The Role of Endogenous Calcitonin Gene-Related Peptide in the Neurotransmitter Quantal Size Increase in Mouse Neuromuscular Junctions

Changes in parameters of spontaneous acetylcholine (ACh) quantal secretion caused by prolonged high-frequency burst activity of neuromuscular junctions and possible involvement of endogenous calcitonin gene-related peptide (CGRP) and its receptors in these changes were studied. With this purpose, miniature endplate potentials (MEPPs) were recorded using standard microelectrode technique in isolated neuromuscular preparations of m. EDL–n. peroneus after a prolonged high-frequency nerve stimulation (30 Hz for 2 min). An increase in the MEPP amplitudes and time course was observed in the postactivation period that reached maximum 20–30 min after nerve stimulation and progressively faded in the following 30 min of recording. Inhibition of vesicular ACh transporter with vesamicol (1 μM) fully prevented this “wave” of the MEPP enhancement. This indicates the presynaptic origin of the MEPP amplitude increase, possibly mediated via intensification of synaptic vesicle loading with ACh and subsequent increase of the quantal size. Competitive antagonist of the CGRP receptor, truncated peptide isoform CGRP_8–37 (1 μM), had no effect on spontaneous secretion parameters by itself but was able to prevent the appearance of enhanced MEPPs in the postactivation period. This suggests the involvement of endogenous CGRP and its receptors in the observed MEPP enhancement after an intensive nerve stimulation. Ryanodine in high concentration (1 μM) that blocks ryanodine receptors and stored calcium release did not influence spontaneous ACh secretion but prevented the increase of the MEPP parameters in the postactivation period. Altogether, the data indicate that an intensive nerve stimulation, which activates neuromuscular junctions and muscle contractions, leads to a release of endogenous CGRP into synaptic cleft and this release strongly depends on the efflux of stored calcium. The released endogenous CGRP is able to exert an acute presynaptic effect on nerve terminals, which involves its specific receptor action and intracellular cascades leading to intensification of ACh loading into synaptic vesicles and an increase in the ACh quantal size.     

Effect of N-acetylaspartylglutamate (NAAG) on non-quantal and spontaneous quantal release of acetylcholine at the neuromuscular synapse of rat

N-Acetylaspartylglutamate (NAAG), known to be present in rat motor neurons, may participate in neuronal modulation of non-quantal secretion of acetylcholine (ACh) from motor nerve terminals. Non-quantal release of ACh was estimated by the amplitude of the endplate membrane hyperpolarization (H-effect) caused by inhibition of nicotinic receptors by (+)-tubocurarine and acetylcholinesterase by armin (diethoxy-p-nitrophenyl phosphate). Application of exogenous NAAG decreased the H-effect in a dose-dependent manner. The reduction of the H-effect by NAAG was completely removed when N-acetyl-beta-aspartylglutamate (betaNAAG) or 2-(phosphonomethyl)-pentanedioic acid (2-PMPA) was used to inhibit glutamate carboxypeptidase II (GCP II), a presynaptic Schwann cell membrane-associated ectoenzyme that hydrolyzes NAAG to glutamate and N-acetylaspartate. Bath application of glutamate decreased the H-effect similarly to the action of NAAG but N-acetylaspartate was without effect. Inhibition of NMDA receptors by dl-2-amino-5-phosphopentanoic acid, (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine (MK801), and 7-chlorokynurenic acid or inhibition of muscle nitric oxide synthase (NO synthase) by N(G)-nitro-l-arginine methyl ester and 3-bromo-7-nitroindazole completely prevented the decrease of the H-effect by NAAG. These results suggest that glutamate, produced by enzymatic hydrolysis of bath-applied NAAG, can modulate non-quantal secretion of ACh from the presynaptic terminal of the neuromuscular synapse via activation of postsynaptic NMDA receptors and synthesis of nitric oxide (NO) in muscle fibers. NAAG also increased the frequency of miniature endplate potentials (mEPPs) generated by spontaneous quantal secretion of ACh, whereas the mean amplitude and time constants for rise time and for decay of mEPPs did not change.     

