Modulation of Calcium Fluxes Across Synaptosomal Plasma Membrane by Local Anesthetics

We have studied the effects of local anesthetics (dibucaine, tetracaine, lidocaine, and procaine) on calcium fluxes through the plasma membrane of synaptosomes. All these local anesthetics inhibit the ATP-dependent calcium uptake by inverted plasma membrane vesicles at concentrations close to those that promote an effective blockade of the action potential. The values obtained for the K0.5 of inhibition of calcium uptake are the following: 23 microM (dibucaine), 0.44 mM (lidocaine), 1.5 mM (procaine), and 0.8 mM (tetracaine). There is a good correlation between these K0.5 values and the concentrations of the local anesthetics that inhibit the Ca2(+)-dependent Mg2(+)-ATPase of these membranes. In addition, except for procaine, these local anesthetics stimulate severalfold the Ca2+ outflow via the Na+/Ca2+ exchange in these membranes. This effect, however, is observed at concentrations slightly higher than those that effectively inhibit the ATP-dependent Ca2+ uptake, e.g., 80-700 microM dibucaine, 2-10 mM lidocaine, and 1-3 mM tetracaine. The results suggest that the Ca2+ buffering of neuronal cytosol is altered by these anesthetics at pharmacological concentrations.     

Studies on the mechanism of action of local anesthetics on proton translocating ATPase from Mycobacterium phlei

We have measured the inhibitory potencies of local anesthetics (procaine, lidocaine, tetracaine and dibucaine) on ATP-mediated H+-translocation, Ca2+-transport and ATPase activity in membrane vesicles from Mycobacterium phlei. Procaine and lidocaine up to 1 mM concentration did not inhibit ATP-dependent H+-translocation, Ca2+-transport and ATPase activity. However, tetracaine and dibucaine at 0.2 mM concentration caused dissipation of the proton gradient, measured by the reversal of the quenching of fluorescence of quinacrine, and inhibition of active Ca2+-transport. Tetracaine (1 mM) inhibited membrane-bound ATPase activity without affecting solubilized F1-ATPase activity. Studies show that these local anesthetics do not prevent the inactivation of F0-F1 ATPase by dicyclohexylcarbodiimide (DCCD). Binding of [14C]DCCD to F0-proteolipid component remained unchanged in the presence of tetracaine indicating that DCCD and tetracaine do not share common binding sites on the F0-proteolipid sector. The inhibition of H+-translocation and membrane-bound ATPase activity by tetracaine was substantially additive in the presence of vanadate.     

Studies on (Na++K+)-activated ATPase: XXXVII. Stabilization by cations of the enzyme-ouabain complex formed with Mg2+ and inorganic phosphate

Dissociation of the (Na⁺+K⁺)-ATPase ouabain complex, formed presence of Mg²⁺ and inorganic phosphate (Complex II), is inhibited by Mg²⁺ (21–45%) and the alkali cations Na⁺ (25–59%) and K⁺ (27–75%) when kidney cortex tissue (bovine, rabbit, guinea pig) is the enzyme source. Choline chloride at 200 mM, equivalent to the highest concentration of NaCl tested, does not inhibit. Dissociation of Complex II from brain cortex (bovine, rat, rabbit) or heart muscle (rabbit) is much less inhibited: 0–11% by Na⁺ and 11–19% by K⁺. The degree of inhibition is not directly related to the size of the dissociation rate constant ($\text{k}{}^{\mathrm{?}}$) of the various complexes, but rather to the extent of interaction between the cation and ouabain binding sites for these tissues.Inhibition curves for Na⁺ and K⁺ are sigmoidal. Half-maximal inhibition for rabbit brain and kidney cortex is at 30–40 mM Na⁺ and 6–10 mM K⁺, and the maximally inhibitory concentrations are 50–150 and 15–20 mM, respectively. Maximal inhibition by Na⁺ or K⁺ for these tissues is the same. For guinea pig kidney cortex Na⁺ and K⁺ are almost equally effective, but 150 mM K⁺ or 200 mM Na⁺ are still not saturating, and inhibition curves indicate high- and low-affinity binding sites for the alkali cations.The inhibition curve for Mg²⁺ is not sigmoidal. In the kidney preparations Mg²⁺ inhibits half-maximally at 0.4-0.5 mM, maximally at 1–3 mM. Maximal inhibition by Mg²⁺ is higher than by Na⁺ or K⁺ for rabbit cortex and lower for guinea pig kidney cortex.There is no competition or additivity among the cations, indicating the existence of different binding sites for Mg²⁺ and the alkali cations.Complex II differs in stability, in the extent of inhibition, in the dependence of inhibition on the cation concentration and in the absence of antagonism between Na⁺ and K⁺, from the ouabain complex formed via phosphorylation by ATP (Complex I). This indicates that the phosphorylation states for the complexes are clearly different.     

