Center of Chemistry and Biochemistry

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Publications

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

Publication . Cunha-Reis, Diana; Ribeiro, Joaquim A.; Almeida, Rodrigo F.M. De; Sebastião, Ana M

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 Gi/o , an effect also dependent on PKC activity. A VPAC2 receptor agonist enhanced both exocytotic VGCC-dependent release through protein Gs -dependent, PKA-dependent and PKC-dependent mechanisms and GABA transporter 1-mediated [3 H]-GABA release through a Gs 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.

2017Journal article

Restricted access

Sphingolipid domains in the plasma membrane of fungal cells : Interplay with membrane proteins and antifungal resistance

Publication . Santos, Filipa C.; Almeida, Rodrigo F. M. de

Plasma membrane (PM) carries out multiple functions a tightly regulated dynamic organization into specialized domains of different size, stability, and composition. Sphingolipids are a major class of lipids of PM being crucial for its structure and function. A specific type of ergosterol-depleted PM domains in fungi, where lipids are tightly packed in a very rigid gel phase, comprises the sphingolipid-enriched domains (SLEDs). In this work, the presence of SLEDs in a mold, Neurospora crassa, was disclosed, as well as their involvement in the response to the antifungal staurosporine. A comparison with the budding yeast Saccharomyces cerevisiae was performed, taking into consideration the known differences between their PM sphingolipidomes. The studies with N. crassa were performed in conidial suspensions along different time points of conidial germination, using several fluorescence spectroscopy techniques and a multiprobe approach. S. cerevisiae living cells were studied in mi-exponential phase. To evaluate the impact of changing sphingolipid composition in the organization of the two main yeast PM compartments, MCC (arginine/ H+ symporter Can1p) and MCP (H+ ATPase Pma1p), the time-resolved fluorescence properties of Can1p tagged with GFP and Pma1p tagged with mRFP, respectively, were studied by FLIM in wt and a mutant strain with a different sphingolipid profile, ipt1∆. IPT1 deletion strongly affects the rigidity of gel domains and Pma1p PM distribution, whereas no significant alterations could be perceived neither in ergosterol-enriched domains nor Can1p distribution. Thus, this work strongly suggests that proper SLEDs hydrophobic chain packing is required for an adequate organization of the MCP, but not MCC. Finally, considering that the PM is crucial for antifungal action, a biophysical connection between SLEDs and antifungal activity was explored in S. cerevisiae.

2023-01Doctoral thesis

Open access