Molecular characterization of a multidrug resistance-associated protein,Mrp2, from the little skate

Multidrug resistance protein Mrp2 (symbol Abcc2) in liver plays a significant role in the biliary excretion of organic anionic conjugates. Mutations in human MRP2 result in defects in excretion of conjugated bilirubin and other cholephiles known as the Dubin-Johnson syndrome. Previous studies indicate that transporters with Mrp2-like functions are present in ancient vertebrates. We have now characterized an Mrp2 ortholog at the molecular level from the liver of the small skate, Raja erinacea, a marine vertebrate that evolved approximately 200 million years ago. The full-length skate Mrp2 cDNA is 6 kb and encodes for a 1,564-amino acid peptide with 56% identity to human Mrp2. Northern blot analysis demonstrated that skate Mrp2 is abundantly expressed in skate liver, intestine, and kidney. Immunoblots reveal a 180-kDa protein in skate liver. Immunofluorescence studies locate skate Mrp2 to the apical membrane of hepatocytes, renal tubules, and intestine. A PDZ-interacting motif is also found at its COOH terminus. Further sequence analysis indicates that transmembrane domains 1, 9, 11, 16, and 17 are the most highly conserved transmembrane domains between skate Mrp2 and mammalian MRP2/Mrp2s. This analysis indicates that Mrp2 orthologs evolved early in vertebrate evolution and that conserved domains may be important determinants of Mrp2 substrate specificity.     

The elasmobranch spiracular organ

Summary The spiracular organ is a lateral line derived receptor associated with the first gill cleft (spiracle). Its functional morphology was studied in the little skate,Raja erinacea, and a shark, the smooth dogfish,Mustelus canis, with light and electron microscopy. The spiracular organ is a tube (skate) or pouch (shark) with a single pore opening into the spiracle. The lumen is lined with patches of sensory hair cells, and filled with a gelatinous cupula. In the little skate, hair cells form synapses with afferents but apparently not with efferent fibers. In both species, the spiracular organs are deformed by flexion of the hyomandibular cartilage at its articulation with the cranium. The hyomandibula is a suspensory element of the jaws; hyomandibular flexion results in jaw protrusion. The little skate spiracular organ is anchored at one end to the cranium and at the other to the hyomandibula so that it is stretched or relaxed during hyomandibular extension and flexion, respectively. InMustelus, the effects of hyomandibular flexion on the spiracular organ are mediated indirectly by the superior post-spiracular ligament which inserts on the distal end of the hyomandibula. Deformation of the dogfish shark cupula during hyomandibular movement was observed. In the little skate, as revealed by transmission electron microscopy, there is a measurable deflection of the hair cell ciliary bundles from spiracular organs fixed with the hyomandibula in the flexed relative to the extended positions. In both species, hyomandibula flexion should result in hair cell depolarization, and sensory afferent excitation, based on the direction of the observed (skate) or expected (shark) deflection of hair cell cilia.     

Morphological variation in the electric organ of the little skate (Leucoraja erinacea) and its possible role in communication during courtship

Skates discharge an electrical current too weak to be used for predation or defense, and too infrequent and irregular to be used for electrolocation. Additionally, skates possess a specialized sensory system that can detect electrical stimuli at the same strength at which they discharge their organs. These two factors are suggestive of a communicative role for the electric organ in skates, a role that has been demonstrated in similarly weakly electric teleosts (e.g., mormyrids and gymnotiforms). There is evidence that the sexual and ontogenetic variations in the electric organ discharge (EOD) in these other weakly electric fishes are linked to morphological variations in electric organs and the electrogenerating cells of the organs, the electrocytes. Little work has been done to examine possible sexual and ontogenetic variations in skate EODs or variations in the electrocytes responsible for those discharges. Electric organs and electrocyte morphology of male and female, and mature and immature little skates, Leucoraja erinacea, are characterized here. Female electric organs were bigger than male electric organs. This is suggestive of a sexually dimorphic EOD waveform or amplitude, which might be used as a sex-specific identification signal during courtship. The shapes of electrocytes that make up the organ were found to be significantly different between mature and immature individuals and, in some cases, posterior membrane surface area of the electrocytes increased at the onset of maturity due to the formation of membrane surface invaginations and papillae. This is evidence that the EOD of skates may differ in its waveform or amplitude or frequency between mature and immature skates, and act as a signal for readiness to mate. This study supports a communicative role during courtship for the weak electric organs of little skates, but studies that characterize skate EOD dimorphisms are needed to corroborate this speculation before conclusions can be drawn about the role the electric organ plays in communication during courtship.     

