Florence Reyl-Desmars

Evidence for an intracellular somatostatin receptor in pancreas: A comparative study with reference to gastric mucosa

Abstract Specific binding sites for 125 I-Tyr 1 somatostatin-14 were comparatively demonstrated in isolated rat pancreatic and gastric parietal cells. In both materials, the sites occurred mostly in cytosol, with apparent affinities of 1×10 −10 M and 3×10 −11 M, respectively, in pancreatic and gastric cells. Somatostatin-14 stimulated cytosolic phosphoprotein phosphatases (PPPases) in pancreas as well as in gastric mucosa with concentrations for half maximal effect consistent with binding affinities. Somatostatin 28 mimicked somatostatin 14 stimulation with a higher efficacy but an equivalent potency. Secretin and cholecystokinin C terminal octapeptide were ineffective. Furthermore, in intact isolated cells, somatostatinic stimulation of PPPases was blocked by 5×10 −4 M dinitrophenol. We therefore suggest that in pancreas as in gastric mucosa, somatostatins inhibitory effect on secretory functions could involve protein dephosphorylation mediated by an intracellular receptor.

Lipopolysaccharide-induced mitochondrial DNA depletion.

Hepatic energy depletion has been described in severe sepsis, and lipopolysaccharide (LPS) has been shown to cause mitochondrial DNA (mtDNA) damage. To clarify the mechanisms of LPS-induced mtDNA damage and mitochondrial alterations, we treated wild-type (WT) or transgenic manganese superoxide dismutase-overerexpressing (MnSOD(+++)) mice with a single dose of LPS (5 mg/kg). In WT mice, LPS increased mitochondrial reactive oxygen species formation, hepatic inducible nitric oxide synthase (NOS) mRNA and protein, tumor necrosis factor-alpha, interleukin-1 beta, and high-mobility group protein B1 concentrations. Six to 48 h after LPS administration (5 mg/kg), liver mtDNA levels, respiratory complex I activity, and adenosine triphosphate (ATP) contents were decreased. In addition, LPS increased interferon-β concentration and decreased mitochondrial transcription factor A (Tfam) mRNA, Tfam protein, and mtDNA-encoded mRNAs. Morphological studies showed mild hepatic inflammation. The LPS (5 mg/kg)-induced mtDNA depletion, complex I inactivation, ATP depletion, and alanine aminotransferase increase were prevented in MnSOD(+++) mice or in WT mice cotreated with 1400W (a NOS inhibitor), (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride, monohydrate (a superoxide scavenger) or uric acid (a peroxynitrite scavenger). The MnSOD overexpression delayed death in mice challenged by a higher, lethal dose of LPS (25 mg/kg). In conclusion, LPS administration damages mtDNA and alters mitochondrial function. The protective effects of MnSOD, NOS inhibitors, and superoxide or peroxynitrite scavengers point out a role of the superoxide anion reacting with NO to form mtDNA- and protein-damaging peroxynitrite. In addition to the acute damage caused by reactive species, decreased levels of mitochondrial transcripts contribute to mitochondrial dysfunction.

MESSENGER RNA EXPRESSION OF SOMATOSTATIN RECEPTOR SUBTYPES IN HUMAN AND RAT GASTRIC MUCOSAE

In several tissues including gastric mucosa, somatostatin displays various biological effects. Five seven-transmembrane-domain somatostatin receptor subtypes (SSTR1-5) have been recently cloned and only SSTR1 has been shown to be present in the human stomach. We used the polymerase chain reaction on reverse transcripts (RT-PCR) to characterize further the SSTRs mRNAs in human and rat gastric mucosae and in the human gastric tumoral cell-line HGTL. The SSTR1-5s mRNAs were found in both human fundic and antral mucosae as well as in the HGT1 cell and rat antrum. The four SSTR2-5s mRNAs but not SSTR1s were detected in the rat fundic mucosa. Furthermore, the use of rat isolated and purified fundic mucosal cells allowed us to localize SSTR2-5 in the parietal cell-enriched fraction, whereas SSTR2 and SSTR5 were the only subtypes found in the endocrine cell-enriched fraction. These results are the first to demonstrate the presence of five SSTRs mRNA subtypes in the stomach.

