Effects of luminal osmolarity on gastric acid secretion in the little skate,Raja erinacea

Summary Isolated gastric mucosa of the skate shows marked changes in acid secretory rate (J _H ), electrical potential difference (PD), and transepithelial resistance (R) with changes in mucosal bathing solution composition and a constant serosal solution. Removal of the 350 mM urea usually present in the mucosal solution reduces acid secretory rate by 25%, while adding urea to 1 M has no significant effect. Complete removal of osmotic solutes (distilled water) inhibits secretion by 78%, isotonic urea (no salts) inhibits by 54%, while isotonic salts alone (no urea) gives control secretory rates. The changes in PD and R are consistent with acid secretory changes. Theory and experience with terrestrial organisms would not predict these changes. The most likely explanation is osmotic swelling and shrinking of the surface cells, and occlusion of the secretory tubules in the swollen condition. Since marine species never encounter hypo- or hyperosmotic conditions due to food ingestion, their surface cells may be water permeable, unlike the situation in terrestrial and fresh water animals.Abbreviations J _H acid secretory rate per square centimetre tissue area - OC oxyntic cell - PBC pit border cell - PD transepithelial electrical potential difference - R transepithelial electrical resistance per square centimetre tissue area - SEC Surface epithelial cell     

Neuropeptide Y-like immunoreactivity in the cardiovascular nerve plexus of the elasmobranchs Raja erinacea and Raja radiata

Summary The distribution of nerves showing neuropeptide Y (NPY)-like immunoreactivity in the cardiovascular system of elasmobranchs and teleosts has been investigated. Two species of teleosts, the rainbow trout (Salmo gairdneri) and the Atlantic cod (Gadus morhua), and three species of elasmobranchs, the spiny dogfish (Squalus acanthias), the little skate (Raja erinacea) and the starry ray (Raja radiata), were used in this study. An innervation of the cardiovascular system by an NPY-like substance was found only in the two species of Raja. A rich innervation was encountered in these skates, with the highest density of fibres in the wall of the ventricle, the conus arteriosus, the coeliac artery and smaller mesenterial vessels. In the vessels, the fibres formed a plexus at the adventitio-mediol border. Few fibres were found in the walls of the dorsal aorta, the sinus venosus and the atrium, and no fibres were observed in the walls of the ventral aorta. Falck-Hillarp fluorescence histochemistry showed the presence of a rich innervation of arteries and arterioles of the gut by catecholamine-containing nerve fibres.     

Site-specific variation in amino acid composition of skate egg capsule (Raja erinacea Mitchell, 1825)

In skate (Raja erinacea Mitchell, 1825) egg capsule, variations in amino acid composition, notably glycine and hydroxyproline, were found to be related to morphologically distinct sites. Components sharing elution characteristics with cross-linking amino acids in collagen and elastin were detected and the relative amounts of these peaks also displayed regional variation. If the skate egg capsule contains collagen, these results suggest it is associated with differing amounts of other protein(s). The varying relative proportions of collagen and non-collagenous protein is predicted to contribute to site-specific differences in physico-chemical properties of the egg capsules.     

Identification of vitellogenin in the little skate (Raja erinacea).

1. Vitellogenin was isolated from mature female skates by selective precipitation with MgCl2/EDTA followed by chromatography on DEAE-cellulose columns. 2. A single monomer of approximately 205 kDa was identified on 6.0% SDS-PAGE gels. 3. In addition, isolation of yolk proteins with ammonium sulfate yielded proteins of 94 and 38 kDa (putative phosvitins) and putative lipovitellins of ca 105, 91 and 67 kDa. 4. In vivo phosphate incorporation in female and male skates implanted with estradiol indicated that vitellogenin was phosphorylated. 5. Total protein phosphate incorporation was significantly higher in females than male skates. 6. In male skates treated with estradiol, phosphate incorporation increased from 2 days after implantation to a maximum at approximately 11 days after implantation. 7. Determination of the rate of disappearance of 32P-labeled protein suggests a half-life of ca 200 hr in normal female skate plasma.     

