Characteristics and mechaninsm of enzyme secretion and in cresase in [Ca^2＋]i in Saikosapoinin（1） stimulateced rat pancreatic acinar cells

AIM:This investigation was to reveal the characteristics and mechanism of enzyme secretion and increase in [Ca^2+]i stimulated by saikosaponin(1)[SA(1)] in rat pancreatic acini.METHODS:Pancreatic acini were prepared from male Wistar rats.Isolated acinar cells were suspended in Eagle‘s MEM solution,After adding drugs,the incubation was performed at 37℃for a set period of time.Amylase of supermatant was assayed using starch-iodide reaction.Isolated acinar single cell was incubated with Fura-2/AM at 37℃,then cells were wasthed and resuspended in fresh sulution and attached to the chamber,Cytoplasm [Ca^2+]i of a single cell was expressed by fluorescence ratio F340/F380 recorded in a Nikon PI Cd^2+ measurement system.RESULTS:Rate course of amylase secretion stimulated by SA(I) in rat pancreatic acini appeared in bell-like shape,The peak amplitude increased depended on SA(I) concentration.The maximum rate responded to 1^10moll/L SA(I) was 13.1-forld of basal and the rate decreased to basal level at 30min.CCK-8 receptor antagonist Bt2-cGMP markedly inhibited amylase secretion stimulated by SA(I)and the dose-effect relationship was similar th that by CCK-8,[Ca^2+]i in a single acinar cell rose to the peak at 5min afer adding 5×10^-6mol/L SA(I) and was 5.1-fold of basal level.In addition,there was a secondary increase after the initial peak.GDP could inhibit both the rate of amylase secretion and rising of [Ca^2+]i stmulated by SA(I) in a single pancreatic acinar cell.     

In vitro growth inhibition of human colonic tumor cells by Verapamil

AIM: To investigate the effects and mechanisms of Verapamil on cultured human colonic tumor (HCT) cells. METHODS: HCT cells were treated with different concentrations of Verapamil, and their proliferation was examined by MTT assay. The areas of sub-diploid peak were measured by flow cytometry, and the DNA ladder was found by agarose gel electrophoresis. The characteristic changes in morphology were observed under light microscopy. The cell nuclei (propidium iodide labeled, Pi-labeled) and cellular distribution and concentration of calcium (Fluo-3-labeled) were studied by using laser confocal scanning microscope. RESULTS: The proliferation of HCT cells was inhibited by different concentrations of Verapamil. With the increase in concentration of Verapamil, the percent of G0-G1 phase cells in HCT cells increased and that of S phase cells decreased. After treating with different concentrations of Verapamil, flow cytometry showed that HCT cells were enlarged in areas of sub-diploid in a dose-dependent manner. Gel electrophoresis results displayed a typical DNA ladder. On staining with Wrights-Giemsa, the typical cellular apoptosis morphologic changes were also observed. Pi-labeled cell nuclei were found markedly changed. In addition, we inspected that the 100 μmol/L Verapamil could increase the intracellular calcium ion concentration [Ca2+]i in HCT cells. CONCLUSION: Verapamil can inhibit proliferation of HCT cells via inducing cell apoptosis.     

Differential Cl- and HCO3- mediated anion secretion by different colonic cell types in response to tetromethylpyrazine

