Ca-dependent regulation by Na, K-pump of post-tetanic sensitization of extrasynaptic choline receptors in Helix lucorum neurons

Posttetanic potentiation (by orthodromic stimulation) of cholinosensitivity in LPa3 and RPa3 Helix lucorum neurons that are command in respect to withdrawal behavior was shown earlier (Pivovarov et al., 1999). Now we studied the regulatory role of the Na,K-pump and intracellular free Ga2+ in the posttetanic potentiation (PTP) of cholinosensitivity in command neurons. Semiintact Helix preparation "CNS-visceral bag" was used in experiments. Acetylcholine-induced inward currents were recorded using two-electrode voltage clamp technique. Acetylcholine was applied to somata of the identified LPa3 and RPa3 neurons with a 10-min interval before and after electrical tetanic stimulation of the n. intestinalis (10.5 mA; 0.1 s; 2/s; 2 min). Ouabain (extracellular application, 70 mcM) blocked the PTP. Intracellular injection of BAPTA (1 mM), chelator of Ca2+ ions, prevented the PTP. The PTP was absent after the ouabain application against the background of preliminary intracellular injection of BAPTA. A conclusion war drawn about Ca-dependent participation of Na,K-pump in posttetanic potentiation of cholinosensitivity in command Helix lucorum neurons of withdrawal behavior.     

Involvement of Na,K-pump in SEPYLRFamide-mediated reduction of cholinosensitivity in Helix neurons

SEPYLRFamide acts as an inhibitory modulator of acetylcholine (ACh) receptors in Helix lucorum neurones. Ouabain, a specific inhibitor of Na,K-pump, (0.1 mM, bath application) decreased the ACh-induced inward current (ACh-current) and increased the leak current. Ouabain decreased the modulatory SEPYLRFamide effect on the ACh-current. There was a correlation between the effects of ouabain on the amplitude of the ACh-current and on the modulatory peptide effect. Ouabain and SEPYLRFamide inhibited the activity of Helix aspersa brain Na,K-ATPase. Activation of Na,K-pump by intracellular injection of 3 M Na acetate or 3 M NaCl reduced the modulatory peptide effect on the ACh-current. An inhibitor of Na/Ca-exchange, benzamil (25 muM, bath application), and an inhibitor of Ca(2+)-pump in the endoplasmic reticulum, thapsigargin (TG, applied intracellularly), both prevented the effect of ouabain on SEPYLRFamide-mediated modulatory effect. Another inhibitor of Ca(2+)-pump in the endoplasmic reticulum, cyclopiazonic acid (applied intracellularly), did not prevent the effect of ouabain on SEPYLRFamide-mediated modulatory effect. These results indicate that Na,K-pump is responsible for the SEPYLRFamide-mediated inhibition of ACh receptors in Helix neurons. Na/Ca-exchange and intracellular Ca(2+) released from internal pools containing TG-sensitive Ca(2+)-pump are involved in the Na,K-pump pathway for the SEPYLRFamide-mediated inhibition of ACh receptors.     

Participation of Na/Ca-exchange and intracellular mobilized Ca2+ in regulating depression of cholino-sensitive Helix lucorum neurons to a cellular analog of habituation

The role the Na/Ca-exchange and intracellular Ca2+ released from Ca(2+)-depots in the modulatory action of Na,K-pump inhibitor ouabain on cholinosensitivity in the command neurons of Helix lucorum was studied in a cellular analogue of habituation. The integral transmembrane inward currents in LPa2, LPa3, RPa3, and RPa2 neurons were recorded in Helix lucorum ganglia preparation using two-electrode voltage clamp technique. The reduction of cholinosensitivity of a neuron was estimated as a depth of the depression of the acetylcholine-induced inward currents during the rhythmic local acetylcholine applications (with the interstimulus interval of 2-4 min) on a somatic membrane. The inhibitor of the Na/Ca-exchange benzamil (the extracellular action, 15-35 mcM) and two specific inhibitors of Ca-ATPase in the sarcoplasmic and endoplasmic reticulum, cyclopiazonic acid and thapsigargin (intracellular injection by spontaneous diffusion, 0.1 mM) prevented the modification of the depression of acetylcholine-induced current by ouabain (100 mcM) during the rhythmic application of acetylcholine. A conclusion is drawn that the inhibitor of the Na,K-pump ouabain modifies the depression of neuron cholinosensitivity in the cellular analogue of habituation via the Na/Ca-exchange and intracellular Ca2+ released from Ca2+ depots.     

