Stimulation Of Transmitter Release Research Articles

The depletion of monoamines blocks the sympathoinhibitory response to cocaine

Recent studies have shown that cocaine decreases, rather than increases sympathetic nerve discharge (SND). Whether these sympathoinhibitory responses are the result of cocaine's actions on monoaminergic transmission (i.e. blockade of neuronal uptake or stimulation of transmitter release) or its local anesthetic actions is not known. The purpose of the present study was to determine the degree to which cocaine's actions on monoaminergic transmission are involved in mediating the sympathoinhibitory response to this drug. We examined the mean arterial pressure, heart rate and splanchnic sympathetic nerve responses elicited by cocaine (I mg/kg, i.v.) in pentobarbital-anesthetized rats depleted of monoamines. Monoamines were depleted by administering reserpine (10 mg/kg, i.p.) either 24, or 48 and 24 h before the experiment. The rats were also given α-methyl- p-tyrosine (200 mg/kg, i.p.) 2 h before the experiment. Vehicle-treated rats served as controls. Depletion of monoamines markedly reduced resting arterial pressure and heart rate and significantly attenuated the pressor response and tachycardia elicited by tyramine (1 mg/kg, i.v.). In control rats, cocaine elicited marked (−64 ± 4%) and prolonged (44 ± 4 min) decreases in SND. The magnitude (−34 ± 11%) and duration (23 ± 6 min) of these responses were significantly attenuated after 1 day of monoamine depletion. After 2 days of depletion, the sympathoinhibitory response was abolished and was replaced by a small, brief increase in SND (l0 3%). The pressor responses were similar in control and depleted rats, while the bradycardic response (−33 ± 4 bpm) was significantly reduced after 1 and 2 days of monoamine depletion to −20 ± 3 and −15 ± 2 bpm, respectively. We conclude that a functionally intact monoaminergic system is essential for the sympathoinhibitory response to cocaine. Whether the pressor responses result from a non-monoaminergic or a reserpine and/or α-methyl- p-tyrosine resistant catecholaminergic mechanism is unknown.

Capsaicin-induced stimulation of polymodal nociceptors is antagonized by Ruthenium Red independently of extracellular calcium

The dual effect of capsaicin on primary afferent neurons, excitation and stimulation of transmitter release, its dependence on extracellular calcium and its modulation by Ruthenium Red have been investigated in the rabbit ear. Injection of capsaicin into the central artery of the isolated perfused ear with intact neuronal connection induced a reflex fall in systemic arterial blood pressure of the anaesthetized rabbit. Addition of Ruthenium Red (0.6–20 μM) to the perfusate of the ear reversibly attenuated this response in a dose-dependent manner. Perfusion of the ear with a Ca 2+-free, 3 mM EGTA-containing physiological salt solution enhanced the capsaicin-evoked depressor reflex but did not prevent the inhibitory action of Ruthenium Red. Perfusion of the isolated rabbit ear with capsaicin (10 μM)-containing physiological salt solution induced the release of substance P-like immunoreactivity which was inhibited by Ruthenium Red (0.6–20 μM) and by omission of extracellular Ca 2+. The results demonstrate that capsaicin-evoked transmitter release is dependent on extracellular calcium while capsaicin-evoked excitation is not reduced in a Ca 2+-free perfusate. Both effects of capsaicin are potently inhibited by Ruthenium Red. The fact that capsaicin-induced excitation of primary afferents is antagonized by Ruthenium Red also in the absence of extracellular Ca 2+ suggests this inhibitory action of Ruthenium Red is not only mediated by inhibition of transmembrane Ca 2+ fluxes.

Effect of chloride ions on giant miniature end-plate potentials at the frog neuromuscular junction.