AIM: Colonic epithelium is known to secrete both CI- and HCO3, but the secretory mechanisms of different colonic cell types are not fully understood. The present study aimed to investigate the differential activation of Cl-and HCO3 secretion by tetramethylpyrazine (TMP) in human crypt-like cell line, T84, and villus-like cell line, Caco-2, in comparison to the TMP-induced secretory response in freshly isolated rat colonic mucosa. METHODS: Colonic epithelial anion secretion was studied by using the short circuit current (Isc) technique. RT-PCR was used to examine the expression of Na^+-HCO3-cotranspoter in different epithelial cell types. RESULTS: TMP produced a concentration-dependent Isc which was increase in both T84 and Caco-2 cells. When extracellular CI was removed, TMP-induced Isc was abolished by 76.6% in T84 cells, but not in Caco-2 cells. However, after both CI and HCO3- were removed, TMP-induced Isc in Caco-2 cells was reduced to 10%. Bumetanide, an inhibitor of Na^+-K^+-Cl-cotranspoter, inhibited the TMP-induced Isc by 96.7% in T84 cells, but only 47.9% in Caco-2 cells. In the presence of bumetanide and 4, 4‘-diisothiocyanostilbene-2, 2‘-disulfonic acid, an inhibitor of Na^+-HCO3- cotransporter, inhibited the TMP-induced current in Caco-2 cells by 93.3% .In freshly isolated rat colonic mucosa, TMP stimulated distinct Isc responses similar to that observed in T84 and Caco-2 cells depending on the concentration used. RT-PCR revealed that the expression of Na^+-HCO3- cotransporter in Caco-2 cells was 4-fold more greater than that in T84 cells. CONCLUSION: TMP exerts concentration-dependent differential effects on different colonic cell types with stimulation of predominant Cl- secretion by crypt cells at a lower concentration, but predominant HCO3- secretion by villus cells at a higher concentration, suggesting different roles of these cells in colonic Cl and HCO3 secretion.     

Lactobacillus plantarum inhibits epithelial barrier dysfunction and interleukin-8 secretion induced by tumor necrosis factor-α

AIM： To determine whether Lactobacillus plantarum can modify the deleterious effects of tumor necrosis factor-α （TNF-α） on intestinal epithelial cells. METHODS： Caco-2 cells were incubated with TNF-α alone or in the presence of L. plantarum. Transepithelial electrical resistance was used to measure epithelial barrier function. Interleukin 8 （IL-8） secretion by intestinal epithelial cells was measured using an ELISA. Cellular lysate proteins were immunoblotted using the anti-extracellular regulated kinase （ERK）, anti-phospho- ERK and anti-IκB-α. RESULTS： A TNF-α-induced decrease in transepithelial electrical resistance was inhibited by L. plantarum. TNF- α-induced IL-8 secretion was reduced by L. plantarum. L. plantarum inhibited the activation of ERK and the degradation of IκB-α in TNF-a-treated Caco-2 cells. CONCLUSION： Induction of epithelial barrier dysfunction and IL-8 secretion by TNF-α is inhibited byL. plantarum. Probiotics may preserve epithelial barrier function and inhibit the inflammatory response by altering the signal transduction pathway.     

Inhibition of tryptase release from human colon mast cells by protease inhibitors

AIM: To investigate the ability of protease inhibitors to modulate tryptase release from human colon mast cells.METHODS: Enzymatically dispersed cells from human colon were challenged with anti-IgE or calcium ionophore A23187 in the absence or presence of tryptase and chymase inhibitors,and tryptase release was determined.RESULTS:IgE dependent tryptase release from colon mast cells was inhibited by up to approximately 37%, 40% and 36.6% by chymase inhibitors Z-Ile-Glu-Pro-Phe-CO2Me (ZIGPFM), N-tosyI-L-phenylalanyl-chloromethyl ketone (TPCK), and α1-antitrypsin, respectively. Similarly, the inhibitors of tryptase leupeptin, N-tosyI-L-lysine chloromethyl ketone (TLCK) and lactoferrin were also able to inhibit anti-IgE induced tryptase release by a maximum of 39.4%,47.6% and 36.6%, respectively. The inhibitory actions of chymase inhibitors, but not tryptase inhibitors on colon mast cells were enhanced by preincubation of them with cells for 20min before challenged with anti-IgE. At a concentration of 10μg/mL, protamine was able to inhibit anti-IgE and calcium ionophore induced tryptase release. However, at 100μg/mL, protamine elevated tryptase levels in supematants.A specific inhibitor of aminopeptidase amastatin had no effect on anti-IgE induced tryptase release. The significant inhibition of calcium ionophore induced tryptase release was also observed with the inhibitors of tryptase and chymase examined. The inhibitors tested by themselves did not stimulate tryptase release from colon mast cells.CONCLUSION:It was demonstrated for the first time that both tryptase and chymase inhibitors could inhibit IgE dependent and calcium ionophore induced tryptase release from dispersed colon mast cells in a concentration dependent of manner, which suggest that they are likely to be developed as a novel class of anti-inflammatory drugs to treat chronic of colitis in man.     

