To explore the importance of the charge of the amino acid in position 15 in influencing the apparent affinities of VIP and secretin for their receptors on pancreatic acinar cells, we tested the synthetic C-terminal 23-peptide fragment of secretin (S_5-27) and two analogues containing substitutions in position 15 for their abilities to interact with secretin-preferring and VIP-preferring receptors on pancreatic acinar cells. In inhibiting the increase in cyclic AMP caused by VIP acting through the VIP-preferring receptors, 15-Lys-S_5-27 was equipotent with 15-Asn-S_5-27, and these analogues were significantly more potent than S_5-27. In inhibiting the increase in cyclic AMP caused by secretin acting through the secretin-preferring receptors, S_5-27 was equipotent with 15-Asn-S_5-27, and these peptides were significantly more potent than 15-Lys-S_5-27. These findings indicate that the affinities of these 23-peptides for the VIPpreferring receptors and for the secretin-preferring receptors were influenced primarily by the absence of a particular charge in position 15 but not by the presence of the opposite charge.

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Vasoactive intestinal polypeptide (VIP) stimulated cyclic AMP formation in the anterior pituitary, hypothalamus, pons and cerebral medulla of rat in vitro.
The concentration of VIP above 10^-7 M significantly raised cyclic AMP level of these tissues with dose-dependency and the maximum stimulation of cyclic AMP formation was obtained at 5 min incubation. This stimulatory effect of VIP was not inhibited by propranolol or somatostatin.
However, no enhancement of hormone release from the anterior pituitary or hypothalamUs into the incubation medium by VIP was observed.
These results suggest that VIP may act through adenylate cyclase in specific areas of rat central nervous system independent of, β-adrenergic receptor, but it may not necessarily be related to hormone release from the anterior pituitary or hypothalamus.

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Vasoactive intestinal polypeptide (VIP) was isolated from porcine intestine in 1970 by Said & Mutt (1970). Characterization showed that it consists of 28 amino acids (Mutt & Said, 1974). Antibodies were raised against the peptide (Said & Faloona, 1975) and radioimmunological determinations revealed high concentrations of VIP in the gastro-intestinal tract (Polak et al., 1974 ; Said, 1975), in neural cell lines (Said & Rosenberg, 1976) and in the central nervous system (Bryant et al., 1976 ; Larsson et al., 1976b ; Said & Rosenberg, 1976). Immunohistochemical studies located the VIP-like immunoreactivity to gastro-intestinal neurons (Bryant et al., 1976; Larsson et al., 1976b ; Fuxe et al., 1977), cerebrovascular nerves (Larsson et al., 1976a) and central neurons (Fuxe et al., 1977 ; Larsson et al., 1976b).
The present paper gives a brief account of the distribution of VIP immunoreactive neurons in the central and peripheral nervous system as revealed by immunohistochemistry.

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As part of study of the phylogeny of VIP, it was observed that the colonic mucosa of the cartilaginous fish, Raja radiata and Raja clavata, contained numerous epithelial cells, obviously of closed type, displaying intense VIP immunoreactivity. Accordingly, high concentrations of VIP were found by radioimmunoassay. At the ultrastructural level, the VIP cells were characterized by the presence of highly electron dense secretory granules.
The colonic mucosa of the ray may be a good source for the isolation of an early VIP.

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The presence of VIP-like immunoreactivity in human neuroblastoma cell line NB-1 was demonstrated by radioimmunoassay of the crude cell extract and by immunocytochemical staining of the cells. Gelflitration profiles of VIP-like immunoreactivity in the cell extract measured by radioimmunoassays using three different region-specific antisera revealed that the immunoreactivity consists of a major molecular form corresponding to porcine VIP having 28 amino acid residues with at least two additional minor forms larger and smaller than the VIP. In addition to VIP-like immunoreactivity, the cell extract was shown to containsubstance P-, neurotensin-and somatostatin-like immunoreactivitiy as well.

