Disturbed cholecystokinin secretion in patients with eating disorders

It has been shown that the gastrointestinal hormone cholecystokinin (CCK) induces satiety and reduces food intake in laboratory animals and humans. In the light of this evidence we studied CCK release in patients suffering from eating disorders. The secretion of CCK into the general circulation was measured in 10 anorectic, in 7 bulimic patients, and in 8 healthy controls before and after a high-caloric liquid testmeal. Baseline CCK values were similar in controls (0.6 +/- 0.2 pmol/l) and bulimics (0.6 +/- 0.1 pmol/l) and were significantly increased in the anorectic group (1.8 +/- 0.4 pmol/l) (p less than or equal to 0.005). After eating peak plasma levels increased to 6.1 +/- 0.9 pmol/l in the anorectic, to 3.8 +/- 0.5 pmol/l in the bulimic and to 2.7 +/- 0.6 pmol/l in the control group. All postprandial CCK values were significantly higher in the anorectic group. The secretion of CCK-8-S, an important peptide in the CCK family, was significantly elevated, too. This disturbed CCK secretion in patients suffering from anorexia nervosa, even if it is a secondary, diet-induced defect, may perpetuate this disorder.     

Orlistat reduces gallbladder emptying by inhibition of CCK release in response to a test meal

BACKGROUND AND AIMS: Orlistat is a covalent inhibitor of digestive lipase derived from lipstatin, the natural product of Streptomyces toxytricini. By blocking the active site of intestinal lipase, orlistat inhibits hydrolysis of dietary triglycerides and thus reduces the intestinal lipid absorption. It is uncertain whether intestinal inhibition of lipase by orlistat also interferes with nutrient-induced CCK release from intestinal I-cells. The aim of the present study was therefore to assess whether oral administration of orlistat inhibits CCK release in response to a test meal and thus causes impaired gallbladder emptying. METHODS: 22 healthy volunteers were given a test meal consisting of 200 ml dairy cream and two teaspoons of chocolate powder (552 kcal=2328 kJ; 56.0 g fat; 5.2 g proteins, 6.6 g carbohydrates), with and without oral application of 120 mg orlistat. Gallbladder volume was determined by ultrasound before and 5, 10, 20, 30 and 40 min after meal ingestion. In parallel, a venous blood sample was collected for the measurement of bioactive CCK. CCK activity was assessed using a bioassay with isolated rat pancreatic acini cells. RESULTS: Oral administration of orlistat significantly impairs gallbladder emptying. After ingestion of the test meal the gallbladder contracted by 78.5% in the control group, whereas the test group with orlistat only showed a contraction of 45.7% (p<0.01). Maximal contraction was reached after 35 to 40 min, the maximal gallbladder emptying was delayed up to 10 min by orlistat. Orlistat induced a significant reduction of bioactive CCK levels in response to a test meal (CCK(max) with orlistat=4.1 pmol/l; CCK(max) without orlistat=7.8 pmol/l). CCK levels were reduced by 47% and the onset of maximal CCK secretion was delayed up to 10 min. CONCLUSION: The inhibition of intestinal lipolytic activity by orlistat results in reduced gallbladder emptying through inhibition of meal-mediated CCK release. We therefore hypothesize that impaired gallbladder motility may represent a risk factor in chronic treatment of severe obesity using orlistat.     

Determination of plasma cholecystokinin (CCK) concentrations by bioassay and radioimmunoassay in man. A critical evaluation.

The present investigation was designed to perform a direct comparison of a rat pancreatic acini bioassay system and a specific CCK radioimmunoassay (antiserum G-160) for the measurement of fasting and meal-stimulated plasma CCK in the presence and absence of the CCK receptor antagonist loxiglumide. The G-160 CCK antiserum is directed against the C-terminal O-sulfated tyrosine residue of the CCK molecule which is essential for full bioactivity of CCK peptides. For plasma extraction prior to bioassay measurement, hydrophobic reverse-phase chromatography on octadecylsilane cartridges was employed and resulted in simultaneous adsorption and elution of both CCK peptides and loxiglumide with recoveries of 87.5 +/- 9% and 75.0 +/- 5.9%, respectively. In the absence of loxiglumide, fasting and meal-stimulated values for CCK-like bioactivity and CCK-immunoreactivity (IR-CCK) were nearly identical (basal values: 1-2 pmol/l; meal-stimulated plateau levels: 4-6 pmol/l). After intravenous infusion of loxiglumide (30 mg/kg/h for 10 min, 10 mg/kg/h thereafter), resulting in plasma steady state levels of 200-300 mumol/l, meal-stimulated CCK-like bioactivity was undetectable, whereas IR-CCK levels were augmented 6.5-fold. In the bioassay system, standard samples containing 50 mumol/l loxiglumide produced complete inhibition of acinar lipase release in response to 50 pmol/l synthetic CCK-8. We conclude, that postprandial circulating non-CCK-like factors do not contribute significantly to the direct receptor-mediated stimulation of exocrine pancreatic secretion. The good agreement of CCK-like bioactivity and IR-CCK levels in the absence of loxiglumide confirms the sensitive and specific recognition of bioactive CCK peptides by the G-160 antiserum and suggests that this antibody exerts binding characteristics probably similar to a pancreatic acinar receptor.     

