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PubMed1990

Stimulation of synaptosomal gamma-aminobutyric acid synthesis by glutamate and glutamine.

gamma-Aminobutyric acid (GABA) synthesis was studied in rat brain synaptosomes by measuring the increase of GABA level in the presence of the GABA-transaminase inhibitor gabaculine. The basal rate of synaptosomal GABA synthesis in glucose-containing medium (25.9 nmol/h/mg of protein) was only 3% of the maximal activity of glutamate decarboxylase (GAD; 804 +/- 83 nmol/h/mg of protein), a result indicating that synaptosomal GAD operates at only a small fraction of its catalytic capacity. Synaptosomal GABA synthesis was stimulated more than threefold by adding 500 microM glutamine. Glutamate also stimulated GABA synthesis, but the effect was smaller (1.5-fold). These results indicate that synaptosomal GAD is not saturated by endogenous levels of its substrate, glutamate, and account for part of the unused catalytic capacity. The greater stimulation of GABA synthesis by glutamine indicates that the GAD-containing compartment is more accessible to extrasynaptosomal glutamine than glutamate. The strong stimulation by glutamine also shows that the rates of uptake of glutamine and its conversion to glutamate can be sufficiently rapid to support GABA synthesis in nerve terminals. Synaptosomes carried out a slow net synthesis of aspartate in glucose-containing medium (7.7 nmol/h/mg of protein). Aspartate synthesis was strongly stimulated by glutamate and glutamine, but in this case the stimulation by glutamate was greater. Thus, the larger part of synaptosomal aspartate synthesis occurs in a different compartment than does GABA synthesis.

4-Aminobutyrate Transaminase↗

PubMed1992

Differentiation between alterations in plasma and mitochondrial membrane potentials in synaptosomes using a carbocyanine dye.

The cationic potentiometric fluorescent probe 3,3'-diethylthiadicarbocyanine iodide [DiS-C2(5)] was used in synaptosomes to assess the relative contributions of plasma and mitochondrial membrane potentials (psi p and psi m, respectively) to overall fluorescence. Addition of synaptosomes to media containing 0.5 microM dye caused a decrease in fluorescence intensity due to dye accumulation, which equilibrated usually within 5 min. Depolarization of mitochondria by combined treatment with cyanide and oligomycin increased fluorescence by 42%, indicating significant prior accumulation of dye into intrasynaptosomal mitochondria. psi p was calculated to be -54 mV and was not altered significantly by prior depolarization of psi m with cyanide and oligomycin (hereafter referred to as "poisoned" synaptosomes). Similarly, the linear relationship between dye fluorescence and psi p was not altered by depolarization of psi m. Valinomycin, a K+ ionophore, caused a psi p-dependent increase in fluorescence in control (nonpoisoned) synaptosomes, but did not alter fluorescence of poisoned synaptosomes except when the extracellular concentration of K+ ([K+]e) was 2 mM, in which case valinomycin hyperpolarized psi p by about 5 mV. The pore-forming antibiotic gramicidin depolarized both psi p and psi m maximally. Under these conditions, Triton X-100 further increased fluorescence by 40%, indicating significant dye binding to synaptosomal components. In poisoned synaptosomes depolarized by 75 mM K+, gramicidin caused a decrease in fluorescence intensity (hyperpolarization of psi p). The organic solvent dimethyl sulfoxide, used as a vehicle for the hydrophobic ionophores, had voltage-dependent effects on psi p and psi m.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Effects of chronic nicotine treatment on the accumulation of [3H]tetraphenylphosphonium by cerebral cortical synaptosomes.

