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Effect of a chronic immobilization stress on colonic transit and the transcription of the gene encoding the CRF receptor in the rat hypothalamus
A954
AGA ABSTRACTS
EXPRESSION OF G-PROTEIN ALPHA SUBUN1TS IN NORMAL RAT COLONOCYTES, AZOXYMETHANE (AOM)-INDUCED COLON TUMORS, AND HUMAN COLON CANCER-DERIVED CELL LINES. M.J.G. Bolt, R.J. Mailloux, R. Wali, B . Frawley, B. Scaglione-Sewell, M. Rasenick*, B.M. Bissonnette, T.A. Brasitus, M.D. Sitrin. Dept. of Medicine, University of Chicago and *Dept. of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, IL , Heterotrimeric regulatory guanine nucleotide-binding proteins (Gproteins) couple many membrane bound receptors to a variety Of intracellular effectors, but vary in expression according tO cell type. We used isolated colonocytes from Sprague-Daw!ey rats to prepare eytosol and • enriched basolateral (BLM) and brush-border membranes (BBM). By use of specific antibodies to G-protein alpha subunits (Ga), western blotting techniques, and laser densitometry, we were able to measure the relative amounts of G a in these cell fractions. Both BLM and BBM were significantly enriched over the initial homogenate(s) when probed with Gai2, God3, Gc~o, Gctq/l 1, Gas, or Gel3; with BLM ranging from 4 to 60 fold and BBM from 1.7 to 7 fold. Relative enrichment of BLM over BBM was 8.5 for Gc~i2, 5.1 for Gctq/ll, 4.2 for Gcd3, and about 2.5 for Gc~o, G a s , Gel3: In contrast, G{xz was found entirely in the cytosolic fraction, differing from the membrane-bound location reported for Gctz in several other cell types. GcO2 was at such low abundance that we could not determine membrane enrichment. However, in human colonic cancer cell lines, CaCo-2, SW480, and HTC-I 1~ expressed large amounts of Goal2. Based on these findings, tumor tissue taken from colons of AOM-dosed Fisher 344 rats, a well-known model of colon carcinogenesis, were examined. All 4 tumors screened expressed large amounts of Goal2, wlaile normal colonic tissue from 3 vehicle-treated animals showed only a trace o f Gc~12. Gccl3, the closest homologue to Gc~12, had a low expression in all groups. Conclusion: The present studies demonstrate that the alpha subunits of G-proteins.are differentially localized to various regions of the rat colonocyte, which may underlie their role in various physiologic processes. Moreover, expression of Gc~12 in colonic tissue is related to cellular transformation, but whether this is a primary or secondary event requires additional study.
• CHARACTERIZATION OF SEROTONIN RECEPTORS INFLUENCING MOTILITY IN THE ISOLATED HUMAN INTESTINE. R.A, Borman, D.E. Burleigh. Department of Pharmacology, Queen Mary & Westfield College, University of London, UK. The aim of the present study was to investigate the receptors responsible for the motile effects of serotonin (5-HT) in the small and large intestine of man. Briefly, segments of small and large intestine were obtained from specimens removed at operations for carcinoma or Crohn's disease. Muscle strips were mounted under isotonic conditions in gassed Krebs solution at 37°C. 5-HT was applied to the tissues in a cumulative manner, and responses tested against a variety of 5-HT receptor antagonists. 5-HT was shown to relax circular muscle strips of human sigmoid e01on with an ECho of 0.24#M (95%C.L. 0.16-0.34, n=15). Unlike methysergide (10/zM), ketanserin (I#M) or ondansetron (10p`M), DAU 6285 (10p`M; a 5-HT4 receptor antagonist) significantly antagonised the response to 5-HT, yielding a pA2 estimate of 6.81_+0.23 (n= 10)~ In the small intestine, 5-HT induced contraction of both circular and longitudinal muscle strips, with an ECso of 58:6nM (95 %C.L. 21.5;86~5). and 34.9nM (95% C.L. 26.3-46.2) respectively. Methysergide was found to be a non-surmountable antagonist of the response to 5-HT in both mdscle layers, 16ut was approximately 1000-fold more potent in longitudinal than circular musdle. Methysergide (10nM) abolished the response to 5-HT in longitudinal mus61e, compared to 10p`M in circular muscle. In addition, ketanserin (30-100nM) was shown to antagonise the response to 5-HT of circular muscle; this antagonism was non-surmountable at I#M ketanserin. This concentration had no effect on the response to 5-HT of human longitudinal muscle. The'response in longitudinal muscle was, however, susceptible to antagonism by application of yohimbine (30-300riM; a 5-HT~ and 5:HT2s receptor antagonist, pA 2 of 8.10_+0.06) or SB 200646A (0.3-3.0p`M; a 5-HT2B and 5-HT2c receptor antagonist; pA2 of 7.17_+0.16). In conclusion, 5-HT has been shown to induce relaxation of human colonic circular mnsele via a receptor of the 5-HT4 sub-typel whereas'it was shown to contract both circular and longitudinal muscle layers of human small intestine. In longitudinal muscle, 5-HT-induced contraction is mediated via a 5-HT2a receptor, however the receptor responsible in circul~" muscle Can not be definitively classified at this time.