Enhancement of intragastric acid stability of a fat emulsion meal delays gastric emptying and increases cholecystokinin release and gallbladder contraction

Preprocessed fatty foods often contain calories added as a fat emulsion stabilized by emulsifiers. Emulsion stability in the acidic gastric environment can readily be manipulated by altering emulsifier chemistry. We tested the hypothesis that it would be possible to control gastric emptying, CCK release, and satiety by varying intragastric fat emulsion stability. Nine healthy volunteers received a test meal on two occasions, comprising a 500-ml 15% oil emulsion with 2.5% of one of two emulsifiers that produced emulsions that were either stable (meal A) or unstable (meal B) in the acid gastric environment. Gastric emptying and gallbladder volume changes were assessed by MRI. CCK plasma levels were measured and satiety scores were recorded. Meal B layered rapidly owing to fat emulsion breakdown. The gastric half-emptying time of the aqueous phase was faster for meal B (72 +/- 13 min) than for meal A (171 +/- 35 min, P < 0.008). Meal A released more CCK than meal B (integrated areas, respectively 1,095 +/- 244 and 531 +/- 111 pmol.min.l(-1), P < 0.02), induced a greater gallbladder contraction (P < 0.02), and decreased postprandial appetite (P < 0.05), although no significant differences were observed in fullness and hunger. We conclude that acid-stable emulsions delayed gastric emptying and increased postprandial CCK levels and gallbladder contraction, whereas acid-instability led to rapid layering of fat in the gastric lumen with accelerated gastric emptying, lower CCK levels, and reduced gallbladder contraction. Manipulation of the acid stability of fat emulsion added to preprocessed foods could maximize satiety signaling and, in turn, help to reduce overconsumption of calories.     

Close arterial infusion of calcitonin gene-related peptide into the rat stomach inhibits aspirin- and ethanol-induced hemorrhagic damage

Afferent neuron-mediated gastric mucosal protection has been suggested to result from the local release of vasodilator peptides such as calcitonin gene-related peptide (CGRP) from afferent nerve endings within the stomach. The present study, therefore, examined whether rat alpha-CGRP, administered via different routes, is able to protect against mucosal injury induced by gastric perfusion with 25% ethanol or acidified aspirin (25 mM, pH 1.5) in urethane-anesthetized rats. Close arterial infusion of CGRP (15 pmol/min) to the stomach, via a catheter placed in the abdominal aorta proximal to the celiac artery, significantly reduced gross mucosal damage caused by ethanol and aspirin whereas mean arterial blood pressure (BP) was not altered. Intravenous infusion of CGRP (50 pmol/min) did not affect aspirin-induced mucosal injury but significantly enhanced ethanol-induced lesion formation. Intravenous CGRP (50 pmol/min) also lowered BP and increased the gastric clearance of [14C]aminopyrine, an indirect measure of gastric mucosal blood flow while basal gastric output of acid and bicarbonate was not altered. Intragastric administration of CGRP (260 nM) significantly inhibited aspirin-induced mucosal damage but did not influence damage in response to ethanol. BP, gastric clearance of [14C]aminopyrine, and gastric output of acid and bicarbonate remained unaltered by intragastric CGRP. These data indicate that only close arterial administration of CGRP to the rat stomach, at doses devoid of a systemic hypotensive effect, is able to protect against both ethanol- and aspirin-induced mucosal damage. As this route of administration closely resembles local release of the peptide in the stomach, CGRP may be considered as a candidate mediator of afferent nerve-induced gastric mucosal protection.     