Inhibition of Na+,K+-ATPase Activity from Rat Hippocampus by Proline

Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities were determined in the synaptic plasma membranes from hippocampus of rats subjected to chronic and acute proline administration. Na⁺,K⁺-ATPase activity was significantly reduced in chronic and acute treatment by 33% and 40%, respectively. Mg²⁺-ATPase activity was not altered by any treatment. In another set of experiments, synaptic plasma membranes were prepared from hippocampus and incubated with proline or glutamate at final concentrations ranging from 0.2 to 2.0 mM. Na⁺,K⁺-ATPase, but not Mg²⁺-ATPase was inhibited (30%) by the two amino acids. In addition, competition between proline and glutamate for the enzyme activity was observed, suggesting a common binding site for these amino acids. Considering that Na⁺,K⁺-ATPase activity is critical for normal brain function, the results of the present study showing a marked inhibition of this enzyme by proline may be associated with the neurological dysfunction found in patients affected by type II hyperprolinemia.     

Potassium-p-nitrophenyl phosphate interactions with (Na+ + K+)-ATPase their relevance to phosphatase activity

The K⁺-dependent p-nitrophenylphosphatase activity catalyzed by purified (Na⁺ + K⁺)-ATPase from pig kidney shows substrate inhibition (K_i about 9.5 mM at 2.1 mM Mg²⁺). Potassium antagonizes and sodium favours this inhibition. In addition, K⁺ reduces the apparent affinity for substrate activation, whereas p-nitrophenyl phosphate reduces the apparent affinity for K⁺ activation. In the absence of Mg²⁺, p-nitrophenyl phosphate, as well as ATP, accelerates the release of Rb⁺ from the Rb⁺ occluded unphosphorylated enzyme. With no Mg²⁺ and with 0.5 mM KCl, trypsin inactivation of (Na⁺ + K⁺)-ATPase as a function of time follows a single exponential but is transformed into a double exponential when 1 mM ATP or 5 mM p-nitrophenyl phosphate are also present. In the presence of 3 mM MgCl₂, 5 mM p-nitrophenyl phosphate and without KCl the trypsin inactivation pattern is that described for the E₁ enzyme form; the addition of 10 mM KCl changes the pattern which, after about 6 min delay, follows a single exponential. These results suggest that (i) the shifting of the enzyme toward the E₁ state is the basis for substrate inhibition of the p-nitrophenulphosphatase acitivy of (Na⁺ + K⁺)-ATPase, and (ii) the substrate site during phosphatase activity is distinct from the low-affinity ATP site.     

Kinetic studies on the (Na+ + K+)-dependent ATPase. Evidence for coexisting sites for Na+, K+ and Mg2+

Na⁺-ATPase activity of a dog kidney (Na⁺ + K⁺)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 μM ATP and 50 μM MgCl₂, but stimulated by 100 mM NaCl in the presence of 30 μM ATP and 3 mM MgCl₂. The $\text{K}{}_{0.5}$ for the effect of MgCl₂ was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na⁺-ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 μM MgCl₂. Similar thimerosal treatment reduced (Na⁺ + K⁺)-ATPase activity by half but did not appreciably affect the $\text{K}{}_{0.5}$ for activation by either Na⁺ or K⁺, although it reduced inhibition by high Na⁺ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na⁺: Na⁺-discharge sites at which Na⁺-binding can drive E₂-P back to E₁-P, thereby inhibiting Na⁺-ATPase activity, and sites activating E₂-P hydrolysis and thereby stimulating Na⁺-ATPase activity, corresponding to the K⁺-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na⁺-discharge and K⁺-acceptance sites. Mg²⁺ may stimulate Na⁺-ATPase activity by favoring E₂-P over E₁-P, through occupying intracellular sites distinct from the phosphorylation site or Na⁺-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na⁺-ATPase reaction and lessen Na⁺-inhibition of the (Na⁺ + K⁺)-ATPase reaction by increasing the efficacy of Na⁺ in activating E₂-P hydrolysis.     

