Sodium flux through the sodium channels of axon membrane fragments isolated from lobster nerves

The efflux of 22Na from vesicles formed by axolemma fragments isolated from lobster nerves was studied in the presence and in the absence of drugs having well-known action on the sodium channels. The vesicles were equilibrated 12-14 h at 4 degrees C with 22Na in lobster solution containing 1 mM ouabain. Afterwards the suspension was divided: one portion was used as control and the others were treated with veratrine (0.025-0.50 mg/ml), tetrodotoxin (1-2,000 nM) in the presence of veratrine, or tetrodotoxin alone. After 3 h at 20-22 degrees C, the suspensions were diluted into nonradioactive solutions and the 22Na efflux followed by a rapid filtration technique. The results revealed that veratrine increases the efflux rate and the additional application of tetrodotoxin abolishes it, e.g., 0.50 mg of veratrine/ml increases the rate, expressed in 10(-2) min(-1), from 0.59 +/- 0.04 (mean +/- SEM; n = 13) to 0.86 +/- 0.05 (n = 13), and the addition of 100 nM tetrodotoxin diminishes it to 0.48 +/- 0.07 (n = 4). This increase and diminution are statistically significant (P less than 0.005), but this is not the case between the control and the veratrine plus tetrodotoxin values (P greater than 0.05). 50% of the diminution is produced by 11.9 +/- 2.4 nM tetrodotoxin. Tetrodotoxin alone produces a slight diminution of the 22Na efflux. Batrachotoxin (0.50 muM) has an action similar to veratrine's. These findings are considered evidence of the presence of functioning sodium channels in the isolated axolemma fragments.     

Different sensitivity of ATP + Mg + Na (I) and Pi + Mg (II) dependent types of ouabain binding to phospholipase A2

Summary The effect of phospholipase A₂ and of related agents on ouabain binding and Na,K-ATPase activity were studied in intact and detergent-treated membrane preparations of rat brain cortex and pig kidney medulla. It was found that phospholipase A₂ (PLA₂) may distinguish or dissociate ouabain binding complexes I (ATP+Mg+Na) and II (P_i+Mg), stimulating the former and inhibiting the latter. Procedures which break the permeability barriers of vesicular membrane preparations, such as repeated freezing-thawing, sonication or hypoosmotic shock failed to mimic the effect of PLA₂, indicating that it was not acting primarily by opening the inside-out oriented vesicles. The detergent digitonin exhibited similar effects on ouabain binding in both ATP+Mg+Na and P_i+Mg media. Other detergents were ineffective.The ability of PLA₂ to distinguish between ouabain binding type I and II can be manifested even in SDS-treated, purified preparations of Na,K-ATPase. The number of ATP+Mg+Na-dependent sites is unchanged, while the P_i+Mg-dependent sites are decreased in number in a manner similar to that seen in original membranes. This inhibition is completely lost in the reconstituted Na,K-ATPase system, where the ATP- as well as P_i-oriented ouabain sites are inhibited by PLA₂.     

Characterization of ouabain-induced phosphoinositide hydrolysis in brain slices of the neonatal rat

The effect of the Na/K-ATPase inhibitor ouabain on phosphoinositide (Ptdlns) hydrolysis was studied in rat brain cortical slices. Ouabain induced a dose-dependent accumulation of inositol phosphates (InsPs) which was much higher in neonatal rats (1570±40% of basal) than in adult animals (287±18% of basal). For this reason, all experiments were conducted with 7 day-old rats. Strophantidin caused a similar stimulation of Ptdlns hydrolysis, although it was less potent than ouabain. The order of potency for ouabain-stimulated InsPs accumulation in brain areas was hippocampus>cortex>brainstem>cerebellum. The effect of ouabain was not blocked by antagonists for the muscarinic, alpha₁-adrenergic and glutamate receptors. Also ineffective were the K⁺ channel blockers 4-aminopyridine and tetraethylammonium, the sodium channel blocker tetrodotoxin, and the calcium channel blocker verapamil, whereas the Na/Ca exchanger blocker amiloride partially antagonized the effect of ouabain. The accumulation of InsPs induced by ouabain was additive to that of carbachol and norepinephrine, as well as to that induced by high K⁺ and veratrine, but not to that of glutamate. Removal of Na⁺ ions from the incubation buffer completely prevented the accumulation of InsPs induced by ouabain. The effect of ouabain was also dependent upon extracellular calcium and was under negative feedback control of protein kinase C. Despite the higher effect of ouabain on Ptdlns hydrolysis of immature rats, the density of [³H]ouabain binding sites, as well as the activity of Na/K-ATPase were higher in adult animals. Furthermore, a poor correlation was found between ouabain-stimulated Ptdlns hydrolysis and [³H]ouabain binding in brain regions. These results suggest an involvement of Na⁺ pump in the hydrolysis of Ptdlns, possibly related to an effect on Na⁺ and Ca²⁺ homeostasis. The immature rat appear to be an useful model for studying the relationship between Na/K-ATPase and inositol metabolism.     

