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A rapid non-radioactive technique for in situ hybridization histochemistry (ISHH) of substance P receptor mRNA in the rat brain
A958
AGA ABSTRACTS
• OVEREXPRESSION OF INSULIN-LIKEGROWTH FACTOR BINDING PROTEIN (IGFBP)-4 RESULTS IN INCREASED PROLIFERATION RATE AND MUC3 EXPRESSION IN HT-29 CELLS. M.R. Corldns. J.A. Vanderhoof, D.H. Slentz, R.G. MacDonald, M.A. Hollingsworth, D.R. Mack. Depts of Pediatrics, Biomedical Sciences, Biochemistry and Molecular Biology, Eppley Research Institute, Creighton University and University of Nebraska Medical Center, Omaha, NE. Insulin-like growth factors (IGFs) stimulate the proliferation of intestinal mucesa in vivo and in many intestinal cell lines in vitro. There are also six IGFBPs that bind specifically to the IGFs. The IGFBPs have been shown under certain conditions to enhance the IGF effect and under others to be inhibitory. HT-29 cells produce IGF-II and IGFBP-4 and express the MUC2 and MUC3 mucins. Increased MUC3 expression may be achieved under growth conditions that induce morphological differentiation in HT-29 cells. This study was designed to examine if there is an association between IGFBP-4, growth rates and mucin expression in HT-29 cells. Full-length rat IGFBP-4 eDNA (provided by S. Shimisald) was ligated into the eukaryotic expression vector pcDNA3. The cells were grown in glucose-containing medium with geneticin for two weeks to isolate clones. In growth experiments, cell numbers were assessed by crystal violet dye binding. Two- to three-fold overexpression of IGFBP-4 was confirmed by ligand blot analysis. Clones and cells transfected with vector alone were grown in medium containing 10% fetal bovine serum and were fed with serum-free medium for two 24 hour periods before analysis. The IGFBP-4 overexpressing clones grew at a faster rate than their controls (68,100 + 5,700 vs. 52,200 __+5,500 cells/day). Northern blot analysis showed that ceils transfeeted with the IGFBP-4 constmct bad increased MUC3 mRNA levels (360%) compared With their controls and similar MUC2 levels. We conclude endogenous IGFBP-4 enhances HT-29 cell proliferation and is associated with increased MUC3 mRNA expression.
• EFFECTS OF THE NONPEPTIDE NEUROTENSIN ANTAGONIST SR 48692 ON RAT AND GUINEA-PIG GJ. MOTILITY. T. Croci, M. Landi, L. Manara, D. Gully l and G. Le Fttr2, SANOFIMIDY S.p.A. Research Centre, Via G.B. Piranesi 38, 20137 Milan (Italy), 1SANOFI Recherche, 195, Route d'Espagne 31036,Toulouse (France) and 2SANOFI Recherche, 32/34 rue Marbeuf, 75008 Paris (France). We investigated the influence of the first non-peptide neurotensin antagonist SR 48692, 2-{[1-7-chloroquinolin-4-yl-)-5(2,6-dimethoxyphenyl)- 1H-pyrazole-3-carbonyl]amino}adamantane-2-carboxylic acid (1), on: gastric emptying of caloric and non-caloric test meals, and small intestinal transit in rats; defecation in rats and guinea-pigs. Methods: Crl:CD®BR, male rats (220-250 g) or CrI(HA)SPF guinea pigs (300-400g) were used. Gastric emptying was assessed in 24h fasted rats either by weighing the stomach contents or measuring a marker (phenol red) concenlration at different times after standard semi-solid meals (BaSO4 or food suspension, 4RF21-Mucedola, Italia), given by~ gavage at the same temperature and viscosity. Intestinal transit (percentage of small intestine traversed by BaSO4) was assessed in conscious fasted rats chronically implanted with an i.d. cannula. Defecation was assessed in fed animals during 2.5 (rats) or 6h (guinea pigs) fecal collection (2); R e s u l t s : SR 48692 (EDS0.0.8 txg/kg, p.o.) dose-dependently increased rat gastric emptying of a caloric meal, but up to 1 mg/kg it had no effect on the non-caloric meal. SR 48692 also dosedependently promoted defecation, 1 g dry weight of feces (AD1) being collected at 1.9 ~tg/kg, p.o. in rats, and 1.5 mg/kg, s.c. in guinea-pigs. SR 48692 increased rat but not guinea-pig fecal water content. SR 48692 had no effect on rat small intestinal transit up to 1 mg/kg, p.o. Conclusions: We suggest that antagonism of endogenous neurotensin accounts for the gastric emptying and defecation promoting actions of SR 48692. References: 1 - Gully D., Canton M., Boigegraln R., Jeanjean F. et al. (1993). Prec. Natl. Acad. Sci. 90, 65-69. 