The C-n.m.r. spectra of inositols and cyclohexanepentols: the validity of rules correlating chemical shifts with configuration

The ¹³C-n.m.r. spectra of the eight diastereomeric inositols and the ten diastereomeric cyclohexanepentols have been recorded and all of the signals have been assigned by a variety of methods. The additive empirical constants used to correlate the configuration of hydroxyl groups with the ¹³C-chemical shifts differ, in the latter group, from those derived for the inositols; the removal of one hydroxyl group had altered the “rules” considerably. A better correlation was found between the spectra of the cyclohexanepentols and those of the inositols derived therefrom, in each case, by the addition of an equatorial hydroxyl group.     

A de novo approach to C-branched inositols: synthesis of a myo-inositol precursor for C-linked glycosyl phosphatidylinositols

C-Linked glycosyl inositols are valuable structure-activity probes because of their greater hydrolytic stability and different conformational behavior compared with their parent O-glycosides. Simple C-branched inositols are synthetic precursors to these and other groups of inositol mimetics. Herein is described a de novo synthesis of C-branched inositols that contain a versatile ethenyl side chain for elaboration into more complex appendages. The approach centers on a stereoselective oxocarbenium ion-allylsilane cyclization and provides C-branched inositols with different stereochemical motifs. The synthesis of C-ethenyl-di-O-isopropylidene-myo-, neo-, epi-, and allo-inositols is discussed.     

Pharmacology of the skate electroretinogram indicates independent ON and OFF bipolar cell pathways

Organization of afferent information into parallel ON and OFF pathways is a critical feature of the vertebrate visual system. All afferent visual information in the vertebrate retina reaches the inner plexiform layer (IPL) via bipolar cells. It is at the bipolar cell level that separation of ON and OFF information first appears for afferent information from cones. This may also hold true for the rod pathway of cold-blooded vertebrates, but not for mammals. The all-rod retina of the skate presents an opportunity to examine such pathways in a retina having but a single class of photoreceptor. Immunocytochemical evidence suggests that both ON and OFF bipolar cells are present in the skate retina. We examined the pharmacology of the skate electroretinogram (ERG) to test the hypothesis that independent ON and OFF bipolar cell pathways are functional as rod afferent pathways from outer to inner plexiform layer in the skate. 100 microM 2-amino-4-phosphonobutyric acid (APB) reversibly blocked the skate ERG b-wave. A small d-wave-like OFF component of the ERG revealed by DC recording of response to a prolonged (10 s) flash of light was reduced or blocked by 5 mM kynurenic acid (KYN). We found that addition of 200 microM picrotoxin to the Ringer''s solution revealed prominent ON and OFF components of the skate ERG while reducing the c-wave. These ON and OFF components were reversibly blocked by 100 microM APB and 5 mM KYN, respectively. Reversible block of the OFF component by KYN was also accomplished in the presence of 500 microM N-methyl-DL-aspartate. From these findings, we conclude that ON and OFF bipolar cells are likely to be functional as parallel afferent interplexiform pathways in the all-rod retina of the skate.     

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.     

Electrophysiological study of central projections of ampullae of lorenzini in skates

The topical representation of individual electroreceptor organs (ampullae of Lorenzini) or groups of them on brain neurons of the thorny skate was investigated. The electroreceptor system was shown to be represented in the anterior lateral lobes of the medulla, the caudal portion of the auricles of the cerebellum, and the midbrain. In the medulla zones of representation of electroreceptors and mechanoreceptors are segregated. No precise somatotopic organization of electroreceptor representation was found in the anterior lateral lobe or in the auricles of the cerebellum. Connections of midbrain neurons with receptors on both ipsilateral and contralateral sides of the body were demonstrated.     

The elasmobranch spiracular organ

The spiracular sense organs of the little skate, Raja erinacea, and the smooth dogfish, Mustelus canis, respond to movements of the hyomandibula-cranial joint. Afferent activity was recorded from the spiracular organ nerve in isolated preparations consisting of at least part of the cranium, the hyomandibula, and the spiracular organ and nerve. Afferents are excited by hyomandibular flexion at its joint with the cranium. Single unit recordings in the little skate revealed a single class of units that were slowly adapting, and had a regular firing pattern. Single unit firing rate increased up to about 70 spikes/s during hyomandibular flexion from a spontaneous rate at rest of 15-20 spikes/s, and could often be silenced by hyomandibular extension. The direction of excitation is consistent with the orientation of the hair cell ciliary bundles observed in morphological studies (Barry et al. 1988). Local deformations of the cupula are sufficient to excite or inhibit primary afferent firing, and volume changes in the spiracular organ as a whole are not necessary. The spiracular organs are relatively insensitive to electrical stimuli, vibration, or water movement. In conclusion, the spiracular organ functions as a sensitive joint receptor.     