1. Frog sartorius muscles were incubated in Ringer solutions with a raised K+ concentration (high K+) and then allowed to recover in medium with a normal K+ concentration. During the recovery period miniature end-plate potentials (m.e.p.p.s) were recorded with intracellular electrodes. In addition, the acetylcholine (ACh) released from muscles in the presence of high K+ was measured by a mass spectrometric method. 2. Incubation in a high-K+ medium induced the appearance of giant miniature end-plate potentials (g.m.e.p.p.s). However, if the Cl- of the medium was substituted by propionate, very few g.m.e.p.p.s were observed. This was due to the absence of Cl- and not to the presence of propionate. The frequency of g.m.e.p.p.s was also greatly depressed when the Cl- concentration was lowered from 120 to 60 mM. 3. The amount of ACh released into a high-K+ medium was the same, regardless of whether Cl- or propionate was the anion. 4. When Cl- was replaced by NO3- or Br-, incubation in high-K+ Ringer solution induced the appearance of g.m.e.p.p.s. However, SO4(2-), like propionate, was unable to substitute for Cl- in this respect. 5. The frequency of g.m.e.p.p.s was correlated with that of m.e.p.p.s during the recovery period. However, when the K+ concentration was raised to 17 mM the frequency of m.e.p.p.s greatly increased, whereas that of the g.m.e.p.p.s did not change significantly. 6. G.m.e.p.p.s disappeared in the presence of curare, but persisted in the presence of tetrodotoxin or in a Ca2+-lacking medium. However, g.m.e.p.p.s failed to appear when the medium had lacked Ca2+ during the stimulation. 7. It is tentatively concluded that g.m.e.p.p.s are associated with Cl--dependent processes which occur after stimulation of transmitter release, and which are linked with the endocytotic retrieval of presynaptic membrane.

Free cytoplasmic Ca2+ and neurotransmitter release: studies on PC12 cells and synaptosomes exposed to alpha-latrotoxin.

The relationship between the free cytoplasmic Ca2+ concentration, [Ca2+]i, and neurotransmitter release was investigated in guinea pig brain synaptosomes and the neurosecretory cell line PC12. Release was induced by alpha-latrotoxin, which acts in both Ca2+ -containing and Ca2+ -free incubation media, or by the classical depolarizing agents high K+ and veratridine, which require extracellular Ca2+. Two complementary approaches were used to reveal changes of [Ca2+]i: (i) direct measurement by a fluorescent Ca2+ indicator (quin2) and (ii) study of the Ca2+ -dependent phosphorylation of a protein, synapsin I, located at the cytoplasmic surface of synaptic vesicles. Depolarizing agents, when applied in Ca2+ -containing medium, induced the [Ca2+]i to increase promptly 3- to 6-fold, drastically increased synapsin I phosphorylation, and caused stimulation of transmitter release. With alpha-latrotoxin, the [Ca2+]i increase was delayed and occurred at a slower rate, the increase of synapsin I phosphorylation was less drastic, and the release response was much more pronounced. In Ca2+ -free medium, depolarizing agents released no transmitter and had no effect on [Ca2+]i or synapsin I phosphorylation, whereas with alpha-latrotoxin these processes were dissociated: considerable stimulation of the release without apparent change of [Ca2+]i and synapsin I phosphorylation. We conclude that the relationship between average [Ca2+]i and transmitter release is not straightforward and, in particular, that the release evoked by alpha-latrotoxin in Ca2+ -free medium is mediated by a factor(s) other than bulk redistribution of Ca2+ from intracellular stores.

The effect of α-latrotoxin on the neurosecretory PC12 cell line: Studies on toxin binding and stimulation of transmitter release

α-Latrotoxin of black widow spider venom was found to bind with high affinity (K A = 1.8·10 9M −1) to specific sites present in discrete number (∼6300/cell, ∼12/aμn2) at the surface membrane of PC12 cells. This binding correlated with (and therefore, probably caused) the secretory response produced by the toxin. Binding was enhanced (∼ 2-fold) in the presence of mM concentrations of various divalent cations (Ca 2+, Mn 2+ and Co 2+) while Ba 2+ and Sr 2+ had a smaller effect and Mg 2+ was inactive. Hypertonicity, concanavalin A and trypsin pretreatment of the cells blocked the binding interaction. The α-latrotoxin-induced stimulation of 3H-dopamine release was massive and occurred very rapidly when cells were exposed to the toxin in a Ca 2+-containing Krebs-Ringer medium, whereas it occurred at a much slower rate in a Ca 2+-free, Mg 2+-containing Ringer. Introduction of Ca 2+ into the latter medium resulted in a shift of the release rate from slow to fast. In contrast, in divalent cation-free medium the response was abolished. The toxin-induced secretory response was unaffected by Na 2+ and Ca 2+ channel blockers (tetrodotoxin and D600) as well as by calmodulin inhibitors (calmidazolium and trifluoperazine). The effects of Ca 2+ and Mg 2+ were found to be concentration-dependent, with half maximal responses occurring at approximately 0.3 and l.5mM for the two divalent cations, respectively. Other divalent cations could substitute for Ca 2+ and Mg 2+, the relative efficacy being Sr 2+ > Ca 2+ ⩾> Ba 2+ > Mn 2+ > Mg 2+ > Co 2+. Moreover, the response occurring at suboptimal concentration of Ca 2+ (0.4 mM) was potentiated by the concomitant addition of either Mg 2+, Mn 2+ or Co 2+. The effect(s) of divalent cations in supporting the α-latrotoxin-induced release response seem(s) to occur primarily at step(s) beyond toxin binding because (a) the stimulatory effects of the various cations on release were not matched by parallel effects on binding (b) Ca 2+ maintained its ability to stimulate fast release even when toxin binding had occurred in a Ca 2+-free medium. Delays in the release responses were observed when cells were exposed to αLTx in Na 2+-free, glucosamine or methylamine-based media, or depolarized with high K 2+ (in the presence of D600) before toxin treatment. Moreover, in these two conditions the ability of Mg 2+ to support the αLTx response was considerably decreased. Taken together with previous data of the literature these results appear consistent with the hypothesis that αLTx acts by activating a ‘non-conventional’ cation channel of low ionic specificity in the plasma membrane. This would result in a stimulated influx of various divalent cations, not only Ca 2+ but also Mg 2+ and the others investigated, which would then be able to stimulate the release response, although with different efficacy.