Lactobacillus plantarum inhibits epithelial barrier dysfunction and interleukin-8 secretion induced by tumor necrosis factor-a

AIM: To determine whether Lactobacillus plantarum can modify the deleterious effects of tumor necrosis factor-a (TNF-a) on intestinal epithelial cells. METHODS: Caco-2 cells were incubated with TNF-a alone or in the presence of L. plantarum. Transepithelial electrical resistance was used to measure epithelial barrier function. Interleukin 8 (IL-8) secretion by intestinal epithelial cells was measured using an ELISA. Cellular lysate proteins were immunoblotted using the anti-extracellular regulated kinase (ERK), anti-phospho- ERK and anti-IκB-a. RESULTS: A TNF-a-induced decrease in transepithelial electrical resistance was inhibited by L. plantarum. TNF- a-induced IL-8 secretion was reduced by L. plantarum. L. plantarum inhibited the activation of ERK and the degradation of IκB-a in TNF-a-treated Caco-2 cells. CONCLUSION: Induction of epithelial barrier dysfunction and IL-8 secretion by TNF-a is inhibited by L. plantarum. Probiotics may preserve epithelial barrier function and inhibit the inflammatory response by altering the signal transduction pathway.     

Herbimycin, a tyrosine kinase inhibitor with Src selectivity, reduces progesterone and estradiol secretion by human granulosa cells

The purpose of the present study was to investigate whether the tyrosine kinase inhibitor herbimycin with some selectivity to block Src would alter the stimulatory effects of follicle-stimulating hormone (FSH) and cyclic adenosine monophosphate (cAMP) on estradiol secretion by human granulosa cells. Granulosa cells were taken from ovaries of premenopausal women undergoing oophorectomy for reasons unrelated to ovarian pathology. Granulosa cells from follicles ranging from 5–20 mm in diameter were subjected to culture. Granulosa cells were cultured with human FSH (2 ng/mL) or cAMP (0–1 mM) and testosterone (1 μM) in the presence and absence of herbimycin (0–2 μM). Media were collected at 24, 48, and 72 h. Accumulation of cAMP, progesterone, and estradiol in the media was determined by radioimmunoassay. Herbimycin dose dependently inhibited the ability of FSH to induce increases in progesterone and estradiol secretion. Although herbimycin increased (p < 0.0001) the accumulation of cAMP in response to FSH, this was evident only at the high concentrations of herbimycin (2 μM). To determine whether herbimycin would inhibit the ability of exogenous cAMP to induce estradiol and progesterone secretion, granulosa cells were incubated with 0–1 mM cAMP in the presence and absence of various doses of herbimycin. Herbimycin inhibited cAMP-induced estradiol and progesterone secretion in granulosa cells. The results from seven experiments indicate that herbimycin inhibits FSH stimulation of estradiol and progesterone secretion and that this inhibition may be, in part, at post-cAMP site(s).     

Discrepancies in the response of the insulin secreting cells of the dog and rat to different adrenergic stimulating agents