2 - Croci T. and Bianchetti A. (1992). J. Pharm. Pharmacol. 44, 358-360
GASTROENTEROLOGY, VoI. IO8, NO. 4
• SOMATOSTATINHAS A DIFFERENTCELLULAR BASISOF ACTIONON COLONICSMOOTHMUSCLECELLS THANGASTRICSMOOTHMUSCLE CELLS. V.D. Corleto, D.H. Coy, R.T. Jensen. NIH, Bethesda, MD, and Tulane University, New Orleans, LA Somatostatin (SS) immunoreactivity is found in neurons projecting into colonic muscle layers as well as in ganglia. SS has been shown to have effects on colonic motility and colonic smooth muscle contractility both in v/re and in vitro. However, it remainsunclear whetherSS effects on the colon are direct or neural-mediated and whether, if direct, its mechanismof action is similarto that shown on gastric smooth muscle cells where it inhibits the action of relaxants. To clarify this; in the present study we have prepared isolated smooth muscle cells from the descending guinea pig colon using collagenase digestion and compared the effects of SS to those on isolated gastric smoothmusclecells from guinea pig. Colonic and gastric resting cell length was 102+1 pm and 93+2 pm, and with both cells, SS-14 and SS-28 whetherwith or without pretense inhibitors had no effect on cell length, With pretense inhibitors present [either phospboramidon(1 pM) and amastatin(10 pM), or thiorphan(1 pM)], in gastric smooth muscle cells SS-14 and SS-28 had no effect on carbachol-induced contraction, whereasin colonic cells each causeda dose-related inhibition(SS-14, ECho 10 nM, SS-28 2.5 nM). In gastric muscle cells, SS-14 and SS-28 both inhibited relaxants demonstrated by the ability of SS to alter VIP's effect on carbachol-induced contraction. In colonic cells SS-28 (1 /tM) caused > 85% inhibition of contraction by CCK-8, bombesin, the phorbel ester, TPA, and the calcium ionopbore, ionomycin, whereas it had no effect on contraction by these agents in gastric cells. These results suggestin colonic cells, sg-28 was inhibiting contractantsby inhibitingbothPKC-induced and Ca2+-induced pathways,whereas in contrast, in previous studiesby us and others it was shown to inhibit relaxantS in gastric smoothmuscle cells at least in part by inhibiting edenylate cyclase. To determine whetherthese different transductionmechanismsmightbe mediated by different SS receptor subtypes,the ability of various octapeptide analogueswith different SS subtype selectivity were examined with each cell type. For colonic muscle cells, the relative potencies for inhibiting carbacbol-induced contraction were: DC-23-99> > > SMS > DC-23-48 > DC-25-20> > NC-8-21, and differed from those in gastric cells for inhibiting VIP-induced relaxation whichwere: DC23-99 > > NC-8-21 = DC-25-20 > > DC- 13-217> SMS= DC-23-48.Theseresults demonstrate that SS interacts directly with both gastric and colonic smooth muscle cells to alter contractility; however its mechanismof action differs. In gastric smooth muscle ceils SS inhibitS relaxants with no effect on contractants whereas in colonic muscle cells SS inhibitS contractantS, This inhibition in colonic cells is mediated by both inhibition of PKC- and CaZ+-activated pathways. The difference in action in these 2 tissues is likely mediated by different SS receptor subtypes.
