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Background & Aims: The type and function of 5-hydroxytryptamine (5-HT) receptors on intestinal muscle cells in humans are not known. 5-HT receptors were characterized pharmacologically and by radioligand binding. Methods: Contraction, relaxation, inositol 1,4,5-triphosphate (IP 3 ) and adenosine 3′,5′-cyclic monophosphate (cAMP) formation, and 5-HT binding were measured in dispersed muscle cells and in cells in which only one receptor type was preserved by selective receptor protection. Results: 5-HT binding was completely inhibited by 5-HT and partially by 5-HT 2A (ketanserin), 5-HT 4 (SDZ-205,557), and 5-HT 1p ( N -acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide; 5-HTP-DP) receptor antagonists. 5-HT caused contraction that was inhibited by ketanserin and augmented by SDZ-205,557 and 5-HTP-DP. In the presence of ketanserin, 5-HT caused relaxation of cholecystokinin-contracted cells that was inhibited by SDZ-205,557 and 5-HTP-DP. 5-HT increased IP 3 , which was inhibited by ketanserin, and cAMP, which was inhibited by SDZ-205,557 and 5-HTP-DP. In cells with only 5-HT 2A receptors, 5-HT caused contraction only, and residual binding was inhibited by ketanserin. In cells with only 5-HT45-HT1p receptors, 5-HT caused only relaxation and residual binding was inhibited by SDZ-205,557 and 5-HTP-DP. Conclusions: 5-HT 2A receptors mediating contraction and 5-HT 4 receptors mediating relaxation coexist on human intestinal muscle cells. The 5-HT 4 receptors are closely similar or identical to 5-HT 1p receptors.

Background & Aims The secretion and function of antral histamine are not known. The aims of this study were to characterize the mechanisms of histamine release from the gastric antrum of humans, dogs, and rats and to determine whether histamine can influence the secretion of somatostatin and gastrin. Methods Somatostatin, gastrin, and histamine secretion from superfused antral segments was measured using radioimmunoassay. Results Superfusion with thioperamide (H 3 antagonist) increased somatostatin and decreased gastrin and histamine secretion in all three species; superfusion with (r)-α-methylhistamine (H 3 agonist) had the opposite effect. The pattern implied that endogenous histamine, acting via H 3 receptors, exerts an inhibitory paracrine influence on somatostatin secretion, which in turn regulates gastrin secretion. Superfusion with somatostatin antibody increased histamine secretion; the increase was not affected by the gastrin antagonist L-365,260, implying that it was not mediated by the concurrent increase in gastrin but by suppression of an inhibitory pathway linking somatostatin and histamine. Superfusion with methacholine alone and in the presence of either the H 3 agonist or antagonist confirmed the existence of reciprocal inhibitory pathways linking somatostatin and histamine. Conclusions Antral histamine in humans, dogs, and rats is linked to antral somatostatin via reciprocal inhibitory paracrine pathways that serve to amplify the regulatory influence of somatostatin.

The present study was designed to identify the mechanism by which distention regulates gastrin secretion.The distal stomach of the isolated vascularly perfused rat was distended to varying degrees for 10-minute periods with an air-filled balloon.Low distention caused a decrease in gastrin (39% +/- 5%; P0.001) and an increase in somatostatin (58% +/- 4%; P0.001) secretion. High distention caused an opposite effect, i.e., an increase in gastrin (49% +/- 11%; P0.01) and a decrease in somatostatin (44% +/- 11%; P0.01) secretion. All responses were abolished by the axonal blocker, tetrodotoxin. Atropine had no effect on the responses to low distention, but converted the pattern of response with high distention to that observed with low distention. Both high and low distention were accompanied by a sustained increase in vasoactive intestinal peptide (VIP) secretion. The selective VIP antagonist, VIP[10-28], abolished both the gastrin and somatostatin responses to low distention.Low gastric distention activates preferentially VIP neurons that stimulate somatostatin and thus inhibit gastrin secretion. Increasing distention leads to progressive recruitment of cholinergic neurons that cause a reversal in the pattern to one of increase in gastrin and decrease in somatostatin secretion.