Summary Our experiments were carried outin vitro on different isolated and perfused pancreas preparations: adult rat pancreas, newborn dog pancreas, uncinate process and splenic portion of adult dog pancreas. The perfusion liquid contained 1.5 g/l glucose. 1) L-isoprenaline (0.05 μM), a stimulating agent of the β-adrenergic receptors, provoked only a slight and brief stimulation of insulin secretion from the rat pancreas. At the same concentration, this substance provoked a considerable stimulation of the insulin secreting cell of the newborn dog pancreas and a still much more marked response from the uncinate process and the splenic portion of adult dog pancreas. The effects of salbutamol (0.09 μM), a stimulating agent of the β-adrenergic receptors, were comparable to those of isoprenaline. The effects of isoprenaline and salbutamol on these preparations were suppressed by propranolol. 2) As to the effects of L-adrenaline on these different preparations, the following results have been recorded: a) Adrenaline (0.011 μM) inhibited insulin secretion by rat pancreas; this effect was suppressed by phenoxybenzamine; it was not modified by propranolol. — b) Adrenaline (0.011 μM) exerted an inhibitory effect on newborn dog pancreas, but only temporarily. Phenoxybenzamine inversed the effects of adrenaline and allowed the appearance of stimulation of insulin secretion; propranolol accentuated the inhibitory effect of adrenaline. — c) On the uncinate process of the adult dog pancreas adrenaline (0.011 μM) provoked a biphasic effect: brief and strong rise of insulin secretion followed by slight inhibition. Phenoxybenzamine enhanced the adrenaline-induced stimulation and transformed the inhibition of the second phase into strong stimulation. Propranolol suppressed the stimulatory effect of adrenaline which then had only an immediate and strong inhibitory effect. Adrenaline at 0.055 μM concentration provoked an extremely brief hypersecretion peak which was less intense than that recorded when using the 5-times weaker concentration; this peak was followed by strong and lasting inhibition. Deceased on january 9, 1977.     

Anti-inflammatory activity of probiotic Bifidobacterium： Enhancement of IL-10 production in peripheral blood mononuclear cells from ulcerative colitis patients and inhibition of IL-8 secretion in HT-29 cells

AIM： To determine the anti-inflammatory activity of probiotic Bifidobacteria in Bifidobacteria-fermented milk （BFM） which is effective against active ulcerative colitis （UC） and exacerbations of UC, and to explore the immunoregulatory mechanisms. METHODS： Peripheral blood mononuclear cells （PBMNC） from UC patients or HT-29 cells were co-cultured with heat-killed probiotic bacteria or culture supernatant of Bifidobacterium breve strain Yakult （BbrY） or Bifidobacterium bifidum strain Yakult （BbiY） to estimate the amount of IL-10 or IL-8 secreted. RESULTS： Both strains of probiotic Bifidobacteria contained in the BFM induced IL-10 production in PBMNC from UC patients, though BbrY was more effective than BbiY. Conditioned medium （CM） and DNA of both strains inhibited IL-8 secretion in HT-29 cells stimulated with TNF-α, whereas no such effect was observed with heat- killed bacteria. The inhibitory effect of CM derived from BbiY was greater than that of CM derived from BbrY. DNAs of the two strains had a comparable inhibitory activity against the secretion of IL-8. CM of BbiY induced a repression of IL-8 gene expression with a higher expression of IκB-ζ mRNA 4 h after culture of HT-29 cells compared to that in the absence of CM.CONCLUSION： Probiotic Bifidobacterium strains in BFM enhance IL-10 production in PBMNC and inhibit IL-8 secretion in intestinal epithelial cells, suggesting that BFM has anti-inflammatory effects against ulcerative colitis.     

Effects of phospholipase A2 inhibitors on Ca2+ oscillations in pancreatic acinar cells

High-affinity cholecystokinin (CCK) receptors were reported to be coupled with phospholipase A2 (PLA2)-arachidonic acid (AA) pathways to mediate Ca2+ oscillations and amylase secretion in rat pancreatic acinar cells. To investigate which types of PLA2 were involved in PLA2-AA pathways, the effects of specific inhibitors for type II and type IV PLA2 on Ca2+ oscillations and amylase secretion were studied in isolated rat pancreatic acini. An inhibitor of type IV (cytosolic) PLA2, AACOCF3 inhibited Ca2+ oscillations elicited by CCK-8 (30 pM) and JMV-180 (100 nM). AACOCF3 inhibited amylase secretion stimulated by JMV-180 and low concentrations of CCK-8 (< or =30 pM). On the other hand, an inhibitor of type II (secretory, nonpancreatic) PLA2 had no effects on Ca2+ oscillations and amylase secretion stimulated by CCK-8 and JMV-180. These results suggest that high-affinity CCK receptors are coupled to cytosolic PLA2 to mediate Ca2+ oscillations and amylase secretion in rat pancreatic acinar cells.     

