Stimulatory and inhibitory effects of TMB-8 on pancreatic enzyme secretion

The putative intracellular calcium antagonist 3,4,5-trimethoxybenzoate 8-(diethylamino)-octyl ester (TMB-8) affects carbachol-induced enzyme secretion from rabbit pancreatic acini in a different way than it does that induced by either the C-terminal octapeptide of cholecystokinin (CCK-8), the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) or the calcium ionophore, A23187. In the presence of TMB-8 the dose-response curve for carbachol-induced amylase release shifts to the right, suggesting competitive antagonism at the muscarinic receptor. The hypothesis that TMB-8 acts as a muscarinic receptor antagonist is supported by the observation that TMB-8 dose-dependently inhibits the carbachol-, but not CCK-8-induced increases in cytosolic free calcium, measured in acinar cells by means of the fluorescent calcium indicator quin2. At a concentration of 100 microM, TMB-8 maximally potentiates the secretory response to suboptimal, but not (supra)optimal, concentrations of CCK-8. At the same concentration the drug also potentiates TPA- and A23187-induced enzyme secretion. Cytosolic free calcium levels and CCK-8-induced increases in cytosolic free calcium remain unaffected by 100 microM TMB-8. The above results strongly suggest that potentiation occurs at or beyond the site of interaction between the diacylglycerol- and the Ca2+-activated pathways. At concentrations beyond 100 microM the potentiating effect of TMB-8 declines and, finally, at a concentration of 500 microM the drug completely abolishes the secretory response to CCK-8 and TPA. Basal enzyme secretion, however, remains unaffected. At 500 microM severe side effects are observed as is shown by Trypan blue uptake, lactic dehydrogenase release and release of trapped quin2. It is concluded that at lower concentrations TMB-8 does not act as a specific intracellular calcium antagonist in pancreatic enzyme secretion and that inhibitory effects obtained with rather high concentrations of this drug should be treated with caution.     

Effects of growth hormone releasing factor on pancreatic secretion in vivo and in vitro

Growth hormone releasing factor (GRF), a 44-residue peptide originally isolated from human pancreatic tumors, shows structural similarities to the members of the secretin-vasoactive intestinal peptide (VIP) peptides. This study was designed to determine the effects of human GRF (hGRF-(1-44] on pancreatic secretion in vivo in conscious dogs and in vitro in dispersed rat pancreatic acini. GRF given i.v. in graded doses in dogs caused a small but significant stimulation of pancreatic HCO3- and protein outputs and potentiated secretin- and cholecystokinin (CCK)-induced pancreatic HCO3- but not protein secretion. When given together with somatostatin, GRF failed to reverse the inhibitory action of this peptide on HCO3- and protein responses to secretin plus CCK in dogs. Studies in vitro dispersed rat pancreatic acini showed that GRF added to the incubation medium of these acini caused an increase in basal amylase release and shifted to the left the amylase dose-response curve to caerulein and urecholine but failed to affect the amylase response to VIP. This study indicates that GRF in vivo stimulates basal and augments secretin- or CCK-induced pancreatic HCO3- secretion and that this is probably due to direct stimulatory action of the peptide on pancreatic secretory cells.     

Multiple effects of Na+ removal on pancreatic secretion in vitro

Summary The effects of removing Na⁺ from the incubation medium on basal and secretagogue induced zymogen release by pancreatic fragments and isolated pancreatic acini were studied by both morphological evaluation and measurement of amylase release. In both fragments and isolated acini, removal of Na⁺ led to an increased basal secretion of zymogen granule contents from acinar cells via exocytosis; secretory material, however, accumulated in acinar and ductular lumina as a result of the lack of fluid secretion necessary to wash out the enzymes. In studies with fragments, after Na⁺ removal there was no significant increase in amylase release into the medium; isolated acini, in contrast, showed an increased amylase release consistent with the shorter distance from the acinar lumen to the bathing medium. Stimulation with either bethanechol or caerulein led to a further depletion of zymogen granules in both preparations; in the absence of Na⁺ secretory product accumulated in intracellular lakes as well as in duct lumens. The hypothesis that Na⁺ influx is important in stimulus-secretion coupling to release intracellular Ca²⁺ was directly tested by measuring ⁴⁵Ca²⁺ efflux. No effect of removing Na⁺ on ⁴⁵Ca²⁺ efflux was seen. It was concluded, therefore, that while Na⁺ is essential for pancreatic fluid secretion, it is not necessary for the secretion of zymogen granule contents into acinar lumina.Supported by NIH grant GM-19998 from the United States Public Health Service     

Effects of sulphydryl reagents on pancreatic glucagon secretion in vitro.

