V-H+ -ATPase translocation during blood alkalosis in dogfish gills: interaction with carbonic anhydrase and involvement in the postfeeding alkaline tide

We investigated the involvement of carbonic anhydrase (CA) in mediating V-H(+)-ATPase translocation into the basolateral membrane in gills of alkalotic Squalus acanthias. Immunolabeling revealed that CA is localized in the same cells as V-H(+)-ATPase. Blood plasma from dogfish injected with acetazolamide [30 mg/kg at time (t) = 0 and 6 h] and infused with NaHCO(3) for 12 h (1,000 microeq.kg(-1).h(-1)) had significantly higher plasma HCO(3)(-) concentration than fish that were infused with NaHCO(3) alone (28.72 +/- 0.41 vs. 6.57 +/- 2.47 mmol/l, n = 3), whereas blood pH was similar in both treatments (8.03 +/- 0.11 vs. 8.04 +/- 0.11 pH units at t = 12 h). CA inhibition impaired V-H(+)-ATPase translocation into the basolateral membrane, as estimated from immunolabeled gill sections and Western blotting on gill cell membranes (0.24 +/- 0.08 vs. 1.00 +/- 0.28 arbitrary units, n = 3; P < 0.05). We investigated V-H(+)-ATPase translocation during a postfeeding alkalosis ("alkaline tide"). Gill samples were taken 24-26 h after dogfish were fed to satiety in a natural-like feeding regime. Immunolabeled gill sections revealed that V-H(+)-ATPase translocated to the basolateral membrane in the postfed fish. Confirming this result, V-H(+)-ATPase abundance was twofold higher in gill cell membranes of the postfed fish than in fasted fish (n = 4-5; P < 0.05). These results indicate that 1) intracellular H(+) or HCO(3)(-) produced by CA (and not blood pH or HCO(3)(-)) is likely the stimulus that triggers the V-H(+)-ATPase translocation into the basolateral membrane in alkalotic fish and 2) V-H(+)-ATPase translocation is important for enhanced HCO(3)(-) secretion during a naturally occurring postfeeding alkalosis.     

Permeabilities of teleost and elasmobranch gill apical membranes: evidence that lipid bilayers alone do not account for barrier function

Teleosts and elasmobranchs faced with considerable osmotic challenges living in sea water, use compensatory mechanisms to survive the loss of water (teleosts) and urea (elasmobranchs) across epithelial surfaces. We hypothesized that the gill, with a high surface area for gas exchange must have an apical membrane of exceptionally low permeability to prevent equilibration between seawater and plasma. We isolated apical membrane vesicles from the gills of Pleuronectes americanus (winter flounder) and Squalus acanthias (dogfish shark) and demonstrated approximately sixfold enrichment of the apical marker, ADPase compared to homogenate. We also isolated basolateral membranes from shark gill (enriched 2.3-fold for Na-K-ATPase) and using stopped-flow fluorometry measured membrane permeabilities to water, urea, and NH(3). Apical membrane water permeabilities were similar between species and quite low (7.4 +/- 0.7 x 10(-4) and 6.6 +/- 0.8 x 10(-4) cm/s for shark and flounder, respectively), whereas shark basolateral membranes showed twofold higher water permeability (14 +/- 2 x 10(-4) cm/s). Permeabilities to urea and NH(3) were also low in apical membranes. Because of the much lower apical to basolateral surface area we conclude that the apical membrane represents an effective barrier. However, the values we obtained were not low enough to account for low water loss (teleosts) and urea loss (elasmobranchs) measured in vivo by others. We conclude that there are other mechanisms which permit gill epithelia to serve as effective barriers. This conclusion has implications for the function of other barrier epithelia, such as the gastric mucosa, mammalian bladder, and renal thick ascending limb.     

The shark rectal gland: a model for the active transport of chloride.

The rectal gland of the spiny dogfish, Squalus acanthias, provides an easily studied model of active chloride transport powered indirectly by Na-K-ATPase. Co-transport of sodium with chloride can be demonstrated in membrane vesicles isolated from basolateral membranes of the gland. Chloride secretion is under the hormonal control of vasoactive intestinal peptide, and possibly other agents, via adenyl cyclase and cyclic AMP. A similar mechanism is probably responsible for the active transport of chloride across other biological membranes.     

