Protein phosphatase inhibitor okadaic acid enhances transmitter release at neuromuscular junctions.

Abdul‐Ghani, Muhammad; Kravitz, Edward A.; Meiri, Halina; Rahamimoff, Rami

doi:10.1073/pnas.88.5.1803

Cited by 36 publications

(17 citation statements)

References 19 publications

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“…Likewise, effects of OA have been described previously on MNT [1,4,6] and chromaffin cells [5]. In particular, Arenson and Gill [4] have shown the coupling of L-type VDCC to neurotransmitter release in the frog MNT as a consequence of the action of OA.…”

Section: Resultsmentioning

confidence: 86%

Coupling of L-type calcium channels to neurotransmitter release at mouse motor nerve terminals

Urbano

Depetris

Uchitel

2001

Pfl�gers Archiv European Journal of Physiology

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Previously, we have presented evidence for the presence of L-type voltage-dependent Ca2+ channels (VDCC) in 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, (acetoxymethyl)ester (BAPTA-AM)-incubated motor nerve terminals (MNTs) of the levator auris muscle of mature mice. The aim of the present work was to study the coupling of these L-type VDCC to neurotransmitter release by inhibiting protein phosphatases. We thus studied the effects of the protein phosphatase inhibitors okadaic acid (OA) and pervanadate on quantal content (QC) of transmitter release with the P/Q-type channels fully blocked. The QC was not significantly different under the three experimental conditions tested: incubation with dimethylsulphoxide (DMSO), ethylene-glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid, (acetoxymethyl)ester (EGTA-AM) and BAPTA-AM. After preincubation with OA (1 microM), but not with pervanadate, QC increased substantially in the BAPTA-AM-incubated (up to 400%) MNT, but not in those incubated with DMSO or EGTA-AM. The OA-induced increment of QC was attenuated greatly (approximately 95% reduction) by preincubation with either nitrendipine (10 microM) or calciseptine (300 nM). The effect of OA (1 microM) and pervanadate (0.1 mM) on spontaneous neurotransmitter release was also studied. After preincubation with OA, but not per-vanadate, miniature end-plate potential (MEPP) frequency increased only in the BAPTA-AM-incubated MNT (up to 700% increment). This response was attenuated (by approximately 80%) by nitrendipine (10 microM) or calciseptine (300 nM). In contrast, neither omega-agatoxin IVA (120 nM) nor omega-conotoxin GVIA (1 microM) affected this OA-induced increment significantly. We also evaluated the relationship between QC and extracellular [Ca2+] ([Ca2+]o) in BAPTA-AM-incubated MNT. Under conditions in which only P/Q-type VDCC were available to participate in neurotransmitter release, QC increased as [Ca2+]o was raised from 0.5 to 2 mM. However, when only L-type VDCC were available, QC increased when [Ca2+]o increased from 0.5 to 1 mM, but decreased significantly at 2 mM. The mean latency for P/Q-type VDCC-mediated EPP was 1.7-1.9 ms; for L-type VDCC-mediated EPP, 1.9-2.5 ms. The rise time of the L-type VDCC mediated EPP was significantly slower than that mediated by P/Q-type VDCC. Preincubation with H-7 (100 microM), a potent inhibitor of protein kinase C (PKC) and adenosine 3',5'cyclic monophosphate (cAMP)-dependent protein kinase (PKA), attenuated the OA-induced increment of both QC and MEPP frequency (50% and 70% decrement, respectively), suggesting the participation of at least these two protein kinases in the coupling of L-type VDCC. In summary, our results show coupling of L-type VDCC to neurotransmitter release when protein phosphatases are inhibited and intracellular [Ca2+] is buffered by the fast chelator BAPTA.

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Section: Resultsmentioning

confidence: 86%

Coupling of L-type calcium channels to neurotransmitter release at mouse motor nerve terminals

Urbano

Depetris

Uchitel

2001

Pfl�gers Archiv European Journal of Physiology

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“…2). Okadaic acid has previously been suggested to enhance synaptic transmission in the neuromuscularjunction by increasing neurotransmitter release (17,18), an effect that was proposed to result from stimulation of Ca2+ influx. Considering its weak effect on the integrated Ca2+ current in the pancreatic cell, this does not appear to be the main mechanism by which this phosphatase inhibitor potentiates exocytosis of the insulin-containing secretory granules.…”

Section: Results As Shown Inmentioning

confidence: 99%

Activation of protein kinases and inhibition of protein phosphatases play a central role in the regulation of exocytosis in mouse pancreatic beta cells.