Doxorubicin and octreotide induce a 40 kDa breakdown product of p53 in human hepatoma and tumoral colon cell lines

The chemotherapeutic drug doxorubicin and the anti-proliferative long-acting somatostatin analogue octreotide, both used in cancer treatment, have been shown to increase the expression of the p53 tumour suppressor protein. In the present study, we demonstrate by Western-blot analysis that, in addition to the p53 protein, these molecules were able to induce the expression of a shorter protein with an apparent molecular mass of 40 kDa (p40), recognized by antibodies raised against the N-terminus of p53. This induction was present in tumoral and non-tumoral cells and did not depend on the status of the endogenous p53 protein. The p40 protein was significantly induced after 3 h of cell treatment with doxorubicin or octreotide, remained stable until 24 h and was located in the nuclear extract. Using reverse primers corresponding to each exon of the p53 gene, only one transcript was amplified by reverse transcriptase-PCR. This suggested that p40 was issued from a post-translational modification and not from an alternative splicing. This protein was not recognized by the PAb421 antibody, suggesting that it was issued from a cleavage of the p53 C-terminal region (p40deltaC). Furthermore, this cleavage was not dependent on caspase activity. In conclusion, these results support the hypothesis that this post-translational modification plays a significant role in the regulation of multiple p53 signalling pathways. These results also suggest that octreotide, a molecule with different signalling pathways, was able as doxorubicin to generate a p53 breakdown product.

A somatostatin receptor negatively coupled to adenylate cyclase in the human gastric cell line HGT-1

Somatostatin receptors are demonstrated in the human derived gastric cell line HGT-1. Using 125I-Tyr11-somatostatin as ligand, two classes of sites were characterized with apparent dissociation constants KD1 = 0.9 X 10(-10) M and KD2 = 4 X 10(-9) M and maximum binding capacities of N1 = 20 and N2 = 556 fmol per mg protein, respectively. These values are close to those previously reported in freshly isolated parietal cells (Reyl, F., Silve, C. and Lewin, M.J.M., Somatostatin receptors on isolated gastric cells. In S. Bonfils et al. (Eds.), Hormone Receptors in Digestion and Nutrition, Elsevier/North-Holland, Amsterdam, 1979, pp. 391-400). Somatostatin binding to the high affinity sites was partially inhibited by the non-hydrolysable guanyl nucleotide analog Gpp(NH)p and by pretreating the cells with islet activating protein (IAP). Furthermore, IAP counteracted the inhibitory effect of somatostatin on histamine stimulation of adenylate cyclase. These findings are interpreted in terms of somatostatin interaction with the 41,000 Da adenylate cyclase GTP-dependent inhibitory subunit, Ni.

Octreotide (SMS 201–995) inhibits the growth of colon peritoneal carcinomatosis in BDIX rats

In order to assess the effect of octreotide, a somatostatin analogue, on the growth of colon peritoneal carcinomatosis, 20 BDIX rats were injected i.p. with 1 x 10(6) colon cancer cells (DHD/K12 tumor cell line) and received octreotide, 65 micrograms/kg s.c. every 12 h (n = 10) or saline (n = 10) for 42 days, starting 3 days after tumor cell injection. Animals were killed at the end of the treatment. The mean volume of ascites was lower in the octreotide group (33.7 +/- 7.6 ml), than in the control group (67.5 +/- 16.3 ml; P < 0.05). The extent of peritoneal carcinomatosis (in five classes according to a previously published classification) was lower in the octreotide group (P < 0.05). Cell proliferation, using the BrdU technique, was markedly inhibited by octreotide (labeling index of tumor cells: 17.0 +/- 0.6% vs. 26.3 +/- 2.2% in controls, P < 0.001). No significant decrease in labeling index was observed in normal colonic mucosa. Two subtypes of somatostatin receptors were found in all tumors, using the 30F3 monoclonal antireceptor antibody. KD and Bmax values were not significantly different in the octreotide and control groups: high affinity, low capacity receptors (KD = 1.4 x 10(-10) M and 0.7 x 10(-10) M, respectively; Bmax = 3.8 and 2.9 pmol/mg protein, respectively); low affinity, high capacity receptors (KD = 1 +/- 0.2 x 10(-9) M and 5.5 +/- 0.05 x 10(-10) M, respectively; Bmax = 27.8 +/- 0.1 and 22.8 +/- 0.05 pmol/mg protein, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Somatostatin inhibition of phosphoinositides turnover in isolated rat acinar pancreatic cells: interaction with bombesin

The effects of somatostatin-14 and bombesin on [3H]inositol phosphate accumulation were studied in 24 h myo-[3H]inositol-prelabeled cultured rat acinar cells. Bombesin, 10 nM, stimulated basal formation of phosphatidyl monophosphate (InsP1), phosphatidyl 4,5-biphosphate (InsP2) and inositol 1,4,5-triphosphate (InsP3) by 128 +/- 5.2%, 147 +/- 10% and 155 +/- 5%, respectively. At 5 s, the ED50 value for InsP3 stimulation was 0.70 +/- 0.2 nM. This stimulation was partly blocked (64 +/- 0.04% inhibition) by 10 ng/ml Bordetella pertussis toxin. In contrast to bombesin, somatostatin, 10 nM, inhibited basal InsP1, InsP2 and InsP3 formation. At 5 s, the inhibition degree for InsP3 was 18 +/- 2.5% and the IC50s values 1 +/- 0.09 nM, 1 +/- 0.12 nM and 0.07 +/- 0.005 nM for InsP1, InsP2 and InsP3, respectively. Bombesin-stimulated InsP3 formation was also inhibited by somatostatin. At 5 s, the inhibition degree was 85 +/- 3.5% at 10 nM and the IC50 value, 0.10 +/- 0.05 nM. Furthermore, somatostatin inhibition of bombesin stimulation was partly blocked (66 +/- 4% inhibition) by Bordetella pertussis toxin. These data therefore suggest that the acinar pancreatic cells contain a somatostatin receptor exerting a negative control on basal and bombesin receptor-stimulated phosphatidyl inositol turnover.