Inositoltriphosphate receptors and ryanodine receptors in regulation of cholinosensitivity of Helix lucorum neurones by Na,K-pump during habituation

Influence of ouabain, the inhibitor of Na,K-pump, on habituation of Helix to tactile stimulation was identical to the ouabain-induced modification of cholinosensitivity reduction in command neurones of defensive behaviour of Helix lucorum in cellular model of habituation. Effects of intracellularly injected ligands of two types of Ca2+ -depot receptors, inositoltrisphosphate (IP3) and ryanodine receptors, on ouabain-induced changes were studied in cellular model of habituation. The antagonist of IP3 receptors heparin (0.1 mM), their agonist IP3 (0.1 mM) and inhibitor of ryanodine-dependent Ca2+ mobilization dantrolen (0.1 mM) prevented the depression of acetylcholine-induced current from the ouabain-evoked modification. The agonist/antagonist of ryanodine receptors ryanodine at two tested concentrations (0.1 mM and 1 mM) did not change the ouabain effect. It is concluded that Ca2+ released from intracellular Ca2+ -depots via IP3 receptors is involved into neuronal mechanism of Na,K-pump regulation of habituation in Helix lucorum to tactile stimulation.     

Modulation of the Na,K-pump function by beta subunit isoforms

To study the role of the Na,K-ATPase beta subunit in the ion transport activity, we have coexpressed the Bufo alpha 1 subunit (alpha 1) with three different isotypes of beta subunits, the Bufo Na,K-ATPase beta 1 (beta 1NaK) or beta 3 (beta 3NaK) subunit or the beta subunit of the rabbit gastric H,K-ATPase (beta HK), by cRNA injection in Xenopus oocyte. We studied the K+ activation kinetics by measuring the Na,K- pump current induced by external K+ under voltage clamp conditions. The endogenous oocyte Na,K-ATPase was selectively inhibited, taking advantage of the large difference in ouabain sensitivity between Xenopus and Bufo Na,K pumps. The K+ half-activation constant (K1/2) was higher in the alpha 1 beta 3NaK than in the alpha 1 beta 1NaK groups in the presence of external Na+, but there was no significant difference in the absence of external Na+. Association of alpha 1 and beta HK subunits produced active Na,K pumps with a much lower apparent affinity for K+ both in the presence and in the absence of external Na+. The voltage dependence of the K1/2 for external K+ was similar with the three beta subunits. Our results indicate that the beta subunit has a significant influence on the ion transport activity of the Na,K pump. The small structural differences between the beta 1NaK and beta 3NaK subunits results in a difference of the apparent affinity for K+ that is measurable only in the presence of external Na+, and thus appears not to be directly related to the K+ binding site. In contrast, association of an alpha 1 subunit with a beta HK subunit results in a Na,K pump in which the K+ binding or translocating mechanisms are altered since the apparent affinity for external K+ is affected even in the absence of external Na+.     

The functional interrelation between Na/K-pump work, Na/Ca metabolism and the chemosensitivity of the neuronal membrane

A correlation between the functions of Na/K-pump, Na/Ca-exchange and chemoreceptors in the membrane has been found. This correlation carries out through intracellular content of cyclic nucleotides. The low doses of transmitters which are unable to activate the chemosensitive ionic channels, have modulatory effect on the above mentioned membrane mechanisms.     

