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Quantification of preprodynorphin (DYN) mRNA in guinea pig gut using a highly sensitive solution hybridization assay
April 1995
• MECHANOSENSOR-MEDIATED INTRACELLULAR CALCIUM RELEASE IN COLONIC SMOOTH MUSCLE CELLS. S.H. Young, H. Ennes, S. Dea and E.A Mayer. CURE: VA/UCLA Gastroenteric Biology Centar/Neuroenteric Biology Group; Depts. of Medicine and Physiology, UCLA and West LAVA Med. Ctr. Los Angeles, CA 90073. Increased internal calcium (Cai) resulting from mechanosensitive activation has been described in a variety of cells, including vascular smooth muscle. We wanted to determine: 1) Can colonic myocytes be activated mechanically? 2) What are the spatiotemporal characteristics of [Ca]i changes; 3) Is activation linked with influx of external calcium and/or membrane depolarization? Methods: Myocytes from longitudinal (L) and circular (C) muscle layers were dissected from the rabbit colon and placed into separate cell cultures for 5-10 days. Cells were loaded with the Ca indicator fura-2, or the potential sensitive dye Di-8-ANEPPS. [Ca]i and membrane potential was determined using a videomicroscopic imaging system (Attofhior, Atto Instruments) at a sampling rate of 2 image pairs/s. Light mechanical stimulation was accomplished by a slight (1-3 bun), short (0.5see) downward movement of a fire-polished glass pipette (2-4l.tm diameter) connected to an electronically controlled micromanipulator (Eppendorf). Results: Mean [Call in individual C myocytes was 125_+13 nM (n=20), not different from L (n=22). Mechanical stimulation increased [Call in L and C to 758+91nM and 774_+98nM respectively. Regionally-induced [Ca]i increases spread with constant amplitude throughout the cell at a propagation velocity of 13_+3urn/s, similar to values found in freshly isolated cells. In 50% of the cells, mechancial stimulation resulted in cell hyperpolarization (<20 mV). When extracellular Ca was removed ( 0 mM Ca, 0.1 mM EGTA) both L and C show normal resting levels (118+20 nM) and produced Ca waves in response to mechanical stimulation (velocity of propagation 21+7 Hm/sec). The amplitude of [Ca]i waves (above baseline [Ca]i) was decreased by 78 % to 170+19nM (p<0.05). Ryanodine (50 ~tM), an inhibitor of the IP3-insensitive Ca release channel, blocked the Ca response in 40% of the cells, while thapsigargin had no effect. Conclusions: i. Mechanosensitive activation of both muscle layers of the colon is mediated by Cai release from ryanodine-sensitive stores, and not by plasma membrane depolarization mediated by mechanosensitive cation channels. 2. Hyperpolarization may be related to activation of Ca-activated K channels. 3. Propagation of Ca waves is regenerative, suggesting a diffusible messenger acting on ryanodine-sensitive Ca stores.
