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.     

Effects of SEPYLRFamide on acetylcholine-induced currents of Helix aspersa neurones: role of ryanodine receptors

The possible participation of ryanodine receptors in the modulatory effects of the endogenous Helix heptapeptide, SEPYLRFamide, on the acetylcholine-induced currents (ACh-currents) of Helix aspersa neurones was studied using the two-electrode voltage clamp technique. SEPYLRFamide (bath application) caused a reduction of the ACh-currents of D1, D2, F1, F2, F76 and F77 neurones. Ryanodine (10 microM; bath application), which modifies ryanodine-controlled Ca(2+) channels, potentiated the inhibitory effect of SEPYLRFamide on the ACh-current. An antagonist of cyclic adenosine diphosphate ribose (cADPR) and ryanodine receptors, ruthenium red (1 mM; intracellular injection), reduced the inhibitory effects of SEPYLRFamide on the ACh-current. Ryanodine (10 microM) did not change the inhibitory effect of SEPYLRFamide on the ACh-current after intracellular injection of ruthenium red. An agonist of ryanodine receptors, caffeine (5 mM; bath application), reduced the ACh-current. Ryanodine (10 microM) did not change the reduction of ACh-currents induced by the first application of caffeine but decreased the reduction of ACh-currents induced by subsequent applications of caffeine. It is proposed that ryanodine receptors are involved in the inhibitory modulatory effects of SEPYLRFamide on somatic cholinergic receptors of Helix aspersa neurones.     

Involvement of ryanodine receptors in EPYLRFamide-mediated reduction of acetylcholine-induced inward currents in helix lucorum identified neurones

The effects of several modulators of ryanodine receptors (RYRs) on the reduction of acetylcholine induced inward current (ACh-current) evoked by EPYLRFamide (5 microM, bath application), the potent N-terminally modified analogue of the endogenous Helix heptapeptide SEPYLRFamide, were investigated. These modulators were applied intracellularly. Inward currents were recorded from identified Helix lucorum LPa2, LPa3, RPa3, RPa2 neurones in ganglia preparations using the two-electrode voltage clamp technique. ACh was applied ionophoretically. BAPTA (0.1 mM), chelator of intracellular Ca(2+), ryanodine (0.1 mM), agonist/antagonist of RYRs and dantrolene (0.1 mM), antagonist of RYRs decrease the effect of EPYLRFamide. Adenosine (1 mM), alpha,beta-methylene ATP (0.1 mM), the nonhydrolisable ATP analogue and cyclic adenosine diphosphate ribose (0.1 mM) (agonists of RYRs) potentiate the modulatory effect of EPYLRFamide. Ruthenium red (1 mM), antagonist of RYRs and caffeine (1 mM), agonist of RYRs do not change the modulatory effect of EPYLRFamide. These data suggest that intracellular Ca(2+) and RYRs are involved in the modulatory effect of EPYLRFamide on ACh-currents. It was concluded that EPYLRFamide decreases ACh-current through elevation of basal intracellular level of a putative endogenous agonist of RYRs which activates RYR-dependent mobilization of Ca(2+) by binding to the adenine nucleotide site of the ryanodine receptor-channel complex and does not bind the site activated by caffeine.     

Effects of SEPYLRFamide on acetylcholine-induced currents of Helix aspersa neurones: role of ryanodine receptors

The possible participation of ryanodine receptors in the modulatory effects of the endogenous Helix heptapeptide, SEPYLRFamide, on the acetylcholine-induced currents (ACh-currents) of Helix aspersa neurones was studied using the two-electrode voltage clamp technique. SEPYLRFamide (bath application) caused a reduction of the ACh-currents of D1, D2, F1, F2, F76 and F77 neurones. Ryanodine (10 μM; bath application), which modifies ryanodine-controlled Ca²⁺ channels, potentiated the inhibitory effect of SEPYLRFamide on the ACh-current. An antagonist of cyclic adenosine diphosphate ribose (cADPR) and ryanodine receptors, ruthenium red (1 mM; intracellular injection), reduced the inhibitory effects of SEPYLRFamide on the ACh-current. Ryanodine (10 μM) did not change the inhibitory effect of SEPYLRFamide on the ACh-current after intracellular injection of ruthenium red. An agonist of ryanodine receptors, caffeine (5 mM; bath application), reduced the ACh-current. Ryanodine (10 μM) did not change the reduction of ACh-currents induced by the first application of caffeine but decreased the reduction of ACh-currents induced by subsequent applications of caffeine. It is proposed that ryanodine receptors are involved in the inhibitory modulatory effects of SEPYLRFamide on somatic cholinergic receptors of Helix aspersa neurones. Accepted: 1 July 1998     

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.     