Characterization of membrane-bound adenosinetriphosphatase activity of sarcolemma-enriched fraction from vascular smooth muscle

Membrane-bound adenosinetriphosphatase (ATPase) activities of the sarcolemma-enriched fraction from bovine aorta were characterized. The membranes, isolated by a sucrose density gradient method, were enriched about 31-fold in sodium- and potassium-stimulated, magnesium-dependent ATPase (Na,K-ATPase) activity, and about 8-fold in 5'-nucleotidase activity compared to the homogenate, suggesting that the isolated membranes were substantially enriched with the sarcolemma. The membranes exhibited about 31, 33 and 42 mumol Pi/mg protein/h of Na,K-ATPase, magnesium-dependent ATPase and calcium-dependent ATPase activities, respectively, in the presence of 4 mmol/l ATP. The sarcolemma-enriched membranes required considerably high concentrations of well-known inhibitors for Na,K-ATPase such as vanadate (more than 1 mumol/l), lanthanum (more than 1 mmol/l) and calcium (10 mmol/l), to induce a significant inhibition in the Na,K-ATPase activity. Treatments of the membrane with physical disruptions and sodium dodecyl sulfate or deoxycholate reduced the total Na,K-ATPase activity, and did not expose fully the ouabain sensitivity of the Na,K-ATPase. These results indicate that there are marked differences in the properties of the ATPase between vascular smooth muscle sarcolemma and cardiac sarcolemma.     

Ouabain resistance of a human trophoblast cell line is not related to its reactivity to ouabain

Ouabain is a specific inhibitor of sodium, potassium-dependent adenosine triphosphatase (Na,K-ATPase), a P-type ion-transporting ATPase which is essential for the maintenance of adequate concentrations of intracellular Na+ and K+ ions. The present study describes the establishment of a ouabain-resistant mutant, TLouaR, from a human trophoblast cell line TL. Morphologically TL and TLouaR are indistinguishable, but, TLouaR is about 1000 times more resistant to the cytotoxic effect of ouabain and > 2000 times to that of bufalin and yet ouabain can retard the growth of the TLouaR cells and in parallel reduce its cloning efficiency in a time- and dose-dependent manner. Furthermore, Na,K-ATPase activity from TLouaR cells is inhibitable by ouabain albeit with lower efficiency. [3H]ouabain binding studies reveal that TLouaR cells have less (P < 0.05) ouabain binding sites (1.7 +/- 0.15 x 10(4)/cell vs. 2.3 +/- 0.115 x 10(4)/cell in the control). However, affinities (dissociation constants Kd) to ouabain for TL and TLouaR cells are not significantly different. Lastly, Na,K-ATPase activity (1.375 +/- 0.25 micromole ATP/min mg protein) of TLouaR cells is significantly higher (P < 0.05) than that of the TL cells (0.895 +/- 0.12 micromole ATP/min x mg protein). These studies show that the interactions between ouabain and Na,K-ATPase can be mediated through different pathways resulting in diverse phenotypic characteristics. In addition, ouabain resistance does not necessarily reflect the lack of response to the digitalis drug. The exact mechanisms of ouabain resistance observed in the present study remain to be determined but the TLouaR cells may be the best tool to uncover the many functional characteristics of Na,K-ATPase.     