Chronic exposure of rats to nicotine increases the number of [3H]nicotine binding sites in the brain; however, it is not clear whether nicotinic cholinergic receptor function is altered as well. In this study, we have used [3H]tetraphenylphosphonium as a probe of synaptosomal membrane potential to investigate whether exposure to nicotine in vivo alters the ability of cerebral cortical synaptosomes to maintain a potential difference and to depolarize in response to in vitro nicotine. Treatment of rats for 14 days with 0.475 mg of nicotine base/day via subcutaneously implanted minipumps resulted in a decrease in the synaptosomal accumulation of [3H]tetraphenylphosphonium in physiological buffer, corresponding to a decrease in estimated membrane potential from -55 mV to -50 mV. The onset of the decrease in membrane potential occurred after 7 days of in vivo nicotine treatment and was significantly correlated with an increase in [3H]nicotine binding to cerebral cortical synaptosomal (P2) membranes. Nicotine, at in vitro concentrations of 3-1,000 microM, decreased [3H]tetraphenylphosphonium accumulation in cerebral cortical synaptosomes from control animals. When compared to accumulation in buffer alone, in vitro nicotine and other nicotinic agonists did not significantly decrease [3H]tetraphenylphosphonium accumulation in cerebral cortical synaptosomes prepared from rats treated with nicotine in vivo. These studies provide evidence that chronic treatment with nicotine results in an average lower membrane potential in cerebral cortical synaptosomes and in functional down-regulation of the depolarization response to nicotinic cholinergic receptor stimulation.

Animals↗

PubMed1993

Reduction of dopamine synthesis inhibition by dopamine autoreceptor activation in striatal synaptosomes with in vivo reserpine administration.

In an attempt to clarify the mechanisms by which dopamine (DA) autoreceptor activation inhibits DA synthesis, the efficacy and potency of the D2 DA agonists bromocriptine, lisuride, and pergolide, and the D1-D2 DA agonist apomorphine were studied in rat striatal synaptosomes, in which the rate of DA synthesis (formation of 14CO2 from L-[1-14C]tyrosine) was increased 103% by treating the animals from which the synaptosomes were obtained with reserpine (5 mg/kg i.p. twice, 24 and 2 h before they were killed), using the striatal total homogenate as the standard synaptosomal preparation. The increase in DA synthesis evoked by reserpine was additive with that produced by treatment of the synaptosomes with dibutyryl cyclic AMP, suggesting that, not a cyclic AMP-dependent, but possibly a Ca(2+)-dependent mechanism was involved. The DA agonists showed a concentration-dependent inhibition of DA synthesis in the control synaptosomes, which was antagonized by the selective D2 DA antagonist (-)-sulpiride. In the synaptosomes with increased rate of DA synthesis obtained from the rats treated with reserpine, the concentration-response curves of DA synthesis inhibition for the other DA agonists were shifted to the right, and the effect of bromocriptine was completely eliminated, whereas bromocriptine antagonized the effect of apomorphine. The increased rate of DA synthesis was not preserved in the striatal P1 + P2 fraction obtained from the reserpine-treated rats, but the effects of the DA agonists were still reduced to the same degree as those in the total homogenate. (-)-Sulpiride did not enhance DA synthesis in synaptosomes from the reserpine-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Chronic ethanol consumption affects filipin-cholesterol complexes and intramembranous particles of synaptosomes of rat brain cortex.

To assess the effect of ethanol on the planar distribution of cholesterol as well as on the surface architecture of presynaptic terminals of rats, synaptosomes isolated from cerebral cortex of rats chronically exposed to alcohol were incubated with filipin, a cytochemical marker for beta-hydroxycholesterol, and analyzed using both conventional (qualitative and quantitative) and freeze-fracture electron microscopy. Synaptosomes incubated in the absence of filipin were used as cytochemical controls. Biochemical determination indicates a 12% increase of cholesterol in synaptosomal membranes from alcohol treated rats. This increase was confirmed by a significant increment in the number of filipin-cholesterol complexes. Synaptosomes of treated rats showed a reduction in the total number of synaptic vesicles (SV) as well as a decrease in the density and total number of intramembranous particles (IMP) per synaptosome. In control rats, most synaptosomal IMP were distributed in clusters whereas in those of rats exposed to alcohol they were distributed at random. These changes in distribution of IMP were also observed in presynaptic terminals analyzed "in situ." These findings indicate that ethanol acts on the presynaptic terminals. The variations in cholesterol content as well as in the density and distribution of IMP appear to be related to alcohol-induced changes in the physicochemical properties of components of the synaptosomal membrane.

