Functional evidence for purinergic inhibitory neuromuscular transmission in the mouse internal anal sphincter

Abstract

transmitter(s) underlying nitric oxide synthase (NOS)-independent neural inhibition in the internal anal sphincter (IAS) is still uncertain. The present study investigated the role of purinergic transmission. Contractile and electrical responses to electrical field stimulation of nerves (0.1-5 Hz for 10 -60 s) were recorded in strips of mouse IAS. A single stimulus generated a 28-mV fast inhibitory junction potential (IJP) and relaxation. The NOS inhibitor N -nitro-L-arginine (L-NNA) reduced the fast IJP duration by 20%. Repetitive stimulation at 2.5-5 Hz caused a more sustained IJP and sustained relaxation. L-NNA reduced relaxation at 1 Hz and the sustained IJP at 2.5-5 Hz. All other experiments were carried out in the presence of NOS blockade. IJPs and relaxation were significantly reduced by the P2 receptor antagonists 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid (PPADS) (100 M), by desensitization of P2Y receptors with adenosine 5Ј-[␤-thio]diphosphate (ADP-␤S) (10 M), and by the selective P2Y1 receptor blocker 2Ј-deoxy-N 6 -methyl adenosine 3Ј,5Ј-diphosphate (MRS2179) (10 M). Relaxation and IJPs were also significantly reduced by the K ϩ channel blocker apamin (1 M). Removal of extracellular potassium (Ko) increased IJP amplitude to 205% of control, whereas return of Ko 30 min later hyperpolarized cells by 19 mV and reduced IJP amplitude to 50% of control. Exogenous ATP (3 mM) relaxed muscles in the presence of TTX (1 M) and hyperpolarized cells by 15 mV. In conclusion, these data suggest that purinergic transmission significantly contributes to NOS-independent neural inhibition in the mouse IAS. P2Y1 receptors, as well as at least one other P2 receptor subtype, contribute to this pathway. Purinergic receptors activate apaminsensitive K ϩ channels as well as other apamin-insensitive conductances leading to hyperpolarization and relaxation.gastrointestinal; enteric; motor inhibition; NANC transmission THE INTERNAL ANAL SPHINCTER (IAS) aids in maintaining fecal continence and permits evacuation of fecal contents during the rectoanal inhibitory reflex (RAIR). To fulfill these functions, the IAS usually exists in a state of contraction that can be relaxed by inhibitory nerves during the defecation reflex. In the present study we examined inhibitory motor innervation to the mouse IAS. The predominant neurotransmitters underlying inhibitory neural responses in the gastrointestinal tract include nitric oxide (NO), VIP, pituitary adenylate cyclase-activating polypeptide, and ATP (or a related compound). There is still controversy regarding the contribution of these neurotransmitters to neural inhibition in the IAS. Whereas some studies suggest that neural inhibition is almost exclusively due to NO in combination with VIP (40), earlier studies of the guinea pig (39), rat (14), and rabbit (32) IAS provided evidence that a purinergic neural pathway contributes as well.Membrane hyperpolarization is an important mechanism contributing to inhibitory motor responses. ...

P2Y1 Receptors Mediate Apamin-Sensitive and -Insensitive Inhibitory Junction Potentials in Murine Colonic Circular Smooth Muscle

“…Apamin-sensitive purinergic IJPs were recorded in the small intestine of guinea pigs (Crist et al, 1992) and lower esophageal sphincter (Zhang et al, 2008) of mice, whereas apaminsensitive and -resistant IJPs, with the latter predominating, were shown in the human colon (Gallego et al, 2006) and mouse internal anal sphincter (McDonnell et al, 2008) and colon (Spencer et al, 1998b). Orientation of the tissues was not specified in the aforementioned reports.…”

Colonic Migrating Motor Complexes, High Amplitude Propagating Contractions, Neural Reflexes and the Importance of Neuronal and Mucosal Serotonin

“…2A). 8,24,99 Under these circumstances the CMMC behaves like a rhythmic peristaltic wave since it involves activation of myenteric descending inhibitory and ascending excitatory nerve pathways. Furthermore, if a fecal pellet is held in the lumen of the isolated guinea-pig distal colon, which rarely exhibits spontaneous CMMCs, 100 then rhythmic orthogradely propagating CMMCs are observed.…”

Purinergic signalling in the gastrointestinal tract and related organs in health and disease

“…ATP hyperpolarised and relaxed the internal anal sphincter of guinea pig [440,562] and rat [171]. P2Y 1 receptors mediated the effects and both apamin-sensitive K + channels and apamininsensitive conductances were involved in hyperpolarisation and relaxation of the mouse internal anal sphincter [466].…”