Changes in the Parameters of Quantal Acetylcholine Release after Activation of PAR1-Type Thrombin Receptors at the Mouse Neuromuscular Junctions

In mature and newly formed neuromuscular synapses of mouse skeletal muscles, miniature endplate potentials (MEPPs) and multiquantal endplate potentials (EPPs) evoked by a single stimulation of the nerve were recorded using intracellular microelectrode technique. The mechanisms underlying the changes in spontaneous and evoked acetylcholine (ACh) release caused by the activation of PAR1-type muscle receptors induced by their peptide agonist TRAP6-NH₂ were studied. It has been shown for the first time that, in either mature or newly formed motor synapses, the activation of PAR1 that lack presynaptic localization causes a sustained increase in the MEPP amplitude due to the increase in the ACh quantal size at the presynaptic level. It was found that phospholipase C (PLC) participates in the signaling mechanism triggered by the PAR1 activation. Exogenously applied brain-derived neurotrophic factor (BDNF) mimics the effect of activation of PAR1 by TRAP6-NH₂. Moreover, an increase in the MEPP amplitude caused by the peptide PAR1 agonist was fully prevented by blocking the BDNF receptors–tropomyosin receptor kinases B (TrkB). Thus, it has been shown for the first time that the increase in ACh quantal size due to the activation of PAR1 in motor synapses is mediated by a complex signaling cascade that starts at the postsynaptic level of the motor synapse and ends at the presynaptic level. It is expected that the activation of PAR1 at the muscle fiber membrane followed by the PLC upregulation results in the release of neurotrophin BDNF as a retrograde signal. Its effect on the presynaptic TrkB receptors triggers the cascade leading to an increase in the quantal size of ACh.     

Non-quantal secretion of acetylcholine from motor nerve endings: Molecular mechanisms, physiological role, and regulation

Results of biochemical and electrophysiological experiments allowing researchers to identify non-quantal release of acetylcholine (ACh), in addition to quantal release, from motor nerve endings, are discussed in the lecture. Based on the analysis of our own data and publications of other experimenters on the dependence of non-quantal secretion on the composition of the ion milieu, sensitivity of this phenomenon to different physiologically active compounds, and peculiarities of its temperature dependence, the authors conclude that non-quantal secretion of the transmitter is an active transport process, and not a passive leakage of ACh from the cytoplasm of the nerve terminal. It is hypothesized that a high-affinity system of choline uptake can play the role of the ACh carrier through the presynaptic membrane. The involvement of non-quantal release in the control of electrogenesis in the muscle fiber and the relation between processes of quantal and non-quantal secretion of the transmitter providing adequate functioning of the nerve terminal in the resting state and in the course of long-lasting high-frequency rhythmic activity are described. Data on the ability of glutamate and nitric oxide to selectively modulate this type of neurosecretion are analyzed. The possible role of non-quantal secretion of ACh in pathogenesis of intoxications of ACh esterase is discussed. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 352–363, July–October, 2007.     

Muscarinic M1 acetylcholine receptors regulate the non-quantal release of acetylcholine in the rat neuromuscular junction via NO-dependent mechanism

