VIP‐ and PACAP‐mediated nonadrenergic, noncholinergic inhibition in longitudinal muscle of rat distal colon: involvement of activation of charybdotoxin‐ and apamin‐sensitive K⁺ channels

Abstract

1 The mediators of nonadrenergic, noncholinergic (NANC) inhibitory responses in longitudinal muscle of rat distal colon were studied.2 An antagonist of pituitary adenylate cyclase activating peptide (PACAP) receptors, PACAP638, concentration-dependently inhibited the rapid relaxation of the longitudinal muscle induced by electrical field stimulation (EFS), resulting in a maximal inhibition of 47% at 3 MM.3 PACAP638 inhibited the relaxation by 75% in the presence of the vasoactive intestinal peptide (VIP) receptor antagonist, VIPIO 28 at 3 Mm, which inhibited the relaxation by 44%.4 An antagonist of large conductance Ca2+-activated K+ channels, charybdotoxin, concentrationdependently inhibited the rapid relaxation of the longitudinal muscle, resulting in a maximal inhibition of 58% at 100 nM.5 An antagonist of small conductance Ca2 +-activated K+ channels, apamin, concentration-dependently inhibited the relaxation (58% at 1 gM).6 Treatment with both K+ channel antagonists resulted in 84% inhibition of the EFS-induced relaxation, which is comparable to the extent of inhibition induced by PACAP638 plus VIPI0O28. 7 The inhibitory effect of VIP1028 and of apamin, but not of charybdotoxin was additive: the same applied to PACAP6-38 and charybdotoxin, but not apamin. 8 Exogenously added VIP (100 nM-1 Mm) induced a slow gradual relaxation of the longitudinal muscle. Charybdotoxin, but not apamin significantly inhibited the VIP-induced relaxation. VIPI0O28, but not PACAP638 selectively inhibited the VIP-induced relaxation.9 Exogenously added PACAP (10-100 nM) also induced slow relaxation. Apamin and to a lesser extent, charybdotoxin, inhibited the PACAP-induced relaxation. PACAP638, but not VIPI0O28 selectively inhibited the PACAP-induced relaxation.10 Apamin at 100 nM inhibited inhibitory junction potentials (ij.ps) induced by a single pulse of EFS. Apamin also inhibited a rapid phase, but not a delayed phase of ij.ps induced by two pulses at 10 Hz. VIPIO28 did not inhibit i.j.ps induced by a single pulse, but significantly inhibited the delayed phase at two pulses. A combination of apamin and VIPIO28 abolished the ij.ps induced by two pulses.11 Both VIP and PACAP induced slow hyperpolarization of the cell membrane of the longitudinal muscle. Apamin inhibited the PACAP-, but not VIP-induced hyperpolarization. 12 From these findings it is suggested that VIP and PACAP are involved in NANC inhibitory responses of longitudinal muscle of the rat distal colon via activation of charybdotoxin-and apaminsensitive K+ channels, respectively.

Mediators of Nonadrenergic, Noncholinergic Relaxation in Sprague Dawley Rat Intestine: Comparison with the Mediators of Other Strains.

“…On the other hand, although VIP and PACAP were suggested to separately, partially mediate NANC relaxation of the distal colon in our previous study [15], participation of both the peptides was not shown in the remaining intestinal regions in the present study (n=3).…”

“…However, the VIP receptor antagonist inhibited the relaxation in the distal colon by 36.4 ± 3.4% (n=14). Charybdotoxin at 100 nM, which exhibited the maximal inhibitory effect on EFSinduced relaxation of the Wistar-ST rat distal colon [15], also inhibited the relaxation by 37.2 ± 5.1% (n=3).…”

“…PACAP 6-38 at 3 µM inhibited the EFS (10 Hz, 10 s)-induced relaxation of longitudinal muscle of the distal colon by 43 % (Table 1). Apamin at 1 µM, which exhibited the maximal inhibitory effect on the EFS (10 Hz, 10 s)-induced relaxation of the Wistar-ST rat distal colon [15], also inhibited the relaxation of the Sprague Dawley rat distal colon by 76.5 ± 9.1 % (n=5). Bath application of exogenous PACAP (10-100 nM) induced slow gradual relaxation of longitudinal muscle of the distal colon.…”

“…Drugs: Apamin, VIP fragment VIP [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] and N G -nitro-Larginine (N 5 -nitro-amidino-L-2,5-diamino-pentanoic acid: L-NOARG) were purchased from Sigma Chemical Co., ST. Louis, U.S.A. VIP, charybdotoxin, PACAP and its fragment PACAP were purchased from The Peptide Institute, Osaka, Japan. Gaseous nitric oxide was dissolved in Tyrode solution just before experiments, as described by Gillespie and Sheng [4], and added to the organ bath in volumes of 0.3-300 µl.…”

“…Pituitary adenylate cyclase activating peptide (PACAP) was also suggested to mediate NANC relaxation in some regions: lower oesophageal sphincter of cats and humans [20], stomach of guinea pigs [14], and colon of guinea pigs [12] and rats [8,15]. We suggested that VIP and PACAP mediate NANC relaxation of longitudinal muscle of the distal colon of Wistar-ST rats via opening of charybdotoxin-and apamin-sensitive K + channels, respectively [15,25]. However, participation of the peptides as well as nitric oxide in NANC relaxation does not seem to be uniform throughout the gastrointestine.…”