GASTROENTEROLOGY, Vol. 108, NO. 4
• EFFECT OF A CHRONIC IMMOBILIZATION STRESS ON COLONIC TRANSIT AND THE TRANSCRIPTION OF T H E GENE ENCODING THE CRF RECEPTOR 1N THE RAT HYPOTHALAMUS. B. Bonaz* and S. Rivest**. *Laboratory of Neurophysiology, INSERM U318, CHU, 38043 Grenoble cedex 09, F R A N C E and ** Laboratory of Molecular Endocrinology, Laval University, Qu6bec, CANADA. Corticotropin-releasing factor (CRF) action:in the CNS is part of the mechanism through which stress alters Colonic motor function in r a t s ( I ). Acute immobilization stress induces transcription of the gene encoding the C R F receptor (CRF-R) in the paraventricular nucleus of the hypothalamus (PVH; parvo-cellular part) (2). Purpose: to study, ir~ rats, the effect of a chronic immobilization stress, 1) on the transcription of the gene encoding for the CRF-R and on the localization of this transcript in CRF perikarya in 'the PVH, 2) on concomitant colonic transit changes. Methods: male SD rats (250g; n--40), with free access to food and water, w e r e submitted to a 90-rain immobilization stress either acutely or chrolllbally during 11 ~onsecutive days. Fecal pellet output was quantified at the end of the 90-rain immobilization session. Rats were killed before, immediately, 1.5, 3, 6 and 12 h after the end of the acute and chronic sessions. Rats were anesthetized and perfused with a solution of 4% parafonnaldehyde:borax: Coronal frozen sections of the brain were cut and the mRNA encoding the rat CRF-R was assayed by in situ hybridization (ISH) using a 35S-k-ibeled riboprobe (2). CRF-R localization within CRF-immunoreactive netitons in the PVH was determined using a combination of imn-/un0cytochemistry and ISH. Results: 1) in acutely stressed rats, mRNA encoding the CRF-R was highly expressed in the parvo-PVH 90 rain and 3 hr after the end of the stress and totally vanished 12 h after the stress. Numerous .CRFimmunoreactive neurons of the p a r v o - P V H expressed the CRF-R transcript. 12:5 + 0.7 pellets were counted at the end of the stress. 2) in chronically stressed rats, a significant decrease of the CRF-R mRNA signal was observed in the parvo-PVH 90 rain and 3 h after the end of the stress session: Very few CRF immunoreactive neurons o f the parvO-PVH expressed the CRF-R transcript. In parallel; a significant decrease of the fecal pellet output was observed (DI: 12.1_+ 0.8; DI 1:7.5 -+ 0.9; P<0.05). Conclusions: a chronic immobilization stress is followed by a sighificant decrease of the transcription of the gene encoding for the CRF-R and of the localization of this transcript in CRF perikarya in the parvo-PVH. These data and the functional modifications of colonic transit argue for an adaptative cellular mechanism involving an attenuated action of CRF within the PVH under chronic stress. (1) Ann.N.Y.Acad.Sci. 697:233-243,1993. (2) J.Neurosci.,1995 (in press).
