4 Hz EMF treated physiological solution depresses Ach-induced neuromembrane current.

The effect of 4 Hz EMF treated physiological solution (PS) on acetylcholine (Ach) sensitivity of the snail neuron was studied. The 4 Hz EMF treated normal PS at room temperature (23 degrees C) has a depressing effect on Ach induced current, while in cold medium (12 degrees C) this effect disappeared. EMF treated, ouabain containing, K-free PS elevates the Ach-induced current at room temperature. It is suggested that the metabotropic effect of EMF treated PS is due to the activation of cGMP-dependent Na:Ca exchange, leading to the decrease of the number of functional active receptors in the membrane, through Na-K pump-induced cell shrinkage, and to increase the receptors affinity to Ach, as the result of decrease of intracellular Ca concentration.

Ouabain-sensitive and insensitive acetylcholine receptors on the membrane of the same neuron in Helix pomatia.

Using internally dialyzed neurons of Helix pomatia as a model, the effect of structural analogs of acetylcholine (ACh) were investigated for their cholinomimetic properties on A- and B-types of ACh-responses. Specifically we analyzed choline esters of N-para- and ortho-alkoxybenzoyl-beta-alanines, (CH(3)O-, C(2)H(5)O-, C(3)H(7)O-, iso-C(3)H(7)O-, C(4)H(9)O-, iso-C(4)H(9)O-, C(5)H(11)O-, iso-C(5)H(11)O-), (in all, 16 combinations). The compounds evoked differing sensitivities of response to factors de-activating the Na-K-pump (ouabain and K-free solution). Most compounds resembled ACh: ionic currents caused by these compounds were inhibited by Na-K pump blockers in the case of A-type responses - B-type responses were insensitive to these factors. Ionic currents induced by choline esters of p-, o-propoxy- and iso-propoxybenzoyl-beta-alanines were insensitive to ouabain and K-free solution in the case of A- and B-type responses. Ionic currents induced by the choline ester of p-butoxybenzoyl-beta-alanine were inhibited by ouabain and K-free solution on both types of neuron. The results allow us to classify choline esters of p-, o-propoxy- and iso-propoxybenzoyl-beta-alanines as N-cholinomimetics, while the choline ester of p-butoxybenzoyl-beta-alanine can be considered as M-cholinomimetic. We conclude that both M- and N-type ACh receptors exist on the membrane of the same neurons.

The electrogenic sodium pump activity in Aplysia neurons is not potential dependent.

We have investigated the potential dependence of the electrogenic sodium pump in Aplysia neurons by recording the potential and current induced by sudden change of the artificial sea water from one containing K+ at various concentrations to K+ -free sea water in the presence or absence of ouabain. Both K+ free sea water and ouabain block sodium transport and result in a significant depolarization due to removal of a maintained outward current that is a result of transport of more Na+ out of the cell than K+ into the cell during pump operation. In the presence of ouabain there is, however, an inward current induced by changing external K+ concentration from zero to some value between 1 and 20 mM, and this current is greater with a greater K+ concentration gradient. The current induced by change from zero to 1 mM K+ does not show any potential dependence, although those currents induced by higher K+ concentrations are potential dependent. We conclude that the activity of the electrogenic sodium pump is not potential dependent, but that the potential independence is obscured if higher concentrations of K+ are used to activate the electrogenic sodium pump.

Changes of hydration of rats' tissues after in vivo exposure to 0.2 Tesla steady magnetic field.

Hydration and [3H]ouabain uptake by different tissues of adult male rats were measured immediately after exposure to homogenous 0.2 T steady magnetic field. A time-dependent decrease of hydration and adaptation, followed by disadaptation, was detected in brain and liver tissues in most of the rats after 3.5-5 h of exposure. The number of functional active ouabain binding receptors, which correlates with cell volume, was also decreased in brain, liver, and spleen and increased in kidney tissue after half an hour of exposure. It is suggested that cell hydration is a second messenger through which the SMF exerts its influence.

The static magnetic field effects on ouabain H3 binding by cancer tissue.

Radioactive labeled ouabain was used for estimating the static magnetic field (SMF) induced cell volume changes. Ouabain is a specific inhibitor of Na+/K+ ATPase, and can be used for estimating its quantity--thus giving information about the cell volume changes. Ouabain binding by cancer and normal glandular tissues of breast cancer patients and normal glandular tissues of healthy women was measured after exposure of tissues to SMF 0.2T. SMF exposure led to a decrease of ouabain binding in both normal and cancer tissues when ouabain concentration in the external medium was 10(-9) M, while in the case of higher concentrations of ouabain (10(-7) M, 10(-6) M) an increase of ouabain binding was seen. The normal glandular tissues of healthy women were sensitive to SMF only at the highest concentrations of ouabain used in our experiments. The SMF-induced decrease of binding at low ouabain concentrations was considered as an evidence for the dehydration effect of SMF. It is suggested that the SMF could influence the cancer cell metabolism through cell hydration changes.