Biosynthesis of the Na,K-ATPase in Madin-Darby canine kidney cells. Activation and cell surface delivery

Madin-Darby canine kidney cells were used to study events in the postsynthetic processing and cell surface delivery of Na,K-ATPase. The photoactivable 2-nitro-5-azidobenzoyl (NAB) derivative of ouabain and an anti-ouabain antibody were employed in experiments designed to determine the time intervals required for newly synthesized Na,K-ATPase to achieve the capacity to bind ouabain and to arrive at the cell surface. Ouabain-binding capacity was assessed in Madin Darby canine kidney cells which were pulse-labeled with [35S]methionine. At various chase intervals cells were disrupted by probe sonication and the resultant vesicles were permeabilized. Vesicles were incubated with NAB-ouabain and, following UV photolysis, solubilized and subjected to immunoprecipitation with an anti-ouabain antibody. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitates revealed that newly synthesized Na,K-ATPase can carry out type II (Mg2+ and Pi supported) ouabain binding throughout the course of its postsynthetic processing. In contrast, the ability to carry out type I (Na+, Mg2+, and ATP-supported) ouabain binding is not attained until 10 min after the completion of the sodium pump's synthesis. Experiments in which intact pulse-labeled cells were incubated with NAB-ouabain revealed that the Na,K-ATPase arrives at the cell surface as soon as 50 min after its synthesis. These results suggest that postsynthetic processing is required before the newly synthesized Na,K-ATPase can display its full repertoire of catalytic functions. This processing seems to be complete prior to the newly synthesized sodium pump's arrival at the cell surface.     

Inhibition of Na,K-ATPase and sodium pump by protein kinase C regulators sphingosine, lysophosphatidylcholine, and oleic acid

The effects and modes of action of certain lipid second messengers and protein kinase C regulators, such as sphingosine, lysophosphatidylcholine (lyso-PC), and oleic acid, on Na,K-ATPase and sodium pump were examined. Inhibition of purified rat brain synaptosome Na,K-ATPase by these lipid metabolites, unlike that by ouabain, was subject to membrane dilution (i.e. inhibition being counteracted by increasing amounts of membrane lipids). Kinetic analysis, using the purified enzyme, indicated that sphingosine and lyso-PC were likely to interact, directly or indirectly, with Na+-binding sites of Na,K-ATPase located at the intracellular face of plasma membranes, a conclusion also supported by studies on Na,K-ATPase and 22Na uptake using the inside-out vesicles of human erythrocyte membranes. The studies also showed that ouabain (but not sphingosine and lyso-PC) increased the affinity constant (K0.5) for K+, whereas sphingosine and lyso-PC (but not ouabain) increased K0.5 for Na+. Sphingosine and lyso-PC inhibited 86Rb uptake by intact human leukemia HL-60 cells at potencies comparable to those for inhibitions of purified Na,K-ATPase and protein kinase C. It is suggested that Na,K-ATPase (sodium pump) might represent an additional target system, besides protein kinase C, for sphingosine and possibly other lipid second messengers.     

Lysine 480 is not an essential residue for ATP binding or hydrolysis by Na,K-ATPase.

Lysine 480 has been suggested to be essential for ATP binding and hydrolysis by Na,K-ATPase because it is labeled by reagents that are thought to react with the ATPase from within the ATP binding site. In order to test this hypothesis, Lys-480 was changed to Ala, Arg, or Glu by site-directed mutagenesis, and the resultant Na,K-ATPase molecules were expressed in yeast cells. The ATPase activity of each of the mutants was similar to the activity of the wild type enzyme indicating that Lys-480 is not essential for ATP hydrolysis. The binding of [3H]ouabain in both ATP-dependent and inorganic phosphate-dependent reactions was used to determine the apparent affinity of each mutant for ATP or Pi. The K0.5(ATP) for ouabain binding to phosphoenzyme formed from ATP was 1-3 microM for Lys-480, Arg-480, and Ala-480, whereas for Glu-480 the K0.5(ATP) was 18 microM. The K0.5(Pi) for ouabain binding to phosphoenzyme formed from inorganic phosphate was 16-28 microM for Lys-480, Arg-480, and Ala-480, but was 74 microM for Glu-480. The Kd for ouabain binding was similar for both the wild type and mutant Na,K-ATPase molecules (3-6 nM). These data indicate that the substitution of an acidic amino acid for lysine at position 480 appears to reduce the affinity of the Na,K-ATPase for both ATP and phosphate. It is concluded that Lys-480 is not essential for ATP binding or hydrolysis or for phosphate binding by Na,K-ATPase but is likely to be located within the ATP binding site of the Na,K-ATPase.     

