Active chloride transport in rabbit thick ascending limb of Henle's loop and elasmobranch rectal gland: chloride fluxes in isolated plasma membranes

Summary To investigate directly whether a sodium-potassium-chloride cotransport system is operating in the mammalian thick ascending limb of Henle's loop (TALH) and in the elasmobranch rectal gland, plasma membrane vesicles were prepared from TALH cells isolated from rabbit kidney outer medulla and from rectal glands ofSqualus acanthias, and chloride uptake was measured by a rapid filtration technique. Chloride uptake into TALH vesicles in the presence of a 25 mM Na₂SO₄, 25 mM K₂SO₄ gradient reached 70% of equilibrium at 2.5 min. In the presence of both sodium and potassium, the 15 s chloride uptake was inhibited 35% by 1 mM bumetanide. When either sodium or potassium was removed from the incubation medium, chloride uptake decreased to the level observed in the presence of 1 mM bumetanide. 0.5 mM SITS had no effect on chloride uptake by the plasma membrane vesicles. This sodium and potassium dependent, bumetanide sensitive chloride uptake was also observed under tracer exchange conditions. Chloride uptake into rectal gland plasma membrane vesicles in the presence of a 50 mM Na₂SO₄, 50 mM K₂SO₄ gradient reached 80% of equilibrium at 2.5 min. 1 mM bumetanide inhibited the 15 s uptake of chloride by 34% and removal of either sodium or potassium from the incubation medium reduced chloride uptake to the level observed in the presence of bumetanide under both gradient and tracer exchange conditions. These studies provide additional support for the hypothesis that a sodium-potassium-chloride cotransport system is operating in these epithelia.Abbreviations SITS 4-acetamido-4-isothiocyanato-stilbene-2,2-disulfonic acid - TALH thick ascending limb of Henle's loop     

Inhibition by mercuric chloride of Na-K-2Cl cotransport activity in rectal gland plasma membrane vesicles isolated from Squalus acanthias

The rectal gland of the dogfish shark is a model system for active transepithelial transport of chloride. It has been shown previously that mercuric chloride, one of the toxic environmental pollutants, inhibits chloride secretion in this organ. In order to investigate the mechanism of action of HgCl(2) at a membrane-molecular level, plasma membrane vesicles were isolated from the rectal gland and the effect of mercury on the activity of the Na-K-2Cl cotransporter was investigated in isotope flux studies. During a 30 s exposure HgCl(2) inhibited cotransport activity in a dose-dependent manner with an apparent K(i) of approx. 50 microM. The inhibition was complete after 15 s, partly reversible by dilution of the incubation medium and completely attenuated upon addition of reduced glutathione. The extent of inhibition by mercury depended on the ionic composition of the medium. The sensitivity of the cotransporter was highest when only the high affinity binding sites for sodium and chloride were saturated. Organic mercurials such as p-chloromercuribenzoic acid and p-chloromercuriphenylsulfonic acid at 100 microM did not inhibit the cotransporter, similarly exposure of the vesicles to 10 mM H(2)O(2) or 1 mM dithiothreitol for 30 min at 15 degrees C did not change cotransport activity. Transport activity was, however, reduced by 45.9+/-2.5% after an incubation with 3 mM N-ethylmaleimide for 20 min. Blocking free amino groups by N-hydroxysuccinimide or biotinamidocapronate-N-hydroxysulfosuccinimide had no effect. Investigations on the sidedness of the plasma membrane vesicles, employing the asymmetry of the (Na+K)-ATPase, demonstrated a right-side-out orientation in which the former extracellular face of the membrane is exposed to the incubation medium. In addition, extracellular mercury (5x10(-5) M) inhibited bumetanide-sensitive rubidium uptake into T84 cells by 48.5+/-7.1% after a 2 min incubation period. This inhibition was reversible in a manner similar to that observed in the plasma membrane vesicles. These studies suggest that in isolated rectal gland plasma membrane vesicles the Na-K-2Cl cotransporter (sNKCC1) exposes functionally relevant mercury binding sites at its external surface. These sites represent probably cysteines, the accessibility and/or sensitivity of which depends on the functional state of the transporter.     