Modulation of gastric distension-induced sensations by small intestinal receptors

Duodenal lipid exacerbates gastrointestinal sensations during gastric distension. Using luminal application of the local anesthetic benzocaine, we investigated the role of intestinal receptors in the induction of these sensations. Nine healthy subjects were studied on five occasions, during which isotonic saline or 20% lipid (2 kcal/min), combined with (duodenal or jejunal) 0.75% benzocaine or vehicle at 2.5 ml/min, was infused intraduodenally before and during gastric distension. Intragastric pressures and volumes, gastrointestinal sensations, and plasma CCK levels were determined. Duodenal lipid combined with vehicle increased gastric volume (in ml: saline, -10 +/- 18; lipid/vehicle, 237 +/- 30) and plasma CCK [mean levels (pmol/l): saline, 2.0 +/- 0. 2; lipid/vehicle, 8.0 +/- 1.6] and, during distensions, induced nausea (scores: saline, 3 +/- 2: lipid/vehicle, 58 +/- 19) and decreased pressures at which fullness and discomfort occurred. Duodenal but not jejunal benzocaine attenuated the effect of lipid on gastric volume, plasma CCK, and nausea during distension (135 +/- 38 and 216 +/- 40 ml, 4.6 +/- 0.6 pmol/l and not assessed, and 37 +/- 12 and 64 +/- 21 for lipid + duodenal benzocaine and lipid + jejunal benzocaine, respectively) and on pressures for sensations. In conclusion, intestinal receptors modulate gastrointestinal sensations associated with duodenal lipid and gastric distension. There is also the potential for local neural mechanisms to regulate CCK release and thereby reduce afferent activation indirectly.     

Glucagon-like peptide 1 (GLP-1) suppresses ghrelin levels in humans via increased insulin secretion

INTRODUCTION: Ghrelin is an orexigenic peptide predominantly secreted by the stomach. Ghrelin plasma levels rise before meal ingestion and sharply decline afterwards, but the mechanisms controlling ghrelin secretion are largely unknown. Since meal ingestion also elicits the secretion of the incretin hormone glucagon-like peptide 1 (GLP-1), we examined whether exogenous GLP-1 administration reduces ghrelin secretion in humans. PATIENTS AND METHODS: 14 healthy male volunteers were given intravenous infusions of GLP-1(1.2 pmol x kg(-1) min(-1)) or placebo over 390 min. After 30 min, a solid test meal was served. Venous blood was drawn frequently for the determination of glucose, insulin, C-peptide, GLP-1 and ghrelin. RESULTS: During the infusion of exogenous GLP-1 and placebo, GLP-1 plasma concentrations reached steady-state levels of 139+/-15 pmol/l and 12+/-2 pmol/l, respectively (p<0.0001). During placebo infusion, ghrelin levels were significantly reduced in the immediate postprandial period (p<0.001), and rose again afterwards. GLP-1 administration prevented the initial postprandial decline in ghrelin levels, possibly as a result of delayed gastric emptying, and significantly reduced ghrelin levels 150 and 360 min after meal ingestion (p<0.05). The patterns of ghrelin concentrations in the experiments with GLP-1 and placebo administration were inversely related to the respective plasma levels of insulin and C-peptide. CONCLUSIONS: GLP-1 reduces the rise in ghrelin levels in the late postprandial period at supraphysiological plasma levels. Most likely, these effects are indirectly mediated through its insulinotropic action. The GLP-1-induced suppression of ghrelin secretion might be involved in its anorexic effects.     

Effect of near physiologic insulin therapy on hypoglycemia counterregulation in type-1 diabetes

OBJECTIVES: The aim of this study was to examine hormonal counterregulation during insulin-induced hypoglycemia in type-1 diabetic patients during long-term near normoglycemic insulin therapy and intensive clinical care. METHODS: Type-1 diabetic patients (age 35.3 +/- 2 years, body mass index 22.8 +/- 1 kg x m(-2), mean diabetes duration 13.6 (11-17 years), mean HbA1c during the last year 6.6 +/- 0.1%) and nondiabetic subjects were studied during (0-120 min) and after (120-240 min) hypoglycemic (3.05 mmol/l) hyperinsulinemic (approximately 330 pmol/l) clamp tests. RESULTS: During hypoglycemia peak plasma concentrations of glucagon (199 +/- 16 vs. 155 +/- 11 ng/l, p < 0.05), epinephrine (4,514 +/- 644 vs. 1,676 +/- 513 pmol/l, p < 0.001), norepinephrine (2.21 +/- 0.14 vs. 1.35 +/- 0.19 nmol/l, p < 0.01) and cortisol (532 +/- 44 vs. 334 +/- 61 nmol/l) were reduced in the diabetic patients. Plasma lactate did not change from baseline values (0.51 +/- 0.06 mmol/l) in diabetic but doubled in healthy subjects (1.13 +/- 0.111 mmol/l, p < 0.001 vs. control). During the posthypoglycemic recovery period plasma concentrations of free fatty acids were higher in diabetic patients at 240 min (1.34 +/- 0.12 vs. 2.01 +/- 0.23 mmol/l, p < 0.05). CONCLUSION: Despite long-term near physiologic insulin substitution and the low incidence of hypoglycemia, hormonal hypoglycemia counterregulation was impaired in type-1 diabetic patients after a diabetes duration of more than 10 years.     

