DNA damage in isolated hamster and rat pancreas cells by pancreatic carcinogens

N-Nitrosobis(2-oxopropyl)amine (BOP), N-nitrosobis(2-hydroxypropyl)-amine (BHP) and N-nitroso(2-hydroxypropyl-2-oxopropyl)amine (HPOP) are pancreatic carcinogens in the Syrian golden hamster (SGH) but do not cause pancreatic tumors in rats. In this study, the ability of these three compounds to induce DNA damage in isolated pancreas cells from both species was determined by alkaline elution analysis. BOP was highly potent in SGH cells, causing DNA damage at concentrations as low as 0.5 micrograms/ml, and HPOP, although less potent than BOP, also caused considerable damage. Isolated SGH pancreas cells are thus able to metabolize BOP and HPOP to DNA-damaging species. Of the three compounds tested, only HPOP at higher doses (25-100 micrograms/ml) induced DNA damage in isolated rat pancreas cells. BHP did not damage rat or SGH pancreas cell DNA at concentrations up to 100 micrograms/ml, apparently due to lack of uptake of this compound by the cells. The observed insensitivity to DNA damage in rat cells is consistent with the resistance of the rat pancreas to carcinogenesis by these three compounds. The sensitivity of SGH pancreas cells to BOP- and HPOP-induced DNA damage correlates with the high carcinogenicity of these compounds for the SGH pancreas.     

Inhibition by heparin and dextran sulfate of stimulated rat pancreatic adenylate cyclase

Heparin inhibited the adenylate cyclase activity of semipurified rat pancreatic plasma membranes stimulated by hormones and by Gpp(NH)p but not by fluoride or when in the persistently active state. When observed, the inhibition was rapid and sustained. It was of a noncompetitive type and never exceeded 20% for secretin. The inhibition of Gpp(NH)p-stimulated activity was more pronounced (48% inhibition at a heparin concentration of 50 μg/ml). For the C-terminal octapeptide of pancreozymin (CCK-8)-stimulated adenylate cyclase, the inhibition amounted to 93% at 50 μg/ml. This inhibition was competitive at low heparin concentration and of a mixed type above 10 μg/ml. Besides, heparin inhibited (I₅₀ = 6 μg/ml) the binding of peptides of the CCK family to their specific receptors without affecting the apparent K_d value of binding. Taken together, these relatively specific effects of heparin gave evidence in favor of the existence of CCK spare receptors. Dextran sulfate was more potent than heparin as an inhibitor of adenylate cyclase activation while chondroitin-4-sulfate and chondroitin-6-sulfate were ineffective. Dansylated pancreatic plasma membranes exhibited characteristics of adenylate cyclase activation by CCK-8 which were similar to those found for untreated membranes exposed to heparin.     

Rapid formation of inositol 1,4,5-trisphosphate in rat pancreatic acini stimulated by carbamylcholine

Cholinergic stimulation of inositol phosphate formation was studied in isolated rat pancreatic acini, prelabelled with myo-[2-3H]inositol. Carbamylcholine increased incorporation of radioactivity into Ins(1,4,5)P3 and InsP4 within 5 s. Increases in [3H]Ins(1,3,4)P3 were delayed with marked stimulation occurring between 10 s and 1 min. Inositol polyphosphate formation was less sensitive to carbamylcholine concentration than was stimulation of amylase release. At a low (0.3 microM) carbamylcholine concentration, no increase in inositol polyphosphate formation was detected, whereas stimulation of amylase release, which was not dependent on extracellular calcium, was observed. Ins(1,4,5)P3 was shown to release actively accumulated 45Ca2+ from isolated rough endoplasmic reticulum membranes to a similar extent as that released from rough endoplasmic reticulum following cholinergic stimulation of pancreatic acini (Richardson, A.E. et al. (1984) Biochem. Soc. Trans. 12, 1066-1067). The data is consistent with Ins(1,4,5)P3 being produced rapidly enough to release sufficient calcium from the rough endoplasmic reticulum to cause an observed increases in cytoplasmic free Ca2+.     

A species difference between hamster and rat in the effect of oestrogens on growth of large preantral follicles

Intact or hypophysectomized 23-day-old hamsters and rats were injected s.c. with 2 mg diethylstilboestrol (DES) or 1 mg oestradiol cyclopentylpropionate (OECP) on Days 23-25 and killed on Day 26. Although serum oestradiol was elevated to the same high levels by OECP, ovarian and uterine weights were increased in the rat by OECP or DES whereas only the uterus responded in the hamster. This correlated with the ability of the oestrogens to increase significantly the number of large preantral and antral follicles in the intact rat but only the number of follicles with 2-3 layers of granulosa cells in the immature hamster. Qualitative study revealed that DES and OECP increased the number of large preantral follicles in the adult hypophysectomized rat but were ineffective in the adult hamster. It is concluded that for the immature and adult hamster oestrogens do not play a major role in the recruitment of large preantral follicles.     