Some characteristics of Na/K-ATPase from rat intestinal basal lateral membranes

Summary Basal lateral membrane vesicles were isolated from rat intestinal epithelial cells. The sodium potassium triphosphatase (Na/K-ATPase) of these plasma membranes has been characterized by (1) the molecular weight of the phosphorylated intermediate, (2) the sensitivity of the phosphorylated intermediate to hydroxylamine, (3) its ouabain binding constants, and (4) its susceptibility to digestion by pronase. The phosphorylated intermediate was shown by SDS polyacrylamide gel electrophoresis to be a protein of 100,000 Daltons apparent mol wt. Its extensive hydrolysis in hydroxylamine demonstrated that it was an acyl phosphate. The isolated basal lateral membranes bound ouabain with a dissociation constant,K _m (1.5×10^–5 m), similar to the inhibitory constantK _I (3×10^–5 m), measured for ouabain inhibition of the Na/K-ATPase activity. The association rate constant measured for ouabain binding at 22°C was 1.3×10³ m ^–1 sec^–1 and is similar to the association rate constants reported for other tissues and species. The high dissociation rate constant, 3.6×10^–2 sec^–1, is consistent with the insensitivity of the rat to ouabain. Digestion of the intact cells by pronase yielded basal lateral membranes in which the Na/K-ATPase had been unaffected. The phosphorylated intermediate ran as a sharp band at 100,000 Daltons on electrophoresis, and the ouabain dissociation constant appeared to be unchanged. In these membranes, protein stains of polyacrylamide gels revealed digestion of the major high mol wt proteins including the major protein at 100,000 Daltons. This suggests that the Na/K-ATPase represent a minor component, less than 1%, of the basal lateral membrane protein. From these characteristics of the phosphorylated intermediate and the ouabain binding constants, we conclude that the Na/K-ATPase of the basal lateral membranes of rat intestinal epithelial cells is similar to that found in other tissues and species. Estimates of the number of pump sites and the turnover number predict rates of Na transport that are consistent with observed values.This paper is dedicated to the memory of Professor David H. Smyth, FRS, who died on September 10, 1979.     

Interaction of thiamine with rat brain synaptosomes

Thiamine has been shown to be bound specifically by a synaptosomal plasmatic membrane and transported inside to the nervous ending. Apparent K[symbol: see text] and Km for processes of binding and transport have been determined as equal 2.34 +/- 0.55 MKM and 3.92 +/- 1.3 MKM, respectively. The thiamine uptake by the isolated nervous endings (synaptosomes) at its physiological concentration is reduced in presence of metabolic inhibitors and partially depends on Mg2+ and Ca2+ ions, that can testify about the interrelation between endogenic thiamine phosphorilation and its transport through the membrane. Thiamine binding with synaptosomes is inhibited by ouabain and neurotoxins such as, latrotoxin and most significantly--with veratridin; tetrodotoxin fail to be efficient practically. In the conditions of synaptic membranes depolarisation their ability to bind thiamine is reduced and output of already uptaken with synaptosomes thiamine is observed.     

