ABSTRACT
BACKGROUND: γ-Aminobutyric acid (GABA) acts on specific neural receptors [A, B and C(Aρ)] to modulate gastrointestinal function. The precise role of GABA receptor activation in the regulation of presynaptic nitric oxide (NO) synthesis in nerve terminals is unknown. METHODS: Rat ileal nerve terminals were isolated by differential centrifugation. Nitric oxide synthesis was analysed using a L-[(3) H]arginine assay. In vitro studies were performed under non-adrenergic non-cholinergic (NANC) conditions on isolated ileal segments. KEY RESULTS: γ-Aminobutyric acid inhibited NO synthesis significantly (n = 6, P < 0.05) [(fmol mg(-1) min(-1)) control: 27.7 ± 1.5, 10(-6) mol L(-1): 19.7 ± 1.3; 10(-5) mol L(-1): 17.5 ± 3.0]. This effect was antagonized by the GABA A receptor antagonist bicuculline and the GABA C receptor antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), but not by the GABA B receptor antagonist SCH 50911. The GABA A receptor agonist muscimol [(fmol mg(-1) min(-1)) control: 27.6 ± 1.0, 10(-6) mol L(-1): 19.1 ± 1.7, n = 5, P < 0.05] and the GABA C receptor agonist cis-4-aminocrotonic acid (CACA) [(fmol mg(-1) min(-1)) control: 29.5 ± 3.2, 10(-3) mol L(-1): 20.3 ± 2.5, n = 6, P < 0.05], mimicked the GABA-effect, whereas the GABA B agonist baclofen was ineffective. Bicuculline reversed the inhibitory effect of muscimol, TPMPA antagonized the effect of CACA. In functional experiments the GABA A and C receptor agonists reduced the NANC relaxation induced by electrical field stimulation in rat ileum by about 40%. After NOS-inhibition by Nε-nitro-L-arginine methyl ester (L-NAME) the GABA A receptor agonist had no effect, whereas the GABA C receptor agonist still showed a residual response. CONCLUSIONS & INFERENCES: γ-Aminobutyric acid inhibits neural NO synthesis in rat ileum by GABA A and GABA C(Aρ) receptor-mediated mechanisms.
Subject(s)
Ileum/innervation , Ileum/physiology , Nitric Oxide/biosynthesis , Synaptosomes/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , GABA Antagonists/metabolism , Male , Muscle Relaxation/physiology , Muscle, Smooth/physiology , Nitric Oxide Synthase/metabolism , Protein Isoforms/metabolism , Rats , Rats, Sprague-Dawley , Rats, Wistar , Receptors, GABA/metabolism , Synapses/metabolismABSTRACT
In enteric synaptosomes of the rat, the role of voltage-dependent Ca(2+) channels in K(+)-induced VIP release and nitric oxide (NO) synthesis was investigated. Basal VIP release was 39 +/- 4 pg/mg, and cofactor-substituted NO synthase activity was 7.0 +/- 0.8 fmol. mg(-1). min(-1). K(+) depolarization (65 mM) stimulated VIP release Ca(2+) dependently (basal, 100%; K(+), 172.2 +/- 16.2%; P < 0.05, n = 5). K(+)-stimulated VIP release was reduced by blockers of the P-type (omega-agatoxin-IVA, 3 x 10(-8) M) and N-type (omega-conotoxin-GVIA, 10(-6) M) Ca(2+) channels by ~50 and 25%, respectively, but not by blockers of the L-type (isradipine, 10(-8) M), Q-type (omega-conotoxin-MVIIC, 10(-6) M), or T-type (Ni(2+), 10(-6) M) Ca(2+) channels. In contrast, NO synthesis was suppressed by omega-agatoxin-IVA, omega-conotoxin-GVIA, and isradipine by ~79, 70, and 70%, respectively, whereas Ni(2+) and omega-conotoxin-MVIIC had no effect. These findings are suggestive of a coupling of depolarization-induced VIP release primarily to the P- and N-type Ca(2+) channels, whereas NO synthesis is presumably dependent on Ca(2+) influx not only via the P- and N- but also via the L-type Ca(2+) channel. In contrast, none of the Ca(2+) channel blockers affected VIP release evoked by exogenous NO, suggesting that NO induces VIP secretion by a different mechanism, presumably involving intracellular Ca(2+) stores.
