Role of the Na+/K+/Cl- transporter in the positive inotropic effect of ouabain in cardiac myocytes

In this study we have characterized the bumetanide-sensitive K+/Na+/Cl- cotransport in cultured rat cardiac myocytes. 1) It carries about 10% of the total K+ influx. 2) It is sensitive to furosemide (Ki0.5 = 10(-6)M) and bumetanide (Ki0.5 = 10(-7)M). 3) It is strongly dependent on the extracellular concentrations of Na+ and Cl-. 4) It carries out influx of both ions, K+ and Na+. A therapeutic concentration of ouabain (10(-7) M) stimulated the bumetanide-sensitive K+ influx (as measured by 86Rb+), in the cultured myocytes, with no effect on the bumetanide-resistant K+ influx, which was mediated mostly by the Na+/K+ pump. Stimulation of the bumetanide-sensitive Rb+ influx by a low ouabain concentration was strongly dependent on Na+ and Cl- in the extracellular medium. A low concentration of ouabain (10(-7) M) was found to increase the steady-state level of cytosolic Na+ by 15%. This increase was abolished by the addition of bumetanide or furosemide. These findings suggest that ouabain, at a low (10(-7) M) concentration, induced its positive inotropic effect in rat cardiac myocytes by increasing Na+ influx into the cells through the bumetanide-sensitive Na+/K+/Cl- cotransporter. In order to examine this hypothesis, we measured the effect of bumetanide on the increased amplitude of systolic cell motion induced by ouabain. Bumetanide or furosemide, added to cultured cardiac myocytes, inhibited the increased amplitude of systolic cell motion induced by ouabain. Neither bumetanide nor furosemide alone has any significant effect on the basal amplitude of systolic cell motion. We propose that stimulation of bumetanide-sensitive Na+ influx plays an essential role in the positive inotropic effect in rat cardiac myocytes induced by low concentration of ouabain.     

Brain Na+, K+-ATPase isoforms: different hypothalamus and mesencephalon response to acute desipramine treatment

We studied Na(+), K(+)-ATPase activity alpha isoforms by performing ouabain inhibition curves in rat hypothalamus and mesencephalon after acute administration of desipramine to rats. In hypothalamus, Ki values for high, intermediate and low affinity populations were 0.075x10(-9) M, 0.58x10(-6) M and 0.97x10(-3) M, with isoform distribution of 55%, 28% and 17%, respectively. In mesencephalon, Ki values for high, intermediate and low affinity populations were 1.80x10(-9) M, 0.56x10(-6) M and 0.21x10(-3) M, with isoform distribution of 28%, 46% and 21%, respectively. Three hours after acute administration of 10 mg/kg desipramine to rats, Na(+), K(+)-ATPase activity in hypothalamus increased significantly 54%, 39% and 51% as assayed respectively in the absence of ouabain or in the presence of 1x10(-9) M, or 5x10(-6) M ouabain, whereas only a trend was recorded in the presence of 1x10(-3) M ouabain. In such conditions, enzyme activity in mesencephalon increased significantly 73%, 54%, 30% and 271%, respectively. Present results showed that desipramine treatment enhances the activity of Na(+), K(+)-ATPase alpha isoforms in rat hypothalamus and mesencephalon, but the extent of this increase differs according to the isoform and the anatomical area studied, suggesting a differential enzyme regulation in response to noradrenergic stimulation.     

Sodium and ouabain induce proliferation of rat thymic lymphocytes via calcium- and magnesium- dependent reactions

When the concentrations of either calcium or of magnesium in the culture medium were increased from the normal 0.6 and 1.0 mM to 1.8 and 2.5 mM respectively mitotic activity of rat thymic lymphocytes increased. Very high (10(-4)M) ouabain concentrations abolished these mitogenic actions whilst lower (10(-7) and 10(-11)M) concentrations had no effect. However in the normal medium these lower concentrations of ouabain were themselves mitogenic. The stimulatory effect of 10(-7)M ouabain was calcium-dependent and oestradiol-blockable and that of 10(-11)M magnesium-dependent and testosterone-blockable. A 10 mM increment in extracellular sodium concentration also stimulated mitosis in these cells in a calcium-dependent manner whilst a 20 mM increment required the presence of magnesium to exert its mitogenic effect. However, when similar osmotic increments were provided by potassium and lithium salts, or sucrose no mitotic stimulation was provoked. Subtle interactions between sodium and the divalent cations are clearly involved in events which lead to mitosis and the steroids oestradiol and testosterone can somehow block these effects.     