Vasoactive intestinal peptide (VIP), secretin, glucagon, gastric inhibitory peptide (GIP), and partial sequences of VIP and secretin were examined for their effect on colonic transport and adenylate cyclase activity. Net water flow was measured gravi- metrically in muscle-stripped, everted sacs of ascending and descending rat colon. VIP and secretin, but not glucagon or GIP, stimulated colonic secretion. The sensitivity of the colonic mucosa to VIP was high: the threshold (10–11 m ) and half-maximal dose (10–10 m ) for the effect of VIP were a 1000 times less than the threshold (10–8 m ) and half- maximal dose (10 –7 m ) for the effect of secretin. A subthreshold concentration of theophylline (2 mM) increased the submaximal responses to VIP or secretin up to but not beyond the maximal response to VIP alone (4.1 ± 0.2 μl mg-1 30 min-1). The combined effect of secretin and a maximal concentration of VIP was not additive. These results indicated that VIP and secretin shared the same receptor sites and/or intermediary mechanisms. VIP14–28 acted as a partial agonist with an efficacy of 0.2 and inhibited significantly the response to VIP. Secretin5–27 showed no secretory activity and did not inhibit the response to secretin. The lactamimide RMI 12330A (10-3 m ) abolished the secretory response to VIP and secretin but had no effect on the secretory response to 10 mM theophylline or on control absorptive flow rate. VIP and secretin, but not glucagon or GIP, stimulated adenylate cyclase activity in homogenates of colon mucosa. The lactamimide RMI 12330A (10-3 m ) inhibited basal adenylate cyclase activity as well as activity stimulated by VIP, secretin, prostaglandin E1, and 5'-guanylyl imidodiphosphate. The results obtained in colon mucosa conform to a pattern of structural and functional homology exhibited by VIP and secretin in several tissues (fat, liver, and pancreatic acinar cells) which sets them apart from glucagon and GIP. The mode of action of VIP and secretin on colonic secretion appears to involve the adenylate cyclasecyclic AMP system. The abundance of VIP in the colon of many species and the high sensitivity of transport mechanisms to VIP shown in this study raise the possibility that VIP may play a local (i.e., paracrine and/or neurocrine) role in the regulation of colonic transport.

The regulation of gastrin and somatostatin secretion by intraluminal chemicals was examined in an isolated vascularly perfused rat stomach, a preparation that maintains the integrity of intramural neural pathways and the polarity of stimulation. Intraluminal perfusion with alkaline solutions (0.1 M NaHCO 3 ) increased gastrin secretion, whereas perfusion with acid solutions (pH 3.5-3.0) inhibited gastrin secretion. Perfusion with 0.5% or 5% peptone solutions stimulated gastrin secretion and inhibited somatostatin secretion. Both these responses were abolished by addition of the axonal blocker, tetrodotoxin (10 −6 M), to the vascular perfusate, from which it was concluded that gastrin and somatostatin responses induced by peptone were mediated by intramural neurons. These neurons were both cholinergic and noncholinergic, because addition of an optimal dose of atropine (10 −7 M) to the vascular perfusate inhibited only partially the gastrin response and converted the somatostatin response from decrease below basal levels—a typical cholinergic effect—to significant increase above basal levels. As shown previously, atropine had an identical effect on gastrin and somatostatin responses to transmural electrical stimulation of the antral region. The results are in accord with a model for the regulation of gastrin secretion by intramural cholinergic and noncholinergic neurons. A direct effect of intraluminal chemicals on gastrin and somatostatin cells was not detected in this study, but is not precluded.

