SEROTONIN INHIBITS CA-2+ CURRENTS IN PORCINE MELANOTROPHS BY ACTIVATING 5-HT1C AND 5-HT1A RECEPTORS

1. We have investigated the effect of serotonin (5-HT) on Ca2+ currents in cultured porcine pituitary intermediate lobe (IL) cells. Electrophysiological recordings were performed in the whole-cell configuration of the patch-clamp technique. All membrane currents other than Ca2+ currents were blocked pharmacologically and by ionic substitution. 2. Two types of Ca2+ currents were recorded in IL cells, differing by their activation and inactivation properties. The first type of Ca2+ current was activated at membrane potentials more positive than - 60 mV and had a transient time course during the 100 ms depolarizing voltage steps. The properties of this current correspond to those of the T-type or low-voltage-activated Ca2+ current. The second type of Ca2+ current had a threshold for activation between - 30 and - 20 mV and showed no sign of inactivation with time during the voltage steps. The properties of this current are similar to those of the L-type or high-voltage-activated Ca2+ current. 3. Current to voltage (I-V) relationships obtained either by conventional 100 ms voltage steps from a holding potential (V(h)) of - 100 mV to various test potentials or by 800 ms voltage ramps from - 100 to + 50 mV matched one another closely and showed two inward current humps corresponding to the activation of the T-type and L-type Ca2+ currents respectively. The ramp protocol was used to characterize the effect of 5-HT on the Ca2+ current I-V relationship. 4. 5-HT (100 nm to 50 muM) reversibly inhibited the amplitude of the Ca2+ current triggered by 100 ms voltage jumps from a V(h) of - 100 mV to a test potential of 0 mV. 5. The effect of 5-HT was dose dependent with a threshold between 10 and 100 nM and a maximal effect at 10 muM. At a concentration of 10 muM. the average inhibition of Ca2+ current by 5-HT was 18.3 +/- 6.5 % (n = 27). 5-HT inhibited Ba2+ current in a similar fashion. 6. When examining the effect of 5-HT on Ca2+ current I-V relationships, we observed a reversible inhibition of the high-threshold component corresponding to the L-type Ca2+ current. We never observed any effect of 5-HT on the T-type current. 7. The effect of 5-HT (10 muM) was antagonized to various extents by mianserin (1 muM) but not by ketanserin (0.1 muM), suggesting the involvement of 5-HT1C receptors. (+/-)-8-Hydroxy-2(N,N-dipropylamino)tetralin (8-OH-DPAT, 100 nM) a selective agonist of the 5-HT1A receptor subtype, also reduced Ca2+ currents in IL cells although to a lesser extent than 5-HT. Renzapride (2 muM), a selective 5-HT4 receptor agonist, did not mimick the effect of 5-HT. 8. 5-HT (10 muM) increased the concentration of free intracellular Ca2+ ([Ca2+]i) as measured with fura-2. 9. The effects of 5-HT (10 muM) or 8-OH-DPAT (100 nM) on Ca2+ or Ba2+ currents were not abolished by adding a high amount (18 mM) of the Ca2+ chelator EGTA to the pipette solution, suggesting that a rise in [Ca2+]i does not mediate the effect of 5-HT or 8-OH-DPAT. The potency of 5-HT (10 muM) to reduce Ca2+ currents was not affected by including 8-bromo cAMP (500 muM) to the pipette solution. 10. Pretreatment of the cultures with pertussis toxin (500 ng ml-1, overnight) abolished the effect of 5-HT. 11. We conclude that cultured porcine IL cells possess functional 5-HT1A and 5-HT1C receptors, the activation of which reversibly inhibits L-type Ca2+ channels. This effect is mediated by a pertussis toxin-sensitive G-protein and does not involve a rise in [Ca2+]i or a change in cAMP levels, but possibly a direct coupling of the receptors to Ca2+ channels.