1 Mechanisms involved in Ca 2 þ sensitization of contractile elements induced by the activation of muscarinic receptors in membrane-permeabilized preparations of the rat proximal and distal colon were studied. 2 In a-toxin-permeabilized preparations from the rat proximal and distal colon, Ca 2 þ induced a rapid phasic and subsequent tonic component. After Ca 2 þ -induced contraction reached a plateau, guanosine 5 0 -triphosphate (GTP) and carbachol (CCh) in the presence of GTP further contracted preparations of both the proximal and distal colon (Ca 2 þ sensitization). 3 Y-27632, a rho-kinase inhibitor, inhibited GTP plus CCh-induced Ca 2 þ sensitization more significantly in the proximal colon than in the distal colon. Y-27632 at 10 mM had no effect on Ca 2 þ -induced contraction or slightly inhibited phorbol-12,13-dibutyrate-induced Ca 2 þ sensitization in either proximal or distal colon. Chelerythrine, a protein kinase C inhibitor, inhibited GTP plus CChinduced Ca 2 þ sensitization in the distal colon, but not in the proximal colon. The component of Ca 2 þ sensitization that persisted after the chelerythrine treatment was completely inhibited by Y-27632. 4 In b-escin-permeabilized preparations of the proximal colon, C3 exoenzyme completely inhibited GTP plus CCh-induced Ca 2 þ sensitization, but PKC(19-31) did not. In the distal colon, C3 exoenzyme abolished GTP-induced Ca 2 þ sensitization. It inhibited CCh-induced sensitization by 50 % and the remaining component was inhibited by PKC(19-31). 5 These results suggest that both protein kinase C and rho pathways in parallel mediate the Ca 2 þ sensitization coupled to activation of muscarinic receptors in the rat distal colon, whereas the rho pathway alone mediates this action in the proximal colon.

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...

ABSTRACT-The origin of Ca2+ necessary for carbachol (CCh)-induced contraction of longitudinal muscle of the proximal colon of rats was studied. CCh induced contraction of the muscle consisting of two phases, phasic and tonic phases, with a concomitant biphasic increase in [Ca 2+ ]i. After removal of Ca 2+ from the bathing solution of the colonic segments, CCh-induced contraction was rapidly inhibited; there was almost complete inhibition 1 min after the removal. Nicardipine, a blocker of voltage-dependent calcium channel, also significantly inhibited CCh-induced contraction. On the other hand, treatment of the colonic segments with thapsigargin, an inhibitor of sarcoplasmic reticulum (SR) Ca 2+-ATPase, did not significantly affect the contraction except causing a slight decrease in the rate of contraction. These results suggest that Ca 2+ entering through voltage-dependent calcium channels, but not released from SR, is essential for CCh-induced contraction of longitudinal muscle of the proximal colon of rats. This strict dependency of the CCh-induced contraction on extracellular Ca 2+ was discussed in relation to the results obtained in the fundus of rats.