Studies on alpha-latrotoxin receptors in rat brain synaptosomes: correlation between toxin binding and stimulation of transmitter release.

alpha-Latrotoxin (alpha-LT), the major component of black widow spider venom, is a high-molecular-weight protein that acts presynaptically by stimulating the release of stored neurotransmitters. The purified toxin was iodinated to high specific radioactivity by the Bolton-Hunter procedure, without appreciable loss of biological activity. By the use of the 125I-toxin, specific receptors were revealed in synaptosome fractions isolated from various regions of the rat brain, but not in nonneural tissues. The density of alpha-LT receptors [which are probably composed of, or include, membrane protein(s)] varies between 0.6 and 0.88 pmol/mg of synaptosome protein, their affinity is very high (KA of the order of 10(10) M-1), their association rate is fast, and their dissociation rate slow. They might belong to a single, homogeneous class. This last conclusion, however, is still uncertain, because results suggesting a possible heterogeneity were obtained by studying the dissociation of the toxin from synaptosomes incubated in high-salt buffer. Experiments in which the binding of alpha-LT and its dopamine release activity in striatal synaptosomes were investigated in parallel in a variety of experimental conditions support the hypothesis that occupation of the high-affinity receptors is the initial step in the alpha-LT activation of the presynaptic response.

Stimulation of transmitter release by guanidine derivatives

Guanidine and its alkylated derivatives stimulate transmitter release in the sciatic nerve-sartorius muscle preparation of the frog, the excitatory nerve-opener muscle preparation of the crayfish cheloped, and the phrenic nerve-diaphragm preparation of the rat. The mechanism underlying this action has been studied. The amplitude of the end-plate potential was increased by guanidine derivatives. Guanidine, methylguanidine, 1,1-dimethylguanidine, ethylguanidine and propylguanidine were almost equally potent with the threshold concentration of 0.1–0.2mM. Formamidine and aminoguanidine were 20–40 times less potent than guanidine. This effect was reversed slowly after washing with normal solution free of drugs. However, the frequency of miniature end-plate potentials was not affected by high concentrations (5–10mM) of guanidine, and no change was observed in membrane potential and input resistance following application of guanidine, methylguanidine and 1,1-dimethylguanidine. The iontophoretically induced acetylcholine potential was suppressed by methylguanidine, 1,1-dimethylguanidine, ethylguanidine and propylguanidine, and was restored quickly upon washing with normal solution. Based on the actions on the end-plate potential and on the iontophoretic acetylcholine potential, the order of the potencies of guanidine derivatives in stimulating transmitter release were estimated as: methylguanidine, 1,1-dimethylguanidine, ethylguanidine, propylguanidine > guanidine > aminoguanidine, formamidine. Amylguanidine and octylguanidine had no presynaptic effect. Increasing external calcium concentration from 1.8 mM to 6 or 12 mM did not prevent the stimulating action of guanidine and 1,1-dimethylguanidine on the end-plate potential. Methylguanidine and 1,1-dimethylguanidine did not seem to affect the time constant of decay of neuromuscular facilitation in the rat. The quantal content of the end-plate potential in the rat preparation was increased by methylguanidine and 1,1-dimethylguanidine, and this increase was due entirely to an increase in the immediately available store of acetylcholine in the nerve terminal. The probability of release remained constant. It is unlikely that this action is due to the inhibition of mitochondrial respiration.