Further studies on F1-ATPase inhibition by local anesthetics

We have measured the inhibitory potencies of several local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and related compounds (chlorpromazine, procainamide and propranolol) on the ATPase activities of bovine heart submitochondrial particles and purified F1 extracted from these particles. All of these agents cause inhibition of ATPase in F1 as well as in submitochondrial particles. A linear relationship is found between the log of the octanol/water partition coefficients and the log of the concentrations required for 50% inhibition of F1. Sedimentation velocity ultracentrifugation and polyacrylamide gel electrophoresis showed that 1.0 mM tetracaine caused partial dissociation of the F1 complex. Complete reversibility of the enzyme inhibitory effects was demonstrated, however. This work shows that local anesthetics can affect protein structure and enzyme activity without the mediation of lipid.     

Inhibition of Rat Brain Na+, K+-ATPase Activity Induced by Homocysteine Is Probably Mediated by Oxidative Stress

The objective of the present study was to investigate the effects of preincubation of hippocampus homogenates in the presence of homocysteine or methionine on Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities in synaptic membranes of rats. Homocysteine significantly inhibited Na⁺, K⁺-ATPase activity, whereas methionine had no effect. Mg²⁺-ATPase activity was not altered by the metabolites. We also evaluated the effect of incubating glutathione, cysteine, dithiothreitol, trolox, superoxide dismutase and GM1 ganglioside alone or incubation with homocysteine on Na⁺, K⁺-ATPase activity. Tested compounds did not alter Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities, but except for trolox, prevented the inhibitory effect of homocysteine on Na⁺, K⁺-ATPase activity. These results suggest that inhibition of this enzyme activity by homocysteine is possibly mediated by free radicals and may contribute to the neurological dysfunction found in homocystinuric patients.     

Further studies on F1-ATPase inhibition by local anesthetics

We have measured the inhibitory potencies of several local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and related compounds (chlorpromazine, procainamide and propranolol) on the ATPase activities of bovine heart submitochondrial particles and purified F₁ extracted from these particles. All of these agents cause inhibition of ATPase in F₁ as well as in submitochondrial particles. A linear relationship is found between the log of the octanol/water partition coefficients and the log of the concentrations required for 50% inhibition of F₁. Sedimentation velocity ultracentrifugation and polyacrylamide gel electrophoresis showed that 1.0 mM tetracaine caused partial dissociation of the F₁ complex. Complete reversibility of the enzyme inhibitory effects was demonstrated, however. This work shows that local anesthetics can affect protein structure and enzyme activity without the mediation of lipid.     

Red cell Na+,K+-ATPase: a method for estimating the extent of inhibition of an enzyme sample containing an unknown amount of bound cardiac glycoside.

Na⁺, K⁺-ATPase activities of the membranes obtained from intact red cells that are exposed to ouabain, digoxin, and digitoxin are inhibited. The extent of inhibition of each enzyme sample can be found by the following two assays: 1) Activity is measured by the addition of enzyme to a buffered solution containing 2 mM ATP, 3 mM Mg²⁺, 1 mM EDTA, 100 mM Na⁺, and 25 mM K⁺. Since little regeneration of the inhibited enzyme occurs under these conditions, the measured activity is that of the partially inhibited enzyme. 2) Enzyme is preincubated for ten minutes in the same solution from which Mg²⁺ and K⁺ are omitted, and then assayed by the addition of Mg⁺ and K⁺. Since the inhibited enzyme is completely regenerated during the preincubation period, the activity measured here serves as a control for that determined in the first assay.     

Amino group modification of (Na++K+)-ATPase

The effects of three amino group reagents on the activity of (Na⁺+K⁺)-ATPase³ and its component K⁺-stimulatedp-nitrophenylphosphatase activity from rabbit kidney outer medulla have been studied. All three reagents cause inactivation of the enzyme. Modification of amino groups with trinitrobenzene sulfonic acid yields kinetics of inactivation of both activities, which depend on the type and concentration of the ligands present. In the absence of added ligands, or with either Na⁺ of Mg²⁺ present, the enzyme inactivation process follows complicated kinetics. In the presence of K⁺, Rb⁺, or Tl⁺, protection occurs due to a change of the kinetics of inactivation toward a first-order process. ATP protects against inactivation at a much lower concentration in the absence than in the presence of Mg²⁺ (P ₅₀ 6 µM vs. 1.2 mM). Under certain conditions (100 µM reagent, 0.2 M triethanolamine buffer, pH 8.5) modification of only 2% of the amino groups is sufficient to obtain 50% inhibition of the ATPase activity. Modification of amino groups with ethylacetimidate causes a nonspecific type of inactivation of (Na⁺+K⁺)-ATPase. Mg²⁺ and K⁺ have no effects, and ATP only a minor effect, on the degree of modification. The K⁺-stimulatedp-nitrophenylphosphatase activity is less inhibited than the (Na⁺+K⁺)-ATPase activity. Half-inhibition of the (Na⁺+K⁺)-ATPase is obtained only after 25% modification of the amino groups. Modification of amino groups with acetic anhydride also causes nonspecific inactivation of (Na⁺+K⁺)-ATPase. Mg²⁺ has no effect, and ATP has only a slight protecting effect. The K⁺-stimulatedp-nitrophenylphosphatase activity is inhibited in parallel with the (Na⁺+K⁺)-ATPase activity. Half-inactivation of the (Na⁺+K⁺)-ATPase activity is obtained after 20% modification of the amino groups.This article is No. 52 in the series Studies on (Na⁺+K⁺)-Activated ATPase.     