Beta-alanine transport in the isolated hepatocytes of the elasmobranch Raja erinacea

Cells were isolated from the liver of the skate and the uptake of beta-alanine followed using [14C]-beta-alanine. The isolated hepatocytes showed good viability, were found to accumulate beta-alanine from the incubation medium, and did so in a manner indicating a transport system involving a saturable carrier. The data for the rate of beta-alanine uptake suggest that this may be a rate-limiting step in the oxidation of the amino acid by the liver. Experiments indicated that the transport system could distinguish beta-alanine from certain structurally similar molecules (L-alanine and taurine, but not gamma-amino butyrate). Cells isolated from fish adapted to a diluted environment (50% seawater) showed no significant change in the uptake rate. However, evidence indicates that, over the range of beta-alanine concentrations occurring in the fish, the uptake rate would be acutely sensitive to small changes in the concentration in the blood, thus forming a self-regulating system for the metabolism of beta-alanine.     

scyllo-inositol and myo-inositol levels in tissues of the skate Raja erinacea.

1. Many organs of the skate Raja erinacea have been found to contain scyllo-inositol levels that are much higher than myo-inositol, the opposite to that found in mammals. 2. Both inositols were found in all skate organs studied. 3. myo-Inosose-2 was found to accompany the inositols in many of these organs.     

The hepatic glutathione transferases of the male little skate, Raja erinacea

Five cytosolic glutathione transferases were isolated from the liver of the male little skate, Raja erinacea, a marine elasmobranch. They were designated E-1 through E-5 in order of their elution from a DEAE-cellulose column with a 0 to 100 mM KCl gradient in 0.01 M Tris (pH 8.0). Each eluted peak of glutathione transferase activity, after concentration, was applied to an affinity column prepared by reaction of epoxy-activated Sepharose 6B with glutathione (GSH). Elution of the various glutathione transferases from this column with GSH resulted in the further purification of each enzyme; the major glutathione transferase, E-4 and E-1, were purified to apparent homogeneity by this procedure. Skate glutathione transferase E-4 is dimeric and the subunits are either very similar or identical in molecular weight (about 26 000 daltons). Enzymes E-2 through E-5 were acidic proteins (pI less than 7.0) and had high specific glutathione transferase activity (0.3--12 mumol/min/mg protein) with benzo[a]pyrene 4,5-oxide (BPO) as substrate, whereas the other enzyme (E-1) had low activity (0.01 mumol/min/mg) with BPO and a basic pI (greater than 9.5). Bilirubin and hematin, non-substrate ligands, bound tightly to homogeneous E-4, with dissociation constants in the micromolar range.     

Immunoglobulin heavy chain gene organization and complexity in the skate, Raja erinacea

Immunoglobulin heavy chain genes from Raja erinacea have been isolated by cross hybridization with probes derived from the immunoglobulin genes of Heterodontus francisci (horned shark), a representative of a different elasmobranch order. Heavy chain variable (VH), diversity (DH) and joining (JH) segments are linked closely to constant region (CH) exons, as has been described in another elasmobranch. The nucleotide sequence homology of VH gene segments within Raja and between different elasmobranch species is high, suggesting that members of this phylogenetic subclass may share one VH family. The organization of immunoglobulin genes segments is diverse; both VD-J and VD-DJ joined genes have been detected in the genome of non-lymphoid cells. JH segment sequence diversity is high, in contrast to that seen in a related elasmobranch. These data suggest that the clustered V-D-J-C form of immunoglobulin heavy chain organization, including germline joined components, may occur in all subclasses of elasmobranchs. While variation in VH gene structure is limited, gene organization appears to be diverse.     

Isolation and characterization of a polarized isolated hepatocyte preparation in the skate Raja erinacea

