Transmembrane electrical potential measurements in rat brain synaptosomes

Deutsch, Carol; Rafałowska, Urszula

doi:10.1016/0014-5793(79)81227-2

Cited by 45 publications

(32 citation statements)

References 30 publications

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“…The release of both cantly greater in the presence than in lower efficacy of a-latrotoxin on transn conditions could be correlated with (Fig. 5, values in parentheses) (15,19,21,(26)(27)(28)(29) or neurobl lizing radioisotopes of the lipophilic c Ph3MeP+ (15,19,21,(27)(28)(29)31). Thei to the extent to which the cations in( significant potential across the inner X mitochondria.…”

Section: Resultsmentioning

confidence: 91%

“…Thus, the failure of a-Latrotoxin and Transmitter Release. The marked effect 4k can be ascribed to the of a-latrotoxin on noradrenaline and 'y-aminobutyrate release ion (27,29) or to omission is only moderately decreased when Ca2" is omitted from the '+ concentration also was incubation fluid. Whether this reflects a true dependency of (1)(2)(3)(4)33).…”

Section: Resultsmentioning

confidence: 99%

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alpha-latrotoxin of black widow spider venom depolarizes the plasma membrane, induces massive calcium influx, and stimulates transmitter release in guinea pig brain synaptosomes.

Nicholls¹,

Rugolo²,

Scott³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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The. effect of a-latrotoxin from black widow. spider venom upon guinea pig cerebral cortical synaptosomes is described. Plasma membranepotential t(A.p), in situ mitochondrial membrane potential (Aqkm) (1-4, 11, 12); moreover, it releases acetylcholine, noradrenaline, dopamine, and y-aminobutyrate from cerebral cortical slices (5-7), synaptosomes (8, 10, 13),. and one neurosecretory cell line (9). A small number ofhigh-affinity binding sites for the toxin are located specifically on the presynaptic plasma. membrane (6, 10), and occupancy of these. correlates well with the subsequent release of neurotransmitter (10). In addition, the toxin is able to create stable conductance channels in protein-free black lipid membranes (14).A number of aspects of a-latrotoxin action are -unclear. In particular, there is no consensus as to the role ofCa2+. Whereas .there is agreement that the release of acetylcholine and yaminobutyrate is independent of external Ca2+ (3, 4, 7, 12, 13), the Ca2e dependency of catecholamine release appears dependent upon the experimental conditions and target membrane (5,(7)(8)(9). In addition, there is a contradiction between the results obtained with neurosecretory PC.12 cells, in which a. primary effect ofthe toxin appears to be a massive.uptake of Ca2+ across the plasma membrane (9), and those obtained with rat cerebral cortical synaptosomes, in which.no effect on Ca2+ flux could be detected (13).The possibility of a-latrotoxin-induced massive Ca2+ uptake across the presynaptic membrane raises a number of unanswered questions about the effects of the toxin on the energy levels in the cell. In particular, it is unclear whether the intraterminal mitochondria would be able to cope with this Ca2+ influx. Indeed swollen and disorganized mitochondria can be observed in electron micrographs of presynaptic expansions both at neuromuscular junctions (1-4, 11) and in cortical slices (7) treated with a-latrotoxin in the presence of external Ca2 . Deenergization of the mitochondria resulting in a collapse of the mitochondrial membrane potential (Aqi(m) would not only prevent mitochondrial synthesis ofATP but also would destroy the ability of the mitochondria to regulate the upper limit of the cytosolic free Ca2+ concentration (for reviews, see refs. 15-17), with a resultant risk of cell death (18).Recent developments in the study of isolated synaptosomes have enabled Ca2+ transport, Aim, and the plasma membrane potential (Ai/5p) to be determined simultaneously (15,(18)(19)(20)(21).In this paper we use these techniques .and introduce the application. of.the tetraphenylphosphonium (Ph4P+)-selective electrode (22) for the monitoring of synaptosomal potentials in suspension. EXPERIMENTAL PROCEDURESSynaptosomes.' Synaptosomes were prepared from the cerebral cortices (including corpus striatum) of Duncan-Hartley strain guinea pigs age 4-8 wk as described (19)(20)(21)(22). The synaptosomes were stored as a pellet in 250 mM suqrose/5 mM 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethane sulfonate (T...

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Section: Resultsmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 99%

alpha-latrotoxin of black widow spider venom depolarizes the plasma membrane, induces massive calcium influx, and stimulates transmitter release in guinea pig brain synaptosomes.

Nicholls¹,

Rugolo²,

Scott³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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“…For this determination an aliquot of homogenate of the cerebral cortex was acidified with 0.4 N perchloric acid and NA in the supernatant was assayed using the radioenzymatic method of Henry et al, (1975). (Deutsch & Rafalowska, 1979;Wood & Wylie, 1983), the equilibrium distribution of propranolol between tissue and incubation medium was estimated to represent a 33 to 50 fold synaptosomal concentration of drug. However, the corresponding distribution ratio for atenolol was in the range 1.0 to 1.4.…”

Section: Pretreatment With 6-hydroxydopaminementioning

confidence: 99%

Depolarization‐induced release of propranolol and atenolol from rat cortical synaptosomes