Effects of PGE2, misoprostol, and enprostil on guinea pig enterocyte adenylate cyclase : clinical implications

Prostaglandins of the E series (PGE) are known to stimulate intestinal water and electrolyte secretions via the activation of the enterocyte adenylate cyclase. Their methylated synthetic analogs misoprostol and enprostil induced diarrhea in 5–13% of the patients in most clinical studies. In order to elucidate the role of PGE-adenylate cyclase interaction in these phenomena, we studied the stimulation of adenylate cyclase by native prostaglandin E2 (PGE2) and synthetic PGE analogs on isolated guinea pig intestinal epithelial cells. PGE2 stimulation of adenylate cyclase was dose-dependent, reaching a maximum for 3×10−4 M, with an EC50 of 3.7×10−6 M. The Hill analysis of the concentration-response curve gave a straight line, with a slope close to 1. The effect of PGE2 was strictly additive to that of 10−5 M forskolin, whereas it was decreased in terms of potency by 10−9 M cholera toxin. Somatostatin-14 markedly inhibited PGE2 stimulation by 37% and 45% with 10−9 M and 10−6 M, respectively. The two PGE methylated analogs misoprostol and enprostil were less potent than PGE2 in stimulating adenylate cyclase in our model. We conclude that (1) the mechanism of PGE2-mediated intestinal water and electrolyte secretions involves the activation of one single class of specific receptors coupled to enterocyte adenylate cyclase via a cholera toxin-sensitive regulatory subunit (Gs); (2) somatostatin is a potent inhibitor of PGE-stimulated adenylate cyclase and thus could be a interest in the treatment of certain PGE-induced secretory diarrheas; and (3) the diarrheogenic effects of misoprostol and enprostil are unlikely to be mediated primarily by adenylate cyclase activation.

Bombesin activation of phospholipase C β 1 in rat acinar pancreatic cells involves the pertussis toxin-sensitive Gαi3 protein

Abstract Bombesin stimulation of inositol 1,4,5-trisphosphate (Ins P 3 ) formation in rat sonicated pancreatic acinar cells was inhibited by an antibody directed against the pertussis toxin (PTX)-sensitive GTP-binding G αi3 protein but not by an anti-G αq-11 antibody. After solubilization and gel filtration, [ 125 I-Tyr 4 ]bombesin binding sites were recovered in a peak of protein of 67 ∼ 90 kDa with a maximal enrichment corresponding to a molecular mass of 83-kDa. Results obtained from the non-hydrolysable GTP analog guanosine-5′-[γ-thio]triphosphate (GTPγS) binding, PTX-stimulated ADP-ribosylation and immunoblotting showed that the 83-kDa fraction contained the G αi3 protein but not the G αq-11 protein. Furthermore, GTPγS increased the bombesin binding dissociation constant ( K D ) from 0.32 to 0.60 nM, while the anti-G αi3 antibody decreased the maximal binding capacity ( B max ) from 50 to 25 fmol/mg protein without affecting the K D . Mixing solubilized bombesin binding sites with a phospholipase C (PLC) preparation from rat pancreas reconstituted a bombesin-stimulated PLC activity which was markedly inhibited by the anti-G αi3 antibody but unaffected by the anti-G αq-11 antibody. In addition, this stimulation was inhibited by an anti-PLC β 1 antibody. This result supports the involvement of the PLC β 1 isoform in bombesin receptor activation.

Somatocrinin stimulates adenylate cyclase-Ns regulatory subunit in a GH3 cell-line: Comparison with VIP

We used a GH3 cell-line to compare the effects of rat GRF (rGRF) and VIP on the adenylate cyclase activity and to determine on what subunit the site of action of these two peptides is. In the GH3 cell-line, VIP was more potent than rGRF to stimulate adenylate cyclase activity. The stimulatory effects of rGRF and forskolin were additive. Cholera toxin decreased the apparent potency of these peptides and pertussis toxin reversed the inhibition by somatostatin of their adenylate cyclase stimulation. We conclude that rGRF acts on the regulatory subunit Ns, different from the regulatory subunit Ni on which somatostatin is suggested to be acting and that, in the GH3 cells, rGRF stimulates adenylate cyclase through VIP-preferring sites.