Increased plasma apoA-IV level is a marker of abnormal postprandial lipemia: a study in normoponderal and obese subjects.

Plasma apolipoprotein A-IV (apoA-IV) levels are found elevated in hypertriglyceridemic patients. However, the relationship between plasma apoA-IV level and postprandial lipemia is not well known and remains to be elucidated. Thus, our objective was to study the relationship between plasma apoA-IV and postprandial TG after an oral fat load test (OFLT). Plasma apoA-IV was measured at fast and during an OFLT in 16 normotriglyceridemic, normoglucose-tolerant android obese subjects (BMI = 34.6 +/- 2.9 kg/m(2)) and 30 normal weight controls (BMI = 22.2 +/- 2.3 kg/m(2)). In spite of not statistically different fasting plasma TG levels in controls and obese patients, the former group showed an altered TG response after OFLT, featuring increased nonchylomicron TG area under the curve (AUC) compared with controls (516 +/- 138 vs. 426 +/- 119 mmol/l x min, P < 0.05). As compared to controls, obese patients showed increased apoA-IV levels both at fast (138.5 +/- 22.4 vs. 124.0 +/- 22.8 mg/l, P < 0.05) and during the OFLT (apoA-IV AUC: 79,833 +/- 14,281 vs. 68,176 +/- 17,463 mg/l x min, P < 0.05). Among the whole population studied, as among the control and obese subgroups, fasting plasma apoA-IV correlated significantly with AUC of plasma TG (r = 0.60, P < 0.001), AUC of chymomicron TG (r = 0.45, P < 0.01), and AUC of nonchylomicron TG (r = 0.62, P < 0.001). In the multivariate analysis, fasting apoA-IV level constituted an independent and highly significant determinant of AUC of plasma TG, AUC of chymomicron TG, AUC of nonchylomicron TG, and incremental AUC of plasma TG. In conclusion, we show a strong link between fasting apoA-IV and postprandial TG metabolism. Plasma fasting apoA-IV is shown to be a good marker of TG response after an OFLT, providing additional information on post-load TG response in conjunction with other known factors such as fasting TGs.     

Effect of bile salts on the plasma concentration of immunoreactive vasoactive intestinal polypeptide in man

The effects of bile salts on the release of immunoreactive vasoactive intestinal polypeptide (IR-VIP) were investigated in men using a specific radioimmunoassay. Plasma IR-VIP was determined after extraction by the acid-acetone method (recovery 75 +/- 5%). Oral administration of 400 mg sodium taurocholate caused a rise in plasma IR-VIP from 18.5 +/- 1.3 pmol/l to 31.1 +/- 2.1 pmol/l after 30 min and 39.0 +/- 1.7 pmol/l after 60 min and return to the initial value after 120 min. Oral administration of chenodeoxycholic acid (CDCA) also increased plasma IR-VIP from a basal level of 14.5 +/- 1.5 pmol/l to 36.3 +/- 1.2 pmol/l after 60 min. Oral administration of ursodeoxycholic acid (UDCA) increased plasma IR-VIP from 11.9 +/- 1.1 pmol/l to 25.6 +/- 1.8 pmol/l after 30 min. Perifusion of 1 mM taurocholate stimulated release of IR-VIP from human duodenal mucosa into the perifusate. These results suggest that bile salts may participate, at least in part, in the release of IR-VIP from the gut.     