Alcoholism↗

PubMed1983

The effect of long-term lithium treatment on the incorporation and distribution of 32P-orthosphosphate into the phospholipids from rat synaptosomes.

Rats were treated with lithium added to their diet for five weeks (40 mmol LiCl/kg diet). Mean plasma concentration was 0.45 mmol Li+/plasma. The investigation was divided into two sections. I) In an in vivo experiment in which the rats were injected with 32P-orthosphosphate for 20 hours, and with carbamoylcholine for 20 min. prior to their death, the distribution of 32P in the synaptosomal phospholipids, phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylcholine (PC), was investigated. II) An in vitro experiment which was carried out in order to establish the effect of carbamoylcholine on the incorporation of 32P into total phospholipids from extracted synaptosomes. In I) the incorporation of 32P-orthosphosphate into PI from carbamoylcholine-stimulated rats was significantly lower than from unstimulated rats, whereas there was no difference between the incorporation of 32P into PI from synaptosomes from control animals and lithium-treated animals. In II) the incorporation of 32P-orthosphosphate was significantly lower in unstimulated synaptosomes from lithium-treated rats than from control rats, while the increase in the 32P-incorporation after stimulation followed the same pattern with regard to synaptosomes from control rats and lithium-treated rats. The results support the idea of lithium exerting an effect on basal synaptosomal activity but not on stimulated synaptosomal activity.

Animals↗

PubMed1989

Effect of chronic renal failure and parathyroid hormone on phospholipid content of brain synaptosomes.

The effects of 21 days of chronic renal failure (CRF) with and without excess parathyroid hormone (PTH) and those of 21 days administration of intact PTH on phospholipids and cholesterol contents of rat brain synaptosomes were examined. CRF and PTH treatment were associated with a significant (P less than 0.01-0.02) reduction in the synaptosomal contents of total phospholipids, phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidylethanolamine (PE). Parathyroidectomy (PTX) prior to the induction of CRF prevented the decrements in the synaptosomal contents of total phospholipids, PI, PS, and PE. The synaptosomal contents of these phospholipids in CRF-PTX rats were not different from those in normal rats despite CRF. There were no significant changes in the cholesterol content of the synaptosomes in the various experimental groups of animals. The data show that CRF affects synaptosomal metabolism of total phospholipids, PI, PS, and PE, and these derangements are due to the state of secondary hyperparathyroidism of renal failure. The decrements in the content of PI, PS, and PE could be, at least in part, responsible for the previously reported abnormalities in the neurotransmitter functions of brain synaptosomes in CRF and could underlie some of the abnormalities in central nervous system dysfunction in uremia.

Animals↗

PubMed2000

Effect of oxidative stress on the uptake of GABA and glutamate in synaptosomes isolated from diabetic rat brain.

It has been suggested that increased oxidative stress might be involved in the pathophysiology of diabetic complications. In this study, we investigated the effect of diabetes on the susceptibility of synaptosomes to oxidative stress (induced by the oxidizing pair ascorbate/Fe(2+)) and on the uptake of the amino acid neurotransmitters gamma-aminobutyric acid (GABA) and glutamate. We found a lower susceptibility of synaptosomes isolated from Goto-Kakizaki (GK) rats, a model of non-insulin-dependent diabetes mellitus, to lipid peroxidation as compared with synaptosomes isolated from Wistar control rats (6.40+/-1.05 and 12.14+/-1.46 nmol thiobarbituric acid reactive substance/mg protein, respectively). The lower susceptibility of GK rat synaptosomes to membrane lipid peroxidation correlates with an increase in synaptosomal vitamin E levels (835+/-58.04 and 624.26+/-50.26 pmol/mg protein in diabetic and normal rats, respectively). In the absence of ascorbate/Fe(2+), no significant differences were observed between the levels of lipid peroxidation of synaptosomes isolated from diabetic and normal rats. Studies of neurotransmitter uptake show that the [(3)H]glutamate uptake was decreased by about 30% in diabetic GK rats as compared with control Wistar rats, whereas the [(3)H]GABA uptake was not significantly different from controls. Under oxidizing conditions, the glutamate uptake in diabetic rats was unaffected, and a decreased GABA uptake (41.39+/-4.41 and 60.96+/-6.4% of control in GK and Wistar rats, respectively) was observed. We conclude that the increased resistance to oxidative stress in GK rat synaptosomes may be due to the increased vitamin E content and that diabetic state and oxidative stress conditions differentially affected the uptake of the neurotransmitters GABA and glutamate.