A straightforward method for analysis of highly phosphorylated inositols in blood cells by high-performance liquid chromatography.

Soluble inositol polyphosphates are found in many cells. The trisphosphate isomers, mainly inositol-1,4,5-trisphosphate, have been extensively studied because of their involvement in signal transduction. However, higher phosphorylated inositols are less frequently studied and their physiological role is poorly understood. Among these, only the myo-inositol-1,3,4,5,6-pentakisphosphate (Ins1,3,4,5,6P5), an important component of bird erythrocytes, has been intensively studied in comparative studies because it is a potent allosteric effector of hemoglobin and decreases its affinity to oxygen. We have developed a procedure for the analysis of inositol polyphosphates and other phosphate compounds in vertebrate blood cells based on a quick and accurate HPLC separation coupled to metal-dye detection. The procedure includes acid extraction of cellular phosphates, acid elimination and concentration of the extract, HPLC separation of phosphate compounds, and quantification by coupled highly sensitive metal-dye detection. The method is especially useful for analyses of highly phosphorylated inositols and for red cell comparative studies. Using the described method we have quantified Ins1,3,4,5,6P5 and the low quantities of InsP6 found in bird erythrocytes. We also identified traces of Ins3,4,5,6P4 and Ins1,3,4,6P4. Moreover, by applying the method in cultured murine macrophages, we have found changes of highly phosphorylated inositols when these cells are activated by lipopolysaccharide.     

Receptive field properties of rod-driven horizontal cells in the skate retina

The large receptive fields of retinal horizontal cells result primarily from extensive intercellular coupling via gap (electrical) junctions; thus, the extent of the receptive field provides an index of the degree to which the cells are electrically coupled. For rod-driven horizontal cells in the dark-adapted skate retina, a space constant of 1.18 +/- 0.15 mm (SD) was obtained from measurements with a moving slit stimulus, and a comparable value (1.43 +/- 0.55 mm) was obtained with variation in spot diameter. These values, and the extensive spread of a fluorescent dye (Lucifer Yellow) from the site of injection to neighboring cells, indicate that the horizontal cells of the all-rod retina of skate are well coupled electrically. Neither the receptive field properties nor the gap-junctional features of skate horizontal cells were influenced by the adaptive state of the retina: (a) the receptive field organization was unaffected by light adaptation, (b) similar dye coupling was seen in both dark- and light-adapted retinae, and (c) no significant differences were found in the gap-junctional particle densities measured in dark- and light-adapted retinas, i.e., 3,184 +/- 286/microns 2 (n = 8) and 3,073 +/- 494/microns 2 (n = 11), respectively. Moreover, the receptive fields of skate horizontal cells were not altered by either dopamine, glycine, GABA, or the GABAA receptor antagonists bicuculline and picrotoxin. We conclude that the rod-driven horizontal cells of the skate retina are tightly coupled to one another, and that the coupling is not affected by photic and pharmacological conditions that are known to modulate intercellular coupling between cone-driven horizontal cells in other species.     

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.     

Electrosensory ampullary organs are derived from lateral line placodes in cartilaginous fishes

Ampullary organ electroreceptors excited by weak cathodal electric fields are used for hunting by both cartilaginous and non-teleost bony fishes. Despite similarities of neurophysiology and innervation, their embryonic origins remain controversial: bony fish ampullary organs are derived from lateral line placodes, whereas a neural crest origin has been proposed for cartilaginous fish electroreceptors. This calls into question the homology of electroreceptors and ampullary organs in the two lineages of jawed vertebrates. Here, we test the hypothesis that lateral line placodes form electroreceptors in cartilaginous fishes by undertaking the first long-term in vivo fate-mapping study in any cartilaginous fish. Using DiI tracing for up to 70 days in the little skate, Leucoraja erinacea, we show that lateral line placodes form both ampullary electroreceptors and mechanosensory neuromasts. These data confirm the homology of electroreceptors and ampullary organs in cartilaginous and non-teleost bony fishes, and indicate that jawed vertebrates primitively possessed a lateral line placode-derived system of electrosensory ampullary organs and mechanosensory neuromasts.     

