Skate Raja Erinacea Research Articles

Feeding hotspots for four northwest Atlantic groundfish species

Abstract Methratta, E.T., and Link, J.S. 2012. Feeding hotspots for four northwest Atlantic groundfish species. — ICES Journal of Marine Science, 69: 1710–1721. We used predator distribution and stomach content data to estimate the annual per capita rate of consumption for four representative predator species from the northwestern Atlantic Ocean, and considered how consumption is influenced by depth, bottom salinity, sediment grain size, location variables, and species-specific diet components. We found that geographic variables and species-specific prey resources were significantly associated with consumption rates, a pattern consistent with predator-prey theory. Prey categories comprised of fish were particularly important for a more mobile predator (silver hake Merluccius bilinearis), whereas benthic invertebrate prey were consistently important for a more sedentary predator (little skate Raja erinacea). Hotspots in consumption rates that overlap with particular prey resources were highlighted by the significance of location variables (longitude) for winter flounder Pseudopleuronectes americanus, silver hake, little skate, and sea raven Hemitripterus americanus. Depth was an important explanatory factor for consumption by little skate, but the explanatory value of abiotic habitat factors was low for the other three species. Greater emphasis on species-specific food habits, migratory patterns, and ecological interactions at the synoptic scales relevant to fisheries is needed for fisheries management.

Reduced ileal expression of OSTα-OSTβ in non-obese gallstone disease

Cholelithiasis is a multifactorial process, and several mechanisms have been postulated. A decreased expression of the ileal apical sodium-dependent bile acid transporter (ASBT) and of the cytosolic ileal lipid binding protein (ILBP) was recently described in female non-obese patients. The role of the recently identified organic solute transporters alpha and beta (OSTalpha, OSTbeta) in gallstone pathogenesis remains unclear. Therefore, we performed analysis of OSTalpha-OSTbeta in gallstone patients according to body weight. Ileal mucosal biopsies were collected during routine colonoscopy from female gallstone carriers (n = 19) and controls (n = 34). OSTalpha-OSTbeta mRNA expression was measured using the LightCycler sequence detection system; protein was analyzed by immunohistochemistry and Western blot. The mRNA expression of OSTalpha-OSTbeta was significantly reduced (OSTalpha: 3.3-fold, P = 0.006; OSTbeta: 2.6-fold, P = 0.03) in normal-weight but not overweight gallstone carriers compared with controls. OSTalpha-OSTbeta protein levels also showed a reduction by 40-67%. The expression of OSTalpha-OSTbeta correlated positively with ASBT (r = 0.65, 0.58, respectively), ILBP (r = 0.77, 0.67), and the farnesoid X receptor (r = 0.58, 0.50). Fibroblast growth factor-19 showed a 2.8-fold reduction (P = 0.06), and liver receptor homolog-1 showed a 2-fold reduction (P = 0.04) in non-obese patients. In conclusion, an impaired function of all three ileal bile acid transporters may lead to low ileal bile acid reabsorption and an altered bile acid pool composition and therefore may contribute to the formation of gallstones in non-obese patients.

Membrane trafficking factors are involved in the hypotonic activation of the taurine channel in the little skate (Raja erinacea) red blood cell

In response to volume expansion, red blood cells of the little skate (Raja erinacea) initially swell and then release small organic compounds and osmotically obligated water in what is called a regulatory volume decrease (RVD) to restore cell volume. One of the major intracellular solutes lost during this process is the non-metabolized beta amino acid taurine. This hypoosmotic-induced increase in cell taurine permeability requires the anion exchanger, skAE1. The abundance of this transporter increases on the surface plasma membrane by a process of exocytosis. The second-messenger pathways involved in exocytosis of skAE1 were investigated with the use of inhibitors which affect membrane trafficking. Hypoosmotic-stimulated taurine uptake was significantly decreased by 42% with wortmannin, a phosphatidylinositol 3-kinase (PI3 kinase) inhibitor. Additional evidence for the involvement of PI3K was obtained with a second inhibitor, LY294002, which decreased the hypoosmotic-stimulated taurine uptake by 28%. The state of actin is also involved, as the actin filament depolymerizer latrunculin B decreased hypoosmotic-stimulated taurine uptake by approximately 40%. Although hypoosmotic conditions did not stimulate changes in the distribution of actin between filamentous and globular forms, latrunculin stimulated a decrease in filamentous actin and increase in globular actin in both isoosmotic and hypoosmotic conditions. Disruptors of other potential cytoskeletal factors (myosin, kinesin, dynein, and microtubules) did not affect taurine uptake. The present results suggest that the exocytosis of skAE1 stimulated by hyposmotic-induced cell volume expansion requires activation of PI3 kinase and is regulated by the state of actin filaments.

