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The Somatostatin receptor subtypes SSTR2 and SSTR5 mediate the inhibition of cell proliferation
April 1995
Hormones and Receptors
• I g X ~ L I L % T I ~ OF II~-E~DROXISTEROID D l ~ a x D R O m n l ~ IM Tile HUKMI ~ . S.Boschi, A.Munarini# P.Mesini*, n.De lasio* and R. De Giorqlo'. Dept. of Olin. Pharmacology, *Central Laboratory, "Inst. of Internal Medicine and Gastroenterology. St.Orsola Hospital, University of Bologna, Bologna, Italy
LOCALIZATION AND PROPERTIES OF NITRIC OXIDE (NO) SYNTHASE IN RAT GASTRIC MUCOSA. J. F. Brown, K. J. Price, J. M. Williams, P. J. Hanson and B. J. R Whittle. Aston University, Aston Triangle, Birmingham and Wellcome Research Laboratories, Beckenham, Kent U.K.
The enzyme llB-hydroxysteroid dehydrogenase 111B-OHSD ) converts, in human, the glucocorticoid cortinol (F} to the correspondent inactive ll-keto-analog cortisone (El and it has been suggested to play a pivotal role for the tissue sensitivity to aldonterone (A}. The specificity of mineralocorticoid receptors (MR), which in vitro exhibit similar affinity for A and F, is dependent upon i n a c t i v a t i o n of glucocorticoid (whose plasma concentration is 100-1000 fold higher thaJl A l by IIB-OHSD, thus enabling A to bind MR. Current evidence indicates that the rat colonic epithelium in A sensitive and possesses MR. A i m of this study was to identify the layer specific localization of IIB-OBSD in the human colon. Surgical specimens of human distal colon were collected fresh and the mucosa and nube~cosa separated from muscle layer. Mucosal and muscle samples were incubated for 2 hours in Krebs-Ringer (pH 7.4) with RADP 10.5 raM), cold F and 3H F (1.4 ~M l . TiBsue samples were extracted with CH2CI 2 and injected into HPLC. Fractions corresponding to F and E were collected and counted. Parallel incubation with the inhibitor B-glycyrrhetinic a c i d IBGA, 10 u M l w e r e p e r f o r m e d . B l a n k samples w e r e run in absence of tissue. Enzyme activities I±SD) were expressed in pmol/h/mg protein. Very high IlB-OHSD activity wan found in fresh colonic mucosa |125.2±20.2; n=4). Thi~ activity was inhibited by ~3A (70±2%}. IIB-OHSD activity wan strongly reduced after freezing of tissue (69.8±7.8; n=4}. By contrast, muscle samples showed low IIB-OHSD activity (11.6±3.33; n=6}, which was inhibited by BGA in a similar fashion. The time course of IIB-OHSD enzymatic activity showed a linear increase lranging from 25.1 to 242.5) between 15 and 160 rain.. Since IIB-OHSD is a bidirectional enzyme, also working as llB-reductase, we investigated this activity. Results showed that the back-conversion of E to F is negligible. Our data demonstrate that IIB-OHSD is predominantly located in the muconal layer of human colon. These findings support the hypothesis of a further site of action for steroids in controlling water and sodium handling.
Ca2+-dependent NO synthase was present in centrifugal elutriator fractions which contained gastric cells of medium size, and which were enriched with mucous cells (BBRC 184, 680, 1992). The present aims were to establish the precise location of NO synthase in the gastric mucosa and to investigate its properties. Localization was investigated by immunohistochemistry (ABC peroxidase method) using a polyclonal antibody raised to the unique N-terminal hexadecapeptide sequence of the rat neuronal NO synthase, and which recognised NO synthase in myenteric plexi. Immunoreactive material was present in surface cells of the corpus epithelium, passed a short way down into the pits, but was absent from the remainder of the mucosa including the mucous neck cells and blood vessels. Staining was abolished by preincubation of the antibody with the peptide against which it was raised. Immunoreactivity to an antibody directed against endothelial NO synthase was present in blood vessels at the base of the mucosa. Soluble NO synthase activity in homogenates of gastric mucosal cells was assayed by the conversion of arginine to citrulline. Activity was inhibited by trifluoperazine (ICs0140/.~M), N~-nitro-L'arginine (ICs0 0.8/~M) and L-canavanine (ICs0 147 FM). The inhibition curves obtained using N°-nitro-L-arginine and L-canavanine were superimposable on those obtained with rat brain enzyme. Preincubation of gastric mucosal cells with the protein phosphatase inhibitor okadaic aci d (1 p.M), or the presence of ATP (5 raM), but not/3y-methylene ATP (5 mM), during homogenization by freezethawing both inhibited NO synthase activity by 47%. Thus, an isoform of NO synthase which resembles the neuronal enzyme in its immunoreactivity, is localized to the surface of the epithelium and is absent from mucous neck cells. Soluble gastric mucosal NO synthase is inhibited by trifluoperazine, N °nitro-L-arginine, L-canavanine and protein phosphorylation.