Nitric oxide (NO), previously demonstrated to participate in the regulation of the resting membrane potential in skeletal muscles via muscarinic receptors, also regulates non-quantal acetylcholine (ACh) secretion from rat motor nerve endings. Non-quantal ACh release was estimated by the amplitude of endplate hyperpolarization (H-effect) following a blockade of skeletal muscle post-synaptic nicotinic receptors by (+)-tubocurarine. The muscarinic agonists oxotremorine and muscarine lowered the H-effect and the M1 antagonist pirenzepine prevented this effect occurring at all. Another muscarinic agonist arecaidine but-2-ynyl ester tosylate (ABET), which is more selective for M2 receptors than for M1 receptors and 1,1-dimethyl-4-diphenylacetoxypiperidinium (DAMP), a specific antagonist of M3 cholinergic receptors had no significant effect on the H-effect. The oxotremorine-induced decrease in the H-effect was calcium and calmodulin-dependent. The decrease was negated when either NO synthase was inhibited by N(G)-nitro-L-arginine methyl ester or soluble guanylyl cyclase was inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. The target of muscle-derived NO is apparently nerve terminal guanylyl cyclase, because exogenous hemoglobin, acting as an NO scavenger, prevented the oxotremorine-induced drop in the H-effect. These results suggest that oxotremorine (and probably also non-quantal ACh) selectively inhibit the non-quantal secretion of ACh from motor nerve terminals acting on post-synaptic M1 receptors coupled to Ca(2+) channels in the sarcolemma to induce sarcoplasmic Ca(2+)-dependent synthesis and the release of NO. It seems that a substantial part of the H-effect can be physiologically regulated by this negative feedback loop, i.e., by NO from muscle fiber; there is apparently also Ca(2+)- and calmodulin-dependent regulation of ACh non-quantal release in the nerve terminal itself, as calmidazolium inhibition of the calmodulin led to a doubling of the resting H-effect.     

Three types of transmitter release from embryonic neurons

Prior to the contact with their target muscle cells in culture, growth cones of many isolated Xenopus embryonic neurons release acetylcholine (ACh) spontaneously. Using patch clamp techniques, this release can be detected by an outside-out patch of muscle membrane placed near the growth cone. Intracellular recording from innervated muscle cells showed spontaneous miniature endplate potentials (MEPPs) of varying amplitudes. Amplitude histograms showed a skewed distribution with multiple peaks, suggesting the existence of subunits in either the quantal packages of ACh released by the nerve terminal or in the postsynaptic muscle response. In addition to the quantal ACh release reflected by MEPPs, nerve terminal also release a large amount of ACh in a non-quantal fashion. This non-quantal ACh release is revealed by the hyperpolarization of the muscle membrane following extracellular application of curare or alpha-bungarotoxin, as well as by denervation of the muscle cell.     

Arachidonic acid metabolites alter G protein-mediated signal transduction in heart. Effects on muscarinic K+ channels

The muscarinic acetylcholine receptor (mAChR)-stimulated, inwardly rectifying K+ current (IK [ACh]) was examined in single bullfrog atrial cells using the whole-cell patch clamp technique. IK[ACh] was activated either by bath addition of 1 microM ACh or via activation of the G protein, Gk, with guanosine-gamma-thiotriphosphate (GTP gamma S). Arachidonic acid (AA) modulated IK[ACh] under both conditions. AA decreased mAChR-stimulated IK[ACh] and increased the rate of decay from the peak current (desensitization). In addition, AA affected GTP gamma S-activated IK[ACh] by modulation of Gk. The effects of AA and its metabolites on Gk were assessed by examining their effects on both the basal rate of Gk activation by GTP gamma S, and the mAChR-mediated increase in activation rate produced by nanomolar ACh. AA increased the basal rate of GTP gamma S-mediated IK[ACh] activation, but reduced the ACh-induced augmentation of this rate. All of the effects of AA on GTP gamma S-mediated IK[ACh] activation were produced by metabolites. A lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), decreased the basal and ACh-enhanced rate of IK[ACh] activation in both the presence and absence of exogenous AA. In contrast, indomethacin (INDO), a cyclooxygenase inhibitor, increased the basal rate of IK[ACh] activation by GTP gamma S in both the presence and absence of exogenous AA, and reversed the effects of AA on the ACh-augmented basal rate. AA metabolites produced via lipoxygenase and cyclooxygenase pathways thus have opposing effects on the signal transduction pathway from mAChR to IK[ACh]. We directly tested a lipoxygenase pathway metabolite, LTC4, on GTP gamma S-mediated IK[ACh] activation and found that it not only overcame the inhibitory effects of NDGA, but also increased both the basal and ACh-augmented rate of IK[ACh] activation. From these data, we propose that AA metabolites modulate the function of Gk by altering its kinetic properties.     