Cell membrane surface expression and tyrosine kinase regulate the osmolyte channel (skAE1) in skate erythrocytes

A regulatory volume decrease response occurs when skate (Raja erinacea) erythrocytes are exposed to hyposmotic medium; they swell and then reduce their volume by releasing organic osmolytes (primarily taurine) and associated water. The response involves the red blood cell anion-exchanger skate anion-exchanger 1 (skAE1), which has been previously shown to be affected by tyrosine phosphorylation, to form tetramers and to change binding affinities to the cytoskeletal proteins, ankyrin and band 4.1. Our recent studies are focused on determining the sequence and mechanism of these events to better understand the activation of skAE1 upon hyposmotic stimulation. Under isosmotic conditions a large portion of skAE1 is found not only on the plasma membranes but also associated intracellularly in detergent-resistant membranes or lipid rafts. We hypothesize that an important step in the hyposmotic-induced increase in taurine transport involves the movement of skAE1 from an intracellular association with lipid rafts into the cell membrane. Inhibition of tyrosine phosphorylation of skAE1 with piceatannol reduces the hyposmotic-induced increase in taurine transport and also inhibits both the decreased binding of skAE1 to band 4.1 and the increased affinity to ankyrin. However, the phosphorylation inhibitor does not block the movement of the transporter into the plasma membrane or the formation of tetramers. This suggests that tyrosine phosphorylation is important in the hyposmotic-induced taurine transport but other steps that do not require phosphorylation play an important role.

Cell and Molecular Biology of Marine Elasmobranchs: Squalus acanthias and Raja erinacea

Elasmobranchs are among the most primitive existing species exhibiting fundamental vertebrate characteristics, such as neural crest, jaws, teeth, and an adaptive immune system. They are also among the earliest-evolved vertebrates with a closed, pressurized circulatory system and related signaling molecules. Although many species are used experimentally, the spiny dogfish shark (Squalus acanthias) and little skate (Raja erinacea) have particular advantages and are the most commonly used elasmobranch biomedical models. These animals display powerful molecular systems for dealing with salt and water homeostasis, cell volume regulation, and environmental and internal osmotic sensing. They have become important unique models in studies of transport-related diseases such as cystic fibrosis and anion or xenobiotic transport. Much of this work has relied on physiological experiments combined with molecular approaches and the advantages of comparative genomic analyses to identify conserved regions representing functional protein domains. Recent work has seen the development of cell cultures and the beginning of expressed sequence tags (EST) and genomic libraries. Other areas in which elasmobranches have played critical roles include immunology and neurobiology. It also appears that sharks have tissue regenerative capability beyond what is commonly seen in mammals. For example, sharks and skates possess a region of renal regeneration, with new tubules being formed continually through adulthood. As comparative functional genomics comes of age, these comparative vertebrate models may play an increasing role in the larger picture of human biomedical research. There is plenty of ocean to share.

Ecological Interactions between Elasmobranchs and Groundfish Species on the Northeastern U.S. Continental Shelf. I. Evaluating Predation

Abstract Elasmobranchs are an important component of the northeastern U.S. continental shelf ecosystem in terms of both biomass and abundance. It has been hypothesized that because of their high biomass these species have a significant negative impact on commercially valuable groundfish via ecological interactions. One of these hypotheses posits that elasmobranchs directly remove such groundfish by predation. We analyzed data from the stomachs of 40,756 spiny dogfish Squalus acanthias, 3,806 smooth dogfish Mustelus canis, 17,618 little skate Raja erinacea, 11,593 winter skate R. ocellata, and 2,571 thorny skate R. radiata collected in bottom trawl surveys conducted principally during spring and autumn 1973–1998 in waters from Cape Hatteras, North Carolina, to Nova Scotia. Except for silver hake Merluccius bilinearis, most groundfish are not common in the diets of the elasmobranchs. Estimates of consumption indicate that both the number and the total biomass of the groundfish that are removed are generally...