O COMPARISON OF SEROTONIN RECEPTORS MEDIATING FLUID SECRETION IN THE ISOLATED HUMAN INTESTINE. R.A. Borman, D.E. Burleigh. Department of Pharmacology, Queen Mary & Westfleld College, University of London, UK. The aim of this study was to characterize the receptors responsible for serotonin (5-HT)-induced fluid secretion in the human small and large intestine. Segments Of terminal ileum, ascending and sigmoid colon were obtained from specimens removed at operations for carcinoma or Crohn's disease. Musclestripped sheets of mucosa complete with submucosa were mounted in Ussing chambers for measurement of short-circuit current (SCC); an indicator of electrognnic fluid secretion. Tissues were exposed to varying concentrations of 5-HT, and responses tested against a selection of 5-HT receptor amagonists. [n the small intestine, serosal application of 5-HT (0.3-100#M) induced a maximum rise in SCC of 65.4_+3.0/~Acm"2 (11=16). Methysergide (10/zM), ketanserin (I#M) or ondansetron (10FM) had no effect on the 5-HT-induced rise in SCC (n=6). Application of DAU 6285 (0.3-10p`M; a selective 5-HT4 receptor antagonis0 caused a rightward shift of the concentration-response curve to 5-HT, with no change in the maximum response to 5-HT. Schild analysis of this antagonism gave a Schild plot with unit slope, allowing a pA~ estimate of 6.17_+0.06 (n>6). In sigmoid -colon, application of 5-HT (1-100p`M) caused a maximum rise in SCC of 35.0-+3.0#Acrea (n=16). The response was unaffected by application of DAU 6285 (10p.M) or ondansetron .(10p.M). Application of lp`M ketanserin, however, was shown to abolish the response to 10p`M 5-HT, and even at a concentration of 30nM led to a significant (88.2_+_4.9%) reduction in the 5-HTinduced rise in SCC (n=10, p<0.05). In ascending colonic mucosa, 5-HT (1-100~tM) induced a maximum rise in SCC of 32.4+2.5#Acre "~ (n = 18). The response was insensitive to application of methysergide (10#M) or ondansetron (10#M). Application of DAU 6285 (10p`M) Caused a rightward shift of the response curve to 5-HT, yielding a pA2 estimate of 5.78-1-0.20 (n=4). In two out of six tissues however, ketansedn (lp.M) was also shown to antagonise the secretory response to 5-HT. In conclusion, 5-HT induces sided electrogenie fluid secretion in the small and large intestine of man. In the ileum, a receptor of the 5-HT4 sub-type mediates this effect, whereas a 5-HT2 receptor appears to mediate secretion in the sigmoid colon. The receptor mediating secretion in ascending colonic mueosa cannot be definitively classed as either of these sub-types at this time, but may reflect a heterogenous receptor population.
AGA ABSTRACTS
EXPRESSION OF G-PROTEIN ALPHA SUBUN1TS IN NORMAL RAT COLONOCYTES, AZOXYMETHANE (AOM)-INDUCED COLON TUMORS, AND HUMAN COLON CANCER-DERIVED CELL LINES. M.J.G. Bolt, R.J. Mailloux, R. Wali, B . Frawley, B. Scaglione-Sewell, M. Rasenick*, B.M. Bissonnette, T.A. Brasitus, M.D. Sitrin. Dept. of Medicine, University of Chicago and *Dept. of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, IL , Heterotrimeric regulatory guanine nucleotide-binding proteins (Gproteins) couple many membrane bound receptors to a variety Of intracellular effectors, but vary in expression according tO cell type. We used isolated colonocytes from Sprague-Daw!ey rats to prepare eytosol and • enriched basolateral (BLM) and brush-border membranes (BBM). By use of specific antibodies to G-protein alpha subunits (Ga), western blotting techniques, and laser densitometry, we were able to measure the relative amounts of G a in these cell fractions. Both BLM and BBM were significantly enriched over the initial homogenate(s) when probed with Gai2, God3, Gc~o, Gctq/l 1, Gas, or Gel3; with BLM ranging from 4 to 60 fold and BBM from 1.7 to 7 fold. Relative enrichment of BLM over BBM was 8.5 for Gc~i2, 5.1 for Gctq/ll, 4.2 for Gcd3, and about 2.5 for Gc~o, G a s , Gel3: In contrast, G{xz was found entirely in the cytosolic fraction, differing from the membrane-bound location reported for Gctz in several other cell types. GcO2 was at such low abundance that we could not determine membrane enrichment. However, in human colonic cancer cell lines, CaCo-2, SW480, and HTC-I 1~ expressed large amounts of Goal2. Based on these findings, tumor tissue taken from colons of AOM-dosed Fisher 344 rats, a well-known model of colon carcinogenesis, were examined. All 4 tumors screened expressed large amounts of Goal2, wlaile normal colonic tissue from 3 vehicle-treated animals showed only a trace o f Gc~12. Gccl3, the closest homologue to Gc~12, had a low expression in all groups. Conclusion: The present studies demonstrate that the alpha subunits of G-proteins.are differentially localized to various regions of the rat colonocyte, which may underlie their role in various physiologic processes. Moreover, expression of Gc~12 in colonic tissue is related to cellular transformation, but whether this is a primary or secondary event requires additional study.
