The properties of spontaneous contractions and their modulation by transmural nerve stimulation (TNS) were investigated in circular smooth muscle preparations isolated from the "pacemaker area" in the flexure region of the guinea-pig colon. Four types of preparation were made; mucosal layer removed preparations, mucosal and submucosal layer removed preparations (submucosal layer removed preparations), longitudinal muscle removed preparations, and preparations with all layers attached (intact preparations). Intact and mucosal layer removed preparations periodically generated two types of phasic contractions; large contractions at a low frequency (about 2 times per min) and small contractions at a higher frequency (about 13 times per min). Submucosal layer removed preparations only produced large contractions, while longitudinal muscle removed preparations only produced small contractions. Both the amplitude and frequency of the large contractions were significantly inhibited by atropine, but were not markedly modulated by N ω -nitro-L-arginine (L-NA) in either intact, mucosal layer removed or submucosal layer removed preparations. Small contractions were not significantly modulated by atropine or L-NA in any type of preparation. In both intact and mucosal layer removed preparations, TNS (1 Hz frequency, for 2-3 min) increased the amplitude and/or frequency of large contractions, but caused no significant change in small contractions. TNS also increased the amplitude and/or frequency of large contractions in submucosal layer removed preparations, but did not significantly modulate small contractions in longitudinal muscle removed preparations. Exogenously applied acetylcholine (>10 -7 M) enhanced, while sodium nitroprusside (>10 -8 M) inhibited, the amplitude and frequency of both types of contraction, in a concentration-dependent manner. The results suggest that the large contractions were elicited by interstitial cells distributed in the myenteric layer (ICC-MP), while the small contractions were elicited by ICC distributed in the submuscular plexus layer (ICC-SMP). Intramural nerves mainly modulated the activity of ICC-MP, while neural regulation of ICC-SMP was weak.

The properties of mechanical responses elicited by stimulation with acetylcholine (ACh) were investigated in circular smooth muscle preparations isolated from the proximal colon of guinea-pig. Application of ACh (10 -8 -10 -6 M) for 3-5 min produced a biphasic response, with an initial contraction followed by a relaxation. Atropine inhibited the initial contraction, while N ω -nitro-L-arginine (L-NA) inhibited the relaxation, suggesting that the former was produced by activation of muscarinic receptors while the latter was produced by an elevated production of nitric oxide (NO). In the presence of atropine, the ACh-relaxation was attenuated by removal of the mucosa and abolished by removal of both submucosal and mucosal layers. The ACh-induced relaxation was also attenuated by either tetrodotoxin (TTX, 3 × 10 -7 M) or hexamethonium (10 -6 M). In the presence of atropine, transmural nerve stimulation (TNS) elicited a biphasic response, with an initial phasic contraction followed by a relaxation. The amplitude of TNS-induced relaxation was significantly reduced by hexamethonium or L-NA and was abolished by TTX. Both ACh and TNS produced relaxation in preparations isolated from the proximal colon, but not in those from the middle part of colon. Immunohistochemistry for neuronal nitric oxide synthase revealed no difference in the distribution of nitrergic nerves between the proximal and middle part of the colon, with nitrergic nerves in both the mucosal and submucosal layers as well as in the smooth muscle and myenteric layers. These results suggest that ACh induces NO production by excitation of postganglionic nerves distributed mainly in the mucosal and submucosal layers. In circular smooth muscle preparations isolated from the middle part of colon, ACh or TNS produced contractile responses alone, with no associated relaxation, suggesting that the ACh-activated postganglionic nitrergic nerves are distributed in the mucosal and submucosal layers of the proximal colon but not in the middle part of the colon.

Mechanical responses of smooth muscle elicited by application of CO2-gas bubbled physiological salt solution (CO2-gas solution) were investigated in isolated stomach antrum and colon preparations of the guinea-pig. Circular smooth muscle preparations of both colon and stomach were spontaneously active with periodic generation of phasic contractions. In colonic preparations, the CO2-gas solution produced a biphasic response, with an initial small transient contraction followed by a sustained inhibition of phasic contractions. Removal of the CO2-gas solution allowed a slow recovery of the spontaneous contractions over a period of about 40 min. The recovery developed with a similar time course irrespective of the length of time exposed to CO2-gas solution. The inhibitory responses elicited by CO2-gas solution were not modulated by atropine, N  -nitro-L-arginine or neostigmine. Atropine-sensitive excitatory responses of smooth muscle elicited by transmural nerve stimulation or exogenously applied acetylcholine were attenuated or abolished in the presence of CO2-gas solution. In stomach preparations, the CO2-gas solution elicited a tri-phasic response, with an initial transient relaxation followed by a transient contraction and then a sustained inhibition of the rhythmic contractions. The peak amplitude of the transient contraction was about 2.5 times larger than the spontaneous phasic contractions. The pH of the CO2-gas solution was reduced to about 6. Application of pH 6 solution again produced a tri-phasic response, as was the case for the CO2-gas solution, however the amplitude of the transient contraction was only about 0.4 times that of the spontaneous contractions. The re-appearance of the abolished phasic contraction was quicker with the pH 6 solution (about 1.8 min) than it was for the CO2-gas solution (about 6 min). The inhibitory responses elicited by the CO2-gas solution could be simulated only partly by the acidified solution, and a possible involvement of additional factors in the inhibition elicited by CO2-gas solution was considered.

In isolated longitudinal muscle tissues of the guinea-pig stomach antrum, recording electrical responses from smooth muscle cells revealed a periodical generation of follower potentials with variable durations. The I-D relationship, made by plotting the duration as a function of the interval before generating follower potential, was linear. Experiments were carried out to investigate the effects of chemicals which had been known to modulate the release of Ca 2+ from the internal stores (2-aminoethoxy-diphenyl-borate, cyclopiazonic acid, caffeine), inhibit mitochondrial metabolic activity (m-chlorophenyl hydrazone, 2-deoxy-Dglucose, potassium cyanide, rotenone), inhibit ATP-sensitive K-channels distributed in mitochondria (glibenclamide, 5-hydroxydecanoic acid) and inhibit the activity of proteinkinase C (chelerythrine), on the I-D relationship of follower potentials. The effects of depolarization on follower potentials were assessed by stimulating tissues with high potassium solution. Experiments were carried out mainly in the presence of nifedipine which minimized the movements of muscles with no modulation of follower potentials. Cycropiazonic acid and caffeine reduced the slope of I-D relationship, with associated reduction of the duration and frequency of follower potentials. 2-Aminoethoxydiphenyl borate reduced the duration and amplitude and increased the frequency of follower potentials, with depolarization of the membrane, and the effects were simulated by high potassium solution. m-Chlorophenyl hydrazone, potassium cyanide, 2-deoxy-D-glucose, rotenone, 5-hydroxydecanoic acid and glibenclamide reduced the slope of I-D relationship, with associated reduction of the frequency of follower potentials. Chelerythrine did not modulate the slope of I-D relationship, with reduced frequency of follower potentials. It seemed likely that the amount of Ca 2+ released from the internal stores and also mitochondrial function had causal relationship to the duration of pacemaker potentials, suggesting that internal Ca-stores and mitochondria are taking the central role for determining the duration of the pacemaker activity. Proteinkinase C did not seem to participate to the function of mitochondria and internal Ca 2+ stores.