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In this study, we explored the effect of the chemical phosphatase 2,3-butanedione monoxime (BDM) on glycine current (IGly) of murine ventromedial hypothalamic neurons. Co-application of 0.01 to 67 mM BDM increased IGly decay rate with little change of the peak amplitude. This effect was both rapid in onset and offset and required the presence of the agonist. Pretreatment with BDM alone did not alter-IGly decay. In addition, dialysis of neurons with 500 microM ATP-gamma-S did not alter the acute effect of BDM. Thus, this effect may result from open channel block rather than BDM-induced dephosphorylation of the receptor/channel protein. In contrast to the acute effect described above, relatively prolonged (i.e., greater than 80 s) pretreatment with BDM reduced peak IGly. The phorbol ester (PDBu), a protein kinase C (PKC) activator, mimicked this effect of BDM. Furthermore, chelerythrine, a specific PKC inhibitor, prevented this effect of BDM on peak IGly. Thus, activation of PKC may mediate this attenuating effect of BDM on IGly. For a sub-population of these pretreated neurons, there was a subsequent potentiation of IGly which followed the initial suppressant effect. This potentiation may be due to a phosphatase effect of BDM, since it was observed more frequently when neurons were also pretreated with the protein kinase inhibitors H7 or chelerythrine. These findings suggest that BDM alters protein kinase activity and acts as a phosphatase to regulate the activity of the glycine receptor/channel complex.

2,3-Butanedione monoxime (BDM) has a negative inotropic effect on smooth muscles as well as the myocardium. Therefore, in the present study we compared the sensitivity to BDM of the cardiovascular system of the spontaneously hypertensive (SHR) and the normotensive Wistar-Kyoto (WKY) rat. While BDM significantly decreased the blood pressure (BP) for both strains, the SHR was significantly more responsive. Specifically, 5, 30, 100, and 200 mg/kg BDM (intravenously) reduced BP of the SHR by 9 +/- 3, 20 +/- 3, 49 +/- 5, and 63 +/- 7 mm Hg, respectively. The same doses of BDM reduced BP of the WKY by 0, 2 +/- 0.4, 18 +/- 3, and 26 +/- 3 mm Hg. The duration of the hypotensive effect of BDM was also greater for the SHR. In vitro, BDM had a greater suppressant effect on the L-type Ca2+ current (ICa(L)) of SHR ventricular myocytes; the IC50 for the suppression of ICa(L) was 17 and 29 mM for SHR and WKY ventricular myocytes, respectively. The beta-adrenergic receptor agonist isoproterenol antagonized the suppressant effect of BDM on ICa(L). Furthermore, BDM significantly reduced the duration of both spontaneous and electrically stimulated action potentials of cultured neonatal rat cardiomyocytes. Intracellular dialysis with the catalytic unit of protein kinase A antagonized BDM's effect on the action potential. These data suggest that suppression of myocardial activity contributes to the hypotensive effect of BDM. In addition, the elevated response to BDM of SHR cardiac myocytes may indicate that the conformation and/or modulation of the L-type Ca2+ channel differ for the SHR and WKY lines of rat.

Ionic currents induced by excitatory amino acids were investigated for freshly isolated murine hypothalamic neurons with whole cell recording techniques. L-glutamate or N-methyl-D-aspartate (NMDA), in combination with glycine, resulted in a rapidly rising current which decayed in the continued presence of agonist. In contrast, kainate currents did not decay. While quisqualate-induced current maintained a steady amplitude in the continued presence of agonist, a rapid decay phase appeared at holding potentials negative to -50 mV. Co-application of 2,3-butanedione monoxime (BDM) reversibly inhibited the currents due to each agonist. Detailed study of BDM suppression of kainate-induced current revealed two components. A component with a rapid onset did not involve phosphatase action since 500 microM ATP-gamma-S or a protein kinase inhibitor (H-7, 200 microM) did not alter current suppression or recovery after BDM. Thus, the probable mechanism for this component of BDM's effect is direct block of the kainate-activated ion channel. However, preincubating neurons with 30 mM BDM reduced their subsequent response to kainate alone. This persistent effect of BDM was not seen for neurons dialyzed with a solution containing ATP-gamma-S during conventional whole cell recording. Furthermore, exposure to H-7 prevented recovery of the kainate response suppressed by preincubation in BDM. These findings suggest that BDM causes sustained suppression of the kainate response of hypothalamic neurons via a "chemical phosphatase" action.

While adult mice receiving picrotoxin (PTX) alone responded with clonic and tonic-clonic seizures, this response was greatly suppressed for mice simultaneously injected with 2,3-butanedione monoxime (BDM). For example, 60% and 10% of the mice convulsed when injected (i.p.) with 3.0 mg/kg PTX alone or PTX plus 205 mg/kg of BDM, respectively. In contrast, a non-oxime analogue of BDM, 2,3-butanedione (BTD), did not have this anticonvulsant effect. In order to explore the basis for the anticonvulsant effect of BDM, we recorded GABA-activated currents (IGABA) of frontal cortical as well as ventromedial hypothalamic neurons before, during and after exposure to this oxime. BDM had a biphasic effect on IGABA. That is, high concentrations (100 microM-40 mM) decreased and lower concentrations (0.01 microM-0.001 microM) potentiated IGABA; these effects of BDM reversed upon washout of the oxime. In contrast, BTD had no effect on IGABA. Finally, when 0.001 microM BDM, 10-30 microM PTX and GABA were co-applied the inhibitory effect of the toxin on IGABA was markedly suppressed. These data suggest that the anticonvulsant effect of oximes involves facilitation of the inhibitory action of GABA.