• CHARACTERIZATION OF SEROTONIN RECEPTORS INFLUENCING MOTILITY IN THE ISOLATED HUMAN INTESTINE. R.A, Borman, D.E. Burleigh. Department of Pharmacology, Queen Mary & Westfield College, University of London, UK. The aim of the present study was to investigate the receptors responsible for the motile effects of serotonin (5-HT) in the small and large intestine of man. Briefly, segments of small and large intestine were obtained from specimens removed at operations for carcinoma or Crohn's disease. Muscle strips were mounted under isotonic conditions in gassed Krebs solution at 37°C. 5-HT was applied to the tissues in a cumulative manner, and responses tested against a variety of 5-HT receptor antagonists. 5-HT was shown to relax circular muscle strips of human sigmoid e01on with an ECho of 0.24#M (95%C.L. 0.16-0.34, n=15). Unlike methysergide (10/zM), ketanserin (I#M) or ondansetron (10p`M), DAU 6285 (10p`M; a 5-HT4 receptor antagonist) significantly antagonised the response to 5-HT, yielding a pA2 estimate of 6.81_+0.23 (n= 10)~ In the small intestine, 5-HT induced contraction of both circular and longitudinal muscle strips, with an ECso of 58:6nM (95 %C.L. 21.5;86~5). and 34.9nM (95% C.L. 26.3-46.2) respectively. Methysergide was found to be a non-surmountable antagonist of the response to 5-HT in both mdscle layers, 16ut was approximately 1000-fold more potent in longitudinal than circular musdle. Methysergide (10nM) abolished the response to 5-HT in longitudinal mus61e, compared to 10p`M in circular muscle. In addition, ketanserin (30-100nM) was shown to antagonise the response to 5-HT of circular muscle; this antagonism was non-surmountable at I#M ketanserin. This concentration had no effect on the response to 5-HT of human longitudinal muscle. The'response in longitudinal muscle was, however, susceptible to antagonism by application of yohimbine (30-300riM; a 5-HT~ and 5:HT2s receptor antagonist, pA 2 of 8.10_+0.06) or SB 200646A (0.3-3.0p`M; a 5-HT2B and 5-HT2c receptor antagonist; pA2 of 7.17_+0.16). In conclusion, 5-HT has been shown to induce relaxation of human colonic circular mnsele via a receptor of the 5-HT4 sub-typel whereas'it was shown to contract both circular and longitudinal muscle layers of human small intestine. In longitudinal muscle, 5-HT-induced contraction is mediated via a 5-HT2a receptor, however the receptor responsible in circul~" muscle Can not be definitively classified at this time.
GASTROENTEROLOGY, Vol. 108, NO. 4
• EFFECT OF A CHRONIC IMMOBILIZATION STRESS ON COLONIC TRANSIT AND THE TRANSCRIPTION OF T H E GENE ENCODING THE CRF RECEPTOR 1N THE RAT HYPOTHALAMUS. B. Bonaz* and S. Rivest**. *Laboratory of Neurophysiology, INSERM U318, CHU, 38043 Grenoble cedex 09, F R A N C E and ** Laboratory of Molecular Endocrinology, Laval University, Qu6bec, CANADA. Corticotropin-releasing factor (CRF) action:in the CNS is part of the mechanism through which stress alters Colonic motor function in r a t s ( I ). Acute immobilization stress induces transcription of the gene encoding the C R F receptor (CRF-R) in the paraventricular nucleus of the hypothalamus (PVH; parvo-cellular part) (2). Purpose: to study, ir~ rats, the effect of a chronic immobilization stress, 1) on the transcription of the gene encoding for the CRF-R and on the localization of this transcript in CRF perikarya in 'the PVH, 2) on concomitant colonic transit changes. Methods: male SD rats (250g; n--40), with free access to food and water, w e r e submitted to a 90-rain immobilization stress either acutely or chrolllbally during 11 ~onsecutive days. Fecal pellet output was quantified at the end of the 90-rain immobilization session. Rats were killed before, immediately, 1.5, 3, 6 and 12 h after the end of the acute and chronic sessions. Rats were anesthetized and perfused with a solution of 4% parafonnaldehyde:borax: Coronal frozen sections of the brain were cut and the mRNA encoding the rat CRF-R was assayed by in situ hybridization (ISH) using a 35S-k-ibeled riboprobe (2). CRF-R localization within CRF-immunoreactive netitons in the PVH was determined using a combination of imn-/un0cytochemistry and ISH. Results: 1) in acutely stressed rats, mRNA encoding the CRF-R was highly expressed in the parvo-PVH 90 rain and 3 hr after the end of the stress and totally vanished 12 h after the stress. Numerous .CRFimmunoreactive neurons of the p a r v o - P V H expressed the CRF-R transcript. 