Bright¹,

Gaffney

Street³

et al. 1985

British J Pharmacology

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The accumulation and release of [3H]‐propranolol and [3H]‐atenolol were examined in synaptosomes from rat cerebral cortex. Synaptosomes accumulated 20 pmol propranolol and 0.6 pmol atenolol mg−1 protein when incubated at 30°C with radiolabeled drugs (0.1 μm). Exposure of propranolol‐loaded synaptosomes to elevated K+, Rb+ or Cs+ evoked a concentration‐dependent increase in propranolol efflux. The action of these ions in releasing propranolol was highly correlated with their ability to produce synaptosomal membrane depolarization, as estimated with the voltage‐sensitive dye diS‐C3‐(5). Elevated K+ also promoted atenolol release from synaptosomes in a concentration‐dependent manner. Veratridine (10 μm) released propranolol and atenolol from synaptosomes and these effects were antagonized by tetrodotoxin (1 μm). Under Ca2+‐free conditions, K+‐induced release of propranolol was reduced by 37% and atenolol release was diminished by 68%. The results support the concept that both polar and non‐polar β‐adrenoceptor blocking drugs may be accumulated by nerve endings for release by membrane depolarization and suggest that neural storage and release of these molecules may influence their concentrations at localized sites of action.

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“…We have succeeded recently in developing a preparation of synaptosomes (20)(21)(22)(23), based on the original procedure ofBooth and Clark (24), in which the values of energy parameters (20,21), concentration of intrasynaptosomal potassium (22), and transmembrane electrical potential (23) are very close to those in intact neurons. In view ofthe postulated function for the highaffinity GABA uptake system in neuronal transmission, we have examined our preparation of synaptosomes for this activity by 'using different and more sensitive methods for measuring GABA concentration.…”

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confidence: 99%

Net uptake of gamma-aminobutyric acid by a high-affinity system of rat brain synaptosomes.

Pastuszko¹,

Wilson²,

Erecińska³

1981

Proc. Natl. Acad. Sci. U.S.A.

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Rat brain synaptosomes isolated on. discontin- 'using different and more sensitive methods for measuring GABA concentration. In this paper we show that synaptosomes carry out rapid net uptake of GABA and that this net-uptake occurs through a high-affinity system. Homoexchange is shown to account for less than 10% ofthe measured [14C]GABA uptake. MATERIALS AND METHODSMale Sprague-Dawley rats (180-220 g) were used throughout the study. Synaptosomes were isolated from the forebrains of rats essentially as described (24). The final synaptosomal pellet was suspended in Krebs-=Henseleit Hepes buffer (140 mM NaCl/5 mM KCV1.3 mM MgSO4/5 mM NaHCOj2.5 mM CaCl2/1 mM Tris phosphate/10 mM Tris Hepes, pH 7.4), containing 10 mM glucose.Synaptosomes, suspended at about 6 mg of protein per ml, were preincubated for 6 min with constant shaking at 250C. The uptake of GABA was measured in three different experimental systems after dilution of the incubation mixture in the Krebs-Henseleit Hepes buffer to a final protein concentration of 1-2 mg/ml (unless indicated otherwise). (i) The synaptosomal suspensions were supplemented with 2.4 AM [14C]GABA (Amersham: specific activity, 224 Ci/mol; 1 -Ci = 3.7 X 10'°b ecquerels), and the radioactivity of the supernatant and of the pellet was measured after rapid centrifugation (Beckman microfuge) of the aliquots of the incubation mixture through a layer of silicone oil (specific gravity 1.03) at appropriate time intervals. (ii) The synaptosomal suspension was supplemented with 2 ,uM nonradioactive GABA, and aliquots were centrifuged through silicone oil into a 3% (wt/vol) solution of perchloric acid. The supernatants were removed, acidified with perchloric acid (3% final concentration), and centrifuged. The perchloric acid-soluble fractions (i.e., synaptosomal pellets and postsynaptosomal supernatants) were diluted 1:2 in 50 mM citrate buffer (pH 2.0), and concentrations of GABA were measured in a Perkin-Elmer amino acid analyzer. (iii) Synaptosomes (5-6 mf of protein per ml) were preincubated for 6 min with 2 juM [ 4C]GABA and then diluted 1:10 into KrebsHenseleit Hepes buffer containing various concentrations of nonradioactive GABA (0-10 AM). Radioactivity was then measured in the supernatants and in the pellets at appropriate time intervals after separation of the synaptosomes from the medium by rapid centrifugation through silicone oil. Radioactivity was determined in a Searle Delta 300 liquid scintillation counter with aqueous counting scintillant (Amersham).Protein was measured by a biuret reaction, with bovine serum albumin as the standard. RESULTS AND DISCUSSIONRadioactive GABA was rapidly taken up by suspensions of synaptosomes, and the initial rate ofuptake was proportional to the protein concentration in the range 0.5-2 mg of protein per ml (Fig. 1). From the initial~linearrates ofGABA uptake at various external concentrations of this amino acid, kinetic constants were calculated by a double-reciprocal plot analysis of the results (Fig. 2). The'Km was found to be 6.25 ,AM a...

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Transmembrane electrical potential measurements in rat brain synaptosomes

Cited by 45 publications

References 30 publications

alpha-latrotoxin of black widow spider venom depolarizes the plasma membrane, induces massive calcium influx, and stimulates transmitter release in guinea pig brain synaptosomes.

alpha-latrotoxin of black widow spider venom depolarizes the plasma membrane, induces massive calcium influx, and stimulates transmitter release in guinea pig brain synaptosomes.

Depolarization‐induced release of propranolol and atenolol from rat cortical synaptosomes

Net uptake of gamma-aminobutyric acid by a high-affinity system of rat brain synaptosomes.

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