Guy De Renzis

3 Ion Transport and Gill Atpases

Publisher Summary This chapter describes the ion transport and gill ATPases in the fish gills. The gill epithelium is located between two liquid compartments of very different ionic composition. The gills are not only the site of entry for selected ions essential to life but also for the extrusion of other ions, such as HCO 3 - , NH 3 + , and H + , which are the ionic forms of metabolic by-products. The magnitude of unidirectional fluxes, as well as the degree and direction of net fluxes, is dependent on the equilibrium established between the fish and its environment. Membranes are gently treated with ionic detergents, such as deoxycholate, dodecyl sulfate, or chaotropic agents, to release proteic constituents that are less strongly associated with phospholipids, (Na + , K + )-ATPase remaining in situ . Euryhaline species are an attractive model to compare branchial physiology from the adaptation medium and to relate physiological and biochemical aspects of ionic transport. The Ca 2+ -ATPase is also a membrane ATPase whose role in the cell is essential and evident, but it could also play a role in transepithelial transport and be implicated in the regulation of plasma Ca 2+ concentration.

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.

Ryanodine Activates Sea Urchin Eggs

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 45Ca 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.

Mediating effect of calcium in HgCl2 cytotoxicity in sea urchin egg: role of mitochondria in Ca2+-mediated cell death

HgCl 2 cytotoxicity was studied in sea urchin eggs by simultaneous measurement of Ca 2+ fluxes and egg respiration. It is shown that mercury chloride increases Ca 2+ influx and net flux in a time- and dose-dependent manner. This action is blocked by the Ca 2+ channel inhibitor verapamil. Accumulation of Ca 2+ is prevented by cyanide, suggesting an involvement of mitochondria in mercury toxicity. Indeed, HgCl 2 stimulates the rate of O 2 consumption by mitochondria. This stimulation corresponds to an uncoupling phenomenon, since it is unaffected by oligomycin. This uncoupling is strictly dependent on external calcium, since the mercury effect does not occur in calcium-free artificial sea water. It is suggested that the observed pathological increase of cellular calcium content induced by HgCl 2 is buffered mainly by mitochondria which results in uncoupling. This leads to inhibition of ATP synthesis, and acceleration in cellular damage leading to cell death. Our results stress the key role of plasmic membrane in heavy metal toxicity. Furthermore, they extend the mediating role of calcium in cell injury and cell death to heavy metals.

Structural modifications induced by TPA (12-O-tetradecanoyl phorbol-13-acetate) in sea urchin eggs☆

We investigated the effect of the phorbol ester TPA (12-O-tetradecanoyl phorbol 13-acetate) on the egg morphology of the sea urchin Arbacia lixula. Our study indicates that TPA alters the cortical region of the egg: the pigment granules migrate toward the surface, while cortical granules detach from the plasma membrane. Cortical granule exocytosis did not occur but the endocytosis process was turned on. Prolonged treatment of the eggs by TPA partially inhibits the cortical granule exocytosis normally triggered by fertilization. We discuss the effects of TPA in terms of its interaction with the Ca2+ pool and cytoskeletal structures. In order to discern the respective roles of pHi and protein kinase C activity in endocytosis process activation, we compared the ultrastructural effects of TPA and ammonia. Finally, the role of pigment vesicles in egg metabolism activation is discussed.

Uptake of valine and alanine by a neutral amino-acid carrier in sea urchin eggs: cyclic variations in the early cleavage stage

SummaryThe characteristics of valine and alanine uptake (respectively, preferential substrates of the L and A or ASC systems in mammalian cells) were studied in sea urchin eggs before and 40 min after fertilization. Substrate concentration dependence showed that in unfertilized eggs alanine is absorbed linearly up to an external concentration of 20mm, whereas valine uptake presented a saturable kinetic with aKm of 6 μm. Competition experiments showed that valine is absorbed by a carriermediated transport resembling the L system. Fertilization develops a new Na-dependent system, resembling the ASC system which is specific for neutral amino acids but does not discriminate between them. This system is superimposed on that of the unfertilized egg. In fertilized eggs, amino-acid transport displayed cyclic variations which have been previously associated with cell cleavage. We have found that eggs prevented from cleavage by treatment with antimitotic undergo a sequence of periodic amino-acid uptake timed with the mitotic cycle of untreated eggs. In addition, artificially activated eggs (A23187) which failed to divide showed a time course of amino-acid uptake similar to that observed in fertilized eggs. Furthermore, these variations are independent of protein synthesis. These results suggest to us that a biological clock exists in the cytoplasm or cortex of sea urchin eggs, which may be involved in timing the cell cycle.

