Patrick Payan

Primary culture of gill epithelial cells from the sea bassDicentrarchus Labrax

SummaryWe have developed the first explant technique that allows the in vitro study of gill physiology and biochemistry in marine species. Gill fragments were cultured at 17° C, in atmospheric Pco2, with nutrient medium (Leibovitz L15), pH 7.8, supplemented with 10% fetal bovine serum and adjusted to the osmolarity of fish plasma (350 mOsm/liter). Coating plates with collagen, gelatin, or polylysin did not improve our results. Decrease in osmotic pressure, removal of bovine serum, or its replacement by fish serum inhibited growth from the explants. Approximately 50% of the explants produced cell growth, and after 4 days of culture a monolayer of contiguous cells was formed. This technique is rapid and does not require the use of enzymes. The cells appeared flat and thin with an epithelioid shape. They looked polygonal with a maximum length of 10 to 50 µm. Evidence that they are unique gill cells is the presence of polymorphic surface crenelations (microplicae), prominent Golgi apparatus, tight junctions and desmosomes. Comparison with in vivo tissue showed them to be epithelial cells having differentiated in a homogeneous population of respiratorylike (pavement) cells. They are polarized with their apical surface facing the culture medium. The development of this culture system represents a new tool for cellular approaches to determine precisely the functions and transport mechanism of gill cells.

Mechanisms regulating intracellular pH in sea urchin eggs

Intracellular pH (pHi) of sea urchin eggs (Paracentrotus lividus) was determined using DMO (dimethyloxazolidinedione) and a rapid filtration technique (P. Payan, J.P. Girard, R. Christen and C. Sardet (1981). Exp. Cell Res. 134, 339-344). Transfer of unfertilized or fertilized eggs from normal sea water into Na+-free artificial sea water leads to a progressive acidification and fall of intracellular Na+ content. A step rise in external Na+ to 10 meq causes a rapid but transient Na+ entry coupled to an excretion of H+, giving rise to a pHi increase. It is shown that the plasma membrane of unfertilized eggs contains a permanent and reversible Na+/H+ exchanger which contributes to the regulation of pHi. This exchange occurs with a 1:1 stoichiometry and is independent of metabolic energy. Proton excretion and sodium entry follow saturable kinetics with respect to external Na+ and are completely inhibited by amiloride. At fertilization, pHi increases from 7.38 to 7.64 and is maintained at this level by two separate mechanisms: (1) a Na+/H+ exchange with the same characteristics as in unfertilized eggs; (2) a H+-excreting system that is dependent on external Na+, amiloride sensitive, and requiring metabolic energy. The relationship between the permanent Na+/H+ exchange involved in pHi regulation and the transient Na+/H+ exchange occurring at fertilization is discussed.

Biochemical relationships between endolymph and otolith matrix in the trout (Oncorhynchus mykiss) and turbot (Psetta maxima).

This paper compares the organic compositions of the otolith and endolymph of trout and turbot. Irrespective of the method of demineralization (0.5 M EDTA or acetic acid), trout otoliths were found to be largely composed of proteins (48%), collagens (23%), and proteoglycans (29%). Collagen was only detectable in the EDTA-insoluble (0.30 microg/mg) and in the acetic acid-soluble fractions (0.53 microg/mg). The same compounds were found in the endolymph but in different proportions (proteins 85%, collagens 12%, and proteoglycans 3%). It was shown that the distribution of these compounds was not uniform within the endolymph. Proteins, collagens, and amino acids were 4, 10, and 3 times, respectively, more concentrated in the proximal (facing the macula) than the distal side whereas proteoglycans were 10 times more concentrated at the distal side. SDS PAGE analyses of proximal and distal samples of endolymph showed similar patterns suggesting that the spatial gradient of protein is quantitative and not qualitative. SDS PAGE comparison of endolymph and otolith samples showed only two proteins with similar molecular weights. We propose that collagen and protein gradients are involved in the organic matrix formation and otolith calcification process. Endolymphs from both trout and turbot display inhibitions of in vitro calcification although these inhibitions were 50 and 80 times, respectively, less than that of the otoliths. The inhibitory factor probably plays a significant role in the regulation of otolith calcification.