Effects of anticonvulsive substances on Na,K-ATPase from the synaptic membranes of animal brain]

The distribution pattern of marker enzymes (Na, K-ATPase, acetylcholinesterase) in three fractions of synaptic membranes (SM) of rat brain were studied. The effects of three anticonvulsive agents on Na, K-ATPase from the total fraction of rat brain SM and purified membrane preparation from ox brain were estimated by different methods. Under optimal conditions (Na/K = 5) diphenylhydantoin (DPH) at a concentration of 0,1 mM activates Na, K-ATPase from the total SM fraction only in the absence of ouabain, whereas carbamazepine and pyrroxane taken at the same concentrations have no effect on Na, K-ATPase, irrespective of the type of the enzyme assay. DPH seems to compete with ouabain. Under non-optimal ionic conditions (Na/K = 250) all the anticonvulsive substances studied inhibit Na, K-ATPase of the total SM fraction. The mixture of hydrophobic agents (propylene glycol and ethanol) used to dissolve carbamazepine inhibits Na, K-ATPase from the total SM fraction only under non-optimal conditions. The inhibiting effect of the anticonvulsive substances under study on Na, K-ATPase from the purified membrane preparations is maximal at the concentration of 10(-6) M; at higher concentrations the effect is less pronounced.     

Calcium-Activated ATPases in Presynaptic Nerve Endings

We studied the properties of calcium-activated ATPases present in preparations of isolated presynaptic nerve ending (synaptosome) and its subfractions from mouse brain. ATPase activity in the preparation was stimulated by Ca2+ and by Mg2+, but not by Na+ and K+, when each was added alone. The substrate specificities were found to be similar. The ATPases hydrolyzed only the high-energy phosphate bond and similar activity was exhibited for all nucleoside triphosphates tested (ATP, CTP, GTP, UTP). Moreover, the enzymes were insensitive to mitochondrial markers and to ouabain, but were inhibited by La3+. La3+ produced uncompetitive inhibition of Ca2+-ATPase in intact synaptosomes. Inhibition by La3+ was greatly increased after lysis of the synaptosomes, suggesting that the active sites of the enzymes may be on the cytosolic face of the membranes. The Ca2+-ATPase activity in synaptosomes was increased by increasing concentrations of external K+, suggesting that Ca2+ influx may be involved The Ca2+-ATPase in synaptosomal plasma membranes and synaptic vesicles had higher specific activities than those of intact synaptosomes and were activated, both in the presence and the absence of Mg2+, by Ca2+ concentrations approximating the intracellular level (10(-7) M). It is concluded that the nonmitochondrial synaptosomal Ca2+-ATPase may play an important role in the regulation of intracellular Ca2+.     

The 24Na-transport increasing effect of veratrine in frog sartorius muscle influenced by the tonicity, composition and temperature of the external environment.

The influence of tonicity, ionic composition and temperature of the incubating medium on the increasing effect of veratrine on 24Na transport in the frog sartorius muscle has been studied. (1) The effect of veratrine applied during 24Na loading on the rate coefficient for sodium loss depended on the tonicity of the medium. The rate of loss of 24Na from muscles loaded in the presence of veratrine was not affected if the muscles had been equilibrated in hypertonic medium. However, when treating the muscles with veratrine in isotonic medium during 24Na loading, we obtained a twofold increase in the rate coefficient for sodium loss. (2) The effect of veratrine applied during the desaturation period on 24Na efflux was also found to depend on the tonicity of the medium. Veratrine applied during the desaturation period increased the 24Na efflux in muscles equilibrated in isotonic Ringer's solution. However, when the muscles were equilibrated in hypertonic medium, veratrine did not influence 24Na efflux, not even after the rate of 24Na loss had been decreased by ouabain. (3) Hypertonic medium inhibited the Li uptake-enhancing effect of veratrine, while in isotonic medium veratrine had a marked enhancing effect. (4) In hypertonic medium lithium inhibited the otherwise characteristic increasing effect of veratrine on 24 Na uptake. (5) The increase of intracellular sodium concentration as a result of incubation in cold, potassium-free Ringer's solution did not influence the 24Na exchange-increasing effect of veratrine in isotonic medium. (6) The increasing effects of 0.1 and 0.5 mM veratrine on 24Na influx had the same degree at room temperature. However, at 5 degrees C 0.5 mM veratrine increased 24Na influx to a greater extent than 0.1 mM. (7) On the basis of our earlier experiments it has been suggested that the site of action of the 24Na uptake-increasing effect of veratrine could be the neural structures in the muscle equilibrated in hypertonic media. The present experiments confirm this suggestion and at the same time demonstrate that there are substantial differences in the mechanism of the sodium transport of veratrine-treated neural and muscle membranes, which become more apparent in hypertonic medium.     

