Synaptic modulation by dopamine of calcium currents in rat pars intermedia

Abstract

Melanotrophs of the rat pars intermedia are innervated by dopaminergic fibers traveling through the pituitary stalk which inhibit secretion via an action on D-2 receptors. As secretion from the melanotroph has been shown to be calcium (Ca2+) dependent, it is possible that dopamine may have an action to inhibit Ca2+ currents in these cells. This possibility was tested by examining the effects of exogenously applied dopaminergic agonists or synaptically released dopamine upon Ca2+ currents recorded under single electrode voltage clamp in intact rat pars intermedia in vitro. Following blockade of sodium and potassium currents in melanotrophs, Ca2+ spikes were elicited with intracellular injection of depolarizing currents; electrical stimulation of the pituitary stalk caused an inhibition of the Ca2+-based action potentials which lasted for several seconds. Using single-electrode voltage-clamp techniques, we recorded inward Ca2+ currents corresponding to the T, N, and L types (see Williams et al., 1990). Stimulation of the pituitary stalk inhibited both the low- and high-threshold peak inward Ca2+ currents elicited from a holding potential of -90 mV. In contrast, when noninactivating Ca2+ currents were elicited from a holding potential of -30 mV, the currents were not altered by stalk stimulation. This pattern of inhibition of the Ca2+ currents was consistent with the preferential inhibition, by stalk stimulation, of the N and T Ca2+ currents, while sparing the L current. We observed that inhibition of Ca2+ currents due to stalk stimulation was completely reversed by bath perfusion of domperidone (1 μM), an antagonist of dopamine at the D-2 receptor. Quinpirole, a D-2 receptor agonist, mimicked the action of pituitary stalk stimulation by inhibiting Ca2+ currents elicited from a holding potential of -90 mV, but not from -30 mV. To examine the role of G-proteins in mediating dopaminergic inhibition of Ca2+ currents in the melanotrophs, rats were pretreated with pertussis toxin, which blocks the actions of some G-proteins. In pertussis toxin-treated rats, only 25% of cells showed any inhibition of Ca2+ currents due to stalk stimulation compared with 100% of controls. In another series of experiments, cells were impaled with electrodes containing GTPγS, a GTP analog which leads to irreversible activation of G-proteins. In these cells, only the noninactivating Ca2+ current was present, indicating that the transient currents normally inhibited by dopamine were already maximally inhibited by GTPγS. These experiments thus indicate that both exogenously applied D-2 agonist and synaptically released dopamine act at a D-2 receptor on the melanotroph to inhibit certain Ca2+ currents via a G-protein mediated mechanism. This is the first characterization of synaptic modulation, by dopamine, of Ca2+ currents in the mammalian nervous system.