Depolarizing agent-induced cyclic AMP accumulation in isolated rat spinal cord

The stimulation of cyclic adenosine 3',5'-monophosphate (cyclic AMP) accumulation by the depolarizing agents K+, ouabain and veratridine, was studied in rat and guinea pig spinal cord tissue slices. Significantly increased accumulation of cyclic AMP was produced by each of the agents in a concentration-dependent manner. Veratridine and ouabain were equipotent (EC50 = 5 x 10(-5)M) and approximately 500 fold more potent than K+ (EC50 = 10(-2)M). Depolarizing agent-induced cyclic AMP accumulation in slices from guinea pig spinal cord was approximately double the response in rat spinal cord. Maximum stimulation occurred within 2.5 min of incubation with these agents and lasted for at least 30 min. Regional studies demonstrated that the maximal accumulation of cyclic AMP occurred to a greater degree in tissue slices from the dorsal section of spinal cord from both rat and guinea pig. Whereas the ouabain and veratridine stimulatory responses are completely dependent on extracellular Ca++, the K+ response is only partially dependent. Stimulation due to ouabain and veratridine is dependent, and K+ is independent, of release of neurohumoral substances such as norepinephrine or adenosine from spinal neurons. These experiments indicate the possible modulatory role of depolarization-linked events in regulating the spinal cord cyclic AMP system.     

Characterization of the stimulation of neuronal Na+, K+-ATPase activity by low concentrations of ouabain

Low concentrations (< 10^?7 M) of ouabain stimulate the activity of Na⁺, K⁺-ATPase in whole homogenates of rat brain. The magnitude of this stimulation varies from 5 to 70%. The concentrations of ouabain which induces maximal stimulation is also highly variable and ranges between 10^?9 to 10^?7 M. The ouabain stimulation disappears following 1:50 dilution and 2 h preincubation or freezing and thawing of the membranes or their treatment with deoxycholate. “Aging” of a preparation of ATPase also results in loss of its ability to be stimulated by ouabain but ouabain inhibition is preserved. No stimulation of enzyme activity by ouabain is observed in rat brain microsomal fraction. The β-adrenergic blocker propranolol does not inhibit the ouabain induced stimulation of ATPase activity. It is suggested that the stimulation of Na⁺, K⁺-ATPase activity by low concentrations of cardiac glycosides if a result of either the displacement of an endogenous ouabain-like compound from the enzyme or an indirect effect by changing membrane surrounding environment of the Na⁺, K⁺-ATPase.     

Inhibition of the cardiac inotropic effect of phenylephrine by a tetramine disulfide with irreversible alpha-adrenoceptor blocking activity.

The newly synthesized alpha-adrenoceptor blocking drug BHC (N,N'-bis[6-(10-methoxybenzyl-amino)-a-hexyl]cystamine) was found to block irreversibly the positive inotropic effect of the sympathomimetic drug phenylephrine on the isolated rat left atrium. BHC was used to test the adrenoceptor interconversion hypothesis which claims that low temperature converts inotropic beta-adrenoceptors in rat atrium and frog ventricle to alpha-adrenoceptors. There was no evidence of adrenoceptor 'interconversion.' In the rat atrium low temperature did not increase the BHC antagonism of phenylephrine and did not cause BHC to inhibit the inotropic effect of noradrenaline or isoprenaline. In the perfused frog heart low temperature did not lead BHC to inhibit the inotropic effect of phenylephrine, adrenaline, or isoprenaline.     

