Pharmacological elevation of cyclic AMP and transmitter release at the mouse neuromuscular junction

1988 ◽  
Vol 66 (2) ◽  
pp. 207-212 ◽  
Author(s):  
W. F. Dryden ◽  
Y. N. Singh ◽  
T. Gordon ◽  
G. Lazarenko
Keyword(s):  
Cyclic Amp ◽  
Neuromuscular Junction ◽  
Cholera Toxin ◽  
Time Course ◽  
Second Messengers ◽  
Phosphodiesterase Inhibitors ◽  
Spontaneous Release ◽  
End Plate ◽  
Release Of Acetylcholine

Intracellular recordings of spontaneous and evoked end-plate potentials have been made at the neuromuscular junction of mouse hemidiaphragms to determine a possible role of cyclic AMP (cAMP) in the release of acetylcholine from presynaptic terminals. Spontaneous release, as determined from the frequency of miniature end-plate potentials, was increased by drugs that inhibit phosphodiesterase: isobutylmethylxanthine (IBMX), SQ 20,009, theophylline, and caffeine; drugs that stimulate adenylate cyclase: forskolin, fluoride, and cholera toxin, and the stable analogue of cAMP: 8-bromo-cAMP but not dibutyryl cAMP. Release increased with time during maintained exposure to the drugs and generally followed a simple exponential time course with time constants ranging from 8 to 17 min at 20 °C, except for SQ 20,009 and cholera toxin which required longer exposure times for effect. The order of potency of the phosphodiesterase inhibitors was IBMX = SQ 20,009 > theophylline = caffeine. This is consistent with an effect mediated by an increase in cAMP concentrations within the nerve terminal. Evoked release, determined from the quantal content of the end-plate potential, was increased to a lesser extent than spontaneous release. The results are discussed with reference to the possible involvement of second messengers in the release of vesicles from nerve terminals in vertebrate synapses.

scholarly journals The Role of Cyclic Nucleotides in the CNS

1977 ◽  
Vol 4 (3) ◽  
pp. 151-195 ◽  
Author(s):  
John W. Phillis
Keyword(s):  
Cyclic Amp ◽  
Synaptic Transmission ◽  
Cyclic Gmp ◽  
Adenosine Receptor ◽  
Cyclic Nucleotides ◽  
Phosphodiesterase Inhibitors ◽  
Adenosine Uptake ◽  
Injection Techniques ◽  
Firm Basis

SUMMARY:On the basis of the information presented in this review, it is difficult to reach any firm decision regarding the role of cyclic AMP (or cyclic GMP) in synaptic transmission in the brain. While it is clear that cyclic nucleotide levels can be altered by the exposure of neural tissues to various neurotransmitters, it would be premature to claim that these nucleotides are, or are not, essential to the transmission process in the pre- or postsynaptic components of the synapse. In future experiments with cyclic AMP it will be necessary to consider more critically whether the extracellularly applied nucleotide merely provides a source of adenosine and is thus activating an extracellularly located adenosine receptor, or whether it is actually reaching the hypothetical sites at which it might act as a second messenger. The application of cyclic AMP by intracellular injection techniques should minimize this particular problem, although possibly at the expense of new difficulties. Prior blockade of the adenosine receptor with agents such as theophylline or adenine xylofuranoside may also assist in the categorization of responses to extracellularly applied cyclic AMP as being a result either of activation of the adenosine receptor or of some other mechanism. Ultimately, the development of highly specific inhibitors for adenylate cyclase should provide a firm basis from which to draw conclusions about the role of cyclic AMP in synaptic transmission. Similar considerations apply to the actions of cyclic GMP and the role of its synthesizing enzyme, guanylale cyclase.The use of phosphodiesterase inhibitors in studies on cyclic nucleotides must also be approached with caution. The diverse actions of many of these compounds, which include calcium mobilization and block of adenosine uptake, could account for many of the results that have been reported in the literature.

scholarly journals Receptor-mediated gonadotropin action in the ovary. Regulatory role of cyclic nucleotide phosphodiesterase(s) in intracellular adenosine 3′:5′-cyclic monophosphate turnover and gonadotropin-stimulated progesterone production by rat ovarian cells

1979 ◽  
Vol 180 (1) ◽  
pp. 201-211 ◽  
Author(s):  
Salman Azhar ◽  
K. M. Jairam Menon
Keyword(s):  
Cyclic Amp ◽  
Kinase Activity ◽  
Phosphodiesterase Inhibitors ◽  
Protein Kinase Activity ◽  
Dibutyryl Cyclic Amp ◽  
Steroid Production ◽  
Progesterone Production ◽  
Ovarian Cells ◽  
Cyclic Amp Accumulation