EPYLRFamide-mediated reduction of acetylcholine-induced inward currents in Helix lucorum-identified neurones: role of NAADP-dependent and IP3-dependent Ca2+ release from internal stores,calmodulin and Ca2+/calmodulin-dependent protein kinase II

The effect of seven compounds intracellularly applied by spontaneous diffusion were investigated on the EPYLRFamide-induced reduction of acetylcholine-induced inward current (ACh-current) recorded from identified neurones from Helix lucorum. Inward currents were recorded from neurones LPa2, LPa3, RPa3 and RPa2 in isolated ganglia preparations using two-electrode voltage clamp technique. ACh was applied ionophoretically. Heparin, an antagonist of IP(3) receptors (IP(3)Rs), and IP(3), the agonist of IP(3)Rs, decreased the effect of EPYLRFamide. Thio-NADP, a blocker of NAADP-induced Ca(2+) release, beta-NAADP, Ca(2+) releaser, R24571, W-7 (both calmodulin antagonists), and KN-62, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase II, did not change the modulatory effect of EPYLRFamide. These data suggest that EPYLRFamide decreases ACh-current through elevation of the basal intracellular level of the putative endogenous agonist of IP(3)Rs which activates release of Ca(2+) from intracellular stores. It is concluded that intracellular free Ca(2+) acts on ACh receptor/ionic channel without activation of calmodulin and Ca(2+)/calmodulin-dependent protein kinase II.     

Regulation of posttetanic increase in choline sensitivity in Helix neurons by Na,K-pump: the role of Na/Ca-exchange and of mobilized calcium

We studied the role of Na/Ca-exchange and intracellular mobilized calcium in ouabain-mediated suppression of potentiation of cholinosensitivity of somatic membrane in Helix LPa3 and RPa3 command neurons of defensive behaviour after electrical orthodromic tetanisation of n. intestinalis. Cholinosensitivity of neurons was assessed by the amplitude of the inward current evoked by acetylcholine. Inhibitor of a Na/Ca-exchange benzamil and specific inhibitor of Ca-ATPase in endoplasmic reticulum thapsigargin prevented the development of the posttetanic potentiation (PTP). PTP did not arise and at joint action of ouabain with benzamil or thapsigargin. It was concluded that Na/Ca-exchange and mobilized calcium are involved in development of PTP of cholinosensitivity in somatic neuronal membrane and its regulation by Na,K-pump.     

The Na,K pump regulates a decrease in the cholinosensitivity of the neurons in the snail Helix lucorum taurica Kryn in a cellular analog of habituation: its dependence on intracellular calcium

In Helix lucorum snail we studied the effects of ouabain, inhibitor of Na,K-pump, on the depression of cholinosensitivity in command neurons of withdrawal behavior and the role of the intracellular free Ca2+. The cellular analog of the negative learning (habituation) was used Transmembrane integral inward currents were recorded from the identified LPa2, LPa3, RPa3, and RPa2 neurons in ganglia preparation using two-electrode voltage clamp technique. Acetylcholine (ACh) was locally applied iontophoretically. Reduction of neuronal cholinosensitivity was estimated as a depth of depression of the ACh-induced inward current during rhythmic local application of ACh (interstimulus interval of 1-3 min) onto the somatic membrane. Bath application of ouabain (0.1 mM) produced an increase in depression in one group of neurons and its decrease in another group. After 60-150 min of spontaneous diffusion of a calcium ion chelator BAPTA (1 mM) from the intracellular microelectrode, ouabain produced only the increase in depression. If CaCl2 (100 mM) was added to the solution of the voltage-recording intracellular microelectrode, 60 min later ouabain produced only the reduction of the depression of the ACh current. The conclusion is drawn that the inhibition of the Na,K-pump by ouabain modifies the depression of neuronal cholinosensitivity in the cellular analog of habituation. The direction of the modulatory effect depends on the basal concentration of the intracellular free Ca2+.     

