Membrane Disordering by Anesthetic Drugs: Relationship to Synaptosomal Sodium and Calcium Fluxes

The effects of membrane perturbants (ethanol, pentobarbital, chloroform, diethylether, phenytoin, cis-vaccenic acid methylester, and cis-vaccenoyl alcohol) on the lipid order of mouse brain synaptic plasma membranes (SPM) were tested by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe of the membrane core and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) as a probe of the membrane surface. The compounds decreased the fluorescence polarization of both probes, indicating that they disordered the membrane lipids. The decrease in polarization was, however, greater for DPH than for TMA-DPH, suggesting a greater effect on the membrane core than on the membrane surface. The voltage-dependent uptake of 24Na and 45Ca was studied in isolated mouse brain synaptosomes as a measure of membrane function. All of the compounds inhibited sodium influx, and their potencies for decreasing sodium uptake and fluorescence polarization of DPH were linearly correlated (r = 0.91). The relationship between changes in sodium influx and TMA-DPH polarization was less consistent (r = 0.66). Synaptosomal calcium uptake was inhibited by most, but not all, of the perturbants, but this inhibition was poorly correlated with changes in fluorescence polarization of DPH (r = 0.36) or TMA-DPH (r = 0.26). These results indicate that the function of synaptic sodium channels is correlated with lipid order in the hydrophobic core of the membrane and that the inhibitory effects of intoxicant-anesthetic drugs on neuronal sodium fluxes may be the result of their capacity to disorder these lipids. In contrast, the effects of drugs on voltage-dependent calcium channels were not clearly related to the capacity of these agents to disorder membrane lipids.     

Reconstitution of neurotoxin-stimulated sodium transport by the voltage-sensitive sodium channel purified from rat brain

Incorporation of the saxitoxin receptor of the sodium channel solubilized with Triton X-100 and purified 250-fold from rat brain into phosphatidylcholine vesicles is described. Fifty to 80% of the saxitoxin receptor sites are recovered in the reconstituted vesicles (KD = 3 nM). Unlike the detergent-solubilized saxitoxin receptor, the reconstituted saxitoxin binding activity is stable to incubation at 36 degrees C. Approximately 75% of the reconstituted saxitoxin receptor sites are externally oriented and 25% are inside-out. The initial rate of 22Na+ uptake into reconstituted vesicles is increased up to 3- to 4-fold by veratridine with a K0.5 of 11 microM. Seventy per cent of this increase is blocked by external tetrodotoxin (TTX) with a Ki of 10 nM. All of the veratridine-stimulated 22Na+ uptake is blocked when TTX is present on both sides of the vesicle membrane, or when tetracaine is added to the external medium. The apparent binding constants for veratridine, saxitoxin, and TTX are essentially identical to those in intact rat brain synaptosomes. The results demonstrate reconstitution of sodium transport, as well as neurotoxin binding and action, from substantially purified sodium channel preparations.     

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.     

Reductions in calcium uptake induced in rat brain synaptosomes by ionizing radiation

Gamma irradiation (60Co) reduced KCl-stimulated voltage-dependent 45Ca2+ uptake in whole-brain, cortical, and striatal synaptosomes. The time course (3, 10, 30, and 60 s) of calcium uptake by irradiated (3 Gy) and nonirradiated synaptosomes, as well as the effect of KCl (15-65 mM), was measured in whole-brain synaptosomes. The fastest and highest rate of depolarization-dependent calcium uptake occurred at 3 s with 65 mM KCl. Irradiation reduced calcium uptake at all incubation times and KCl concentrations. Bay K 8644 enhancement of KCl-stimulated calcium influx was also reduced by radiation exposure. Nimodipine binding to dihydropyridine (DHP) L-type calcium channel receptors was not altered following radiation exposure. These results demonstrate an inhibitory effect of ionizing radiation on the voltage-sensitive calcium channels in rat brain synaptosomes that are not mediated by DHP receptors.     

Ethanol inhibits veratridine-stimulated sodium uptake in synaptosomes

The effects of ethanol and pentobarbital on voltage-sensitive sodium channels in whole brain (rat) synaptosomes were studied using isotopic flux measurements. Incubation of synaptosomes with ethanol or pentobarbital $\text{in}$$\text{vitro}$ inhibited veratridine-stimulated ²²Na⁺ uptake. The effect of ethanol is dose-dependent, occurs at sublethal, pharmacologically relevant concentrations and is fully reversible. These results suggest that ethanol and pentobarbital directly interfere with sodium channel function in nervous tissue. Alterations in sodium channel function may be a possible mechanism for the central nervous system (CNS) depressant action of ethanol and related compounds.     