A RAPID NON-RADIOACTIVE TECHNIQUE FOR IN SITU HYBRIDIZATION HISTOCHEMISTRY (ISHH) OF SUBSTANCE P RECEPTOR mRNA IN THE RAT BRAIN. A. Cruz, HF Wrzos, R Polavarapu. and A Ouyang Department of Medicine, The Milton S. Hershey Medical Center, College of Medicine- The Pennsylvania State University, Hershey, PA Substance P is found widely in the mammalian central and peripheral nervous system. It is important in the central and enteric control of gut motility. The only published studies of the distribution of SP receptor (SPR) mRNA by in situ hybridization histochemistry (ISHH) involve the use of 35S-labeled oligonucleotides (Macho 1993, Kiyama 1993). The digoxigenin labeling technique is non radioactive and uses a stable probe. It is generally felt to be less sensitive than the radiolabeled techniques and may be less useful for low abundance message. Aims: to develop the digoxigenin-labeled technique to localize SPR mRNA in the rat central nervous system and to compare the distribution with that seen using a 35S-labeled riboprobe. The use of riboprobe for study of the distribution of SPR has not been previously published. Methods: Brains were quickly removed from anesthetized rats, frozen in isopentane, and 15 B sections were cut, thaw-mounted on Vectabond coated slides and stored at -20oc. A digoxigenin-UTP-labeled riboprobe was prepared from the 1234 bp insert of SPR cDNA (from J. Krause). Hybridization and washes were performed at 52oc. Sections were incubated with antidigoxigenin antibody at 4oc overnight and hybridization immediately detected by a colorimetric method using nitroblue tetrazolium (NBT) and X-phosphatase using the Genius system (Boehringer Mannheim). For 35S-labeled riboprobe studies, the same insert of SPR cDNA was used. Preparation of slides and hybridization conditions were similar, except the results were detected after 21 days of incubation after coating the slides with emulsion. Results: Using the digoxigenin-laheled riboprobe, NK-1 receptor was detected in the rat brain. The greatest hybridization was observed in the frontal cortex-motor area, pyriform cortex, candate nucleus, putamen, hippoeampus, and thalamic nucleL The distribution was the same as seen using 35S-labeled riboprobe. The distribution of SPR mRNA in our studies was similar to that described by Maeno et al. using oligonucleotides. Conclusion: The digoxigenin-labeled method successfully localizes SPR mRNA in the rat brain. The advantage of this method is that it is rapid, sensitive and non-radioactive, and the probe remains stable. Supported by NIH grant R01-DK34818.
AGA ABSTRACTS
• OVEREXPRESSION OF INSULIN-LIKEGROWTH FACTOR BINDING PROTEIN (IGFBP)-4 RESULTS IN INCREASED PROLIFERATION RATE AND MUC3 EXPRESSION IN HT-29 CELLS. M.R. Corldns. J.A. Vanderhoof, D.H. Slentz, R.G. MacDonald, M.A. Hollingsworth, D.R. Mack. Depts of Pediatrics, Biomedical Sciences, Biochemistry and Molecular Biology, Eppley Research Institute, Creighton University and University of Nebraska Medical Center, Omaha, NE. Insulin-like growth factors (IGFs) stimulate the proliferation of intestinal mucesa in vivo and in many intestinal cell lines in vitro. There are also six IGFBPs that bind specifically to the IGFs. The IGFBPs have been shown under certain conditions to enhance the IGF effect and under others to be inhibitory. HT-29 cells produce IGF-II and IGFBP-4 and express the MUC2 and MUC3 mucins. Increased MUC3 expression may be achieved under growth conditions that induce morphological differentiation in HT-29 cells. This study was designed to examine if there is an association between IGFBP-4, growth rates and mucin expression in HT-29 cells. Full-length rat IGFBP-4 eDNA (provided by S. Shimisald) was ligated into the eukaryotic expression vector pcDNA3. The cells were grown in glucose-containing medium with geneticin for two weeks to isolate clones. In growth experiments, cell numbers were assessed by crystal violet dye binding. Two- to three-fold overexpression of IGFBP-4 was confirmed by ligand blot analysis. Clones and cells transfected with vector alone were grown in medium containing 10% fetal bovine serum and were fed with serum-free medium for two 24 hour periods before analysis. The IGFBP-4 overexpressing clones grew at a faster rate than their controls (68,100 + 5,700 vs. 52,200 __+5,500 cells/day). Northern blot analysis showed that ceils transfeeted with the IGFBP-4 constmct bad increased MUC3 mRNA levels (360%) compared With their controls and similar MUC2 levels. We conclude endogenous IGFBP-4 enhances HT-29 cell proliferation and is associated with increased MUC3 mRNA expression.