• COLONIC SMOOTH MUSCLE OF RATS IS A MAJOR TARGET OF INSULIN-LIKE GROWTH FACTOR-I IN V1VO AND IN VITRO. J.M. Zeeh. F. Procaccino, P. Hoffinann, T.T. Zittel*, V.E. Eysselein and J.A. MeRoberts. Div. of Gastroenterology, Harbor-UCLA Medical Center, Torrance, CA, *Tiibingen University, Dept. of General Surgery, Tfibingen, FRG Introduction: Insulin-like growth factor-I (IGF) is an important mediator of gastrointestinal growth and tissue integrity. We have recently shown an increase in IGF binding sites after colonic injury exclusively in the museularis propria. In this study we investigated the ~ of exogenous IGF-I on colonic smooth muscle (SM) in vivo and isolated rat colonic smooth muscle cells (SMC) in primary culture. Methods: Sprague-Dawley rats were treated with rhlGF-I via subcutaneous osmotic minipumps at a dose of 2.3g mg/day/kg for 14 days. The body weight of all animals was monitored before and after treatment. Colons were removed, rinsed and the wet weight and total length was measured. Crosssections were stained for H&E and the thickness of the muscularis propria and the musculafis mucosa was measured. Cell proliferation in the musenlaris propria was determined immtmohistoehemienlly using an antibody against PCNA. Growth rate of SMC in primary cultures isolated from rat colon was determined in a defined media containing transferrin and selenium (basal media) in the presence or absence of IGF-I (10-s M), EGF (10"s M) and FGF (200 ng/ml). Results: Body weight of IGF-treated animals was significantlyhigher compared to controls (365 q- 6g vs. 341 ± 4g). Both colon wet weight and length inerensed significantly in treated animals (2.5 4- 0.1g vs. 1.9 4- 0.1g and 19.2 q- 0.4cm vs. 15.2 ± 0.5era, respectively). The thickness of the longitudinal and circular SM was markedly increased in treated animals (26 4- 2p.m vs. 13 q- lpm and 94 q6/am vs. 64 4- 6pro, respectively). PCNA staining in the museularis propria showed a significantly higher number of positive cells in IGF-treated animals compared to controls (23 4-3 vs. 9 4- 2 per 10~m section). The thickness of the muscularis mucosa did not change. In in vitro studies, growth rate of SMC when treated with IGF alone (0.15 4- 0.02 doublings/day) was not statisticallydifferent from that of cells in basic media (0.06 4- 0.01). However, IGF in combination with EGF and FGF caused a significant increase (0.51 4- 0.04) in the growth rate compared to EGF in combination with FGF (0.33 4- 0.03) and to IGF alone. Conclusion: IGF has profound growth stimulatory effcets on colonic smooth muscle in vivo. Growth experiments with SMC in primary culture support our in vivo findings but suggest that IGF acts along with other growth factors such as EGF and FGF.
Hormones and Receptors
A1019
QUANTIFICATION OF PREPRODYNORPHIN (DYN) mRNA IN GUINEA PIG GUT USING A HIGHLY SENSITIVE SOLUTION HYBRIDIZATION ASSAY V. Yuferov. J.A. Culpepper-Morgan, LH. Claye, K.S. LaForge, and M.J. Kreek. Biology of Addictive Diseases Laboratory, The Rockefeller University, New York, N Y and Norwalk Hospital, Norwalk, CT. Kappa opioid agonists delay gastrointestinal transit (GIT) in the guinea pig and mouse but not in the rat (Culpepper-Morgan, Life Sci 42:2073, 1988). Guinea pig brain contains more high affinity kappa-1 opioid receptors than rat brain (Zukin~ Proc Natl Aead Sci. 85:4061, 1988). Dynorphin is released by in vitro preparations of guinea pig small intestine during peristalsis (Donnerer, Brit J Pharrn. 83:919, 1984). Interspecies differences affect motility responses to kappa agonists; also regional intraspecies differences in gut DYN mRNA levels may affect regional gut motility, in this study absolute and relative amounts o f D Y N mRNA were quantified for selected regions of guinea pig gut and solid organs by a highly sensitive solution hybridization RNase protection TCA-precipitation assay using a newly cloned guinea pig dynorphin gene-derived specific probe. The DYN mRNA content was determined by hybridization of tissue extracts to labeled cRNA. DYN mRNA content expressed as picograms per ~tg of total RNA, which was determined using an 18S ribosomal RNA hybridization probe, ; shown below (*: p < 0.01): Fundus
0.09 ± 0.01
Cecum
0.10 ± 0.03
Duodenum
0.09 =k 0.01
Recatm*
0.17 ± 0.02
Jejunum
0.09 ± 0.01
Spleen
0.11 ± 0.02
Kidney 0.10 ± 0.02 asurable m all re~ous of the guinea pig gut as well as the spleen and kidne' ,. The levels were about 1/8th the level found in the adrenal gland (0.74 pg/~tg). The distal rectum, contained more DYN mRNA than the proximal stomach, small bowel, and cecum (Supported in part by NIH D A D50-05130). Ileum
0.12 ± 0.01