Mechanism of the rate-determining step of the Na(+),K(+)-ATPase pump cycle

The kinetics of the E(2) --> E(1) conformational change of unphosphorylated Na(+),K(+)-ATPase from rabbit kidney and shark rectal gland were investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 degrees C). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize initially the E(2) conformation. When rabbit kidney enzyme was mixed with NaCl alone, tris ATP alone or NaCl, and tris ATP simultaneously, a fluorescence decrease was observed. The reciprocal relaxation time, 1/tau, of the fluorescent transient was found to increase with increasing NaCl concentration and reached a saturating value in the presence of 1 mM tris ATP of 54 +/- 3 s(-1) in the case of rabbit kidney enzyme. The experimental behavior could be described by a binding of Na(+) to the enzyme in the E(2) state with a dissociation constant of 31 +/- 7 mM, which induces a subsequent rate-limiting conformational change to the E(1) state. Similar behavior, but with a decreased saturating value of 1/tau, was found when NaCl was replaced by choline chloride. Analogous experiments performed with enzyme from shark rectal gland showed similar effects, but with a significantly lower amplitude of the fluorescence change and a higher saturating value of 1/tau for both the NaCl and choline chloride titrations. The results suggest that Na(+) ions or salt in general play a regulatory role, similar to that of ATP, in enhancing the rate of the rate-limiting E(2) --> E(1) conformational transition by interaction with the E(2) state.     

Inositol lipid turnover and adenosine 3,5 cyclic monophosphate in the salt-secreting rectal gland of the dogfish (Scyliorhinus canicula)

The rectal gland of the dogfish is rich in inositol lipids. Total phospholipids from the gland contained 9.1 mol% of phosphatidylinositol (PtdIns), 1.0 mol% of phosphatidylinositol 4-phosphate (PtdIns4P) and 0.9 mol% of phosphatidylinositol 4,5-biphosphate (PtdIns4,5P2). [32P]Orthophosphate was readily incorporated into PtdIns, phosphatidic acid (PtdA) and especially into PtdIns4P and PtdIns4,5P2 in salt gland slices incubated in elasmobranch Ringer with glucose and no other additions over a 2 hr period. The calcium ionophore A23187 stimulated incorporation into PtdIns and PtdA, but not into PtdIns4P or PtdIns4,5P2. Oxygen uptake by rectal gland slices was maximally stimulated by 0.08mM forskolin, 2.5mM 8-chlorophenylthio cyclic AMP, 2.0mM dibutyryl cyclic AMP and 0.25mM theophylline. Stimulated oxygen uptake was inhibited by 0.1mM ouabain in all cases. Incorporation of [32P]orthophosphate into PtdIns, PtdA, PtdIns4P and PtdIns4,5P2 was inhibited by 0.08mM forskolin and 2.0mM dibutyryl cyclic AMP over a 2 hr period. The results are discussed in relation to the control of salt secretion by the rectal gland.     

Shark rectal gland vasoactive intestinal peptide receptor: cloning, functional expression, and regulation of CFTR chloride channels

Vasoactive intestinal peptide (VIP) is a secretagogue that mediates chloride secretion in intestinal epithelia. We determined the relative potency of VIP and related peptides in the rectal gland of the elasmobranch dogfish shark and cloned and expressed the VIP receptor (sVIP-R) from this species. In the perfused rectal gland, VIP (5 nM) stimulated chloride secretion from 250 +/- 66 to 2,604 +/- 286 microeq x h(-1) x g(-1); the relative potency of peptide agonists was VIP > PHI = GHRH > PACAP > secretin, where PHI is peptide histidine isoleucine amide, GHRH is growth hormone-releasing hormone, and PACAP is pituitary adenylate cylase activating peptide. The cloned sVIP-R from shark rectal gland (SRG) is only 61% identical to the human VIP-R1. It maintains a long, extracellular NH2 terminus with seven cysteine residues, and has three N-glycosylation sites and eight other residues implicated in VIP binding. Two amino acids considered important for peptide binding in mammals are not present in the shark orthologue. When sVIP-R and the CFTR chloride channel were coexpressed in Xenopus oocytes, VIP increased chloride conductance from 11.3 +/- 2 to 127 +/- 34 microS. The agonist affinity for activating chloride conductance by the cloned receptor was VIP > GHRH = PHI > PACAP > secretin, a profile mirroring that in the perfused gland. The receptor differs from previously cloned VIP-Rs in having a low affinity for PACAP. Expression of both sVIP-R and CFTR mRNA was detected by quantitative PCR in shark rectal gland, intestine, and brain. These studies characterize a unique G protein-coupled receptor from the shark rectal gland that is the oldest cloned VIP-R.     