Calcium permeability of synaptosomes induced by alpha-latrotoxin

The paper deals with characteristics of ionic alpha-latrotoxin-induced permeability of rat brain synaptosomes. It has been shown that the addition of alpha-latrotoxin to synaptosomes in the Ca2+-containing media resulted in an extensive and rapid uptake of 45Ca2+ in synaptosomes. alpha -Latrotoxin was not able to enhance the 22Na+ and 86Rb+ uptake or efflux in the Ca2+-containing and Ca2+- and Mg2+-free media. The dye di-O-C3 was used to monitor the membrane potential changes as a consequence of alpha-latrotoxin treatment of synaptosomes. It has been found that alpha-latrotoxin increased synaptosomal fluorescence in the Ca2+-containing media, but failed to induce any increase of fluorescence in Ca2+- and Mg2+-free media. It has been also shown that the calcium uptake induced by alpha-latrotoxin depends on free calcium concentration in synaptosomes. Toxin-induced calcium flows are shown to be of the vector character.     

22Na+ Uptake and Catecholamine Secretion by Primary Cultures of Adrenal Medulla Cells

The uptake of 22Na+ and secretion of catecholamines by primary cultures of adrenal medulla cells under the influence of a variety of agonists and antagonists were determined. Veratridine, batrachotoxin, scorpion venom, and nicotine caused a parallel increase in 22Na+ uptake and Ca2+-dependent catecholamine secretion. Ba2+, depolarizing concentrations of K+, and the Ca2+ ionophore Ionomycin stimulated secretion of catecholamines but did not increase the uptake of 22Na+. Tetrodotoxin inhibited both 22Na+ uptake and catecholamine secretion evoked by veratridine, batrachotoxin, and scorpion venom, but had no effect on 22Na+ uptake and catecholamine secretion caused by nicotine. On the other hand, histrionicotoxin, which blocks the acetylcholine receptor-linked ion conductance channel, blocked nicotine-stimulated 22Na+ uptake and catecholamine secretion, but only partially inhibited veratridine-stimulated catecholamine secretion and had no effect on veratridine-stimulated 22Na+ uptake. The combination of veratridine plus tetrodotoxin, which has been shown to prevent nicotine-stimulated secretion of catecholamines by adrenal medulla cells, also prevented nicotine-stimulated 22Na+ uptake by the primary cultures. These studies demonstrate the presence of tetrodotoxin-sensitive Na+ channels in adrenal medulla cells which are functionally linked to Ca2+-dependent catecholamine secretion. However, These channels are not utilized for Na+ entry upon activation of nicotinic receptors; in this case Na+ entry occurs through the receptor-associated ion conductance channel.     

Reductions of Γ-Aminobutyric Acid and Glutamate Uptake and (Na++ K+)-ATPase Activity in Brain Slices and Synaptosomes by Arachidonic Acid

Arachidonic acid, a major polyunsaturated fatty acid of membrane phospholipids in the CNS, reduced the high-affinity uptake of glutamate and gamma-aminobutyric acid (GABA) in both rat brain cortical slices and synaptosomes. alpha-Aminoisobutyric acid uptake was not affected. Intrasynaptosomal sodium was increased concomitant with decreased (Na+ + K+)-ATPase activity in synaptosomal membranes. The reduction of GABA uptake in synaptosomes could be partially reversed by alpha-tocopherol. The inhibition of membrane-bound (Na+ + K+)-ATPase by arachidonic acid was not due to a simple detergent-like action on membranes, since sodium dodecyl sulfate stimulated the sodium pump activity in synaptosomes. These data indicate that arachidonic acid selectively modifies membrane stability and integrity associated with reductions of GABA and glutamate uptake and of (Na+ + K+)-ATPase activity.     

Sodium channels in presynaptic nerve terminals. Regulation by neurotoxins

Regulation of Na+ channels by neurotoxins has been studied in pinched- off nerve endings (synaptosomes) from rat brain. Activation of Na+ channels by the steroid batrachotoxin and by the alkaloid veratridine resulted in an increase in the rate of influx of 22Na into the synaptosomes. In the presence of 145 mM Na+, these agents also depolarized the synaptosomes, as indicated by increased fluorescence in the presence of a voltage-sensitive oxacarbocyanine dye [diO-C5(3)]. Polypeptide neurotoxins from the scorpion Leiurus quinquestriatus and from the sea anemone Anthopleura xanthogrammica potentiated the stimulatory effects of batrachotoxin and veratridine on the influx of 22Na into synaptosomes. Saxitoxin and tetrodotoxin blocked the stimulatory effects of batrachotoxin and veratridine, both in the presence and absence of the polypeptide toxins, but did not affect control 22Na influx or resting membrane potential. A three-state model for Na+ channel operation can account for the effects of these neurotoxins on Na+ channels as determined both by Na+ flux measurements in vitro and by electrophysiological experiments in intact nerve and muscle.     