Studies on the interaction of chick brain microsomal (Na+ + K+)-ATPase with copper

Chick brain microsomal ATPase was strongly inhibited by Cu²⁺. (Na⁺ + K⁺)-ATPase was more susceptible to low levels of Cu²⁺ than Mg²⁺-ATPase. The inhibition of (Na⁺ + K⁺)-ATPase could be partially protected from Cu²⁺ in the presence of ATP in the preincubation period. When Cu²⁺ (6 μM) was preincubated with the enzyme in the absence of ATP, only sulfhydryl-containing amino acids (d-penicillamine and l-cysteine) could reverse the inhibition. At lower concentrations of Cu²⁺ (< 1.4 μM), in the absence of ATP during preincubation, the inhibition could be completely reversed by the addition of 5 mM l-phenylalanine and l-histidine as well as d-penicillamine and l-cysteine.Kinetic analysis of action of Cu²⁺ (1.0 μM) on (Na⁺ + K⁺)-ATPase revealed that the inhibition was uncompetitive with respect to ATP. At a low concentration of K⁺ (5 mM), V with Na⁺ was markedly decreased in the presence of Cu²⁺ and K_m was about twice that of the control. However, at high K⁺ concentration (20 mM), the K_m for Na⁺ was not affected. At both low (25 mM) and high (100 mM) Na⁺, Cu²⁺ displayed non-competitive inhibition of the enzyme with respect to K⁺.On the basis of these data, we suggest that Cu²⁺ at higher concentrations (> 6 μM) inactivates the enzyme irreversibly, but that at lower concentrations (< 1.4 μM), Cu²⁺ interacts reversibly with the enzyme.     

Inhibition of Na+/K+-ATPase and Mg2+-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na⁺/K⁺-ATPase and Mg²⁺-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu²⁺, Zn²⁺, Fe²⁺ and.Co²⁺) and heavy metals (Hg²⁺and Pb²⁺) ions were studied. All investigated metals produced a larger maximum inhibition of Na⁺/K⁺-ATPase than Mg²⁺-ATPase activity. The free concentrations of the key species (inhibitor, MgATP^{2 ?} , MeATP^{2 ?} ) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu²⁺/Fe²⁺ or Hg²⁺/Pb²⁺caused additive inhibition, while Cu²⁺/Zn²⁺ or Fe²⁺/Zn²⁺ inhibited Na⁺/K⁺-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu²⁺/Fe²⁺ or Cu²⁺/Zn²⁺ inhibited Mg²⁺-ATPase activity synergistically, while Hg²⁺/Pb²⁺ or Fe²⁺/Zn²⁺ induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na⁺/K⁺-ATPase activity by reducing the maximum velocities (V_max) rather than the apparent affinity (K_m) for substrate MgATP^2-, implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg²⁺-ATPase activity by Zn²⁺, Fe²⁺ and Co²⁺ as well as kinetic analysis indicated two distinct Mg²⁺-ATPase subtypes activated in the presence of low and high MgATP^{2 ?} concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na⁺/K⁺-ATPase with various potencies. Furthermore, these ligands also reversed Na⁺/K⁺-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg²⁺-induced inhibition was not obtained.     

Inhibition of red cell Ca-ATPase by vanadate

1. 1. The Mg²⁺- plus Ca²⁺-dependent ATPase (Ca²⁺-ATPase) in human red cell membranes is susceptible to inhibition by low concentrations of vanadate.