ATP-dependent GSH and glutathione S-conjugate transport in skate liver: role of an Mrp functional homologue

Multidrug resistance-associated proteins 1 and 2 (Mrp1 and Mrp2) are thought to mediate low-affinity ATP-dependent transport of reduced glutathione (GSH), but there is as yet no direct evidence for this hypothesis. The present study examined whether livers from the little skate (Raja erinacea) express an Mrp2 homologue and whether skate liver membrane vesicles exhibit ATP-dependent GSH transport activity. Antibodies directed against mammalian Mrp2-specific epitopes labeled a 180-kDa protein band in skate liver plasma membranes and stained canaliculi by immunofluorescence, indicating that skate livers express a homologous protein. Functional assays of Mrp transport activity were carried out using (3)H-labeled S-dinitrophenyl-glutathione (DNP-SG). DNP-SG was accumulated in skate liver membrane vesicles by both ATP-dependent and ATP-independent mechanisms. ATP-dependent DNP-SG uptake was of relatively high affinity [Michaelis-Menten constant (K(m)) = 32 +/- 9 microM] and was cis-inhibited by known substrates of Mrp2 and by GSH. Interestingly, ATP-dependent transport of (3)H-labeled S-ethylglutathione and (3)H-labeled GSH was also detected in the vesicles. ATP-dependent GSH transport was mediated by a low-affinity pathway (K(m) = 12 +/- 2 mM) that was cis-inhibited by substrates of the Mrp2 transporter but was not affected by membrane potential or pH gradient uncouplers. These results provide the first direct evidence for ATP-dependent transport of GSH in liver membrane vesicles and support the hypothesis that GSH efflux from mammalian cells is mediated by members of the Mrp family of proteins.     

Purification, structural characterization, and myotropic activity of a peptide related to des-Arg(9)-bradykinin from an elasmobranch fish, the little skate, Leucoraja erinacea

A bradykinin (BK)-related peptide was isolated from heat-denaturated plasma from an elasmobranch fish, the little skate, Leucoraja erinacea after incubation with porcine pancreatic kallikrein. The primary structure of the peptide (H-Gly-Ile-Thr-Ser-Trp-Leu-Pro-Phe-OH; skate BK) shows limited structural similarity to the mammalian B1 receptor agonist, des-Arg(9)-BK. The myotropic activities of synthetic skate BK, and the analog skate [Arg(9)]BK, were examined in isolated skate vascular and intestinal smooth muscle preparations. Skate BK produced a concentration-dependent constriction of the mesenteric artery (EC(50)=4.37x10(-8)M; maximum response=103.4+/-10.23% of the response to 60mM KCl) but the response to skate [Arg(9)]BK was appreciably weaker (response to 10(-6)M=73.0+/-23.4% of the response to 60mM KCl). Neither the first branchial gill arch nor the ventral aorta responded to either purified peptide. Skate BK also produced a concentration-dependent constriction of intestinal smooth muscle preparations (EC(50)=2.74x10(-7)M; maximum response 31.0+/-12.2% of the response to 10(-5)M acetylcholine). Skate [Arg(9)]BK was without effect on the intestinal preparation. The data provide evidence for the existence of the kallikrein-kinin system in a phylogenetically ancient vertebrate group and the greater potency of skate BK compared with the analog skate [Arg(9)]BK suggests that the receptor mediating vascular responses resembles the mammalian B1 receptor more closely than the B2 receptor.     

Hepatic microsomal NADPH-cytochrome P-450 reductase from little skate, Raja erinacea. Comparison of thermolability and other molecular properties with a mammalian enzyme

Components of little skate (an elasmobranch) and rabbit hepatic microsomal cytochrome P-450 dependent monooxygenase systems were examined for differences which might explain the decreasing xenobiotic-metabolizing activity of little skate microsomes assayed at temperatures above 30 degrees C. The proportion of saturated fatty acids in microsomal lipids and the habitat temperature are both lower in skate as compared to rabbit, which is consistent with the known adaptive pattern. The more thermolabile enzyme of the skate system in microsomal preparations is NADPH-cytochrome P-450 reductase. The optimal assay temperature for purified skate reductase (30 degrees C) is 10 degrees C lower than that for the purified rabbit reductase. The purified skate reductase differs from rabbit reductase in monomeric molecular weight, in peptides produced by partial proteolysis, in immunochemical properties, but not in flavin content.     