The effect of sulphydryl reagents on glucagon secretion of isolated Wistar rats islets was studied. Chloromercuribenzene-p-sulphonic acid (CMBS) is a strong stimulator of glucagon secretion, whereas 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) markedly inhibited the hormone release. The effect of CMBS could not be suppressed by 20 mM glucose, but in the presence of 1 mM 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulphonic acid (SITS), and is independent of the glucagon content of islets. The results let us assume that sulphydryl-groups of pancreatic A-cells are involved in the regulation of glucagon secretion.     

The effect of luminal ghrelin on pancreatic enzyme secretion in the rat

Ghrelin, a 28-amino-acid peptide produced predominantly by oxyntic mucosa has been reported to affect the pancreatic exocrine function but the mechanism of its secretory action is not clear. The effects of intraduodenal (i.d.) infusion of ghrelin on pancreatic amylase outputs under basal conditions and following the stimulation of pancreatic secretion with diversion of pancreato-biliary juice (DPBJ) as well as the role of vagal nerve, sensory fibers and CCK in this process were determined. Ghrelin given into the duodenum of healthy rats at doses of 1.0 or 10.0 microg/kg increased pancreatic amylase outputs under basal conditions or following the stimulation of pancreatic secretion with DPBJ. Bilateral vagotomy as well as capsaicin deactivation of sensory fibers completely abolished all stimulatory effects of luminal ghrelin on pancreatic exocrine function. Pretreatment with lorglumide, a CCK(1) receptor blocker, reversed the stimulation of amylase release produced by intraduodenal application of ghrelin. Intraduodenal ghrelin at doses of 1.0 or 10.0 microg/kg increased plasma concentrations of CCK and ghrelin. In conclusion, ghrelin given into the duodenum stimulates pancreatic enzyme secretion. Activation of vagal reflexes and CCK release as well as central mechanisms could be implicated in the stimulatory effect of luminal ghrelin on the pancreatic exocrine functions.     

Role of leptin in the control of postprandial pancreatic enzyme secretion.

Leptin released by adipocytes has been implicated in the control of food intake but recent detection of specific leptin receptors in the pancreas suggests that this peptide may also play some role in the modulation of pancreatic function. This study was undertaken to examine the effect of exogenous leptin on pancreatic enzyme secretion in vitro using isolated pancreatic acini, or in vivo in conscious rats with chronic pancreatic fistulae. Leptin plasma level was measured by radioimmunoassay following leptin administration to the animals. Intraperitoneal (i.p.) administration of leptin (0.1, 1, 5, 10, 20 or 50 microg/kg), failed to affect significantly basal secretion of pancreatic protein, but markedly reduced that stimulated by feeding. The strongest inhibition has been observed at dose of 10 microg/kg of leptin. Under basal conditions plasma leptin level averaged about 0.15 +/- 0.04 ng/ml and was increased by feeding up to 1.8 +/- 0.4 ng/ml. Administration of leptin dose-dependently augmented this plasma leptin level, reaching about 0.65 +/- 0.04 ng/ml at dose of 10 microg/kg of leptin. This dose of leptin completely abolished increase of pancreatic protein output produced by ordinary feeding, sham feeding or by diversion of pancreatic juice to the exterior. Leptin (10(-10)-10(-7) M) also dose-dependently attenuated caerulein-induced amylase release from isolated pancreatic acini, whereas basal enzyme secretion was unaffected. We conclude that leptin could take a part in the inhibition of postprandial pancreatic secretion and this effect could be related, at least in part, to the direct action of this peptide on pancreatic acini.     