The transient outward K+ current (Ito) was assessed in single rat ventricular myocytes with the whole-cell patch-clamp technique. Extracellular application of the chemical phosphatase 2,3-butanedione monoxime (BDM) inhibited Ito in a concentration-dependent manner. The IC50 was 14 mM. The on-set of this effect occurred within 20 s after BDM application. Ito recovered almost completely at 2 min after washout of BDM. Application of 20 mM BDM shifted the steady-state inactivation curve of Ito by 9 +/- 2 mV (n = 8) in the negative direction. Addition of 5 microM isoproterenol enhanced Ito amplitude by 16.2 +/- 1.8%. This concentration of isoproterenol partially reversed the BDM-induced inhibition of Ito. Furthermore, application of 10 mM 8-bromoadenosine 3':5'-cyclic monophosphate enhanced the amplitude of Ito and also significantly reversed the BDM-induced suppression of Ito. In contrast, intracellular dialysis with guanosine 3':5'-cyclic monophosphate (cGMP, 1-10 mM) did not affect the BDM-induced inhibition of Ito. The inward rectifier K+ current (Ik1) was relatively insensitive to BDM; i.e., 20 mM BDM inhibited Ito and Ik1 to 35.5 +/- 4.3% (n = 8) and 92.9 +/- 4.0% (n = 4) of the control, respectively. These results indicate that BDM suppressed Ito but not Ik1 of rat ventricular myocytes. We attribute the BDM suppression of Ito to dephosphorylation of the channel protein.

2,3-Butanedione monoxime, also known as diacetyl monoxime, is a nucleophilic agent which dephosphorylates acetylcholinesterase poisoned with organophosphates. This "chemical phosphatase" activity stimulated studies of the effect of 2,3-butanedione monoxime on phosphorylation-dependent cellular processes. As a result of these studies, we know that the drug affects a number of mechanisms including muscle contraction, ionic current flow and synaptic transmission. Furthermore, it may be used as a component of cardioplegic solutions since it protects cardiac tissue exposed to certain ischaemic conditions. While this MiniReview reveals the diversity of its cellular actions, there continues to be unresolved questions regarding its molecular mechanism.

1. Voltage-activated currents through calcium channels in primary cultures of murine dorsal root ganglion cells (DRG) were studied with the whole-cell and cell-attached patch recording techniques. 2. The chemical phosphatase 2,3-butanedione monoxime (BDM) reversibly reduced the amplitude of L-type calcium current (ICa) in a dose-dependent manner; at a concentration of 20 mM, BDM caused a 47% suppression of ICa. 3. Application of 10 mM-8-bromo-cyclic AMP or 50 microM-isoprenaline onto DRG treated with BDM completely restored ICa to the pre-BDM level. 4. In striking contrast, bath application of Bay K 8644 (0.5-5 microM) had no effect on the BDM-suppressed ICa. As expected, Bay K 8644 alone caused a two- to threefold increase of the maximal ICa and shifted its I-V relationship to the left. Interestingly, if a cell was first exposed to Bay K 8644 further treatment with 20 mM-BDM resulted in 100% suppression of ICa. This suggests that Bay K 8644 changes the conformation of the calcium channel to one which is more sensitive or more accessible to the action of the phosphatase. 5. Pre-treatment of DRG with an activator of protein kinase C, 12-O-tetradecanoyl-phorbol-13-acetate, did not antagonize BDM's effect on ICa. 6. The depressant action of BDM on ICa was distinct from that of nifedipine in that it did not exhibit use dependence. 7. When single calcium channel currents were recorded in cell-attached patches (barium as the charge carrier), bath application of BDM reduced the percentage of time that the channel spent in the open state. 8. Superfusion with 8-bromo-cyclic AMP restored the ensemble macroscopic 'ICa' to the pre-BDM amplitude. This was due to a dramatic enhancement of the frequency of channel openings. 9. We suggest that BDM acts through the cytoplasm to alter cyclic AMP-dependent protein kinase modulation of neuronal L-type calcium channels. The brief, high-frequency openings which 8-bromo-cyclic AMP activates in the presence of BDM may reflect a rapid phosphorylation-dephosphorylation sequence which controls channel gating.

1. The amplitude of endplate potentials was increased by concentrations of butanedione monoxime (BDM, 5-20 mM) that typically caused muscle paralysis. 2. Although BDM slowed the decay of spontaneous miniature endplate currents, the effect was insufficient to explain most of the large increase in amplitude of endplate potentials. 3. The quantal content of endplate potentials was increased by BDM in a reversible, concentration-dependent manner. 4. The frequency of miniature endplate potentials was not changed by 10 mM BDM in the presence of normal or raised potassium concentrations, indicating that BDM does not change quantal content by a direct effect on calcium channels or on steady-state intracellular calcium concentration. 5. A change in the time course of the extracellularly recorded nerve terminal action potential caused by BDM was similar to the change produced by 4-aminopyridine (4-AP). 6. The increase in quantal content produced by BDM was only slightly reduced in the presence of 1 mM tetraethylammonium (TEA) but was significantly reduced in the presence of 0.5 to 1 mM 4-AP. 7. It was concluded that BDM blocks a 4-AP-sensitive potassium conductance in motor nerve terminals and, by increasing the duration of the action potential in this way, increases evoked transmitter release.