Tyrosine kinase involvement in apamin‐sensitive inhibitory responses of rat distal colon

Abstract

Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated from ovine hypothalamus and shown to stimulate adenylate cyclase (Miyata et al. 1989). Later, PACAP was also shown to be widely distributed in the gastrointestinal tract (Sundler et al. 1991). Katsoulis & Schmidt (1996) and Schw orer et al. (1992) reported that PACAP relaxed the smooth muscle preparations of guineapig ileum and taenia coli, rat fundus, ileum and colon, pig ileum, and human colon. We have previously suggested that PACAP mediates non-adrenergic, non-cholinergic (NANC) inhibitory responses of longitudinal muscle of rat distal colon via the activation of apamin-sensitive K¤ channels (Kishi et al. 1996). PACAP has also been shown to be released by nerve stimulation and to be responsible for the apaminsensitive component of electrical field stimulation (EFS)-induced relaxation and inhibitory junction potentials (IJPs) of guinea-pig taenia coli (Jin et al. 1994a;McConalogue et al. 1995b). These findings strongly suggest that PACAP is a member of a group of potential neurotransmitters responsible for NANC relaxation, which activates apamin-sensitive K¤ channels in some regions of the gastrointestinal tract. However, the mechanism by which PACAP activates apaminsensitive K¤ channels is still unknown. Increases in cyclic AMP concentrations result in the stimulation of large conductance Ca¥-activated K¤ channels in rat aorta (Sadoshima et al. 1988) and rabbit trachea (Kume 1. It has been suggested that pituitary adenylate cyclase activating peptide (PACAP) may be involved in the non-adrenergic, non-cholinergic (NANC) inhibitory response of longitudinal muscle of rat distal colon. In this study, we have investigated the intracellular mechanism of PACAP-induced relaxation in this muscle. 2. PACAP induced an apamin-sensitive relaxation of the longitudinal muscle. The tyrosine kinase inhibitors genistein at 10 ìÒ and tyrphostin 25 at 30 ìÒ, but not the cyclic AMPdependent protein kinase inhibitor Rp-8-bromoadenosine-3',5'-cyclic monophosphorothioate at 30 ìÒ significantly inhibited the PACAP-induced relaxation to 60% and 25% of control values, respectively. PACAP did not increase the cyclic AMP content of the muscle. 3. Tyrphostin 25 at 10 ìÒ significantly inhibited the relaxation of longitudinal muscle induced by electrical field stimulation (EFS), to 50% of control values. Apamin at 1 ìÒ, an antagonist of small conductance Ca¥-activated K¤ channels, also inhibited the relaxation, to 42% of control values. The inhibitory effects of tyrphostin 25 and apamin were not additive (44% of control values). 4. PACAP induced an apamin-sensitive, slow hyperpolarization of the cell membrane of the muscle. Tyrphostin 25 at 3 ìÒ inhibited this PACAP-induced hyperpolarization. Tyrphostin 25 at 10 ìÒ and genistein at 10 ìÒ inhibited the apamin-sensitive inhibitory junction potentials induced by a single pulse of EFS. 5. The PACAP-induced relaxation of longitudinal muscle occurred with a concomitant decrease in intracellular Ca¥ levels ([Ca¥]é). Tyr...

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

“…It has been suggested that PACAP induces the relaxation via opening of apamin-sensitive K + channels in the colon of rats [8] and the tenia coli of guinea pigs [12] and mediated i.j.ps via opening of the channels in the caecum of guinea pigs [17]. We also suggested that PACAP mediates about half NANC relaxation in the distal colon of Wistar-ST rats via opening of apamin-sensitive K + channels [15,25]. A few reports also suggest association of ATP-mediated relaxation in the human colon [3] and ATP-mediated i.j.ps in the guinea pig colon [29] with apamin-sensitive K + channels.…”

“…However, no participation of VIP in the relaxation was suggested in the distal colon of Sprague Dawley rats and in any regions other than the distal colon of all the three strains. In the present study, effect of VIP [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] was examined on the relaxation induced by EFS only at 10 Hz for 10s, because VIP 10-28 inhibited relaxations of longitudinal muscle of the Wistar-ST rat distal colon induced by EFS at 0.1 and 10 Hz by a similar extent about 45% [15] and the antagonist did not affect the relaxation in Sprague Dawley rats induced by EFS at 10 Hz for longer duration, 20-90 s [21]. The present results indicate that VIP participates NANC relaxation only in the distal colon of Wistar and Wistar-ST, but not Sprague Dawley rats.…”

“…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.…”

Signal Transduction Pathways Involved in the Mechanical Responses to Protease-Activated Receptors in Rat Colon

“…Since tyrosine kinase has been reported to be involved in apamin-sensitive relaxation of longitudinal colonic muscle (Takeuchi et al, 1999) as well as in the PAR-evoked gastric contraction in rats (Saifeddine et al, 1996), we also tested the effects of PAR-1 and PAR-2 activating peptides in the presence of genistein, a tyrosine kinase inhibitor. Genistein (1-10 M) decreased the amplitude of the spontaneous contractions and significantly reduced in a concentration-dependent manner the contractile and the relaxant effects on the longitudinal muscle in response to PAR-1 activating peptide.…”