We have investigated the effects of sigma ligands [1,3-di(2-tolyl)guanidine (DTG) and (1)-pentazocine] on the electrical activity of cultured frog pituitary melanotrope cells by using the patch-clamp technique. DTG and (1)-pentazocine (10 mM each) induced a reversible depolarization associated with an increase in membrane resistance and action potential firing. In voltage-clamp experiments, DTG and (1)-pentazocine elicited inward currents whose intensity augmented with membrane depolarization. The currents vanished or reversed between -90 and -100 mV, at values close to the K1 equilibrium potential(EK15 -102 mV). DTG (2–500 mM) and (1)-pentazocine (0.2-200 mM) reduced the outward delayed rectifier K1 current [IK(V)] in a dose-dependent manner with EC50 of 64 and 37 mM,respectively. In contrast, naloxone (50 mM) and pirenzepine (10mM) did not affect the sigma ligand-induced inhibition of IK (V).Addition of guanosine-59-O-(3-thiophosphate) in the pipettesolution irreversibly sustained the DTG-induced current whereas guanosine-59-O-(2-thiodiphosphate) virtually suppressed the response. Cholera toxin-pretreatment (1 mg/ml; 18hr) abolished the inward current and the inhibition of IK (V)induced by sigma ligands. In contrast, pretreatment with pertussis toxin (1 mg/ml; 18 hr) had no effect. Taken together, these data indicate that DTG and (1)-pentazocine activate the electrical activity of cultured frog melanotrope cells by reducing both a tonic K1 current and a voltage-dependent [IK (V)] K1conductance through the activation of a cholera toxin-sensitive G-protein.

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.

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.