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Endothelin Activates Large-Conductance K⁺ Channels in Rat Lactotrophs: Reversal by Long-Term Exposure to Dopamine Agonist¹

Program in Neuroscience, Department of Biological Science, Florida State University, Tallahassee, Florida 32306

Address all correspondence and requests for reprints to: Marc E. Freeman, Department of Biological Science, Florida State University, Tallahassee, Florida 32306-4075. E-mail: freeman{at}neuro.fsu.edu

Endothelin-1 (ET-1) inhibits PRL secretion from cultured rat lactotrophs. However, ET-1 stimulates PRL secretion after cultured lactotrophs have been exposed for 48 h to dopamine or D₂ dopamine agonists. In the present study, we have used cell-attached and inside-out patch recordings to establish an ionic basis for these effects. Bath application of 20 nM ET-1 to untreated lactotrophs evoked a robust and persistent activation of large-conductance K⁺ channels in cell-attached patches. This effect of ET-1 had a long latency to onset, was maintained for as long as ET-1 was present, and required at least 10 min of washing in control saline before complete recovery was achieved. The stimulatory effect of 20 nM ET-1 on these channels was markedly attenuated in the presence of the selective ET_A receptor antagonist BQ-610 (200 nM), or after pertussis toxin (200 ng/ml, 16 h) pretreatment.

The unitary slope conductance of the ET-1 activated channels in cell attached patches was 165 and 95 pS when the recording electrodes contained 150 and 5.4 mM KCl, respectively. These channels were voltage-sensitive and their activity increased upon patch depolarization. Previously activated channels in cell-attached patches became quiescent immediately upon patch excision into Ca²⁺-free bath saline. Exposure of the intracellular surface to 0.1 µM Ca²⁺ restored the activity of these channels similar to the level seen before patch excision. In addition, preincubating the cells with the membrane-permeable Ca²⁺-chelator BAPTA-AM, or using Ca²⁺-free solution in the recording pipettes, prevented the activation of these channels by ET-1. The ET-1 activated large-conductance Ca²⁺-dependent K⁺ (BK_Ca) channels were blocked by 20 mM tetraethylammonium but were insensitive to the K⁺ channel blockers apamin (1 µM), charybdotoxin (200 nM), or iberiotoxin (200 nM). Acute application of 10 µM dopamine and 20 nM ET-1 caused activation of BK_Ca channels with indistinguishable kinetic properties, although the effect of dopamine occurred with shorter latency. After 48-h exposure to the specific D₂ dopamine receptor agonist (±)-2-(N-phenyl-N-propyl) amino-5-hydroxytetralin hydrochloride (PPHT, 500 nM), bath application of 20 nM ET-1 resulted in inhibition of spontaneously active BK_Ca channels.

These data suggest that both the stimulatory and inhibitory effects of ET-1 on PRL secretion are mediated, at least in part, by actions on BK_Ca channels, and that long term exposure to dopamine or D₂ agonists alters the signaling pathways from the ET_A receptor to BK_Ca channels.

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