Cellular and molecular mechanisms of nitric oxide-induced heart muscle relaxation.

1. The nitric oxide (NO) donor S-nitro-N-acetyl-penicillamine (SNAP) inhibits Helix aspersa heart activity and relaxes muscles. 2. K-free saline and ouabain both depress SNAP-induced relaxation in most experiments, but in a few preparations they either had no effect or potentiated SNAP-induced relaxation. 3. Na-K pump reactivation following preincubation in K-free saline leads to the pronounced transient relaxation of heart muscle, the magnitude of which depends on the duration of preincubation. 4. 0.1 mM SNAP inhibited the ouabain sensitive part of 86Rb uptake, which reflects Na-K pump activity. This inhibition is potentiated by phospholipase C. 5. SNAP increased cGMP levels in the heart. 6. These results indicate that SNAP-induced relaxation depends on Na and Ca gradients across the membrane, which suggests that Na:Ca exchange is involved in the mechanisms of SNAP-induced relaxation. It is postulated that SNAP elicits its inhibitory effect on the heart through a cGMP-dependent Na:Ca exchange.

Metabotropic GABA receptors regulate acetylcholine responses on snail neurons.

1. The A type of acetylcholine response of Helix neurons is downmodulated by low concentrations of GABA that do not elicit any measurable change in membrane potential or conductance. 2. We find that these physiological actions are associated with an increase in both intracellular cyclic AMP levels and 45Ca2+ influx. 3. The modulation of the acetylcholine response by GABA is blocked when the neurons are injected with EGTA to prevent a rise in intracellular Ca2+ concentration or when tolbutamide, an inhibitor of protein kinase A, is applied. 4. These results are consistent with the effects of GABA being mediated by a metabotropic GABA receptor that is activated at very low GABA concentrations and mediates modulation of the acetylcholine response via regulation of intracellular Ca2+ and cyclic AMP levels.

Low concentrations of ouabain stimulate Na/Ca exchange in neurons.

1. The effects of low concentrations of ouabain on 22Na efflux, 86Rb influx, 45Ca uptake and cyclic AMP levels were studied in snail ganglia. Ouabain, at concentrations below that which inhibits the Na-K pump as monitored by 86Rb influx, activated "reverse mode" Na/Ca exchange, as indicated by an increased 22Na efflux and 45Ca influx. 2. With electrophysiologic recordings ouabain, in the presence of K(+)-free saline to block Na/K transport, caused a membrane hyperpolarization. These concentrations of ouabain also caused elevation of intracellular cyclic AMP levels. 3. We suggest that the ouabain-induced stimulation of Na efflux is due to a stimulation of reverse Na/Ca exchange. since Na/Ca exchange is electrogenic, these observations are most consistent with ouabain stimulation of Na/Ca exchange in a reversed direction (intracellular Na for extracellular Ca). 4. The effect on Na/Ca exchange may be secondary to a rise in intracellular cyclic AMP.

Magnetic fields alter electrical properties of solutions and their physiological effects.

Calcium chloride and snail physiological salt solutions were exposed to static magnetic fields (2.3-350 mT), and the physical properties of the solutions as well as their biologic effects were studied. Our preliminary observations show that these fields alter physicochemical properties of CaCl2 solutions and the functional effects of physiological solutions. Experiments on CaCl2 solutions demonstrated field-dependent changes of electrical conductivity, with the magnitude and the direction of conductivity change being a function of both concentration and field intensity. The changes in conductivity were maintained for periods in excess of 1 h after exposure. Conductivity changes were not found after exposure of physiological solutions to static magnetic fields, but changes of biological consequence did occur. Other experiments showed that there were several changes in cellular function observed in ganglia and isolated neurons of Helix pomatia when the perfusing medium was changed from the normal physiologic solution to the same solution after exposure to magnetic fields. These changes include membrane depolarization and increased action potential discharge, reduced uptake of Ca into cells, altered content of cyclic nucleotides in ganglia, and increased volume of isolated cell bodies. A change in hydration of calcium ions may be one of the consequences of magnetic-field exposure, and in physiological solutions this change may have functional consequences.

The effect of osmotic gradients on the outward potassium current in dialyzed neurons of Helix pomatia.