The regulatory role of cyclic nucleotide phosphodiesterase(s) and cyclic AMP metabolism in relation to progesterone production by gonadotropins has been studied in isolated rat ovarian cells. Low concentrations of choriogonadotropin (0.4–5ng/ml) increased steroid production without any detectable increase in cyclic AMP, when experiments were carried out in the absence of phosphodiesterase inhibitors. The concentration of choriogonadotropin (10ng/ml) that stimulated progesterone synthesis maximally resulted in a minimal increase in cyclic AMP accumulation and choriogonadotropin binding. Choriogonadotropin at a concentration of 10ng/ml and higher, however, significantly stimulated protein kinase activity and reached a maximum between 250 and 1000ng of hormone/ml. Higher concentrations (50–2500ng/ml) of choriogonadotropin caused an increase in endogenous cyclic AMP, and this increase preceded the increase in steroid synthesis. Analysis of dose–response relationships of gonadotropin-stimulated cyclic AMP accumulation, progesterone production and protein kinase activity revealed a correlation between these responses over a wide concentration range when experiments were performed in the presence of 3-isobutyl-1-methylxanthine. The phosphodiesterase inhibitors papaverine, theophylline and 3-isobutyl-1-methylxanthine each stimulated steroid production in a dose-dependent manner. Incubation of ovarian cells with dibutyryl cyclic AMP or 8-bromo cyclic AMP mimicked the steroidogenic action of gonadotropins and this effect was dependent on both incubation time and nucleotide concentration. Maximum stimulation was obtained with 2mm-dibutyryl cyclic AMP and 8-bromo cyclic AMP, and this increase was close to that produced by a maximally stimulating dose of choriogonadotropin. Other 8-substituted derivatives such as 8-hydroxy cyclic AMP and 8-isopropylthio cyclic AMP, which were less susceptible to phosphodiesterase action, also effectively stimulated steroidogenesis. The uptake and metabolism of cyclic [3H]AMP in ovarian cells was also studied in relation to steroidogenesis. When ovarian cells were incubated for 2h in the presence of increasing concentrations of cyclic [3H]AMP, the radioactivity associated with the cells increased almost linearly up to 250μm-cyclic [3H]AMP concentration in the incubation medium. The 3H label in the cellular extract was recovered mainly in the forms ATP, ADP, AMP, adenosine and inosine, with cyclic AMP accounting for less than 1% of the total tissue radioactivity. Incubation of cyclic AMP in vitro with ovarian cells resulted in a rapid breakdown of the nucleotide in the medium. The degradation products in the medium have been identified as AMP, adenosine and inosine. The rapid degradation of cyclic AMP by phosphodiesterase(s) makes it difficult to correlate changes in cyclic AMP concentrations with steroidogenesis. These observations thus provide an explanation for the previously observed lack of cyclic AMP accumulation under conditions in which low doses of choriogonadotropin stimulated steroidogenesis without any detectable changes in cyclic AMP accumulation.

STUDIES ON THE POSSIBLE ROLE OF CYCLIC AMP IN SOME ACTIONS OF CHOLERA TOXIN*

1971 ◽  
Vol 185 (1) ◽  
pp. 376-385 ◽  
Author(s):  
D. E. Schafer ◽  
W. D. Lust ◽  
J. B. Polson ◽  
J. Hedtke ◽  
B. Sircar ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Cholera Toxin

scholarly journals Agonist-specificity in the role of Ca2+-induced Ca2+ release in hepatocyte Ca2+ oscillations

1993 ◽  
Vol 291 (1) ◽  
pp. 169-172 ◽  
Author(s):  
A Sanchez-Bueno ◽  
P H Cobbold
Keyword(s):  
Angiotensin Ii ◽  
Cyclic Amp ◽  
Time Course ◽  
Signalling Pathway ◽  
Cellular Mechanisms ◽  
Working Hypothesis ◽  
Open Conformation ◽  
Agonist Concentration

Ca(2+)-mobilizing hormones induce oscillations in the cytoplasmic concentration of free Ca2+ (‘free Ca’) (spikes) in many cells. In hepatocytes the frequency of spiking depends on agonist dose, but the time course of an individual spike does not change with agonist concentration. Interestingly, the time course of individual spikes does depend on the hormone species, but the cellular mechanisms underlying this agonist-specificity are not understood. Here we show that ryanodine, which blocks the muscle Ca2+ channel responsible for Ca(2+)-induced Ca2+ release (‘CICR’) in the open conformation, has almost no effect on phenylephrine-induced spikes, but does, in contrast, inhibit vasopressin- or angiotensin II-induced spikes. We also show that ryanodine has no effect either on the increase in frequency or on the elevated peak free Ca induced by increased cyclic AMP on phenylephrine spikes. In contrast, ryanodine truncates the prolonged falling phases of spikes induced by vasopressin or angiotensin II in the presence of elevated cyclic AMP. A working hypothesis is proposed in which vasopressin- or angiotensin II-induced spikes consist of an Ins(1,4,5)P3-mediated symmetrical spike, identical in time course and mechanism with those induced by phenylephrine, followed by a ‘tail’ that represents CICR. The data hint at the existence of a novel signalling pathway.