• THE SOMATOSTATIN RECEPTOR SUBTYPES SSTR2 AND SSTR5 MEDIATE THE INHIBITION OF CELL PROLIFERATION. L. Bnscail, J.-P. Est~ve, N. Saint-Laurent, V, Bertrand, E. Chastre*, J.C. Vaillant*, C. Gespach*, T Reisine§, A-M. O'Carroll ¥ , H. Kaltho~, G.I. Bell ~[§, A.V Schally Ta~, N. Vaysse, C. Susini. INSERM U151, CHU Rangueil, Toulouse, France; * INSERM U55, Paris, France.§ School of Medicine, Philadelphia, PA, ¥ NIMH~ Bethesda, MD, ~[Univ. of Kiel, Germany, ~§ HHMI, Chicago, IL; tlY Tulane Univ., New Orleans, LA. The biological effects of somatostatin are mediated by specific receptors which five subtypes have been cloned (SSTR1 to 5). We investigated in this study the implication of the 5 subtypes in the regulation of cell proliferation We evaluated the effects of the stable analogue RC-160 on cell proliferation of CHO cells stably transfected with SSTR1 to 5 subtypes. RC160 inhibited serum-induced proliferation of CHO cells expressing SSTR2 and SSTR5 (ECs0:53 and 150 pM respectively), but had no effect on growth of cells expressing SSTR1, 3 and 4. In SSTR2-expressing ceils, orthovanadate suppressed the growth inhibitory effect of RC-160. Only in this cellular clone, the analogue inhibited insulin-induced proliferation and caused a rapid stimulation of a tyrosine phosphatase activity at doses (EC50: 4.6 pM) similar to those required to inhibit both binding (IC50:170 pM) and growth, implicating tyrosine phosphastase as a transducer of growth inhibitory signal in SSTR2-expressing cells. In SSTR5-expressing cells, RC-160 inhibited CCK-stimulated intracellular calcium concentration at doses (ECs0:0.3 nM), similar to those necessary to inhibit both binding (ICs0:21 nM) and CCK-induced cell proliferation (ECs0:1 nM). This suggests that the phosphoinositides/calcium pathway could be implicated in the antiproliferative effect of the analogue in SSTR5-expressing cells. We secondly investigated the mRNA expression of SSTR1 to 5 in human digestive tract tumors using the RT-PCR method: Tumors (n) SSTR1 SSTR2 SSTR3 SSTR4 SSTR5 Stomach (2) + + + Colon adenoma (2) + + + Colon cancer (11) + (2) + (2) + (3) + (4) + (7) Exocrine pancreas (a) + (2) + (a) Endocrine pancreas (4) + (2) + (1) + (2) + (3) Liver metastasis (1) + We conclude that both SSTR2 and SSTR5 mediate the inhibition of cell growth induced by the sornatostatin analogue RC-160 by distinct mechanisms. These two receptors are expressed in most of GI tract tumors that could he promising for the therapeutic potential of the somatostatin analogue.