Urea and water permeability in dogfish (Squalus acanthias) gills

We used a perfused gill preparation from dogfish to investigate the origin of low branchial permeability to urea. Urea permeability (14C-urea) was measured simultaneously with diffusional water permeability (3H2O). Permeability coefficients for urea and ammonia in the perfused preparation were almost identical to in vivo values. The permeability coefficient of urea was 0.032 x 10(-6) cm/sec and of 3H2O 6.55 x 10(-6) cm/sec. Adrenalin (1 x 10(-6) M) increased water and ammonia effluxes by a factor of 1.5 and urea efflux by a factor of 3.1. Urea efflux was almost independent of the urea concentration in the perfusion medium. The urea analogue thiourea in the perfusate had no effect on urea efflux, whereas the non-competitive inhibitor of urea transport, phloretin, increased efflux markedly. The basolateral membrane is approximately 14 times more permeable to urea than the apical membrane. We conclude that the dogfish apical membrane is extremely tight to urea, but the low apparent branchial permeability may also relate to the presence of an active urea transporter on the basolateral membrane that returns urea to the blood and hence reduces the apical urea gradient.     

Type IV carbonic anhydrase is present in the gills of spiny dogfish (Squalus acanthias)

Physiological and biochemical studies have provided indirect evidence for a membrane-associated carbonic anhydrase (CA) isoform, similar to mammalian type IV CA, in the gills of dogfish (Squalus acanthias). This CA isoform is linked to the plasma membrane of gill epithelial cells by a glycosylphosphatidylinositol anchor and oriented toward the plasma, such that it can catalyze the dehydration of plasma HCO(3)(-) ions. The present study directly tested the hypothesis that CA IV is present in dogfish gills in a location amenable to catalyzing plasma HCO(3)(-) dehydration. Homology cloning techniques were used to assemble a 1,127 base pair cDNA that coded for a deduced protein of 306 amino acids. Phylogenetic analysis suggested that this protein was a type IV CA. For purposes of comparison, a second cDNA (1,107 base pairs) was cloned from dogfish blood; it encoded a deduced protein of 260 amino acids that was identified as a cytosolic CA through phylogenetic analysis. Using real-time PCR and in situ hybridization, mRNA expression for the dogfish type IV CA was detected in gill tissue and specifically localized to pillar cells and branchial epithelial cells that flanked the pillar cells. Immunohistochemistry using a polyclonal antibody raised against rainbow trout type IV CA revealed a similar pattern of CA IV immunoreactivity and demonstrated a limited degree of colocalization with Na(+)-K(+)-ATPase immunoreactivity. The presence and localization of a type IV CA isoform in the gills of dogfish is consistent with the hypothesis that branchial membrane-bound CA with an extracellular orientation contributes to CO(2) excretion in dogfish by catalyzing the dehydration of plasma HCO(3)(-) ions.     

The putative mechanism of Na(+) absorption in euryhaline elasmobranchs exists in the gills of a stenohaline marine elasmobranch, Squalus acanthias

We recently cloned an NHE3 orthologue from the gills of the euryhaline Atlantic stingray (Dasyatis sabina), and generated a stingray NHE3 antibody to unequivocally localize the exchanger to the apical side of epithelial cells that are rich with Na(+)/K(+)-ATPase (A MRC). We also demonstrated an increase in NHE3 expression when stingrays are in fresh water, suggesting that NHE3 is responsible for active Na(+) absorption. However, the vast majority of elasmobranchs are only found in marine environments. In the current study, immunohistochemistry with the stingray NHE3 antibody was used to localize the exchanger in the gills of the stenohaline marine spiny dogfish shark (Squalus acanthias). NHE3 immunoreactivity was confined to the apical side of cells with basolateral Na(+)/K(+)-ATPase and was excluded from cells with high levels of vacuolar H(+)-ATPase. Western blots detected a single protein of 88 kDa in dogfish gills, the same size as NHE3 in stingrays and mammals. These immunological data demonstrate that the putative cell type responsible for active Na(+) absorption in euryhaline elasmobranchs is also present in stenohaline marine elasmobranchs, and suggest that the inability of most elasmobranchs to survive in fresh water is not due to a lack of the gill ion transporters for Na(+) absorption.     