Animals↗

PubMed1993

Abnormal norepinephrine metabolism in rat brain synaptosomes in phosphate depletion.

Abnormalities in the function of the central nervous system exist in phosphate depletion (PD). It is possible that this is due to an adverse effect of PD on the metabolism of neurotransmitters, such as norepinephrine (NE), in brain synaptosomes. We examined the effects of PD, produced by restriction of dietary phosphate intake on NE metabolism of brain synaptosomes. Synaptosomes from PD rats had significantly reduced NE content, uptake and release, elevated Km, but normal Vmax of tyrosine hydroxylase, normal Km and Vmax of monoamine oxidase, elevated resting levels of cytosolic calcium ([Ca2+]i), higher delta [Ca2+]i in response to KCl, higher delta [Ca2+]i/basal [Ca2+]i ratio, lower ATP content and reduced activity of Na(+)-K(+)-ATPase as compared to synaptosomes from pair-weighed rats. Treatment of PD rats with verapamil corrected all the synaptosomal derangements except for the elevated Km of tyrosine hydroxylase and NE content. Verapamil did not affect the metabolism of PW rats. The data demonstrate that PD causes significant derangements in NE metabolism of brain synaptosomes. Observations in the present study and in others indicate that these derangements in NE metabolism are due to the PD-induced abnormalities in the homeostasis of synaptosomal [Ca2+]i, ATP and phospholipids and in the activities of Na(+)-K(+)-ATPase and Ca(2+)-ATPase.

Animals↗

PubMed1997

Role of Ca2+ in metabolic inhibition-induced norepinephrine release in rat brain synaptosomes.

Ischemia and simulated ischemic conditions induce enhanced release of norepinephrine (NE) in the brain and the heart. Although studies with neuronal preparations demonstrated a rise in [Ca2+]i under energy-depleted conditions, such release of NE in the heart appears to be predominantly Ca2+ independent. Since Ca2+ overload occurs in ischemia or energy depletion and since a rise in [Ca2+]i triggers exocytosis without membrane depolarization, we tested the possibility, using brain synaptosomes, that increased NE release could be, at least in part, a consequence of raised [Ca2+]i. Brain synaptosomes were incubated with Krebs-Henseleit medium, and ischemia was mimicked by treatment with metabolic inhibitors. NE content in incubation medium (supernatant) and synaptosomes was analyzed chromatographically. Treatment with metabolic inhibitors reduced ATP content by 75% and increased [Ca2+]i by more than fourfold within minutes. Metabolic inhibition elicited NE release, which started within 10 minutes and reached a maximum after 30 minutes, with a corresponding 55% reduction in synaptosomal NE content after 40 minutes. NE release, together with a marked increase in [Ca2+]i, was also induced in energy-depleted synaptosomes by Ca2+ repletion after incubation with the Ca(2+)-free medium. Effects on NE release of various interventions to prevent Ca2+ overload were tested. Omission of Ca2+ from the incubation medium or loading synaptosomes with the Ca2+ chelator BAPTA-AM (20 and 100 mumol/L) prevented NE release, indicating a Ca(2+)-dependent mechanism. Inhibition of Ca2+ channels with omega-conotoxin, cadmium, or nifedipine had no effect on NE release during energy depletion. In contrast, nickel and 3,4-dichlorobenzamil, Na(+)-Ca2+ exchange inhibitors, dose-dependently inhibited NE release. In conclusion, this study provides evidence that under energy-depleted conditions, Ca2+ overload in synaptosomes of noradrenergic neurons from the brain is an important mechanism for the enhanced release of NE and that a reversal of Na(+)-Ca2+ exchange may be the key pathway leading to intraneuronal Ca2+ overload.