Effects of internal Na and external K concentrations on Na/K coupling of Na,K-pump in frog skeletal muscle

Summary To clarify the dependency of the Na/K coupling of the Na,K-pump on internal Na and external K concentrations in skeletal muscle, the ouabain-induced change in membrane potential, the ouabain-induced change in Na efflux and the membrane resistance were measured at various internal Na and external K concentrations in bullfrog sartorius muscle.Upon raising the internal Na concentration from 6 mmol/kg muscle water to 20 mmol/kg muscle water, the magnitude of the ouabain-induced change in membrane potential increased about eightfold and the magnitude of the ouabain-induced change in Na efflux increased about fivefold while the membrane resistance was not significantly changed. As the external K concentration increased from 1 to 10mm, the magnitude of the ouabain-induced change in membrane potential decreased (1/5.5 fold), while the magnitude of the ouabain-induced change in Na efflux increased (about 1.5-fold). The membrane resistance decreased upon raising the external K concentration from 1 to 10mm (1/2-fold). These observations imply that the values of the Na/K coupling of the Na,K-pump increases upon raising the internal Na concentration and decreases upon raising the external K concentration.     

Activation of the Na,K-pump by isoproterenol, methylxanthines, and iodoacetate in erythrocytes of the frogRana temporaria

Our preliminary studies have shown that the Na,K-pump in frog erythrocytes is activated by isoproterenol (ISP), phosphodiesterase blocker (3-isobutyl-methylxantine, IBMX), and by iodoacetate (MIA). The aim of the present study was to determine a mechanism responsible for the effect of MIA on the Na,K-pump activity in frog red blood cells as well as the role of G proteins and intracellular messengers in modulation of active K⁺ transport induced by ISP. An additive stimulation of active K⁺ (⁸⁶Rb) transport in frog erythrocytes was found after exposure of the cells to MIA in a combination with ISP or IBMX. The treatment of the red blood cells with 1 mM MIA for 1 or 2 h was associated with a significant decrease in intracellular Na⁺ concentration, on average, by 13 and 20%, respectively, suggesting a direct action of MIA on the Na,K-pump. Incubation of cells in the presence of dibutyryl-cAMP (1 mM) or adenylate cyclase activator forskolin (0.1 mM) caused stimulation of the active K⁺ influx by 21.8 and 27.9%, respectively. AlF ₄ ^- and cholera toxin able to increase cell cAMP levels via G protein interactions had no effect on the total and IPS-induced K⁺ influx in frog erythrocytes. The treatment of the red blood cells with sodium nitroprusside that increases cGMP concentration in cells also had no effect on the K⁺ influx. The stimulatory influence of ISP on the Na,K-pump was reduced with increase of the intracellular Na⁺ concentration. ISP increased affinity of the Na,K-pump to Na⁺ (the Mihaelis constant K_M = 34.4 ± 5.1 in control and 25.3 ± 2.8 mM in the presence of ISP,p < 0.01), but did not change maximal velocity (8.1 ± 0.6 and 7.7 ± 0.3 mmol/1/h in the control and ISP-treated cells, respectively). The results obtained indicate the presence of several different signal pathways involved in regulation of the Na,K-pump activity in frog erythrocytes.     

Hypoxia-induced increase in intracellular calcium concentration in endothelial cells: role of the Na(+)-glucose cotransporter.

Hypoxia is a common denominator of many vascular disorders, especially those associated with ischemia. To study the effect of oxygen depletion on endothelium, we developed an in vitro model of hypoxia on human umbilical vein endothelial cells (HUVEC). Hypoxia strongly activates HUVEC, which then synthesize large amounts of prostaglandins and platelet-activating factor. The first step of this activation is a decrease in ATP content of the cells, followed by an increase in the cytosolic calcium concentration ([Ca(2+)](i)) which then activates the phospholipase A(2) (PLA(2)). The link between the decrease in ATP and the increase in [Ca(2+)](i) was not known and is investigated in this work. We first showed that the presence of extracellular Na(+) was necessary to observe the hypoxia-induced increase in [Ca(2+)](i) and the activation of PLA(2). This increase was not due to the release of Ca(2+) from intracellular stores, since thapsigargin did not inhibit this process. The Na(+)/Ca(2+) exchanger was involved since dichlorobenzamil inhibited the [Ca(2+)](i) and the PLA(2) activation. The glycolysis was activated, but the intracellular pH (pH(i)) in hypoxic cells did not differ from control cells. Finally, the hypoxia-induced increase in [Ca(2+)](i) and PLA(2) activation were inhibited by phlorizin, an inhibitor of the Na(+)-glucose cotransport. The proposed biochemical mechanism occurring under hypoxia is the following: glycolysis is first activated due to a requirement for ATP, leading to an influx of Na(+) through the activated Na(+)-glucose cotransport followed by the activation of the Na(+)/Ca(2+) exchanger, resulting in a net influx of Ca(2+).     

