Leonard B. Kirschner

The mechanism of sodium chloride uptake in hyperregulating aquatic animals

SUMMARY The emphasis in this review will be on Na+ absorption across the skin and gills of vertebrates and the gills of crustaceans. However, some recent studies of Cl– uptake, especially in crustaceans, will also be described.

Studies on gill ATPase of rainbow trout (Salmo gairdneri)

Abstract 1. 1. Microsomal ATPase activities from gills of fresh water- and salt water-adapted rainbow trout (Salmo gairdneri) were examined at temperatures of 13 °C and 37 °C. 2. 2. The alkali metal-stimulated activity measured at 13 °C was enhanced by preincubating the reaction mixture, minus ATP, for 30 min at 37 °C. This procedure nearly eliminated “baseline” Mg2+-ATPase activity. The salt water “baseline” showed a greater temperature sensitivity than that of fresh water. 3. 3. The enzyme from salt water fish required both Na+ (100 mM) and K+ (20 mM) for maximal activation at 13 °C. The K m for Na+ was 7 mM and for K+, 0.8 mM. 5·10−4 M ouabain completely inhibited for alkali metal stimulation ( K i = 1.3·10 −5 M ). Na+ alone was ineffective in stimulating activity at 13 and 37 °C. 4. 4. The enzyme from fresh water fish required only Na+ (200 mM) for maximal stimulation at 13 °C, although a small amount of K+ stimulation was sometimes seen. The K m for Na+ was 25 mM. This activity was unaffected by 5·10−4 M ouabain; 5·10−3 M ouabain inhibited only about 30%. At 37 °C, K+, in addition to Na+, was required for maximal activity. This K+ stimulation was inhibited by 5·10−4 M ouabain while the Na+ stimulation remained relatively insensitive to the inhibitor. 5. 5. The fresh water enzyme required 2.5 mM Mg2+ ( K m = 0.9 mM ) for optimal activity at 13 °C and the salt water enzyme required [Mg 2+ ] = 5 mM (K m = 1.0 mM ) . 6. 6. The fresh water enzyme showed maximal activity over wide ranges of pH (6.6–8.0) whereas the salt water enzyme showed a distinct optimum at pH 7.1. 7. 7. The alkali metal-activated ATPase activity was greater in the gills of salt water fish than those adapted to fresh water.

DI- and triphosphoinositide metabolism in swine erythrocyte membranes

Abstract When membranes isolated from swine erythrocytes were incubated with ATP and Mg2+ there was rapid net synthesis of di- and triphosphoinositides which lasted about an hour. Initial levels in the washed membranes were: diphosphoinositide 0.27 nmole per mg dry weight and triphosphoinositide 2.56 nmoles per mg dry weight. After an hour at 37° in the presence of ATP and Mg2+ (5 mM) the concentrations rose to 1.52 and 3.55 nmoles/mg. Synthesis was nearly completely inhibited by Ca2+ (1 mM) and thiol reagents. After net synthesis was completed turnover of diphosphoinositide and triphosphoinositide could be demonstrated by incubation with [32P]ATP. Prelabeled diphosphoinositide (but not triphosphoinositide) also lost isotope when incubated with nonradioactive ATP. Turnover rates in the steady state were of the order of 1 nmole . mg−1 . h−1 for both compounds. They were some-what higher during the early phase of net synthesis. Neither the phase of net synthesis nor steady state turnover showed a requirement for Na+ or K+. Both were unaffected by ouabain. Rates of synthesis of diphosphoinositide and triphosphoinositide were 2 orders of magnitude slower than hydrolysis of ATP by the Na+ + K+-activated ATPase in the same preparations.

HCO3−-Dependent ATPase activity in the gills of rainbow trout Salmo gairdneri

Abstract 1. 1. A HCO3− activated ATPase is present in both fresh water and sea water adapted rainbow trout gills. The apparent Km for HCO3− is 0·016 M. 2. 2. Both the ATPase and active Cl− uptake by intact gills are inhibited by SCN−, but by apparently independent mechanisms. 3. 3.Cl− is not required for activation of the ATPase; at [Cl−] greater than 0·010 M, enzyme activity is inhibited 4. 4. The intracellular location of the HCO3− ATPase is unclear, but preliminary evidence suggests it is both mitochondrial and microsomal.

OSMOTIC AND IONIC REGULATION IN AMBYSTOMA TIGRINUM.

