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.     

A primitive ATP receptor from the little skate Raja erinacea

P2Y ATP receptors are widely expressed in mammalian tissues and regulate a broad range of activities. Multiple subtypes of P2Y receptors have been identified and are distinguished both on a molecular basis and by pharmacologic substrate preference. Functional evidence suggests that hepatocytes from the little skate Raja erinacea express a primitive P2Y ATP receptor lacking pharmacologic selectivity, so we cloned and characterized this receptor. Skate hepatocyte cDNA was amplified with degenerate oligonucleotide probes designed to identify known P2Y subtypes. A single polymerase chain reaction product was found and used to screen a skate liver cDNA library. A 2314-base pair cDNA clone was generated that contained a 1074-base pair open reading frame encoding a 357-amino acid gene product with 61-64% similarity to P2Y(1) receptors and 21-37% similarity to other P2Y receptor subtypes. Pharmacology of the putative P2Y receptor was examined using the Xenopus oocyte expression system and revealed activation by a range of nucleotides. The receptor was expressed widely in skate tissue and was expressed to a similar extent in other primitive organisms. Phylogenetic analysis suggested that this receptor is closely related to a common ancestor of the P2Y subtypes found in mammals, avians, and amphibians. Thus, the skate liver P2Y receptor functions as a primitive P2Y ATP receptor with broad pharmacologic selectivity and is related to the evolutionary forerunner of P2Y(1) receptors of higher organisms. This novel receptor should provide an effective comparative model for P2Y receptor pharmacology and may improve our understanding of nucleotide specificity among the family of P2Y ATP receptors.     

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.     

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.     

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.     

Relaxin from an oviparous species, the skate (Raja erinacea)

An acid-acetone extract prepared from ovaries of the skate, Raja erinacea, contained a weakly crossreacting molecule when tested in a pig relaxin radioimmunoassay. The material was isolated and purified to homogeneity by ion exchange chromatography, molecular exclusion chromatography, and HPLC. Analytical tests proved the molecule to consist of two chains and to have a molecular weight of 7,500. Sequence analyses of the A and B chains yielded the following sequence: Glu-Glu-Lys-Met-Gly-Phe-Ala-Lys-Lys-Cys-Cys-Ala-Ile-Gly-Cys-Ser-Thr-Glu- Asp-Phe-Arg-Met-Val-Cys and Arg-Pro-Asn-Trp-Glu-Glu-Arg-Ser-Arg-Leu-Cys-Gly-Arg-Asp-Leu-Ile-Arg-Ala- Phe- Ile-Tyr-Leu-Cys-Gly-Gly-Thr-Arg-Trp-Thr-Arg-Leu-Pro-Asn-Phe-Gly-Asn-Tyr- Pro-Ile-Met respectively. Skate relaxin has 0.2% of the activity of B29 pig relaxin in the symphysis pubis assay and 0.5% in the mouse uterine muscle strip contraction inhibition assay.     

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     

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.     

Isolation and characterization of lactate dehydrogenase from white fin muscles of the skate Raja clavata

Lactate dehydrogenase (LDH) from white driving muscle of skate Raja clavata was purified by the differential precipitation of ammonium sulphate between 52 and 55% saturation. Only one protein band with the LDH activity was obtained by nondenaturing electrophoresis. The same result was obtained by the SDS-electrophoresis. The relative molecular weight calculated by this method in the presence of DS-Na was 34 kDa; Km was 29 +/- 7 and 71 +/- 16 microM for NAD.H and pyruvate, respectively. The reaction was maximally activated by 0.8-6.0 mM pyruvate and inhibited in the regions above this level. Dilution of LDH below concentration of 1 microgram/ml reduced the enzyme activity. The pH-optimum for the LDH activity ranged 7.0-8.0.     

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.     

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.     