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Secretory actions of VIP (vasoactive intestinal peptide), secretin and CCK-PZ (Cholecystokinin-pancreozymin) were compared in the isolated and perfused kitten pancreas. Perfusion of the isolated kitten pancreas with a solution containing 20 mU/ml CCK-PZ resulted in gradual hyperpolarization of the pancreatic acinar cell. CCK-PZ induced a dose-dependent increase in the pancreatic protein output and a small but definite increase in the flow of pancreatic juice which was also dose dependent upon CCK-PZ. Secretin and VIP induced a dose-dependent increase in the flow but a negligible increase in the pancreatic protein output. The estimated ED₅₀ for VIP was 1.5 nM which was 5 times as large as the estimated ED₅₀ for secretin. A considerable amount of VIP, which corresponded roughly to the activity of exogenous 0.6 nM VIP, was spontaneously released from the isolated kitten duodenum into the portal vein, and this VIP release was nullified during vascular perfusion with a Ca-deficient solution. With respect to these and other results, we will propose a view concering a possible role of VIP in controlling the function of the execrine pancreas.

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A modification of a technique to isolate kidney tubule fragments and pancreatic acinar cells has been used to prepare a suspension of pancreatic duct fragments from rats with pancreatic lipomatosis due to pretreatment with penicillamine and a copperfree diec. This suspension contains almost pure fractions of pancreatic duct fragments. The accumulation of 3', 5'-cyclic adenosine monophosphate (cAMP) in response to secretin, VIP and theophylline was studied in these isolated pancreatic duct fragments. In the absence of theopylline, secretin increased the level of cAMP in a dose-dependent manner with a maximum at 10^-6M. With supramaximal doses the concentration of cAMP decreased. Vasoactive intestinal peptide (VIP) also increased the formation of cAMP. However, VIP was 10 times less effective than secretin on a molar basis. The addition of various concentrations of VIP to a submaximal dose of secretin did not alter cAMP levels as compared to the levels observed with the same concentration of secretin alone. Theophylline (5×10^-3 and 10^-2M) stimulated cAMP accumulation and 5×10^-3M theophylline potentiated the response to secretin and VIP. These data lend support to the hypothesis that cAMP is the intracellular mediator of the action of secretin and VIP on the pancreatic duct cells.

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The effects of synthetic chicken VIP (an octacosapeptide corresponding to the entire sequence of chicken VIP) on pancreatic blood flow and on systemic arterial pressure was examined in dogs. The relationship between pancreatic blood flow and systemic arterial pressure in response to synthetic chicken VIP was also investigated. Pancreatic blood flow increased promptly and significantly with exogenous synthetic chicken VIP. Synthetic chicken VIP administered in graded doses resulted in dose-dependent increases in both the peak rate of the pancreatic blood flow and the duration of the response of the pancreatic blood flow. Graded doses of synthetic chicken VIP also elicited both a graded increase in the systemic systolic pressure and a decrease in the systemic diastolic pressure. Therefore, synthetic chicken VIP given in graded doses resulted in a dose-dependent augmentation of the maximum pulse pressure. Pancreatic blood flow increased despite a fall in the systemic arterial pressure in response to synthetic chicken VIP. This increase in blood flow lasted significantly longer than the changes in the systemic arterial pressure. It was clarified in this study that synthetic chicken VIP has a biological effect on both the pancreatic blood flow and the systemic arterial pressure. The results of our experiments also suggest that synthetic chicken VIP itself may directly induce vasodilatation of the local vessels in the pancreas.

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Vasoactive Intestinal Polypeptide (VIP) has been demonstrated in the female genitourinary tract, localized in neurons which seem to innervate vessels and nonvascular smooth muscle. The present work has demonstrated that the concentration of immunoreactive VIP varies between different mammalian species and within th genital tract of the same species. Using various in vivo and in vitro preparations the peptide was found to have a dose-dependent inhibitory effect on the myoelectrical activity and contractility of the uterine muscle, and to increase myometrial blood flow. The findings support the hypothesis that VIP may play a physiological role in the local control of uterine motility and blood flow.