The influence of hindlimb unloading on the effectiveness of modulation of the mediator secretion via the autoreceptor system

In experiments on neuromuscular synapses of rat fast (m. Extensor digitorum longus, EDL) and slow (m. soleus) skeletal muscles, changes in the intensity of spontaneous quantal mediator secretion in response to the activation of presynaptic cholinoreceptors by the nonhydrolyzable acetylcholine analogue carbachol and to an increase in K+ concentration in the control group of animals and in animals subjected to different terms of unloading of hindlimbs have been compared. The intensity of spontaneous secretion of mediator quanta was evaluated from the mean frequency of miniature endplate potentials. In the control group of animals, the frequency of miniature endplate potentials by the action of carbachol increased by 363% in m. EDL and by 62% in m. soleus. The frequency of miniature endplate potentials in the synapses of m. EDL was more sensitive to K(+)-induced depolarization too. The bearing unloading of hindlimbs abolished the sensitivity of spontaneous secretion to carbachol in the synapses of m. EDL, whereas in m. soleus it was unchanged. However, the preservation of sensitivity of nerve endings of fast muscle to K(+)-induced depolarization allows one to assume that the hindlimb unloading leads to a decrease in the number of functioning presynaptic receptors.     

Glutamate regulation of non-quantal release of acetylcholine in the rat neuromuscular junction

Glutamate, previously demonstrated to participate in regulation of the resting membrane potential in skeletal muscles, also regulates non-quantal acetylcholine (ACh) secretion from rat motor nerve endings. Non-quantal ACh secretion was estimated by the amplitude of endplate hyperpolarization (H-effect) following blockade of skeletal muscle post-synaptic nicotinic receptors by (+)-tubocurarine and cholinesterase by armin (diethoxy-p-nitrophenyl phosphate). Glutamate was shown to inhibit non-quantal release but not spontaneous and evoked quantal secretion of ACh. Glutamate-induced decrease of the H-effect was enhanced by glycine. Glycine alone also lowered the H-effect, probably due to potentiation of the effect of endogenous glutamate present in the synaptic cleft. Inhibition of N-methyl-d-aspartate (NMDA) receptors with (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine (MK801), dl-2-amino-5-phosphopentanoic acid (AP5) and 7-chlorokynurenic acid or the elimination of Ca2+ from the bathing solution prevented the glutamate-induced decrease of the H-effect with or without glycine. Inhibition of muscle nitric oxide synthase by NG-nitro-l-arginine methyl ester (l-NAME), soluble guanylyl cyclase by 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and binding and inactivation of extracellular nitric oxide (NO) by haemoglobin removed the action of glutamate and glycine on the H-effect. The results suggest that glutamate, acting on post-synaptic NMDA receptors to induce sarcoplasmic synthesis and release of NO, selectively inhibits non-quantal secretion of ACh from motor nerve terminals. Non-quantal ACh is known to modulate the resting membrane potential of muscle membrane via control of activity of chloride transport and a decrease in secretion of non-quantal transmitter following muscle denervation triggers the early post-denervation depolarization of muscle fibres.     

Modification of frequency augmentation-potentiation by GTPγS in the frog neuromuscular junction

The effect of modifiers of guanine nucleotide-binding proteins (G proteins) on the frequency augmentation-potentiation of transmitter release were studied in the frog neuromuscular junction. Using Genetransfer^R as a carrier the mean quantal content of the endplate potential increased by penetration of GTPγS into the presynaptic nerve terminal. Neither GTPγS alone nor carrier alone had any effect. The relationship of log (mean quantal content) versus stimulation frequency changed from a single linear to a dual linear function, suggesting that the immediately releasable pool was modified. GDPβS + carrier also had similar effects, but was less potent. Aluminium fluoride was without effect. Extracellularly recorded presynaptic nerve action potentials remained unchanged with GTPγS + carrier. Also, GTPγS + carrier did not affect the action potential nor the cytosolic Ca²⁺ concentration in differentiated NG108–15 hybrid cells. It is suggested that some smg-type G protein-dependent processes are involved in determining frequency augmentation-potentiation.     