Bile protein secretion in the rat stimulated by taurocholate: effect of chloroquine

The biliary protein excretion during sodium taurocholate induced choleresis was studied in normal rats and in rats treated with the lysosomotropic agent, chloroquine. The analysis of the protein component in bile was made on SDS-polyacrilamide gel, and the individual polypeptides were quantitated by densitometry. The excretion of bile polypeptides was compared with that of lysosomal acid phosphatase. The biliary excretion of polypeptides of molecular mass lower than and equal to 54 kDa was markedly stimulated by taurocholate-induced choleresis. Chloroquine treatment of rats diminished the biliary excretion of such polypeptides and also inhibited their excretion induced by taurocholate. The behaviour of these polypeptides was well correlated to that of the lysosomal marker. The biliary excretion of polypeptide bands of a higher molecular mass (up to 140 kDa) did not show major changes during taurocholate-induced choleresis in any of the groups. The results indicate that biliary excretion of proteins in the rat may be either stimulated by taurocholate or may be independent of the bile salt. The former requires the functional integrity of chloroquine-sensitive hepatocyte compartments, which may involve the lysosomes.     

Effects of pancreatic polypeptide on the pancreatic exocrine secretion stimulated by secretin and cholecystokinin in the conscious pig

Fourteen castrated male Large White pigs, weighing 42.5 +/- 1.0 kg, were fitted with pancreatic and duodenal fistulae for pancreatic secretion studies. Moreover, catheters were placed in a carotid artery for blood sampling and in a jugular vein for peptide infusion. Pancreatic juice was automatically restituted to the animals and continuously sampled for analysis on experimental days. Following an 8-day recovery period, perfusion studies were performed after an overnight fast. After a 30-min basal period, sustained pancreatic flow and protein output were obtained and maintained throughout the assay with secretin (36 pmol/kg/h) and CCK-8 (600 pmol/kg/h) infusion. Then, 200, 400, 600, 800 or 1200 pmol/kg/h of porcine pancreatic polypeptide (PP) were infused for 60 min. Secretin + CCK infusion was continued for 1 h after PP infusion was stopped. Each dose of PP was given on a separate day. Neither pancreatic flow nor bicarbonate output were affected whatever the dose of infused PP. On the contrary, protein concentration and output decreased with the lowest dose of PP (200 pmol/kg/h) and the diminution was more pronounced with the other doses. With 600 pmol/kg/h as well as with 800 and 1200 pmol/kg/h of PP, pancreatic protein output fell to about 20% of values obtained with secretin + CCK. Plasma levels of PP were below or similar to postprandial values for 200, 400 and 600 pmol/kg/h and they were significantly larger with 800 and 1200 pmol/kg/h. Protein concentration and output returned to values obtained with secretin + CCK infusion after cessation of PP infusion. In conclusion, porcine PP given in physiological doses to the pig decreases pancreatic protein output whereas pancreatic flow remains unaffected.     

The influence of age on human pancreatic growth hormone releasing hormone stimulated growth hormone secretion

63 non-obese healthy subjects aged 18 to 95 years were investigated for age-dependence of GHRH-stimulated GH-secretion. In addition, priming of GH-secretion with three oral doses of propranolol (3 x 80 mg, the last dose 2 hours prior to the second GHRH-bolus) was carried out in 15 subjects below 40 years and 13 subjects older than 70 years. We found that mean maximal incremental GH-levels were inversely correlated with chronological age (r = -0.44, P = 0.001) of the probands. Propranolol premedication caused a significant rise of both basal and peak GHRH-induced relative increases in all subjects tested, whereas GHRH-induced relative increases of GH remained unchanged. In a well selected group of non-obese healthy subjects stimulated GH-secretion is found to undergo an aging process that is supposed to be of pituitary and suprapituitary origin. Priming GH-secretion with a beta-Blocker is possible both in young and very old healthy subjects and is likely to affect the basal GH secretory tone and not GHRH-stimulated GH-secretion.     