C-MET PROTO-ONCOGEN IN RAT PANCREAS. GENE EXPRESSION DURING ONTOGENY AND REGENERATION AFTER ACUTE PANCREATITIS INDUCED BY CAERULEIN. E. Calve, G. Pelletier, C. Boucher, J. Morisset. University of Sherbrooke and Research Station, Lennoxville, Quebec, Canada. The Hepatocyte Growth Factor (HGF) has several activities on epithelial cells, including mitoganesis, dissociation of epithelial sheets, stimulation of motility and promotion of matrix invasion. HGF is the ligand of a dimeric transmembrane tyrosine kinase receptor encoded by the Met proto-oncogane (c-Met). Earlier, we demonstrated that HGF mRNA was overexpressed during pancreas development and maturation as well as after pancreatic aggression but the expression of its receptor still remains unknown under these circumstances. The aim of this study was to characterize c-Met mRNA expression in rat pancreas during development and during regeneration after pancreatectomy or acute pancreatitis. Pancreas were excised from 19d fetus and at days 1, 3, 5, 7, 9, 11, 13, 17, 21, 25, 35, 45 and 90 after birth. For regeneration studies, adult male rats were sacrificed at 1, 2, 3 and 6 days following 90% panereatectomy (Ptx). Acute panereatitis (AP) was induced in adult by eaemlein 12 pgkg"t, 3 x day for 2 days. Total RNA was extracted with the guanidine thiocyanate method and Northern blots were performed using 20 p.g of total RNA and labelled 32p-c-Met cRNA probe: The abundance of transcripts were estimated by densitometric scanning, and a 18S ribosomal eDNA probe was used as a control to measure total RNA loading. Pancreatic e-Met mRNAs levels were higher between f19 and post-natal day 11, and decreased to a very low level at day 35 which was maintained until the adult age. After Ptx, the level of c-Met gene transcription increased from the first day post-ptx to reach its maximum level on the 2nd day, and a return to control values at day 6. In the AP model, c-Met mRNA levels were maximal during the first two days of induction and then immediately dropped to control values at 3 days. In conclusion ; c-Met pancreatic gane expression is age dependent with the highest expression before weaning. Overexpression of c-Met mRNA up to fetal levels was observed after Ptx. Finally, increases in c-Met mRNA were evident during acute pancreatitis induction. These data demonstrate for the first time that overexpression of c-Met may be involved in pancreatic development and maturation as well as during pancreas regeneration after tissue aggression. Funded by NSERC Canada and FCAR, Quebec.
A955
Hormones and Receptors
• I g X ~ L I L % T I ~ OF II~-E~DROXISTEROID D l ~ a x D R O m n l ~ IM Tile HUKMI ~ . S.Boschi, A.Munarini# P.Mesini*, n.De lasio* and R. De Giorqlo'. Dept. of Olin. Pharmacology, *Central Laboratory, "Inst. of Internal Medicine and Gastroenterology. St.Orsola Hospital, University of Bologna, Bologna, Italy
LOCALIZATION AND PROPERTIES OF NITRIC OXIDE (NO) SYNTHASE IN RAT GASTRIC MUCOSA. J. F. Brown, K. J. Price, J. M. Williams, P. J. Hanson and B. J. R Whittle. Aston University, Aston Triangle, Birmingham and Wellcome Research Laboratories, Beckenham, Kent U.K.
The enzyme llB-hydroxysteroid dehydrogenase 111B-OHSD ) converts, in human, the glucocorticoid cortinol (F} to the correspondent inactive ll-keto-analog cortisone (El and it has been suggested to play a pivotal role for the tissue sensitivity to aldonterone (A}. The specificity of mineralocorticoid receptors (MR), which in vitro exhibit similar affinity for A and F, is dependent upon i n a c t i v a t i o n of glucocorticoid (whose plasma concentration is 100-1000 fold higher thaJl A l by IIB-OHSD, thus enabling A to bind MR. Current evidence indicates that the rat colonic epithelium in A sensitive and possesses MR. A i m of this study was to identify the layer specific localization of IIB-OBSD in the human colon. Surgical specimens of human distal colon were collected fresh and the mucosa and nube~cosa separated from muscle layer. Mucosal and muscle samples were incubated for 2 hours in Krebs-Ringer (pH 7.4) with RADP 10.5 raM), cold F and 3H F (1.4 ~M l . TiBsue samples were extracted with CH2CI 2 and injected into HPLC. Fractions corresponding to F and E were collected and counted. Parallel incubation with the inhibitor B-glycyrrhetinic a c i d IBGA, 10 u M l w e r e p e r f o r m e d . B l a n k samples w e r e run in absence of tissue. Enzyme activities I±SD) were expressed in pmol/h/mg protein. Very high IlB-OHSD activity wan found in fresh colonic mucosa |125.2±20.2; n=4). Thi~ activity was inhibited by ~3A (70±2%}. IIB-OHSD activity wan strongly reduced after freezing of tissue (69.8±7.8; n=4}. By contrast, muscle samples showed low IIB-OHSD activity (11.6±3.33; n=6}, which was inhibited by BGA in a similar fashion. The time course of IIB-OHSD enzymatic activity showed a linear increase lranging from 25.1 to 242.5) between 15 and 160 rain.. Since IIB-OHSD is a bidirectional enzyme, also working as llB-reductase, we investigated this activity. Results showed that the back-conversion of E to F is negligible. Our data demonstrate that IIB-OHSD is predominantly located in the muconal layer of human colon. These findings support the hypothesis of a further site of action for steroids in controlling water and sodium handling.