Ämmälä¹,

Eliasson²,

Bokvist³

et al. 1994

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

180

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The mechanisms that regulate insulin secretion were investigated using caitance measurements of exocytosis in single (3 MATERIALS AND METHODS Preparation of Cells. Normal mouse 13 cells were used throughout the study. The mice were stunned by a blow against the head and killed by cervical dislocation and decapitation. The pancreas was quickly removed and pancreatic islets were isolated by collagenase digestion. Single cells were prepared as described (7), plated on Corning tissue culture dishes, and maintained in tissue culture for up to 3 days in RPMI 1640 tissue culture medium, containing 5 mM glucose and supplemented with streptomycin (100 pug/ml) and penicillin (100 international units/ml).Electrophysiology. Whole-cell membrane currents were recorded from individual (3 cells by using the perforatedpatch whole-cell configuration as described (3,8). Perforation required a few minutes and the voltage clamp was regarded as satisfactory when the series conductance exceeded 40 nS. The holding potential was -70 mV and depolarizations were 500 ms long and went to 0 mV. Exocytosis was measured as changes in cell capacitance (Cm,) as described elsewhere (9). The interval between each measurement of Cm was 100 ms. Depolarizations were applied at low frequency (0.3-0.5 min'). It was ascertained that stable capacitance responses were obtained before addition of a compound so that the observed changes are not just longterm trends. Effects on the Ca2+ current were assessed by measuring both the peak amplitude of the Ca2+ current (Ica) and the integrated Ca2+ current ([QCa = f ICa(t)dtI)-Solutions. The extracellular medium consisted of 118 mM NaCl, 20 mM tetraethylammonium chloride, 5.6 mM KCl, 1.2 mM MgCl2, 2.6 mM CaCl2, and 5 mM Hepes (pH 7.4 with NaOH). Glucose was included in the extracellular solution at a concentration of 5 mM to provide the metabolic fuel required to energize the secretory process. However, subsequent experiments have indicated that secretion proceeds well in the complete absence of the sugar. The pipette solution contained 76 mM Cs2SO4, 10 mM NaCl, 10 mM KCl, 1 mM MgCl2, and 5 mM . Electrical contact was established by the addition of amphotericin B (Sigma) to the pipette solution. Briefly, a stock solution containing 6 mg of amphotericin dissolved in 100 Al of dimethyl sulfoxide was prepared. Twenty microliters of this stock solution was then added to 5 ml of the pipette solution, yielding a final concentration of 0.24 mg/ml. The tip of the pipette was filled with amphotericin-free solution and the pipette was then back-filled with amphotericin-containing solution. Okadaic acid (Molecular Probes), the phorbol ester phorbol 12-myristate 13-acetate (PMA), and forskolin (both from Sigma) were dissolved in dimethyl sulfoxide (final concentration of dimethyl sulfoxide, 0.02-0.1%). In Figs. 1 and 4, the cells were pretreated with forskolin for >15 min. This protocol was used as it was difficult to maintain the recording long enough to permit multiple additions and to Abbreviations: PMA, phorb...

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“…1999; Turner et al . 1999), and inhibition of phosphatases has a similar effect (Abdul‐Ghani et al . 1991; Ammala et al .…”

mentioning

confidence: 92%

Physiological regulation of Munc18/nSec1 phosphorylation on serine‐313

Craig

Evans

Morgan

2003

Journal of Neurochemistry

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Increased protein phosphorylation enhances exocytosis in most secretory cell types, including neurones. However, the molecular mechanisms by which this occurs and the specific protein targets remain unclear. Munc18-1/nSec1 is essential for exocytosis in neurones, and is known to be phosphorylated by protein kinase C (PKC) in vitro at Ser-313. This phosphorylation has been shown to decrease its affinity for syntaxin, and to alter the kinetics of exocytosis in chromaffin cells. However, there are no data on the physiological regulation of Ser-313 phosphorylation. Using phospho-Ser-313-specific antisera, we demonstrate here that Ser-313 is phosphorylated in intact and permeabilized chromaffin cells in response to histamine and Ca 2+ respectively. Furthermore, Ser-313 is rapidly and transiently phosphorylated in intact synaptosomes in response to depolarization by KCl treatment or by 4-aminopyridine, and by the metabotropic glutamate receptor agonist dihydroxyphenylglycine. PKC was identified as the kinase, and PP1 and PP2B as the phosphatases responsible for regulating Ser-313 phosphorylation. As phosphorylation of nSec1 on Ser-313 affects the rate of transmitter release in chromaffin cells, the demonstration here that this phosphorylation event occurs in neurones suggests that synaptic neurotransmitter release may be similarly regulated by nSec1 phosphorylation. Furthermore, such changes in release kinetics are associated with long-term potentiation and depression, thus implicating nSec1 phosphorylation as a potential regulatory mechanism underlying presynaptic plasticity.

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Protein phosphatase inhibitor okadaic acid enhances transmitter release at neuromuscular junctions.

Cited by 36 publications

References 19 publications

Coupling of L-type calcium channels to neurotransmitter release at mouse motor nerve terminals

Coupling of L-type calcium channels to neurotransmitter release at mouse motor nerve terminals

Activation of protein kinases and inhibition of protein phosphatases play a central role in the regulation of exocytosis in mouse pancreatic beta cells.

Physiological regulation of Munc18/nSec1 phosphorylation on serine‐313

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