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Three patients with the watery diarrhea-hypokalemia-achlorhydria (WDHA) syndrome were studied. All had watery diarrhea, hypokalemia and hypercalcemia. Plasma vasoactive intestinal polypeptide (VIP) levels determined by radioimmunoassay were markedly elevated in these patients, indicating that they had VIP-producing tumors. Plasma VIP levels determined serially after the operation indicate that its determination is useful in estimating the effect of a treatment.
As for multiple endocrine neoplasia type 1 (MEN1), two out of the three cases belonged to this category. Patient 1 had a brother with insulinoma, and in case 2, even though there was no family history, the autopsy revealed not only multiple tumors of the pancreas but also pituitary adenomas, chief cell hyperplasia of the parathyroid glands, thyroid adenomas and adrenocortical adenomas.
VIP and other hormones in the tumors as well as in the plasma were examined extensively in these cases. In case 1, VIP, gastrin and calcitonin were produced in the tumor and only plasma VIP levels were elevated. In case 2, with multiple tumors, tumor 1 produced VIP, glucagon, pancreatic polypeptide, gastrin and calcitonin, and tumor 2, VIP, pancreatic polypeptide, gastrin and β-melanocyte stimulating hormone. In this case, plasma VIP, pancreatic polypeptide and glucagon levels were elevated. In case 3, VIP and calcitonin were produced in the tumor, and plasma VIP and calcitonin levels were elevated.
These results indicate that (1) VIP is a good tumor marker for the WDHA syndrome due to VIP-producing tumors ;(2) patients with the WDHA syndrome are sometimes associated with MEN1 ; and (3) VIP-producing tumors are multiple hormone-producing tumors, and VIP predominantly elevated in the plasma results inthe WDHA syndrome, although other hormones such as pancreatic polypeptide, glucagon and calcitonin are sometimes found to be elevated in plasma without contributing to the clinical features.

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The present experiments were undertaken to investigate the effect of alteration in on extracellular calcium concentration and of somatostatin on cholecystokinin-(CCK)-and caerulein-induced insulin and glucagon release from the isolated perfused rat pancreas. In control studies using perfusate containing 2.5 mM CaCl₂ and 50 mg/dl glucose, CCK and caerulein caused insulin and glucagon release in a dose-related fashion. During perfusion with calcium free medium, insulin release was markedly inhibited. Subsequent introduction of 2.5 mM CaCl₂ to the medium restored insulin response toward control levels. Extracellular calcium depletion, however, had no effect on CCK-or caerulein-induced glucagon release. The output of glucagon in the absence of calcium was comparable to that seen in the control experiments. On the other hand, somatostatin abolished the increase in glucagon secretion, but not the increase in insulin secretion, when perfused simultaneously with CCK or caerulein. However, pretreatment for 10 min with somatostatin blocked even insulin secretion. The effects of somatostatin on hormonal discharge are suggested to be related to an alteration in the handling of or response to calcium. Recently, somatostatin has also been shown to inhibit calcium uptake by islets. Thus, the present results indicate a differential sensitivity of CCK-and caerulein-stimulated alpha and beta cell to extracellular calcium depletion and to the effect of somatostatin.

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The role of endogenous glucagon and somatostatin in insulin release was studied. Islets isolated from adult rats were incubated for 60 min in 3.3, 8, 3 and 16.7 mM glucose with anti-glucagon antiserum (AGA) or anti-somatostatin antiserum (ASA) which was produced in rabbits. For the control experiments the antiserum was replaced by normal rabbit serum (NRS). Insulin release from islets treated with AGA was suppressed in 3.3 and 8.3 mM glucose as compared to islets treated with NRS, while insulin release was not affected in 16.7 mM glucose. On the other hand, insulin release from islets treated with ASA was enhanced in 3.3 and 8.3 mM glucose as compared to that from islets treated with NRS, whereas in 16.7 mM glucose it was not different from the control. These observations indicate that endogenous glucagon and somatostatin play a physiological role in the regulation of insulin release, but their regulation of insulin release is deranged at non-physiologically high glucose concentrations.