A bile alcohol sulfate as a major component in the bile of the small skate (Raja erinacea)

The nature of bile alcohols and bile acids in gall-bladder and hepatic bile from perfused livers of the small skate (Raja erinacea) has been investigated. The main bile alcohol sulfate was isolated by thin-layer chromatography and analyzed by fast atom bombardment mass spectrometry and 13C NMR. Following solvolysis and purification on Lipidex-DEAP, the bile alcohol profile was measured by capillary gas-liquid chromatography-electron impact mass spectrometry. Based on these studies and on comparison with authentic scymmnol sulfate and scymnol, the main bile alcohol was identified as 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 xi,26,27-hexol sulfate. The mean +/- SD concentration in gallbladder bile from five different skates was 24.6 +/- 8.7 mmol/l. Only 0.1 mmol/l of cholic acid and its conjugates was found in a pool of skate bile. The main bile alcohol sulfate in the bile of the small skate seems to be a metabolic end product, present in a concentration comparable to that of bile salts in mammals.     

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.     

Anion transport in basolateral (sinusoidal) liver plasma-membrane vesicles of the little skate (Raja erinacea).

The mechanism(s) of [35S]sulphate transport was investigated in basolateral liver plasma-membrane vesicles of the little skate elasmobranch, Raja erinacea. Imposition of an intravesicular alkaline pH gradient (pH 8.0 in/pH 6.0 out) stimulated sulphate uptake 5-10-fold compared with pH-equilibrated (pH 8.0 in = out) conditions and 2-3-fold over equilibrium sulphate uptake (overshoot). This pH-gradient-stimulated sulphate uptake was temperature-dependent, saturable with increasing concentrations of sulphate and could be inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone and the anion-transport inhibitors 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid (DIDS) and probenecid, cis-Inhibition of pH-gradient-driven sulphate uptake was observed with sulphate, oxalate, cholate and bromosulphophthalein, but not with chloride and taurocholate. In addition, sulphate and oxalate trans-stimulated [35S]sulphate uptake under pH-equilibrated conditions. Although also stimulated by an inside-alkaline pH gradient, transmembrane transport of [3H]cholate was not inhibited by DIDS, suggesting that its pH-gradient-driven uptake is not mediated by an anion-transport 'carrier'. In conclusion, these studies indicate that a basolateral plasma-membrane sulphate-transport system has evolved in skate hepatocytes and is similar to that in mammalian liver cells. This archaic anion-exchange system co-transports certain organic anions such as oxalate and has developed early in vertebrate evolution.     

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.     

Plasma estradiol, testosterone, and progesterone levels during the ovulatory cycle of the skate (Raja erinacea)

Amounts of estradiol, testosterone, and progesterone in plasma were measured during the reproductive cycle of female Raja erinacea. Estradiol titers correlated directly with follicle size in females undergoing ovarian recrudescence, while highest concentrations were found in females with preovulatory follicles. These data indicate that as follicles grow, their steroidogenic capacity increases. In mature, nonspawning females, titers of estradiol and testosterone varied markedly. Progesterone was not detected in peripheral plasma of skates that did not produce eggs during the observation period. In females producing eggs, estradiol and testosterone predominated during the follicular phase of each spawning cycle. While estradiol and testosterone were elevated, progesterone was not detectable in the peripheral circulation. As ovulation and formation of capsules approached, plasma estradiol and testosterone declined to near baseline levels. Circulating progesterone rose sharply two days before encapsulation of ovulated eggs and remained elevated for only two days. On the day of encapsulation, concentrations of plasma progesterone had fallen to nearly baseline levels. Progesterone titers remained low throughout egg retention and oviposition. These measurements demonstrate that progesterone titers are elevated at specific times during the reproductive cycle of the skate and clearly suggest that progesterone is critically involved in events occurring at ovulation, encapsulation, and possibly oviposition.     

Osmotic stress stimulates the organic osmolyte channel in Xenopus laevis oocytes expressing skate (Raja erinacea) AE1

The aim of this study was to determine whether hypo-osmolarity, which activates taurine transport through the volume-sensitive organic osmolyte channel in skate (Raja erinacea) erythrocytes, also activates the organic osmolyte channel activity of skate AE1 (skAE1) expressed in oocytes. When Xenopus laevis oocytes expressing skAE1 were incubated in hypo-osmotic ND 96 (210 mOsm) media, taurine was transported at a significantly higher rate than when incubated in ND 96 (235 mOsm), which is iso-osmotic to Xenopus plasma. Therefore, hypo-osmotic stress is part of the activation mechanism of the organic osmolyte channel in skAE1 expressing oocytes.     

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.