1. The effect of outward and inward water flows through the membrane on outward potassium currents of dialyzed Helix pomatia neurons was studied. 2. An outward water flow increased the peak and sustained outward potassium currents and accelerated the kinetics of their activation. An inward water flow had quite opposite effects--it decreased the peak and sustained potassium currents and delayed the kinetics of their activation. 3. The analysis of the effect of water flow on the conductance of potassium channels showed that an outward water flow increased both the potassium conductance at a given potential (gk) and the maximum potassium conductance (gkmax). An inward water flow again had the opposite effect--it decreased the potassium conductance at given potential and the maximum potassium conductance. 4. Neither an outward nor an inward water flow significantly affected the fraction of open potassium channels at a given potential [n infinity(V)]. 5. These data suggest that in dialyzed neurons the changes of outward potassium current during water flow through the membrane are due mainly to the changes in single-channel conductance and the time constant of current activation.

Ouabain blocks some rapid concentration-induced clamp acetylcholine responses on Helix 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.

The effects of cAMP, Ca2+, and phorbol esters on ouabain-induced depression of acetylcholine responses in Helix neurons.

1. Using internal perfusion and concentration-clamp procedures applied to Helix neurons, the effects of cAMP, Ca2+, and phorbol esters on ouabain-induced depression of acetylcholine Cl-dependent responses were determined. 2. Intracellular cAMP (10(-4) M) depressed those acetylcholine responses which were blocked by ouabain but had no effect on ouabain-insensitive acetylcholine responses. In the presence of elevated intracellular cAMP, ouabain had no further depressant effect on these acetylcholine responses. Both elevated cAMP and ouabain reduced the acetylcholine response without altering the current-voltage curves. 3. An increase in intracellular Ca2+ concentration depressed the amplitude of current induced by application of acetylcholine in neurons with ouabain-sensitive responses and shifted the dose-response relationship to the right. However, elevated Ca2+ did not reduce the maximal response induced by acetylcholine, nor did it prevent the reduction of that response by ouabain. 4. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent stimulator of protein kinase C activity, caused depression of both the ouabain-sensitive and the ouabain-insensitive acetylcholine responses. The inhibitory effect of TPA was markedly enhanced after addition of ATP to the intracellular medium and was greatly reduced by cooling to 5 degrees C. The blocking effect of ouabain, however, reexamined in the presence of TPA. 5. These observations are consistent with the hypothesis that the depression of acetylcholine induced Cl--responses in Helix neurons is a result of an increase in intracellular cAMP concentration but is unrelated to activation of protein kinase C or increases in intracellular Ca2+.

Low concentrations of neuroactive peptides modulate cholinergic transmission and cyclic AMP levels in Helix aspersa.

1. FMRFamide and the Catch relaxing peptide (CARP) at 0.01-0.1 nM modulate acetylcholine (ACh) induced currents of identified neurons of Helix aspersa, but have no direct effect on membrane current and conductivity. 2. Both FMRFamide and CARP noncompetitively inhibit ACh Cl- responses while having either no effect or increasing ACh Na+/K+ response. 3. The inhibitory effect of FMRFamide and CARP on the ACh Cl- response was eliminated following pretreatment with forskolin (20 microM), an activator of adenylate cyclase. 4. Ascaris peptide (ASC) and a synthetic CARP analogue NORL-CARP, in which the amino acid methionine is replaced by either leucine or norleucine, at 1-10 microM, showed no effect on responses to ACh. 5. FMRFamide and CARP, at 10 nM, increased cAMP levels to 240% and 148% respectively above resting basal cAMP levels, while ASC and NORL-CARP had no significant effective. 6. Our results suggest that FMRFamide and CARP, in low concentrations, modulate ACh responses of Helix neurons, possibly through changes in cAMP levels. They also indicate the importance of the presence of methionine in these neuroactive peptides.

The changes in CiNa of sheep cardiac Purkinje fibres in hyperosmotic solutions.

1. The changes in intracellular sodium ion concentration (CiNa) of sheep cardiac Purkinje fibres in hyperosmotic solutions were studied using Na-sensitive liquid ion-exchanger microelectrodes. 2. CiNa was increased in hyperosmotic solutions containing different concentrations of sucrose from 0 to 300 mM. 3. The changes in resting membrane potential (RMP) in hyperosmotic solutions had no regularity. In most of the experiments there was hyperpolarization of the membrane but in a few cases a depolarization or no change of RMP were also observed. 4. The N-shape of I-V relations of the fibres became more pronounced in hyperosomotic solutions.

Effects of water flow on transmembrane ionic currents in neurons of Helix pomatia and in squid giant axons.

1. The effects of water flow through the membrane produced by an osmotic gradient on the ionic currents in Helix neurons and in squid giant axons were studied. 2. Outward water flow had a marked effect on the ionic currents. 3. Cell volume diminution in hypertonic solution was accompanied by a decrease in the number of functioning ionic channels in the neurons. 4. Decrease of the tonicity of the external 10(-8) M TTX-containing solution leads to a transient recovery of the action potentials of the squid.