12:5 + 0.7 pellets were counted at the end of the stress. 2) in chronically stressed rats, a significant decrease of the CRF-R mRNA signal was observed in the parvo-PVH 90 rain and 3 h after the end of the stress session: Very few CRF immunoreactive neurons o f the parvO-PVH expressed the CRF-R transcript. In parallel; a significant decrease of the fecal pellet output was observed (DI: 12.1_+ 0.8; DI 1:7.5 -+ 0.9; P<0.05). Conclusions: a chronic immobilization stress is followed by a sighificant decrease of the transcription of the gene encoding for the CRF-R and of the localization of this transcript in CRF perikarya in the parvo-PVH. These data and the functional modifications of colonic transit argue for an adaptative cellular mechanism involving an attenuated action of CRF within the PVH under chronic stress. (1) Ann.N.Y.Acad.Sci. 697:233-243,1993. (2) J.Neurosci.,1995 (in press).
O COMPARISON OF SEROTONIN RECEPTORS MEDIATING FLUID SECRETION IN THE ISOLATED HUMAN INTESTINE. R.A. Borman, D.E. Burleigh. Department of Pharmacology, Queen Mary & Westfleld College, University of London, UK. The aim of this study was to characterize the receptors responsible for serotonin (5-HT)-induced fluid secretion in the human small and large intestine. Segments Of terminal ileum, ascending and sigmoid colon were obtained from specimens removed at operations for carcinoma or Crohn's disease. Musclestripped sheets of mucosa complete with submucosa were mounted in Ussing chambers for measurement of short-circuit current (SCC); an indicator of electrognnic fluid secretion. Tissues were exposed to varying concentrations of 5-HT, and responses tested against a selection of 5-HT receptor amagonists. [n the small intestine, serosal application of 5-HT (0.3-100#M) induced a maximum rise in SCC of 65.4_+3.0/~Acm"2 (11=16). Methysergide (10/zM), ketanserin (I#M) or ondansetron (10FM) had no effect on the 5-HT-induced rise in SCC (n=6). Application of DAU 6285 (0.3-10p`M; a selective 5-HT4 receptor antagonis0 caused a rightward shift of the concentration-response curve to 5-HT, with no change in the maximum response to 5-HT. Schild analysis of this antagonism gave a Schild plot with unit slope, allowing a pA~ estimate of 6.17_+0.06 (n>6). In sigmoid -colon, application of 5-HT (1-100p`M) caused a maximum rise in SCC of 35.0-+3.0#Acrea (n=16). The response was unaffected by application of DAU 6285 (10p.M) or ondansetron .(10p.M). Application of lp`M ketanserin, however, was shown to abolish the response to 10p`M 5-HT, and even at a concentration of 30nM led to a significant (88.2_+_4.9%) reduction in the 5-HTinduced rise in SCC (n=10, p<0.05). In ascending colonic mucosa, 5-HT (1-100~tM) induced a maximum rise in SCC of 32.4+2.5#Acre "~ (n = 18). The response was insensitive to application of methysergide (10#M) or ondansetron (10#M). Application of DAU 6285 (10p`M) Caused a rightward shift of the response curve to 5-HT, yielding a pA2 estimate of 5.78-1-0.20 (n=4). In two out of six tissues however, ketansedn (lp.M) was also shown to antagonise the secretory response to 5-HT. In conclusion, 5-HT induces sided electrogenie fluid secretion in the small and large intestine of man. In the ileum, a receptor of the 5-HT4 sub-type mediates this effect, whereas a 5-HT2 receptor appears to mediate secretion in the sigmoid colon. The receptor mediating secretion in ascending colonic mueosa cannot be definitively classed as either of these sub-types at this time, but may reflect a heterogenous receptor population.