The gastric secretory response to prompt intravenous injection of pentagastrin was investigated in 3 normal subjects and 3 patients with duodenal ulcer. The highest responses to pentagastrin and histamine were correlated. Intravenous pentagastrin in a dose of 0.5 μg per kg could thus achieve the same extent of discrimination between individuals with a 10-fold economy in dosage over subcutaneous pentagastrin. Sensitivity to pentagastrin, expressed in the D50, was higher in patients with duodenal ulcer. Unexpectedly, the highest response to intravenous pentagastrin was only 40% of the peak response to subcutaneous histamine. Accordingly, intravenous pentagastrin acted as a partial agonist with an efficacy of about 0.4. The effect of prompt injection of pentagastrin contrasted with the effect of slow intravenous infusion of pentagastrin, as well as with the effects of gastrin-17 given by either prompt injection or slow infusion. A model is proposed to account for these and related findings in other species. The secretory receptor for gastrin and its analogues is viewed as consisting of two sites: a high affinity stimulatory site and a low affinity inhibitory site. Partial agonism after prompt intravenous injection would result from blood and tissue transients which are successively too high (partially inhibitory) and low (stimulatory). The disparate effects of analogues and the differences between species would be determined by the relative affinities of the two sites. The overflow of stimulant into inhibitory sites would account for the reversal of response at high doses observed in numerous studies.

The effect of peptide hormones and other small intestinal secretory stimulants on ion and water transport by ascending and descending rat colon was investigated in vitro using a muscle-stripped everted open sac preparation. Net water flux was measured gravimetrically at 30min intervals for 150 min, each sac serving as its own control. Water flux rate was constant over the entire period and equal in ascending (15.6 ± 1.1) and descending (14.9 ± 1.1 μl hr-1 mg-1 of dry weight) colon. Both segments responded identically to all test substances. Neither glucagon 10-5 m nor pentagastrin 10-5 m, singly or in combination, had a significant effect on net water flux. In contrast, theophylline 10-2 M and dibutyryl cyclic AMP 10-3 M reduced net flux significantly by 23% (P

Smooth muscle cells were isolated from segments of human antrum obtained at operation. The presence, activity, and specificity of receptors to acetylcholine, the C-termina1 octapeptide of cholecystokinin, methionine—enkephalin, and vasoactive intestinal peptide were examined. Muscarinic cholinergic receptors sensitive to low concentrations of atropine, cholecystokinin receptors sensitive to prog1umide and dibutyryl cyclic guanosine monophosphate, enkepha1in receptors sensitive to naloxone were demonstrated and found to mediate contraction. Vasoactive intestinal peptide receptors were demonstrated also and found to mediate relaxation. The stoichiometry and specificity of responses were closely similar to those previously found in isolated gastric smooth muscle cells of the guinea pig. The existence of high-affinity functional receptors on smooth muscle cells, together with the presence of the peptides in nerve terminals adjacent to these cells, makes it possible for these peptides to act as neurotransmitters.

The contractile action of cholecystokinin (CCK) on smooth muscle of the gut is either direct (gallbladder and gastric fundus) or both direct and neurally mediated (small intestine). These regional differences were used to characterize pharmacologically CCK receptors on smooth muscle cells and neurons of the gastric fundus, gallbladder, and ileum of the guinea pig. In circular and longitudinal ileal smooth muscle, tetrodotoxin was used to separate direct from neurally mediated contractile effects. Cholecystokinin receptors on smooth muscle cells were found in all locations. The muscle cells displayed a decreasing order of sensitivity to the C-terminal octapeptide of cholecystokinin as expressed in the median doses, and to the selective cholecystokinin antagonist, proglumide, as expressed in the inhibitory dissociation constants. The median doses of the octapeptide of cholecystokinin ranged from 5.5 nM in gallbladder muscle to 185 nM in circular ileal muscle; the corresponding inhibitory dissociation constants of proglumide ranged from 180 to 437 microM [corrected]. Cholecystokinin receptors on cholinergic neurons were confined to circular and longitudinal ileal muscle; the neurons were 80-300 times more sensitive to the octapeptide of cholecystokinin (D50's 0.5 and 2.3 nM) than the corresponding muscle cells, and 19-21 times more sensitive to proglumide (inhibitory dissociation constants, 20 microM [corrected]). The results provide clear evidence of cellular heterogeneity of cholecystokinin receptors (i.e., difference in sensitivity between muscle cells and neurons from the same location) as well as regional heterogeneity (i.e., difference in sensitivity between muscle cells from various locations).