2. 2. Several natural activators of Ca²⁺-ATPase (Mg²⁺, K⁺, Na⁺ and calmodulin) modify inhibition by increasing the apparent affinity of the enzyme for vanadate.

3. 3. Among the ligands tested, K⁺, in combination with Mg²⁺, had the most pronounced effect on inhibition by vanadate.

4. 4. Under conditions optimal for inhibition of Ca²⁺-ATPase, the K for vanadate was 1.5 μM and inhibition was nearly complete at saturating vanadate concentrations.

5. 5. There are similarities between the kinetics of inhibition of red cell Ca²⁺-ATPase and (Na⁺ + K⁺)-ATPase prepared from a variety of sources; however, (Na⁺ + K⁺)-ATPase is approx. 3 times more sensitive to inhibition by vanadate.

Trifluoperazine inhibition of calmodulin-sensitive Ca2+-ATPase and calmodulin insensitive (Na+ + K+)- and Mg2+-ATPase activities of human and rat red blood cells

Trifluoperazine dihydrochloride-induced inhibition of calmodulin-activated Ca²⁺-ATPase and calmodulin-insensitive (Na⁺ + K⁺)- and Mg²⁺-ATPase activities of rat and human red cell lysates and their isolated membranes was studied. Trifluoperazine inhibited both calmodulin-sensitive and calmodulin-insensitive ATPase activities in these systems. The concentration of trifluoperazine required to produce 50% inhibition of calmodulin-sensitive Ca²⁺-ATPase was found to be slightly lower than that required to produce the same level of inhibition of other ATPase activities. Drug concentrations which inhibited calmodulin-sensitive ATPase completely, produced significant reduction in calmodulin-insensitive ATPases as well. The data presented in this report suggest that trifluoperazine is slightly selective towards calmodulin-sensitive Ca²⁺-ATPase but that it is also capable of inhibiting calmodulin-insensitive (Na⁺ + K⁺)-ATPase and Mg²⁺-ATPase activities of red cells at relatively low concentrations. Thus the action of the drug is not due entirely to its interaction with calmodulin-mediated processes, and trifluoperazine cannot be assumed to be a selective inhibitor of calmodulin interactions under all circumstances.     

Modulations of rabbit erythrocyte ATPase activities induced by in vitro and in vivo exposure to ethanol

Alcohol intake is associated with numerous degenerative disorders, and the detrimental effects of alcohol may be due to its influence on plasma membrane and cellular transport systems. The aim of the present study was to compare in vitro and in vivo effects of ethanol on rabbit erythrocyte ATPase activities and correlate them with ethanol-induced oxidative stress. Age-matched male rabbits were given 5% ethanol in 2% sucrose solution, for 6 weeks ad libitum; control animals were given tap water. Daily intake of ethanol was 5 g/kg body weight; this experimental regimen resulted in an average serum ethanol concentration of 16.77 ± 2.00 mM/l. After this period, blood was collected, serum ethanol concentration was determined and erythrocyte membranes were prepared according to the method of Post et al. Activities of Na⁺/K⁺- and Mg²⁺-ATPases were determined. Thiobarbituric acid-reactive substance (TBARS) assay was used to detect levels of lipid peroxidation, a major indicator of oxidative stress. In vitro ethanol inhibits both Na⁺/K⁺-ATPase and Mg²⁺-ATPase, but Na⁺/K⁺-ATPase is more sensitive to the ethanol-induced inhibition. Increasing concentration of ethanol increased TBARS production, but significant difference was attained only at 5 and 12.5 mM of ethanol. Chronic ethanol consumption induced significant increase in Na⁺/K⁺- and Mg²⁺-ATPase activity, and TBARS production. Our results suggest that increased ATPase activity induced by chronic ethanol consumption is due to oxidative, induced modification of membrane phospholipids and proteins, which are responsible for inhibition of ATPase activity. Increased production of TBARS induced by in vitro exposure to ethanol is not the only factor that influences ATPases activity. Further research is needed to elucidate this relationship.     