Effects of human corticotropin releasing factor (CRF) on gastric and pancreatic secretion in vivo and in vitro

Human CRF given IV inhibited dose-dependently pentagastrin- but not histamine-induced gastric acid secretion. When added to the incubation medium of the isolated gastric glands, CRF did not alter the formation of HCl under basal conditions or after stimulation with histamine or DBcAMP. CRF caused a small but significant increase in pancreatic HCO3 and protein secretion. It augmented CCK-induced pancreatic protein and secretin-induced HCO3 secretion in vivo but failed to affect basal or stimulated (CCK and urecholine) amylase release by the in vitro dispersed pancreatic acini. This study indicates that CRF inhibits gastric and stimulates pancreatic secretion in vivo but not in vitro and these effects are indirect involving, at least in part, alterations in the pancreatic circulation.     

The in vitro and in vivo effects of mefloquine on Trypanosoma brucei brucei.

Blood stream forms of Trypanosoma brucei brucei were grown over mouse kidney (MK) cells in minimum essential medium with various concentrations of mefloquine. The drug was observed to inhibit multiplication of the parasites in vitro. Groups of male albino mice were treated with mefloquine at 24, 48 and hours after T. b. brucei infection. Mefloquine at 0.03 mg/kg body weight administered for 4 consecutive days cleared the infection. No trypanosomes were detected in the blood of these mice for 90 days and over after the clearance of parasite from the blood. The doses for both the in vitro and in vivo therapy, were well below those prescribed for humans.     

Acute orexin effects on insulin secretion in the rat: in vivo and in vitro studies

Orexin-A and orexin-B are members of a family of newly described orexigenic hypothalamic neuropeptides. Scanty data are available suggesting the involvement of orexins in regulation of the secretion of pituitary hormones and in control of energy homeostasis. Present studies aimed to explain whether orexins affect blood insulin concentration and insulin secretion in the rat. To check this possibility, adult female rats were subcutaneously injected with different doses (1 or 2 nmol) of orexin-A or orexin-B. A bolus administration of orexin-A resulted in an increase in blood insulin (up to min 120) and glucose (60 min after injection) concentration. The higher dose of orexin-B, on the other hand, exerted effect on insulin secretion only at min 60 of experiment and neither doses changed blood glucose level. Only orexin-A stimulated insulin secretion in an in vitro perfusion system of the rat pancreas preparation, while orexin-B was less effective. The results demonstrate that orexins belong to a group of neuropeptides influencing insulin secretion and acting directly on the pancreas. Direct, at least partial, effect of orexin on insulin secretion may be connected with the regulation of metabolism by this peptide.     

Two hypotheses on the feedback regulation of pancreatic enzyme secretion

We review the mechanisms underlying the feedback regulation of pancreatic enzyme secretion in response to a meal. Pancreatic enzyme secretion in the rat and pig is known to be regulated by a negative feedback mechanism mediated by intestinal trypsin and chymotrypsin. Such a mechanism has recently been noted in humans. The presence of these enzymes in the small intestine suppresses pancreatic enzyme secretion, whereas their removal increases it. Two novel peptides have been proposed to account for the stimulation of pancreatic enzyme secretion in response to feeding trypsin inhibitor. One was assumed to be present in rat pancreatic juice and the other to be spontaneously secreted from the rat small intestine. In either case, trypsin and trypsin inhibitors do not directly interact with the luminal surface of the small intestine, but their actions are mediated by a trypsin-sensitive, cholecystokinin-releasing peptide. This is a novel explanation of the well-recognized stimulation of pancreatic enzyme secretion in response to dietary protein intake.     