• EFFECTS OF THE NONPEPTIDE NEUROTENSIN ANTAGONIST SR 48692 ON RAT AND GUINEA-PIG GJ. MOTILITY. T. Croci, M. Landi, L. Manara, D. Gully l and G. Le Fttr2, SANOFIMIDY S.p.A. Research Centre, Via G.B. Piranesi 38, 20137 Milan (Italy), 1SANOFI Recherche, 195, Route d'Espagne 31036,Toulouse (France) and 2SANOFI Recherche, 32/34 rue Marbeuf, 75008 Paris (France). We investigated the influence of the first non-peptide neurotensin antagonist SR 48692, 2-{[1-7-chloroquinolin-4-yl-)-5(2,6-dimethoxyphenyl)- 1H-pyrazole-3-carbonyl]amino}adamantane-2-carboxylic acid (1), on: gastric emptying of caloric and non-caloric test meals, and small intestinal transit in rats; defecation in rats and guinea-pigs. Methods: Crl:CD®BR, male rats (220-250 g) or CrI(HA)SPF guinea pigs (300-400g) were used. Gastric emptying was assessed in 24h fasted rats either by weighing the stomach contents or measuring a marker (phenol red) concenlration at different times after standard semi-solid meals (BaSO4 or food suspension, 4RF21-Mucedola, Italia), given by~ gavage at the same temperature and viscosity. Intestinal transit (percentage of small intestine traversed by BaSO4) was assessed in conscious fasted rats chronically implanted with an i.d. cannula. Defecation was assessed in fed animals during 2.5 (rats) or 6h (guinea pigs) fecal collection (2); R e s u l t s : SR 48692 (EDS0.0.8 txg/kg, p.o.) dose-dependently increased rat gastric emptying of a caloric meal, but up to 1 mg/kg it had no effect on the non-caloric meal. SR 48692 also dosedependently promoted defecation, 1 g dry weight of feces (AD1) being collected at 1.9 ~tg/kg, p.o. in rats, and 1.5 mg/kg, s.c. in guinea-pigs. SR 48692 increased rat but not guinea-pig fecal water content. SR 48692 had no effect on rat small intestinal transit up to 1 mg/kg, p.o. Conclusions: We suggest that antagonism of endogenous neurotensin accounts for the gastric emptying and defecation promoting actions of SR 48692. References: 1 - Gully D., Canton M., Boigegraln R., Jeanjean F. et al. (1993). Prec. Natl. Acad. Sci. 90, 65-69. 2 - Croci T. and Bianchetti A. (1992). J. Pharm. Pharmacol. 44, 358-360
GASTROENTEROLOGY, VoI. IO8, NO. 4
• SOMATOSTATINHAS A DIFFERENTCELLULAR BASISOF ACTIONON COLONICSMOOTHMUSCLECELLS THANGASTRICSMOOTHMUSCLE CELLS. V.D. Corleto, D.H. Coy, R.T. Jensen. NIH, Bethesda, MD, and Tulane University, New Orleans, LA Somatostatin (SS) immunoreactivity is found in neurons projecting into colonic muscle layers as well as in ganglia. SS has been shown to have effects on colonic motility and colonic smooth muscle contractility both in v/re and in vitro. However, it remainsunclear whetherSS effects on the colon are direct or neural-mediated and whether, if direct, its mechanismof action is similarto that shown on gastric smooth muscle cells where it inhibits the action of relaxants. To clarify this; in the present study we have prepared isolated smooth muscle cells from the descending guinea pig colon using collagenase digestion and compared the effects of SS to those on isolated gastric smoothmusclecells from guinea pig. Colonic and gastric resting cell length was 102+1 pm and 93+2 pm, and with both cells, SS-14 and SS-28 whetherwith or without pretense inhibitors had no effect on cell length, With pretense inhibitors present [either phospboramidon(1 pM) and amastatin(10 pM), or thiorphan(1 pM)], in gastric smooth muscle cells SS-14 and SS-28 had no effect on carbachol-induced contraction, whereasin colonic cells each causeda dose-related inhibition(SS-14, ECho 10 nM, SS-28 2.5 nM). In gastric muscle cells, SS-14 and SS-28 both inhibited relaxants demonstrated by the ability of SS to alter VIP's effect on carbachol-induced contraction. In colonic cells SS-28 (1 /tM) caused > 85% inhibition of contraction by CCK-8, bombesin, the phorbel ester, TPA, and the calcium ionopbore, ionomycin, whereas it had no effect on contraction by these agents in