Dogfish Rectal Gland Research Articles

The effects of dietary sodium loading on the activity and expression of Na, K-ATPase in the rectal gland of the European Dogfish ( Scyliorhinus canicula)

cDNA fragments of both the α- and β-subunits of the Na, K-ATPase and a cDNA fragment of the secretory form of Na–K–Cl cotransporter from the European dogfish ( Scyliorhinus canicula) were amplified and cloned using degenerate primers in RT-PCR. These clones were used along with a sCFTR cDNA from the related dogfish shark, Squalus acanthias to characterise the expression of mRNAs for these ion transporters in the dogfish rectal gland subsequent to an acute feeding episode. Following a single feeding event where starved dogfish were fed squid portions (20 g squid/kg fish), there was a delayed and transient 40-fold increase in the activity of Na, K-ATPase in crude rectal gland homogenates. Increases in enzyme activity were apparent 3 h after the feeding event and peaked at 9 h before returning to control values within 24 h. These increases in activity were accompanied by small and transient decreases in plasma sodium and chloride concentrations lasting up to 3 days. Significant increases in the expression of mRNAs for α- and β-subunits of the Na, K-ATPase, the Na–K–Cl cotransporter and CFTR chloride channel were detected but not until 1–2 days after the feeding event. It is concluded that the transient increase in Na, K-ATPase activity is not attributable to increases in the abundance of α- and β-subunit mRNAs but must be associated with some, as yet unknown, post-transcriptional activation mechanism.

Basolateral potassium channels and epithelial ion transport.

Basolateral potassium channels and epithelial ion transport.

Effects of extracellular sodium concentration on the activity of Na,K-ATPase in dogfish rectal gland epithelial cells.

Effects of extracellular sodium concentration on the activity of Na,K-ATPase in dogfish rectal gland epithelial cells.

CAMP-activated Cl- channels in primary cultures of spiny dogfish (Squalus acanthias) rectal gland

Whole cell and single-channel patch-clamp techniques were used to identify and characterize the Cl- currents responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion in the rectal gland of the spiny dogfish (Squalus acanthias). During whole cell recordings, in cultured rectal gland cells forskolin (10 microM) and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (400 microM) stimulated a 28-fold increase in Cl- conductance (n = 10). This cAMP-activated conductance pathway had a linear current-voltage (I-V) relationship that was time and voltage independent. Substitution of 235 meq Cl- with I- in the bath inhibited the cAMP-activated current at both positive and negative voltages (64%). Glibenclamide (60 microM) abolished the cAMP-stimulated current, and its effect was irreversible (n = 3). During cell-attached recording, increased cellular cAMP activated single Cl- channels in nine previously quiet patches. These channels had a linear I-V relationship with an average single-channel conductance of 5.1 +/- 0.2 pS (n = 6). Similar properties were observed in excised inside-out patches, permitting further characterization of the single-channel properties. Excised quiescent patches could be activated by the addition of ATP and protein kinase A. Replacing bath Cl- with I- inhibited both inward and outward currents (n = 3). In three inside-out patches, glibenclamide (300 microM) reversibly reduced open probability by 74%, with no effect on single-channel current amplitude. Similar results were obtained in four outside-out recordings. These results suggest that increased cellular cAMP in dogfish rectal gland activates a small linear Cl- channel that resembles human cystic fibrosis transmembrane conductance regulator in its biophysical and pharmacological properties.

Renin-like Activity, Angiotensin I-Converting Enzyme-like Activity, and Osmoregulatory Peptides in the Dogfish Rectal Gland

Renin-like activity (RLA) and angiotensin I-converting enzyme-like activity (ACELA), two key enzymes of the renin-angiotensin cascade (RAS), were sought in the dogfish rectal gland. RLA was 1.1 ± 0.2 ng Ang I/mg protein/hr after incubation with porcine angiotensinogen and 0.8 ± 0.1 ng Ang I/mg protein/hr after incubation with homologous plasma. ACELA was 7.22 ± 1.08 and 8.87 ± 1.9 nmol hippurate generated/min/mg protein respectively, at 0 and 37°. The presence of these enzymes may indicate the presence of an endogenous RAS-like system in the rectal gland. Angiotensin II (Ang II) and atrial natriuretic peptide (ANP) binding sites were demonstrated autoradiographically in the subcapsular region of the gland, suggesting a possible interaction of the two hormones in the blind outer ends of the rectal gland tubules. Immunoreactivities toward Ang II, ANP, bombesin, vasoactive intestinal polypeptide (VIP), glucagon, and somatostatin were differentially localized in the rectal gland within three concentric zones with potentially different functional activities. In the capsule, there was a strong positive ir-glucagon reaction and a slightly weaker reaction for ir-somatostatin and VIP. In the blind outer ends of the tubeles (in the subcapsular zone), strong immunoreactivity was present toward all the tested peptides except glucagon and somatostatin. In the inner zone and in the central canal, only a weak immunoreactivity toward Ang II and glucagon was observed.