Abstract 1. 1. Larval and adult A. tigrinum maintain NaCl balance in artificial pond water even when starved. 2. 2. Sodium is lost in the urine and across the body surface at a mean rate of 1·3 μg-equiv/10 g per hr. Urinary loss represents 60–90 per cent of the total efflux in larvae and 55 per cent of the total in adults. 3. 3. sodium loss is balanced by uptake across the body surface against a large gradient of electrochemical potential. Chloride is also actively transported inward. 4. 4. Osmotic influx of water occurs at the rate of 26 per cent of body weight per day in larvae, 32 per cent of body weight per day in adults. The osmotic steady state is preserved by excretion of an equivalent volume of dilute urine. 5. 5. Sodium and chloride concentrations were lower in adult than in larval urine.

ATPase activity in gills of euryhaline fish

Abstract 1. 1. Gills of eels and flounders were assayed for their content of Na/K-stimulated and total ATPase activities. Specimens adapted to fresh water had levels comparable to those reported for fresh-water-adapted Fundulus heteroclitus . 2. 2. No differences were noted in eels or flounders adapted to sea water. 3. 3. An increase in levels of the Na/K-stimulated enzyme does not seem to be required by all fish making the transition from fresh to sea water.

Fluoride inhibition of sodium extrusion from swine erythrocytes and its metabolic correlates

Abstract Fluoride, like iodoacetamide, inhibits active sodium extrusion from swine erythrocytes. The latter acts by inhibiting metabolism of adenosine (provided as a substrate) with the result that cellular ATP disappears within 2 hours. However, fluoridetreated cells contain 85% as much ATP as control cells, and P 32 incorporation into ATP is only slightly depressed. Lactic acid production is decreased by fluoride only during the early part of an experiment. Fluoride, in concentrations capable of blocking ion transport, inhibits an ATPase known to be required for this process. The data suggest that inhibition of sodium extrusion is the result of failure of this enzyme, not because ATP synthesis stops. Human erythrocytes provided with adenosine also have near normal ATP levels. Lactic production is decreased only moderately by fluoride in concentrations known to block glycolysis completely.

Di- and triphosphoinositide metabolism in intact swine erythrocytes.

Intact swine erythrocytes incubated for more than 3 h at 37° contained 16.8 nmoles diphosphoinositide per ml packed cells and 43.7 nmole triphosphoinositide/ml. Shorter incubation times gave lower recoveries suggesting that these compounds break down on storage. Kinetic analysis showed that the rates of synthesis were 2.4–6.6 nmoles · ml−1·−1 for diphosphoinositide, and 3.0–10.8 nmoles·ml−1·h−1 for triphosphoinositide. Both the quantities recovered and reaction rates were comparable to those found in isolated membranes after the latter had been incubated with ATP for 1 h. Synthesis of diphosphoinositide and triphosphoinositide was faster in dog than in swine erythrocytes. Polyphosphoinositide synthesis was not appreciably changed by exposing the cells to ouabain (10−3 M), fluoride (5.10−3 M) or a K+-free solution. Synthesis was also 2 orders of magnitude slower than Na+ extrusion from these cells.

Calcium and Electric Potential across the Clam Mantle

An excised clam mantle develops a potential difference, shell side positive, when both surfaces are bathed by tap water or physiological saline solution. The magnitude and sign of the potential are sensitive to the calcium concentration in the bath solution. Bubbling carbon dioxide through the solution bathing the shell side increased the potential.

The role of the hypothalamo-neurohypophysial system in maintaining hydromineral balance in larval salamanders (Ambystoma tigrinum)

Abstract 1. 1. Hypophysectomy and hypophysectomy combined with electrolytic lesions in the preoptic nuclei of the hypothalamus did not change sodium fluxes in larval Ambystoma tigrinum . Operated animals were able to maintain hydromineral balance in pond water and to adapt to very dilute solutions as well as controls. 2. 2. Glomerular filtration rate (GFR) was reduced in salt-loaded animals and increased when they were replaced in pond water. But these changes were about the same in operated-lesioned animals as in controls. Therefore, the hypothalamo-hypophysial system is not important in controlling GFR. 3. 3. Water reabsorption in the excretory system (kidney plus urinary bladder) of normal animals was stimulated by salt-loading and dehydrating. Operated animals did not show this effect, and we conclude that renal antidiuresis in these animals is under hypothalamo-hypophysial control. Arginine vasotocin may be the active hormone in this phenomenon.