Branchial mitochondria-rich cells in the dogfish Squalus acanthias

In marine teleost fishes, the gill mitochondria-rich cells (MRCs) are responsible for NaCl elimination; however, in elasmobranch fishes, the specialized rectal gland is considered to be the most important site for salt secretion. The role of the gills in elasmobranch ion regulation, although clearly shown to be secondary, is not well characterized. In the present study, we investigated some morphological properties of the branchial MRCs and the localization, and activity of the important ionoregulatory enzyme Na(+)/K(+)-ATPase, under control conditions and following rectal gland removal (1 month) in the spiny dogfish, Squalus acanthias. A clear correlation can be made between MRC numbers and the levels of Na(+)/K(+)-ATPase activity in crude gill homogenates (r(2)=-0.69). Strong Na(+)/K(+)-ATPase immunoreactivity is also clearly associated with the basolateral membrane of these MRCs. In addition, the dogfish were able to maintain ionic balance after rectal gland removal. These results all suggest a possible role of the dogfish gill in salt secretion. MRCs were, however, unresponsive to rectal gland removal in terms of changes in number, fine structure and Na(+)/K(+)-ATPase activity, as might be expected if they were compensating for the loss of salt secretion by the rectal gland. Thus, the specific role that these MRCs play in ion regulation in the dogfish remains to be determined     

Gallbladder epithelial cell hydraulic water permeability and volume regulation

The hydraulic water permeability (Lp) of the cell membranes of Necturus gallbladder epithelial cells was estimated from the rate of change of cell volume after a change in the osmolality of the bathing solution. Cell volume was calculated from computer reconstruction of light microscopic images of epithelial cells obtained by the "optical slice" technique. The tissue was mounted in a miniature Ussing chamber designed to achieve optimal optical properties, rapid bath exchange, and negligible unstirred layer thickness. The control solution contained only 80% of the normal NaCl concentration, the remainder of the osmolality was made up by mannitol, a condition that did not significantly decrease the fluid absorption rate in gallbladder sac preparations. The osmotic gradient ranged from 11.5 to 41 mosmol and was achieved by the addition or removal of mannitol from the perfusion solutions. The Lp of the apical membrane of the cell was 1.0 X 10(-3) cm/s . osmol (Posm = 0.055 cm/s) and that of the basolateral membrane was 2.2 X 10(-3) cm/s . osmol (Posm = 0.12 cm/s). These values were sufficiently high so that normal fluid absorption by Necturus gallbladder could be accomplished by a 2.4-mosmol solute gradient across the apical membrane and a 1.1-mosmol gradient across the basolateral membrane. After the initial cell shrinkage or swelling resulting from the anisotonic mucosal or serosal medium, cell volume returned rapidly toward the control value despite the fact that one bathing solution remained anisotonic. This volume regulatory response was not influenced by serosal ouabain or reduction of bath NaCl concentration to 10 mM. Complete removal of mucosal perfusate NaCl abolished volume regulation after cell shrinkage. Estimates were also made of the reflection coefficient for NaCl and urea at the apical cell membrane and of the velocity of water flow across the cytoplasm.     

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 microM-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 1mM 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).     

Stimulation of NaCl secretion in the rectal gland of the dogfish Squalus acanthias

1. The rectal gland of the dogfish (Squalus acanthias) secretes NaCl when stimulated by a hormone related to vasointestinal peptide. 2. Patch clamp and microelectrode techniques are used to examine the changes in membrane conductances occurring with hormonal stimulation. 3. The conductance of the "resting" cell is dominated by basolateral K+ channels. 4. Hormonal stimulation "opens" apical Cl- channels. 5. This opening of apical chloride channels appears to be mediated by cAMP-dependent phosphorylation of pre-existing closed channels.     