Inhibition of Na,K-ATPase by Cadmium: Different Mechanisms in Different Species

The mechanism of action of Cd on Na,K-ATPase was investigated in two "classical" model systems, the shark rectal gland and rabbit kidney outer medulla. In lyophilized plasma membranes from dogfish rectal gland Cd inhibited Na,K-ATPase activity after 30 min of preincubation with an I 50 of 1.3 × 10 −5 M. K-Dependent p-nitrophenylphosphatase (pNPPase) activity was inhibited 50% by Cd at 9.4 × 10 −6 M. Neither Na nor K altered the interaction of the enzyme with Cd. Decreasing the ATP concentration, however, lowered the apparent sensitivity of Na,K-ATPase to Cd. The inhibitory effect was also significantly reduced when the Mg concentration present during the preincubation was increased from 0.5 to 6.0 mM. The apparent Cd sensitivity of the K-dependent pNPPase is lower at 10 mM Mg than at 1 mM Mg. In initial rate experiments 4 × 10 −5 M Cd increased the apparent K m of the enzyme for Mg significantly from 0.88 ± 0.29 mM to 1.73 ± 0.3 mM whereas the V max (167 ± 32 μmol/min × mg protein compared to 140 ± 16 μmol/min × mg protein) remained essentially unchanged. In lyophilized rabbit kidney outer medulla, Cd was found to inhibit Na,K-ATPase activity with an I 50 of 1.9 × 10 −5 M. K-Dependent pNPPase was inhibited 50% under identical conditions by Cd at a nominal concentration of 2.1 × 10 −4 M. Increasing K concentrations protected the enzyme from the inhibitory action of Cd as indicated by a 10-fold decrease in sensitivity of pNPPase when the K concentration was increased from 1 to 20 mM. K, 20 mM, delayed also the onset of inhibition by about 15 min at 37°C. These studies suggest that the mode of action of Cd on Na,K-ATPase varies in different species. In rectal gland Cd competes with a Mg site (or sites) critically involved in ATP and pNPP hydrolysis, whereas in rabbit renal medulla Cd interacts with a potassium-binding site. Differences in the protein sequence, protein conformation, and/or in the kind of protein membrane-lipid interaction might contribute to this diversity observed in the inhibitory mechanisms.

Identification and localization of a dogfish homolog of human cystic fibrosis transmembrane conductance regulator.

Chloride channels in the apical plasma membrane of cells in the dogfish rectal gland have served as a model system for the study of regulation of chloride flux by changes in intracellular cyclic AMP levels. Similar regulation by cyclic AMP has been described for channels in cells of human secretory epithelia where defective regulation by cyclic AMP-dependent protein phosphorylation is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). We have isolated a cDNA clone from the rectal gland encoding a protein that is 72% identical to the human CFTR. One of the major phosphorylation sites in CFTR is absent in the dogfish protein. The dogfish protein has, however, four additional putative substrate sites for the cyclic AMP-dependent protein kinase. A peptide antibody, which was raised against an amino acid sequence common to both the human and dogfish CFTR sequences, recognizes proteins with similar molecular masses (160 kDa) in the dogfish gland and in mammalian lung. Immunolocalization studies with this antibody show that the putative dogfish CFTR is localized to the apical membrane of cells lining the lumen of the rectal gland.

PCMBS-induced swelling of dogfish ( Squalus acanthias) rectal gland cells: role of the Na +,K +-ATPase and the cytoskeleton