Mechanisms of up-regulation of single calcium channels by serotonin in Helix pomatia neurons

Action of serotonin (5-HT) on single Ca(2+) channel activity was studied in identified neurons of snail Helix pomatia. Only one type of Ca(2+) channels of 5 pS unitary conductance was determined under patch-clamp cell-attached mode. Kinetic analysis have shown a monotonically declining distribution of channel open times (OT) with mean time constant of 0.2 ms. The distribution of channel closed times (CT) could be fitted by double-exponential curve with time constants 1 and 12 ms. We established that 5-HT acts on Ca(2+) channel activity indirectly via cytoplasm. 5-HT prolonged the OT (up to 0.3 ms) and shortened the CT proportionally for both constants to 0.4 and 6 ms correspondingly. A conclusion is made that enhancement of Ca(2+) macro-current by 5-HT is determined by kinetic changes, increase of the number of active channels, and increase of the probability of OT. At the same time the transmitter did not affect the unitary channel conductance.     

Charge translocation by the Na,K-pump: II. Ion binding and release at the extracellular face

Summary In the first part of the paper, evidence has been presented that electrochromic styryl dyes, such as RH 421, incorporate into Na, K-ATPase membranes isolated from mammalian kidney and respond to changes of local electric field strength. In this second part of the paper, fluorescence studies with RH-421-labeled membranes are described, which were carried out to obtain information on the nature of charge-translocating reaction steps in the pumping cycle. Experiments with normal and chymotrypsin-modified membranes show that phosphorylation by ATP and occlusion of Na⁺ are electroneutral steps, and that release of Na⁺ from the occluded state to the extracellular side is associated with translocation of charge. Fluorescence signals observed in the presence of K⁺ indicate that binding and occlusion of K⁺ at the extracellular face of the pump is another major electrogenic reaction step. The finding that the fluorescence signals are insensitive to changes of ionic strength leads to the conclusion that the binding pocket accommodating Na⁺ or K⁺ is buried in the membrane dielectric. This corresponds to the notion that the binding sites are connected with the extracellular medium by a narrow access channel (ion well). This notion is further supported by experiments with lipophilic ions, such as tetraphenylphosphonium (TPP⁺) or tetraphenylborate (TPB^–), which are known to bind to lipid bilayers and to change the electrostatic potential inside the membrane. Addition of TPP⁺ leads to a decrease of binding affinity for Na⁺ and K⁺, which is thought to result from the TPP^–-induced change of electric field strength in the access channel.Deceased (September 13, 1990).     

The role of Na,K-ATPase alpha subunit serine 775 and glutamate 779 in determining the extracellular K+ and membrane potential-dependent properties of the Na,K-pump

The roles of Ser775 and Glu779, two amino acids in the putative fifth transmembrane segment of the Na,K-ATPase alpha subunit, in determining the voltage and extracellular K+ (K+(o)) dependence of enzyme-mediated ion transport, were examined in this study. HeLa cells expressing the alpha1 subunit of sheep Na,K-ATPase were voltage clamped via patch electrodes containing solutions with 115 mM Na+ (37 degrees C). Na,K-pump current produced by the ouabain-resistant control enzyme (RD), containing amino acid substitutions Gln111Arg and Asn122Asp, displayed a membrane potential and K+(o) dependence similar to wild-type Na,K-ATPase during superfusion with 0 and 148 mM Na+-containing salt solutions. Additional substitution of alanine at Ser775 or Glu779 produced 155- and 15-fold increases, respectively, in the K+(o) concentration that half-maximally activated Na,K-pump current at 0 mV in extracellular Na+-free solutions. However, the voltage dependence of Na,K-pump current was unchanged in RD and alanine-substituted enzymes. Thus, large changes in apparent K+(o) affinity could be produced by mutations in the fifth transmembrane segment of the Na,K-ATPase with little effect on voltage-dependent properties of K+ transport. One interpretation of these results is that protein structures responsible for the kinetics of K+(o) binding and/or occlusion may be distinct, at least in part, from those that are responsible for the voltage dependence of K+(o) binding to the Na,K-ATPase.     