Effect of carbachol on spontaneous transmitter release from rat motor nerve endings depending on extracellular potassium concentration

The effect of carbachol (10 µM) on the frequency of miniature end-plate potentials (MEPP) was studied in experiments on the Wistar rat soleus muscle during a change in extracellular potassium concentration from 2 to 15 mM. Between the range of potassium concentrations from 2 to 7.5 mM the cholinomimetic had no effect on spontaneous transmitter release. In higher potassium concentrations carbachol caused an increase in the frequency of MEPP. This facilitatory effect increased in strength with an increase in potassium concentration; at 15 mM the frequency of MEPP was increased up to 160%. The results confirmed the previous hypothesis that the action of the mimetic on spontaneous transmitter release, relaized through presynaptic acetylcholine receptors, depends on the initial level of polarization of nerve endings.S. V. Kurashov Kazan' State Medical Institute. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 470–475, July–August, 1984.     

Effect of 2-(4-Phenylpiperidino)cyclohexanol on Acetylcholine Release and Subcellular Distribution in Rat Striatal Slices

These experiments measured the effect of 2-(4-phenylpiperidino)cyclohexanol (AH5183) on the release of acetylcholine (ACh) and its subcellular distribution in slices of rat striatum incubated in vitro. The AH5183, a drug that blocks the uptake of ACh by isolated synaptic vesicles, reduced the release of ACh from slices stimulated to release transmitter in response to K+ depolarization. Tissue stimulated in the presence of AH5183 contained more ACh in a nerve terminal cytoplasmic fraction than did tissue stimulated in the drug's absence, but stimulation in AH5183's presence reduced the amount of ACh measured in fractions containing synaptic vesicles. The depletion of ACh caused by stimulating tissue in the presence of AH5183 was more evident in the fraction of nerve terminal ACh occluded within synaptic vesicles as isolated by gradient centrifugation (fraction D) than it was in other nerve terminal occluded stores. It is concluded that the synaptic vesicles isolated as fraction D under the present experimental conditions likely contain releasable transmitter. The AH5183 also depressed the spontaneous release of ACh from incubated slices of striatum and this effect was evident in the presence or the absence of medium Ca2+. It is suggested that this effect might indicate that the process of spontaneous ACh release measured neurochemically results, in part, from an AH5183-sensitive carrier-mediated process.     

Amphetamine effects on acetylcholine release and mammalian neuromuscular transmission

The mechanisms by which (+)-amphetamine biphasically modifies neuromuscular transmission were studied in the rat phrenic nervediaphragm preparation. Low to moderate amphetamine concentrations (30–300 μM) enhanced twitch height and potentiated the nerve stimulated release of acetylcholine (ACh) by up to 4.8-fold from the phrenic nerve. Higher amphetamine concentrations depressed muscle twitch and ACh release. Using a cannulated diaphragm preparation, amphetamine enhanced the twitch response to nerve stimulation but markedly depressed the contractions elicited by a pulsed injection of ACh. Amphetamine-induced enhancement of ACh release was prevented by pretreatment of animals with α-methyl-p-tyrosine, suggesting that amphetamine may be acting indirectly by releasing catecholamines. These results support the hypothesis that amphetamine enhancement results from a presynaptic increase in ACh release and the blocking actions are mediated by a postsynaptic inhibitory effect.     

A retrograde signal is involved in activity-dependent remodeling at a C. elegans neuromuscular junction

We have characterized how perturbations of normal synaptic activity influence the morphology of cholinergic SAB motor neurons that innervate head muscle in C. elegans. Mutations disrupting components of the presynaptic release apparatus, acetylcholine (ACh) synthesis or ACh loading into synaptic vesicles each induced sprouting of SAB axonal processes. These sprouts usually arose in the middle of the normal innervation zone and terminated with a single presynaptic varicosity. Sprouting SAB neurons with a similar morphology were also observed upon reducing activity in muscle, either by using mutants lacking a functional nicotinic ACh receptor subunit or through muscle-specific expression of a gain-of-function potassium channel. Analysis of temperature-sensitive mutants in the choline acetyltransferase gene revealed that the sprouting response to inactivity was developmentally regulated; reduction of synaptic activity in early larval stages, but not in late larval stages, induced both sprouting and addition of varicosities. Our results indicate that activity levels regulate the structure of certain synaptic connections between nerve and muscle in C. elegans. One component of this regulatory machinery is a retrograde signal from the postsynaptic cell that mediates the formation of synaptic connections.     