Animals↗

PubMed1989

Calculation of membrane potential in synaptosomes with use of a lipophilic cation (tetraphenylphosphonium).

To estimate membrane potential in synaptosomes with the use of tetraphenylphosphonium (TPP+), an equation relating the amount of TPP+ accumulated in synaptosomes with membrane potential was derived from the following two assumptions. (1) TPP+ molecules were distributed into plasma membranes, mitochondria and cytosol of synaptosomes. (2) TPP+ achieves a Nernst equilibrium across both the synaptosomal and inner mitochondrial membranes. We propose three methods for calculation of membrane potential using this equation. The concentration of TPP+ was measured under various controlled conditions with an electrode selective for TPP+. The amount of TPP+ accumulated in synaptosomes was determined by measuring the difference between its initial concentration and the concentration after addition of synaptosomes, and membrane potential was estimated by the three methods. The resting potential of synaptosomes was estimated to be -75 to -90 mV by all of these methods. Membrane potentials under various controlled conditions were calculated, and the characteristics of the methods for estimation of membrane potential and those of membrane potential obtained by the methods are discussed.

Animals↗

PubMed1986

[Properties of ATP-dependent H+-transport in rat brain synaptosomes].

Properties of the ATP-dependent H+-transport system in rat brain synaptosomes were examined using the acridine orange fluorescence quenching method. ATP-dependent H+-accumulation assessed by the quenching of acridine orange fluorescence was observed with the synaptosomes treated with hypotonic solution (hypotonic shock-synaptosomes), but not with the intact synaptosomes. With hypotonic shock-synaptosomes, H+-transport was activated in the presence of Cl- or Br-. However, this transport activity was markedly reduced in the presence of Mes-, NO3-, I- or SCN-. On the other hand, H+-transport activity was less effective with cations other than K+ in the following decreasing order of potency: K+ greater than Cs+ greater than Na+ greater than Li+. The H+-transport activity was inhibited by 0.3 mM ethacrynic acid, 10 microM 4-acetamide-4'-isothiocyanostilbene-2,2'-disulfonic acid or 1 mM 4-aminopyridine to 39.2%, 36.3%, or 33.1% of the control, respectively, but was not inhibited by 1 mM ouabain, 500 microM vanadate, 10 microM picrotoxin and 100 microM gamma-aminobutyric acid. These results suggest that the ATP binding site of the synaptosomal ATP-dependent H+-transport system exists on the internal surface of synaptosomal plasma membranes and that the H+-transport system is stimulated by the presence of Cl- or Br- and by K+ movement through the K+ channel.

Adenosine Triphosphate↗

PubMed1982

Effects of morphine, codeine and codeine-epoxide on calcium uptake into the synaptosomes isolated from naive and tolerant rats.

We studied the effects of morphine, codeine and codeine-7,8-oxide (codeine-epoxide) on the stimuli-induced 45Ca2+ uptake into the synaptosomes isolated from naive and tolerant rats and clarified the relationship between pharmacological responses of opiates and synaptosomal 45Ca2+ uptake. In vitro additions of morphine and codeine-epoxide inhibited the synaptosomal 45Ca2+ uptakes induced by two stimuli, that is, high KCl and veratrine in a concentration-dependent manner; and the inhibitions could be reversed by naloxone. However, the inhibitory action of codeine was less than that of morphine and codeine-epoxide. Since the potency ratios of the anti-nociceptive action of opiates are higher in the order of morphine greater than codeine-epoxide greater than codeine, the inhibitory effect of opiates on synaptosomal 45Ca2+ uptake may partly relate to their antinociceptive action. On the other hand, opiates significantly increased synaptosomal 45Ca2+ uptake when animals were rendered tolerant to their antinociceptive action, and data showed that the elevation of stimuli-induced 45Ca2+ uptake into the synaptosomes isolated from tolerant animals may reflect the degree of antinociceptive tolerance. Our results support the hypothesis that some of the pharmacological effects of opiates may be attributable to its ability to affect calcium accumulation in synaptosomes.

