Functional evidence for purinergic inhibitory neuromuscular transmission in the mouse internal anal sphincter.
The neurotransmitter(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(omega)-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 microM), by desensitization of P2Y receptors with adenosine 5'-[beta-thio]diphosphate (ADP-betaS) (10 microM), and by the selective P2Y1 receptor blocker 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate (MRS2179) (10 microM). Relaxation and IJPs were also significantly reduced by the K(+) channel blocker apamin (1 microM). Removal of extracellular potassium (K(o)) increased IJP amplitude to 205% of control, whereas return of K(o) 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 microM) 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 apamin-sensitive K(+) channels as well as other apamin-insensitive conductances leading to hyperpolarization and relaxation.
McDonnell
Bronagh
B
Dept. of Physiology and Cell Biology, Univ. of Nevada, Reno, Reno, NV 89557, USA.
Hamilton
Ross
R
Fong
Miranda
M
Ward
Sean M
SM
Keef
Kathleen D
KD
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D001003:Anal Canal / Q000294:innervation*; D000818:Animals; D001030:Apamin / Q000494:pharmacology; D004558:Electric Stimulation; D017615:Enteric Nervous System / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D053444:Inhibitory Postsynaptic Potentials; D051379:Mice; D008807:Mice, Inbred BALB C; D009046:Motor Neurons / Q000378:metabolism; D009119:Muscle Contraction; D009126:Muscle Relaxation; D009130:Muscle, Smooth / Q000294:innervation*; D009433:Neural Inhibition / Q000187:drug effects; D009469:Neuromuscular Junction / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D019335:Nitroarginine / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D026902:Potassium Channel Blockers / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists; D011687:Purines / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1; D009435:Synaptic Transmission / Q000187:drug effects; D013779:Tetrodotoxin / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology
D004791:Enzyme Inhibitors; C111914:N(6)-methyl-2'-deoxyadenosine 3',5'-diphosphate; C546383:P2ry1 protein, mouse; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D058919:Purinergic P2 Receptor Antagonists; D011687:Purines; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D013873:Thionucleotides; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D019335:Nitroarginine; D001030:Apamin; D009569:Nitric Oxide; C030812:adenosine 5'-O-(2-thiodiphosphate); D013779:Tetrodotoxin; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D019001:Nitric Oxide Synthase; D011188:Potassium