Mercury toxicity in the shark (Squalus acanthias) rectal gland: apical CFTR chloride channels are inhibited by mercuric chloride

In the shark rectal gland, basolateral membrane proteins have been suggested as targets for mercury. To examine the membrane polarity of mercury toxicity, we performed experiments in three preparations: isolated perfused rectal glands, primary monolayer cultures of rectal gland epithelial cells, and Xenopus oocytes expressing the shark cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In perfused rectal glands we observed: (1) a dose-dependent inhibition by mercury of forskolin/3-isobutyl-1-methylxanthine (IBMX)-stimulated chloride secretion; (2) inhibition was maximal when mercury was added before stimulation with forskolin/IBMX; (3) dithiothrietol (DTT) and glutathione (GSH) completely prevented inhibition of chloride secretion. Short-circuit current (Isc) measurements in monolayers of rectal gland epithelial cells were performed to examine the membrane polarity of this effect. Mercuric chloride inhibited Isc more potently when applied to the solution bathing the apical vs. the basolateral membrane (23 +/- 5% and 68 +/- 5% inhibition at 1 and 10 microM HgCl2 in the apical solution vs. 2 +/- 0.9% and 14 +/- 5% in the basolateral solution). This inhibition was prevented by pre-treatment with apical DTT or GSH; however, only the permeant reducing agent DTT reversed mercury inhibition when added after exposure. When the shark rectal gland CFTR channel was expressed in Xenopus oocytes and chloride conductance was measured by two-electrode voltage clamping, we found that 1 microM HgCl2 inhibited forskolin/IBMX conductance by 69.2 +/- 2.0%. We conclude that in the shark rectal gland, mercury inhibits chloride secretion by interacting with the apical membrane and that CFTR is the likely site of this action.     

Chloride secretagogues stimulate inositol phosphate formation in shark rectal gland tubules cultured in suspension.

Neuroendocrine activation of transepithelial chloride secretion by shark rectal gland cells is associated with increases in cellular cAMP, cGMP, and free calcium concentrations. We report here on the effects of several chloride secretagogues on inositol phosphate formation in cultured rectal gland tubules. Vasoactive intestinal peptide (VIP), atriopeptin (AP), and ionomycin increase the total inositol phosphate levels of cultured tubules, as measured by ion exchange chromatography. Forskolin, a potent chloride secretagogue, has no effect on inositol phosphate formation. The uptake of 3H-myo-inositol into phospholipids is very slow, preventing the detection of increased levels of inositol trisphosphate. However, significant increases in inositol monophosphate (IP1) and inositol biphosphate (IP2) were measured. The time course of VIP- and AP-stimulated IP1 and IP2 formation is similar to the effects of these agents on the short-circuit current responses of rectal gland monolayer cultures. In addition, aluminum fluoride, an artificial activator of guanine nucleotide-binding proteins, stimulates IP1 and IP2 formation. We conclude that rectal gland cells contain VIP and AP receptors coupled to the activation of phospholipase C. Coupling may be mediated by G-proteins. Receptor-stimulated increases in inositol phospholipid metabolism is one mechanism leading to increased intracellular free calcium concentrations, an important regulatory event in the activation of transepithelial chloride secretion by shark rectal gland epithelial cells.     

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 micron) 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.     

Ionic strength and the polyvalent cation receptor of shark rectal gland and artery