The effect of somatostatin on colonic secretion induced by 10 −8 m vasoactive intestinal peptide (VIP), 10 −2 m theophylline, and 2 × 10 −3 m dibutyryl cyclic AMP was studied in muscle-stripped everted open rat colon sacs. The secretory response to VIP, measured as the decrease in net absorptive flow rate (microliters 30 min −1 mg −1 of dry weight), was maximal and equalled the responses to theophylline or dibutyryl cyclic AMP. Somatostatin (10 −5 m) blocked completely the secretory response to VTP but only partially the secretory response to theophylline or dibutyryl cyclic AMP. This difference in the extent of inhibition suggested that somatostatin exerted an inhibitory effect both before and after the point of generation of intracellular cyclic AMP. In order to test the hypothesis that one component of the action of somatostatin involved inhibition of the production of cyclic AMP, measurements of this nucleotide were made in isolated rat colon cells. Control levels of cyclic AMP measured by radioimmunoassay (12.6 ± 1.6 pmoles per 10 6 cells) were not affected by 10 −5 m somatostatin. VIP (5 × 10 −8 m) increased cyclic AMP levels 2-fold ( P

The nature of histamine receptors in gallbladder muscle and examined using specific histamine-receptor agonists and antagonists. The H2-receptor antagonist, metiamide, augmented the contractile response to histamine indicating that gallbladder muscle possessed stimulatory H1 receptors and inhibitory H2 receptors. The independent inhibitory character of H2 receptors was confirmed by (1) induction of relaxation with histamine after H1-receptor blockade and the suppression of this relaxation with metiamide, and (2) induction of relaxation with a specific H2-receptor agonist, 4-methyl histamine, and the suppression of this relaxation with metiamide. Further, blockade of H2 but not of H1 receptors augmented the response to the octapeptide of cholecystokinin. The nature of this effect was such that the apparent affinity of the octapeptide for its own receptor was increased. The finding raised the possibility that in their native unoccupied state, H2 receptors may modify the response to hormonal agents.

Two main candidates have been proposed for the role of relaxant neurotransmitter in the intestine: (a) the purine nucleotide, 5′-adenosine triphosphate (ATP) and (b) the neuropeptide, vasoactive intestinal peptide (VIP). The candidacy of VIP is favored by its precise location in nerve fibers that innervate circular smooth muscle and tenia coli. We have used a photoaffinity analog of ATP, 3′- O -(4-Benzoyl)benzoyl ATP, that binds irreversibly to ATP receptors and inactivates them in the presence of light, and a specific VIP antiserum to examine the claims of VIP and ATP as relaxant neurotransmitters in tenia coli of the guinea pig. Both VIP and ATP caused dose-dependent, tetrodotoxin-insensitive relaxation of tenia coli. The effect of ATP was equipotent to that of its stable isostere α,β-methylene ATP and resistant to degradation by adenosine deaminase, indicating interaction of ATP with purinergic-P 2 receptors. Photoactivated 3′- O -(4-Benzoyl)benzoyl adenosine triphosphate selectively inhibited relaxation induced by ATP but had no effect on relaxation induced by VIP or by field (i.e., neural) stimulation. Vasoactive intestinal peptide antiserum (final dilution 1:60), on the other hand, inhibited relaxation caused by VIP and by field stimulation but had no effect on relaxation caused by ATP. Neither normal rabbit serum nor preneutralized VIP antiserum had any effect on relaxation induced by ATP, VIP, or field stimulation. Inhibition of neurally induced relaxation by VIP antiserum ranged from 52% ± 7% (p