Chemical composition of saccular endolymph and otolith in fish inner ear: lack of spatial uniformity

Fish otoliths provide a record of age, growth, and environmental influences. In both trout and turbot, spatial chemical investigation of the endolymph surrounding the otolith (sagitta) showed a lack of uniformity. Proteins, PO(3-)(4), and Mg(2+) were significantly more concentrated in the proximal (facing the macula) than distal zone, whereas the opposite was observed for K(+) and total CO(2) (totCO(2)). Na(+) concentration ([Na(+)]) was 20% higher in the proximal zone in trout but not in turbot. Total Ca and Cl(-) contents were uniformly distributed in both species. We propose that the endolymphatic gradients of protein and totCO(2) concentration within the endolymph are involved in the otolithic biocalcification process. Microchemical analyses of otolith sections by wavelength dispersive spectrometry showed a lack of spatial uniformity in the K/Ca and Na/Ca ratios, whereas the Sr/Ca ratio was uniform. There is a clear relationship between endolymph and otolith [K(+)], but the interpretation of the results for [Na(+)] needs further investigation. Thus the lack of uniformity in the otolith composition must be taken into account when investigating otolith microchemistry.Fish otoliths provide a record of age, growth, and environmental influences. In both trout and turbot, spatial chemical investigation of the endolymph surrounding the otolith (sagitta) showed a lack of uniformity. Proteins, [Formula: see text], and Mg2+ were significantly more concentrated in the proximal (facing the macula) than distal zone, whereas the opposite was observed for K+ and total CO2(totCO2). Na+ concentration ([Na+]) was 20% higher in the proximal zone in trout but not in turbot. Total Ca and Cl- contents were uniformly distributed in both species. We propose that the endolymphatic gradients of protein and totCO2 concentration within the endolymph are involved in the otolithic biocalcification process. Microchemical analyses of otolith sections by wavelength dispersive spectrometry showed a lack of spatial uniformity in the K/Ca and Na/Ca ratios, whereas the Sr/Ca ratio was uniform. There is a clear relationship between endolymph and otolith [K+], but the interpretation of the results for [Na+] needs further investigation. Thus the lack of uniformity in the otolith composition must be taken into account when investigating otolith microchemistry.

Initial effect of sodium bicarbonate on intracellular pH depends on the extracellular nonbicarbonate buffering capacity.

ObjectiveThe effect of sodium bicarbonate on intracellular pH under conditions close to those in vivo, with both bicarbonate and nonbicarbonate buffering systems, is unknown. We postulated that this effect depends on the nonbicarbonate buffering capacity because the alkali-induced back-titration of these buffers results in a concentration-dependent release of CO2 in the extracellular space, leading to a decrease in intracellular pH. DesignThe study was conducted in two stages. First, human hepatocytes were perfused with pH 7 bicarbonate-buffered medium (5 mM HCO3−, 20 torr Pco2) containing no nonbicarbonate buffer or small amounts (5 mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid [HEPES]) or large amounts (20 mM HEPES) of nonbicarbonate buffer. Second, the changes in intracellular pH of hepatocytes placed in acidotic human blood (pH 7, 5 mM HCO3−, 20 torr Pco2) at three hematocrits (40%, 20%, and 5%) were measured. SettingResearch laboratory at a medical university. SubjectsCryopreserved human hepatocytes thawed the day before the experiments. InterventionsSodium bicarbonate was infused for 10 mins to increase the HCO3− concentration from 5 to 30 mM. In the second part, 20 mM sodium bicarbonate was added directly to the blood bathing the cells. Measurements and Main Results The intracellular pH was measured with the pH-sensitive fluorescent dye bis-carboxyethyl carboxy-fluorescein in its esterified form, acetoxy-methyl ester, by using a single-cell imaging technique. Gas analyses were performed before and during the sodium bicarbonate load. Sodium bicarbonate caused a decrease in intracellular pH with all media except the artificial medium containing no HEPES. This decrease was small in media with low nonbicarbonate buffering capacity (5 mM HEPES and 5% hematocrit blood) and large in media with high nonbicarbonate buffering capacity (20 mM HEPES and 40% hematocrit blood). The change in intracellular pH was linked closely to the change in Pco2 caused by the sodium bicarbonate. ConclusionsThe effect of sodium bicarbonate on intracellular pH depends on changes in Pco2 in the medium bathing the cells. The increase in Pco2 is correlated with the extracellular nonbicarbonate buffering capacity because of the release of H+ ions coming from the back-titration of these buffers. We conclude that sodium bicarbonate may exacerbate cell acidosis under buffering conditions close to those in vivo and that the initial changes in cell pH caused by sodium bicarbonate depend on the extracellular nonbicarbonate buffering capacity.