Effects of the calcium ionophore A-23187 on the regulation of sugar transport in muscle

The distribution of (¹⁴C)-3-0-methyl-D-glucose and of (⁴⁵Ca) was followed in perifused left atria and intact hemidiaphragms of the rat. The carboxylic calcium ionophore A-23187 affected sugar and Ca²⁺ influx in parallel, with low concentrations inhibiting and higher ones stimulating influx under basal conditions. The stimulation of sugar transport by insulin, high concentrations of adrenaline or ouabain, or by K⁺-free medium was antagonized by the calcium ionophore. Likewise, A-23187 counteracted the depression of sugar transport caused by low concentrations of ouabain or adrenaline. These results support a role of Ca²⁺ in the regulation of sugar transport in muscle. However, increased influx of Ca²⁺ cannot explain all the effects of A-23187. It is suggested that the ionophore may also act by releasing Ca²⁺ from intracellular storage and binding sites.     

Ouabain- and potassium-induced stimulation of amylase release in fragments and acini of rabbit pancreas

Ouabain increases the enzyme secretion from the isolated rabbit pancreas and pancreatic fragments, but not from isolated pancreatic acini. The increase occurs after a delay of 45-60 min and is not accompanied by an increase in lactate dehydrogenase release. The stimulatory effect of ouabain (10(-5) M) is dependent on the presence of extracellular calcium, and is not antagonized by 10(-4) M atropin, 10(-4) M propranolol, 10(-5) M phentolamine, 10(-3) M dibutyryl-cyclic GMP, 10(-6) M tetrodotoxin, 10(-4) M verapamil or 10(-4) M D-600. Elevation of the extracellular potassium concentration to 120 mM in the presence of 10(-4) M atropin also increases the enzyme secretion from rabbit pancreatic fragments. The increase is again dependent on the presence of extracellular calcium and is resistant to adrenergic blockade and to tetrodotoxin, verapamil or D-600. Forskolin also stimulates a Ca2+-dependent release of amylase from pancreatic fragments but not from pancreatic acini. In the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IMX), ouabain (10(-5) M) and K+ (120 mM) cause an immediate increase in the cyclic AMP content of pancreatic fragments which does not occur in the absence of extracellular calcium. In pancreatic acini, the cAMP production is only slightly increased by ouabain. In the absence of IMX, the cAMP levels in fragments or acini are not detectably altered by ouabain or K+. The results suggest that the stimulation of enzyme secretion by ouabain and high K+ is an indirect effect, mediated by the release of an endogenous transmitter from non-cholinergic, non-adrenergic nerves in the intact preparations. The release and/or the effect of the transmitter appears to be mediated primarily by Ca2+ and secondarily by cyclic AMP.     

Cadmium stimulation of Na-independent organic acid transport in the kidney tubules

The influence of Cd++ (as well as of Hg++ and Cu++) on the uptake of an organic acid (fluorescein) in superficial proximal tubules of the surviving rat kidney was studied at 20 degrees C, when the active transport of fluorescein does not depend on the external Na. In contrast to mercury and copper, cadmium stimulated the uptake of fluorescein from the beginning of incubation. The minimal effective concentration of Cd++ was 5 X 10(-6)M, the relative effect of Cd++ on the uptake being the same within the concentration range from 5 X 10(-6) to 10(-3) M. A 60 minutes pre-incubation with Cd++ at 20 degrees C resulted in a significant increase in the stimulatory effect of acetate on the fluorescein transport. The stimulation of the fluorescein transport by cadmium was prevented by ouabain or by omissing Na from the incubating medium, although neither ouabain nor the absence of Na affected the transport of fluorescein under these conditions. It is supposed that the stimulation by Cd++ of the fluorescein transport may result from the activated oxidation of NAD-linked substrates due to acceleration of the active transepithelial transport of Na ions.     