Ca2+-dependent NO synthase was present in centrifugal elutriator fractions which contained gastric cells of medium size, and which were enriched with mucous cells (BBRC 184, 680, 1992). The present aims were to establish the precise location of NO synthase in the gastric mucosa and to investigate its properties. Localization was investigated by immunohistochemistry (ABC peroxidase method) using a polyclonal antibody raised to the unique N-terminal hexadecapeptide sequence of the rat neuronal NO synthase, and which recognised NO synthase in myenteric plexi. Immunoreactive material was present in surface cells of the corpus epithelium, passed a short way down into the pits, but was absent from the remainder of the mucosa including the mucous neck cells and blood vessels. Staining was abolished by preincubation of the antibody with the peptide against which it was raised. Immunoreactivity to an antibody directed against endothelial NO synthase was present in blood vessels at the base of the mucosa. Soluble NO synthase activity in homogenates of gastric mucosal cells was assayed by the conversion of arginine to citrulline. Activity was inhibited by trifluoperazine (ICs0140/.~M), N~-nitro-L'arginine (ICs0 0.8/~M) and L-canavanine (ICs0 147 FM). The inhibition curves obtained using N°-nitro-L-arginine and L-canavanine were superimposable on those obtained with rat brain enzyme. Preincubation of gastric mucosal cells with the protein phosphatase inhibitor okadaic aci d (1 p.M), or the presence of ATP (5 raM), but not/3y-methylene ATP (5 mM), during homogenization by freezethawing both inhibited NO synthase activity by 47%. Thus, an isoform of NO synthase which resembles the neuronal enzyme in its immunoreactivity, is localized to the surface of the epithelium and is absent from mucous neck cells. Soluble gastric mucosal NO synthase is inhibited by trifluoperazine, N °nitro-L-arginine, L-canavanine and protein phosphorylation.
• THE SOMATOSTATIN RECEPTOR SUBTYPES SSTR2 AND SSTR5 MEDIATE THE INHIBITION OF CELL PROLIFERATION. L. Bnscail, J.-P. Est~ve, N. Saint-Laurent, V, Bertrand, E. Chastre*, J.C. Vaillant*, C. Gespach*, T Reisine§, A-M. O'Carroll ¥ , H. Kaltho~, G.I. Bell ~[§, A.V Schally Ta~, N. Vaysse, C. Susini. INSERM U151, CHU Rangueil, Toulouse, France; * INSERM U55, Paris, France.§ School of Medicine, Philadelphia, PA, ¥ NIMH~ Bethesda, MD, ~[Univ. of Kiel, Germany, ~§ HHMI, Chicago, IL; tlY Tulane Univ., New Orleans, LA. The biological effects of somatostatin are mediated by specific receptors which five subtypes have been cloned (SSTR1 to 5). We investigated in this study the implication of the 5 subtypes in the regulation of cell proliferation We evaluated the effects of the stable analogue RC-160 on cell proliferation of CHO cells stably transfected with SSTR1 to 5 subtypes. RC160 inhibited serum-induced proliferation of CHO cells expressing SSTR2 and SSTR5 (ECs0:53 and 150 pM respectively), but had no effect on growth of cells expressing SSTR1, 3 and 4. In SSTR2-expressing ceils, orthovanadate suppressed the growth inhibitory effect of RC-160. Only in this cellular clone, the analogue inhibited insulin-induced proliferation and caused a rapid stimulation of a tyrosine phosphatase activity at doses (EC50: 4.6 pM) similar to those required to inhibit both binding (IC50:170 pM) and growth, implicating tyrosine phosphastase as a transducer of growth inhibitory signal in SSTR2-expressing cells. In SSTR5-expressing cells, RC-160 inhibited CCK-stimulated