Hepatocytes of the small skate (Raja erinacea) were isolated by collagenase perfusion and evaluated by a variety of functional and morphologic criteria. Cell yield was 1.45 X 10(8) +/- 1.3 X 10(7) cells per isolation, and as long as 8 h after isolation 98% of the hepatocytes excluded Trypan blue and no leakage of lactate dehydrogenase (LDH) or cell associated potassium could be detected. Oxygen consumption averaged 1.6 +/- 0.5 nmol/min/mg cell protein, was not stimulated by 1 mM succinate, and also remained stable for up to 8 h following isolation. However, 2,4,-dinitrophenol (5 X 10(-5) M) produced a 55% increase in oxygen utilization while ouabain, (1 mM) or sodium removal decreased oxygen consumption by 31 +/- 6 and 33 +/- 7%, respectively, indicating that a significant portion of the cells energy utilization is coupled to the activity of plasma membrane Na+, K+-ATPase. Light microscopic studies showed that the individual hepatocytes had diameters of 28 +/- 5 microns and contained large lipid droplets. Electron microscopy revealed groups of three to five cells with normal ultrastructure and tight junctions and desmosomes surrounding a single bile canaliculus. These studies indicate that skate hepatocytes can be isolated in high yield that retain their structural polarity in the form of clusters of cells formed around a single bile canaliculus. These hepatocytes remain morphologically intact and metabolically stable for a prolonged period of time.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical and morphological differences in the kidney zones of the elasmobranch, Raja erinacea mitch

The histological investigation of the kidney of the skate Raja erinacea revealed a thin cap of dorsal bundles, which contain segments of single nephrons that are arranged separately in a countercurrent manner, and a large ventral zone, where the second proximal segments (PII) and parts of the lower nephron are located. This zonation is apparent in fresh, unfixed material and makes it possible to separate small tissue samples under a dissecting microscope. The osmolality in both zones does not differ. The dorsal bundle zone had a lower urea concentration and a higher sodium concentration than the ventral zone. The differences in the mean concentrations of the tissue samples indicate a gradient for the two substances along the bundles. Determinations of amounts of water and solutes per mg solute-free, dry tissue of the two zones, showed that the amounts of water, total osmolytes, Na and K were greater in the bundle zone than in the ventral zone, while the amount of urea was identical in the two zones. This indicates that the lower urea concentration in the bundle zone is established through an accumulation of Na and water in the interstitium. The countercurrent arrangement of very early and late segments of single renal tubules supports the concept of passive reabsorption of urea in the kidney of the marine elasmobranch.     

Barrier membranes at the outer surface of the brain of an elasmobranch,Raja erinacea

Summary This report gives the results of the first electron-microscopic examination of the cell layers covering the outer brain surface and the inner surface of the cartilaginous skull in the skate, Raja erinacea. The perivascular glial blood-brain barrier — a characteristic of elasmobranchs — extends to the outer surface of the brain. This outer barrier layer is surrounded, in turn, by a subarachnoid compartment (depth: 30–40 m), containing loose connective tissue and blood vessels; by an arachnoid-like epithelium (10–15 cell layers), impermeable to horseradish peroxidase; and, by perimeningeal fluid, a fluid with a slow turnover rate and a protein composition different from plasma. The inside of the skull, facing the perimeningeal fluid, is covered by a multilayered (10–15 layers) cuboidal epithelium, also impermeable to horseradish peroxidase. Closely apposed cells in the luminal layer of this epithelium have apical microvilli and numerous vesicular profiles, containing material of moderate electron density. These observations may explain, in terms of structure, the regulated protein content of perimeningeal fluid and the restricted exchange of solutes between brain and perimeningeal fluid in elasmobranchs.     

Effects of L-alanine on membrane potential, potassium (86Rb) permeability and cell volume in hepatocytes from Raja erinacea

Isolated hepatocytes from the elasmobranch Raja erinacea were examined for their regulatory responses to a solute load following electrogenic uptake of L-alanine. The transmembrane potential (Vm) was measured with glass microelectrodes filled with 0.5 M KCl (75 to 208 M omega in elasmobranch Ringer's solution) and averaged -61 +/- 16 mV (S.D.; n = 68). L-Alanine decreased (depolarized) Vm by 7 +/- 3 and 18 +/- 2 mV at concentrations of 1 and 10 mM, respectively. Vm did not repolarize to control values during the 5-10 min impalements, unless the amino acid was washed away from the hepatocytes. The depolarizing effect of L-alanine was dependent on external Na+, and was specific for the L-isomer of alanine, as D- and beta-alanine had no effect. Hepatocyte Vm also depolarized on addition of KCN or ouabain, or when external K+ was increased. Rates of 86Rb+ uptake and efflux were measured to assess the effects of L-alanine on Na+/K+-ATPase activity and K+ permeability, respectively. Greater than 80% of the 86Rb+ uptake was inhibited by 2 mM ouabain, or by substitution of choline+ for Na+ in the incubation media. L-Alanine (10 mM) increased 86Rb+ uptake by 18-49%, consistent with an increase in Na+/K+ pump activity, but had no effect on rubidium efflux. L-Alanine, at concentrations up to 20 mM, also had no measurable effect on cell volume as determined by 3H2O and [14C]inulin distribution. These results indicate that Na+-coupled uptake of L-alanine by skate hepatocytes is rheogenic, as previously observed in other cell systems. However, in contrast to mammalian hepatocytes, Vm does not repolarize for at least 10 min after the administration of L-alanine, and changes in cell volume and potassium permeability are also not observed.     

