Effectiveness of GABAB antagonists in inhibiting baclofen-induced reductions in cytosolic free Ca concentration in isolated melanotrophs of rat

Abstract

1 The purpose of the present experiments was to assess the activities of GABA_B receptor antagonists in mammalian isolated melanotrophs. 2 Cytosolic free Ca concentration ([Ca²⁺]_i) in rat melanotrophs in primary culture was monitored with the fluorescent probe, fura-2. 3 (−)-Baclofen lowered [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ of 0.96 μm. The reduction in [Ca²⁺]_i produced by (−)-baclofen at a maximally effective concentration (100 μm) was similar to that produced by the classic transmitter inhibitory to melanotroph secretion, dopamine, at a corresponding concentration (100 nm), or by perifusion with a nominally Ca-free solution. 4 The GABA_B receptor antagonists, 3-aminopropyl(diethoxymethyl)phosphinic acid (CGP 35348), 2-hydroxy saclofen, phaclofen and 4-amino-3-(5-methoxybenzo[b]furan-2-yl) butanoic acid (9H), had inhibitory effects on the reduction in [Ca²⁺]_i produced by (−)-baclofen (3 μm). Of the antagonists tested, CGP 35348 was the most potent with an IC₅₀ of 60 μm, compared to 120 to 400 μm for the others. CGP 35348 acted competitively. 5 CGP 35348 alone had no effect on basal [Ca²⁺]_i, or on the changes in [Ca²⁺]_i produced by dopamine (10 nm) or the specific GABA_A receptor agonist, muscimol (10 μm). 6 The evidence indicates that of the antagonists tested, CGP 35348 offers the greatest promise for pharmacological analysis of the functional significance of the GABA_B receptors in melanotrophs.