EGF-Induced increase in diacylglycerol,choline release,and DNA synthesis is extracellular calcium dependent

Previous studies have demonstrated a strict extracellular Ca²⁺ dependence for the G₀ to G₁ and G₁ to S transition in growth factor-treated T51B rat liver cells that is associated with increased levels of protein kinase C activity. Consequently, we have examined these cells for changes in phospholipid-derived second messengers in response to epidermal growth factor (EGF) and thrombin in order to determine which signals are generated during the initiation of the G₀ to G₁ transition. Thrombin is coupled to a phosphoinositide hydrolyzing phospholipase C, as we have found a rapid Ca²⁺-independent increase in the levels of inositol 1,4,5-trisphosphate (Ins[1,4,5]P₃), inositol 1,4-bisphosphate (Ins[1,4]P₂), and inositol 4-monophosphate (Ins[4]P), as well as a concomitant, transient elevation in diacylglycerol. No changes in either intracellular or extracellular choline metabolites, or an increase in DNA synthesis, were found in response to thrombin. By contrast, treatment of T51B cells with EGF results in a slower, more prolonged extracellular Ca²⁺-dependent increase in both [³H]-glycerol radiolabeled diacylglycerol, and diacylglycerol mass, an increase in choline release into the extracellular medium, and eventually a substantial DNA synthesis. We were, however, unable to detect any changes in phosphatidylinositol (Ptdlns) turnover, either by accumulation of inositol phosphates or by changes in phospholipids in response to EGF. These results indicate that DNA synthesis can readily occur in the absence of stimulated Ptdlns turnover, and that Ptdlns turnover is not sufficient in itself or necessary to induce DNA synthesis and is not necessary for a Ca²⁺-dependent increase in diacylglycerol. Moreover, we have demonstrated that the extracellular Ca²⁺-dependent increase in diacylglycerol levels in response to EGF is associated with an increase in extracellular choline release, which is indicative of an activation of a phosphatidylcholine-linked phospholipase D. These results suggest that diacylglycerol sources other than Ptdlns's may be important in the extracellular Ca²⁺-dependent regulation of EGF-mediated cell replication. © 1995 Wiley-Liss, Inc.     

Comparative study of the functional expression of the Na/K-pump in human lymphocytes, activated by phytohemagglutinin, phorbol ester, ionomycin, and interleukin-2

Rubidium (Rb) influxes via Na/K pump and ouabain-resistant pathways, and protein, RNA and DNA syntheses have been studied in human blood lymphocytes during the cell transit from quiescence to proliferation. In lymphocytes, stimulated either by phytohemagglutinin (PHA), phorbol 12,13-dibutyrate (PDBu) calcium ionophore, ionomycin, or by PDBu and interleukin-2 (IL-2), the late stages of the G0/G1-->S transit are accompanied by sustained 3-fold increased ouabain-sensitive Rb influxes. Using a two-pulse activation by a brief (1 h) PDBu/ionomycin treatment, followed by incubation with PDBu or IL-2 for 48 h, it has been found that both IL-2 and IL-2 receptor (IL-2R) are necessary for a long-term enhancement of Na/K pump. When present at the early stage of PDBu and ionomycin induction, cyclosporin A (CsA, 1 microgram/ml) inhibits the late increase in pump-mediated Rb influxes. However, when applied after the competence induction, CsA produced no effect on the flux increase at the progression stage. It is concluded that in activated human lymphocytes the functional expression of Na/K pump may by associated with IL-2-dependent progression of competent cells in the cell cycle.     

Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements

Summary Membrane fragments containing a high density of Na, K-ATPase can be noncovalently labeled with amphiphilic styryl dyes (e.g., RH 421). Phosphorylation of the Na,K-ATPase by ATP in the presence of Na⁺ and in the absence of K⁺ leads to a large increase of the fluorescence of RH 421 (up to 100%). In this paper evidence is presented that the styryl dye mainly responds to changes of the electric field strength in the membrane, resulting from charge movements during the pumping cycle: (i) The spectral characteristic of the ATP-induced dye response essentially agrees with the predictions for an electrochromic shift of the absorption peak. (ii) Adsorption of lipophilic anions to Na, K-ATPase membranes leads to an increase, adsorption of lipophilic cations to the decrease of dye fluorescence. These ions are known to bind to the hydrophobic interior of the membrane and to change the electric field strength in the boundary layer close to the interface. (iii) The fluorescence change that is normally observed upon phosphorylation by ATP is abolished at high concentrations of lipophilic ions. Lipophilic ions are thought to redistribute between the adsorption sites and water and to neutralize in this way the change of field strength caused by ion translocation in the pump protein. (iv) Changes of the fluorescence of RH 421 correlate with known electrogenic transitions in the pumping cycle, whereas transitions that are known to be electrically silent do not lead to fluorescence changes. The information obtained from experiments with amphiphilic styryl dyes is complementary to the results of electrophysiological investigations in which pump currents are measured as a function of transmembrane voltage. In particular, electrochromic dyes can be used for studying electrogenic processes in microsomal membrane preparations which are not amenable to electrophysiological techniques.Deceased (September 13, 1990).     

Regulation of calcium currents and secretion by magnesium in crustacean peptidergic neurons

The effect of varying the external Mg²⁺ concentration on Ca²⁺ currents through voltage-operated Ca²⁺ channels has been examined with the patch-clamp technique in acutely isolated neuronal somata from the X-organ-sinus gland (XOSG) of the crab,Cardisoma carnifex. Neurons from this neurosecretory system were selected for morphology associated with crustacean hyperglycemic hormone (CHH) content. In parallel, the effects of Mg²⁺ concentration on K⁺-evoked secretion of CHH from isolated, intact XOSGs have been assayed by ELISA. At physiological Ca²⁺ levels the high-voltage-activated Ca²⁺ currents were attenuated with increasing Mg²⁺ concentration, with 50% inhibition at 75 mM. Mg²⁺ block was voltage-dependent, relief from block occurring with increasing depolarization. Thus, in 24 mM Mg²⁺ inhibition of the Ca²⁺ current was 55% at –10 mV and 30% at +20 mV. Secretion of CHH varied almost linearly with the log of Mg²⁺ concentration; in 2.4 mM Mg²⁺ it was double that in 24 mM Mg²⁺ and almost completely inhibited in 100 mM. Thus, Mg²⁺ produces a parallel inhibition of Ca²⁺ currents and CHH secretion and may play a role as a physiological modulator of neuronal activity and secretion in the XOSG of these crabs.     

The effect of the internal Na concentration on the electrogenicity of the insulin-stimulated Na,K-pump in frog skeletal muscles

Insulin induced a hyperpolarization of the membrane by stimulating the Na,K-pump in frog skeletal muscles. The Na,K-pump activity was dependent on the internal Na concentration. As the internal Na concentration was raised from 5 mmol/kg muscle water to 18 mmol/kg muscle water, the magnitude of the insulin-induced increase in the ouabain-sensitive Na efflux (an index of the Na,K-pump activity) rose by 5-fold and the magnitude of the insulin-induced hyperpolarization rose by 8.5-fold. On the other hand, the specific membrane resistance was not significantly changed by a rise in the internal Na concentration. The Na/K coupling of the Na,K-pump was calculated at low, normal or high internal Na concentration by using the values of the insulin-induced changes in the ouabain-sensitive Na efflux and the membrane potential. As a result of the calculation, it was suggested that in frog skeletal muscles the Na/K coupling would increase with a rise of the internal Na concentration.     

The effect of oxytocin on potential-dependent calcium current in snail neurons, Helix pomatia.

1. The effect of external application of oxytocin on inward calcium current in dialyzed snail neurons has been investigated under clamp conditions. 2. External application of oxytocin in a dose-dependent manner (Kd 0.9 microM) inhibits inward calcium current in dialyzed neurons of the snail, Helix pomatia. 3. Inhibition of calcium current developed with the time constant of about 2 min. The degree of restoration of calcium current after oxytocin washout depends on duration of oxytocin action. 4. It has been suggested that inhibition of calcium current by oxytocin occurs in two stages, the initial one is more fast and reversible and the second one--more slow and irreversible. The participation of soluble second messengers in the inhibitory effect of oxytocin on calcium current is discussed.