IHC and scoring were conducted as described. Primary antibodies of inducible NOS (iNOS) (mouse, Abcam, ab129372), neuronal NOS (nNOS) (rabbit, Abcam, ab3511), Cav1.2 (mouse, Abcam, ab84814), Cav1.3 (mouse, Abcam, ab85491), IP[sub]3R1 (rabbit, Abcam, ab125076), IP[sub]3R2 (rabbit, Millipore, ab9074), IP[sub]3R3 (rabbit, Millipore, ab9076), and RyR2 (rabbit, Chemicon, ab9080) were diluted at 1:100.
From automatic plotting of dissolution and amplification curves in ABI 7500 and base sequence results of Invitrogen, Cav1.2, Cav1.3, three subtypes of IP[sub]3 Rs, RyR2, iNOS, and nNOS were positive in the achalasia and control groups (esophageal circular muscle, EC), but other subtypes were negative [Figure 1]a.
Mechanistically, S[O.sub.2] and its derivatives induced the [K.sub.ATP] and B[K.sub.Ca] channels activation through increasing the expressions of Kir6.1, Kir6.2, SUR2B, and B[K.sub.Ca] channel subunits [alpha] and [beta]1 in rat aortic rings, while S[O.sub.2] and its derivatives inhibited the L-type calcium channel through decreasing the expressions of Cav1.2 and Cav1.3
If the concentration of SO[sub]2 is >500 [micro]mol/l, the vasodilatory effects are independent of the endothelial system but related to K[sub]ATP channels and the inhibition of L-type calcium channels.,, In contrast, if the concentration of SO[sub]2 is <450 [micro]mol/L, the vasorelaxant mechanism depends on the big-conductance calcium-activated K + (BKCa) and cyclic guanosine monophosphate. SO[sub]2 induced the K[sub]ATP and BKCa channel activation via upregulating the expressions of Kir6.1, Kir6.2, SUR2B, and BKCa channel subunits a and [sz]1 in rat aortic rings, while SO[sub]2 inhibited the L-type calcium channel by downregulating the Cav1.2 and Cav1.3