DISTRIBUTION OF IONOCYTES IN THE SACCULAR EPITHELIUM OF THE INNER EAR OF TWO TELEOSTS (ONCORHYNCHUS MYKISS AND SCOPHTHALMUS MAXIMUS)

Abstract.The saccular membranes of trout (Oncorhynchus mykiss) and turbot (Scophthalmus maximus) were examined to characterize specialized epithelial cells that might be responsible for ion exchange. The approach for localizing cell types was new for this tissue, as observations were made with a stereomicroscope and a light microscope in order to have a general view of the epithelium. No important differences between the two species were seen. The saccular tissue is a monolayer epithelium (except for the macula neural zone) surrounded by a layer of connective tissue invaded by many blood vessels. The use of the fluorescent probe DAPSMI and zinc iodide/osmium fixation-coloration defined two areas in which ionocytes were present. In the first, large ionocytes were grouped into a nearly complete, crowned meshwork around, but separated from, the macula. In the second area, opposite the macula, the ionocytes were smaller, cubical, and grouped in patches. Cells rich in Na+, K+-ATPase and carbonic anhydrase II were present in both areas. Contrary to previous studies in mammals and fish, ionocytes were also found in the epithelium of the saccule.

Changes in intracellular cations following fertilization of sea urchin eggs: Na+H+ and Na+K+ exchanges

Sodium and potassium contents, as well as H+ excretion were measured at various times after fertilization in eggs of the sea urchin Paracentrotus, using a rapid filtration technique (Payan, P, Girard, U P, Christen, R & Sardet, C, Exp cell res 134 (1981) 339 [1]). Sodium content changes with a biphasic time course due to the appearance of two exchange mechanisms: 1. A transient Na+/H+ exchange with a l/l stoichiometry causes a rapid rise in intracellular sodium. This exchange lasts about 3 min. and is not inhibited by poisoning the eggs with NaCN. 2. An Na+/K+ exchange, detectable within minutes of fertilization causes sodium content of the egg to fall and settle below unfertilized level. This exchange is energy-dependent, reduced in low K+ (2 mM), totally inhibited by harmaline (10(-4) M) and only partially inhibited by ouabain (10(-2) M). Potassium content rises rapidly after fertilization by an undefined mechanism and is further increased via the Na+/K+ exchange. The Na+/Ka+ exchange increases the cytoplasmic K+/Na+ ratio of the egg from 6 to 12 in the first hour after fertilization. When fertilization occurs in low K+ SW, where the Na+/K+ exchange is reduced, the ratio Na+/K+ decreases and the embryos develop with a preponderance of ectodermal structures (animalization).

Na+ movements and their oscillations during fertilization and the cell cycle in sea urchin eggs.

