Molecular and pharmacological evidence for a facilitatory functional role of pre-synaptic GLUK2/3 kainate receptors on GABA release in rat trigeminal caudal nucleus.

BACKGROUND: Gamma-aminobutyric acid (GABA) and glutamate (GLU) are involved in nociceptive signals processing in the trigeminal system. In this study, we investigated the influence of excitatory transmission on GABA release in nerve terminals isolated from the rat trigeminal caudal nucleus (TCN). METHODS: We utilize biochemical (superfused synaptosomes loaded with [(3) H]GABA) and morphological (immunofluorescence experiments with specific antibody) techniques. RESULTS: Our results show that GLU potentiates the release of [(3) H]GABA evoked by 9, 15 and 30 mM [K(+)](e); 15 mM [K(+)](e)-evoked [(3) H]GABA release was also reinforced by domoate and kainate (KA), two naturally occurring GLU-receptor agonists. The enhancement of 15 mM [K(+)](e)-evoked [(3) H]GABA release produced by 100 µM KA was abolished by NBQX, a mixed AMPA/KA receptor antagonist, but was not affected by GYKI52466, a selective AMPA receptor antagonist. ATPA, a selective agonist for KA receptors containing the GLUK1 subunit, had no effect on depolarization-induced [(3) H]GABA release, and UBP310, which selectively antagonizes these same receptors, failed to reverse the KA-induced potentiation of 15 mM [K(+)](e)-evoked [(3) H]GABA release. The KA-induced potentiation was also unaffected by concanavalin A (10 µM), a positive allosteric modulator of GLUK1- and GLUK2-containing KA receptors. Immunofluorescence experiments revealed that GABAergic nerve terminals in the TCN differentially expressed GLUK subunits, with GLUK2/3-positive terminals being twice more abundant than GLUK1-containing synaptosomes. CONCLUSIONS: These findings indicate that pre-synaptic KA receptors facilitating GABA release from TCN nerve terminals mainly express GLUK2/GLUK3 subunits, supporting the notion that different types of KA receptors are involved in the various stages of pain transmission.

KV7 channels regulate muscle tone and nonadrenergic noncholinergic relaxation of the rat gastric fundus.

Voltage-dependent type 7 K+ (KV7) channels play important physiological roles in neurons and muscle cells. The aims of the present study were to investigate the motor effects of KV7 channel modulators in the rat gastric fundus and the expression of KV7 channels in this tissue. Muscle tone and electrical field stimulation (EFS)-evoked relaxations of precontracted longitudinal muscle strips of the rat gastric fundus were investigated under nonadrenergic noncholinergic conditions by organ bath studies. Gene expression was studied by real-time PCR and tissue localization of channels was investigated by immunohistochemistry. The KV7 channel blocker XE-991 induced concentration-dependent contractions, with mean pD2 and Emax of 5.4 and 48% of the maximal U46619-induced contraction, respectively. The KV7 channel activators retigabine and flupirtine concentration-dependently relaxed U46619-precontracted strips, with pD2s of 4.7 and 4.4 and Emax of 93% and 91% of the maximal relaxation induced by papaverine, respectively. XE-991 concentration-dependently inhibited retigabine-induced relaxation with a pIC50 of 6.2. XE-991 and DMP-543, another KV7 channel blocker, increased by 13-25% or reduced by 11-21% the relaxations evoked by low- or high-frequency EFS, respectively. XE-991 also reduced the relaxation induced by vasoactive intestinal polypeptide (VIP) by 33% of controls. Transcripts encoded by all KV7 genes were detected in the fundus, with 7.4 and 7.5 showing the highest expression levels. KV7.4 and 7.5 channels were visualized by confocal immunofluorescence in both circular and longitudinal muscle layers. In conclusion, in the rat proximal stomach, KV7 channels appear to contribute to the resting muscle tone and to VIP- and high-frequency EFS-induced relaxation. KV7 channel activators could be useful relaxant agents of the gastric smooth muscle.