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.

The effects of short-chain fatty acids on the neuronal membrane functions of Helix pomatia. III. 22Na efflux from the cells.

The effect of short-chain fatty acids on both ouabain-sensitive and ouabain-insensitive fractions of 22Na efflux from the neurons of Helix pomatia was studied. Fatty acids, having fewer than 10 carbon atoms in the hydrocarbon chain, increased the ouabain-sensitive 22Na efflux from the neurons, while fatty acids, having more than 9 carbon atoms, inhibited the 22Na efflux in comparison with that in normal physiological solution. All the fatty acids used had an inhibiting effect on the ouabain-insensitive 22Na efflux from the cells independent on the number of carbon atoms in the hydrocarbon chain. These studies indicate that these short-chain fatty acids can be effective modulators of both ouabain-sensitive and ouabain-insensitive fractions of Na efflux from the cells.

The effects of short-chain fatty acids on the neuronal membrane functions of Helix pomatia. II. Cholinoreceptive properties.

We have examined the effects of short-chain fatty acids on acetylcholine (ACh)-induced transmembrane currents using internally dialyzed neurons of Helix. Decenoic acid, which increased the fluidity of excitable membranes, caused dramatic changes in the voltage sensitivity of ACh currents consisting of an ACh-induced increase in membrane permeability for K+ and Na+ ions and a shift of the Erev of these ACh responses to more positive potentials. Valeric acid, which did not change the membrane fluidity, had no effect on this type of ACh response. Changes of the ENa and ECl had no effect on the size of the decenoic acid-induced shift of the Erev. But the influence of decenoic acid on the voltage sensitivity of ACh-induced currents almost disappeared after the change of the EK by the reduction of the internal K concentration. Decenoic acid had no effect on ACh responses in which K+ ions were not involved in the generation of ACh-induced currents. The results suggest that decenoic acid-induced changes in membrane fluidity modulate cholinoreceptive properties of the neuronal membrane by the inhibition of the K+ carrier involved in the generation of ACh responses.

The effects of short-chain fatty acids on the neuronal membrane functions of Helix pomatia. I. Electrical properties.

The effects of short-chain fatty acids on the membrane excitability, current-voltage (I-V) characteristics, and cell volume of Helix pomatia neurons were studied. 2-Decenoic acid (DA), having 10 carbon atoms in the hydrocarbon chain, suppressed the excitability of bursting neurons RPa1 (Sakharov and Salanki, 1969) for 30-60 min, while valeric acid (VA), having 5 carbon atoms, had no significant effect on excitability. DA had three different effects on the excitability of beating neurons: in some neurons DA suppressed excitability as in bursting neurons; in a second type of neuron DA had a negligible effect on excitability; and in the neuron located near RPa1 DA had a pentylentetrazol (PTZ)-like effect, i.e., it converted the discharge of the neuron from beating to bursting. DA decreased the peak value of the current, inducing a negative-resistance region in the I-V curve of the bursting neuron without any change in the level of the voltage at which the current reaches its maximal value. DA inhibited the hyperpolarization induced by activation of the Na+ pump, tested after preliminary enrichment of neurons with Na+ ions by incubation in a potassium-free solution for 20 min. DA caused a swelling of the neuron by about 10% which was independent of the Na+ pump. In all the above-mentioned cases VA had no significant effect.

Further study of the correlation between Na-pump activity and membrane chemosensitivity.

Using internally dialyzed neurons of Helix, we have examined the effects of sodium-pump activity and intracellular ATP concentration on transmembrane currents induced by acetylcholine (ACh) and gamma-aminobutyric acid (GABA). We also report on the effects of pump activity and levels of intracellular ATP on binding by Helix ganglia of 3H-alpha-bungarotoxin (3H-alpha-BT) and 3H-GABA. Both ouabain-containing and potassium-free solutions depressed the neurotransmitter-induced transmembrane current of one type of dialyzed neurons. An increase in the intracellular ATP concentration led to a depression of ACh-induced currents and to the disappearance of the blocking effect of ouabain on these currents. Intracellular ADP had a similar but smaller effect on transmitter-induced currents, and intracellular AMP was ineffective. The depressing effect of internal ATP on ACh-induced currents was absent in the presence of an inhibitor of membrane phosphorylation (dinitrophenol). The binding of tritium-labeled alpha-BT and GABA to the membranes was depressed by both ouabain-containing and K-free solutions and also by compounds (theophylline and NaF) which increase the levels of intracellular ATP. The results suggest that the Na pump modulates the affinity of ACh and GABA membrane receptors by the regulation of the phosphorylated state of membrane receptors.