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To investigate the physiological role of glucagon in the maintenance of euglycemia, the effect of the neutralization of glucagon by an antiserum specific for glucagon on blood glucose and C-peptide immunoreactivity (CPR) levels was studied in theb asal as well as in the hyperglucagonemic states caused by hypoglycemia or an intravenous infusion of arginine in rats. The antiserum employed in this study was raised in rabbits by subcutaneous injection of a conjugate of BSAand the C-terminal fragment of glucagon. Since the antiserum was found to contain 896 ng/ml of antibody-bound glucagon, the antigen-stripped glucagon antibody was prepared. Its affinity constant and binding capacity were 2.57×10¹⁰ M^-1 and 147 nM, respectively.
After the administration of 1 ml of the antiserum in 24-hr-fasted rats, no free immuno reactive glucagon (IRG) was detected during the experiment for 60 min. Likewise, antiserum treatment prevented a rise in the plasma IRG level following an arginine infusion and insulin-induced hypoglycemia. Plasma CPR levels tended to be lower in antiserum-treated rats than in the control in any conditions covered by the present study. However, changes in blood glucoselevels the both groups were comparable. These results demonstrate that in the presence of glucagon antibodids euglycemia can be maintained with a reduced supply of insulin.

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Synthetic bombesin (100ng/min) was infused under pentobarbital anesthesia in normal dogs and in insulin deprived depancreatized dogs 4 days after surgery. The release of gut glucagon-like immunoreactive materials (gut GLI) calculated as the difference between the values measured using cross-reacting antiserum and a so-called pancreatic glucagon specific antiserum was markedly stimulated by bombesin infusion. Plasma glucagon immunoreactivity (GI) measured by pancreatic glucagon specific antiserum also showed a small increase, whereas plasma glucose decreased significantly with a transient rise in insulin. The plasma glucose level did not decrease in depancreatized dogs. Gut GLI response in the regional mesenteric vein to 5% glucose administered into the loop of the ileum was strongly augmented by bombesin infusion. It is concluded that (1) bombesin infusion decreased blood glucose level in normal dogs but not in depancreatized dogs.(2) Bombesin infusion markedly augmented the release of GLI from the intestine.(3) Bombesin also stimulated the release of glucagon which was probably of gastrointestinal origin.(4) Insulin release was stimulated transiently by bombesin infusion. Thus, a competition of gut GLI with glucagon at the glucagon receptor site may be an explanation of the reduction in blood glucose.

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Since the secretion of pancreatic glucagon is largely influenced by changes in the blood glucose level, the response of extrapancreatic glucagon to glycemic changes was investigated in man and dogs. Neither insulin-induced hypoglycemia nor arginine infusion caused a rise in plasma glucagon in two patients with total pancreatectomy, but plasma glucagon increased transiently in one of the two patients after oral glucose load. Glucose-induced hyperglycemia did not alter plasma glucagon in the portal vein in a group of 4 depancreatized dogs. When blood glucose fell lower than 50 mg/100 ml after iusulin infusion in a group of 7 depancreatized dogs, plasma glucagon in the portal vein did not increase significantly. The administration of 2-deoxyglucose did not cause any changes in plasma glucagon in the portal vein in a group of 5 pancreatectomized dogs. After the ligation of the hepatic artery, blood glucose decreased gradually in a group of 5 pancreatectomized dogs with portocaval shunt, but plasma glucagon in the vena cava was not altered significantly. It is concluded from the present study that regulation of extrapanacreatic glucagon differs from that of pancreatic glucagon.