Action of a beta-bungarotoxin on autonomic ganglia and adrenergic neurotransmission.

A beta-bungarotoxin was isolated from the venom of Bungarus multicinctus by column chromatography on Sephadex G-50 and SP-Sephadex. The toxin produced presynaptic effects on neuromuscular transmission with characteristics similar to those described by others. In a sympathetic ganglion, the toxin increased spontaneous acetylcholine (ACh) release and decreased ACh release evoked by preganglionic nerve stimulation. The toxin did not block the response of isolated ileum to cholinergic nerve stimulation, did not block the release of noradrenaline from the adrenergic nerve terminals of a nictitating membrane preparation, and did not alter the responses of smooth and cardiac muscle preparations to noradrenaline. It is suggested that the specificity of beta-bungarotoxin for certain nerve terminals is related either to selective binding of the toxin or to the selective presence of a necessary substrate for its action. An attempt to show selective binding of 125I-toxin to cholinergic nerve terminals in skeletal muscle was not successful.     

Effect of clonidine on neuromuscular transmission and the nicotinic acetylcholine receptor

The effects of clonidine on neuromuscular transmission were investigated in the mouse phrenic nerve-diaphragms and chicken biventer cervicis. Clonidine inhibited the indirect twitch response dose-dependently and reversibly without an effect on the direct response of the muscles to electrical stimulation and KCl. This effect was antagonized effectively by diaminopyridine but not by yohimbine, phentolamine or physostigmine. The quantal content was not affected although the amplitudes of end-plate potential (epp) and spontaneous miniature epp (mepp) were markedly depressed. Clonidine also decreased the slope of the ACh dose-response curve and maximal response in denervated mouse diaphragms as well as the carbachol response in the chinck muscle. In the latter, ACh response was not depressed by clonidine probably because of its inherent anticholinesterase activity. Clonidine facilitated the fading of ACh-contracture either in mouse or chick muscle. It is concluded that clonidine impairs the neuromuscular transmission by a noncompetitive blockade of ACh receptors, most likely affecting the ACh channel but not the recognition site of the ACh receptor. Its inhibitory effect is not mediated by alpha 2-adrenoceptor, suggesting that there is no alpha 2-adrenoceptor on the motor nerve terminal to modulate the transmitter release.     

3H]acetylcholine synthesis in cultured ciliary ganglion neurons: effects of myotube membranes

Avian ciliary ganglion neurons in cell culture were examined for the capacity to synthesize acetylcholine (ACh) from the exogenously supplied precursor, choline. Relevant kinetic parameters of the ACh synthetic system in cultured neurons were found to be virtually the same as those of the ganglionic terminals in the intact iris. Neurons were cultured in the presence of and allowed to innervate pectoral muscle; this results in an capacity for ACh synthesis. In particular, the ability to increase ACh synthesis upon demand after stimulation is affected by interaction with the target. This effect is shown to be an acceleration of the maturation of the cultured neurons. Lysed and washed membrane remnants of the muscle target were able to duplicate, in part, this effect of live target tissue on neuronal transmitter metabolism. Culture medium conditioned by muscle, and by the membrane remnants of muscle, was without significant effect. Thus, substances secreted into the medium do not play a major role in this interaction. Neurons cultured with either muscle or muscle membrane remnants formed large, elongate structures on the target membrane surface. These were not seen in the absence of the target at the times examined. This morphological difference in terminal-like structures may parallel the developmental increases in size and vesicular content of ciliary ganglion nerve terminals in the chick iris, and may relate to the increased ACh synthetic activity. The results suggest that direct contact with an appropriate target membrane has a profound, retrograde influence upon neuronal metabolic and morphological maturation.     