EFFECTS OF INSULIN-LIKE GROWTH FACTOR-I ON COLONIC EPITHELIAL PROLIFERATION AND ON MUCOSAL INJURY IN RAT COLITIS. J.M. Zeeh, F. Procaccino, P. Hoffmann, V.E. Eysselein and J.A. MeRoberts. Harbor-UCLA Medical Center, Div. of Gastroenterology, Torrance, CA Introduction: Insulin-like growth faetor-I (IGF-I) has trophic effects on the intestine. Recent studies have shown that IGF-I induces marked intestinal growth in catabolic rats and enhances mucosal adaptation after jejunoileal resection. The aim of this study was to investigate the effects of exogenous IGF on colonic epithelial proliferation and to determine if it ameliorates mucosal injury in experimental colitis in rats. Methods: In two groups of 10 animals, osmotic pumps were implanted subcutaneously to deliver either rhlGF-I at a dose of 600p.g/day or vehicle. Colitis was induced at the same time by instillation of 2,4,6trinitrobenzenesulfonic acid (TNB, 50 mg/kg) in ethanol as an enema. Animals were sacrificed after 7 days. Tissue damage was examined microscopically and expressed as percentage of tissue with ulcerations or erosions and compared to controls. An additional two groups of rats without colitis underwent the same treatment with IGF or vehicle and were injected with bromodeoxyuridine (BrdU) 1 hour prior to sacrifice. Crosssections of uninflamed colon in IGF-treated animals were examined immunohistoehemically using antibodies against BrdU and PCNA. Results: Proliferation staining in colonic tissue from uninflamed animals revealed significantly more BrdU-positiv¢ cells per colonic crypt compared to controls (4.2 + 0.1 vs. 3.0 + 0.1, p=0.0004). PCNA staining also showed more positive cells per crypt in IGF-treated animals (11.2 + 0.5 vs. 8.4 + 0.5, p=0.0068). However, IGF-treatment had no significant effect on microscopic damage such as erosions and ulcerations in animals with TNB-induced colitis compared to controls. Conclusions: Exogenous IGFI has growth promoting effects on the colonic epithelium but does not ameliorate or prevent mucosal injury in experimental colitis in rats.
• MECHANOSENSOR-MEDIATED INTRACELLULAR CALCIUM RELEASE IN COLONIC SMOOTH MUSCLE CELLS. S.H. Young, H. Ennes, S. Dea and E.A Mayer. CURE: VA/UCLA Gastroenteric Biology Centar/Neuroenteric Biology Group; Depts. of Medicine and Physiology, UCLA and West LAVA Med. Ctr. Los Angeles, CA 90073. Increased internal calcium (Cai) resulting from mechanosensitive activation has been described in a variety of cells, including vascular smooth muscle. We wanted to determine: 1) Can colonic myocytes be activated mechanically? 2) What are the spatiotemporal characteristics of [Ca]i changes; 3) Is activation linked with influx of external calcium and/or membrane depolarization? Methods: Myocytes from longitudinal (L) and circular (C) muscle layers were dissected from the rabbit colon and placed into separate cell cultures for 5-10 days. Cells were loaded with the Ca indicator fura-2, or the potential sensitive dye Di-8-ANEPPS. [Ca]i and membrane potential was determined using a videomicroscopic imaging system (Attofhior, Atto Instruments) at a sampling rate of 2 image pairs/s. Light mechanical stimulation was accomplished by a slight (1-3 bun), short (0.5see) downward movement of a fire-polished glass pipette (2-4l.tm diameter) connected to an electronically controlled micromanipulator (Eppendorf). Results: Mean [Call in individual C myocytes was 125_+13 nM (n=20), not different from L (n=22). Mechanical stimulation increased [Call in L and C to 758+91nM and 774_+98nM respectively. Regionally-induced [Ca]i increases spread with constant amplitude throughout the cell at a propagation velocity of 13_+3urn/s, similar to values found in freshly isolated cells. In 50% of the cells, mechancial stimulation resulted in cell hyperpolarization (<20 mV). When extracellular Ca was removed ( 0 mM Ca, 0.1 mM EGTA) both L and C show normal resting levels (118+20 nM) and produced Ca waves in response to mechanical stimulation (velocity of propagation 21+7 Hm/sec). The amplitude of [Ca]i waves (above baseline [Ca]i) was decreased by 78 % to 170+19nM (p<0.05). Ryanodine (50 ~tM), an inhibitor of the IP3-insensitive Ca release channel, blocked the Ca response in 40% of the cells, while thapsigargin had no effect. Conclusions: i. Mechanosensitive activation of both muscle layers of the colon is mediated by Cai release from ryanodine-sensitive stores, and not by plasma membrane depolarization mediated by mechanosensitive cation channels. 2. Hyperpolarization may be related to activation of Ca-activated K channels. 3. Propagation of Ca waves is regenerative, suggesting a diffusible messenger acting on ryanodine-sensitive Ca stores.