(1) 0.1–1.0 mM p-chloromercuribenzene sulfonate (pCMBS) and some other organic mercurials produce a swelling of slices of dogfish shark ( Squalus acanthias) rectal glands, with an uptake of cell Na + and a loss of K +. In contrast, 1 mM N-ethylmaleimide (NEM) does not swell rectal gland cells (RGC), while affecting cell cations. (2) The slow entry of [ 203Hg]pCMBS is linearly related to its external concentration (10 μ-1 mM) and a small accumulation of pCMBS (apparent gradient about 3) in the cells occurs in 2 h. Cell 203Hg rapidly washes out of the cells (fast rate constant 0.153 · min −1; slow rate constant 0.0067 · min −1), and this efflux is accelerated by 1 mM dithiothreitol. Thus, a major portion of pCMBS inter-acts rather loosely with cell components. (3) pCMBS and NEM share: (a) a negligible effect on the efflux of 86Rb + and of [ 14C]urea; (b) a gradual inhibition of the cell Na +,K +-ATPase activity. (4) NEM as well as agents lowering cell glutathione accelerate and increase the pCMBS-induced cell swelling. Conditions inhibiting the Na +,K +-ATPase (ouabain, absence of Na +) have the same effect. (5) pCMBS, but not NEM produce a disappearance of the F-actin-phalloidin fluorescence independent of cell volume changes, particularly at the basolateral RGC membrane. (6) The data are consistent with the following set of events: (a) pCMBS (but not NEM) affects the cell membrane by increasing the efflux of the cell osmolyte taurine (Ziyadeh et al. (1988) Biochim. Biophys. Acta 943, 43–52 and unpublished data); (b) on entry into the cells, pCMBS and NEM interact with cell -SH, including those of the Na +,K +-ATPase; this action produces the observed changes in cell cations. Also, pCMBS, but not NEM, decrease F-actin at the membrane; (c) the inhibition of the Na +,K +-ATPase activity together with the decreased resistance of the cell membrane to stretch (absence of F-actin) produces the observed pCMBS-induced cell swelling by osmotic forces (intracellular non-diffusible anions).

Activation of taurine efflux in hypotonically stressed elasmobranch cells: Inhibition by stilbene disulfonates

AbstractHypotonic stress activates taurine efflux from elasmobranch cells. The transport mechanism involved in this efflux is not known. In this study we show that the hypotonic‐activated taurine efflux from skate (Raja erinacea) erythrocytes and dogfish (Squalus acanthias) rectal gland tubules is inhibited by stilbene disulfonates. These results suggest that the anion transporter (Band‐3) is involved in the efflux activation mechanism.

Immunohistochemical Localization of Vasoactive Intestinal Peptide in the Dogfish Rectal Gland

Immunohistochemical Localization of Vasoactive Intestinal Peptide in the Dogfish Rectal Gland

Immunohistochemical evidence for neural mediation of VIP activity in the dogfish rectal gland

Vasoactive intestinal peptide (VIP) has been shown to increase chloride secretion from the rectal gland of the spiny dogfish, Squalus acanthias. Immunohistochemistry was used to localize the distribution of immunoreactive VIP (iVIP). Rectal glands were perfused with either buffered acrolein or paraformaldehyde/glutaraldehyde, sectioned (20 μm) and processed by either avidin-biotin complex (ABC) or peroxidase anti-peroxidase (PAP) methods. At the light microscopic level, iVIP was observed in thick fibers which traversed the fibromembranous capsule of the rectal gland. In the parenchyma, smaller iVIP-containing fibers were noted within connective tissue and in close approximation to tubule cells. At the ultrastructural level, iVIP axons in the fibromembranous capsule were unmyelinated. Immunoreactive fibers within the parenchyma frequently terminated on the basal side of tubule cells. Within the glands, iVIP bouton terminals were observed and contained vesicles of different sizes, with reaction product in dense core vesicles (60–120 nm). We conclude that iVIP is distributed in nerve fibers throughout the dogfish rectal gland. The anatomic distribution suggests that VIP may act as a neurotransmitter in this model of chloride ion transport.

The “small” conductance chloride channel in the luminal membrane of the rectal gland of the dogfish (Squalus acanthias)

Besides the "larger" Cl- channel, with a single channel conductance of about 45 pS, a "small" channel was observed in the luminal membrane of the dogfish rectal gland. In cell excised (inside out) patches with NaCl solution on both sides, the latter channel had a single channel conductance of 11 +/- 1 pS (n = 21), and its current-voltage relationship was linear in the voltage range +90 to -90 mV. The open state probability increased moderately with negative clamp potentials. Ionic replacement studies revealed a high selectivity of Cl- over gluconate, sulfate, and iodide, whereas bromide was permeable to some extent. Also the channel is impermeable for Na+. The Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate did not affect this "small" conductance Cl- channel. It can be concluded that the luminal membrane of stimulated rectal gland cells possesses two types of Cl- channels, which differ markedly in their characteristics.

Oxygen consumption of dogfish rectal gland after in vitro and in vivo zinc treatments

Zinc treatments inhibit dogfish rectal gland respiration. In vitro exposure causes inhibition at concentrations higher than 15 ppm. Acute in vivo exposure also produces inhibition, while no effects are observed after subacute treatment. Results are discussed in relation to previous work on other tissues.