Ouabain blocks some rapid concentration-induced clamp acetylcholine responses onHelix neurons

1. The effects of ouabain, a potent inhibitor of Na(+)-K+ ATPase, were determined on the transmembrane responses of internally dialyzed Helix neurons to rapid acetylcholine (ACh) application using the "concentration clamp" technique. 2. Ouabain selectively depressed "A"-type responses to ACh, which are due to a selective increase in membrane permeability to chloride. In contrast, the "B"-type responses, due primarily to an increase in monovalent cation permeability, was unaffected. 3. The blockade of the Cl- responses was not associated with a change of the reversal potential of the response. Ouabain depressed the maximal response without shifting the dose-response curve. 4. Ouabain caused an increase in the time constant of decay of the ACh current, but the value in the presence of ouabain was not different from that of a lower concentration of ACh determined so as to give a response of the same peak amplitude. Therefore, the effect of ouabain is not on the process of receptor desensitization directly.     

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.     

On the Mechanism of Ouabain-Induced Release of Acetylcholine from Synaptosomes

Ouabain (5 x 10(-8)-5 x 10(-4) M) was confirmed to cause a dose-dependent increase in [3H]acetylcholine ([3H]ACh) release, cytosolic free Ca2+ concentration ([Ca2+]i), and 22Na+ uptake in cerebrocortical synaptosomes of rats in the presence of extracellular Ca2+. Ouabain also caused a dose-dependent decrease in membrane potential. In a low-Na+ (10 mM) medium, ouabain failed to increase [3H]ACh release and [Ca2+]i. Tetrodotoxin (10(-6) M) had no effect on the ouabain-induced increase in both [3H]ACh release and [Ca2+]i but abolished the increase in 22Na+ uptake and partially inhibited the depolarizing effect. Verapamil (10(-6)-5 x 10(-4) M) inhibited the ouabain-induced increase in both [3H]ACh release and [Ca2+]i in a dose-dependent manner. Removal of extracellular Ca2+ abolished the effect of ouabain on [Ca2+]i but not on [3H]ACh release and 22Na+ uptake, regardless of the presence or absence of EGTA. In the absence of extracellular Ca2+, 10 mM Mg2+ blocked ouabain-induced [3H]ACh release, which was resistant to verapamil. These results suggest that ouabain can increase ACh release from synaptosomes without the preceding increases in intracellular Ca2+ and/or Na+ content. It seems likely that the removal of extracellular Ca2+ unmasks mechanisms of ouabain action different from those operating in the presence of Ca2+.     

Identification of a GM1/Sodium–Calcium exchanger complex in the nuclear envelope of non-neuronal cells

Stationary level of reactive oxygen species (ROS) in cerebellum granule cells of 12-day-old-rats was measured using three fluorescent dyes characteristic of different location within the neuronal cell: BODIPY 581/591 (for LOO.radicals), DCF-DA (for H202) and DHR123 (OH-radicals in mitochondria). When the neurons were activated by N-methyl- d -aspartate (NMDA) a dose- and time-dependent rise of the fluorescent signal was registered with each of the three dyes; the former dye provided the smallest and the latter the largest response. 3-HPG, a ligand for metabotropic receptors decreases ROS fluorescence and suppressed the NMDA-induced effect. NMDA and kainic acid presented simultaneously cumulatively increased ROS levels. Ouabain, specific inhibitors of Na/K-pump induced a considerable increase in ROS fluorescence, which was decreased by 2.5–5 m m KCl, 50 mkM Vanadate or 10 mkM D-AP5, an inhibitor of NMDA-activated ionic channels. The K0.5 for activation of ROS generation by Ouabain was more than 250 mkM, which is much higher than that for inhibition of Na/K-ATPase or its rubidium pumping activity. The data show that the Na/K-pump protein regulates ROS production by NMDA-receptors and that the E1(Na) conformation of the Na/K-pump being less sensitive to ouabain may be responsible for the effects. The data illustrate functional interaction between ionotropic and metabotropic receptors and Na/K-ATPase.
Acknowledgements:   Supported by DAAD, Grant 325-sm, Germany.     