Comparison of enkephalin and atropine in the inhibition of vagally stimulated gastric and pancreatic secretion and gastrin and pancreatic polypeptide release in dogs

Enkephalins have been detected in vagal nerves and myenteric plexus neurons but no study has been performed to determine their action on vagally stimulated gastric and pancreatic secretion. In this study we infused IV methionine-enkephalin (Met-enk) alone, naloxone (a pure opiate antagonist) alone, or their combination before, during and after vagal stimulation in 4 dogs with esophageal, gastric and pancreatic fistulas. For the comparison, atropine was given before, during and after vagal stimulation in the same animals. Vagal stimulation was obtained by 15 min sham-feeding, which produced an increase in gastric H⁺ output to a peak of about 75% of the maximal response to pentagastrin and pancreatic protein secretion amounting to about 71% of the maximal response to caerulein. It was accompanied by a significant rise in serum gastrin and pancreatic polypeptide (PP) levels. Met-enk inhibited significantly both gastric H⁺ and pancreatic protein secretion and reduced plasma PP but not gastrin levels. Similar effects were obtained after the administration of atropine. The effects of Met-enk were partly reversed by the addition of naloxone. We conclude that (1) enkephalin suppresses vagally stimulated gastric and pancreatic secretion and plasma PP release; (2) these secretory effects of enkephalin seem to be mediated by opiate receptors and could be explained by its inhibitory action on acetylcholine release (“anticholinergic” action) in the stomach and the pancreas.     

The effect of oleate on pancreatic and bile secretion in the conscious rat

The effects of sodium oleate infused into either the duodenum or the terminal ileum on bile and pancreatic secretion were examined in the conscious rat. Rats were prepared with cannulae draining pure bile and pancreatic juice separately, and with an ileal and two duodenal cannulae. A 40 mM taurocholate solution containing 7 mg/ml bovine trypsin was infused into the duodenum throughout the experiment to replace diverted bile-pancreatic juice to maintain the normal regulation of pancreatic secretion. The intraduodenal infusion of sodium oleate significantly increased pancreatic juice flow, protein, and bicarbonate outputs, whereas it did not affect bile secretion. Intravenous infusion of proglumide (300 mg/kg/hr) did not inhibit pancreatic secretion stimulated by intraduodenal infusion of sodium oleate. An intravenous infusion of atropine (100 micrograms/kg/hr) attenuated protein and fluid secretions but not that of bicarbonate in response to intraduodenal oleate. In contrast, the intraileal infusion of oleate had no effect on pancreatic secretion, whereas it decreased bile flow, bicarbonate, and bile salt outputs. In conclusion, sodium oleate introduced in the duodenum stimulates pancreatic secretion but oleate in the terminal ileum inhibits bile secretion.     

The effect of hypophysectomy on pancreatic islet hormone and insulin-like growth factor I content and mRNA expression in rat

The growth arrest after hypophysectomy in rats is mainly due to growth hormone (GH) deficiency because replacement of GH or insulin-like growth factor (IGF) I, the mediator of GH action, leads to resumption of growth despite the lack of other pituitary hormones. Hypophysectomized (hypox) rats have, therefore, often been used to study metabolic consequences of GH deficiency and its effects on tissues concerned with growth. The present study was undertaken to assess the effects of hypophysectomy on the serum and pancreatic levels of the three major islet hormones insulin, glucagon, and somatostatin, as well as on IGF-I. Immunohistochemistry (IHC), in situ hybridization (ISH), radioimmunoassays (RIA), and Northern blot analysis were used to localize and quantify the hormones in the pancreas at the peptide and mRNA levels. IHC showed slightly decreased insulin levels in the cells of hypox compared with normal, age-matched rats whereas glucagon in cells and somatostatin in cells showed increase. IGF-I, which localized to cells, showed decrease. ISH detected a slightly higher expression of insulin mRNA and markedly stronger signals for glucagon and somatostatin mRNA in the islets of hypox rats. Serum glucose concentrations did not differ between the two groups although serum insulin and C-peptide were lower and serum glucagon was higher in the hypox animals. These changes were accompanied by a more than tenfold drop in serum IGF-I. The pancreatic insulin content per gram of tissue was not significantly different in hypox and normal rats. Pancreatic glucagon and somatostatin per gram of tissue were higher in the hypox animals. The pancreatic IGF-I content of hypox rats was significantly reduced. Northern blot analysis gave a 2.6-, 4.5-, and 2.2-fold increase in pancreatic insulin, glucagon, and somatostatin mRNA levels, respectively, in hypox rats, and a 2.3-fold decrease in IGF-I mRNA levels. Our results show that the fall of serum IGF-I after hypophysectomy is accompanied by a decrease in pancreatic IGF-I peptide and mRNA but by partly discordant changes in the serum concentrations of insulin and glucagon and the islet peptide and/or mRNA content of the three major islet hormones. It appears that GH deficiency resulting in a low IGF-I state affects translational efficiency of these hormones as well as their secretory responses. The maintenance of normoglycemia in the presence of reduced insulin and elevated glucagon serum levels, both of which would be expected to raise blood glucose, may result mainly from the enhanced insulin sensitivity, possibly due to GH deficiency and the subsequent decrease in IGF-I production.     