intracellular calcium concentration at doses (ECs0:0.3 nM), similar to those necessary to inhibit both binding (ICs0:21 nM) and CCK-induced cell proliferation (ECs0:1 nM). This suggests that the phosphoinositides/calcium pathway could be implicated in the antiproliferative effect of the analogue in SSTR5-expressing cells. We secondly investigated the mRNA expression of SSTR1 to 5 in human digestive tract tumors using the RT-PCR method: Tumors (n) SSTR1 SSTR2 SSTR3 SSTR4 SSTR5 Stomach (2) + + + Colon adenoma (2) + + + Colon cancer (11) + (2) + (2) + (3) + (4) + (7) Exocrine pancreas (a) + (2) + (a) Endocrine pancreas (4) + (2) + (1) + (2) + (3) Liver metastasis (1) + We conclude that both SSTR2 and SSTR5 mediate the inhibition of cell growth induced by the sornatostatin analogue RC-160 by distinct mechanisms. These two receptors are expressed in most of GI tract tumors that could he promising for the therapeutic potential of the somatostatin analogue.
C-MET PROTO-ONCOGEN IN RAT PANCREAS. GENE EXPRESSION DURING ONTOGENY AND REGENERATION AFTER ACUTE PANCREATITIS INDUCED BY CAERULEIN. E. Calve, G. Pelletier, C. Boucher, J. Morisset. University of Sherbrooke and Research Station, Lennoxville, Quebec, Canada. The Hepatocyte Growth Factor (HGF) has several activities on epithelial cells, including mitoganesis, dissociation of epithelial sheets, stimulation of motility and promotion of matrix invasion. HGF is the ligand of a dimeric transmembrane tyrosine kinase receptor encoded by the Met proto-oncogane (c-Met). Earlier, we demonstrated that HGF mRNA was overexpressed during pancreas development and maturation as well as after pancreatic aggression but the expression of its receptor still remains unknown under these circumstances. The aim of this study was to characterize c-Met mRNA expression in rat pancreas during development and during regeneration after pancreatectomy or acute pancreatitis. Pancreas were excised from 19d fetus and at days 1, 3, 5, 7, 9, 11, 13, 17, 21, 25, 35, 45 and 90 after birth. For regeneration studies, adult male rats were sacrificed at 1, 2, 3 and 6 days following 90% panereatectomy (Ptx). Acute panereatitis (AP) was induced in adult by eaemlein 12 pgkg"t, 3 x day for 2 days. Total RNA was extracted with the guanidine thiocyanate method and Northern blots were performed using 20 p.g of total RNA and labelled 32p-c-Met cRNA probe: The abundance of transcripts were estimated by densitometric scanning, and a 18S ribosomal eDNA probe was used as a control to measure total RNA loading. Pancreatic e-Met mRNAs levels were higher between f19 and post-natal day 11, and decreased to a very low level at day 35 which was maintained until the adult age. After Ptx, the level of c-Met gene transcription increased from the first day post-ptx to reach its maximum level on the 2nd day, and a return to control values at day 6. In the AP model, c-Met mRNA levels were maximal during the first two days of induction and then immediately dropped to control values at 3 days. In conclusion ; c-Met pancreatic gane expression is age dependent with the highest expression before weaning. Overexpression of c-Met mRNA up to fetal levels was observed after Ptx. Finally, increases in c-Met mRNA were evident during acute pancreatitis induction. These data demonstrate for the first time that overexpression of c-Met may be involved in pancreatic development and maturation as well as during pancreas regeneration after tissue aggression. Funded by NSERC Canada and FCAR, Quebec.
A955