Na+ transport and the mechanism of action of this heavy metal are studied in unfertilized and fertilized sea urchin eggs, in which amino acid transport systems comparable to that described in mammalian somatic cells have been characterized. These transport systems called "L" for leucine, "ASC" for alanine, serine, cysteine, and "A" for alanine are differentiated mainly by their Na+-dependency and by the amino acids transported. The carrier-mediated amino acid uptake is reduced in a dose- and time-dependent manner by HgCl2, with a 50% inhibitory concentration (IC50) in the micromolar range. The mechanisms of inhibition of the Na+-independent (L system) and the Na+-dependent (A or ASC system) transport components are different: in unfertilized eggs, HgCl2 directly interferes with the L amino acid carrier leading to a decrease of its affinity for amino-acids, whereas in fertilized eggs the inhibition of the Na+-dependent uptake of amino acids may result from an elevation of Na+ content induced both by an inhibition of the Na+ pump and by an increase in Na+ permeability. It is also shown that the action of HgCl2 on amino acid diffusion differs between unfertilized and fertilized eggs. Our findings are discussed in the context of the role of membrane in xenobiotic toxicity.

[Immunochemical and immunohistochemical study of calpastatin, an endogenous calpain inhibitor, in the masseter muscle of the rabbit]. / Studio immunochimico ed immunoistochimico della calpastatina, inibitore endogeno delle calpaine, nel muscolo massetere di coniglio.

The calpains-calpastatin system (calcium-activated neutral proteases and endogenous inhibitor) seems to be, in the skeletal muscle, a fine enzymatic system of myofibrillar turnover regulation, in normal as well as pathological conditions (for ex., dystrophic muscle). The purpose of the research is to establish in qualitative and quantitative terms whether the level of calpastatin can evidence differences between a muscle in normal activity conditions and one having dysfunctional alterations experimentally induced. So the masseter muscle of rabbit in normal conditions and with experimentally modified occlusal plane has been used. Our results confirm the presence of the 68 KDa calpastatin in the masseter muscle. The presence of the inhibitor in the same subcellular structures in which the calpains have been detected (myofibrillars, sarcolemma, endomysial connective) has been confirmed. Finally, variations in calpastatin amount in the muscle of animals experimentally treated with respect to the controls have been found. Thus, calpastatin seems to act as a marker of muscle dysfunctions connected to occlusal plane alteration and to loss of vertical dimention.

The branchial chloride pump in the goldfish Carassius auratus: relationship between Cl-/HCO3- and Cl-/Cl-- exchanges and the effect of thiocyanate.

1. The effect of thiocyanate on chloride and sodium fluxes across the gill was studied in the goldfish Carassius auratus. At low external chloride concentrations, addition of SCN- to the bathing solution markedly inhibited chloride influx and efflux, the net flux being reversed, SCN- injection was without effect. SCN- had no effect on sodium fluxes when injected or added to the external medium. 2. The inhibition of chloride influx by SCN- was of a mixed type involving simultaneous modifications of the affinity constant of the carrier for Cl- and of the maximal Cl- influx. The affinity constant of the carrier for SCN- was 10 times lower than that for Cl-. 3. The gill of the goldfish was found to be practically impermeable to SCN-. 4. In the presence of external SCN-, the Cl-/HCO3- exchange was reversed: Cl- was lost against HCO3- which is absorbed. This suggests an obligatory exchange. 5. Exchange diffusion for chloride was also demonstrated. 6. A kinetic model is proposed to explain chloride and bicarbonate transport across the gill of Carassius auratus.

A new technique for the determination of low CO2 concentrations in liquids.

A new method is described by which total CO2 concentration as low as 0.05 mM can be determined in 2 ml samples of liquid. The principle is based on the measurement of CO2 escape from the liquid into the gas phase following acidification and vigorous stirring of samples. This escape is recorded as pressure variations at constant volume and temperature by means of an electronic manometer connected to the reaction vessel. The precision, which depends on the amount of total CO2 present, ranges from 5 to 15 muM.

[Development of some cholinergic components in rat sympathetic ganglion]. / Sviluppo di alcune componenti del sistema colinergico in un ganglio simpatico di ratto.

