VPAC1 and VPAC2 receptor activation on GABA release from hippocampal nerve terminals involve several different signalling pathways

Abstract

Background and purpose: Vasoactive intestinal peptide (VIP) is an important modulator of hippocampal synaptic transmission that influences both GABAergic synaptic transmission and glutamatergic cell excitability through activation of VPAC₁ and VPAC₂ receptors. Presynaptic enhancement of GABA release contributes to VIP modulation of hippocampal synaptic transmission.

Experimental approach: We investigated which VIP receptors and coupled transduction pathways were involved in VIP enhancement of K⁺ -evoked [³ H]-GABA release from isolated nerve terminals of rat hippocampus.

Key results: VIP enhancement of [³ H]-GABA release was potentiated in the presence of the VPAC₁ receptor antagonist PG 97-269 but converted into an inhibition in the presence of the VPAC₂ receptor antagonist PG 99-465, suggesting that activation of VPAC₁ receptors inhibits and activation of VPAC₂ receptors enhances, GABA release. A VPAC₁ receptor agonist inhibited exocytotic voltage-gated calcium channel (VGCC)-dependent [³ H]-GABA release through activation of protein G_i/o , an effect also dependent on PKC activity. A VPAC₂ receptor agonist enhanced both exocytotic VGCC-dependent release through protein G_s -dependent, PKA-dependent and PKC-dependent mechanisms and GABA transporter 1-mediated [³ H]-GABA release through a G_s protein-dependent and PKC-dependent mechanism.

Conclusions and implications: Our results show that VPAC₁ and VPAC₂ VIP receptors have opposing actions on GABA release from hippocampal nerve terminals through activation of different transduction pathways. As VPAC₁ and VPAC₂ receptors are located in different layers of Ammon's horn, our results suggest that these VIP receptors underlie different modulation of synaptic transmission to pyramidal cell dendrites and cell bodies, with important consequences for their possible therapeutic application in the treatment of epilepsy.

Figure 1

VIP bidirectionally modulates K⁺‐evoked [³H]‐GABA release from hippocampal nerve terminals. (A–C) Time course of individual [³H]‐GABA release experiments in which the effect of VIP (1 nM) was tested in the absence of added drugs (A) or in the presence of either the selective VPAC₁ receptor antagonist PG 97‐269 (100 nM, B) or the selective VPAC₂ receptor antagonist PG 99‐465 (100 nM, C). Nerve terminals were labelled with [³H]‐GABA as described and release of [³H]‐GABA was evoked by two 2 min pulses of 25 mM KCl as indicated by the horizontal bars. VIP was added to the test chambers before S₂, as indicated by the horizontal bar, whereas it was not added to the parallel control chambers. Each point represents the mean results of one experiment performed in duplicate chambers. (D) Averaged S₂/S₁ ratios observed when testing the action of VIP applied alone or in the presence of the VPAC₁ and VPAC₂ receptor selective antagonists on K⁺‐evoked [³H]‐GABA release. Evoked release was calculated by integration of the peak, after subtraction of the basal [³H]‐GABA release, and S₂/S₁ ratios were obtained. Each bar represents the mean ± SEM of the results obtained in 4–12 experiments performed in duplicate. ★ P < 0.05, significantly different from the S₂/S₁ ratio in the corresponding control conditions; Student's t‐test. (E) Averaged effects for the enhancement of K⁺‐evoked [³H]‐GABA release caused by VIP in hippocampal nerve terminals in the absence and in the presence of VPAC₁ or VPAC₂ receptor selective antagonists, or the broad‐range VIP receptor blocker [Ac‐Tyr¹, D‐Phe²] GRF (1‐29). The effect of VIP was calculated by comparing the S₂/S₁ ratio obtained in test (presence of VIP during S₂) and in control conditions. Each bar represents the mean ± SEM of results obtained from n=4–12 experiments. ★ P < 0.05, significantly different from 1nM VIP alone; ANOVA, followed by Dunnett's multiple‐comparison test.

Figure 2

Selective VPAC₁ and VPAC₂ receptor activation has opposing actions on K⁺‐evoked [³H]‐GABA release from hippocampal nerve terminals. (A–C) Time course of individual [³H]‐GABA release experiments in which the effect of VIP (1 nM, A), the VPAC₁ receptor agonist [K¹⁵, R¹⁶, L²⁷] VIP (1‐7)/GRF (8‐27) (10 nM, B) and the VPAC₂ receptor agonist RO 25‐1553 (10 nM, C) was tested. Nerve terminals were labelled with [³H]‐GABA as described and release of [³H]‐GABA was evoked by two 2 min pulses of 25 mM KCl as indicated by the vertical bars. VIP or VIP receptor agonists were added to the test chambers before S₂, as indicated by the horizontal bar, whereas were not added to the parallel control chambers. Each point represents the mean results of one experiment performed in duplicate chambers. (D) Averaged S₂/S₁ ratios observed when testing the action of VIP, [K¹⁵, R¹⁶, L²⁷] VIP (1‐7)/GRF (8‐27) or RO 25‐1553 on K⁺‐evoked [³H]‐GABA release. The S₂/S₁ ratios were calculated as described . Each bar represents the mean ± SEM of the results obtained in 6–12 experiments performed in duplicate. ★ P < 0.05, significantly different from S₂/S₁ ratio in control conditions; ANOVA, followed by Dunnett's multiple‐comparison test. (E) Concentration–response curves for the effect of [K¹⁵, R¹⁶, L²⁷] VIP (1‐7)/GRF (8‐27) or RO 25‐1553 on K⁺‐evoked [³H]‐GABA in hippocampal nerve terminals. The effect of VIP receptor agonists was calculated by comparing the S₂/S₁ ratio obtained in test (presence of agonists during S₂) and in control conditions. Each point represents the mean ± SEM of results obtained in 6–10 experiments.