Functional analysis of the musculo‐skeletal system of the gill apparatus in Heptranchias perlo (Chondrichthyes: Hexanchidae)

Musculo‐skeletal morphology is an indispensable source for understanding functional adaptations. Analysis of morphology of the branchial apparatus of Hexanchiform sharks can provide insight into aspects of their respiration that are difficult to observe directly. In this study, I compare the structure of the musculo‐skeletal system of the gill apparatus of Heptranchias perlo and Squalus acanthias in respect to their adaptation for one of two respiratory mechanisms known in sharks, namely, the active two‐pump (oropharyngeal and parabranchial) ventilation and the ram‐jet ventilation. In both species, the oropharyngeal pump possesses two sets of muscles, one for compression and the other for expansion. The parabranchial pump only has constrictors. Expansion of this pump occurs only due to passive elastic recoil of the extrabranchial cartilages. In Squalus acanthias the parabranchial chambers are large and equipped by powerful superficial constrictors. These muscles and the outer walls of the parabranchial chambers are much reduced in Heptranchias perlo , and thus it likely cannot use this pump. However, this reduction allows for vertical elongation of outer gill slits which, along with greater number of gill pouches, likely decreases branchial resistance and, at the same time, increases the gill surface area, and can be regarded as an adaptation for ram ventilation at lower speeds.     

Inhibition of chloride secretion by BaCl2 in the rectal gland of the spiny dogfish, Squalus acanthias

In the rectal gland of the spiny dogfish (Squalus acanthias), chloride enters the cell via a cotransport system together with sodium and potassium in a 2 Cl-: 1 Na+: 1 K+ stoichiometry. The system is energized by the electrochemical potential for sodium directed into the cell. Sodium is extruded from the cell by Na-K-ATPase located on the basolateral cell membrane. Chloride leaks into the lumen following a favorable electrical gradient. Potassium is thought to recirculate across the basolateral cell membrane. Since barium ions inhibit the efflux of potassium from cells we used barium chloride to explore the role of potassium in the process of stimulated secretion of chloride by the gland. The secretion of chloride was stimulated with theophylline 2.5 X 10(-4)M and dibutyryl cyclic AMP 5 X 10(-5)M. Ba++ inhibited the secretion of chloride in a way that was reversible and dose dependent. The reduction in secretion was associated with a parallel fall in transglandular electrical potential. Inhibition was half maximal at a concentration of Ba++ of 10(-3)M. The reduction in efflux of potassium produced by Ba++ presumably decreases the potassium diffusion potential, thus reducing the electronegativity of the cell and dissipating the driving force for chloride across the apical cell membrane. Recirculation of K+ across the basolateral border of the cell would thus be essential for the maintenance of chloride secretion by the gland.     

Properties of single- and double-barreled Cl channels of shark rectal gland in planar bilayers

Chloride channels from the apical plasma membrane fraction of rectal gland of Squalus acanthias were characterized by incorporation into planar bilayers in the presence of cAMP-PK/ATP. In a total of 80 bilayer preparations, 21 Cl-selective channels were observed as single channels and 13 as pairs. This was a significantly greater number of double Cl channels than expected from a binomial distribution. The double Cl channels were divided into two groups based on kinetic and voltage-dependent behavior. One group had properties identical to the single channels (gb1) while the other was consistent with a double-barreled channel (gb2) with coordinated activity between proto-channels. The single-channel slope conductances of gb1 and gb2 from -60 to +20 mV with a 250/70 mM KCl gradient were 41 and 75 pS, respectively. With symmetrical 250 mM KCl, the I-V relation of gb1 showed outward rectification with 47.8 +/- 6.6 pS at cis negative potentials and 68.9 +/- 6.1 pS at cis positive potentials. gb1 was open from 70 to 95% at all electrochemical potentials from -80 to +40 mV. gb2 was steeply voltage dependent between -80 and -20 mV. Both gb1 and gb2 were insensitive to Ca (from 100 nm to 1 microM), blocked by 0.1 mM DIDS and highly selective for chloride. These data suggest that double-barreled Cl channels are related to the family of small, outwardly rectifying Cl channels of epithelial membranes.     

Cyclic AMP regulation of active chloride transport in the rectal gland of marine elasmobranchs.

Active transport of chloride in the salt secreting rectal gland of the dogfish Squalus acanthias is markedly stimulated by the addition of theophylline (0.01 to 5 mM) or dibutyryl cyclic AMP (0.05 mM) during in vitro perfusion. The effect occurs promptly and may persist for two hours. Specific hormonal effectors have not yet been identified. The isolated perfused rectal gland thus offers a unique opportunity to examine the cellular mechanisms of active chloride secretion.     