The importance of limitations in aerobic metabolism, glycolysis, and membrane excitability for the development of high-frequency fatigue in isolated rat soleus muscle

We investigated the role of limitations in aerobic metabolism, glycolysis, and membrane excitability for development of high-frequency fatigue in isolated rat soleus muscle. Muscles mounted on force transducers were incubated in buffer bubbled with 5% CO(2) and either 95% O(2) (oxygenated) or 95% N(2) (anoxic) and stimulated at 60 Hz continuously for 30-120 s or intermittently for 120 s. Cyanide (2 mM) and 2-deoxyglucose (10 mM) were used to inhibit aerobic metabolism and both glycolysis and aerobic metabolism, respectively. Excitability was reduced by carbacholine (10 microM), a nicotinic ACh receptor agonist, or ouabain (10 microM), an Na(+)-K(+) pump inhibitor. Membrane excitability was measured by recording M waves. Intracellular Na(+) and K(+) contents and membrane potentials were measured by flame photometry and microelectrodes, respectively. During 120 s of continuous stimulation, oxygenated and anoxic muscles showed the same force loss. In oxygenated muscles, cyanide did not alter force loss for up to 90 s, whereas 2-deoxyglucose increased force loss (by 19-69%; P < 0.01) from 14 s of stimulation. In oxygenated muscles, 60 s of stimulation reduced force, M wave area, and amplitude by 70-90% (P < 0.001). Carbacholine or ouabain increased intracellular Na(+) content (P < 0.001), induced a 7- to 8-mV membrane depolarization (P < 0.001), and accelerated the rate of force loss (by 250-414%) during 30 s of stimulation (P < 0.001). Similar effects were seen with intermittent stimulation. In conclusion, limitations in glycolysis and subsequently also in aerobic metabolism, as well as membrane excitability but not aerobic metabolism alone, appear to play an important role in the development of high-frequency fatigue in isolated rat soleus muscle.     

Identification of two molecular forms of (Na+,K+)-ATPase in rat adipocytes. Relation to insulin stimulation of the enzyme

Two molecular forms of the (Na+,K+)-ATPase catalytic subunit have been identified in rat adipocyte plasma membranes using immunological techniques. The similarity between these two forms and those in brain (Sweadner, K. J. (1979) J. Biol. Chem. 254, 6060-6067) led us to use the same nomenclature: alpha and alpha(+). The K0.5 values of each form for ouabain (determined by inhibition of phosphorylation of the enzyme from [gamma-32P]ATP) were 3 X 10(-7)M for alpha(+) and 1 X 10(-5)M for alpha. These numbers correlate well with the K0.5 values for the two ouabain-inhibitable components of 86Rb+/K+ pumping in intact cells (1 X 10(-7) M and 4 X 10(-5)M). Quantitation of the Na+ pumps in plasma membranes demonstrated a total of 11.5 +/- 0.2 pmol/mg of membrane protein, of which 8.5 +/- 0.3 pmol/mg, or 75%, was alpha(+). Insulin stimulation of 86Rb+/K+ uptake in rat adipocytes was abolished by ouabain at a concentration sufficient to inhibit only alpha(+)(2-5 X 10(-6)M). Immunological techniques and ouabain inhibition of catalytic labeling of the enzyme from [gamma-32P]ATP demonstrated that alpha(+) was present in skeletal muscle membranes as well as in adipocyte membranes, but was absent from liver membranes. Since insulin stimulates increased Na+ pump activity in adipose and muscle tissue but not in liver, there is a correlation between hormonal regulation of (Na+,K+)-ATPase and the presence of alpha(+). We propose that alpha(+) is the hormonally-sensitive version of the enzyme.     

