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The effects on acetylcholine release from the guinea-pig colon of the N-type calcium channel blocker omega-conotoxin GVIA (omega-conotoxin), the L-type calcium channel blocker nifedipine and the putative blocker of T-type channels, flunarizine, have been investigated. Endogenous basal acetylcholine release and electrically (1 Hz, 1 ms, 450 mA)-evoked overflow in the presence of cholinesterase inhibitor were studied. omega-Conotoxin (1-10 nM) and nifedipine (0.03-3 microM) dose-dependently inhibited basal and electrically-evoked acetylcholine release. Maximal inhibition of basal or electrically-evoked acetylcholine release was about 40% for nifedipine and about 75% for omega-conotoxin. The potency of nifedipine was inversely related to the external calcium concentration: its EC50 value in low-calcium medium (0.5 mM) was as low as 12 nM. Flunarizine inhibited acetylcholine release only at concentrations higher than 0.2 microM. Our results are consistent with an involvement of N- and L-type calcium channels in the control of the endogenous acetylcholine release from the guinea-pig colon.

The possible role of opioid systems in the adaptive changes which follow chronic sympathetic denervation in the guinea-pig colon has been studied by comparing the effects of the opioid agonist morphine in control animals and after chronic sympathetic denervation. Supersensitivity to the inhibitory effects of morphine on the peristaltic reflex was observed after chronic sympathetic denervation, while the potency against acetylcholine release was unmodified. Our results suggest that a modification of the opioid system occurs after sympathetic denervation in the guinea-pig colon. Supersensitivity to endogenous opioids at a site different from that regulating acetylcholine release could account for the counter-regulation of intestinal motility after chronic sympathetic denervation.

beta-Casomorphins are a family of opioid peptides originally isolated from beta-casein. In view of a possible physiological significance of these milk-derived compounds, the effects of bovine beta-casomorphin-5 (beta-CM-5), beta-casomorphin-4 (beta-CM-4) and D-Ala2-beta-casomorphin-4-NH2 (D-Ala2-beta-CM-4-NH2) have been investigated on the peristaltic reflex in the guinea-pig isolated colon and compared with morphine. beta-CM-5 and D-Ala2-beta-CM-4-NH2 each dose-dependently inhibited the velocity of propulsion of an intraluminal bolus; beta-CM-4 was ineffective. IC50 values were 0.30, 5.21 and 0.29 microM for morphine, beta-CM-5 and D-Ala2-beta-CM-4-NH2, respectively. The potency ratios vs morphine were 0.06 and 0.96 for beta-CM-5 and D-Ala2-beta-CM-4-NH2, respectively. Blockade of the peristaltic reflex by beta-CM-5 or D-Ala2-beta-CM-4-NH2 was reversed by the opioid antagonist naloxone. D-Ala2-beta-CM-4-NH2 also dose-dependently inhibited resting acetylcholine output (IC50 = 5.69 microM; potency ratio vs morphine: 0.63). In conclusion, certain beta-casomorphins inhibit intestinal propulsion and cholinergic neurotransmission in the guinea-pig colon, probably by acting at opioid receptors.

In segments of guinea-pig colon, treated with 1 microM hyoscine, a 5-HT desensitization procedure that decreased the potency of 5-HT 41-fold in relaxing the longitudinal musculature, failed to modify the non-adrenergic GABA-induced inhibitory responses, suggesting that the action of GABA in this preparation is not 5-HT-mediated. These results are at variance with those obtained in the guinea-pig ileum where 5-HT seems to play a role in GABA-induced cholinergic contractions.

To study cholinergic function in the guinea-pig colon, resting and electrically evoked 3H release after preincubation with [3H] choline has been compared in colonic and ileal myenteric plexus preparations. Fractional spontaneous colonic 3H release was significantly higher than ileal 3H release, while the reverse was true for electrically evoked 3H outflow. Electrically evoked 3H outflow in the colon was linearly related to stimulation frequency (0.2-3 Hz range) and current intensity (300-600 mA range), while 3H outflow per pulse was inversely related to stimulation frequency. Electrically evoked 3H outflow was prevented in Ca2(+)-free solution, indicating that it probably mirrored neuronal exocytotic [3H]acetylcholine release. Both noradrenaline and clonidine concentration-dependently inhibited electrically evoked 3H outflow, clonidine being more potent but less efficacious than noradrenaline. For both noradrenaline and clonidine, the potency and efficacy for inhibition of 3H outflow were close to the values previously reported for the inhibition of electrically evoked endogenous acetylcholine output from colonic preparations. In conclusion, these data indicate that 3H release after incubation with [3H]choline is a valid alternative to measurement of endogenous acetylcholine output to study colonic cholinergic neuronal function in the guinea-pig.

Concentrations of dilazep which were ineffective in altering the muscular tone of the guinea-pig taenia caeci (0.03, 0.3 microM) or the phasic mechanical activity of the rabbit proximal ileum (0.03 microM) markedly potentiated the inhibitory action of adenosine on both these parameters. Dilazep, 0.3 microM or greater, dose-dependently inhibited the mechanical activity of the proximal ileum. This inhibitory action was probably mediated by more than one mechanism, as shown by the fact that theophylline (50, 100 microM) antagonized the effect at lower dilazep concentrations (up to 3 microM) leaving essentially unchanged the response to higher concentrations (6, 10 microM). Similarly, the responses to low doses of dilazep were reduced after desensitization of the organ to adenosine, whilst the responses to higher doses were unaffected by this procedure. In a Ca2+-free, high-K+ medium, dilazep (1-10 microM) caused a parallel shift to the right of the Ca2+-induced contractions of the guinea-pig taenia caeci. Adenosine showed only slight Ca2+-antagonistic properties within the mM range of concentrations. These findings suggest that, at the higher concentration tested, dilazep exhibits Ca2+-antagonistic properties unrelated to its adenosine-mediated mode of action.