Effects of dehydration on water relations and survival of lumbricid earthworm egg capsules

Earthworm egg capsules of five species were compared with regard to survival and water relations upon exposure to controlled dehydration at 20°C. Cocoons of the investigated species all contained about 3.5 g water·g^-1 dry weight when fully hydrated. Approximately 18% of this does not readily freeze upon cooling to -40°C and is referred to as osmotically inactive water. Cocoons exposed to desiccation lose a large proportion of the osmotically active water over 1–4 days until water in the cocoon fluids has equilibrated with surrounding water vapour. The amount of osmotically inactive water, on the other hand, is only reduced by 10–20%. Dendrobaena octaedra was the species most tolerant to drought, its tolerance limit coinciding with loss of practically all osmotically active water. For the five species investigated, there seemed not to be any clear correlation between drought tolerance and microhabitat. Previous investigations have suggested a very close relation between tolerance to dehydration and to subzero temperatures in overwintering earthworm cocoons. Survival at a given level of dehydration at room temperature is less than at temperatures below 0°C, and the tolerance of room temperature dehydration is not closely correlated with cold hardiness across the range of the species studied.Abbreviations dw dry weight - DSC differential scanning calorimetry - fw _pd fresh weight of partially dehydrated cocoons - OAW osmotically active water - OIW osmotically inactive water - Osm osmolality - water potential - R universal gas constant - T absolute temperature - V specific volume of water     

The excretion pattern of biliverdin and bilirubin in bile of the small skate (Raja erinacea)

Summary Bile pigment composition (biliverdin, bilirubin and their conjugates) was analyzed in stored gallbladder bile and newly synthesized hepatic bile from the small skate (Raja erinacea). During a five day period of captivity, gallbladder volume remained relatively constant while bilirubin and biliverdin content increased two to three fold. Biliverdin which accounted for 50% of the pigments did not increase as a percentage of tetrapyrroles during this period. The relative proportion of bilirubin and its conjugates also remained constant, averaging 65% for bilirubin monoglucuronide, 30% for bilirubin diglucuronide and 5% for unconjugated bilirubin as measured by HPLC methods. Intravenous administration of biliverdin resulted in significant increases in the biliary excretion of both biliverdin and all bilirubin tetrapyrroles. Insignificant quantities of³H-biliverdin were detected in hepatic bile following the intravenous administration of³H-bilirubin. These studies indicate that the small skate excreted both biliverdin and bilirubin conjugates in bile and that the biliverdin was not produced by in vitro oxidation of bilirubin or its metabolites.     

The Hearing Sensitivity of the Little Skate, Raja erinacea: A Comparison of Two Methods

We determined the hearing sensitivity of the little skate, Raja erinacea using two methods: Behavioral conditioning and the auditory brainstem response (ABR). This marks the first time that the hearing in any member of the Rajiformes has been examined and the first time that the ABR method has been used with an elasmobranch. We obtained audiograms of R. erinacea using each method and were found to be statistically similar. The best hearing sensitivity for R. erinacea was between 100 and 300Hz. We compared the audiograms to audiograms obtained from other species of elasmobranchs. This analysis showed that R. erinacea, a bottom-dwelling elasmobranch, has less sensitive hearing than the lemon shark, Negaprion brevirostris, and the bull shark, Carcharhinus leucas, a free-swimming, raptorial elasmobranch. However, R. erinacea showed sensitivity comparable to that of the horn shark, Heterodontus francisi, another bottom-dwelling elasmobranch; both species feed primarily on benthic prey. These findings are in agreement with Corwin's hypothesis (1978) that hearing sensitivity is correlated with feeding behavior. An examination of the macula neglecta of R. erinacea found a total count of 10000 hair cells, which is within the range of other bottom-dwelling elasmobranchs.     