Alkaloid neurotoxins-dependent sodium transport in insect synaptic nerve-ending particles

1. Sodium uptake associated with the activation of voltage-sensitive sodium channels by alkaloid activators, batrachotoxin, veratridine, and aconitine in presynaptic nerve terminals isolated from the central nervous system of cockroach (Periplaneta americana) was investigated. 2. Batrachotoxin (K0.5, 0.2 microM) was full agonist as for most effective activator of Na+ uptake; veratridine (K0.5, 2.5 microM) and aconitine (K0.5, 7.6 microM) produced a maximal stimulation of 22Na+ uptake that were 71% and 43% respectively of that produced by batrachotoxin. 3. Veratridine-dependent 22Na+ uptake was completely inhibited by tetrodotoxin (I0.5, 11 nM), a specific inhibitor of the nerve membrane sodium channels. 4. The present study describes appropriate conditions for measuring neurotoxins--stimulated sodium transport in insect central nervous system synaptosomes. The data show that voltage-sensitive sodium channels as defined by specific activation by the alkaloid neurotoxins are qualitatively distinct in insect synaptosomes than those previously described for vertebrate brain synaptosomes, cultured neuronal cell, nerve membrane vesicles and neuroblastoma cells.     

Characterization of the electrogenic sodium channel from rat brain membranes using neurotoxin-dependent 22Na uptake

The sodium channel was studied in osmotically-sensitive membrane preparations from rat brain and in innervated and chronically denervated rat soleus and extensor digitorum longus muscles. These experiments were undertaken in order to define a set of parameters for sodium channel function at the subcellular level to be used as a measure of retention of channel integrity upon subsequent isolation of the channel. Various neurotoxins and drugs were employed to control the permeability of the brain membranes to 22Na and the sodium-conductance properties of the muscles. Batrachotoxin (ED50 = 0.2 micrometer), veratridine (ED50 = 1 micrometer), or grayanotoxin I (ED50 = 30 micrometers) stimulated 22Na uptake in brain membranes is inhibited in an apparently uncompetitive manner by the sodium channel blocking agents tetrodotoxin and saxitoxin in a simple competitive manner by Ca2+ and in a partial or allosteric competitive manner by lidocaine and procaine. This 22Na uptake assay, which can be equated to a measure of equilibrium toxin binding, shows dependence on the concentration of the membranes and is sensitive to pH, temperature, ionic strength, and the ionic composition of the media. Parallel biophysical studies on sodium channels in rat muscle show that the properties of the sodium channel are similarly affected by these agents.     

N 6-Cyclohexyladenosine Inhibits Veratridine-Stimulated Na Uptake by Rat Brain Synaptosomes

The effect of the stable adenosine analogue, N⁶-cyclohexyladenosine, on ²²Na uptake by rat brain synaptosomes stimulated by veratridine was investigated. In the presence of N⁶-cyclohexyladenosine, both the initial rate and the maximum sodium uptake were decreased. The inhibitory effect of N⁶-cyclohexyladenosine on sodium uptake by synaptosomes after 5 s of incubation with ²²Na was concentration-dependent, antagonized by 1,3-dipropyl-8-p-sulfophenylxanthine, and attenuated by increasing the concentration of veratridine. The possibility that the adenosine analogue, by activating a xanthine-sensitive adenosine receptor, can operate inhibition of the voltage-dependent sodium channels is discussed.     

Purification and pharmacological properties of eight sea anemone toxins from Anemonia sulcata, Anthopleura xanthogrammica, Stoichactis giganteus, and Actinodendron plumosum

Eight different polypeptide toxins from sea anemones of four different origins (Anemonia sulcata, Anthopleura xanthogrammica, Stoichactis giganteus, and Actinodendron plumosum) have been studied. Three of these toxins are new; the purification procedure for the five other ones has been improved. Sea anemone toxins were assayed (i) for their toxicity to crabs and mice, (ii) for their affinity for the specific sea anemone toxin receptor situated on the Na+ channels of rat brain synaptosomes, and (iii) for their capacity to increase, in synergy with veratridine, the rate of 22Na+ entry into neuroblastoma cells via the Na+ channel. Some of the toxins are more active on crustaceans, whereas others are more toxic to mammals. A very good correlation exists between the toxic activity to mice, the affinity of the toxin for the Na+ channel in rat brain synaptosomes, and the stimulating effect on 22 Na+ uptake by neuroblastoma cells. The observation has also been made that the most cationic toxins are also the most active on mammals and the least active on crustaceans. Toxicities (LD50) to mice of the most active sea anemone toxins and of the most active scorpion toxins are similar, and sea anemone toxins at high enough concentrations prevent binding of scorpion toxins to their receptor. However, scorpion toxins have affinities for the Na+ channel which are approximately 60 times higher than those found for the most active sea anemone toxins. Three sea anemone toxins appear to be more interesting than toxin II from A. sulcata (the "classical" sea anemone toxin) for studies of the Na+ channel structure and mechanism when the source of the channel is of a mammalian origin. Two of these three toxins can be radiolabeled with iodine while retaining their toxic activity; they appear to be useful tools for future biochemical studies of the Na+ channel.     