Dysfunctional inhibitory muscarinic receptors mediate enhanced histamine release in isolated human bronchi

In human airways mucosal mast cells are under the control of inhibitory muscarinic receptors. The described experiments tested, whether the inhibitory potency of two muscarinic receptor agonists (oxotremorine, acetylcholine) becomes impaired in advanced chronic obstructive pulmonary disease (COPD). Isolated human bronchi obtained from 26 patients with lung cancer were separated into two groups. Group 1 patients suffered from moderate COPD (mean FEV1 56%; range 34-71%; mean pack years of cigarette smoking 50, range 20-96; one non-smoker). Group 2 patients had no or only a mild form of COPD; mean FEV1 was 82% (62-97%) and the number of pack years 22 (6-45; 3 non-smoker). The calcium ionophore A23187 induced a maximal histamine release of 4100+/-870 pmol/g/5 min in group 1 bronchi, in contrast to only 1730+/-240 pmol/g/5 min in group 2 bronchi (p<0.02). Oxotremorine (1 nmol/L) reduced the stimulated histamine release by 81+/-5% in group 2 bronchi, but did not produce a significant effect in group 1 bronchi (11+/-14%). In conclusion, the present experiments show an enhanced histamine release in advanced COPD, which can be explained by a dysfunction of inhibitory muscarinic receptors.     

Physiological plasma concentrations of cholecystokinin stimulate pancreatic enzyme secretion and gallbladder contraction in man

The present study was undertaken to determine whether infusion of cholecystokinin (CCK) to plasma concentrations comparable to those found after a meal stimulates pancreatic enzyme secretion and gallbladder contraction. Plasma CCK concentrations were measured by radioimmunoassay using antibody T204, which binds to all carboxyl-terminal CCK-peptides containing the sulfated tyrosine region. Ingestion of a standardized test meal in 7 normal subjects induced significant increases in plasma CCK from 2.0 +/- 0.2 pmol/l to levels between 4.6 +/- 0.6 and 7.3 +/- 1.0 pmol/l (p less than 0.05-p less than 0.0005). Infusion of 2.5 pmol/kg X h CCK 33 resulted in significant increases in plasma CCK from 2.0 +/- 0.2 to 3.9 +/- 0.3 pmol/l (p less than 0.0005). This infusion of CCK induced significant increases in trypsin secretion from 0.5 +/- 0.1 to 1.4 +/- 0.2 KU/15 min (p less than 0.005) and in bilirubin output from 1.6 +/- 0.7 to 30.3 +/- 8.0 mumol/15 min (p less than 0.05). It is concluded that physiological plasma concentrations of CCK stimulate pancreatic enzyme secretion and gallbladder contraction in man.     

Do normal plasma neurotensin concentrations increase ileal output?

Infusions of neurotensin increase ileal secretion in experimental animals, and the volume of ileal effluent in patients with ileostomies. The aim of the present study was to determine whether normal postprandial plasma concentrations of neurotensin increase the volume of fluid leaving the ileum. Basal and peak postprandial plasma neurotensin concentrations were 23 (17-36) and 39 (25-43) pmol/l (median and range) respectively in five subjects with ileostomies and 15 (3-27) and 32 (15-82) pmol/l respectively in nine normal subjects. Infusion of neurotensin for 30 min at a rate of 6.3 pmol/kg/min into six patients with ileostomies increased ileostomy output about 10-fold, and produced a significant decrease in the concentration of solid material, but plasma neurotensin concentrations rose to 237 (82-422) pmol/l during infusion at this rate. Infusion of neurotensin at 2.3 pmol/kg/min, producing plasma levels of 60 (16-108), had no significant effect the amount or nature of ileostomy effluent. We conclude that normal postprandial plasma concentrations of neurotensin are unlikely to influence the volume of fluid leaving the ileum.     

Effect of pancreatic enzyme supplementation on postprandial plasma cholecystokinin secretion in patients with pancreatic insufficiency

To test the hypothesis, based on studies in healthy man and dog, that patients with impaired digestion due to severe pancreatic insufficiency have impaired postprandial cholecystokinin (CCK) secretion that can be improved by the addition of pancreatic enzymes, we have studied plasma CCK responses to a test meal with and without addition of pancreatic enzymes in 10 patients with pancreatic insufficiency and steatorrhea, in 8 patients with chronic pancreatitis without steatorrhea, and in 6 healthy subjects. The patients with steatorrhea had a significantly (P less than 0.001) lower integrated plasma CCK response to the meal (177 +/- 23 pM.150 min) than the healthy subjects (468 +/- 41 pM.150 min), while patients with chronic pancreatitis without steatorrhea had an intermediate integrated postprandial CCK secretion (327 +/- 101 pM.150 min). Addition of pancreatic enzymes to the meal significantly augmented the integrated CCK response in both the patients with steatorrhea to 483 +/- 72 pM.150 min (P less than 0.01) and in those without steatorrhea to 480 +/- 85 pM.150 min (P less than 0.05). These values were not significantly different from those in the healthy subjects (521 +/- 86 pM.150 min). Integrated CCK secretion in the three groups during bombesin infusion was similar (patients with steatorrhea 134 +/- 23 pM.20 min, patients without steatorrhea 131 +/- 33 pM.20 min, and healthy subjects 146 +/- 28 pM.20 min), indicating a normal capacity to secrete CCK in response to a humoral stimulus. These data are in agreement with the suggestions from previous studies that digestion of nutrients by pancreatic enzymes plays an important role in the regulation of plasma CCK secretion after feeding.     