• COLONIC SMOOTH MUSCLE OF RATS IS A MAJOR TARGET OF INSULIN-LIKE GROWTH FACTOR-I IN V1VO AND IN VITRO. J.M. Zeeh. F. Procaccino, P. Hoffinann, T.T. Zittel*, V.E. Eysselein and J.A. MeRoberts. Div. of Gastroenterology, Harbor-UCLA Medical Center, Torrance, CA, *Tiibingen University, Dept. of General Surgery, Tfibingen, FRG Introduction: Insulin-like growth factor-I (IGF) is an important mediator of gastrointestinal growth and tissue integrity. We have recently shown an increase in IGF binding sites after colonic injury exclusively in the museularis propria. In this study we investigated the ~ of exogenous IGF-I on colonic smooth muscle (SM) in vivo and isolated rat colonic smooth muscle cells (SMC) in primary culture. Methods: Sprague-Dawley rats were treated with rhlGF-I via subcutaneous osmotic minipumps at a dose of 2.3g mg/day/kg for 14 days. The body weight of all animals was monitored before and after treatment. Colons were removed, rinsed and the wet weight and total length was measured. Crosssections were stained for H&E and the thickness of the muscularis propria and the musculafis mucosa was measured. Cell proliferation in the musenlaris propria was determined immtmohistoehemienlly using an antibody against PCNA. Growth rate of SMC in primary cultures isolated from rat colon was determined in a defined media containing transferrin and selenium (basal media) in the presence or absence of IGF-I (10-s M), EGF (10"s M) and FGF (200 ng/ml). Results: Body weight of IGF-treated animals was significantlyhigher compared to controls (365 q- 6g vs. 341 ± 4g). Both colon wet weight and length inerensed significantly in treated animals (2.5 4- 0.1g vs. 1.9 4- 0.1g and 19.2 q- 0.4cm vs. 15.2 ± 0.5era, respectively). The thickness of the longitudinal and circular SM was markedly increased in treated animals (26 4- 2p.m vs. 13 q- lpm and 94 q6/am vs. 64 4- 6pro, respectively). PCNA staining in the museularis propria showed a significantly higher number of positive cells in IGF-treated animals compared to controls (23 4-3 vs. 9 4- 2 per 10~m section). The thickness of the muscularis mucosa did not change. In in vitro studies, growth rate of SMC when treated with IGF alone (0.15 4- 0.02 doublings/day) was not statisticallydifferent from that of cells in basic media (0.06 4- 0.01). However, IGF in combination with EGF and FGF caused a significant increase (0.51 4- 0.04) in the growth rate compared to EGF in combination with FGF (0.33 4- 0.03) and to IGF alone. Conclusion: IGF has profound growth stimulatory effcets on colonic smooth muscle in vivo. Growth experiments with SMC in primary culture support our in vivo findings but suggest that IGF acts along with other growth factors such as EGF and FGF.
