Transmitter release by non-receptor activation of the alpha-subunit of guanine nucleotide regulatory protein in rat striatal slices.

The effects of 5 mM NaF + 10 microM AlCl3, a direct activator of guanine nucleotide-binding proteins (G proteins), on the release of [3H]dopamine ([3H]DA), [3H]gamma-aminobutyric acid ([3H]GABA), and [3H]acethylcholine ([3H]ACh) were investigated in slices of rat striatum. When the tissue was exposed to NaF + AlCl3 the release of [3H]DA, [3H]GABA, and [3H]ACh was enhanced significantly. In a calcium-free solution the release of [3H]GABA and [3H]DA was increased by NaF+AlCl3 much more than in the presence of [Ca2+]o. In slice preparations taken from reserpinized animals, in which the vesicular storage of [3H]DA was therefore prevented, NaF + AlCl3 had no effect on [3H]DA release. HPLC analysis of the radioactivity of the perfusate showed that, in the presence of NaF + AlCl3, the content of dihydroxyphenylacetic acid (DOPAC) in perfusate samples increased significantly, while in pargyline-treated animals only the DA content was increased. Inhibition of DA carriers by nomifensine or low temperature prevented the effect of NaF + AlCl3. N-ethylmaleimide (NEM) preincubation did not modify the effect of NaF + AlCl3 on [3H]DA release Neomycin (0.1 mM), a phospholipase C (PLC) inhibitor, significantly decreased the effect of NaF + AlCl3 on [3H]DA and [3H]GABA release. The internal concentration of Ca2+ in synaptosomes was enhanced by NaF + AlCl3 in normal solution. However, [Ca2+]i was not influenced by NaF + AlCl3 in Ca(2+)-free medium. It is concluded that a non-receptor-mediated activation, by NaF + AlCl3, of the alpha-subunit of a G protein, results in a [Ca2+]o-independent release of DA and GABA, but not that of ACh.

Detection of intracellular free Na+ concentration of synaptosomes by a fluorescent indicator, Na(+)-binding benzofuran isophthalate: the effect of veratridine, ouabain, and alpha-latrotoxin.

A novel fluorescent Na+ indicator, Na(+)-binding benzofuran isophthalate (SBFI), was used to follow changes in the intracellular free Na+ concentration ([Na+]i) of synaptosomes. The dye, when loaded into synaptosomes in the form of its acetoxymethyl ester, was responsive to changes of [Na+]. Calibration was made using the 340/380 nm excitation ratio when the cytoplasmic Na+ concentration was equilibrated with different concentrations of extracellular Na+ in the presence of 2 microM gramicidin D. The basal value of [Na+]i in synaptosomes in the presence of 140 mM extracellular Na+ was found to be 10.9 +/- 1.8 mM. Veratridine, which opens potential-dependent Na+ channels, caused a sudden increase in [Na+]i in a concentration-dependent manner (1-20 microM), whereas the effect of ouabain (20 and 50 microM), the inhibitor of the plasma membrane Na+,K(+)-ATPase, was more gradual. The rise in the fluorescence intensity upon addition of veratridine was prevented completely by 2 microM tetrodotoxin. alpha-Latrotoxin, the black widow spider toxin, caused an increase in the fluorescence intensity, which became evident 1 min after the addition of the toxin. The rate of increase was proportional to the concentration of the toxin (0.19-1.5 nM). This report confirms our earlier finding demonstrating a Na(+)-dependent component in the action of alpha-latrotoxin, and shows that changes in [Na+]i in synaptosomes can be followed by SBFI.

Effect of alpha-latrotoxin on acetylcholine release and intracellular Ca2+ concentration in synaptosomes: Na(+)-dependent and Na(+)-independent components.

We studied the effect of alpha-latrotoxin (alpha LTX) on [14C]acetylcholine ([14C]ACh) release, intracellular Ca2+ concentration ([Ca2+]i). plasma membrane potential, and high-affinity choline uptake of synaptosomes isolated from guinea pig cortex. alpha LTX (10(-10)-10(-8) M) caused an elevation of the [Ca2+]i as detected by Fura 2 fluorescence and evoked [14C]ACh efflux. Two components in the action of the toxin were distinguished: one that required the presence of Na+ in the external medium and another that did not Displacement of Na+ by sucrose or N-methylglucamine in the medium considerably decreased the elevation of [Ca2+]i and [14C]ACh release by alpha LTX. The Na(+)-dependent component of the alpha LTX action was obvious in the inhibition of the high-affinity choline uptake of synaptosomes. Some of the toxin action on both [Ca2+]i and [14C]ACh release remained in the absence of Na+. Both the Na(+)-dependent and the Na(+)-independent components of the alpha LTX-evoked [14C]ACh release partly required the presence of either Mg2+ or Ca2+. The nonneurotransmitter [14C]choline was released along with [14C]ACh, but this release did not depend on the presence of either Na+ or Ca2+, indicating nonspecific leakage through the plasma membrane. We conclude that there are two factors in the release of ACh from synaptosomes caused by the toxin: (1) cation-dependent ACh release, which is related to (a) Na(+)-dependent divalent cation entry and (b) Na(+)-independent divalent cation entry, and (2) non-specific Na(+)- and divalent cation-independent leakage.

