Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), has been widely investigated in terms of its pharmacological action, but less is known about its direct effect on ion channels. Here, the effect of diclofenac on voltage-dependent transient outward KC currents (IA) in cultured rat cerebellar granule cells was investigated using the whole-cell voltage-clamp technique. At concentrations of 10 5e10 3 M, diclofenac reversibly increased the IA amplitude in a dose-dependent manner and significantly modulated the steady-state inactivation properties of the IA channels, but did not alter the steady-state activation properties. Furthermore,diclofenac treatment resulted in a slightly accelerated recovery from IA channel inactivation.Intracellular application of diclofenac could mimic the effects induced by extracellular application, although once the intracellular response reached a plateau, extracellular application of diclofenac could induce further increases in the current. These observations indicate that diclofenac might exert its effects on the channel protein at both the inner and outer sides of the cell membrane. Our data provide the first evidence that diclofenac is able to activate transient outward potassium channels in neurons. Although further work will be necessary to define the exact mechanism of diclofenac-induced IA channel activation, this study provides evidence that the nonsteroidal anti-inflammatory drug, diclofenac, may play a novel neuronal role that is worthy of future study.

Our previous study revealed that 4-aminopyridine (4-AP), a specific blocker of A-type current, could also inhibit inward Na+ currents (INa) with a state-independent mechanism in rat cerebellar granule cells. In the present study, we report an inhibitory effect of 4-AP on voltage-gated and tetrodotoxin (TTX)-sensitive INa recorded from cultured rat myoblasts. 4-AP inhibited INa amplitude in a dose-dependent manner between the concentrations of 0.5 and 10 mM without significant alteration in the activation or inactivation kinetics of the channel. By comparison to the 4-AP-induced inhibitory effect on cerebellum neurons, the inhibitory effect on myoblasts was enhanced through repetitive pulse and inflected by changing frequency. Specifically, the lower the frequency of pulse, the higher the inhibition observed, suggesting that block manner is inversely use-dependent. Moreover, experiments adding 4-AP to the intracellular solution indicate that the inhibitory effects are localized inside the cell. Additionally, 4-AP significantly modifies the properties of steady-state activation and inactivation kinetics of the channel. Our data suggest that the K+ channel blocker 4-AP inhibits both neuron and myoblast Na+ channels via different mechanisms. These findings may also provide information regarding 4-AP-induced pharmacological and toxicological effects in clinical use and experimental research.

Activation of potassium(K+)cu rrents plays a critical role in the control of prog rammed cell death Because pituitary adenylate cyclase-activating polypeptide(PACAP)has been shown to inhibit the apoptotic cascade in the cerebellar cortex du ring development, we hav einvestigated the effect of PACAP on K’cu rrents in cultu red cerebellar g ranule cells using the patch-clamp technique in the whole-cell configu ration Two types of outward K_cu rrents, a transient K+cu rrent(k)and a delayed rectifier K’cu rrent(/K)were characterized using two different voltage protocols and specific inhibitors of K’channels Application of PACAP induced a reversible reduction of the /K amplitude, but did not affect/A, while the PACAP-related peptide vasoactive intestinal polypeptide had no effect on either types of K+ cu rrents Repeated applications of PACAP induced g radual attenuation of the electrophysiological response In the presence of guanosine 5'-rthio1rlphosphate(GTPh, S), PACAP provoked a marked and i rreversible/K depression, whereas cell dialysis with guanosine 5'-bthiodiphosphate GDPl3S totally abolished the effect of PACAR P re-treatment of the cells with pertussis toxin did not modify the effect of PACAP on/K In contrast, cholera toxin suppressed the PACAP-induced inhibition of/K Exposu re of g ranule cells to dibutyryl cyclic adenosine monophosphate(dbcAMP)mimicked the inhibitory effect of PACAP on/K Addition of the specific protein kinaseAinhibitorH89inthepatch pipettesolution preventedthe reductionof/Kinducedbyboth PACAPand dbcAMR PACAPprovoked a sustainedincreaseofthe restingmembranepotentialin cerebellarg ranule cells cultu red eitherin high orlowKCI-containingmedium, and this long-ferm depolarizing effect of PACAP was mimicked by the/K specific blocker tetraethylammonium chloride fTEA)In addition, pre-incubation of g ranule cellswithTEA suppressedthe effect of PACAP on resting membrane potential TEAmimickedthe neu roprotective effect of PACAP against ethanol-induced apoptotic cell death, and the increase of caspase-3 activity observed after exposu re of g ranule cells to ethanol was also significantly inhibited by TEA Taken together, the present results demonstrate that, in rat cerebellar g ranule cells, PACAP reduces the delayed outward rectifier K’cu rrent by activating a type 1 PACAP(PACl)receptorcoupledtothe adenylyl cyclase/protein kinaseApathwayth rougha choleratoxin-sensitiveGsprotein Our dataalso showthat PACAP and TEA induce long-term depolarization of the resting membrane potential promote cell su rvival and inhibit caspase-3 activity, suggesting that PACAP-evoked inhibition of/K contributes to the anti-apoptotic effect of the peptide on cerebellar granule cells