A Retrospective of Recombinant P2Y Receptor Subtypes and Their Pharmacology

Since the first cloning of P2Y receptor sequences in 1993 it has become apparent that this family of G-protein-coupled receptors is omnipresent. At least 25 individual sequences entered in the GenBank sequence database encode P2Y receptors from a variety of species ranging from the little skate Raja erinacea to man. In man, six receptor subtypes have been cloned and found to be functionally active (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y12). In this article a review of the P2Y receptor subtypes is presented considering both their sequences and the pharmacological profiles of the encoded receptors expressed in heterologous expression systems.

Bile salt export pump is highly conserved during vertebrate evolution and its expression is inhibited by PFIC type II mutations.

Bile secretion is a fundamental function of the liver of all vertebrates and is generated by ATP-dependent transport proteins at the canalicular membrane of hepatocytes, particularly by the bile salt export pump BSEP. To determine the evolutionary origin and structure-function relationship of this transport mechanism, a liver cDNA library from the marine skate Raja erinacea, a 200 million-year-old vertebrate, was screened for BSEP orthologues. A full-length clone was isolated that encodes for 1,348 amino acids and shares 68.5% identity to human BSEP. Northern blot analysis revealed a 5-kb transcript only in skate liver. Expression of skate Bsep in Sf9 cells demonstrated a sixfold stimulation of ATP-dependent taurocholate transport compared with controls, with a Michaelis-Menten constant of 15 microM, which is comparable to rat Bsep. Sequences at the site of published mutations in human BSEP are also conserved in skate Bsep. When two of these mutations were introduced into the skate Bsep cDNA, this resulted in defective expression of the mutant proteins in Sf9 cells. These studies demonstrate that Bsep is a liver-specific ATP-dependent export pump that is highly conserved throughout evolution and provide insights into critical determinants for the function of this transporter in higher vertebrates.

Expression cloning of two genes that together mediate organic solute and steroid transport in the liver of a marine vertebrate.

Uptake of organic solutes and xenobiotics by mammalian cells is mediated by ATP-independent transporters, and four families of transporters have now been identified. To search for novel organic solute transporters, a liver cDNA library from an evolutionarily primitive marine vertebrate, the little skate Raja erinacea, was screened for taurocholate transport activity by using Xenopus laevis oocytes. In contrast to the organic anion transporters identified to date, a transport activity was identified in this library that required the coexpression of two distinct gene products, termed organic solute transporter alpha and beta (Ostalpha, Ostbeta). Ostalpha cDNA encodes for a protein of 352 aa and seven putative transmembrane (TM) domains. Ostbeta contains 182 aa and has at least one and perhaps two TM domains. There is no significant sequence identity between Ostalpha and Ostbeta, and only low identity with sequences in the databases; however, Ostalpha bears a resemblance to some G protein-coupled receptors, and Ostbeta exhibits 22% amino acid identity with the C-terminal TM and intracellular domains of protocadherin-gamma, a cell surface glycoprotein. Xenopus oocytes injected with the cRNA for both Ostalpha and Ostbeta, but not each separately, were able to take up taurocholate, estrone sulfate, digoxin, and prostaglandin E(2), but not p-aminohippurate or S-dinitrophenyl glutathione. Transport was sodium-independent, saturable, and inhibited by organic anions and steroids, including the major skate bile salt, scymnol sulfate. These results identify an organic anion transporter composed of a putative seven-helix TM protein and an ancillary membrane polypeptide.

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.

Molecular identification of a primitive P2Y ATP receptor in liver from the little skate Raja erinacea

Molecular identification of a primitive P2Y ATP receptor in liver from the little skate Raja erinacea

Hepatic Toxicity and Persistence of ser/thr Protein Phosphatase Inhibition by Microcystin in the Little Skate Raja erinacea