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To test the possibility that insulitis might play an etiological role in the pathogenesis of insulin dependent diabetes, functions of 3 kinds of islet constituting cells (A, B and PP cells) were estimated by quantifying secretory responses of glucagon-, Cpeptide-and pancreatic polypeptide-producing cells to hyperglycemia and hypoglycemia. In insulin dependent diabetes, all 3 hormonal responses were severely impaired to the same extent. On the other hand, 3 islet cell functions were uniformly but less severly impaired in insulin independent diabetics without a diabetic family history. These results suggest that A, B and PP cells of islet of Langerhans are evenly destroyed in parallel fashion at least in insulin dependent diabetes and in some insulin independent diabetes, suggesting insulitis as a possible cause of these types of diabetes.

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Significant amounts of glucagon immunoreactivity (GI) and glucagon-like immunoreactivity (GLI) were found in two portions of the canine brain-the hypothalamus and brain stem. Ratios of GLI to GI were low in the hypothalamus, and variable but low in the brain stem. Dilution curves of the extracts from both regions showed similar patterns to that of glucagon in radioimmunoassays using specific (30K) and cross-reacting (YG9) antisera. The hypothalamic extract contained a larger amount of 3, 500 MW fraction comparable to glucagon and a lesser amount of 9, 000 MW fraction ressembling proglucagon. While displacement of ¹²⁵I-glucagon from the rat liver cell membrane by the main fraction of the hypothalamus was compatible with that of glucagon, activation of adenylate cyclase by the hypothalamus extract was higher than glucagon and that by the extract of the brain stem was lower than glucagon.
Although purificatiou was not sufficient and specific binding of circulating glucagon to the brain has not been overlooked, the hypothalamic extract at least contains material immunologically and probably biologically resembling glucagon.

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Motilin-like immunoreactivities were examined in extracts of duodena of pig, human, dag, rat, cat, rabbit and guinea pig with a combination of radioimmunoassays using three different antisera possessing distinct region-specificities. Antisera GP-1103 and R-1104 were rather C-terminal specific, but the immunological feature of GP-1103 was different from that of R-1104. In the case of antiserum GP-1103, the Glu residue in position 15 in the motilin moleculeparticipated in antigen-antibody interaction, but not in the case of R-1104. Antiserum MBR-02was N-terminal specific. with any of the three assay systems, only a single major peak was detected in the elution profile from BioGel P-10 column of motilin-like immunoreactivity in the porcine duodenal extract, of which the elution volume corresponded to that for synthetic porcine motilin. On the other hand, the gelfiltration profiles of the human and canine duodenal extracts consisted of two or three peaksof the immunoreactivity. The major peak emerged in the elution volume identicalwith that for synthetic porcine motilin and the other two peaks were eluted earlier. The peak which was eluted in the volume corresponding to that for BSA marker was detectable only by the assay system with N-terminal specific antiserumMBR-02. The immunoreactivity of the major peak component in the human, canine or rat extract was found to be lower than that in the porcine extract. The present data may indicate the existence of heterogeneity of motilin-like immunoreactivity and species variation of the struc ture, of tne immunoreactive motilin.

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Plasma motilin levels were measured by dextran-charcoal radioimmunoassay in normal subjects and in patients with diabetes mellitus and certain other disorders: Fasting plasma motilin in normal subjects determined by a single blood sampling varied considerably within a range of 50 to 545 pg/m/. Repeated blood samplings revealed cyclic fluctuations in plasma motilin during fasting, with a peak appearing between an interval of 100 to 120 min which was accompanied by gastric contraction as determined with a balloon inserted into the stomach. Meal ingestion caused a transient but significant rise in plasma motilin, followed by a progressive decrease with an absence of the secretory episode. On the other hand, plasma motilin levels decreased, without an initial increase, following oral and intravenous glucose loading.
Fasting plasma motilin levels were significantly elevated in patients with diabetes mellitus, and chronic liver diseases but within normal limits in patients with acromegaly and hyperthyroidism. Repeated blood samplings showed cyclic fluctuation in plasma motilin levels in diabetic patients. Levels rose after meal ingestion to a peak higher than that in normal subjects and remained higher until 120 min.
In summary, 1) fasting plasma motilin levels in normal subjects varied considerably because of cyclic fluctuations, 2) meal ingestion caused a transient rise in plasma motilin, followed by a decrease, whereas glucose administration produced only a decrease, 3) plasma motilin levels were elevated in patients with diabetes mellitus.