Effects of cadmium and mercury on Na(+)-K+, ATPase and uptake of 3H-dopamine in rat brain synaptosomes.

Effects in vivo of cadmium (Cd), mercury (Hg) and methylmercury (CH3Hg) on Na(+)-K+ ATPase and uptake of 3H-dopamine (DA) in rat brain synaptosomes were studied. These heavy metals significantly inhibited the Na(+)-K+ ATPase activity in a dose-dependent manner. Similarly, inhibition of DA uptake by synaptosomes was also observed in rats treated with these metals. Intraperitoneal route of metal administration was found to be more effective than per os treatment. Mercuric compounds compared to Cd elicited a higher inhibition of Na(+)-K+ ATPase and DA uptake in rat brain synaptosomes.     

Effect of gamma radiation on sodium channels in different conformations in neuroblastoma cells

We studied the dose-response relationship between gamma radiation and batrachotoxin-stimulated sodium influx in neuroblastoma cells in tissue culture. We also tested the hypothesis that changes in sodium channel conformation may alter the radiosensitivity of the channel. We found that gamma radiation inhibited toxin-stimulated 22Na uptake at doses beyond a threshold of 200-300 Gy. No effects were seen following doses below 100 Gy. This inhibition of sodium permeability was seen when the cells were irradiated with sodium channels in the closed or inactivated, nonconducting states. However, when the channels were in the toxin-opened, conducting state, gamma radiation had no effect at doses up to 2000 Gy. Our results support earlier electrophysiological studies that showed that high doses of ionizing radiation are required to produce a measurable decrease in sodium permeability. In addition, our data suggest that by changing the sodium channel conformation, batrachotoxin appears to alter radiosensitive chemical bonds in the gating or ion-conducting portion of the channel.     

Effect of aluminium on lipid peroxidation of human high density lipoproteins

We investigated the effect of aluminium (Al 3+ ) on lipid peroxidation and physico-chemical properties of high density lipoproteins (HDL) isolated from human plasma. Our results demonstrated that Al 3+ enhances lipid peroxidation of human HDL as shown by the significant increase in lipid hydroperoxides in Al-treated HDL with respect to control HDL. The oxidative effect was higher at acid pH (pH 5.5) with respect to pH 7.4. Moreover, a stimulating effect of Al 3+ on iron-induced lipid peroxidation of HDL was demonstrated. The study of the effect of Al 3+ on the physico-chemical properties of HDL, using the fluorescence polarization (Pf) of the probes TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene iodide) and DPH (1,6-diphenyl-1,3,5-hexatriene), showed a significant decrease of Pf in Al-treated HDL with respect to control. These results suggest that Al 3+ induces a decrease of molecular order at the lipoprotein surface. Moreover, the study of tryptophan (Trp) fluorescence demonstrated that aluminium induces structural modifications of HDL apoproteins and on HDL physico-chemical properties. The effect of Al 3+ on lipid peroxidation of HDL was observed at aluminium concentrations similar to those observed in the brain of patients affected by neurological diseases. Aluminium-induced oxidative damage of HDL could be involved in the development of neurological diseases.     

Effect of Aluminium on Lipid Peroxidation of Human High Density Lipoproteins

We investigated the effect of aluminium (Al 3+ ) on lipid peroxidation and physico-chemical properties of high density lipoproteins (HDL) isolated from human plasma. Our results demonstrated that Al 3+ enhances lipid peroxidation of human HDL as shown by the significant increase in lipid hydroperoxides in Al-treated HDL with respect to control HDL. The oxidative effect was higher at acid pH (pH 5.5) with respect to pH 7.4. Moreover, a stimulating effect of Al 3+ on iron-induced lipid peroxidation of HDL was demonstrated. The study of the effect of Al 3+ on the physico-chemical properties of HDL, using the fluorescence polarization (Pf) of the probes TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene iodide) and DPH (1,6-diphenyl-1,3,5-hexatriene), showed a significant decrease of Pf in Al-treated HDL with respect to control. These results suggest that Al 3+ induces a decrease of molecular order at the lipoprotein surface. Moreover, the study of tryptophan (Trp) fluorescence demonstrated that aluminium induces structural modifications of HDL apoproteins and on HDL physico-chemical properties. The effect of Al 3+ on lipid peroxidation of HDL was observed at aluminium concentrations similar to those observed in the brain of patients affected by neurological diseases. Aluminium-induced oxidative damage of HDL could be involved in the development of neurological diseases.     