Mouse brain synaptosomal sodium channels: activation by aconitine, batrachotoxin, and veratridine, and inhibition by tetrodotoxin

Batrachotoxin, veratridine and aconitine, activators of the voltage-dependent sodium channel in excitable cell membranes, increase the rate of 22Na+ uptake by mouse brain synaptosomes. Batrachotoxin was both the most potent (K0.5, 0.49 microM) and most effective activator of specific 22Na+ uptake. Veratridine (K0.5, 34.5 microM) and aconitine (K0.5, 19.6 microM) produced maximal stimulations of 22Na+ uptake that were 73% and 46%, respectively, of that produced by batrachotoxin. Activation of 22Na+ uptake by veratridine was completely inhibited by tetrodotoxin (I50, 6 nM ), a specific blocker of nerve membrane sodium channels. These results identify appropriate conditions for measuring sodium channel-dependent 22Na+ flux in mouse brain synaptosomes. The pharmacological properties of mouse brain synaptosomal sodium channels described here are distinct from those previously described for sodium channels in rat brain synaptosomes and mouse neuroblastoma cells.     

Characterization of the Actions of Toxins II-9.2.2 and II-10 from the Venom of the Scorpion Centruroides noxius on Transmitter Release from Mouse Brain Synaptosomes

Toxic peptides II-9.2.2 and II-10, purified from Centruroides noxius venom, bear highly homologous N-terminal amino acid sequences, and both toxins are lethal to mice. However, only toxin II-10 is active on the voltage-clamped squid axon, selectively decreasing the voltage-dependent Na+ current. Here, we have tested toxins II-9 and II-10 on synaptosomes from mouse brain: both toxins increased the release of gamma-[3H]aminobutyric acid ([3H]GABA). Their effect was completely blocked by tetrodotoxin or by the absence of external Na+. Also, both toxins increased Na+ permeability in isolated nerve terminals. Besides the observation that toxin II-9 is active on synaptosomes, the effect of toxin II-10 in this preparation is opposite to that observed in the squid axon. Thus, our results reflect functional differences between the populations of Na+ channels in mouse brain synaptosomes and in the squid axon. The release of GABA evoked by these toxins from synaptosomes required external Ca2+ and was blocked by Ca2+ channel blockers (verapamil and Co2+). This latter observation is in sharp contrast to the releasing action of veratrine, which evoked release even in the absence of external Ca2+. Furthermore, the action of both C. noxius toxins was potentiated by veratrine, a result suggesting they have different mechanisms of action. Among drugs that release neurotransmitters by increasing Na+ permeability, it is noteworthy that scorpion toxins are the only ones yet reported to have a strict requirement for external Ca2+.     

Kinetic parameters of Na,K-ATPase in the gills of crabs adapted to freshened sea water]

The dependence of Na,K-ATPase activity on concentrations of ATP, Na+, K+, Mg2+ and ouabain in the membrane preparations of crab gills was studied. The first group of crabs was adapted to freshened (25%) and the second one--to normal (100%) sea water. A 40-day adaptation of crabs to the freshened sea water results in an increase of maximal activity of Na,K-ATPase, but does not affect the enzyme affinity for ATP, Na+, K+, Mg2+ and ouabain, as well as its cooperative properties. It is assumed that adaptation of crabs to freshened sea water is accompanied by an accumulation of Na, K-ATPase in the epithelial cell membranes or crab gills without causing any qualitative changes of the enzyme.     