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 glycyrrhizin and glycyrrhetinic acid on (Na+ + K+)-ATPase of renal basolateral membranes in vitro

Studies were made on the direct effects of glycyrrhizin and its aglycone, glycyrrhetinic acid on the activities of (Na+ + K+)-ATPase and (Ca2+ + Mg2+)-ATPase, a membrane bound Na+ and Ca2+-extrusion pump enzyme of the basolateral membranes (BLM) of canine kidney. Glycyrrhetinic acid inhibited the activity of the Na+-pump enzyme dose-dependently (IC50 = 1.5 x 10(-4) M), but had no effect on that of the Ca2+-pump enzyme of kidney BLM and homogenates. Glycyrrhizin also inhibited the Na+-pump enzyme activity but had less effect (IC50 = 2 x 10(-3) M). The effects of these compounds were due to competitive inhibition with ATP binding to the enzyme (Ki = 12 microM) and so were different from that of ouabain, which inhibits the Na+-pump by binding to its extracellular K+-binding site. The direct effect of glycyrrhetinic acid on the membrane may be important role in the multiple actions of licorice.     

Autoradiographic localisation of [3H]ouabain bound to cultured epithelial cell monolayers of MDCK cells

[3H]Ouabain binding to intact MDCK (cultured monolayers of dog kidney) cells of 60 serial passages is dependent upon ouabain concentration, time and medium K+. By utilising high K+ incubations to estimate non-specific [3H]ouabain-binding, the concentration of ouabain giving half maximal specific binding was estimated to be 1.0 . 10(-7) M and the total maximum binding to be 2.33 . 10(5) sites/cell. Ouabain inhibition of (Na+, K+)-pump function was monitored by the cellular uptake of 86Rb over 5 min. The larger fraction of 86Rb uptake was ouabain sensitive and the ouabain concentration giving half-maximal inhibition was 2 . 10(-7) M. The cellular distribution of the (Na+ + K+)-ATPase was investigated using [3H]ouabain autoradiography of intact freeze-dried epithelial monolayers of MDCK cells grown upon millipore filter supports. Binding of [3H]ouabain is localised over the lateral cellular membranes. Autoradiographic silver grain density is close to background levels over both the apical and basal (attachment) membranes.     

The pharmacological characteristics of two types of cholinoreceptors in the membrane of dialyzed neurons

1. The ramped voltage clamp technique was developed as a rapid means of studying the effects of certain nicotinic and muscarinic agents on ionic involvement and conductance changes during acetylcholine (ACh) responses of Helix pomatia neurons. 2. Atropine was found to be a potent cholinolytic on A-type neurons, ACh responses of which are blocked by ouabain and mediated by Na+ and Cl- permeabilities, while d-tubocurarine blocked B-type ACh responses which are insensitive to ouabain and mediated by Na+ and K+ permeabilities. 3. Nicotinic agent butyrylcholine was found to be a potent cholinomimetric on B-type cells. 4. The results suggest that ACh receptors on A-type cells are more "muscarinic" while those on B-type cells are more "nicotinic". 5. It was also suggested that both muscarinic and nicotinic ACh receptors may coexist in the Helix neuronal membrane and the possibility of ACh interacting with one of them is determined by the level of phosphorylation of the membrane proteins.     

alpha-Adrenoceptor stimulation-mediated negative inotropism and enhanced Na(+)/Ca(2+) exchange in mouse ventricle