Extracellular carbonic anhydrase in the dogfish, Squalus acanthias: a role in CO2 excretion.

In Pacific spiny dogfish (Squalus acanthias), plasma CO(2) reactions have access to plasma carbonic anhydrase (CA) and gill membrane-associated CA. The objectives of this study were to characterise the gill membrane-bound CA and investigate whether extracellular CA contributes significantly to CO(2) excretion in dogfish. A subcellular fraction containing membrane-associated CA activity was isolated from dogfish gills and incubated with phosphatidylinositol-specific phospholipase C. This treatment caused significant release of CA activity from its membrane association, a result consistent with identification of the dogfish gill membrane-bound CA as a type IV isozyme. Inhibition constants (K(i)) against acetazolamide and benzolamide were 4.2 and 3.5 nmol L(-1), respectively. Use of a low dose (1.3 mg kg(-1) or 13 micromol L(-1)) of benzolamide to selectively inhibit extracellular CA in vivo caused a significant 30%-60% reduction in the arterial-venous total CO(2) concentration difference, a significant increase in Pco(2) and an acidosis, without affecting blood flow or ventilation. No effect of benzolamide on any measure of CO(2) excretion was detected in rainbow trout (Oncorhynchus mykiss). These results indicate that extracellular CA contributes substantially to CO(2) excretion in the dogfish, an elasmobranch, and confirm that CA is not available to plasma CO(2) reactions in rainbow trout, a teleost.     

Elasmobranch tissue culture: in vitro growth of brain explants from a shark (Rhizoprionodon) and dogfish (Squalus)

Brain explants from a Caribbean sharpnose shark (Rhizoprionodon porosus) and six spiny dogfish (Squalus acanthias) were cultured at 22 degrees C in Leibovitz L-15 medium, 1000 mOsmol, with 350 mM urea, 20 mM added NaCl, and either 2% fetal bovine serum, 2% bull shark (Carcharhinus leucas) serum, or 2% spiny dogfish serum. Cell outgrowth from explants appeared after 1 month. Cells were subcultured once with 20% survival, and were viable at termination of the trials 6 months later. Cell morphology appeared normal throughout the experiment.     

Subcellular location of glutamine synthetase and urea cycle enzymes in liver of spiny dogfish (Squalus acanthias)

Glutamine synthetase and glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase, both of which are present in high concentrations in liver of urea-retaining elasmobranchs, have been found to be located exclusively in the mitochondria in liver from the representative elasmobranch Squalus acanthias. This observation is consistent with the view that the function of this unique carbamoyl-phosphate synthetase is related to urea synthesis, and that the initial nitrogen-donating substrate for urea synthesis in these species is glutamine rather than ammonia. The urea cycle enzymes, ornithine carbamoyltransferase and arginase, are also located in the mitochondria, whereas argininosuccinate synthetase and argininosuccinate lyase are located in the cytosol. Glutamine synthetase and arginase are mitochondrial enzymes in uricotelic species, but are normally found in the cytoplasm in ureotelic species. the properties of the elasmobranch arginase, however, are characteristic of arginases from ureotelic species (e.g. the Km for arginine is 1.2 mM, and the enzyme has an Mr congruent to 100,000).     

Urea tolerance of myofibrillar proteins of two elasmobranchs: Squalus acanthias and Raja tengu

Some biochemical properties of actomyosin and myosin from elasmobranchs, Squalus acanthias and Raja tengu are compared with those of a freshwater (Cyprinus carpio) and a marine teleost (Seriola quinquiradiata). Whereas Ca2+-ATPase of teleost actomyosins are more stable in the absence of urea, the reverse is true for elasmobranchs up to 1.0 M urea. In contrast to that of teleosts, the Mg2+-ATPase of S. acanthias actomyosin shows an activation in the presence of urea, where as that of R. tengu persists. Below 1.0 M urea, there is low incorporation of DTNB into thiols of elasmobranch myosins, and losses in alpha-helicity are reversible up to 5.0 M urea. The results, thus, demonstrate that for a certain concentration of urea, elasmobranch myofibrillar proteins may exhibit a group specific tolerance to urea.