The inhibitory effect of the melatonin receptor antagonist luzindole on voltage-activated transient outward K current (I) was K(A) investigated in cultured rat cerebellar granule cells using the whole cell voltage-clamp technique. At the concentration of 1 mM to 1 mM,luzindole reversibly inhibited I in a concentration-dependent manner. In addition to reducing the current amplitude of I,luzindole K(A) K(A) accelerated the fast inactivation of I channels and shifted the curves of voltage-dependent steady-state activation and inactivation of K(A) I by 16.6 mV and 27.0 mV, respectively. The inhibitory effect of luzindole was neither use-dependent nor voltage-dependent, K(A) suggesting that the binding affinity of luzindole to I channels is state-dependent. Including luzindole in the pipette solution, or K(A) extracellular application of 4 P-PDOT, an antagonist of melatonin receptors, did not change the luzindole-induced inhibitory effect on the I current, indicating that luzindole exerts its channel blocking inhibitory action at the extracellular mouth of the channel, and that the K(A)effect is not due to action of the melatonin receptors. Our data are the first demonstration that luzindole is able to block transient outward 1 K channels in rat cerebellar granule cells in a state-dependent manner, likely associated with extracellular interaction of the drug with the I inactivation gate.

Swelling-activated Cl? currents, I(Cl,swell), were measured during hyposmotic shock in white Leghorn embryonic chick heart cells using the whole-cell recording of patch-clamp technique. Genistein, an inhibitor of protein tyrosine kinase (PTK), suppressed I(Cl,swell). Under isosmotic condition phorbol 12-myristate 13-acetate (PMA), an activator of PKC, elicited the Cl? current similar to that in hyposmotic solution, whereas hyposmotic shock did not elicit I(Cl,swell) in chelerythrine chloride(an inhibitor of PKC)-treated cells. Confocal microscopy experiments using FITC-phalloidin as a fluorescent label of F-actin showed that the actin network was moved from cortical region of the cell to the center after hyposmotic shock as compared with the image under isosmotic condition. When the cells were treated with cytochalasin B (CB) or cytochalasin D (CD) under isosmotic condition the disruption of the F-actin integrity was observed, and I(Cl,swell) was not elicited. With combination treatment of CB with PMA, hyposmotic solution could not elicited I(Cl,swell). The results suggested that the role of PTK, probably receptor tyrosine kinase, for regulation of I(Cl,swell) appeared to be at upstream site related to the role of F-actin. Then PKC signal pathway was activated somehow and finally change in the polymerization state of cytoskeleton led to activate the swelling-activated Cl? channels. These results demonstrate clearly that PTK, PKC and F-actin are important factors for regulation of I(Cl,swell), in embryonic chick heart cells as compared with often controversial results reported in different cell types.