We examined the release of endogenous adenyl purines such as adenosine triphosphate (ATP), ADP, AMP and adenosine from the caudal artery (CA), saphenous artery (SA), renal artery (RA), mesenteric artery (MA), pulmonary artery (PA) and thoracic aorta (TA) of rats, using high-performance liquid chromatography fluorescence detection . Noradrenaline in duced the release of adenyl purines from these blood vessels. The total amount of adenyl purines release induced by noradrenaline from the CA was considerably larger than that from the TA. The rank order of the amount of adenyl purines released from the six blood vessels was CA > SA > RA > MA > PA≥TA. The noradrenaline-induced release of adenyl purines from the CA was significantly reduced by the removal of the endothelium. Noradrenaline also induced the release of adenyl purines from cultured endothelial cells of the CA and TA. The total amount of adenyl purines released from the former blood vessel was much larger than that from the latter. These results suggest the existence of vascular endothelial cells that are able to release ATP by an α₁-adrenoceptor mediated mechanism, and that these cells are not homogeneously distributed in the vasculature .

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To clarify the exact mechanisms of the pharmacological effects of glucagon on gastro intestinal motility, the following experiments were performed on the conscious and anesthetized dogs. 1) During phase I of interdigestive migrating contractions (IMC), glucagon (5-50μg/kg, drip infusion for 5 minutes) induced phasic contractions in the duodenum, jejunum and ileum, but not in the antrum. These excitatory responses were also observed in the truncal vagotomized dogs. These contractions were abolished by atropine or hexamethonium in the conscious dogs, and also by tetrodotoxin in the anesthetized dogs. 2) Glucagon inhibited cisapride-induced contractions only in the antrum in the conscious dogs .After pre-treatment with hexamethonium, glucagon inhibited these contractions in the duodenum, jejunum and ileum as well as in the antrum. After pre-treatment with tetrodotoxin in the anesthetized dogs, glucagon did not affect acetylcholine-induced contractions in any region. 3) Glucagon inhibited spontaneous phase III contractions and erythromycin-induced phase III-like contractions in the antrum, but did not inhibit either contractions in the other regions in the conscious dogs. These paradoxical effects of glucagon between the antrum and intestine were similar to those involved in the blockade of 5-hydroxytryptamine 3 receptors. After pre-treatment with hexamethonium, glucagon inhibited these contractions in the duodenum, jejunum and ileum as well as in the antrum . In conclusion: 1) Glucagon latently inhibits cholinergic motor activities in the antrum and intestine not directly, by binding to either receptor on the smooth muscle cells, but through postganglionic cholinergic neurons and possibly through 5-hydroxytryptamine neurons. 2) On the other hand, in the intestine the reverse effects through preganglionic cholinergic neurons involving nicotinic and muscarinic receptors are more potent . 3) As a result, glucagon inhibits antral contractions and does not affect intestinal contractions in a conscious state.

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The role of newly synthesized ATP in cyclic GMP-induced relaxation was studied in membrane-permeabilized longitudinal muscle preparations of the rat proximal colon . Cyclic GMP and 8-bromo cGMP induced concentration-dependent relaxation of α-toxin permeabilized preparations which were precontracted by 3μM Ca²⁺ in the presence of 4 mM ATP and 5 mM phosphocreatine (PC). The relaxation by 8-bromo cGMP was inhibited by Rp-8-pCPT-cGMPS, an inhibitor of cyclic GMP-dependent protein kinase . The relaxation was inhibited by removal of PC from the bathing solution, in spite of the presence of ATP. The relaxation was also inhibited by dinitrofluorobenzene (DNFB), a selective inhibitor of creatine kinase. The removal of PC or treatment with DNFB is known to produce accumulation of ADP within smooth muscle cell, however, ADP_βS did not affect the relaxation.After irreversible inhibition of endogenous creatine kinase by DNFB in β-escin permeabilized preparations, treatment of the preparations with exogenous creatine kinase restored the relaxation. In the presence of ADP and PC but without ATP, 8-bromo cGMP induced the relaxation to the similar extent to that in the presence of ATP and PC . These results suggest that ATP newly synthesized from ADP and PC by creatine kinase is essential for cyclic GMP-induced relaxation of the smooth muscle preparations obtained from the proximal colon of rats.

