Key points• Vasoactive intestinal polypeptide (VIP)-ergic neuromuscular transmission (NMT) was examined in the internal anal sphincter of wild-type mice and compared with that of VIP −/− mice.• Relaxation and hyperpolarization during brief trains of electrical field stimulation (EFS; 4 s) were mediated by purinergic and nitrergic NMT.• During longer stimulus trains, a non-purinergic, non-nitrergic (NNNP) relaxation and hyperpolarization slowly developed and persisted for several minutes beyond the end of the stimulus train.• The NNNP NMT was abolished by VIP receptor antagonists, absent in the VIP −/− mouse internal anal sphincter and mimicked by exogenous VIP.• These data suggest that NNNP NMT gives rise to ultraslow relaxation and hyperpolarization that is mediated by VIP. In vivo, this pathway may be activated with larger rectal distensions, leading to a more prolonged anal relaxation.Abstract There is evidence that vasoactive intestinal polypeptide (VIP) participates in inhibitory neuromuscular transmission (NMT) in the internal anal sphincter (IAS). However, specific details concerning VIP-ergic NMT are limited, largely because of difficulties in selectively blocking other inhibitory neural pathways. The present study used the selective P2Y 1 receptor antagonist MRS2500 (1 μM) and the nitric oxide synthase inhibitor N G -nitro-L-arginine (L-NNA; 100 μM) to block purinergic and nitrergic NMT to characterize non-purinergic, non-nitrergic (NNNP) inhibitory NMT and the role of VIP in this response. Nerves were stimulated with electrical field stimulation (0.1-20 Hz, 4-60 s) and the associated changes in contractile and electrical activity measured in non-adrenergic, non-cholinergic conditions in the IAS of wild-type and VIP −/− mice. Electrical field stimulation gave rise to frequency-dependent relaxation and hyperpolarization that was blocked by tetrodotoxin. Responses during brief trains of stimuli (4 s) were mediated by purinergic and nitrergic NMT. During longer stimulus trains, an NNNP relaxation and hyperpolarization developed slowly and persisted for several minutes beyond the end of the stimulus train. The NNNP NMT was abolished by VIP6-28 (30 μM), absent in the VIP −/− mouse and mimicked by exogenous VIP (1-100 nM). Immunoreactivity for VIP was co-localized with neuronal nitric oxide synthase in varicose intramuscular fibres but was not detected in the VIP −/− mouse IAS. In conclusion, this study identified an ultraslow component of inhibitory NMT in

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