Microcystin-induced ser/thr protein phosphatase (PP) inhibition and toxicity were examined in the little skate (Raja erinacea), an evolutionarily primitive marine vertebrate. As in mammals, PP inhibition and toxicity were exclusively hepatocellular, but were much more persistent in the skate. A dose of 63 μg/kg given iv to adult male skates resulted in the near complete inhibition of hepatic PP activity at 24 h. PP activity was still 95% inhibited 7 days after dosing in skates given 125 μg/kg microcystin. Mortality occurred at doses of 500 μg/kg or more. Hepatic lesions were only seen in animals with fully inhibited PP activity in liver. The histological changes seen at 125 μg/kg were mild periportal inflammatory changes increasing in severity together with hepatocyte necrosis at higher doses of microcystin. Microcystin persisted and could be detected in plasma up to 7 days after dosing. This finding shows that, in the skate, as in mammals, the liver is the only organ capable of uptake of microcystin, since there was no significant inhibition of PP activity in the rectal gland and small decreases in PP activity of the kidney that were not time or dose dependent. In vitro microcystin caused dose-dependent inhibition of PP activity in isolated skate hepatocytes, while it was without effect in cultured rectal glands. Uptake of microcystin and the accompanying inhibition of PP activity in skate hepatocytes was prevented by the addition of a series of organic dyes and bile acids. The spectrum of inhibitors of microcystin uptake in skate is similar to that seen in the rat, indicating common features of the carrier(s) in these diverse species.

The aryl hydrocarbon receptor in early vertebrates

The Ah receptor (AhR), which mediates effects of dioxins and related halogenated aromatic hydrocarbons, belongs to the emerging bHLH-PAS family of transcription factors. A reverse-transcription-polymerase chain reaction (RT-PCR) method using degenerate primers was used to search for the AhR in early vertebrates, including bony, cartilaginous, and jawless fish. AhR cDNA sequences were obtained for the killifish Fundulus heteroclitus, smooth dogfish Mustelus canis, the little skate Raja erinacea and the sea lamprey Petromyzon marinus. Further studies revealed evidence for a second AhR gene (AHR2) in Fundulus and Mustelus. The fish AhR1 and AhR2 genes are as different from each other as each is from mammalian AhRs, suggesting that they are the result of an ancient gene duplication, possibly occurring prior to the divergence of lineages leading to present-day mammals and fish. These studies demonstrate the conservation of AhR structure over more than 500 million years of vertebrate evolution. A search of the GenBank protein sequence database revealed a possible AhR homolog in the nematode Caenorhabditis elegans. Phylogenetic analysis of fish AhR sequences in comparison to mammalian AhRs and other members of the bHLH-PAS gene family provides an evolutionary perspective to the study of dioxin toxicity and Ah receptor function.

Characterization of Progesterone-Binding Moieties in the Little SkateRaja erinacea

In this study we report evidence of a [3H]progesterone-binding moiety in the liver and oviduct of the little skateRaja erinacea.It is characterized by high affinity, low capacity, and DNA-cellulose-binding activity. Furthermore Western blot analysis revealed that monoclonal antibodies against the chicken progesterone receptor (PR) subunits A and B cross-reacted with a 110-kDa band in the liver and a 80-kDa band in the oviduct. When analyzed by DEAE-Sepharose ion-exchange column chromatography, [3H]progesterone-binding molecules resolved into two peaks, one nonadherent and one adherent to the column. The liver adherent peak eluted in a linear gradient at a NaCl concentration of about 0.07 M and resolved on Western blot as a single band of a 110 kDa. The oviduct adherent peak eluted at about 0.14 M NaCl and resolved on Western blot as a single band of 80 kDa. Competition studies showed that the progesterone-binding moiety in the cytosol was specific for progesterone. On the contrary, the nuclear component is not specific for progesterone; it also binds testosterone and estradiol 17β in the oviduct, and progesterone, testosterone, dihydrotestosterone, estradiol 17β, mibolerone, and R5020 in the liver. The [3H]progesterone-binding activity was monitored in both liver and oviduct of females in different reproductive stages. Females were separated into three groups: laying, nonlaying, and immature. [3H]Progesterone-binding activity levels were higher in the liver of immature than of nonlaying skates, and it was undetectable in laying skates. [3H]Progesterone binding was higher in the oviduct of laying and nonlaying skates than of immature skates. This PR-binding moiety has many characteristics of a true receptor: high affinity, low capacity, binds to DNA, and cross-reacts with antibodies against chicken PR. However, while the cytosolic form of this progesterone-binding component was quite specific for P, nuclear extracted material was nonspecific. If these progesterone-binding components are homologous with the PR A and PR B forms of other vertebrates, as we believe, it is clear that there are species differences that probably relate to phylogenetic level and physiology of the organism.