The Caenorhabditis elegans mRNA 5'-capping enzyme. In vitro and in vivo characterization

Eukaryotic mRNA capping enzymes are bifunctional, carrying both RNA triphosphatase (RTPase) and guanylyltransferase (GTase) activities. The Caenorhabditis elegans CEL-1 capping enzyme consists of an N-terminal region with RTPase activity and a C-terminal region that resembles known GTases, However, CEL-1 has not previously been shown to have GTase activity. Cloning of the cel-1 cDNA shows that the full-length protein has 623 amino acids, including an additional 38 residues at the C termini and 12 residues at the N termini not originally predicted from the genomic sequence. Full-length CEL-1 has RTPase and GTase activities, and the cDNA can functionally replace the capping enzyme genes in Saccharomyces cerevisiae. The CEL-1 RTPase domain is related by sequence to protein-tyrosine phosphatases; therefore, mutagenesis of residues predicted to be important for RTPase activity was carried out. CEL-1 uses a mechanism similar to protein-tyrosine phosphatases, except that there was not an absolute requirement for a conserved acidic residue that acts as a proton donor. CEL-1 shows a strong preference for RNA substrates of at least three nucleotides in length. RNA-mediated interference in C. elegans embryos shows that lack of CEL-1 causes development to arrest with a phenotype similar to that seen when RNA polymerase II elongation activity is disrupted. Therefore, capping is essential for gene expression in metazoans.     

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.     

Control of pancreatic enzyme secretion: a critique on the role of calcium.

Secretion of digestive enzymes by the exocrine pancreas is thought to be initiated by a rise in cytosol Ca²⁺ activity. It is not yet clear whether influx of extracellular calcium or release of calcium from an intracellular site is responsible for this event. Review of the literature provides evidence that both extracellular and intracellular calcium regulate secretion and that under different circumstances each may be an important source of ‘messenger’ calcium.     

Distribution of neurotransmitters and their effects on glucagon secretion from the in vitro normal and diabetic pancreatic tissues

The distribution of adrenergic, cholinergic and amino acid neurotransmitters and/or their enzymes were examined in both the normal and diabetic pancreatic tissues in rat using immunohistochemistry to determine whether changes in the pattern of distribution of nerves containing these neurotransmitters will occur as a result of diabetes mellitus. In addition to this, the effect of noradrenaline (NA), adrenaline (ADR), acetylcholine (ACh) and gamma-amino butyric acid (GABA) on glucagon secretion from the isolated normal and diabetic pancreatic tissues was also investigated. Pancreatic fragments from the tail end of normal and diabetic rats were removed and incubated with different concentrations (10(-8)-10(-4) M) of these neurotransmitters. Glucagon secretion into the supernatant was later determined by radioimmunoassay. NA at 10(-6) M evoked a three-fold increase in glucagon secretion from normal pancreatic tissue fragments. In diabetic pancreatic tissue, NA at 10(-6) M was able to increase glucagon secretion 1.5 times the value obtained from diabetic basal. ADR (10(-8) M) increased glucagon secretion slightly but not significantly in normal pancreatic tissue. ADR inhibited glucagon secretion from diabetic pancreas at all concentrations. ACh (10(-8) M) induced a five-fold increase in glucagon secretion from normal pancreatic tissue. In a similar way, ACh evoked a two-fold increase in glucagon secretion from diabetic pancreas at 10(-4) M. In normal pancreatic tissue, GABA produced a slight but not significant increase in glucagon secretion at 10(-4) M. In contrast to this it inhibited glucagon secretion from diabetic pancreatic tissue fragments at all concentrations. In summary, tyrosine hydroxylase- and choline acetyltransferase-positive nerves are equally well distributed in both normal and diabetic rat pancreas. There was an increase in the number of glucagon positive cells and a decrease in the number of GABA-positive cells in diabetic pancreas. NA and ACh have a potent stimulatory effect on glucagon secretion from normal pancreatic tissue fragments, whereas ADR and GABA produced a small but not significant increase in glucagon secretion from normal pancreas. NA and GABA stimulated glucagon secretion from diabetic pancreas. In contrast, ADR and ACh inhibited glucagon secretion from diabetic pancreas. Neurotransmitters vary in their ability to provoke glucagon secretion from either normal or diabetic pancreas.     

Insulin secretion reactivation of pancreatic islets from starved rats in vitro.

Starvation of Wistar rats induced a shift of glucose threshold for insulin secretion of isolated islets above 5 mM, which can be restored by pretreatment of the tissue with glucose, mannose, glyceraldehyde, an theophylline, but not with acetylcholine or lactate. The improved insulin secretion is not connected with an enhanced glucose utilization.