Isoquinoline alkaloids

Representatives of eleven different classes of isoquinoline alkaloids inhibit Na⁺, K⁺-ATPase and Mg²⁺-ATPase in rat brain microsomal preparations. In most cases the Na⁺, K⁺-ATPase is more sensitive than Mg²⁺-ATPase to inhibition by the alkaloids. The classes of alkaloids can be ranked according to potency of inhibition of Na⁺, K⁺-ATPase. Protoberberines are most effective, followed in decreasing order by benzophenanthridines, benzylisoquinolines, aporphines, tetrahydroprotoberberines, pavines, protopines, isoquinolines, tetrahydrobenzylisoquinolines, morphinanes, and tetrahydroisoquinolines. As specific representatives of each of the first four classes of alkaloids, berberine, sanguinarine, papaveroline and 1,2,10,11-tetrahydroxyaporphine, respectively, prove most valuable in kinetic studies because they exhibit the greatest inhibitory action on brain Na⁺, K⁺-ATPase. Kinetic analyses plotted in double reciprocal form reveal that berberine and 1,2,10,11-tetrahydroxyaporphine are simple linear competitive inhibitors with respect to ATP, whereas sanguinarine and papaveroline are simple linear noncompetitive inhibitors. These four representative alkaloids exhibit nonlinear competitive inhibition with respect to Na⁺-activation. Additionally, these alkaloids significantly inhibit rat brain microsomal K⁺-activatedpNPPase. The results demonstrate that certain members of several classes of isoquinoline alkaloids markedly affect various cation-dependent phosphohydrolases in vitro.     

A comparative study on the effects of different bile salts on mucosal ATPase and transport in the rat jejunum in vivo

The effects of deoxycholate, taurocholate and cholate on transport and mucosal ATPase activity have been investigated in the rat jejunum in vivo using closed-loop and perfusion techniques.In the closed-loops, 5 mM deoxycholate selectively inactivated (Na⁺ + K⁺)-ATPase, and net secretion of Na⁺ induced by 2.5 mM deoxycholate was due to reduced lumen to plasma flux of the ion; deoxycholate (2.5 mM) produced marked inhibition of 3-O-methylglucose transport. Luminal disappearance rates of deoxycholate (60.5±2.9 % per g wet wt of gut) greatly exceeded those of taurocholate (4.3±1.0).In the perfusion studies 1 mM deoxycholate induced net secretion of water, Na⁺ and Cl⁻, and inhibited active glucose transport; concomitantly “total” ATPase, (Na⁺ + K⁺)-ATPase, and Mg²⁺-ATPase were inhibited. At higher concentrations (5 mM) deoxycholate stimulated Mg²⁺-ATPase activity. Taurocholate and cholate at 1 mM had no effect on transport or (Na⁺ + K⁺)-ATPase. Mucosal lactase, sucrase and maltase activities were not affected by 1 mM deoxycholate, taurocholate or cholate.These results suggest that deoxycholate inhibits sodium-coupled glucose transport by inhibition of (Na⁺ + K⁺)-ATPase at the lateral and basal membranes of the epithelial cell, rather than from an effect at the brush-border membrane level.     

Studies of (Na+ + K+)-sensitive ATPase activity in pig lymphocytes. Effects of concanavalin A

(Na⁺ + K⁺)-ATPase activity is demonstrated in plasma membranes from pig mesenteric lymph nodes. After dodecyl sulfate treatment plasma membranes have an 18-fold higher (Na⁺ + K⁺)-ATPase activity, while their ouabain-insensitive Mg²⁺-ATPase is markedly lowered. A solubilized (Na⁺ + K⁺)-ATPase fraction, obtained by Lubrol WX treatment of the membranes, has very high specific activity (21μmol P_i/h per mg protein). Concanavalin A has no effect on these partially purified (Na⁺ + K⁺)-ATPase, while it inhibits (40%) this activity in less purified fractions which still contain Mg²⁺-ATPase activity.     

Effects of local anesthetics on cytokinesis and polar lobe formation in fertilized eggs of Ilyanassa obsoleta

Summary

We have examined the ability of fertilized eggs of Ilyanassa obsoleta to form polar lobe constrictions and undergo cytokinesis in the presence of several local anesthetics and compared these effects with those of drugs known to affect microtubules. Procaine, lidocaine (Xylocaine), mepivacaine, tetracaine, and dibucaine all delay the beginning of polar lobe constrictions at low concentrations and in the order of their lipid solubilities. All of the anesthetics are effective at lower concentrations in the absence of extracellular Ca²⁺. Procaine and tetracaine ‘lock’ cells for several hours halfway through the constriction of the polar lobe neck and prevent subsequent cytokinesis, effects similar to those of the microtubule agents, colchicine and nocodazole. Procaine has no effect on membrane potential, ψ_m, or on intracellular chloride activity, (Cl)_c, as determined with ion-selective microelectrodes. This suggests that procaine does not inhibit cellular shape changes by affecting the ionic activities of the predominant intracellular cation (K⁺) or anion (Cl^?).