Excitatory Amino Acids and Serotonin Uptake Blockers Reveal two Physiologically Distinct Serotonin Systems in the Retina of the Skate, Raja Erinacea

The retina of the skate (Raja erinacea) contains at least 2 types of cell (amacrines and bipolars) that can be visualized with an antiserum against serotonin. We have employed serotonin immunocytochemistry in combination with pharmacological manipulation of retinal tissue to analyze physiological properties of serotonergic amacrine cells and serotonin-accumulating bipolar cells. Excitatory amino acids (NMDA, aspartate) had no detectable effects on serotonin-immimoreactivity in bipolar cells but decreased staining in amacrine cells. High K+ Ringer increased staining in bipolar cell somata, however, it depleted the inner plexiform layer of the retina of serotonin. Zimelidine, a serotonin uptake inhibitor, completely blocked serotonin accumulation by bipolar cells but had no effect on amacrine cells, whereas incubation of the retinas in fluoxetine (Prozac), a different inhibitor of serotonin uptake, did not block serotonin accumulation into bipolar cells which was actually enhanced in some cases. We conclude that amacrine and bipolar cells of the skate retina employ two different serotonin uptake carrier systems, thus generating two distinct pharmacological components that are capable of interacting with each other as they compete for extracellular serotonin. Similar mechanisms may exist in the vertebrate CNS and further examination of the interaction of these systems could provide important insights into the action and possible side effects of serotonin-related drugs.

Effect of MS-222 on response to light and rate of metabolism of the little skate Raja erinacea

The effect of tricaine methanesulfonate (MS-222) on the standard (SMR) and routine (RMR) metabolic rates of Raja erinacea was estimated from oxygen-consumption measurements. Data were gathered from a computerized, flow-through respirometry system. Individual trials were run for 96 h at 10 °C on anesthetized and untreated fish. The resulting rates, SMR = 20.1 ± 1.99 SE and RMR = 48.3± 2.5 SE mg O2 kg−1 h−1, for an unanesthetized skate of 0.5 kg standardized weight, are the lowest reported for any elasmobranch. Periodogram analysis revealed a significant light-response component to the oxygen consumption of these fish, indicating a pattern of nocturnal and crepuscular activity. This activity pattern was disrupted in skates subjected to anesthesia. The use of low-dosage MS-222 in conjunction with the respirometry trials provides positive preliminary evidence that this technique may be useful in giving quick, accurate estimates of SMR in the more intractable elasmobranch fishes.

Active microtubule-dependent secretion of a fluorescent bile salt derivative in skate hepatocyte clusters.

Fluorescence microscopy and video image analysis were used to study the transport of a fluorescent bile acid derivative [N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-7 beta-amino-3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oyl-2-aminoethanesulfonate (NBD-TC)] in isolated clusters of hepatocytes from the little skate Raja erinacea. Analysis of images of hepatocyte clusters that were incubated in medium with 0.5-1 microM NBD-TC showed that the fluorescent derivative accumulated in the cells and that the clusters retained a patent canalicular lumen as well as the ability to actively transport the bile acid derivative from the cells into the lumen; i.e., the lumen-to-cell fluorescence ratio greatly exceeded unity. NBD-TC uptake by hepatocytes was inhibited by several organic anions, of which taurocholate was the most effective. Uptake was also blocked by metabolic inhibitors and by incubation in the cold. Neither Na replacement nor increased medium K, which depolarizes the membrane electrical potential [potential difference (PD)], affected NBD-TC accumulation by hepatocytes. Transport of NBD-TC into the canalicular lumen was inhibited by incubation in the cold and was substantially reduced by high-K medium; these blocks were removed by warming and transfer to normal-K medium, respectively. Treatment of hepatocytes with 20-40 microM nocodazole, a drug that reversibly depolymerizes microtubules, reduced cellular NBD-TC accumulation and blocked its secretion into the canalicular lumen; nocodazole effects were reversed by washing the hepatocyte clusters in drug-free medium. Thus uptake of NBD-TC by skate hepatocytes is active and carrier mediated but not dependent on the PD or Na gradient. NBD-TC secretion from cell to canalicular lumen also appears to be active and carrier mediated. Canalicular secretion appears to be driven at least in part by the PD and is highly dependent on an intact microtubular system in this marine species.

Characterization and function of ATP receptors on hepatocytes from the little skate Raja erinacea.