Abstract We have analysed in detail the Na + content and Na + influx during fertilization and first divisions of the sea urchin egg ( Paracentrotus lividus ) using a filtration technique devised to eliminate rapidly contamination by the Na + of external sea water. In the first 5 min following fertilization the egg fills up with Na + (+ 30%). Thereafter Na + is extruded and the Na + content stabilizes at about 60% of the unfertilized egg level by the second cleavage (2 h). The initial increase in Na + content is due to a large increase in Na + influx already detected at 20 sec. The Na + influx reaches its maximum at 1 min and its minimum at 5 min. H + excretion follows the same kinetics. A second increase in Na + influx is noted 5–10 min after fertilization; it reaches its maximum at prophase metaphase (30 min) and its minimum during cleavage (60 min). These oscillations in Na + influx were observed for the first three divisions. Fertilization also immediately stimulates the Na + efflux which remains elevated throughout the cell cycle and is responsible for the depletion of the Na + content of the embryos. Activation of the eggs by weak amine bases (5 mM NH 4 Cl) which bypasses the early cortical reaction produces only a depletion in the Na + content of the egg similar to that produced by fertilization. NH 4 Cl also increases the Na + influx soon after fertilization, although no transient variations are noted.

Composition of Biomineral Organic Matrices with Special Emphasis on Turbot (Psetta maxima) Otolith and Endolymph

The soluble organic matrix (OM) of various biominerals (red coral skeleton, oyster shell, sea urchin test, turbot otolith, chicken eggshell) was extracted after demineralization with acetic acid. The protein content of the OM varies strongly from 0.02 to 1.6 µg/mg biomineral whereas proteoglycans present less variations (from 0.7 to 1.4 µg/mg biomineral). Electrophoresis of biominerals OM shows differences in their protein pattern although several bands are present in all matrices. OM of all biominerals shows carbonic anhydrase activity but no activity was detectable in the endolymph. OM of all biominerals also displays an anticalcifying activity. After separation of the OM extracts by chloroform-methanol, 80% of the anticalcifying activity was found in the methanol phase except in the urchin test. After OM precipitation with trichloracetic acid, 70% of the activities was found in the supernatants. Partial biochemical characterization suggests that the anticalcifying factor is a polyanionic and water-soluble molecule, which could be proteoglycans. The endolymph surrounding the otolith also displays an anticalcifying activity although its inhibitous activity was 50 times lower than that of the otolith OM. However, the anticalcifying activity of the endolymph is assumed by a proteic structure (80% activity precipitated with TCA treatment). Our results suggest that both carbonic anhydrase and anticalcifying activities are widespread and play a significant role in the regulation of biomineral formation. Results are discussed in relation to the calcification process that takes place at the fluid-mineral interface.

Daily variations of endolymph composition: relationship with the otolith calcification process in trout

SUMMARY Ionic and organic parameters of the otolith calcification process in the trout Oncorhynchus mykiss were analysed in plasma and endolymph over the day:night cycle. Plasma pH remained constant and total CO2 concentration was significantly lower (by 21%) during the day than at night. Calcifying parameters (total CO2, total calcium concentration) were measured in the proximal and distal endolymphs and were unchanged in the latter during the day:night cycle, but fluctuated in the former. Non-collagenous protein and collagen concentrations in endolymph were higher (1.5- and 10-fold, respectively) during the day than at night. As there was no change in total calcium concentration, we propose that Ca2+ increases during the dark period and was maximal by the end of the night when the total CO2 concentration has also increased (by 14%). Measurements of endolymph pH in situ revealed significant differences between samples from proximal and distal endolymph (7.38 and 7.87, respectively), but no variation between values obtained during the day and at night. Thus, the saturation state of aragonite (Sa) in the proximal endolymph should fluctuate around unity during the day:night cycle, and CaCO3 precipitation should occur when supersaturation is reached. The electrophoretic pattern of proximal endolymph showed variations in both major and minor components. Immunoblotting of endolymph, using a rabbit antiserum raised against the otolith soluble organic matrix revealed an increase in the expression of two proteins (65 kDa and 75 kDa) during the day period. We propose that organic matrix and calcium carbonate deposition on the otolith vary antiphasically: organic matrix deposition begins by the end of the day period, when the concentration of organic precursors is maximal in the endolymph, whereas CaCO3 precipitation starts once the solubility of CaCO3 is exceeded.