scholarly journals Human platelets are defective in processing of cholera toxin

1983 ◽  
Vol 212 (3) ◽  
pp. 669-678 ◽  
Author(s):  
R J Hughes ◽  
P A Insel
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Mammalian Cells ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity

Cholera toxin is unable to elevate cyclic AMP levels in intact human platelets despite being very efficacious in this respect in other mammalian cells; in the presence of 0.5 mM-isobutylmethylxanthine, we found that 3-6nM-cholera toxin over 3h at 37 degrees C elevated platelet cyclic AMP from 33 +/- 13 to 39 +/- 12pmol/mg of protein (means +/- S.D.; n = 12). We have investigated the basis for this lack of response. 125I-labelled cholera toxin bound to platelets both saturably and with high affinity (Kd congruent to 60pM; Bmax. congruent to 50fmol/mg of protein). Incubation of platelets with the putative cholera toxin receptor monosialoganglioside GM1 enhanced 125I-labelled cholera toxin binding at least 40-fold but facilitated only a minimal (less than or equal to 3-fold) elevation of platelet cyclic AMP levels. In contrast, dithiothreitol-activated cholera toxin markedly stimulated adenylate cyclase activity in platelet membranes. Platelet cytosol both enhanced stimulation of adenylate cyclase activity by activated cholera toxin (A1 subunit) and supported stimulation by the A1-A2 subunit of cholera toxin. Neither GTP nor NAD+, both necessary for response to cholera toxin, was lacking in intact platelets. However, we found that platelets were unable to cleave cholera toxin to the active A1 subunit (as assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis). By contrast, murine S49 lymphoma cells were able to generate the A1 subunit with a time course that closely resembled the kinetics of toxin-mediated cyclic AMP accumulation in these cells. Thus we conclude that human platelets are defective in their ability to process surface-bound cholera toxin. These results indicate that binding of cholera toxin to surface receptors is necessary, but not sufficient, for expression of the toxin effect and the generation of the A1 subunit of the toxin may be rate-limiting for expression of cholera toxin response.

Different VAMP/Synaptobrevin Complexes for Spontaneous and Evoked Transmitter Release at the Crayfish Neuromuscular Junction

1998 ◽  
Vol 80 (6) ◽  
pp. 3233-3246 ◽  
Author(s):  
Shao-Ying Hua ◽  
Dorota A. Raciborska ◽  
William S. Trimble ◽  
Milton P. Charlton
Keyword(s):  
Neuromuscular Junction ◽  
Transmitter Release ◽  
Neurotransmitter Release ◽  
Action Potentials ◽  
Neuromuscular Junctions ◽  
Spontaneous Release ◽  
Intracellular Ca ◽  
Crayfish Neuromuscular Junction ◽  
Evoked Transmitter Release

Hua, Shao-Ying, Dorota A. Raciborska, William S. Trimble, and Milton P. Charlton. Different VAMP/synaptobrevin complexes for spontaneous and evoked transmitter release at the crayfish neuromuscular junction. J. Neurophysiol. 80: 3233–3246, 1998. Although vesicle-associated membrane protein (VAMP/synaptobrevin) is essential for evoked neurotransmitter release, its role in spontaneous transmitter release remains uncertain. For instance, many studies show that tetanus toxin (TeNT), which cleaves VAMP, blocks evoked transmitter release but leaves some spontaneous transmitter release. We used recombinant tetanus and botulinum neurotoxin catalytic light chains (TeNT-LC, BoNT/B-LC, and BoNT/D-LC) to examine the role of VAMP in spontaneous transmitter release at neuromuscular junctions (nmj) of crayfish. Injection of TeNT-LC into presynaptic axons removed most of the VAMP immunoreactivity and blocked evoked transmitter release without affecting nerve action potentials or Ca2+ influx. The frequency of spontaneous transmitter release was little affected by the TeNT-LC when the evoked transmitter release had been blocked by >95%. The spontaneous transmitter release left after TeNT-LC treatment was insensitive to increases in intracellular Ca2+. BoNT/B-LC, which cleaves VAMP at the same site as TeNT-LC but uses a different binding site, also blocked evoked release but had minimal effect on spontaneous release. However, BoNT/D-LC, which cleaves VAMP at a different site from the other two toxins but binds to the same position on VAMP as TeNT, blocked both evoked and spontaneous transmitter release at similar rates. The data indicate that different VAMP complexes are employed for evoked and spontaneous transmitter release; the VAMP used in spontaneous release is not readily cleaved by TeNT or BoNT/B. Because the exocytosis that occurs after the action of TeNT cannot be increased by increased intracellular Ca2+, the final steps in neurotransmitter release are Ca2+ independent.

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