Lack of involvement of [Ca2+]i in the external Ca(2+)-independent release of acetylcholine evoked by veratridine, ouabain and alpha-latrotoxin: possible role of [Na+]i.

Synaptosomes were challenged by veratridine, ouabain and alpha-latrotoxin, and the release of 14C-acetylcholine (ACh) was measured in the absence of external Ca2+. We wished to test whether Ca2+ mobilized from internal stores triggered the ACh release that was independent of external Ca2+. We found that none of the agents altered the [Ca2+]i in a Ca(2+)-free medium. Buffering the intracellular Ca2+ concentration with BAPTA did not prevent the increase in release of 14C-ACh by veratridine or ouabain in the absence of Ca2+, however, it greatly reduced the release evoked in a Ca(2+)-containing medium. In parallel samples the release of ACh and the change in the internal Na+ concentration ([Na+]i) were measured. It was found that veratridine, ouabain and alpha-latrotoxin all enhanced [Na+]i in a concentration-dependent manner and a good quantitative relationship existed between the increase in [Na+]i and the release of ACh.

Parameters not influenced by vesamicol: membrane potential, calcium uptake, and internal calcium concentration of synaptosomes.

In our previous study vesamicol, an inhibitor of the acetylcholine transporter of the cholinergic vesicles, inhibited veratridine-evoked external Ca(2+)-dependent acetylcholine release from striatal slices but did not influence acetylcholine release observed in Ca(2+)-free medium (4). Here we examined if the effect of veratridine on membrane potential, Ca(2+)-uptake, and intracellular Ca2+ concentration of synaptosomes was altered by vesamicol in parallel with the inhibition of acetylcholine release. The depolarizing effect of 10 microM veratridine (from 67 +/- 2.3 mV resting membrane potential to 50.7 +/- 2.5 mV) was not significantly influenced by vesamicol (1-20 microM). Vesamicol (1-20 microM) had no effect on either the overall curve of the veratridine-evoked 45Ca2+ uptake or the amount of Ca2+ taken up by synaptosomes. Veratridine caused a rise in intrasynaptosomal Ca2+ concentration as measured by Fura2 fluorescence, and the same increase both in characteristics and in magnitude was observed in the presence of vesamicol (20 microM). The K(+)-evoked (40 mM) increase of Ca2+ uptake and of intracellular calcium concentration were also unaltered by vesamicol. In high concentration (50 microM) vesamicol inhibited both the fall in membrane potential and the elevated Ca2+ uptake by veratridine, indicating a possible nonspecific effect on potential-dependent Na+ channels at this concentration. Vesamicol, in lower concentration (20 microM) when neither of the above parameters was changed, completely prevented veratridine-evoked increase of [14C]acetylcholine release. This was observed only when vesamicol was present in the media throughout the experiment after loading the preparation with [14C]choline.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+o-independent veratridine-evoked acetylcholine release from striatal slices is not inhibited by vesamicol (AH5183): mobilization of distinct transmitter pools.

The effect of 2-(4-phenylpiperidino)cyclohexanol (AH5183 or vesamicol), a compound known to block the uptake of acetylcholine (ACh) into cholinergic synaptic vesicles, on the release of endogenous and [14C]ACh from slices of rat striatum was investigated. ACh release was evoked either by electrical stimulation or by veratridine. The effect of electrical stimulation was entirely dependent on external Ca2+. By contrast, veratridine (40 microM) also enhanced ACh release in the absence of Ca2+. Indeed, with veratridine two components were clearly distinguished: one dependent on external Ca2+ and the other not. Vesamicol inhibited [14C]ACh release evoked by both veratridine and electrical stimulation in the presence of external Ca2+, provided it was added to the tissue prior to loading with [14C]choline. With the same treatment vesamicol only slightly affected the release of endogenous ACh. Under the same conditions the Ca2(+)-independent [14C]ACh release evoked by veratridine was not prevented by vesamicol. The differential responsiveness to vesamicol suggests that ACh pools involved in Ca2+o-dependent ACh release are different from those mobilized during Ca2+o-independent ACh release.