Facilitation of spontaneous acetylcholine release induced by activation of cAMP in rat neuromuscular junctions

Regulation of neurotransmitter release is thought to involve modulation of the release probability by protein phosphorylation. Activation of the cAMP-protein kinase A (PKA) pathway has been shown to facilitate synaptic transmission in mammalian neuromuscular synapses, although the relevant phosphorylation targets are mostly unknown. We found that the inhibitor of the phosphodiesterase aminophylline (1 mM AMIN), the membrane-permeable analog of cAMP, 8-Br-cAMP (5 mM) and, the direct adenylate cyclase activator, forskolin (20 microM), induced an increase of miniature end-plate potentials (MEPPs) frequency in rat neuromuscular junctions. We investigated the possible involvement of the voltage-dependent calcium channels (VDCC), since these proteins are known to be phosphorylated by PKA. But this possibility was ruled out, since the increase in MEPPs frequency was not attenuated by the VDCC blocker Cd2+ (100 microM) and it was observed when AMIN was studied on hyperosmotic response, which is independent of [Ca2+]o and of Ca2+ influx through the VDCC. The lack of action of AMIN on MEPPs frequency when [Ca2+]i was diminished by exposing the preparations to zero Ca2+-EGTA solution (isotonic condition) or when nerve terminals were loaded with a permeant Ca2+ chelator (BAPTA-AM) (hypertonic condition), indicate that cAMP-mediated presynaptic facilitation is a function of nerve terminal Ca2+ concentration. We also found that AMIN exerted a comparable increase in MEPPs frequency in control and high K+ (10 and 15 mM), suggesting a single mechanism of action for spontaneous and K+-induced secretion.     

Mechanism of acetylcholine-induced inhibition of Ca current in bullfrog atrial myocytes

The mechanism of the anti-beta-adrenergic action of acetylcholine (ACh) on Ca current, ICa, was examined using the tight-seal, whole-cell voltage clamp technique in single atrial myocytes from the bullfrog. Both isoproterenol (ISO) and forskolin increased ICa dose dependently. After ICa had been enhanced maximally by ISO (10(-6) M), subsequent application of forskolin (50 microM) did not further increase ICa, suggesting that ISO and forskolin increase ICa via a common biochemical pathway, possibly by stimulation of adenylate cyclase. ACh (10(-5) M) completely inhibited the effect of low doses of forskolin (2 x 10(-6) M), as well as ISO, but it failed to block the effects of high doses of forskolin (greater than 5 x 10(-5) M). Intracellular application of cyclic AMP (cAMP) also increased ICa. ACh (10(-5) M) failed to inhibit this cAMP effect, indicating that the inhibitory action of ACh occurs at a site proximal to the production of cAMP. ACh (10(-5) M) also activated an inwardly rectifying K+ current IK(ACh). Intracellular application of a nonhydrolyzable GTP analogue, GTP gamma S (5 X 10(-4) M), activated IK(ACh) within several minutes; subsequent application of ACh (10(-5) M) did not increase IK(ACh) further. These results demonstrate that a GTP-binding protein coupled to these K+ channels can be activated maximally by GTP gamma S even in the absence of ACh. Intracellular application of GTP gamma S also strongly inhibited the effect of ISO on ICa in the absence of ACh. Pertussis toxin (IAP) completely prevented both the inhibitory effect of ACh on ICa and the ACh-induced activation of IK(ACh). GTP gamma S (50 microM-1 mM) alone did not increase ICa significantly; however, when ISO was applied first, GTP gamma S (5 x 10(-4) M) gradually inhibited the ISO effect on ICa. These results indicate that ACh antagonizes the effect of ISO on ICa via a GTP-binding protein (Gi and/or Go). This effect may be mediated through a direct inhibition by the alpha-subunit of Gi which is coupled to the adenylate cyclase.