Effects of C-terminal fragments of cholecystokinin on exocrine and endocrine secretion from isolated perfused rat pancreas

The ability of various C-terminal fragments of cholecystokinin (CCK) to increase pancreatic exocrine and endocrine secretion was examined in the isolated perfused rat pancreas. CCK octapeptide (CCK-8) induced biphasic dose-response curves for stimulation of pancreatic juice and amylase secretion. Maximal pancreatic juice and amylase output were obtained with 100 pM CCK-8. Concentrations of CCK-8 that caused pancreatic exocrine secretion also increased insulin release in the presence of 8.3 mM glucose. The tetrapeptide of CCK also simultaneously stimulated both exocrine and endocrine secretion, but was about 100,000 times less potent than CCK-8. By contrast both deca- and tetradecapeptide of CCK at a concentration of 100 pM stimulated secretion of pancreatic juice and amylase, and elicited insulin release comparably to CCK-8. The complete CCK-8 sequence was required as deamidated CCK-8 was without effects on exocrine and endocrine pancreatic secretion at a concentration of 100 pM. The present observations suggest that the structural requirements for CCK-induced insulin secretion are the same as those for CCK-induced exocrine secretions, and that the amino acids in position 5-8 and the amidated residue on the C-terminus are required for physiological activity of CCK on both the exocrine and endocrine pancreas. It is concluded that C-terminal fragments of CCK with eight or more amino acid residues are potent potentiators of insulin release as well as pancreatic exocrine stimulants.     

Pancreatic stellate cells produce acetylcholine and may play a role in pancreatic exocrine secretion

The pancreatic secretagogue cholecystokinin (CCK) is widely thought to stimulate enzyme secretion by acinar cells indirectly via activation of the vagus nerve. We postulate an alternative pathway for CCK-induced pancreatic secretion. We hypothesize that neurally related pancreatic stellate cells (PSCs; located in close proximity to the basolateral aspect of acinar cells) play a regulatory role in pancreatic secretion by serving as an intermediate target for CCK and secreting the neurotransmitter acetylcholine (ACh), which, in turn, stimulates acinar enzyme secretion. To determine whether PSCs (i) exhibit CCK-dependent ACh secretion and (ii) influence acinar enzyme secretion, primary cultures of human and rat PSCs were used. Immunoblotting and/or immunofluorescence was used to detect choline acetyltransferase (ACh synthesizing enzyme), vesicular ACh transporter (VAChT), synaptophysin, and CCK receptors 1 and 2. Synaptic-like vesicles in PSCs were identified by EM. ACh secretion by PSCs exposed to 20 pM CCK was measured by LC-MS/MS. Amylase secretion by acini [pretreated with and without the muscarinic receptor antagonist atropine (10 μM) and cocultured with PSCs] was measured by colorimetry. PSCs express ACh synthesizing enzyme, VAChT, synaptophysin, and CCK receptors; exhibit CCK-dependent ACh secretion; and stimulate amylase secretion by acini, which is blocked by atropine. In conclusion, PSCs express the essential elements for ACh synthesis and secretion. CCK stimulates ACh secretion by PSCs, which, in turn, induces amylase secretion by acini. Therefore, PSCs may represent a previously unrecognized intrapancreatic pathway regulating CCK-induced pancreatic exocrine secretion.     

Proglumide analogues CR 1409 and CR 1392 inhibit cholecystokinin-stimulated insulin release more potently than exocrine secretion from the isolated perfused rat pancreas

