Effects of the Gβγ-binding inhibitor NSC8668 (Gβγ⁻; 10 μM) on LH release responses stimulated by (A, B) GnRH2 (100 nM) or (C, D) GnRH3 (100 nM). LH release profiles are shown in the left panel (A, C; gray solid square, inhibitor alone; red solid diamond, GnRH2 alone; blue solid diamond, GnRH3 alone; open circle, GnRH + inhibitor) and quantified net LH responses are shown on the right (B, D; mean ± standard error of the mean). Duration of LH release response quantification is indicated by the vertical dotted lines. LH release responses were expressed as a percentage of the pretreatment values (% pretreatment, average of the first 5 perifusion fractions collected; black horizontal bar; 4.08 ± 0.14 ng/mL). Pooled responses are shown from 4 experiments (n = 8) using individual cell preparations from goldfish with gonads that were at a sexually matured/prespawning stage (April). The solid gray horizontal bar indicates the duration of the inhibitor treatment, whereas the red (GnRH2) or blue (GnRH3) horizontal bar represents the 5-minute exposure to either GnRH. Treatments that were significantly different from one another are identified by different symbols (** vs ‡; analysis of variance followed by Tukey’s honest significant difference multiple comparisons; P < 0.05).