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The effect of various nutrients and hormones on motilin release was examined in normal volunteers and postvagotomy patients to investigate the mechanism of motilin secretion in the postprandial state. The ingestion of a mixed meal, protein and fat elevated the plasma motilin level, but the oral glucose load and arginine infusion lowered it. Although gastrin infusion showed no effect on the plasma motilin concentration, the infusion of insulin or glucagon inhibited motilin release in vivo. The patients, who underwent a selective or truncal vagotomy, also revealed a rise in plasma motilin after meal ingestion, suggesting that motilin could be released even in the absence of gastric acid and vagal stimulus. The perifusion experiments demonstrated that motilin release from human duodenal mucosa into the perfusate was stimulated markedly by low pH and 15 mm taurocholate, but not affected by the perifusion of 20mm glucose, 20mm arginine, 100 mU/l insulin or 30nM glucagon.
These results indicate that the motilin release induced by meal ingestion depend upon the balance of food components and that in normal subjects duodenal acidification, bile acid and unknown factors contained in the mixed meal may participate at least in part in postprandial motilin release. The inhibitory effect of glucose on motilin release may be attributable to a certain mediator mechanism which remains to be studied.

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Motilin has been called an interdigestive hormone, suspected to induce the interdigestive myoelectric complex (IDMC) in dog (Itoh et al., 1978). Somatostatin (GH-RIH) has been shown to inhibit motilin release in man (Mitznegg et al., 1977). The following study was designed to look at the effect of GH-RIH on blood levels of motilin and on the IDMC in dog.

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In order to investigate the possible involvement of gastrointestinal hormones in functional disorders of the digestive tract, serum motilin, neurotensin and gastrin levels and their response to oral intake of fat and glucose were examined in patients with irritable colon syndrome and dumping syndrome. The following results were obtained.(1) Basal serum motilin levels were higher in patients with irritable colon syndrome than in normal subjects, and remained high after ingestion of either 50 g of butter or 50 g of glucose.(2) No consistent response in serum neurotensin levels was found in patients with irritable colon syndrome or in normal subjects.(3) An immediate increase in serum gastrin levels was found in response to fat ingestion both in patients with irritable colon syndrome and in normal subjects, but there was no difference between these two groups.(4) In a patient with typical dumping syndrome, a markedly high level of fasting serum motilin was found, and the level increased further after the oral intake of glucose. These findings suggest that motilin may be involved in the irritable colon syndrome and dumping syndrome.

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Among biological actions of motilin on gastrointestinal motility, its action to induce phase III, activity front, of interdigestive myoelectric complex (IMC) appears to be of physiological significance. Synthetic motilin in a dose as little as 0.06μg/kg-h was shown to induce phase III in dog. A cyclic increase in fasting plasma immunoreactive motilin concentration (PIMC) occurred in phase III in dog and in a later part of phase II in man, suggesting strongly that there is an intimate relationship between cyclic increase in fasting PIMC and IMC. The observation also suggested that gastric acid secretion increases in phase II and phase III of IMC. The cyclic increase in the acid secretion may be related to a coincidental cyclic increase in PIMC. Ingestion of a meat meal results in a significant decrease in PIMC and abolishes the cyclic increase in PIMC, while IMC changes to digestive pattern after a meal. The significant decrease in PIMC is not attributed to the 3 known gut hormones including gastrin, octapeptide of CCK and secretin.

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