Phosphatases modulate transmission and serotonin facilitation at synapses: Studies with the inhibitor okadaic acid

Swain, James E.; Robitaille, Richard; Dass, Gavin R.; Charlton, Milton P.

doi:10.1002/neu.480220806

Cited by 35 publications

(30 citation statements)

References 61 publications

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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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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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“…When we applied OA to phasic synapses, transmitter release increased by ϳ33% (Fig. 5E), but at tonic excitor synapses on the crayfish leg-opener muscle, OA caused an increase of 300% (Swain et al, 1991). This observation may indicate that in both phasic and tonic synapses, transmitter release is controlled by phosphorylation of critical substrates; however, under normal conditions, more of these are phosphorylated in phasic synapses than in tonic synapses.…”

Section: Phasic and Tonic Synapsesmentioning

confidence: 92%

“…Control recordings indicate that this concentration of DMSO does not alter LFD (data not shown). Drug concentration was chosen empirically based on dose-effect trials and on previous published concentrations used in crayfish [Rp-cAMPS, rapamycin (Beaumont et al, 2001), staurosporine, GF, PMA (Friedrich et al, 1998), OA (Swain et al, 1991;Lin and Fu, 2005), Sp-cAMPS, FK-506 (J. E. Swain and M. P. Charlton, unpublished observation)] or in other invertebrates such as Aplysia (FK-506) (Sharma et al, 2003), Mytilus (calcineurin autoinhibitory peptide) (Yamada et al, 2004), and Drosophila (FK-506) (Kuromi et al, 1997).…”

Section: Methodsmentioning

confidence: 99%

“…For instance, Swain et al (1991) showed that inhibition of phosphatases with OA greatly enhanced transmitter release at synapses of the crayfish claw-opener muscle. We hypothesized that LFD is caused by a reduction in phosphorylation of key proteins.…”

Section: Phosphorylation and Lfdmentioning

confidence: 99%

“…Because phosphorylation of synaptic substrates plays a key role in regulating the strength of synaptic transmission in both Crustacean (Dixon and Atwood, 1989a;Swain et al, 1991;Beaumont et al, 2001) and mammalian nervous systems (Tokuda and Hatase, 1998), we tested the hypothesis that LFD at phasic synapses at crayfish leg extensor muscles involves changes in phosphorylation. We found that LFD does not involve reduction in quantal amplitude or presynaptic action potentials.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Phosphorylation-Dependent Low-Frequency Depression at Phasic Synapses of a Crayfish Motoneuron

Silverman‐Gavrila¹,

Orth²,

Charlton³

2005

J. Neurosci.

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Extremes in presynaptic differentiation can be studied at the crayfish leg extensor muscle where, on the same muscle fiber, one motoneuron makes "phasic" depressing synapses that have a high probability of neurotransmitter release and another motoneuron makes "tonic," low-probability, facilitating synapses. The large motor axons permit intracellular access to presynaptic sites. We examined the role of phosphorylation during low-frequency depression ( This study shows that phosphorylation-dependent mechanisms are involved in LFD and suggests that exocytosis is controlled by conditions that shift the balance between phosphorylated and unphosphorylated substrates. The shift can occur by alteration in the relative activities of protein kinases and phosphatases.

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Physiological functions of FMRFamide‐like peptides (FLPs) in crustaceans

Mercier

Friedrich

Boldt

2003

Microscopy Res & Technique

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Phosphatases modulate transmission and serotonin facilitation at synapses: Studies with the inhibitor okadaic acid

Cited by 35 publications

References 61 publications

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

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

Phosphorylation-Dependent Low-Frequency Depression at Phasic Synapses of a Crayfish Motoneuron

Physiological functions of FMRFamide‐like peptides (FLPs) in crustaceans

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