Vol. 294, Issue 1, 270-279, July 2000

Nitric Oxide from Enteric Nerves Acts by a Different Mechanism from Myogenic Nitric Oxide in Canine Lower Esophageal Sphincter¹

Edwin E. Daniel, Jennifer Jury, Anne Marie Salapatek², Tim Bowes³, Annette Lam³, Shoba Thomas³, Michelle Ramnarain³, Vicky Nguyen³ and Valerie Mistry³

Department of Medicine, McMaster University, Hamilton, Ontario, Canada

In canine lower esophageal sphincter, myogenic constitutive nitric-oxide (NO) synthase (NOS) in plasma membrane limits tone by opening large conductance Ca²⁺-dependent K⁺ channels (BK_Ca channels) and hyperpolarizing the membrane. We examined whether K_V channels were involved and whether NO from enteric nerves and from NO donors used the same mechanisms. With nerves inactive, 100 nM iberiotoxin, like N-nitro-L-arginine (L-NOARG), increased tone but less. 4-Aminopyridine (4-AP) at 5 mM behaved similarly. Tetraethyl ammonium (TEA) at 20 mM equaled the effect of L-NOARG and occluded any tone increase from any combination of these agents. More than iberiotoxin or 4-AP, TEA decreased relaxations in response to sodium nitroprusside (SNP) or 3-morpholino-sydnonimine (Sin-1) by ~50%. In whole-cell patch-clamp recordings, TEA and 4-AP reduced outward K⁺ currents additively by >90% at depolarization of +90 mV. Thus, K⁺ channels in addition to BK_Ca channels are opened by myogenic NO, and exogenous NO had relaxing effects both related and unrelated to K⁺ channel openings. TEA (20 mM) increased tone but did not inhibit relaxations to electrical field stimulation (EFS) of enteric nerves. 4-AP relaxed tone, an effect that was abolished and reversed by L-NOARG. 4-AP apparently released NO and acetylcholine from nerves. The putative Cl channel blocker niflumic acid (NFA; 30-100 µM) dose dependently reduced tone, but tone, restored by 10⁶ M carbachol or 20 mM TEA, was still relaxed by EFS and by SNP. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) at 500 to 1000 µM did not inhibit relaxation to EFS or SNP. The addition of TEA (20 mM) to DIDS (1000 µM) induced tonic and phasic activity and markedly inhibited relaxations to EFS. DIDS plus TEA reduced the relaxations to SNP like TEA alone. Reduction in extracellular [Cl] by isethionate substitution reduced tone but did not reduce relaxations when tone was restored. The combination of reduced extracellular [Cl] and TEA did not abolish relaxation to EFS until DIDS was added. Thus, multiple K⁺ channels are opened by myogenic NO, and openings of these channels, as well as DIDS-sensitive, undefined mechanisms, are induced when NO is released from nerves or SNP.