Analgesics↗

PubMed1983

Relationship between synaptosomal calcium uptake and antinociceptive action of morphine.

We studied the relationship between inhibition of 45Ca2+ uptake and the antinociceptive action induced by morphine and effects of papaverine and 1,1-diphenyl-3-piperidinobutanol hydrochloride (Aspaminol) on both these actions of morphine. Addition of these drugs in the incubation medium significantly inhibited glutamate-stimulated synaptosomal 45Ca2+ uptake. The inhibition curves of morphine and Aspaminol on glutamate-stimulated synaptosomal 45Ca2+ uptake were linear, but that of papaverine was not. The inhibition of morphine on synaptosomal 45Ca2+ uptake was reversed by addition of naloxone. The inhibition of synaptosomal 45Ca2+ uptake induced by morphine was increased by the simultaneous addition of Aspaminol, but the inhibition induced by both morphine and papaverine was not increased to more than that by papaverine alone. Since morphine-antinociception was potentiated by Aspaminol and blocked by papaverine, these results support that the inhibition of synaptosomal calcium uptake plays an important role in the production of morphine-antinociception. However, since the inhibition of synaptosomal 45Ca2+ uptake by morphine was less than that by both Aspaminol and papaverine, and papaverine blocked morphine-antinociception, notwithstanding that 10(-4) M of papaverine alone completely inhibited glutamate-stimulated 45Ca2+ uptake into synaptosomes, it may be difficult to account for the antinociceptive action of morphine by the inhibition of 45Ca2+ uptake only.

Analgesia↗

PubMed1998

[Protective effects of schisanhenol against oxygen free radical induced injury of rat cerebral mitochondria and synaptosomes].

The cerebral tissue injury induced by ischemia and reperfusion or trauma has been considered to be due to over production of oxygen free radicals (OFRs). The aim of this study was to evaluate the effects of schisanhenol (Sal) on Fe(2+)-cysteine (Cys) induced injury of rat cerebral mitochondria and synaptosomes in vitro. Incubation of cerebral mitochondria or synaptosomes with Fe(2+)-Cys at 37 degrees C resulted in an increase of malondialdehyde (MDA) formation and decrease of ATPase activity. Sal(10(-4) mol.L-1) completely inhibited Fe(2+)-Cys induced increase of MDA formation of mitochondria and synaptosomes as well as the loss of ATPase activity of mitochondria. The swelling of mitochondria and reduction of membrane fluidity of mitochondria and synaptosomes induced by Fe(2+)-Cys were also prevented by Sal. Sal(10(-5) mol.L-1) was shown to significantly inhibit the decrease of synaptosomal GSH content induced by H2O2. The electron micrographs also showed that Sal markedly reduced the pathological damage of mitochondria and synaptosomes induced by Fe(2+)-Cys. The results suggest that Sal has protective action against Fe(2+)-Cys induced injury of rat cerebral mitochondria and synaptosomes.

Adenosine Triphosphatases↗

PubMedDate not supplied

[Interactions between liposomes and synaptosomes].