The dogfish shark Squalus acanthias regulates plasma osmolality and extracellular volume by secreting a fluid from its rectal gland which has a higher NaCl and lower urea concentration than plasma. We have previously identified the presence of a calcium-sensing receptor or polyvalent cation sensing receptor (CaSR) on vascular smooth muscle of the rectal gland artery (RGA) and rectal gland tubules (RGT). Activity of the CaSR is influenced by changes in ionic strength. This led us to speculate that the ingestion of invertebrate sea animals increased plasma ionic strength, resulting in inhibition of the receptor, relaxation of RGA, and reversal of inhibition of chloride secretion by the RGT. In contrast, ingestion of fish could diminish ionic strength and have the opposite effect. To study the effect of changes in extracellular ionic strength, shark Ringers solutions were adjusted to three different ionic strengths with NaCl, but the osmolarities were kept constant by varying the concentration of urea. High ionic strength inhibited and low ionic strength enhanced the response to increasing external Ca2+ from 2.5 to 4.7 mM in RGT. The increase in cytosolic Ca2+ ([Ca2+]i) of cells in low, normal, and high ionic strength Ringers solution was 344 +/- 60, 201 +/- 26, and 114 +/- 15 nmol/L, respectively. The [Ca2+]i responses of RGA to external Ca2+ in Ringers of three different ionic strengths were 323 +/- 43, 231 +/- 14, and 56 +/- 11 nmol/L, respectively. Activation of the CaSR by spermine was reduced by more than 50% by high ionic strength in both RGT and RGA. Whether the small changes in shark plasma ionic strength that occur after a shark ingests marine animals with lower and higher ionic strength modulates salt secretion by the rectal gland is not yet known.     

Role of the cytoskeleton in secretion of chloride by shark rectal gland

The salt gland of the spiny dogfish, Squalus acanthias, can be stimulated to secrete chloride by two different endogenous peptides: cardiac natriuretic peptide (CNP) and the neurotransmitter, vasoactive intestinal peptide (VIP). We examined the role of the actin cytoskeleton and of myosin light chains in this process by perfusing isolated rectal glands with and without an inhibitor of actin filament organization (cytochalasin D) and an inhibitor of myosin light chain kinase (ML-7). Cytochalasin D, 10(-6) M, reduced secretion stimulated by a 1-min bolus of CNP (5x10(-7) M) by 50-60%. In the presence of 10(-2) M procaine (which blocks neural release of VIP), cytochalasin D completely prevented CNP stimulation. In contrast, cytochalasin D did not inhibit stimulation of the rectal gland by VIP or by forskolin. Similarly, 5x10(-6)M ML-7 almost completely inhibited direct stimulation of rectal gland secretion by CNP, but did not alter chloride secretion induced by VIP or forskolin. Finally, the average time between hormonal injection and activation of secretion was 2 min longer for CNP than for VIP, consistent with the hypothesis that a contractile cellular function involving the cytoskeleton is important in CNP-induced chloride secretion, but less so when secretion is stimulated by VIP.     

A preliminary investigation into the roles played by the rectal gland and kidneys in the osmoregulation of the striped dogfish Poroderma africanum.

Presented here are the results of a preliminary investigation into ionic and osmotic regulation by the kidneys and rectal gland of the striped dogfish, Poroderma africanum. Fish with ligated rectal glands showed an increase in blood concentration of sodium and chloride within a short time period, reaching a maximum after four days. The blood concentration of the two ions then decreased over the following ten days. Control animals showed relatively unchanged blood-sodium and chloride levels, over the entire 14-day period. After salt loading, both control animals and those with ligated rectal glands showed initial rise in blood sodium and chloride levels, but these returned towards initial values within seven hours of injection. Comparison of the two groups indicates that the rectal gland may control blood-chloride levels more so than -sodium, although its action as a salt regulator does not seem very pronounced in either case. Urine and rectal gland fluid, were collected as a compound fluid, from normal fish, and the estimated cloacal salt loss is discussed. Urine from normal fish was also collected separately and was analysed for its contribution to salt loss. Results are discussed and compared with previous relevant findings.     

Regulation by cations of adenylate cyclase activity in chicken tissues

The effects of magnesium and sodium ions on adenylate cyclase activity in plasma membranes from chicken heart and eggshell gland mucosa were studied. It was found that the increase in magnesium chloride concentration from 5 to 40 mM results in the stimulation (4.1-fold) of the adenylate cyclase activity. The increase in sodium chloride concentration up to 150 mM stimulated the enzyme activity 2-fold. The stimulation of adenylate cyclase by magnesium and sodium ions was less pronounced in the eggshell gland. GTP did not activate adenylate cyclase. The activating effect of magnesium and sodium ions was accompanied by the attenuation of the enzyme sensitivity to NaF, guanylyl imidodiphosphate and isoproterenol. Activation by guanylyl imidodiphosphate was completely abolished in the presence of 40 mM magnesium chloride. It is assumed that high concentrations of the salt promote subunit dissociation of the adenylate cyclase regulatory protein and its interaction with the catalytic subunit in the presence of endogenous nucleotides. The differences in the adenylate cyclase sensitivity to cations in chicken heart and eggshell gland mucosa correlate with the amount of pertussis toxin substrate.     

