Jean Morisset

MEK/ERK signaling pathway regulates the expression of Bcl-2, Bcl-XL, and Mcl-1 and promotes survival of human pancreatic cancer cells

Background and aims: Growth factors are well known for their participation in the regulation of cell proliferation and survival. However, the intracellular signaling pathways by which growth factors promote survival are still poorly understood. In the present study, using the MIA PaCa‐2 cell line, a well‐established model of pancreatic cancer cells, we analyzed the roles of ERK1/2 activities in the regulation of cell survival and investigated some of the mechanisms involved. Methods: The ability of the MEK inhibitor PD98059 to modulate survival of the MIA PaCa‐2 cells was evaluated, and the responses were correlated with expression of Bcl‐2 homologs and caspases 1, 3, 6, 8, and 9 activities. Results. Herein, we showed that inhibition of ERK1/2 activities caused (1) a G1 arrest; (2) a down‐regulation of the expression levels of the anti‐apoptotic homologs Bcl‐2, Mcl‐1, and Bcl‐XL without affecting the pro‐apoptotic levels of Bax and Bak; (3) a promotion of caspases 3, 6, 8, and 9 activities; (4) a stimulation of PARP cleavage; and (5) a programmed cell death by apoptosis. Conclusion: Our data suggest that activation of the ERK pathway functions to protect pancreatic tumor cells from apoptosis as well as to regulate their progression in the cell cycle. J. Cell. Biochem. 79:355–369, 2000.

Somatostatin: a potential antigrowth factor for the exocrine pancreas

The increases in DNA synthesis and total DNA content after caerulein treatment support the trophic effect of this CCK analog on the pancreas. Over a 15 day caerulein treatment, pancreatic growth plateaued after 5 days and somatostatin is believed to be responsible for this phenomenon. The present study was undertaken to test this possibility. Rats were treated for 2 or 4 days with caerulein (1 microgram X kg-1), somatostatin antiserum plus caerulein or caerulein plus somatostatin (600 micrograms X kg-1). Caerulein increased all parameters studied after 2 and 4 days; pancreatic hyperplasia was established after 2 days. The somatostatin antiserum significantly enhanced the effect of caerulein, especially on DNA synthesis and contents after 2 and 4 days. The trophic effect of caerulein was significantly reduced by somatostatin dramatically so with respect to hyperplasia. The effects of the somatostatin antiserum and those of somatostatin on stimulated pancreatic growth support the hypothesis that somatostatin may be considered an endogenous growth inhibitory factor for the pancreas.

Localization of Cholecystokinin Receptor Subtypes in the Endocine Pancreas

This study was undertaken to clarify the controversy in the literature about pancreatic localization of the cholecystokinin (CCK) CCKA and CCKB receptors. With antibodies used by other investigators, we first established their specificity by Western blotting, indirect immunofluorescence, and confocal microscopy with each antibodys peptide antigen. Co-localization assays between the CCK receptors and the pancreatic hormones insulin, glucagon, and somatostatin revealed that the CCKA RAbs 1122 and R1-2 recognized insulin and glucagon cells in rat, pig, and human pancreas but not in the somatostatin cells. Conversely, the three CCKB RAbs tested, 9262, 9491, and GR4, identified the somatostatin cells. Abs 9491 and GR4 occasionally co-localized with glucagon, a feature that never occurred with Ab 9262. Finally, the specificity of Ab 9262 for the pancreatic CCKB R was confirmed in six different species. It co-localized with somatostatin but never with glucagon in these species. Our data suggest the use of Abs 1122 and 9262 to specifically identify and localize pancreatic CCKA and CCKB receptors, respectively. Confusion in the literature may result from the lack of specificity of most antibodies used, as established in this study.

Effects of chronic administration of somatostatin on rat exocrine pancreas

We studied the effects of somatostatin on synthesis of pancreatic DNA, RNA and protein and on pancreatic weight and contents of DNA, protein, amylase and chymotrypsinogen in rats. In short term synthesis studies, rats were injected with 100 micrograms . kg-1 somatostatin or 0.15 M NaCl (control) at times 0, 8 and 16 h. Eight rats from each treatment group were killed 2, 4, 8, 12, 16, 20 and 24 h after beginning treatment. Incorporation rates in vivo of [3H]thymidine into DNA, [3H]uridine into RNA and [14C]phenylalanine into total protein were significantly depressed by somatostatin. In long term studies, four groups of 12 rats were injected every 8 h for 5 days with 0.15 M NaCl or 11, 33 or 100 micrograms . kg-1 somatostatin. Body weight was unaffected but pancreatic contents of DNA, protein and enzymes were significantly decreased by somatostatin. Administration of somatostatin inhibits DNA, RNA and protein synthesis in exocrine pancreas with resulting decreases in DNA and enzyme contents.

Influence of age and sex on basal secretion of growth hormone (GH) and on GH-induced release by porcine GH-releasing factor pGRF(1-29NH2) in growing pigs.