The effect of methacholine, theophylline, and cyclic adenyl and guanyl nucleotides on gastrin secretion from antral and proximal duodenal mucosa of the guinea pig was studied. Both 2 mM dibutyryl (db) cAMP and 5 mM theophylline produced significant increases in gastrin secretion, 4.3 ± 0.7 (P

The nicotinic receptor agonist, 1,1-dimethyl-4-phenylpiperazinium was used to examine the intramural neural regulation and the functional linkage of gastrin and somatostatin in the isolated vascularly perfused rat stomach. 1,1-Dimethyl-4-phenylpiperazinium (10−5 to 10−4M) caused a biphasic, dosedependent increase in gastrin secretion. The gastrin response was abolished by hexamethonium but only partly inhibited (35% at the higher dose of 1,1-dimethyl-4-phenylpiperazinium) by atropine. The same dose of atropine inhibited the maximal gastrin response to the muscarinic agonist, methacholine, by 80%. From this it was concluded that 1,1-dimethyl-4-phenylpiperazinium activates intramural cholinergic and noncholinergic neurons to cause gastrin secretion. 1,1-Dimethyl-4-phenylpiperazinium also caused a biphasic, dose-dependent increase in somatostatin secretion. The somatostatin response was completely inhibited by atropine and hexamethonium. Since muscarinic cholinergic agonists cause inhibition of somatostatin secretion, the observed net increase in somatostatin suggests that 1,1 dimethyl-4-phenylpiperazinium activated predominantly a noncholinergic stimulatory neuron. It is hypothesized that this neuron is identical to the gastrin-stimulating noncholinergic neuron and that it releases bombesin, a stimulant of gastrin and somatostatin secretion known to be present in gastric mucosal nerve terminals. The results of this study are consistent with a previously proposed model according to which gastrin secretion is regulated by the activity of two interdependent intramural neurons: a cholinergic neuron that causes gastrin secretion via inhibition of somatostatin and a noncholinergic neuron that releases a synergistic, direct stimulant of gastrin secretion, most likely bombesin.

The lower esophageal sphincter response to prompt intravenous injection of pentagastrin alone, and in combination with glucagon, was investigated in four healthy volunteers. The highest observed response to pentagastrin was 305% of resting pressure. The progress of the dose–response curve before decline set in appeared to indicate that a response maximally equal to that for gastrin might have been attained. Glucagon significantly inhibited resting and pentagastrin-stimulated lower esophageal sphincter pressure. Kinetic analysis disclosed that inhibition by glucagon was of the mixed type with a change both in affinity for, and efficacy of, pentagastrin. Endogenous glucagon released by infusion of alanine had no effect on the resting pressure. It was concluded that glucagon, like secretin, does not play a role in regulating the competence of the sphincter in the resting state. Its role during digestion remains to be determined.

The clearance rates of synthetic human gastrin-17-I were measured in man, dog, and cat. Half-life of disappearance and acid secretory potency (D50) were also measured in man and dog. The clearance rates in dog and cat were, respectively, 3 and 8 times more than in man. Accordingly, the half-life of gastrin-17 in the dog (3.5 min) was 3 times shorter than in man (9.5 to 10.5 min). The D50 for acid secretion was proportional to the clearance rate and yielded approximately similar increments of serum gastrin, indicating an equal sensitivity to gastrin-17 at cellular level in the three species. An inverse allometric relation between clearance rate and body weight was consistent with the known greater efficiency of metabolic and eliminatory processes in species of small size. Recent studies of the disposal of other gastrointestinal hormones indicate that the concepts developed theoretically for secretory stimulants and confirmed experimentally for gastrin-17 may have wider applicability.