Effect of anisotonic media on cell volume and electrolyte fluxes in slices of the dogfish (Squalus acanthias) rectal gland

Osmotic responses of slices of dogfish rectal gland to hypotonic (urea-free) and hypertonic media were studied. Transfer of tissue from isotonic (890 mosM) to hypotonic (550 mosM) saline produced an osmotic swelling associated with a slow net uptake of cell K+ (and Cl−) and a slow, two-component efflux of urea. Media made hypertonic (1180 mosM) by addition of urea or mannitol produced osmotic shrinkage with a net loss of KCl. The cell osmotic responses in hypotonic media were lower than predicted for an ideal osmometer. No volume regulatory responses were seen subsequent to the initial osmotic effects. The cation influx in hypotonic media lacked specificity: in the presence of 0.5 mM ouabain or in K+-free media a net influx of Na+ was found. At steady state, the cell membrane potential evaluated from the Nernst potentials of K+ and triphenylmethyl phosphonium+, was independent of medium tonicity, suggesting the membrane potential as a determinant in the cellular osmotic response. Zero-time86Rb+ fluxes were measured:86Rb+ influx was not affected by hypotonicity, implying an unchanged operation of the Na+−K+-ATPase. On the other hand,86Rb+ efflux was significantly reduced at hypotonicity; this effect was transient, the efflux returning to the control value once the new steady state of cell volume had been reached. A controlled efflux system is therefore involved in the cell osmotic response. The absence of the volume regulatory phenomenon suggests that the cells are not equipped with a volume-sensing mechanism.

Amphotericin B and the elasmobranch rectal gland: implications for the relationship between oxygen consumption and ion transport.

In slices of the dogfish rectal gland, amphotericin B produced increases in oxygen consumption and ouabain binding similar to those produced by cyclic AMP, except that the increases induced by the latter were inhibited by furosemide whereas those resulting from the addition of amphotericin B were unaffected by the diuretic. Unlike cyclic AMP, however, amphotericin B failed to stimulate secretion by the isolated perfused gland. These results support the suggestion that the increases in ouabain binding and oxygen consumption produced by cyclic AMP result simply from an increased sodium entry into the cells, and as such can be mimicked by amphotericin B, whereas secretion rate itself depends on some additional, cyclic AMP-induced process. The implications of this for determinations of the relationship between rates of oxygen consumption and ion transport are discussed.

Freeze-fracture and morphometric analysis of occluding junctions in rectal glands of elasmobranch fish.

The structure of occluding junctions in secretory and ductal epithelium of salt-secreting rectal glands from two species of elasmobranch fish, the spiny dogfish Squalus acanthias and the stingray Dasyatis sabina, was examined by thin-section and freeze-fracture electron microscopy. In both species, occluding junctions between secretory cells are shallow in their apical to basal extent and are characterized by closely juxtaposed parallel strands. Average strand number in the dogfish was 3.5 +/- 0.2. with a mean depth of 56 +/- 5 nm; in the stingray a mean of 2.0 +/- 0.2 strands encompassed an average depth of 18 +/- 3 nm. In contrast, the linear extent of these junctions was remarkably large due to the intermeshing of the narrow apices of the secretory cells to form the tubular lumen. Morphometric analysis gave values of 66. 8 +/- 2.5 and 74.9 +/- 4.6 m/cm2 for the length of junction per unit of luminal surface area in the dogfish and stingray, respectively. This junctional morphology is similar to that generally described for "leaky" epithelia. In comparison, the stratified ductal epithelium which carries the NaCl-rich secretion to the intestine is characterized by extensive occluding junctions which extend 0.6-0.8 mum in depth and consist of a mean of 12 strands arranged in an anastomosing network, an architectural pattern typical of "tight" epithelia. The length density of these junctions in the dogfish rectal gland was 7.6 +/- 0.1 m/cm2. The junctional architecture of the rectal gland secretory epithelium (few strands, large junctional length densities) is similar to that described for several other hypertonic secretory epithelia [20, 34] and is compatible with the recent model for salt secretion in rectal glands [39] and in other C1- secretory epithelia which posits a conductive paracellular pathway for trans-epithelial Na+ secretion from intercellular space to the lumen to form the NaCl-rich secretory product.

Molecular weight and stoichiometry of the sodium- and potassium-activated adenosine triphosphatase subunits.