Isolation of Raja erinacea basolateral liver plasma membranes: characterization of lipid composition and fluidity.

Developing a method for isolating skate (Raja erinacea) basolateral liver plasma membranes, as well as characterizing the lipid composition and fluidity of these membranes, was the primary purpose of this study. Membranes were isolated using self-generating Percoll gradients. Marker enzyme studies indicate that this preparation is highly enriched in the basolateral domain of the liver plasma membrane and largely free of contamination by intracellular organelles or canalicular membranes. Further, these membranes contain the agency responsible for Na(+)-dependent alanine transport. This finding indicates that this membrane preparation will be useful for the study of skate liver plasma membrane transport processes. The lipid composition and fluidity (as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene) of the skate basolateral liver plasma membrane shows little variation among preparations. Further, DPH anisotropy plotted as a function of temperature yields a straight line (r = 0.99) which indicates that there is no lipid phase change in these membranes from 4 degrees to 37 degrees C. The membrane preparation does contain substantial phospholipase A2 activity. The function of this enzyme is, in part, to modify membrane lipid composition and fluidity in response to temperature variations; therefore, this finding suggests that in situ lipid metabolizing enzymes may play a central role in the adaptation of skate basolateral liver plasma membranes to changes in the ambient temperature.     

Bilirubin metabolism in the spiny dogfish, Squalus acanthias, and the small skate, Raja erinacea.

1. The main bilirubin conjugate in bile of spiny dogfish (Squalus Acanthias) and small skate (Raja Erinacea) is bilirubin monoglucuronide. 2. Microsomal preparations from dogfish and small skate liver have similar bilirubin UDPglucuronyltransferase (UDPGT) activity and catalyze the conjugation of bilirubin with glucose from UDPglucose. 3. The activity of bilirubin glucosidation (UDPGT) was 0.5 times UDPG1T activity in dogfish and 0.15 times in skate liver microsomes. 4. Sodium cholate increased UDPGT and UDPG1T activities in dogfish and skate liver microsomal preparations only minimally, but the detergent markedly increased thermolability of UDPGT in skate liver microsomes.     

Solute effects on mitochondria from an elasmobranch (Raja erinacea) and a teleost (Pseudopleuronectes americanus)

Varying osmolarity with sucrose/KCl media resulted in similar effects on the oxidation of glutamate by mitochondria isolated from the livers of an elasmobranch, Raja erinacea, and a teleost, Pseudopleuronectes americanus. In both species trimethylamine oxide (TMAO) inhibited mitochondrial oxidation of glutamate. Urea penetrated the inner mitochondrial membrane of both species and equilibrated with a ratio ureai/ureao of unity. Urea had little effect on the oxidation of glutamate in both species at concentrations as high as 760 mM. Addition of urea (urea/TMAO, 2:1) did not overcome the detrimental effects of TMAO in the mitochondria of either species. In the case of the elasmobranch, the osmolarity of the urea/TMAO media giving the optimal rate of respiration was hypoosmotic with respect to the intracellular osmolarity. The rate of glutamate oxidation steadily declined as osmolarity increased above this value. Assuming the osmotic profile obtained with the urea/TMAO (2:1) medium resembled most closely the in vivo situation, higher rates of oxidation or organic solutes at low osmolarity would help deplete the cell of these solutes and could contribute to cell volume regulation during hypoosmotic stress. It is suggested that two broad classes of intracellular solutes can be defined based on their effects on mitochondrial respiration. Solutes such as K+, C1-, and TMAO penetrate the inner mitochondrial membrane slowly or not at all. Increasing concentrations of these solutes result in lower rates of oxidation. This capacity may be important in regulating intracellular levels of organic solutes during osmotic stress. Solutes such as urea rapidly penetrate the cell and inner mitochondrial membrane reducing the mitochondrial volume changes associated with osmotic stress. The known detrimental effects of urea on protein structure may prevent its exclusive use as an intracellular osmotic effector.