NANC inhibitory neuromuscular transmission in the hamster distal colon

Abstract

The neurotransmitter(s) that generate the inhibitory junctional potential (IJP) in the circular muscle of hamster distal colon and their mechanisms have not been elucidated. The aim of the present study, therefore, was to determine the contributing roles of the non-adrenergic, non-cholinergic (NANC) inhibitory transmitter(s) including nitric oxide (NO), adenosine 5′-triphosphate (ATP) and vasoactive intestinal polypeptide (VIP) in the generation of IJP in the hamster distal colon. For this purpose, the effects of the corresponding blockers of these putative NANC inhibitory mediators have been investigated using microelectrode technique. Intracellular membrane potential recordings were made from smooth muscle cells at 35 °C in Tyrode's solution that contained atropine (0.5 μM), guanethidine (3 μM) and nifedipine (0.5 μM). Single electrical stimuli (0.5 ms, 50 V) as well as trains of two and five pulses (20 Hz at the same duration and voltage) elicited NANC IJP consisted of initial fast (IJP-F) followed by a slow hyperpolarization (IJP-S). The response had been abolished by tetrodotoxin (TTX, 0.3 μM). The nitric oxide synthase (NOS) inhibitor, N G-nitro-L-arginine methyl ester (L-NAME; 200 μM) blocked IJP-S but enhanced IJP-F. The later had been blocked with suramin, a universal P2 receptor antagonist, or with CBF3GA, a P2Y receptor antagonist at dose-dependent fashions. The IJP-F had been markedly inhibited by desensitization of P2Y receptor with its putative agonist 2-methylthio-ATP (2-meSATP, 50 μM for 30 min). IJP-F was sensitive to the P2Y1 receptor specific antagonist A3P5PS (10 μM) and to the G-protein inhibitor, pertussis toxin (PTX, 400 ng/ml for 2 h) as well as to the small and intermediate Ca 2+ sensitive K + channels blocker, apamin (0.3 μM). IJP-S was blocked by the guanylate cyclase (GC) inhibitor, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 μM) and was partially sensitive to apamin. Exogenously applied ATP (100 μM–1 mM) produced typical hyperpolarization that was blocked by suramin, CBF3GA and 2-meSATP desensitization; while exogenously applied NO (3–10 μM) produced slowly developing hyperpolarization that was not blocked by L-NAME but ODQ. In the presence of both purinergic and nitrergic inhibitors, stimulation using a train of eight pulses at 25 Hz evoked a small slow hyperpolarization that was sensitive to the VIP antagonist (VIP 6–28, 1 μM). Exogenous application of VIP (1–10 μM) produced similar response that was not evident in the presence of VIP 6–28. These data indicate that NANC IJP that is generated in the circular muscle cells of hamster distal colon is mediated by ATP and NO via P2Y1/P2Y2 receptor and GC-dependent pathways, respectively. A masked role for VIP is also indicated.