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.

The effects of adenosine on the voltage-sensitive delayed-rectifier Kq I. currents and hyperpolarization-activated cationic inward K current Ih. were studied in cultured frog melanotrophs using the whole-cell configuration of the patch-clamp technique. The A1 receptor agonist R-N6-phenylisopropyl-adenosine R-PIA; 50 mM. reversibly increased I. Perfusion of dibutyryl-cAMP 1 mM. in the external K solution did not modify the R-PIA-induced enhancement of IK. Pretreatment of melanotrophs with pertussis toxin 1 mgrml; 12 h.totally abolished the R-PIA-evoked response. Application of hyperpolarizing voltage pulses from y60 to y120 mV to melanotrophs induced a two-component inward current corresponding to an I-like conductance. This conductance was characterized by a high Kq hselectivity and a low Naq permeability and was resistant to etrodotoxin 1 mM.. R-PIA had no effect on I. The present study hdemonstrates that in frog melanotrophs adenosine inhibits the electrical activity by activating IK through an A1 receptor subtype coupled to a pertussis toxin-sensitive pathway independent of the cAMPrPKA system. This study also demonstrates the existence of a Ih conductance in frog melanotrophs which is not modulated by A1 receptors. q1998 Elsevier Science B.V. All rights reserved.

The effects of 4-aminopyridine (4-AP), a specific blocker of outward K current, on voltage-activated transient outward K current 1(I) and inward Na current (I) were investigated on cultured rat cerebellar granule cells using the whole cell voltage-clamp K(A) Na 1technique. At the concentration of 1–5 mM, 4-AP inhibited both I and I. It reduced the amplitude of peak Na current without K(A) Na significant alteration of the steady-state activation and inactivation properties. The inhibitory effect was not enhanced by repeated 1depolarizing pulses (0.5 or 0.1 Hz), suggesting that the binding affinity of 4-AP on Na channels is state-independent. In contrast, the 1 effect of 4-AP on Na channels appeared to be voltage-dependent, the weaker inhibition occurred at more depolarization. Moreover, 4-AP 1 slowed both the activation and inactivation kinetics of Na current. These effects were similar to those induced by a-scorpion toxin and 1 sea anemone toxins on Na channels in other cell model. Our data demonstrate for the first time that 4-AP is able to block not only 11 1 A-type K channels, but also Na channels in rat cerebellar granule cells. It is concluded that the inhibition exerted by 4-AP on Na current likely differs from that provoked by local anesthetics. The possibility that the binding site of neurotoxin receptor 3 may be involved is discussed.