Nitric oxide not carbon monoxide mediates nonadrenergic noncholinergic relaxation in the murine internal anal sphincter.
OBJECTIVE
Inhibitory reflexes in the internal anal sphincter (IAS) are controlled by inhibitory nonadrenergic, noncholinergic innervation (i-NANC). We investigated the roles of 3 different neurohumoral agonists as possible i-NANC neurotransmitters: carbon monoxide (CO), nitric oxide (NO), and vasoactive intestinal peptide (VIP).


METHODS
IAS smooth muscle strips were isolated from wild-type (WT), heme oxygenase (HO)-2 knockout (HO-2-/-) and neuronal NO synthase (nNOS) knockout (nNOS-/-) mice. Relaxation of IAS was induced by CO, NO, VIP, and electrical field stimulation (EFS) in the presence and absence of neurohumoral inhibitors (tin protoporphyrin IX [SnPP IX] for CO synthesis, N(omega)-nitro-L-arginine [L-NNA] for NO synthesis, and VIP(10-28) for VIP receptor). Western blot and immunohistochemistry were used to test the presence and localization of HO (for CO synthesis) types 1 (HO-1) and 2 (HO-2), neuronal NO synthase (nNOS, for NO synthesis), and VIP.


RESULTS
All 3 neurohumoral agonists produced relaxation (with no difference between WT and HO-2-/- IAS), but CO was over 100 times less potent than NO and VIP. EFS produced relaxation in WT and HO-2-/- IAS with the same intensity. L-NNA and nNOS deletion (approximately 80%) and VIP(10-28) (approximately 15%) significantly inhibited the relaxations, whereas SnPP IX had no effect. Positive immunoreactivities for HO-2, nNOS, and VIP were found in the myenteric plexus of WT IAS. HO-2-/- IAS did not express immunoreactivity for HO-2.


CONCLUSIONS
i-NANC relaxations of mouse IAS are primarily mediated via NO (by nNOS activity) and partly via VIP. CO directly relaxes the mouse IAS but does not play any significant role in the i-NANC relaxation.
Rattan
Satish
S
Department of Medicine, Division of Gastroenterology and Hepatology, and Division of Emergency Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA. Satish.Rattan@Jefferson.edu
Regan
Raymond F
RF
Patel
Chirag A
CA
De Godoy
Márcio A F
MA
D000109:Acetylcholine / Q000378:metabolism*; D001003:Anal Canal / Q000166:cytology / Q000294:innervation / Q000378:metabolism; D000818:Animals; D002248:Carbon Monoxide / Q000494:pharmacology*; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004837:Epinephrine / Q000378:metabolism*; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000378:metabolism; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D008665:Metalloporphyrins / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D009126:Muscle Relaxation / Q000502:physiology*; D009130:Muscle, Smooth / Q000187:drug effects* / Q000378:metabolism; D009569:Nitric Oxide / Q000494:pharmacology*; D052248:Nitric Oxide Synthase Type I / Q000235:genetics / Q000378:metabolism; D019335:Nitroarginine / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D011524:Protoporphyrins / Q000494:pharmacology; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology
D004791:Enzyme Inhibitors; D008665:Metalloporphyrins; D010446:Peptide Fragments; D011524:Protoporphyrins; D019335:Nitroarginine; D009569:Nitric Oxide; D014660:Vasoactive Intestinal Peptide; D002248:Carbon Monoxide; C032628:tin protoporphyrin IX; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse; D006419:Heme Oxygenase (Decyclizing); C105245:heme oxygenase-2; D006162:Guanylate Cyclase; D000109:Acetylcholine; D004837:Epinephrine