Nitric oxide from enteric nerves acts by a different mechanism from myogenic nitric oxide in canine lower esophageal sphincter

In canine lower esophageal sphincter, myogenic constitutive nitric-oxide (N O) synthase (NOS) in plasma membrane limits tone by opening large conductan ce Ca2+-dependent K+ channels (BKCa channels) and hyperpolarizing the membr ane. We examined whether K-V channels were involved and whether NO from ent eric nerves and from NO donors used the same mechanisms. With nerves inacti ve, 100 nM iberiotoxin, like N-nitro-L-arginine (L-NOARG), increased tone b ut less. 4-Aminopyridine (4-AP) at 5 mM behaved similarly. Tetraethyl ammon ium (TEA) at 20 mM equaled the effect of L-NOARG and occluded any tone incr ease from any combination of these agents. More than iberiotoxin or 4-AP, T EA decreased relaxations in response to sodium nitroprusside (SNP) or 3-mor pholino-sydnonimine (Sin-1) by similar to 50%. In whole-cell patch-clamp re cordings, TEA and 4-AP reduced outward K+ currents additively by >90% at de polarization of +90 mV. Thus, K+ channels in addition to BKCa channels are opened by myogenic NO, and exogenous NO had relaxing effects both related a nd unrelated to K+ channel openings. TEA (20 mM) increased tone but did not inhibit relaxations to electrical field stimulation (EFS) of enteric nerve s. 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 mu M) dose dependently reduced tone, but tone, restored by 10(-6) M carbachol or 20 mM TEA, was still rel axed by EFS and by SNP. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) at 500 to 1000 mu M did not inhibit relaxation to EFS or SNP. The ad dition of TEA (20 mM) to DIDS (1000 mu M) induced tonic and phasic activity and markedly inhibited relaxations to EFS. DIDS plus TEA reduced the relax ations to SNP like TEA alone. Reduction in extracellular [Cl-2] by isethion ate substitution reduced tone but did not reduce relaxations when tone was restored. The combination of reduced extracellular [Cl-2] and TEA did not a bolish relaxation to EFS until DIDS was added. Thus, multiple K+ channels a re opened by myogenic NO, and openings of these channels, as well as DIDS-s ensitive, undefined mechanisms, are induced when NO is released from nerves or SNP.