Postnatal development of some cholinergic components has been studied in the rat superior cervical ganglion. Choline-acetyltransferase, the enzyme localized exclusively at the preganglionic nerve terminals, follows the same developmental pattern of the postganglionic alpha-bungarotoxin-acetylcholine receptors. These results suggest that pre- and postganglionic structures develop simultaneously and parallel. The ontogeny of acetylcholinesterase, a cholinergic enzyme localized at both pre- and postganglionic level, has been also investigated.

[Effect of preganglionic activation on nicotinic cholinergic receptors linked to alpha-bungarotoxin in the rat superior cervical ganglion]. / Effetto della attivazione pregangliare sui recettori colinergici nicotinici leganti alpha-bungarotossina nel ganglio cervicale superiore di ratto.

alpha-Bungarotoxin binds to nicotinic cholinergic sites in the rat superior cervical ganglion. In physiological conditions the receptors are relatively independent upon preganglionic signals, as suggested by experiments of preganglionic denervation. In the present paper we show that the receptors are also uneffected by the afferent nerve fibres when preganglionic activation is induced by Reserpine administration or by cold exposure.

Activation of polyphosphoinositide metabolism at artificial maturation of Patella vulgata oocytes.

The metabolism of polyphosphoinositides (PPI) has been investigated during the meiosis reinitiation of the oocytes of a prosobranch mollusk, the limpet Patella vulgata. Meiosis reinitiation which leads to germinal vesicle breakdown (GVBD) and metaphase-1 spindle formation was artificially induced by treating the prophase-blocked oocytes with 10 mM NH4Cl, pH 8.2. This treatment, which results in a rise in intracellular pH, triggered a general increase in polyphosphoinositide synthesis. Determinations of phosphorus content showed that maturation induced a 30 to 50% increase in both phosphatidylinositol (PI) and phosphatidylinositol-1 monophosphate (PIP) concentrations. Incorporations of 32PO4 and [3H]inositol have been measured in three classes of polyphosphoinositides: PI, PIP, and phosphatidylinositol 4,5-bisphosphate (PIP2). By comparing incorporation rates of the radiolabeled precursors into PPI before and after meiosis reinitiation, we found that artificial maturation by ammonia induced a 50-fold increase in the turnover of these lipids. No significant burst of inositol 1,4,5-trisphosphate (IP3) was observed after maturation. We suggest that modifications in PPI metabolism occurring at maturation of Patella oocytes might ensure the formation of an important stock of PPI that would be available for the profuse production of IP3, the messenger responsible for the Ca2+ signal at fertilization.

Effect of arachidonic acid on Na+/H+ exchange and neutral amino acid transport in sea urchin eggs.

We have investigated the role of arachidonic acid (AA) in regulation of sea urchin egg metabolism activated after fertilization. We show in this paper that addition of exogenous AA to fertilized eggs triggered a transitory stimulation of three ionic events related to the Na+/H+ exchange, H+ excretion, and increase in Nai and in pHi. AA also induced a complete inhibition of neutral amino acid uptake. We found that alterations in this Na(+)-dependent amino acid uptake induced by AA came from modifications in the intracellular sodium concentration. We discuss how AA or derivates may play a role in regulating intracellular free calcium concentration and pH.

Cl--HCO3--ATPase in gills of the rainbow trout: evidence for its microsomal localization.

The intracellular localization of a Cl--HCO3--ATPase, which is inhibited by SCN-, was studied in the gills of the rainbow trout, Salmo gairdneri. This activity can be measured in the absence of contamination by mitochondria (i.e., in the absence of succinate dehydrogenase or cytochrome c oxidase activities). The distribution of the 5'-nucleotidase and of the ATPase stimulated by Cl- and HCO3- after sucrose density gradient centrifugation of the microsomal fraction was compared. Because those activities cannot be separated, it is postulated that the anion-stimulated ATPase is located in the plasma membrane. The activation of this microsomal anion ATPase by chloride has been studied extensively. The possible role of the Cl--HCO3--ATPase of trout gills in the Cl-/HCO3- exchange and in the regulation of the internal acid-base balance is discussed.