Activation of amino acid uptake at fertilization in the sea urchin egg: Requirement for proton compartmentalization during cytosolic alkalosis

The comparative importance of the release of intracellular ionic calcium, Na+/H+ exchange and cytosolic alkalosis as activator signals was studied on the development of amino acid uptake at fertilization in sea urchin eggs. We show that, once stimulated, the rate of valine uptake is greatly dependent upon intracellular pH. Suppression of the Na+/H+ exchange at the time of activation, by applying ionophore (A23187) in sodium-free artificial sea water (ONaASW), inhibits the development of valine influx. This cannot be restored by a further (30 min later) alkalosis by transferring eggs into sea water. Suppressing the alkalosis in the presence of Na+/H+ exchange at fertilization by simultaneous addition of acid into sea water results in activation of the amino acid carrier which exhibits an increased rate of transport as soon as the eggs are replaced in sea water at pH 8.0. The absence of alkalosis in eggs activated in ONaASW can be counterbalanced either by adding NH4Cl 10 mM or by transfer into ASW at pH 9.0 at activation. Ammonia-treated eggs absorbed amino acid as controls, whereas eggs in sea water at pH 9.0 failed to develop a valine uptake system, suggesting that ammonia can completely replace the effect of Na+/H+ exchange. Furthermore, addition of NH4Cl immediately before fertilization conceals the Na+/H+ exchange but stimulates valine uptake as in controls. These data suggest that: the occurrence of the intracellular calcium increase alone is not sufficient for the develpment of the amino acid transport system; cell alkalinization at fertilization derives from the cytoplasmic membrane-located Na+/H+ exchange and an inward movement of protons into a cortical acidic compartment, which is discussed.

Effect of Cytochalasin B on the Development of Membrane Transports in Sea Urchin Eggs after Fertilization

Investigations were made on the role of the cytoskeleton in the onset of ionic events following fertilization of sea urchin eggs. Events which depend upon phosphoinositide metabolism, such as the cortical reantion and acid release are affected by cytochalasin B (CB) after fertilization but not after activation of eggs with the ionophore A23187. These findings suggest that the sequence of events following sperm‐egg attachment depends on the cytoskeleton. CB also inhibits the Na+ pump and alanine uptake when added before insemination and during the following 30 min. These results argue for a role of the egg cortex cytoskeleton in activation of the Na+ pump by fertilization. We propose that the inhibitory effect of CB on the development of amino‐acid uptake after fertilization may result from an increase in the Na+ content of the egg resulting from Na+ pump suppression rather than from direct blockage of the carrier.

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.

Ca2+ release from intracellular stores by thapsigargin in sea urchin eggs: Relationship to larval development and relevance in egg activation

Thapsigargin (Tg), an inhibitor of microsomal Ca2+ ATPase, is used as a tool to study the changes in Ca2+ sequestration in sea urchin eggs and their relationship to embryonic development. Micromolar amounts of Tg inhibit ATP‐dependent Ca2+ sequestration in a dose‐dependent and non‐reversible manner, depending on the bulk of biological material used. IC5O values are 1 nmol/L and 1–10μmol/L, respectively, in the cortical Ca2+ stores (isolated cortices preparation) and in digitonin‐permeabilized eggs, a preparation giving access to the deeper reticulum compartment. Micromolar Tg does not induce Ca2+ release from 45Ca pre‐loaded cortices but leads to a loss of 25% of the total Ca2+ content from the cortical area. Using microspectrofluorimetry of fura‐2‐loaded eggs, we found that 10 μmol/L Tg induced a moderate rise in cytosolic Ca2+ activity as compared with the fertilization‐induced Ca2+ transient whether eggs were fertilized or not. Early events related to fertilization as, for example, elevation of the fertilization envelope, proton excretion and sustained increase of amino acid uptake, are triggered by 10μmol/L Tg but with a delayed onset relative to sperm‐induced effects. The present findings indicate that although it triggers most fertilization‐related events, Tg cannot be considered as a true mitotic agent in sea urchin eggs. When added after fertilization, Tg affects cleavage and the further embryonic development giving rise to abnormalities comparable to the animalized larvae obtained with other compounds responsible for the inhibition of reticular Ca2+ sequestration.