The effects of proglumide-related cholecystokinin (CCK) receptor antagonists CR 1409 and CR 1392 on CCK-octapeptide (CCK-8)-stimulated immunoreactive insulin (IRI) release and exocrine secretion were examined simultaneously in the isolated perfused rat pancreas. The CR 1409, at concentrations of 10-100 nM, significantly inhibited CCK-8 (100 pM) stimulation on IRI release but failed to inhibit the stimulatory effect of CCK-8 on both pancreatic juice flow and protein secretion. Increasing concentrations of CR 1409 inhibited both CCK-8-stimulated IRI release and exocrine secretion. Half-maximal inhibition was observed with approximately 2 nM for IRI release and 1 microM for protein secretion. When a higher dose (1 nM) of CCK-8 was used, the inhibitory effect of 10 nM CR 1409 on CCK-8-stimulated IRI release was abolished, whereas 10 microM CR 1409 retained significant inhibitory effect. Furthermore, 1 microM carbachol-induced IRI release was not altered by the addition of 10 microM CR 1409. The CR 1392 also had an inhibitory effect on both CCK-8-stimulated IRI release and exocrine secretion. The concentration of CR 1392 that caused half-maximal inhibition of CCK-8-stimulated IRI release was 300 times lower than that of exocrine secretion. In addition, 1 microM carbachol-stimulated IRI release was not altered by 100 microM CR 1392. Thus, the inhibitory effects of CR 1409 and CR 1392 on IRI release were mediated through the interaction at the CCK receptor and were more potent than those on juice and protein secretion. This study suggests, therefore, that CCK receptors on B cells might be different from those on acinar cells in terms of their relative affinities for antagonists.     

Inhibition of cell proliferation by the somatostatin analogue RC-160 is mediated by somatostatin receptor subtypes SSTR2 and SSTR5 through different mechanisms.

Effects of the stable somatostatin analogue RC-160 on cell proliferation, tyrosine phosphatase activity, and intracellular calcium concentration were investigated in CHO cells expressing the five somatostatin receptor subtypes SSTR1 to -5. Binding experiments were performed on crude membranes by using [125I-labeled Tyr11] somatostatin-14; RC-160 exhibited moderate-to-high affinities for SSTR2, -3, and -5 (IC50, 0.17, 0.1 and 21 nM, respectively) and low affinity for SSTR1 and -4 (IC50, 200 and 620 nM, respectively). Cell proliferation was induced in CHO cells by 10% (vol/vol) fetal calf serum, 1 microM insulin, or 0.1 microM cholecystokinin (CCK)-8; RC-160 inhibited serum-induced proliferation of CHO cells expressing SSTR2 and SSTR5 (EC50, 53 and 150 pM, respectively) but had no effect on growth of cells expressing SSTR1, -3, or -4. In SSTR2-expressing cells, orthovanadate suppressed the growth inhibitory effect of RC-160. This analogue inhibited insulin-induced proliferation and rapidly stimulated the activity of a tyrosine phosphatase in only this cellular clone. This latter effect was observed at doses of RC-160 (EC50, 4.6 pM) similar to those required to inhibit growth (EC50, 53 pM) and binding to the receptor (IC50, 170 pM), implicating tyrosine phosphatase as a transducer of the growth inhibition signal in SSTR2-expressing cells. In SSTR5-expressing cells, the phosphatase pathway was not involved in the inhibitory effect of RC-160 on cell growth, since this action was not influenced by tyrosine and serine/threonine phosphatase inhibitors. In addition, in SSTR5-expressing cells, RC-160 inhibited CCK-stimulated intracellular calcium mobilization at doses (EC50, 0.35 nM) similar to those necessary to inhibit somatostatin-14 binding (IC50, 21 nM) and CCK-induced cell proliferation (EC50, 1.1 nM). This suggests that the inositol phospholipid/calcium pathway could be involved in the antiproliferative effect of RC-160 mediated by SSTR5 in these cells. RC-160 had no effect on the basal or carbachol-stimulated calcium concentration in cells expressing SSTR1 to -4. Thus, we conclude that SSTR2 and SSTR5 bind RC-160 with high affinity and mediate the RC-160-induced inhibition of cell growth by distinct mechanisms.     