Comparative analysis of feeding habits and dietary niche breadth in skates: the importance of body size,snout length,and depth

Skates (Elasmobranchii, Rajiformes) are a morphologically conservative group of bentophagous chondrichthyan fishes with a high degree of endemism, that occur on marine soft bottoms. Subtle morphological aspects and bathymetric distribution are traits that vary among skate species that could have implications for their feeding ecology. We test how body size, snout length and bathymetric distribution influence the feeding habits and dietary niche breadth in skates using data on 71 species taken from the literature. We hypothesized that snout length has an effect on diet composition. We also hypothesized that dietary niche breadth increases with increasing depth range and decreases with increasing body size of skate species. Generalized additive models for location scale and shape were fitted with taxonomic level (genera nested within family) included as a random effect term in each model. A model selection approach to test the level of support for alternative models was applied. We found that skate species that forage on large prey have the largest body size and skate species with the smallest body size prey on small and medium-sized invertebrates. The results indicated that body size has an effect on feeding habits of skates, whereas an effect of snout length was not supported. Bathymetric variables have an effect on the diet of skates. Our prediction that dietary niche breadth increases with increasing depth range and decreases with increasing body size of skate species was supported in part: in a first phase the relationship between dietary niche breadth and body size is positive, then in a second phase, including species larger than 1000 mm total length, the relationship become negative.     

Adaptation properties of the ERG in the grasshopper,Romalea microptera

The grasshopper ERG displays a rapid recovery of responsivity following the onset of a background light. Although observed earlier in skate and frog, this phenomenon has not previously been seen in an invertebrate. Furthermore, a period of hyperresponsivity exists in early dark adaptation and resembles that found in skate and frog. Thus, recovery in the light and hyperresponsivity in the dark seem to be corollaries of each other. Finally, spectral sensitivity of the ERG is determined and two peaks are found: one at 510 nm and the other at 360 nm. The former appears to be a rhodopsin-mediated sensitivity but the latter does not and they are not clearly separated by chromatic adaptation.     

Questioning assumptions of trophic behavior in a broadly ranging marine predator guild

We evaluated whether existing assumptions regarding the trophic ecology of a poorly‐studied predator guild, northwest (NW) Atlantic skates (family: Rajidae), were supported across broad geographic scales. Four hypotheses were tested using carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope values as a proxy for foraging behavior: 1) species exhibit ontogenetic shifts in habitat and thus display a shift in ¹³C with differential use of the continental shelf; 2) species exhibit ontogenetic prey shifts (i.e. from smaller to larger prey items) and become enriched in ¹⁵N; 3) individuals acquire energy from spatially confined local resource pools and exhibit limited displacement; and 4) species exhibit similarly sized and highly overlapping trophic niches. We found some evidence for ontogenetic shifts in habitat‐use (δ¹³C) for thorny and little skate and diet (δ¹⁵N) of thorny and winter skate and hypothesize that individuals exhibit gradual trophic niche transition, especially in δ¹⁵N space, rather than a clear and distinct shift in diet throughout ontogeny. Spatial isoscapes generated for little, thorny, and winter skate highlighted distinct spatial patterns in isotopic composition across the coastal shelf. For little and thorny skate, patterns mimicked expected spatial variability in the isotopic composition of phytoplankton/POM, suggesting limited displacement and utilization of spatially confined resource pools. Winter skate, however, exhibited a much narrower range of δ¹³C and δ¹⁵N values, suggesting individuals may use resources from a more confined latitudinal range. Although high total trophic niche overlap was observed between some species (e.g. little and thorny skate), sympatric species (e.g. little and winter skate) exhibited a degree of trophic niche separation. These findings offer new insight into the trophic dynamics of a poorly‐studied, vulnerable group of predators, and highlight a need to re‐examine assumptions pertaining to aspects of their ecology.     

Skate Bathyraja spp. egg predation in the eastern Bering Sea

Predation on skate eggs by snails was examined for three skate species at seven nursery sites in three regions (north, middle and south) of the eastern Bering Sea. Mean predation levels were 6·46% for the Alaska skate Bathyraja parmifera , 2·65% for the Aleutian skate Bathyraja aleutica and 22·25% for the Bering skate Bathyraja interrupta . Predation levels were significantly higher at the middle and north sites than the south sites for all species combined. Predation levels decreased with increasing egg-case densities at all nursery sites, and the highest predation levels occurred where egg-case densities were very low. Predated egg-case density increased with increasing snail densities across all nursery sites examined. The Oregon triton Fusitriton oregonensis was the most abundant snail species at all nursery sites and displayed ability to drill holes in the egg case of B. parmifera. Holes left by predatory snails in egg cases of B. parmifera were significantly larger, and of different shape at the middle site compared to the south site. Holes in B. parmifera were also significantly larger than those in egg cases of B. interrupta across all sites examined. Egg cases of B. aleutica possess surface spines that cover the egg case and may inhibit predation by snails at a greater rate than that of the B. parmifera and B. interrupta , which have a smoother egg-case surface.