The aim of the present study was to investigate the relative importance of the different putative nonadrenergic noncholinergic (NANC) mediators and their interplay with cholinergic nerves in the rabbit internal anal sphincter (IAS). IAS preparations were mounted in organ baths for recording of isometric tension. Transmural field stimulation (TMS; 5-s trains; supramaximal voltage, 140-160 V; 0.4-ms impulse duration) was applied every 2 min with frequencies varying from 0.2 to 32 Hz. TMS induced frequency-dependent relaxations that amounted to 89.3 +/- 2.2% (n = 7). N omega-nitro-L-arginine (L-NNA; 10(-7)-10(-4) M; 8 Hz) reduced relaxations and this effect was partially inhibited by preincubation with L-arginine (10(-4) M). The effect of L-NNA was attenuated by atropine preincubation. Apamin (10(-6) M) shifted the frequency-response curve to the right but left maximal relaxations in response to TMS unaffected. In the presence of L-NNA (10(-4) M) and atropine (10(-6) M), the action (area between the frequency-response curve with or without a substance) of apamin was more pronounced, but, despite the presence of both L-NNA and apamin, some relaxation still remained. The frequency-response curve (control) was significantly shifted to the right by carbachol (10(-6) M). Concentration-response experiments showed that the response to exogenous nitric oxide (NO; 10(-7)-10(-4) M) was unaffected by carbachol (10(-6) M) preincubation, whereas responses to vasoactive intestinal polypeptide (VIP) and ATP were significantly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)