The aim of this study was to determine the effect of age and sex on basal secretory patterns of growth hormone (GH) and growth hormone-releasing factor (GRF) induced GH release. Eighteen pigs (9 castrated males and 9 females) were stimulated with pGRF(1-29)NH2 at 7,11,15,19 and 23 weeks of age. Blood samples were taken from each animal via jugular vein cannulae every 20 min, from 6 hr before to 5 hr after iv GRF administration at a dose of 4 micrograms/kg. GH baseline levels, amplitude of the GH peaks, area under the GH peaks and the overall mean of GH serum levels decreased (P less than .001) with age in both sexes. Age also had a marked effect on GRF-induced GH release: the amplitude of GH peaks and area under the GH peaks decreased (P less than .001) with age. The GH response to pGRF(1-29)NH2 varied considerably, depending on the timing of the episodic endogenous secretion of GH. An immediate response (less than 30 min) was observed when GRF was injected at the end of a trough period or at the beginning of a peak, but there was no immediate response when GRF was injected at the end of a peak or at the beginning of a trough period. Our results show that both endogenous GH secretion and pGRF(1-29)NH2-induced GH release declines with age, suggesting a decreased sensitivity of the somatotroph cells to GRF with age; and that the high variability of the GH response to pGRF(1-29)NH2 stimulation depends greatly on the timing of the episodic endogenous GH release, thus implying a possible episodic endogenous somatostatin secretion by the hypothalamus.

Distribution of Muscarinic Receptors in the Digestive Tract Organs

We have studied the binding of the potent muscarinic antagonist, (3H)-quinuclidinyl benzilate [(3H)-QNB], in order to characterize muscarinic receptors of the digestive tract organs and evaluate their population. In each tissue examined, the binding sites were saturable and stereoselective; (3H)-QNB could also be displaced by known muscarinic agonists and antagonists. Along the digestive tract, the stomach (for stomach, fundus, antrum) and the colon possess the highest number of binding sites while the secretory (parotid, mandibular and pancreas), with a comparable amount of sites, are among those with less receptors. This study presents for the first time a complete distribution of the muscarinic receptors in organs of the digestive system evaluated under identical experimental conditions.

Muscarinic receptor of rat pancreatic acini: [3H]QNB binding and amylase secretion

Binding studies were performed on rat pancreatic dispersed acini with (--)-[3H]QNB, in parallel with secretory studies to establish that the muscarinic receptors characterized are involved in enzyme secretion. Scatchard analysis of [3H]QNB saturation isotherm gave an apparent KD value of 3.09 X 10(-10) M with binding capacities of 2605 fmol/mg of DNA. The true KD calculated from the ratio of the kinetic parameters was 64 pM. The receptor population can be divided into two classes for the agonist binding sites: a population of high affinity sites and one of low affinity sites. With carbamylcholine, maximal stimulation of amylase release occurred with 40% receptor occupation while occupation of the remaining 60% caused a progressive decrease in stimulation of amylase release. Moreover, the dissociation constant of the high affinity sites for carbamylcholine (1.78 X 10(-6) M) is very close to the ED50 of carbamylcholine (5.81 X 10(-7) M) found for the stimulation of enzyme secretion. These data suggest that the physiological response of the pancreatic acini to a muscarinic agonist would involve the high affinity site population.

Negative control of human pancreatic secretion: physiological mechanisms and factors.

The negative control of pancreatic exocrine secretion in man occurs during the interdigestive and postprandial periods of the digestive cycle. The physiological mechanisms involved include negative feedback mechanisms, well described and accepted in animals, and controlled by the cholecystokinin- and secretin-releasing factors of pancreatic and duodenal origin, along with the active pancreatic proteases present in the upper gut. The presence of these factors and their efficacy in humans, however, have their supporters and detractors, with a possibility for reconciliation among opponents. Besides these releasing factors, hormones, mostly from the intestine, are also involved in this inhibitory process of pancreatic secretion. Somatostatin, peptide YY, pancreatic polypeptide, glucagon, ghrelin, and leptin were described as potentially involved from studies mostly performed on animals. Finally, bile and bile salts have mixed responses on this inhibition, and their effects seem to be at the intestine level with gastrointestinal hormones involved. Future studies will have to be performed in humans to determine the presence of cholecystokinin- and secretin-releasing factors and their role. Finally, the demonstrated modulatory action of hormones and bile acids in other species needs to be confirmed in humans.

Endogenous cholecystokinin, the major factor responsible for dietary protein-induced pancreatic growth.

This study was undertaken to clarify the role of endogenous cholecystokinin (CCK) in induction of pancreatic growth stimulated by a high protein diet. Rats with i.v. jugular cannulae in place and kept in Bollman cages were adapted to 5% casein diet for 9 days and switched to 70% casein for 2 days. MK-329, a CCK receptor antagonist, and SMS 201–995, a somatostatin agonist, were continuously infused at 0.5 mg/kg/h and 5 pg/kg/h, respectively, starting at the onset of feeding 70% casein. The 5 and 70% casein control groups were infused with saline. Feeding 70% casein significantly stimulated pancreatic hyperplasia and tissue hypertrophy. MK-329 and SMS 201–995 totally prevented 70% casein-induced increases in pancreatic weight and total RNA and DNA contents. The results indicate that endogenous CCK is the major factor responsible for pancreatic growth induced by a high protein diet.

Folyamines and pancreatic growth induced by caerulein

Activation of polyamine metabolism may be important to initiation of pancreatic cell growth. We are reporting that such activation did occur during pancreatic growth initiation by caerulein, a cholecystokinin analog. Maximal increases in total putrescine (319%), spermidine (63%) and spermine (50%) were observed 12, 96 and 96 hr respectively after the beginning of the caerulein treatment. This time period coincides with pancreatic hypertrophy and hyperplasia as characterized by increased cell mass and DNA content. Rates of pancreatic weight and DNA content increases were significantly correlated with total spermidine and spermine contents. These data suggest that polyamine biosynthesis is closely associated with pancreatic growth.