Hormones and Receptors
A1019
QUANTIFICATION OF PREPRODYNORPHIN (DYN) mRNA IN GUINEA PIG GUT USING A HIGHLY SENSITIVE SOLUTION HYBRIDIZATION ASSAY V. Yuferov. J.A. Culpepper-Morgan, LH. Claye, K.S. LaForge, and M.J. Kreek. Biology of Addictive Diseases Laboratory, The Rockefeller University, New York, N Y and Norwalk Hospital, Norwalk, CT. Kappa opioid agonists delay gastrointestinal transit (GIT) in the guinea pig and mouse but not in the rat (Culpepper-Morgan, Life Sci 42:2073, 1988). Guinea pig brain contains more high affinity kappa-1 opioid receptors than rat brain (Zukin~ Proc Natl Aead Sci. 85:4061, 1988). Dynorphin is released by in vitro preparations of guinea pig small intestine during peristalsis (Donnerer, Brit J Pharrn. 83:919, 1984). Interspecies differences affect motility responses to kappa agonists; also regional intraspecies differences in gut DYN mRNA levels may affect regional gut motility, in this study absolute and relative amounts o f D Y N mRNA were quantified for selected regions of guinea pig gut and solid organs by a highly sensitive solution hybridization RNase protection TCA-precipitation assay using a newly cloned guinea pig dynorphin gene-derived specific probe. The DYN mRNA content was determined by hybridization of tissue extracts to labeled cRNA. DYN mRNA content expressed as picograms per ~tg of total RNA, which was determined using an 18S ribosomal RNA hybridization probe, ; shown below (*: p < 0.01): Fundus
0.09 ± 0.01
Cecum
0.10 ± 0.03
Duodenum
0.09 =k 0.01
Recatm*
0.17 ± 0.02
Jejunum
0.09 ± 0.01
Spleen
0.11 ± 0.02
Kidney 0.10 ± 0.02 asurable m all re~ous of the guinea pig gut as well as the spleen and kidne' ,. The levels were about 1/8th the level found in the adrenal gland (0.74 pg/~tg). The distal rectum, contained more DYN mRNA than the proximal stomach, small bowel, and cecum (Supported in part by NIH D A D50-05130). Ileum
0.12 ± 0.01
EFFECTS OF INSULIN-LIKE GROWTH FACTOR-I ON COLONIC EPITHELIAL PROLIFERATION AND ON MUCOSAL INJURY IN RAT COLITIS. J.M. Zeeh, F. Procaccino, P. Hoffmann, V.E. Eysselein and J.A. MeRoberts. Harbor-UCLA Medical Center, Div. of Gastroenterology, Torrance, CA Introduction: Insulin-like growth faetor-I (IGF-I) has trophic effects on the intestine. Recent studies have shown that IGF-I induces marked intestinal growth in catabolic rats and enhances mucosal adaptation after jejunoileal resection. The aim of this study was to investigate the effects of exogenous IGF on colonic epithelial proliferation and to determine if it ameliorates mucosal injury in experimental colitis in rats. Methods: In two groups of 10 animals, osmotic pumps were implanted subcutaneously to deliver either rhlGF-I at a dose of 600p.g/day or vehicle. Colitis was induced at the same time by instillation of 2,4,6trinitrobenzenesulfonic acid (TNB, 50 mg/kg) in ethanol as an enema. Animals were sacrificed after 7 days. Tissue damage was examined microscopically and expressed as percentage of tissue with ulcerations or erosions and compared to controls. An additional two groups of rats without colitis underwent the same treatment with IGF or vehicle and were injected with bromodeoxyuridine (BrdU) 1 hour prior to sacrifice. Crosssections of uninflamed colon in IGF-treated animals were examined immunohistoehemically using antibodies against BrdU and PCNA. Results: Proliferation staining in colonic tissue from uninflamed animals revealed significantly more BrdU-positiv¢ cells per colonic crypt compared to controls (4.2 + 0.1 vs. 3.0 + 0.1, p=0.0004). PCNA staining also showed more positive cells per crypt in IGF-treated animals (11.2 + 0.5 vs. 8.4 + 0.5, p=0.0068). However, IGF-treatment had no significant effect on microscopic damage such as erosions and ulcerations in animals with TNB-induced colitis compared to controls. Conclusions: Exogenous IGFI has growth promoting effects on the colonic epithelium but does not ameliorate or prevent mucosal injury in experimental colitis in rats.