In continuing experiments to determine the basis of presynaptic modulation, rat cortical synaptosomes were employed and transmembrane calcium fluxes were determined with a metalocromophoric dye, Arsenazo III. The intracellular free Ca2+ concentrations show complex fluctuations in time and space in response to a variety of stimuli, acting as a pluripotent signal for many neuronal functions. Depolarisation of synaptosomes by K+ 20 mM determined calcium influx in cortical synaptosomes, mainly mediated by the Q type Ca2+ channels, with a minor participation of the N type channels. The administration protocol for the polyamines, revealed that agmatine, spermine, spermidine, putresceine and cadaverine reduce, in variable proportions, the K(+)-induced Ca2+ influx, in synaptosomal preparations. The results plead for the ability of agmatine and spermine to modulate Ca2+ fluxes in synaptosomes, both from the interior and exterior side. Moreover, rigidization of the synaptosomal membrane (by cholesterol-enriched liposomes administration) do not significantly influences K(+)-induced Ca2+ influx in synaptosomal preparations.

Animals↗

PubMed1992

Antagonism of synaptosomal calcium channels by subtypes of omega-agatoxins.

Venom of the funnel web spider Agelenopsis aperta inhibits the binding of 125I-omega-conotoxin GVIA (omega-CgTx) to calcium channels in chick brain synaptosomal membranes. Fractionation of the venom by liquid chromatography shows that this inhibitory activity is associated primarily with a diverse class of peptide toxins called omega-agatoxins (omega-Aga). Using binding inhibition as an assay, we purified and identified the novel, 76-amino acid toxin, omega-Aga-IIIA. Inhibition of 125I-omega-CgTx binding to chick synaptosomal membranes by omega-Aga-IIIA and omega-Aga-IIA is correlated with block of potassium-stimulated 45Ca entry into synaptosomes; omega-Aga-IA neither inhibits 125I-omega-CgTx binding nor 45Ca entry under identical conditions. omega-Aga-IIA and omega-Aga-IIIA are 20-30-fold more potent than omega-CgTx as antagonists of synaptosomal calcium channels. However, whereas omega-CgTx completely blocks 45Ca entry into synaptosomes at saturating concentrations, the omega-agatoxins maximally block only 60-70% of 45Ca entry. Pretreatment of synaptosomes with omega-Aga-IIIA occludes block of 45Ca entry by omega-CgTx. The results indicate that, while the omega-agatoxins bind to the entire population of omega-CgTx-sensitive calcium channels in chick synaptosomal membranes, they exert only a partial block of 45Ca flux. Such block could occur via two distinct mechanisms. Toxin binding may alter the kinetics of a homogeneous population of channels, resulting in lower overall conductance upon depolarization. Alternatively, the omega-agatoxins may bind to two distinct channel subtypes, only one of which is blocked as a result of toxin occupation.

Agatoxins↗

PubMed1991

[3H]tetraphenylphosphonium accumulation in cerebral cortical synaptosomes as a measure of nicotine-induced changes in membrane potential.

The effect of nicotine on synaptosomal membrane potential in P2 preparations of rat cerebral cortex was investigated using a membrane permeant, lipophilic cation, [3H]tetraphenylphosphonium ([3H]TPP+). [3H]TPP+ accumulated in synaptosomes in a time-dependent manner and its accumulation was decreased when the extracellular potassium concentration was increased and in the presence of the sodium channel toxin, veratridine. Nicotine (1-1000 microM) decreased the accumulation of [3H]TPP+ in both P2 synaptosomal preparations and in synaptosomes purified using Percoll gradients. This effect of nicotine was mimicked by other nicotinic agonists (1,1-dimethyl-4-phenylpiperazinium iodide, cytisine, suberyldicholine and acetylcholine) and was partially blocked by 10 microM mecamylamine and 30 microM hexamethonium. Atropine (1 microM) and the removal of calcium from the incubation mixture both enhanced the effect of nicotine while the addition of physostigmine (10 microM) reduced the nicotine-induced decrease in [3H]TPP+ accumulation, evidence that acetylcholine released from the synaptosomes by nicotine may produce hyperpolarization of synaptosomes via stimulation of presynaptic muscarinic receptors. It is concluded that the effect of nicotine on [3H]TPP+ accumulation is mediated by nicotine stimulation of a ganglionic-type nicotinic cholinergic receptor and that this method of determining synaptosomal membrane potential will provide a functional measure of presynaptic nicotinic receptor activation.

Animals↗