Inhibitory effect of pancreastatin on pancreatic exocrine secretion in the conscious rat

The effect of newly discovered pancreastatin on pancreatic secretion stimulated by a diversion of bile-pancreatic juice (BPJ) from the intestine was examined in the conscious rat. Exogenous pancreastatin infusion (20, 100 and 200 pmol/kg.h) inhibited pancreatic protein and fluid outputs during BPJ diversion in a dose-dependent manner. Pancreastatin did not affect plasma cholecystokinin (CCK) concentrations. Pancreastatin (100 pmol/kg.h) inhibited CCK-stimulated pancreatic secretion, but did not inhibit secretin-stimulated pancreatic secretion. Pancreastatin alone, however, did not affect basal pancreatic secretion. In contrast, pancreastatin (10(-10)-10(-7)M) did not suppress CCK-stimulated amylase release from isolated rat pancreatic acini. These results indicate that pancreastatin has an inhibitory action on exocrine function of the pancreas. This action may not be mediated by direct mechanisms and nor via an inhibition of CCK release. It is suggested that pancreastatin may play a role in the regulation of the intestinal phase of exocrine pancreatic 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.     

Influence of conjugated and conventional linoleic acid on tumor growth and lipid peroxidation in pancreatic adenocarcinoma in hamster

Conventional linoleic acid (LA) is regarded as a promotor of carcinogenesis. However, the effect of its conjugated derivative on cancer is still unknown. Therefore we investigated the influence of conventional and conjugated LA on tumor growth and lipid peroxidation in a solid model of pancreatic adenocarcinoma in Syrian hamsters. 60 male hamsters were randomized in 4 groups (Gr.) (n=15). Gr. 1 and 2 received 0.5 ml 0.9% sodium chloride subcutaneously (s.c.) once a week while Gr. 3 and 4 were injected 10 mg N-nitrosobis-2-oxopropylamine (BOP)/kg body weight weekly for 12 weeks to induce pancreatic cancer. Gr. 1 and 3 received a diet containing conventional LA, Gr. 2 and 4 were fed a diet of conjugated LA. After 29 weeks all animals were sacrificed, pancreas was weighed and examined macroscopically and histologically. The level of lipid peroxidation and activities of glutathion peroxidase and superoxide dismutase were determined in tumor-free as well as in pancreatic carcinoma tissue. Different diets did not influence the incidence of pancreatic carcinoma, however, pancreas weight was increased by conjugated LA compared to conventional LA. Furthermore both diets decreased the activity of glutathion peroxidase and increased the level of lipid peroxidation in pancreatic intratumoral tissue. The content of conjugated LA in dietary did not influence pancreatic tumor growth in a solid model of pancreatic adenocarcinoma in Syrian hamsters.     

Effect of lorglumide (CR-1409) on pancreatic secretory and trophic response to caerulein in newborn rats.

The authors investigated whether lorglumide a specific CCK-receptor antagonist affects the pancreatic actions of caerulein in female newborn Wistar rats. Pancreatic secretory response (expressed as the decrease in specific trypsin activity in the pancreas) was studied in 11-day-old rats following acute administration of saline (control), caerulein (0.3, 1, or 3 micrograms/kg s.c.) either without or with lorglumide (10 mg/kg s.c.). Lorglumide was given 15 min before caerulein. In chronic studies rats were treated 3x/day for 10 days from the day of birth (Day 1) with caerulein and lorglumide as above. On Day 11 the rats were decapitated and exsanguinated, their pancreas removed and analyzed. Acute administration of caerulein induced a dose-dependent depletion of specific trypsin activity from the pancreas and this was antagonized by lorglumide. Chronic treatment with each dose of the peptide increased total pancreatic trypsin content. Besides, the 3 micrograms/kg dose caused to increase pancreatic protein, DNA, and amylase content and to increase plasma corticosterone level. Chronic administration of lorglumide did not influence normal pancreatic growth, while it strongly inhibited the increase in trypsin content evoked by caerulein. However, lorglumide, given alone or in combination with caerulein, induced a significant increase in pancreatic amylase content without affecting plasma corticosterone level.