The molecular weight and stoichiometry of the subunits of the sodium- and potassium-activated adenosine triphosphatase. (Na,K)-ATPase, have been examined in four highly purified preparations of this enzyme: dog kidney, eel electroplax, dogfish rectal gland, and brine shrimp nauplius. The molecular weights of the subunits were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 10 different total acrylamide concentrations (%T) and by a gradient SDS-PAGE system, standardized with 16 molecular weight marker proteins of known primary structure. The molecular weight of the large (Na,K)-ATPase subunit (alpha subunit) was found to be reliably estimated by SDS-PAGE, whereas the small subunit (beta subunit) was not. The molecular weight of the alpha subunit was 97,000, 97,700, 104,200, and 97,800 for the dog, eel, dogfish, and brine shrimp (Na,K)-ATPase, respectively. The molecular weight of the beta subunit is overestimated by SDS-PAGE. Mass ratio analysis of the alpha and beta subunits separated by SDS-PAGE was determined by three techniques: total amino acid quantitation, gel scanning and dye elution of Coomassie blue-stained gels, and UV gel scanning. The former method, which provides the most reliable estimate of the mass ratio, gives an alpha/beta mass ratio of 2.41, 2.33, 2.91, and 2.44 for the dog, eel, dogfish, and brine shrimp preparations, respectively. Results of the mass ratio studies, the molecular weight analysis, and prior cross-linking studies clearly demonstrate that the quaternary structure of the (Na,K)-ATPase is an alpha 2 beta 2 tetramer in all four species. Based on all of the above data, a more reliable molecular weight estimate of the beta subunit is calculated as 40,200, 43,000, 35,800, and 40,100 for the dog, eel, dogfish, and brine shrimp preparations, respectively. These results now confirm that the molecular weight of the protein portion of the alpha subunit is near 100,000 and that of the beta subunit is near 40,000 and that the (Na,K)-ATPase holoenzyme is an alpha 2 beta 2 tetramer of molecular weight 274,000 to 280,000, regardless of species.

The mechanism of cyclic AMP stimulation of secretion in the dogfish rectal gland

1. Cyclic AMP induces pronounced increases in ouabain binding and ouabain-sensitive oxygen consumption in rectal gland slices but does so in the absence of any change in the Na−K-ATPase activity of tissue homogenates. It is suggested that this apparent paradox results from the presence of inactive or concealed ‘latent sites’ in the membrane of the unstimulated gland which become exposed or activated in the presence of cAMP. 2. Evidence suggests that, in the absence of cAMP, the exposure and activation of these latent sites is largely determined by the rate of sodium entry into the cells. It appears that sodium can enter the cells either by a simple passive diffusion down the electrochemical gradient or via a furosemide-sensitive, chloride-coupled pathway. 3. Experiments have shown that the cAMP effect on ouabain binding and oxygen consumption is absent in conditions designed to eliminate the gradient for sodium entry into the cells. Similarly, no cAMP effect on these parameters is observed in the absence of external chloride or in the presence of furosemide. Furthermore, cAMP has been shown to produce a marked increase in sodium entry into the rectal gland cell and this enhanced entry is entirely furosemide-sensitive. 4. It appears therefore that cAMP has no direct effect on the recruitment of latent sites per se but instead has a specific, stimulatory action on the furosemide-sensitive entry of sodium which, in turn, leads to an increase in functional sodium pump sites and an enhanced sodium efflux. Chloride efflux appears to be increased in a passive manner as a result of the increased gradient on this ion subsequent to an enhanced entry via the cAMP-stimulated, furosemide-sensitive pathway.

Oxygen consumption in the rectal gland of the dogfishScyliorhinus canicula and the effects of cyclic AMP

Oxygen consumption was measured in slices of dogfish (Scyliorhinus canicula) rectal gland tissue and compared with spleen and kidney. Concentrations of dibutyryl cAMP and theophylline that have previously been shown to stimulate secretion from the perfused gland and to produce a large increase in ouabain binding in this tissue, greatly increased oxygen consumption in the rectal gland but were without effect on oxygen consumption in the spleen and the kidney. Addition of theophylline alone increased rectal gland oxygen consumption and no further increase was detected on addition of cAMP at the concentration used (0.05 mmol l−1). Ouabain (10−4 M) significantly decreased oxygen consumption in all three tissues studied and completely abolished the increase in oxygen consumption of the rectal gland produced as a result of the addition of cAMP and theophylline. These findings are discussed in relation to the apparent specific effect of cAMP on the rectal gland and its possible mode of action in this tissue.

Hormonal regulation of active chloride transport in the dogfish rectal gland

Active transport of chloride is modulated by cyclic AMP in the rectal gland of Squalus acanthias. Vasoactive intestinal peptide (VIP) specifically activates the production of cyclic AMP by the gland and stimulates the secretion of chloride. Somatostatin inhibits VIP-induced secretion but has no effect alone. Both these peptides are present in the dogfish shark and may play an important role in electrolyte homeostasis.