d-Amphetamine inhibits inwardly rectifying potassium channels in Xenopus oocytes expression system.

Effects of d-amphetamine on the renal outer medullary potassium (ROMK1) channels were tested in the Xenopus oocytes expression system. Xenopus oocytes were injected with mRNA coding for wild-type or mutant ROMK1 channels. Giant inside-out patch-clamp recordings were performed. d-Amphetamine inhibited the activity of ROMK1 channels in a manner that was concentration-dependent but voltage-independent. ROMK1 channels are regulated by intracellular pH (pH i) and protein kinase A (PKA). d-Amphetamine decreased the activity of wild-type and pH i gating residue mutant (K80M) channels over a range of pH i values. However, d-amphetamine failed to reduce channel activity in the presence of PKA inhibitors (H89 and KT 5720) and had no inhibitory effect on the mutants of PKA-phosphorylation sites (S44A, S219A, or S313A), mutants that mimicked the negative charge carried by a phosphate group bound to a serine (S44D, S219D, or S313D), or mutant channels with a positive charge (S219R). These findings suggest that d-amphetamine inhibits ROMK1 channels independently of the pH i. The effects of d-amphetamine on ROMK1 channels may be due to a conformational change induced by PKA-mediated phosphorylation, but not to charge-charge interactions.

Effects of sodium azide, barium ion, d-amphetamine and procaine on inward rectifying potassium channel 6.2 expressed in Xenopus oocytes.

BACKGROUND/PURPOSE: Inward rectifying potassium channel 6.2 (Kir6.2DelataC26 channel) is closely related to ATP-sensitive potassium channels. Whether sodium azide, barium ion, d-amphetamine or procaine acts directly on the Kir6.2DeltaC26 channel remains unclear. We studied the effects of these compounds on Kir6.2DeltaC26 channel expressed in Xenopus oocytes. METHODS: The coding sequence of a truncated form of mouse Kir6.2 (GenBank accession number NP_034732.1), Kir6.2(1-364) (i.e. Kir6.2DeltaC26), was subcloned into the pET20b(+) vector. Plasmid containing the correct T7 promoter-Kir6.2(1-364) cDNA fragment [Kir6.2/pET20b(+)] was then subject to NotI digestion to generate the templates for in vitro run-off transcriptions. The channel was expressed in Xenopus laevis oocytes. Two-electrode voltage clamping was used to measure the effects of sodium azide, barium ion, d-amphetamine and procaine on Kir6.2DeltaC26 channel current. RESULTS: Sodium azide activated and barium ion and d-amphetamine inhibited the Kir6.2DeltaC26 channel. Procaine did not have any significant effect on the Kir6.2DeltaC26 channel. CONCLUSION: Kir6.2DeltaC26 channel expressed in Xenopus oocytes can be used as a pharmacological tool for the study of inward rectifying potassium channels.

Bursts of potential elicited by d-amphetamine in central snail neuron: effect of sodium azide.

Effects of sodium azide (NaN(3)) on spontaneously generated action potential and bursts of potential elicited by d-amphetamine (d-amphetamine-elicited BoP) were studied on the right parietal 4 (RP4) neuron of the snail Achatina fulica Ferussac in vitro. Sodium azide altered the spontaneous action potential of RP4 neuron in a concentration-dependent manner. In lower concentrations, neither NaN(3) (30, 100, 300 microM; 1 and 3 mM) nor d-amphetamine (135 microM) affect the resting membrane potential, amplitude and frequency of RP4 neurons, while in the higher concentrations NaN(3) (30 mM) did abolish the spontaneous action potential on RP4 neurons and depolarized the RP4 neurons reversibly. At lower concentration, NaN(3) (30 microM) facilitated the d-amphetamine-elicited BoP. The BoP elicited by NaN(3) (30 microM) and d-amphetamine (135 microM) were decreased following treatment with KT5720 (protein kinase A inhibitor), or intracellular injection of EGTA [ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. However, the BoP was not affected by applying U73122 (1-[6-[((17beta)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione) or neomycin (phospholipase inhibitors). Voltage clamp studies revealed that NaN(3) (30 microM) did not alter the total fast inwards currents (70 msec.) and the steady-state outwards currents (5 sec.). It appeared that the BoP elicited by NaN(3) (30 microM) and d-amphetamine (135 microM) was mainly due to protein kinase A-related messenger system and intracellular calcium. It is concluded that d-amphetamine-elicited BoP was not mainly due to inhibition of the function of mitochondria in the neuron while the function of mitochondria did alter the BoP elicited by amphetamine.

Effects of penicillin on procaine-elicited bursts of potential in central neuron of snail, Achatina fulica.

Effects of penicillin on changes in procaine-elicited bursts of potential (BoP) were studied in a central neuron (RP4) of snail, Achatina fulica Ferussac. Procaine elicited BoP in the RP4 neuron while penicillin elicited depolarization of the neuron. Penicillin decreased the BoP elicited by procaine in a concentration-dependent manner. The effect of penicillin on the procaine-elicited BoP was not altered in the preparations treated with ascorbate or L-NAME (N-nitro-L-arginine methyl ester). However, the inhibitory effect of penicillin on the procaine-elicited BoP was enhanced with a decrease in extracellular sodium ion. Sodium ion was one of the important ions contributing to the action potential of the neuron. Two-electrode voltage-clamp studies revealed that penicillin decreased the fast sodium inward current of the neuron. It is concluded that penicillin inhibited the BoP elicited by procaine and sodium ion altered the effect of penicillin on procaine-elicited BoP.

Effects of rolipram on induction of action potential bursts in central snail neurons.

Effects of rolipram, a selective inhibitor of phosphodiesterases (PDE) IV, on induction of action potential bursts were studied pharmacologically on the RP4 central neuron of the giant African snail (Achatina fulica Ferussac). Oscillations of membrane potential bursts were elicited by rolipram and forskolin. The bursts of potential elicited by rolipram were not inhibited after administration with (a) calcium-free solution, (b) high-magnesium solution (30 mM) or (c) U73122. However, the bursts of potential elicited by rolipram were inhibited by pretreatment with KT-5720 (10 microM). Voltage-clamp studies revealed that rolipram decreased the total inward current and steady-state outward currents of the RP4 neuron. The negative slope resistance (NSR) was not detectable in control or rolipram treated RP4 neurons. TEA elicited action potential bursts and an NSR at membrane potential between -50 mV and -30 mV. It is suggested that the bursts of potential elicited by rolipram were not due to (1) synaptic effects of neurotransmitters; (2) NSR of steady-state I-V curve; (3) phospholipase activity of the neuron. The rolipram-elicited bursts of potential were dependent on the phosphodiesterases inhibitory activity and the cAMP signaling pathway in the neuron.