Figure 3

VIP receptor‐mediated modulation of [³H]‐GABA release involves both VGCC‐dependent and GAT‐1 carrier‐dependent release mechanisms. Averaged effects on [³H]‐GABA release elicited by VIP (1 nM, A), the VPAC₁ receptor agonist [K¹⁵, R¹⁶, L²⁷] VIP (1‐7)/GRF (8‐27) (10 nM, B) and the VPAC₂ receptor agonist RO 25‐1553 (10 nM, C) tested alone or in the presence of either the VGCC inhibitor (CdCl₂) or the GAT‐1 inhibitor (SKF89976A) in hippocampal nerve terminals. The effect of VIP or selective agonists was calculated by comparing the S₂/S₁ ratio obtained in test (presence of VIP or VIP receptor agonists during S₂) and in control conditions. Each bar represents the mean ± SEM of results obtained in 4–12 experiments. ★ P < 0.05, significantly different from VIP or selective agonists alone; ANOVA, followed by Dunnett's multiple‐comparison test.

Figure 4

VIP receptor‐mediated modulation of [³H]‐GABA release involves both PKA‐dependent, PKC‐dependent and CaMKII‐dependent mechanisms. Averaged effects on [³H]‐GABA release elicited by VIP (1 nM, A), the VPAC₁ receptor agonist [K¹⁵, R¹⁶, L²⁷] VIP (1‐7)/GRF (8‐27) (10 nM, B) and the VPAC₂ receptor agonist RO 25‐1553 (10 nM, C) tested alone or in the presence of either the PKA inhibitor (H‐89), the PKC inhibitor (GF109203X) or the CaMKII inhibitor (KN‐62) in hippocampal nerve terminals. The effect of VIP or selective agonists was calculated by comparing the S₂/S₁ ratio obtained in test (presence of VIP or VIP receptor agonists during S₂) and in control conditions. Each bar represents the mean ± SEM of results obtained in 4–12 experiments. ★ P < 0.05, significantly different from VIP or selective agonists alone; ANOVA, followed by Dunnett's multiple‐comparison test.

Figure 5

Differential G‐protein coupling and downstream transduction mechanisms are involved in VPAC₁ and VPAC₂ receptor‐mediated modulation of [³H]‐GABA release. Averaged effects on [³H]‐GABA release elicited by VIP (1 nM, A and B), the VPAC₁ receptor agonist [K¹⁵, R¹⁶, L²⁷] VIP (1‐7)/GRF (8‐27) (10 nM, C) and the VPAC₂ receptor antagonist RO 25‐1553 (10 nM, D) tested alone, in the presence of cholera toxin (ChTx) or Pertussis toxin (PTx) together with either the PKA inhibitor (H‐89) or the PKC inhibitor (GF109203X) in hippocampal nerve terminals. The effect of VIP or selective agonists was calculated by comparing the S₂/S₁ ratio obtained in test (presence of VIP or VIP receptor agonists during S₂) and in control conditions. Each bar represents the mean ± SEM of results obtained in 3–12 experiments. ★ P < 0.05, significantly different from VIP or selective agonists alone or, VIP or agonists in the presence of either ChTx or PTx; ANOVA, followed by Sidak's multiple‐comparison test.

Figure 6

VPAC₂ receptor‐mediated enhancement of [³H]‐GABA release through VGCC‐dependent and GAT‐1 carrier‐dependent mechanisms involves different protein kinases. Averaged effects on [³H]‐GABA release elicited by VPAC₂ receptor antagonist RO 25‐1553 (10 nM) tested alone, in the presence of VGCC inhibitor (CdCl₂) or the GAT‐1 inhibitor (SKF89976A) together with either the PKA inhibitor (H‐89) or the PKC inhibitor (GF109203X) in hippocampal nerve terminals. The effect of RO 25‐1553 (10 nM) was calculated by comparing the S₂/S₁ ratio obtained in test (presence of RO 25‐1553 during S₂) and in control conditions. Each bar represents the mean ± SEM of results obtained in 3–9 experiments. ★ P < 0.05, significantly different from RO 25‐1553 in the absence of other drugs (first column), or VIP or agonists in the presence of either CdCl₂ or SKF89976A (second column): ANOVA, followed by Sidak's multiple‐comparison test .

Figure 7

Schematic representation of the main mechanisms involved in VPAC₂‐mediated VIP enhancement of GABA release from hippocampal nerve terminals. AC, adenylate cyclase; G_sα, G_s protein α subunit; VPAC₂ R, VPAC₂ receptor.

Figure 8

Schematic representation of the main mechanisms involved in VPAC₁‐mediated inhibition of GABA release from hippocampal nerve terminals. G_iα,G_i protein α subunit; G_iβγ,G_i protein β–γ dimer; VPAC₁ R, VPAC₁ receptor 1.