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Modulation of L-type Ca²⁺ channels by acetylcholine (ACh) was studied in enzymatically isolated guinea-pig tracheal smooth muscle cells (TSMCs). ACh reversibly inhibited whole-cell L-type Ca²⁺ current measured with Ba²⁺ ions as charge carriers (I_Ba). With pipette solution containing 0.1mM EGTA, 1μM ACh induced transient inhibition of I_Bafollowed by sustained inhibition (67.0±3.7% of the control, n=19). When intracellular Ca²⁺concentration ([Ca²⁺] _i) was fixed at 50nM by BAPTA-Ca²⁺ buffer in the pipette, the transient inhibition was abolished whereas the sustained inhibition (66.0±7.8%, n=6) still occurred, suggesting that the transient inhibition was attributed to inactivation of the channels induced by increase in [Ca²⁺] _i. The sustained inhibition was abolished when [Ca²⁺] _i was fixed at zero. The sustained inhibition of I_Ba by 1μM ACh was observed in the presence of 10μM AF-DX 116, whereas it was not observed in the presence of 1μM4-DAMP. ACh did not inhibit I_Ba, in the presence of 1mM GDP-β-S in the pipette, whereas the drug irreversibly inhibited the current in the presence of 0.1mM GTP-γ-S in the pipette.Pretreatment of TSMCs with pertussis toxin did not altered the effects of ACh. Application of neither 1-oleoyl-2-acetyl-sn-glycerol (1μM) nor phorbol 12-myristate 13-acetate (1μM) reduced T_Ba. These results suggest that the sustained inhibition of I_Ba by ACh is mediated by Ca²⁺ - requiring and protein kinase C-independent mechanisms existing in the downstream of G-protein coupled with M₃ receptors.

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The physiological or pharmacological role of glucagon in the postprandial regulation of gastrointestinal motility has not yet been clarified. To clarify it, the following experiments were performed on conscious dogs. Antral, duodenal, jejunal and ileal contractile activities were monitored by chronically implanted strain gauge force transducers without restraint. The serum gastrin concentration in response to ingestion was measured by radioimmunoas say. 1) When glucagon (5-50μg/kg, drip infusion for 5 minutes) was administered before ingestion of meal or 2 hours after ingestion, it inhibited postprandial motility dose-dependently in the antrum, while enhancing it in the duodenum, jejunum and ileum. 2) At the same time, glucagon inhibited the meal-induced elevation of the serum gastrin concentration. 3) On the other hand, glucagon did not inhibit the contractions induced by pentagastrin (4μg/kg, s.c.) or those induced by acetylcholine chloride (0.5 mg/kg, drip infusion for 10 minutes) in any region. 4) These glucagon-induced inhibitory effects in postprandial antral motility were not affected by phentolamine (0.5 mg/kg, i.v.) or nitro-L-arginine-methyl ester (L-NAME) (3 mg/kg/hr, drip infusion for 30 minutes). These results suggest that: 1) Glucagon inhibits the postprandial elevation of the serum gastrin concentration and thus inhibits postprandial antral motility. 2) On the other hand, in the intestine, glucagoninduced inhibitory responses might be reversed by glucagon-induced excitatory responses through preganglionic cholinergic motor neurons. 3) The mechanism of inhibition of gastrin release was not definite in my experiments, but one of the candidates may be activation of somatostation release from the D cells by glucagon.

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To investigate the relationship between postprandial electrogastrographic changes and parasympathetic nervous activity, 10 healthy adult males (20 to 29 years old) volunteered for EGG recording in the following conditions: 1) butylscopolamine bromide (scopolamine) administration to block parasympathetic nervous activity ; 2) after food intake ; and 3) during a postprandial period after the parasympathetic blockade. Power spectral analysis of EGG was performed according to Akaike's autoregressive model. When the parasympathetic nervous activity was blocked, there were no changes in the dominant frequency of the EGG. During the postprandial period, the dominant frequency in EGG increased significantly, and postprandial dip (transient frequency decrease after the food intake) was observed in 8 of the 10 subjects. During the postprandial period after scopolamine administration, the dominant frequency of EGG did not increase, and postprandial dip was observed in only two subjects. These results suggest that the parasympathetic nervous activity is involved in occurrence of postprandial gastric motor function and postprandial electrogas trographic changes.

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