gastric cells. These results suggestin colonic cells, sg-28 was inhibiting contractantsby inhibitingbothPKC-induced and Ca2+-induced pathways,whereas in contrast, in previous studiesby us and others it was shown to inhibit relaxantS in gastric smoothmuscle cells at least in part by inhibiting edenylate cyclase. To determine whetherthese different transductionmechanismsmightbe mediated by different SS receptor subtypes,the ability of various octapeptide analogueswith different SS subtype selectivity were examined with each cell type. For colonic muscle cells, the relative potencies for inhibiting carbacbol-induced contraction were: DC-23-99> > > SMS > DC-23-48 > DC-25-20> > NC-8-21, and differed from those in gastric cells for inhibiting VIP-induced relaxation whichwere: DC23-99 > > NC-8-21 = DC-25-20 > > DC- 13-217> SMS= DC-23-48.Theseresults demonstrate that SS interacts directly with both gastric and colonic smooth muscle cells to alter contractility; however its mechanismof action differs. In gastric smooth muscle ceils SS inhibitS relaxants with no effect on contractants whereas in colonic muscle cells SS inhibitS contractantS, This inhibition in colonic cells is mediated by both inhibition of PKC- and CaZ+-activated pathways. The difference in action in these 2 tissues is likely mediated by different SS receptor subtypes.
A RAPID NON-RADIOACTIVE TECHNIQUE FOR IN SITU HYBRIDIZATION HISTOCHEMISTRY (ISHH) OF SUBSTANCE P RECEPTOR mRNA IN THE RAT BRAIN. A. Cruz, HF Wrzos, R Polavarapu. and A Ouyang Department of Medicine, The Milton S. Hershey Medical Center, College of Medicine- The Pennsylvania State University, Hershey, PA Substance P is found widely in the mammalian central and peripheral nervous system. It is important in the central and enteric control of gut motility. The only published studies of the distribution of SP receptor (SPR) mRNA by in situ hybridization histochemistry (ISHH) involve the use of 35S-labeled oligonucleotides (Macho 1993, Kiyama 1993). The digoxigenin labeling technique is non radioactive and uses a stable probe. It is generally felt to be less sensitive than the radiolabeled techniques and may be less useful for low abundance message. Aims: to develop the digoxigenin-labeled technique to localize SPR mRNA in the rat central nervous system and to compare the distribution with that seen using a 35S-labeled riboprobe. The use of riboprobe for study of the distribution of SPR has not been previously published. Methods: Brains were quickly removed from anesthetized rats, frozen in isopentane, and 15 B sections were cut, thaw-mounted on Vectabond coated slides and stored at -20oc. A digoxigenin-UTP-labeled riboprobe was prepared from the 1234 bp insert of SPR cDNA (from J. Krause). Hybridization and washes were performed at 52oc. Sections were incubated with antidigoxigenin antibody at 4oc overnight and hybridization immediately detected by a colorimetric method using nitroblue tetrazolium (NBT) and X-phosphatase using the Genius system (Boehringer Mannheim). For 35S-labeled riboprobe studies, the same insert of SPR cDNA was used. Preparation of slides and hybridization conditions were similar, except the results were detected after 21 days of incubation after coating the slides with emulsion. Results: Using the digoxigenin-laheled riboprobe, NK-1 receptor was detected in the rat brain. The greatest hybridization was observed in the frontal cortex-motor area, pyriform cortex, candate nucleus, putamen, hippoeampus, and thalamic nucleL The distribution was the same as seen using 35S-labeled riboprobe. The distribution of SPR mRNA in our studies was similar to that described by Maeno et al. using oligonucleotides. Conclusion: The digoxigenin-labeled method successfully localizes SPR mRNA in the rat brain. The advantage of this method is that it is rapid, sensitive and non-radioactive, and the probe remains stable. Supported by NIH grant R01-DK34818.