Hormonal regulation of hepatocytes, via cytosolic Ca2+ signaling, is well established in higher life forms but has not been investigated in elasmobranchs. We therefore examined Ca2+ signaling in hepatocytes isolated from the little skate, Raja erinacea. In hepatocyte populations, ATP induced a rapid, biphasic increase in Ca2+, as it does in mammalian hepatocytes. Other hormones that act on mammalian hepatocytes, such as vasopressin, angiotensin, and phenylephrine, induced no such Ca2+ increase. The initial phase of the ATP-induced Ca2+ increase was seen even in Ca(2+)-free medium, whereas the late sustained phase of the increase was not. Similar dose-response curves were obtained by stimulation with ATP, ADP, UTP, and 2-methylthio-ATP. In contrast, AMP, adenosine, beta, gamma-methyl-ATP, CTP, and GTP induced little or no Ca2+ increase. In single hepatocytes, ATP, ADP, UTP, and 2-methylthio-ATP each induced a sustained increase in Ca2+ at high concentrations, but at low concentrations induced Ca2+ oscillations. A maximal concentration of ATP (100 microM) caused a marked, transient increase in bile flow in the isolated perfused skate liver, whereas 100 microM adenosine had no such effect. These findings demonstrate that skate hepatocytes possess P2 nucleotide receptors that link to intracellular plus extracellular Ca2+ mobilization, which in turn regulates bile secretion. The broad specificity of the response to ATP and related compounds suggests either that multiple types of P2 receptors are expressed by skate hepatocytes or else that these cells possess a single primitive nucleotide receptor from which other P2 subtypes subsequently evolved.

Swelling-activated anion conductance in skate hepatocytes: regulation by cell Cl- and ATP.

Cell swelling activates an outwardly rectifying anion conductance in mammalian cells. The channel responsible for this conductance mediates volume-regulatory efflux of organic osmolytes such as taurine. We observed a similar conductance in hepatocytes from the skate Raja erinacea. Whole cell Cl- conductance was increased > 100-fold by a 2-fold increase in hepatocyte volume. The conductance was outwardly rectifying and had a relative cation permeability of approximately 0.2. Cation permeability was increased by reductions in patch pipette CsCl concentration, suggesting that the channel pore contains saturable anion and cation binding sites with different anion and cation affinities. The conductance had a broad anion selectivity and a relative taurine permeability of 0.17. Activation of the conductance required intracellular ATP or a nonhydrolyzable ATP analogue. Elevation of intracellular Cl- from 20 to 155 mM reduced current activation while the rate and extent of cell swelling were unaffected. Reduction of intracellular Cl- concentration to 5-10 mM caused spontaneous current activation without cell swelling. These results suggest that increases in cell Cl- levels increase the volume set point of the channel. We propose that the main function of the outwardly rectifying anion channel is nonselective transport of organic solutes.

Two classes of bipolar cell in the retina of the skate Raja erinacea.

We have used immunoreactions against serotonin and protein kinase C to visualize two distinct classes of bipolar cell in the all-rod retina of the skate, Raja erinacea. To enhance the immunoreaction in serotonin-accumulating bipolar cells, prior to fixation, some retinas were incubated in Ringer's solution containing serotonin and pargyline. We found the somata of serotonin-accumulating bipolar cells to be located slightly distal to the midline of the inner nuclear layer. With increasing eccentricity from the visual streak, the size of the perikarya increases, concomitant with a decline in density of their distribution. Dendrites emanate from stout primary stalks and branch out before reaching the outer plexiform layer. Axons are bistratified within the inner plexiform layer with ramifications at the border of strata 1 and 2 and in stratum 4. The overall morphology of serotonin-accumulating bipolar cells is similar to that of serotonin-accumulating OFF bipolar cells of other non-mammalian vertebrates. Protein kinase C immunoreactive cells display the typical appearance of rod bipolar cells. Somata of protein kinase C immunoreactive bipolar cells are spindle-shaped and located distal to the serotonin-accumulating bipolar cells. Dendrites of these bipolars do not ramify before reaching the outer plexiform layer. Thin axons of protein kinase C immunoreactive bipolar cells end in large, club-shaped terminals in stratum 5 of the inner plexiform layer, bearing a striking similarity to axon terminals of mammalian ON rod bipolar cells. Our findings suggest that the all-rod retina of the skate contains at least two distinct vertical pathways including an OFF bipolar cell pathway in addition to a classical rod ON bipolar pathway.