Copeptin levels are independent of ingested nutrient type after standardised meal administration – the CoMEAL study

Abstract

Context/objective: To clarify ambiguous published data, we determined whether standardized nutrient intake influences serum copeptin concentrations.

Materials/methods: Thirty healthy volunteers underwent oral glucose tolerance testing (OGTT) and mixed-meal tolerance testing (MMTT), respectively drinking 300?ml/237?ml of glucose-containing or fat/protein/carbohydrate-containing fluid. Copeptin was measured 30?min pre-(“baseline”)–180?min post-fluid intake.

Results: Median [25th–75th percentile] copeptin fell from 4.9 [3.6–8.3]/4.9 [3.6–7.1] pmol/l at OGTT/MMTT baselines to 3.2 (2.8–5.9)/4.1 (2.7–6.1) pmol/l at post-OGTT/post-MMTT nadirs (150?min/120?min; p?<?0.001, linear mixed-effect modeling).

Discussion/conclusions: Regardless of nutrient type ingested, copeptin did not increase, suggesting values can be interpreted independently of prandial status.     

Multiple sources of 1,2-diacylglycerol in isolated rat pancreatic acini stimulated by cholecystokinin. Involvement of phosphatidylinositol bisphosphate and phosphatidylcholine hydrolysis

Changes in the cellular content of 1,2-diacylglycerol (DAG) in isolated rat pancreatic acini in response to agonist stimulation were studied using a sensitive mass assay. When acini were stimulated by 10 nM COOH-terminal cholecystokinin-octapeptide (CCK8), the increase in DAG was biphasic, consisting of an early peak at 5 s and a second, larger, gradual increase that was maximal by 15 min. The basal level of DAG in acini was 1.04 nmol/mg of protein, which was increased to 1.24 nmol/mg of protein at 5 s and 2.76 nmol/mg of protein at 30 min. In comparison, the increase in DAG stimulated by 30 pM CCK8, a submaximal concentration for amylase release, was monophasic, increasing without an early peak but sustained to 60 min. Other Ca2+-mobilizing secretagogues such as carbamylcholine and bombesin increased DAG in acini, whereas vasoactive intestinal peptide, which acts to increase cAMP, had no effect. Phorbol ester and Ca2+ ionophore also stimulated DAG production. Analysis of the mass level of inositol 1,4,5-trisphosphate (1,4,5-IP3) showed that the generation of 1,4,5-IP3 stimulated by 10 nM CCK8 peaked at 5 s, a finding consistent with the early peak of DAG. The basal level was 4.7 pmol/mg of protein, which was increased to 144.6 pmol/mg of protein at 5 s by 10 nM CCK8. The levels of 1,4,5-IP3 then returned toward basal in contrast to the gradual and sustained increase of DAG. The dose dependencies of 1,4,5-IP3 and DAG formation at 5 s with respect to CCK8 were almost identical. This suggests that phosphatidylinositol 4,5-bisphosphate hydrolysis is a major source of the early increase in DAG but not of the sustained increase in DAG. Therefore, a possible contribution of phosphatidylcholine hydrolysis to DAG formation was examined utilizing acini prelabeled with [3H]choline. CCK8 (1 nM) maximally increased [3H]choline metabolite release by 133% of control at 30 min. Separation of these metabolites by thin layer chromatography showed that the products of CCK8-stimulated release were almost entirely phosphorylcholine, indicating the activation of a phospholipase C specific for phosphatidylcholine. By comparison, 1 nM CCK8 stimulated [3H]ethanolamine metabolite release from [3H]ethanolamine-labeled acini by only 22% of control. These data suggest that CCK stimulates both phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine hydrolysis; the latter may contribute to the sustained generation of DAG and hence the maintained activation of protein kinase C.     