Erythrocyte sodium pump stimulation by ouabain and an endogenous ouabain-like factor

Cardiac glycosides inhibit the sodium pump. However, some studies suggest that nanomolar ouabain concentrations can stimulate the activity of the sodium pump. In this study, using the Na(+)/K(+)-ATPase of human erythrocytes, we compared the effect of digoxin, ouabain and an ouabain like-factor (OLF), on (86)Rb uptake. Ouabain concentrations below 10(-9) M significantly stimulate Rb(+) uptake, and the maximal increase above base-line values is 18 +/- 5% at 10(-10) M ouabain. No stimulation is observed in the same conditions by digoxin. OLF behaved like ouabain, producing an activation of Rb(+) flux at concentrations lower than 10(-9) M ouabain equivalents (14 +/- 3% at 10(-10) M). Western blot analysis revealed the presence of both alpha(1) and alpha(3) pump isoforms in human erythrocytes. Our data confirm the analogies between OLF and ouabain and suggest that Na(+)/K(+)-ATPase activation may be related to the alpha(3) isoform. In addition, we investigated whether ouabain at different concentrations was effective in altering the intracellular calcium concentration of erythrocytes. We found that ouabain at concentration lower than 10(-9) M did not affect this homeostasis.     

Effect of inhibition of Na+/K(+)-ATPase on the vasopressin-induced increase in intracellular Na+ concentration in vascular smooth muscle cells.

The effect of inhibition of Na+/K(+)-ATPase by ouabain on the arginine vasopressin (AVP)-induced increase in intracellular Na+ concentration [( Na+]i) was examined in cultured rat vascular smooth muscle cells (VSMC) by the direct measurement of [Na+]i using a fluorescent indicator dye. AVP at a concentration of 1 x 10(-9) M or higher increased [Na+]i in a dose-dependent manner in cultured rat VSMC. The preincubation of cells with 1 x 10(-4) M ouabain for 1 hr at 37 degrees C did not affect the basal [Na+]i but enhanced the 1 x 10(-6) M AVP-induced increase in [Na+]i. The preincubation was not necessary because similar results were obtained after the simultaneous administration of AVP and ouabain. The treatment with ouabain did not affect the intracellular pH changes induced by AVP. These results therefore indicate that the inhibition of Na+/K(+)-ATPase enhances the AVP-induced increase in [Na+]i by decreasing cellular Na+ efflux in cultured rat VSMC.     

Effect and mechanisms underlying scorpion toxin action from Androctonus australis garzonii on atrial natriuretic peptide in rat atria: an in vitro study

Scorpion envenomation is considered public health problem in Northern African countries. The mechanisms of cardiac dysfunction following scorpion envenomation are not fully understood. This study examined the effect and mechanisms underlying scorpion toxin action from Androctonus australis garzonii on atrial natriuretic peptide (ANP) release from rat atrium using in vitro organ perifusion. Male Sprague Dawley rats were used in this study. Three experiments were conducted. In experiment 1, atrial tissues were exposed either to Krebs-bicarbonate buffer medium (control) or to scorpion toxin (10(-8) M to 10(-6) M). In experiment 2, animals were chemically sympathectomized with a single intraperitoneal injection of 6-hydroxydopamine (6-OHDOPA) at a dose of 40 microg/g 24 h before sacrifice. Vehicle-treated rats served as control. Atrial tissues were collected and perifused in the presence of 10(-6) M scorpion toxin. In experiment 3, atrial tissues were exposed to 10(-6) M scorpion toxin either in the absence or presence of 10(-6) M propranolol (a beta-adrenoceptor blocker), or 10(-6) M tetrodotoxin (a sodium channel blocker). ANP levels released in the perifusion medium were determined by radioimmunoassay. The scorpion toxin at 10(-6) M induced a significant (p<0.01) increase (106%) in ANP levels. This effect was decreased (20%) by 6-OHDOPA. Propranolol and tetrodotoxin significantly (p<0.01) inhibited 55% and 60%, respectively, the toxin-induced ANP release. The data show that the North African scorpion toxin from Androctonus australis garzonii increases the ANP release in rat atrium through stimulation of sympathetic cardiac nerves and sodium channels activation.     

Effect of ouabain on mechanical and electrical properties of rat and guinea pig hearts at 180 C.