Summary In 2 normal subjects, dose-response curves for the effect on acid secretion of single intravenous injections of gastrin II showed a good fit to a previously proposed model of secretion. The En5o's in micrograms per kilogram were similar in the 2 subjects, but were some 3 times lower than for subcutaneous gastrin. In 1 subject, dose-response curves for combinations of histamine, mechothane, or atropine with intravenous gastrin showed an equally good fit to the model. The maximal responses calculated for the synergistic or inhibitory combination were similar to the maximal response calculated for gastrin alone. It was concluded that in man the limit of secretion was determined by the functional size of the stomach and not by the nature of the stimulant (s). Analysis of published data on man suggests that similar conclusions as for gastrin apply to histamine.

An in vitro unilateral preparation of gastric mucosa is described in which only the serosal side is bathed with a nutrient solution. The preparation permits direct measurement of secretory flow rate and ionic concentration and a continuous recording of transmucosal potential difference. Analysis of amphibian secretion obtained in this manner disclosed striking similarities to the in vivo mammalian secretion. Thus, [Na] and [H] varied inversely, while [K] and [H] varied directly. Secretion became hypo-osmotic at intermediate acidities, but tended to osmotic equilibrium at high flow rates. Neither the early [K] transients nor the late decline in [K] were observed in vitro. The transmucosal potential difference, after correction for junction potentials, was shown not to have altered with the development of secretion. With recent developments in the study of mammalian gastric secretion in vitro, this technique can now be adapted to the direct study of mammalian active and passive secretory mechanisms.

Pancreatic secretion was obtained from the cannulated main ducts of man and dog and analyzed for ionic and protein content. In the dog, as [HCO3] increased, [Cl] and [Ca] decreased while [Na] and [K] increased linearly from their equilibrium levels with interstitial fluid concentrations. In both species, the slope ( − 0.86 to − 0.87) of the inverse linear relation between [HCO3] and [Cl] was significantly less than − 1. The relation between flow rate and anion concentration was described by a family of curves, the course of each curve being determined by the Cl content of its constituent samples. These findings were shown to be consistent with explicit formulations given to each of three models for pancreatic exocrine secretion (“two-component,” “unicellular,” and “exchange-diffusion” models). The same findings, however, served to exclude certain variants of these models in the case of man and dog, such as a 1:1 exchange of Cl for HCO3 or a unicellular model based on a constant sum of the anions.

The undissociated acidity of gastric juice represents the difference between titratable acidity and H+ ion concentration as measured by the glass electrode. An estimate of titratable acidity was obtained by extending the titration to that end point at which the positive charges contributed by Na+, K+, H+ ions and buffered protons matched the negative charges contributed by Cl-. In addition, equations were derived based on the significant correlation between H+ ion concentration and titratable acidity which permit direct estimation of undissociated acidity from either value. The positive relation noted between undissociated acidity and the concentration of nonpepsin protein suggests that the latter is the main buffer in gastric juice.

The effect of cholecystokinin (CCK), octapeptide-CCK, caerulein, and pentagastrin on guinea pig gallbladder muscle contraction was investigated in vitro. Concentration-response curves were constructed for each peptide and shown to obey Michaelis–Menten type kinetics. Maximal responses were similar indicating that the efficacies of all four peptides were equal. In this instance, therefore, the C50’s—that is, the concentrations required to elicit a given (half-maximal) response—could be held to represent molar potency. Accordingly, it was calculated that the octapeptide fragment of CCK and caerulein were, respectively, 10 and 2 times more potent than CCK while pentagastrin was 1800 times less potent. Response could not be altered by anticholinergic, anti-adrenergic, and depolarizing agents, or tetrodotoxin, from which it was concluded that CCK and related-peptides exerted their action directly on gallbladder muscle.

Five carboxyl-terminal fragments of secretin ranging in size from 6 to 21 amino acid residues were tested for pancreatic secretory activity in the rat. None of the fragments displayed activity when given alone but each displayed significant activity when given after secretin. This apparent activity was shown to be the result of displacement of secretin bound to the walls of the injection catheter. The activity was abolished by dissolving secretin in 2% bovine serum albumin. The finding emphasizes the ease with which secretin can bind to plastic surfaces and consequently the need to reevaluate previous dose-response studies and the caution required in the design of future studies.