Use of forskolin to study the relationship between cyclic AMP formation and bone resorption in vitro.

The effect of the adenylate cyclase activator forskolin on bone resorption and cyclic AMP accumulation was studied in an organ-culture system by using calvarial bones from 6-7-day-old mice. Forskolin caused a rapid and fully reversible increase of cyclic AMP, which was maximal after 20-30 min. The phosphodiesterase inhibitor rolipram (30 mumol/l), enhanced the cyclic AMP response to forskolin (50 mumol/l) from a net cyclic AMP response of 1234 +/- 154 pmol/bone to 2854 +/- 193 pmol/bone (mean +/- S.E.M., n = 4). The cyclic AMP level in bones treated with forskolin (30 mumol/l) was significantly increased after 24 h of culture. Forskolin, at and above 0.3 mumol/l, in the absence and the presence of rolipram (30 mumol/l), caused a dose-dependent cyclic AMP accumulation with an calculated EC50 (concentration producing half-maximal stimulation) value at 8.3 mumol/l. In 24 h cultures forskolin inhibited spontaneous and PTH (parathyroid hormone)-stimulated 45Ca release with calculated IC50 (concentration producing half-maximal inhibition) values at 1.6 and 0.6 mumol/l respectively. Forskolin significantly inhibited the release of 3H from [3H]proline-labelled bones stimulated by PTH (10 nmol/l). The inhibitory effect by forskolin on PTH-stimulated 45Ca release was significant already after 3 h of culture. In 24 h cultures forskolin (3 mumol/l) significantly inhibited 45Ca release also from bones stimulated by prostaglandin E2 (1 mumol/l) and 1 alpha-hydroxycholecalciferol (0.1 mumol/l). The inhibitory effect of forskolin on spontaneous and PTH-stimulated 45Ca release was transient. A dose-dependent stimulation of basal 45Ca release was seen in 120 h cultures, at and above 3 nmol of forskolin/l, with a calculated EC50 value at 16 nmol/l. The stimulatory effect of forskolin (1 mumol/l) could be inhibited by calcitonin (0.1 unit/ml), but was insensitive to indomethacin (1 mumol/l). Forskolin increased the release of 3H from [3H]proline-labelled bones cultured for 120 h and decreased the amount of hydroxyproline in bones after culture. Forskolin inhibited PTH-stimulated release of Ca2+, Pi, beta-glucuronidase and beta-N-acetylglucosaminidase in 24 h cultures. In 120 h cultures forskolin stimulated the basal release of minerals and lysosomal enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Specificity of siderophore receptors in membrane vesicles of Bacillus megaterium.

Membrane vesicles of Bacillus megaterium strains SK11 and Ard1 bound the ferrischizokinen and ferriferrioxamine B siderhores (iron transport cofactors). An approximately equimolar uptake of both labels of [3H, 59Fe]ferrischizokinen indicated binding of the intact chelate. Binding reached equilibrium in 2 to 5 min, was temperature independent, and was unaltered by the addition of several energy sources. A 91% dissociation of bound [Fe]ferrischizokinen was achieved in 60 s by the addition of excess ferrischizokinen. Ferriaerobactin, a siderophore which is structurally related to ferrischizokinen, caused no detectable release of bound [59Fe]ferrischizokinen. Of several other ferrigydroxamates tested, only ferriferrichrome A achieved the release (11%) of [Fe]ferrischizokinen. Rapid dissociation (92%) of bound [59Fe]ferriferrioxamine B by the addition of ferriferrioxamine B was observed, and a 67% release of [59Fe]ferriferrioxamine B was caused by ferriA2265, its structural relative. Ferrischizokinen, ferriferrichrome A, and ferrirhodotorulic acid produced a 6, 25, and 29% dissociation, respectively, of [59Fe]ferriferrioxamine B; ferriaerobactin caused no dissociation. [59Fe]ferriaerobactin was bound by the membranes, but its dissociation was not effected by unlabeled ferriaerobactin, suggesting no specific receptors for this chelate. The respective binding affinity constants and maximal binding capacities of membrane vesicles of strain SK11 were 2 x 10(7) M-1 and 280 pmol per mg of protein for ferrischizokinen and 7 x 10(7) M-1 and 37 pmol per mg of protein for ferriferrioxamine B. These values in strain Ard1 were, respectively, 1.4 x 10(7) M-1 and 186 pmol per mg of protein for ferrischizokinen and 11 x 10(7) M-1 and 23 pmol per mg of protein for ferriferrioxamine B. Separate, specific binding sites (receptors) for ferrischizokinen and ferriferrioxamine B exist on the vesicles. The ferrischizokinen receptors have a lower affinity but a higher binding capacity (eightfold) than that shown by the ferriferrioxamine B receptor. These receptors may be components of independent transport systems.     