Mechanisms underlying the negative inotropic response to alpha-adrenoceptor stimulation in adult mouse ventricular myocardium were studied. In isolated ventricular tissue, phenylephrine (PE), in the presence of propranolol, decreased contractile force by approximately 40% of basal value. The negative inotropic response was similarly observed under low extracellular Ca(2+) concentration ([Ca(2+)](o)) conditions but was significantly smaller under high-[Ca(2+)](o) conditions and was not observed under low-[Na(+)](o) conditions. The negative inotropic response was not affected by nicardipine, ryanodine, ouabain, or dimethylamiloride (DMA), inhibitors of L-type Ca(2+) channel, Ca(2+) release channel, Na(+)-K(+) pump, or Na(+)/H(+) exchanger, respectively. KB-R7943, an inhibitor of Na(+)/Ca(2+) exchanger, suppressed the negative inotropic response mediated by PE. PE reduced the magnitude of postrest contractions. PE caused a decrease in duration of the late plateau phase of action potential and a slight increase in resting membrane potential; time courses of these effects were similar to that of the negative inotropic effect. In whole cell voltage-clamped myocytes, PE increased the L-type Ca(2+) and Na(+)/Ca(2+) exchanger currents but had no effect on the inwardly rectifying K(+), transient outward K(+), or Na(+)-K(+)-pump currents. These results suggest that the sustained negative inotropic response to alpha-adrenoceptor stimulation of adult mouse ventricular myocardium is mediated by enhancement of Ca(2+) efflux through the Na(+)/Ca(2+) exchanger.     

Differential involvement of vacuolar H(+)-ATPase in the refilling of thapsigargin- and agonist-mobilized Ca(2+) stores

Our objective was to evaluate the role of vacuolar H(+)-ATPase and proton gradients in the refilling of Ca(2+) stores in fura-2-loaded pancreatic acinar cells. Once depleted with a high level of ACh, the Ca(2+) stores were replenished with a Ca(2+)-containing solution. The degree of refilling was estimated with a second release in response to either ACh (ACh-releasable store) or thapsigargin (thapsigargin-releasable store), a specific inhibitor of the endoplasmic reticulum Ca(2+) pumps. Both the protonophore nigericin and folimycin, a specific inhibitor of the vacuolar H(+)-ATPase, reduced reuptake into the ACh-mobilized stores but not into the thapsigargin-releasable pools. These treatments effectively dissipated the subcellular pH gradients (revealed by confocal observation of the distribution of a marker for acidic compartments), and did not impair the [Ca(2+)](i) response to ACh in control cells. Our results indicate that thapsigargin and ACh release heterogeneous Ca(2+) stores which are differently operated by vacuolar proton ATPase.     

Sarcolemmal cation channels and exchangers modify the increase in intracellular calcium in cardiomyocytes on inhibiting Na+-K+-ATPase

Although inhibition of the sarcolemmal (SL) Na(+)-K(+)-ATPase is known to cause an increase in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) by stimulating the SL Na(+)/Ca(2+) exchanger (NCX), the involvement of other SL sites in inducing this increase in [Ca(2+)](i) is not fully understood. Isolated rat cardiomyocytes were treated with or without different agents that modify Ca(2+) movements by affecting various SL sites and were then exposed to ouabain. Ouabain was observed to increase the basal levels of both [Ca(2+)](i) and intracellular Na(+) concentration ([Na(+)](i)) as well as to augment the KCl-induced increases in both [Ca(2+)](i) and [Na(+)](i) in a concentration-dependent manner. The ouabain-induced changes in [Na(+)](i) and [Ca(2+)](i) were attenuated by treatment with inhibitors of SL Na(+)/H(+) exchanger and SL Na(+) channels. Both the ouabain-induced increase in basal [Ca(2+)](i) and augmentation of the KCl response were markedly decreased when cardiomyocytes were exposed to 0-10 mM Na(+). Inhibitors of SL NCX depressed but decreasing extracellular Na(+) from 105-35 mM augmented the ouabain-induced increase in basal [Ca(2+)](i) and the KCl response. Not only was the increase in [Ca(2+)](i) by ouabain dependent on the extracellular Ca(2+) concentration, but it was also attenuated by inhibitors of SL L-type Ca(2+) channels and store-operated Ca(2+) channels (SOC). Unlike the SL L-type Ca(2+)-channel blocker, the blockers of SL Na(+) channel and SL SOC, when used in combination with SL NCX inhibitor, showed additive effects in reducing the ouabain-induced increase in basal [Ca(2+)](i). These results support the view that in addition to SL NCX, SL L-type Ca(2+) channels and SL SOC may be involved in raising [Ca(2+)](i) on inhibition of the SL Na(+)-K(+)-ATPase by ouabain. Furthermore, both SL Na(+)/H(+) exchanger and Na(+) channels play a critical role in the ouabain-induced Ca(2+) increase in cardiomyocytes.