Franklin H. Epstein

Na‐K‐Cl Cotransport in Chloride‐transporting Epitheliaa

The elasmobranch rectal gland has served as a useful model to study features of Na-K-Cl cotransport that are common to many chloride-transporting epithelia. These include: (1) dependence on a Na+ gradient created by Na-K-ATPase; (2) high intracellular Cl- concentration; (3) characteristic inhibitor profile including inhibition by loop diuretics and barium but not by amiloride, SITS, DIDS, or carbonic anhydrase inhibitors; and (4) remarkable energy efficiency of transepithelial transport (25-30 NaCl/l 02). The mechanism by which this is accomplished is clarified by kinetic analysis of experiments with isolated perfused rectal glands of Squalus acanthias in which perfusate concentrations of Na and Cl are systematically varied. These show a Hill coefficient of one for Na+ and two for Cl-, suggesting that one Na+, one K+, and two Cl- interact with the cotransport carrier. Nitrate can substitute for Cl- to some extent, and it itself weakly transported. The loop diuretic bumetanide behaves like a competitive inhibitor of Cl-. The teleological significance of the neutral cotransport of two Cl- with one Na+ and one K+ is that it enables transporting epithelia like the rectal gland, cornea, salivary gland, and thick ascending limb of Henles loop to double the efficiency of their Na-K-ATPase pump.

Disparate mechanisms for hypoxic cell injury in different nephron segments. Studies in the isolated perfused rat kidney.

Hypoxic injury was evaluated morphologically in the proximal tubule and in the medullary thick ascending limb of isolated rat kidneys perfused for 90 min without O2 or with various metabolic inhibitors. Inhibition of mitochondrial respiration (with rotenone, antimycin, oligomycin) or of intermediary metabolism (with monofluoroacetate, malonate, 2-deoxyglucose) caused reduction in renal oxygen consumption, renal function, and ATP content comparable with those elicited by oxygen deprivation. Metabolic inhibition produced hypoxiclike injury in the first portions of the proximal tubule, S1 and S2 (clubbing of microvilli, mitochondrial swelling), and the extent of damage was correlated with the degree of ATP depletion. In the third portion of the proximal tubule, S3, hypoxiclike damage (cytoplasmic edema or fragmentation) occurred most consistently when both aerobic and anaerobic metabolism were inhibited simultaneously. In the medullary thick ascending limb, none of the metabolic or mitochondrial inhibitors used could reproduce the injury of oxygen deprivation. Thus, the proximal tubule and the thick ascending limb have markedly different responses to cellular energy depletion, suggesting disparate mechanisms for hypoxic injury along the nephron.

Sodium-potassium pump, ion fluxes, and cellular dehydration in sickle cell anemia.

We studied the role of the sodium-potassium pump in erythrocytes of 12 patients with sickle cell anemia (SS). Ouabain-binding sites per cell and pump-mediated Rb/K uptake were significantly higher in SS patients than in white or black controls. Ouabain-resistant Rb/K influx was also greater than in normal controls or patients with sickle cell trait. Deoxygenation of SS erythrocytes increased ouabain-sensitive Rb/K influx without altering ouabain binding, presumably as the consequence of an increase in the passive influx of sodium. Deoxygenation increased mean corpuscular hemoglobin concentration (MCHC) by 5.5%, and studies of the density distribution of SS cells indicated an increase in highly dense fractions known to contain sickled erythrocytes. Ouabain prevented the rise in MCHC and reduced the percentage of dense cells. These findings indicate a magnified role for the sodium-potassium pump in the pathophysiology of SS erythrocytes and suggest that its inhibition might prove useful in therapy.

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(-5) M and 10(-4) M, 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.

HORMONAL CONTROL OF SECRETION IN SHARK RECTAL GLAND

Although elasmobranch fishes, including sharks and rays, are said to have originally developed in fresh water, they have lived for millions of years in the salty ocean where they are subjected to osmotic stresses that menace the composition of their body fluids. The shark swims in a sea containing about 500 mEq/L of sodium chloride, or 1,000 mOsm/L. The serum sodium of the shark is higher than that of man, approximately 260-290 mEq/L, but sodium and its associated anions in the extracellular fluid of the shark amount to only half the external osmotic pressure of the ocean. In order to counterbalance the hypertonicity of the sea, which if unopposed would rapidly dehydrate the animal, elasmobranchs synthesize urea, which circulates in high concentration in the blood and also permeates all cells. The concentration of mineral salts plus urea precisely balances the osmotic concentration of seawater, preventing the depletion of body water. Nevertheless, sodium chloride tends to difXuse inward through the gills and is also absorbed when food is swallowed. Thus, the shark must combat a tendency to become hypernatremic. The mechanism that elasmobranchs have devised for getting rid of unwanted increments of salt is the rectal gland.. The rectal gland of the spiny dogfish shark, Squalus acanthias, is a relatively simple structure that looks like a human appendix. It is composed of tubules packed tightly together looking superficially in histologic section like mammalian kidney cortex, and well supplied with capillaries. Electron microscopy reveals a relatively uniform population of cells with extensive basolateral infoldings. These are rich in (Na+K) -ATPase, as demonstrated autoradiographically by the binding of radioactive ouabain in histological ~ e c t i o n . ~ It has a single artery, vein, and central duct. It is therefore a simple matter to isolate the gland, remove it from the shark and perfuse it in the laboratory at the temperature of seawater with a shark Ringers solution resembling an ultrafiltrate of shark plasma.

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.

Cyclic AMP stimulates ouabain-insensitive ion movement in shark rectal gland

SummaryWhen the active sodium-potassium pump (Na−K-ATPase) of shark rectal glands is blocked by ouabain, the concentration of intracellular ions changes in the direction of equilibrium with extracellular fluid. These changes were examined when isolated perfused glands were in the basal state and also when they were stimulated to secrete with cAMP and theophylline, to see whether stimulation affected the passive movement of sodium, potassium and chloride across cell membranes. In basal glands 10−4M ouabain induced an increase of 30 meq/l in intracellular [Na+] and a decrease in intracellular [K+] of about 50 meq/l after 30 min, while intracellular [Cl−] was unchanged. In stimulated glands, these movements were exaggerated. The increase in intracellular [Na+] averaged 112 meq/l, and the decrease in intracellular [K+], 96 meq/l (P<0.01), while mean intracellular [Cl−] rose by 80 meq/l. Furosemide, 10−4M, partially reversed the accelerated changes in intracellular electrolytes seen after ouabain was added to stimulated glands. These results are consistent with an action of cAMP upon a ouabaininsensitive cotransport of sodium, potassium and chloride in the rectal gland, analogous to that described in avian erythrocytes.

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

Abstract. 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, 10–6xa0M, reduced secretion stimulated by a 1-min bolus of CNP (5×10–7xa0M) by 50–60%. In the presence of 10–2xa0M 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, 5×10–6M 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 2xa0min 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.

Secretion of nitrate by rectal gland of Squalus acanthias.

1. Rectal glands secrete nitrate at 30% of their capacity to secrete chloride. 2. Nitrate secretion is directly related to its concentration at constant chloride concentrations. 3. Chloride has a biphasic effect on nitrate secretion. 4. Hill coefficients at chloride < 100 mM are equal to 1, while at 100 mM indicate inhibition of nitrate by chloride. 6. Lineweaver-Burk plots at chloride < 100 indicate a single site, while at 100 mM indicate inhibition of nitrate by chloride. 7. Bumetanide inhibits nitrate secretion. 8. The data suggest that nitrate interacts with one of the two chloride sites of the chloride transporter.