Oxyntomodulin inhibits pancreatic secretion through the nervous system in rats

Glicentin (GLIC), oxyntomodulin (OXM), and peptide YY (PYY) released in blood by ileocolonic L-cells after meals may inhibit pancreatic secretion. Whereas OXM interacts with glucagon and tGLP-1 receptors, OXM 19-37, a biologically active fragment, does not. The purpose of this study was to measure the effect of OXM, OXM 19-37, GLIC, tGLP-1, and PYY on pancreatic secretion stimulated by 2 deoxyglucose (2DG), electrical stimulation of the vagus nerves (VES), acetylcholine and cholecystokinin octapeptide (CCK8) in anesthetized rats. The effect of OXM was also studied in dispersed pancreatic acini. Plasma oxyntomodulin-like immunoreactivity (OLI) was measured by radioimmunoassay after the exogenous infusion of OXM and after an intraduodenal meal. OXM 19-37, infused at doses mimicking postprandial plasma levels of OLI, decreased pancreatic secretion stimulated by 2DG, VES, or CCK8. Similar effects were found with OXM and GLIC. OXM 19-37 did not change the pancreatic stimulation induced by acetylcholine in vivo, or CCK-induced amylase release in isolated acini. Vagotomy completely suppressed the inhibitory effect of OXM 19-37 on CCK8-stimulated pancreatic secretion. PYY inhibited the effect of 2DG, but not that of CCK8, whereas tGLP-1, even in pharmacologic doses, had no effect on stimulated pancreatic secretion. OXM, OXM 19-37, but not tGLP-1, inhibit pancreatic secretion at physiologic doses, through a vagal neural indirect mechanism, different from that used by PYY, and probably through a GLIC-related peptide-specific receptor.     

Effect of somatostatin on cholecystokinin-induced amylase release in rat pancreatic acini.

The effect of somatostatin on cholecystokinin-induced amylase release was investigated in isolated rat pancreatic acini. Acini were isolated by enzymatic digestion and incubated in a HEPES buffered Ringer's solution with testing reagents for 30 minutes at 37 degrees C. The activity of released amylase, cAMP, and inositol phosphate formation were measured. Intracellular calcium concentration ([Ca2+]i) was also checked. Somatostatin 14 and octreotide, a somatostatin analog, inhibited CCK-stimulated amylase release in a concentration-dependent manner. The inhibitory effect of octreotide on CCK-induced amylase release was not shown when the acini were treated with 8-Br-cAMP, irrespective of the presence of IBMX. Forskolin potentiated CCK-induced amylase release and this effect was blocked by octreotide treatment; although CCK-8 (3 x 10(-11) M) failed to stimulate cAMP formation, octreotide significantly inhibited basal cAMP formation in the acini. The increase of [Ca2+]i in response to CCK was inhibited by octreotide. However, CCK-induced inositol phosphate formation was not changed by 10(-9) M octreotide. Octreotide had no effect on CCK-stimulated tyrosine phosphorylation, and tyrosine phosphatase inhibitors (NaF and Na2WO4) did not influence the effect of octreotide on CCK-induced amylase release. From these results, we conclude that octreotide inhibits CCK-induced amylase release by inhibiting basal cAMP formation and decreasing the [Ca2+]i stimulated by CCK.     

Endogenous somatostatin inhibits interaction of insulin and cholecystokinin on exocrine secretion of isolated, perfused rat pancreas

INTRODUCTION: Although somatostatin inhibits pancreatic exocrine secretion, the inhibitory mechanism of endogenous somatostatin is not clearly understood. AIM: To investigate the effect of endogenous somatostatin on the interaction between endogenous insulin and exogenous cholecystokinin (CCK) in exocrine secretion of the totally isolated, perfused rat pancreas. METHODOLOGY: Endogenous releases of somatostatin and insulin were induced by 18 mM glucose. Streptozotocin (75 mg/kg) or cysteamine (300 mg/kg) was injected into rats 24 hours before the experiment to deplete insulin or somatostatin in the pancreas. RESULTS: Glucose (18 mM) enhanced CCK (10 pM)-stimulated secretions of fluid and amylase in the normal pancreas, which was further elevated by a somatostatin antagonist. Exogenous insulin (100 nM) also enhanced CCK-stimulated secretions in the streptozotocin-treated pancreas, which was also markedly increased by the somatostatin antagonist. The glucose (18 mM)-enhanced CCK-stimulated secretions were much higher in the cysteamine-treated pancreas than in the normal pancreas, which was dose-dependently reduced by exogenous somatostatin (30, 100 pM). However, endogenous or exogenous somatostatin did not modify the pancreatic responses to CCK alone. CONCLUSION: Endogenous somatostatin inhibits the interaction of endogenous insulin and CCK on pancreatic exocrine secretion in the rat rather than reducing the action of CCK alone or endogenous release of insulin.     