Molecular forms of cholecystokinin in human plasma during infusion of bombesin

Bombesin is a tetradecapeptide with stimulatory actions on several gastrointestinal functions. Infusion of bombesin (60 pmol/kg. 20 min) into 7 normal subjects induced significant increases in plasma cholecystokinin (CCK) as measured with 2 sequence-specific radioimmunoassays. Employing antibody 1703, specific for carboxyl-terminal CCK-peptides containing at least 14 amino acid residues, plasma CCK concentrations rose from 0.8 +/- 0.2 pmol/l to 9.9 +/- 1.7 pmol/l (p less than 0.005), while using antibody T204, specific for the sulfated tyrosine region of CCK, plasma CCK levels increased from 2.9 +/- 0.5 pmol/l to 12.4 +/- 1.3 pmol/l (p less than 0.005). Plasma samples obtained from 3 subjects during bombesin infusion were fractionated by Sephadex column chromatography. Fractionation revealed 4 molecular forms of CCK: peak I eluted in the void volume and comprised 0-7% of CCK-like immunoreactivity, peak II eluted at 35% and comprised 8-41% of CCK-like immunoreactivity, peak III eluted at 50% and comprised 44-61% of CCK-like immunoreactivity, and peak IV eluted at 75% and comprised 15-27% of CCK-like immunoreactivity. Radioimmunoassay with a carboxyl-terminal CCK-antibody fully cross-reacting with gastrin did not reveal additional molecular forms of CCK. Since both the carboxyl-terminus and the sulfated tyrosine region are required for biological activity of CCK, it is likely that all these molecular forms of CCK possess biological activity.     

Priming effect of glucagon-like peptide-1 (7-36) amide, glucose-dependent insulinotropic polypeptide and cholecystokinin-8 at the isolated perfused rat pancreas.

The priming effect of glucagon-like peptide-1 (7-36) amide (GLP-1 (7-36) amide), glucose-dependent insulin-releasing polypeptide (GIP) and cholecystokinin-8 (CCK-8) on glucose-induced insulin secretion from rat pancreas was investigated. The isolated pancreas was perfused in vitro with Krebs-Ringer bicarbonate buffer containing 2.8 mmol/l glucose. After 10 min this medium was supplemented with GLP-1 (7-36) amide, GIP or CCK-8 (10, 100, 1000 pmol/l) for 10 min. After an additional 10 min period with 2.8 mmol/l glucose alone, insulin secretion was stimulated with buffer containing 10 mmol/l glucose for 44 min. In control experiments the typical biphasic insulin response to 10 mmol/l glucose occurred. Pretreatment of the pancreas with GIP augmented insulin secretion: 10 pmol/l GIP enhanced only the first phase of the secretory response to 10 mmol/l glucose; 100 and 1000 pmol/l GIP stimulated both phases of hormone secretion. After exposure to CCK-8, enhanced insulin release during the first (at 10 and 1000 pmol/l CCK-8) and the second phase (at 1000 pmol/l) was observed. Priming with 100 pmol/l GLP-1 (7-36) amide significantly amplified the first and 1000 pmol/l GLP-1 (7-36) amide both secretion periods, 10 pmol/l GLP-1 (7-36) amide had no significant effect. All three peptide hormones influenced the first, quickly arising secretory response more than the second phase. Priming with forskolin (30 mM) enhanced the secretory response to 10 mM glucose plus 0.5 nM GLP-1 (7-36) amide 4-fold. With a glucose-responsive B-cell line (HIT cells), we investigated the hypothesis that the priming effect of GLP-1 (7-36) amide is mediated by the adenylate cyclase system. Priming with either IBMX (0.1 mM) or forskolin (2.5 microM) enhanced the insulin release after a consecutive glucose stimulation (5 mM). This effect was pronounced when GLP-1 (7-36) amide (100 pM) was added during glucose stimulation. Priming capacities of intestinal peptide hormones may be involved in the regulation of postprandial insulin release. The incretin action of these hormones can probably, at least in part, be explained by these effects. The priming effect of GLP-1 (7-36) amide is most likely mediated by the adenylate cyclase system.