The effects of ouabain 10(-6) M on rat and guinea pig hearts have been studied at 18 degrees C, in order to reduce almost fully both the Na+, K+-dependent ATPase activity and the ouabain induced inhibition of this enzyme. In isolated guinea pig hearts the positive inotropic response to ouabain obtained at 32 degrees C disappeared at 18 degrees C. On the contrary, the contractile strength of rat hearts was slightly reduced by ouabain and in the same manner at both temperatures. Current and voltage clamp experiments carried out at 18 degrees C in ventricular fibres revealed that ouabain 10(-6) M decreased both the action potential overshoot and the fast sodium current in rat and guinea pig, by reduction of the membrane sodium conductance. Ouabain did not change the calcium current in guinea pig preparations, whereas in rat heart muscle this current was reduced. The effects of ouabain on both the action potential plateau and outward repolarizing current indicated some inconsistencies from preparation to preparation and cannot therefore be considered as significant. The persistence of the ouabain induced alterations of g Na (in rat and guinea pig) and calcium current (in rat) at 18 degrees C supports the hypothesis of two ouabain cell receptors in heart muscle.     

3H]dizocilpine binding to N-methyl-D-aspartate (NMDA) receptor is modulated by an endogenous Na+, K+-ATPase inhibitor. Comparison with ouabain

An endogenous Na+, K+-ATPase inhibitor termed endobain E has been isolated from rat brain which shares several biological properties with ouabain. This cardiac glycoside possesses neurotoxic properties attributable to Na+, K+-ATPase inhibition, which leads to NMDA receptor activation, thus supporting the concept that Na+/K+ gradient impairment has a critical impact on such receptor function. To evaluate potential direct effects of endobain E and ouabain on NMDA receptors, we assayed [3H]dizocilpine binding employing a system which excludes ionic gradient participation. Brain membranes thoroughly washed and stored as pellets ('non-resuspended' membranes) or after resuspension in sucrose ('resuspended' membranes) were employed. Membrane samples were incubated with 4 or 10 nM ligand with or without added endobain E or ouabain, in the presence of different glutamate plus glycine combinations, with or without spermidine. [3H]dizocilpine basal binding and Na+, K+- and Mg2+-ATPase activities proved very similar in 'non-resuspended' or 'resuspended' membranes. Endobain E decreased [3H]dizocilpine binding to 'resuspended' membranes in a concentration-dependent manner, attaining roughly 50% binding inhibition with the highest endobain E concentration assayed. Among tested conditions, only in 'resuspended' membranes, with 4 nM ligand and with 1x10(-8) M glutamate plus 1x10(-5) M glycine, was [3H]dizocilpine binding enhanced roughly +24% by ouabain (1 mM). After Triton X-100 membrane treatment, which drastically reduces Na+, K+-ATPase activity, the effect of ouabain on binding was lost whereas that of endobain E remained unaltered. Results indicate that not only membrane preparation but also treatment and storage are crucial to observe direct endobain E and ouabain effects on NMDA receptor, which are not attributable to changes in Na+, K+-ATPase activity or to Na+/K+ equilibrium alteration.     

Insulin stimulation of (Na+,K+)-adenosine triphosphatase-dependent 86Rb+ uptake in rat adipocytes

Insulin stimulated the uptake of 86Rb+ (a K+ analog) in rat adipocytes and increased the steady state concentration of intracellular potassium. Half-maximal stimulation occurred at an insulin concentration of 200 pM. Both basal- and insulin-stimulated 86Rb+ transport rates depended on the concentration of external K+, external Na+, and were 90% inhibited by 10(-3) M ouabain and 10(-3) M KCN, indicating that the hormone was activating the (Na+,K+)-ATPase. Insulin had no effect on the entry of 22Na+ or exit of 86Rb+. Kinetic analysis demonstrated that insulin acted by increasing the maximum velocity, Vmax, of 86Rb+ entry. Inhibition of the rate of Rb+ uptake by ouabain was best described by a biphasic inhibition curve. Scatchard analysis of ouabain binding to intact cells indicated binding sites with multiple affinities. Only the rubidium transport sites which exhibited a high affinity for ouabain were stimulated by insulin. Stimulation required insulin binding to an intact cell surface receptor, as it was reversible by trypsinization. We conclude that the uptake of 86Rb+ by the (Na+,K+)-ATPase is an insulin-sensitive membrane transport process in the fat cell.     