Ionizing Radiation Alters the Properties of Sodium Channels in Rat Brain Synaptosomes

The effect of ionizing radiation on neuronal membrane function was assessed by measurement of neurotoxin-stimulated 22Na+ uptake by rat brain synaptosomes. High-energy electrons and gamma photons were equally effective in reducing the maximal uptake of 22Na+ with no significant change in the affinity of veratridine for its binding site in the channel. Ionizing radiation reduced the veratridine-stimulated uptake at the earliest times measured (3 and 5 s), when the rate of uptake was greatest. Batrachotoxin-stimulated 22Na+ uptake was less sensitive to inhibition by radiation. The binding of [3H]saxitoxin to its receptor in the sodium channel was unaffected by exposure to ionizing radiation. The effect of ionizing radiation on the lipid order of rat brain synaptic plasma membranes was measured by the fluorescence polarization of the molecular probes 1,6-diphenyl-1,3,5-hexatriene and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene. A dose of radiation that reduced the veratridine-stimulated uptake of 22Na+ had no effect on the fluorescence polarization of either probe. These results demonstrate an inhibitory effect of ionizing radiation on the voltage-sensitive sodium channels in rat brain synaptosomes. This effect of radiation is not dependent on changes in the order of membrane lipids.     

The steroid-binding subunit of the Na/K-ATPase as a progesterone receptor on the amphibian oocyte plasma membrane

Progesterone acts at a plasma membrane receptor on the Rana oocyte to initiate meiosis. A cascade of lipid messengers occurs within seconds, followed by sequential changes in membrane phospholipid composition. We now show that progesterone binding to the plasma membrane increases continuously over the first 4 h. Subsequently, about 60% of the total plasma membrane and > 90% of membrane-bound progesterone, ouabain binding sites, and Na/K-ATPase activity are internalized. Until the completion of membrane internalization, oocytes must be continuously exposed to nanomolar concentrations of exogenous progesterone for meiosis to continue. The membrane-bound progesterone remains unchanged, whereas microinjected [(3)H]progesterone is rapidly metabolized. We find that progesterone and the plant steroid ouabain compete for one of two ouabain binding sites on the oocyte surface. Ouabain blocks progesterone action and inhibits subsequent meiosis if added at any time during the first 4-5 h. Western blots of SDS/PAGE extracts of isolated oocyte plasma membranes contain a -110 kDa band which binds an antibody to the steroid-binding c-terminal domain in rat and human PR. The number of binding sites and K(d) for progesterone binding to the plasma membrane is comparable to those for low-affinity ouabain binding to the alpha-subunit of the Na/K-ATPase (112 kDa). Our results suggest that progesterone binding to the ouabain binding site on the N-terminal region of the alpha-subunit of Na/K-ATPase may modulate early plasma membrane events over the first 4-6 h. Progesterone may thus act in part through the plasma membrane Na/K-ATPase signaling system.     

Effect of dopamine and dopamine mimetics on Na, K-ATPase of corpus striatum synaptosomes

Dopamine (DA) and DA-mimetics (apomorphine, midantan, piribedil) have a dual effect on Na, K-ATPase of the rat brain striate synaptosomes: activating at micromolar concentrations and inhibitory at higher concentrations (less than or equal to 30 microM). In the presence of Ca2+ (1 mM EGTA + 2.5 mM Ca2+) DA activating effect completely disappears and the inhibitory effect becomes even more pronounced. In the presence of cAMP (50 microM) which has no effect of its own on Na, K-ATPase, DA activation maximum is shifted towards lower concentrations, and the inhibitory effect remains unchanged. The above mentioned effects of DA persist in the presence of ouabain (1 mM), i.e. during measuring of Na, K-ATPase activity by an "ouabain" method, with DA activation maximum shifted towards higher concentrations.     

Cytoskeletal regulation of the sensitivity of brain synaptosomes to the depolarizing action of veratrine

Using a radioactive permeant cation 3H-tetraphenylphosphonium, the sensitivity of rat brain synaptosomes to depolarizing action of veratrine, which specifically opens the sodium channels, was compared before and after destruction of microtubules and microfilaments. Depolymerization of microtubules with colchicin and vinblastine decreased an apparent affinity of veratrine to its receptor in the channel, while destruction of microfilaments with cytochalasin B had the opposite effect. Colchicine did not change allosteric interactions between the receptor for veratrine and that for scorpion venom in the sodium channel evaluated by the ability of scorpion venom to facilitate veratrine-induced depolarization of synaptosomes. It is suggested that two main cytoskeleton subsystems control the state of sodium channels in the nerve ending.