Evoked effects of cholesterol binding on integral proteins and lipid fluidity of dog brain synaptosomal plasma membranes

Binding of cholesterol into dog brain synaptosomal plasma membranes (SPM) within the limits of concentration used (0.5-5 microM) follows an exponential curve described by the general formula y = a.ebx. This curve, which represents the total binding (specific and nonspecific), acquires sigmoid character in the presence of 100 microM cholesterol glucoside, with a Hill coefficient of h = 2.98 +/- 0.18. The specific activity of the Na+/K+-transporting ATPase and Ca2+-transporting ATPase rose after a 2-h preincubation of SPM with cholesterol (up to 5 microM) or its glucoside (up to 50 microM) to at least 50% above their original values. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) increased with cholesterol glucoside (50 microM) incorporation. Cholesterol (5 microM) had no effect on the DPH fluorescence polarization. Arrhenius plots of Na+/K+-transporting ATPase activity exhibited a break point at 23.2 +/- 1.1 degrees C in control SPM, which was elevated to 29.5 +/- 1.4 degrees C in SPM treated with cholesterol glucoside (50 microM) and abolished in SPM treated with cholesterol (5 microM). The allosteric properties of SPM-bound Na+/K+-transporting ATPase inhibited by F- and Ca2+-transporting ATPase inhibited by Na+ (as reflected by changes in the Hill coefficient) were modulated by cholesterol. It could be stated that cholesterol glucoside (50 microM) produced an increased packing of the bulk lipids, while cholesterol (5 microM) increased the fluidity of the lipid microenvironment of both Na+/K+-transporting ATPase and Ca2+-transporting ATPase.     

Calcium modulates fatty acid dynamics in rat liver plasma membranes

Modulation of free fatty acid binding in isolated rat liver plasma membranes was evaluated using the fluorescent fatty acids trans-parinaric and cis-parinaric acid as analogues for saturated and unsaturated fatty acids, respectively. Binding of trans-parinarate but not cis-parinarate was inhibited by physiological levels of Ca2+. The effect was reversed by addition of excess EGTA. Calcium decreased the aqueous to lipid partition coefficient, Kp, of trans-parinaric acid for liver plasma membranes while increasing the Kp for cis-parinaric acid. In addition, Ca2+ also altered the fluorescence lifetime, the quantum yield, and the relative partitioning of trans-parinaric and cis-parinaric acid into fluid and solid phases. Calcium and EGTA did not affect the binding of 1,6-diphenyl-1,3,5-hexatriene. The effect of Ca2+ on the liver plasma membrane structure was to increase the rigidity of the membrane, primarily the solid domain. The fluorescence polarization of trans-parinarate, cis-parinarate, and 1,6-diphenyl-1,3,5-hexatriene at 24 degrees C in liver plasma membranes in the absence of Ca2+ was 0.295 +/- 0.008, 0.253 +/- 0.007, and 0.284 +/- 0.005, respectively. Calcium (2.4 mM) increased the polarization of these probe molecules in liver plasma membranes by 8-10%. EGTA (3.4 mM) reversed or abolished the increase in polarization. Thus, the fluorescent fatty acids trans-parinarate and cis-parinarate may be used to monitor fatty acid binding by isolated membranes, to evaluate factors such as Ca2+ which modulate fatty acid binding, and to investigate the microenvironment in which the fatty acids residue. The data suggest that Ca2+ may be an important regulator of fatty acid uptake by the liver plasma membrane, and thereby interact with intermediary metabolism of lipids at a step not involving lipolytic or synthetic enzymes.     

Effect of ionizing radiation on the kinetic parameters of rat cerebral cortex Na, K-ATPase

A study was made of the effect of ionizing radiation of 10.3 and 180.6 mC/kg on kinetic parameters of the processes of activation of Na,K-ATPase of rat brain cortex by Mg-ATP-substrate and Na+ and K+ ions. The obtained results prompt an assumption that a conformational rearrangement occurs under the effect of ionizing radiation which is not identical after relatively small and lethal radiation doses.