Ionic composition of the rectal contents and excreta of the reduviid bug Triatoma infestans

We have investigated the chemical composition of the rectal contents, faeces and urine of the blood-sucking bug Triatoma infestans. This is the environment in which the important disease-causing organism, Trypanosoma cruzi, lives. Directly after feeding of Triatoma infestans, the pH of the excreta switched from an acidic to an alkaline pH and, 1 day later, back to a slightly acidic pH. The osmolality varied in the initial excreta and in the rectal contents on the day following the meal between 300 and 460 mosmol/kg H(2)O, but after an additional day it increased to 350-970 mosmol/kg H(2)O. Determinations by ion capillary electrophoresis showed that sulphate and phosphate dominated the rectal contents in unfed bugs. After feeding, the first four drops of fluid excreta were mainly a sodium chloride solution (>150 mM for each). One to 10 days after feeding strong individual variations in the concentrations of individual ions were evident, especially for potassium and sodium. Mean concentrations of chloride remained at about 70 mM; sulphate and phosphate showed an increase within the first 1 or 2 days and then reached a level of about 160 and 210 mM, respectively. The rectal contents of long-term starved bugs contained high concentrations of phosphate and potassium; sulphate and sodium were slightly lower.     

The effect of mercury on chloride secretion in the shark (Squalus acanthias) rectal gland

1. Mercuric chloride inhibited chloride secretion in a dose dependent way in isolated perfused rectal glands. The effect was readily apparent at a concentration of 10⁻⁶ M and profound and irreversible at 10⁻⁴ M.2. The dithiol dithiothreitol (DTT) 10⁻² M completely prevented the effect of 10⁻⁶ M mercuric chloride, reduced that at 10⁻⁵M and 10⁻⁴M, and made the inhibition at the latter concentration reversible.3. Two organic mercurials, mersalyl and meralluride, that are effective diuretics in the mammalian kidney, and p-chloromercuribenzoyl sulfonic acid (PCMBS), that has no diuretic activity, had no effect on chloride secretion by the rectal gland.4. The effect of mersalyl was not modified by lowering the pH of the solution perfusing the glands.5. These results indicate that inorganic mercury and organic mercurials do not share the same mechanism of action.6. The absence of an effect of organic mercurials on chloride transport in the rectal gland suggests that its effect on another chloride transporting epithelia, the thick ascending limb of the loop of Henle, is not mediated by inhibition of the chloride cotransporter or Na⁺, K⁺-ATPase, common to both epithelia.     

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.     

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.     

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.     

Changes in chloride secretion rate and vascular perfusion in the rectal gland of the European lesser-spotted dogfish in response to environmental and hormonal stimuli

Chloride secretion rates of rectal glands taken from the European lesser-spotted dogfish Scyliorhinus canicula adapting to 70% and 120% sea water (SW) were significantly greater and less than, respectively, those in the control 100% SW group. C-type natriuretic peptide (CNP) significantly increased chloride secretion rates above basal values in 100% SW although angiotenisn II (ANG II) had no effect. Perfusion of the secretory epithelia in rectal glands from 70% SW lesser-spotted dogfish was significantly higher than in rectal glands from 100% and 120% SW lesser-spotted dogfish. Perfusion of rectal glands with ANG II had no effect on perfusion of the secretory epithelia, although CNP perfusion induced significantly greater perfusion of the secretory epithelia than all other treatments. It remains to be determined if a reduction in environmental salinity induces an increase in plasma concentration of CNP and hence an increase in rectal gland activity.     

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.     

Oxygen cost of chloride transport in perfused rectal gland ofSqualus acanthias

Summary In the stimulated state, with glucose as substrate, oxygen uptake by the isolated perfused rectal gland is directly related to the rate of chloride secretion. Lactate production is negligible under aerobic conditions in the stimulated gland. A stoichiometric relationship exists between chloride transport and oxygen consumption, with a Cl/O₂ ratio of about 301, resembling that reported for sodium in mammalian kidneys. This ratio remains constant under varying degrees and modes of stimulation. The ratio does not change when the gland is induced to secrete chloride against varying electrochemical gradients by altering the concentration of urea in the perfusate.Established Investigator of the American Heart Association.