Do endogenous prostaglandins modulate noradrenergic transmission in the rat isolated perfused vas deferens?

The effects of the prostaglandin synthetase inhibitors aspirin, indomethacin and meclofenamic acid have been studied on the response of the rat isolated perfused vas deferens. None of these drugs, up to a concentration of 5 x 10(-5) M affected either phase of the biphasic constrictor response to 30 s periods of field stimulation. In the same preparations an inhibition of the response to field stimulation was seen in the presence of prostaglandin E1 at concentrations of 1 to 5 ng ml-1. All three prostaglandin synthetase inhibitors, at 5 x 10(-5) M, caused significant reduction of prostaglandin biosynthesis by homogenates of rat vas deferens. It is, therefore, suggested that stimuli which activate directly the noradrenergic nerves in the rat vas do not activate simultaneously a release of endogenous prostaglandins.     

Ouabain binding in rectal gland ofSqualus acanthias

In an attempt to examine the mechanisms of activation of (Na, K)-ATPase when epithelial transport is stimulated, the binding of ouabain to rectal gland tissue was measured before and after stimulation with dibutyryl cAMP and theophylline. Stimulation significantly altered the characteristics of ouabain binding to slices of Squalus acanthias rectal gland and to isolated rectal gland cells, accelerating the rate of binding and increasing the amount of ouabain bound at equilibrium when low concentrations of ouabain (10(-9) to 10(-7) M) were present in the medium. Scatchard plots of ouabain binding were nonlinear, suggesting at least two classes of binding sites, one of higher and one of lower affinity. Stimulation with cAMP and theophylline appeared to increase the affinity of the high-affinity site. Ouabain binding was increased by cAMP and theophylline even in the presence of furosemide (10(-4) M) or bumetanide (10(-5) M), and when Li+ was substituted for Na+, or NO3- for Cl- -maneuvers known to inhibit rectal gland secretion. The changes in ouabain binding induced by cAMP and theophylline do not appear, therefore, to be secondary to secretory activity but may reflect a change in the configuration, environment or location of existing enzyme so as to enhance its activity. Stimulation of ouabain binding cannot be demonstrated in whole homogenates of rectal gland, indicating that intact cells are necessary for the cyclic AMP-induced increase in ouabain binding to become manifest.     

Evidence for a beta-adrenoceptor-mediated regulation of human natural killer cells

Pretreatment of lymphocytes (16 hr, 37 degrees C) with adrenaline at final concentrations of 10(-7) to 10(-9) M, followed by removal of the drug, increased natural killer (NK) cell activity vs K562 leukemic cells in a 4-hr 51Cr-release assay. The most efficient concentration of adrenaline was 10(-8) M; mean increase of NK activity over base-line activity for all donors examined was 30%. However, the individual response to adrenaline pretreatment was variable; in some donors, the effect was equal to maximal interferon (IFN) stimulation. Effects of adrenaline pretreatment were consistently reduced to base-line activity by co-incubation with the nonselective beta-adrenoceptor antagonist propranolol at 100-fold higher concentrations. The enhancing effect of adrenaline (10(-8) M) pretreatment was also observed after 1-hr pretreatment; this effect was prevented by simultaneous incubation with propranolol but was not affected by dex-propranolol. Direct addition of adrenaline to lymphocyte/target cell mixtures was inhibitory at 10(-6) M adrenaline concentration. The inhibitory effect of adrenaline in this assay was again completely prevented by propranolol and unaffected by dex-propranolol. The observed stimulatory effect of adrenaline pretreatment could not be ascribed to IFN production. Data presented indicate a dual effect of adrenaline on NK cell activity and suggest both a positive and a negative beta-adrenoceptor-mediated regulation of human NK cells.