Effects of porcine gastrin-releasing peptide on amylase release, 2-deoxyglucose uptake, and alpha-aminoisobutyric acid uptake in mouse pancreatic acini

The effects of synthetic porcine gastrin-releasing peptide (pGRP), a recently isolated gut hormone, were studied in isolated mouse pancreatic acini. pGRP was found to exert direct effects on amylase release, 2-deoxyglucose ( [3H] 2DG) uptake, and alpha-aminoisobutyric acid (AIB) uptake. The stimulatory effect of pGRP on amylase release was significant at 100 pM, and maximal at 1 nM. Higher concentrations of pGRP exerted a smaller stimulatory effect on amylase release. pGRP also increased [3H]2DG uptake, exerting a detectable effect at 300 pM, and a maximal effect at 30 nM. In contrast to its stimulatory effect on amylase release and [3H]2DG uptake, pGRP inhibited AIB uptake. A significant inhibitory effect on AIB uptake occurred at 100 pM, and a maximal inhibitory effect occurred at 3 nM. Dose-response curves of pGRP for amylase release and AIB uptake were found to be biphasic. Bombesin was also found to stimulate amylase release with a biphasic dose-response curve in mouse acini. Both cholecystokinin (CCK) octapeptide and the cholinergic analog carbachol exerted similar effects in isolated mouse acini. However, the effects of pGRP were not inhibited by either dibutyryl cyclic guanosine 3',5'-monophosphate or atropine, whereas the effects of CCK octapeptide were inhibited by dibutyryl cyclic guanosine 3',5'-monophosphate and the effects of carbachol were inhibited by atropine. These results indicate that pGRP can mimic the biological effects of CCK and acetylcholine, but that its actions are probably mediated via a separate class of receptors in mouse acini.     

Comparison between the synthetic cholecystokinin analogues caerulein and Thr28NlE31CCK25-33(CCK9) with regards to plasma bioactivity, degradation rate and stimulation of pancreatic exocrine function.

A new synthetic analogue of cholecystokinin, Thr28Nle31CCK25-33(CCK9) is compared with caerulein with regards to plasma bioactivity, degradation rate, side effects, and stimulation of pancreatic secretion. 24 healthy male volunteers were intubated with a double lumen Lagerl?f-tube. 30 min after correct positioning of the tube subjects received a continuous intravenous infusion of synthetic secretin (1 U/kg) together with either ceruletide (61.5 pM/kg) or CCK9 (30 pM/kg) both for 45 min. 30 pM/kg of CCK9 have been shown by others to cause maximal enzyme secretion (1). 61.5 pM/kg (= 100 ng) of caerulein are probably supramaximal but used by many centers for direct pancreatic function tests. Plasma CCK was measured by bioassay which compares amylase release of isolated rat pancreatic acini stimulated by plasma extracts with known standards of CCK8. Lipase, amylase, trypsin, chymotrypsin, and bicarbonate were measured in 15 min fractions after the onset of CCK infusion. Both drugs caused a similar stimulation of pancreatic enzyme secretion during infusion of the respective analogue which declined after termination. After the onset of CCK9 infusion plasma bioactivity reached a plateau around 20 pM at 15 min. Values declined after 30 min. After termination of infusion bioactivity rapidly declined within 3 min but still remained slightly elevated after further 15 min as compared to basal values (3.9 vs 0.6 pM). Plasma kinetics of caerulein were quite similar. However, despite a dose which was only twice as high as compared to CCK9, plasma bioactivity was six times higher with plateau values of about 120 pM.(ABSTRACT TRUNCATED AT 250 WORDS)