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Trafficking of the receptor for gastrin releasing peptide in swiss 3T3 cells after exposure to agonist
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Abstracts fi'om the 11 th hzternational Symposium on Regulatory Peptides
TRAFFICKING OF THE RECEPTOR FOR GASTRIN RELEASING PEPT1DE IN SWISS 3T3 CELLS AFTER EXPOSURE TO AGONIST. N.W. Bunnett, G. Dulai, L. Slice, J.H. Walsh, E.F. Grady, University of California, San Francisco, and University of California, Los Angeles. Endocytosis of receptors may contribute to desensitization and resensitization of cellular responses. Internalization of the receptor for gastrin releasing peptide (GRP-R) has been directly observed in transfected cell lines. Here we extend these studies to Swiss 3T3 cells, which naturally express the GRP-R and which respond to GRP by growth. Swiss 3T3 cells were enriched for high expression of GRP-R by FACS using cyanine 3-GRP (cy3-GRP). There were -10,000 binding sites/cell prior to sorting, and ~80,000 binding sites/cell in the sorted population, as determined by Scatchard analysis using 125I-GRP. Cells were incubated with 200 nM cy3-GRP or 50 nM GRP for 2 h at 4"C and warmed to 37°C for 0-120 rain. They were washed, fixed in 4% paraformaldehyde, and observed by confocal microscopy. Some cells were processed for immunofluorescence using an antiserum to the C-terminus of the mouse GRP-R, and a monoclonal antibody to the human transferrin receptor. At 40C, cy3-GRP was detected at the cell-surface. After 5 min at 37°C, numerous small vesicles containing cy3-GRP were observed immediately beneath the plasma membrane. By 10 min, prominent vesicles with cy3-GRP were evenly distributed throughout the cell. After 30 min, in some cells, a perinuclear staining pattern occurred which was more prominent after 60 min, and was present in most cells after 90 min. We then examined the distribution of the GRP-R. At 4°C, weak surface GRP-R immunoreactivity was evident. After 30s at 37°C, GRP-R was visible in very small vesicles. By 5 rain, the GRP-R was present in small and larger vesicles. By 10-15 min, GRP-R immunoreactivity was intense, and present in vesicles of even size distributed randomly throughout the cells. After 30 min, the GRP-R was in a small perinuclear sorting region in some cells. By 60 min, many fine vesicles as well as a perinuclear staining for GRP-R were apparent. At 90-120 min, most cells had some GRP-R immunoreactivity by the nucleus~ in very fine vesicles, and on the cell surface. After 2, 5 and 10 rain at 37*C, the GRP-R was colocalized with the transferrin receptor, thus it is present in a recffcling compartment. We measured the rate of GRP-R internalization in binding experiments with 125I-GRP, using an acid wash to separate surface from internalized peptide. Binding of 125I-GRP to Swiss 3T3 cells at 40C, followed by an acid wash showed 90% of surface counts were internalized after 20 min at 37°C. Sucrose and phenylarsine oxide inhibited internalization. Thus, GRP induces rapid internalization of the GRP-R into a recycling compartment in Swiss 3T3 cells. Unlike transfected rat kidney epithelial cells, which recover surface GRP-R 30 min after internalization, Swiss 3T3 cells process internalized GRP-R at a much slower rate. Supported by NSF IBN9510314.
CENTRAL NERVOUS SYSTEMEFFECTSOF PACAP 1-38 ON GASTROINTESTINALTRANSIT IN THE RAT
M. Burlage, U.R. F61sch, W.E. Schmidt. 1st Department of Medicine, Christian Albrechts University 24105 Kiel, Germany Pituitary Adenylate Cyclase Activating Polypeptide (PACAP 1-38) is a novel member of the glucagon/secretin/VIP peptide family and acts as neurotransmitter. PACAP is expressed in neurons of the esophagus, stomach, small and large bowel of sheep, rat and man. In vitro PACAP contracts guinea pig ileum and relaxes human longitudinal sigmoidal muscle strips. The purpose of this study was to examine if intracerebroventricularly (icv) injected PACAP affects gastrointestinal motility in awake rats. METHODS: Male Sprague-Dawley rats (250 - 300 g) were equipped with intracerebroventricular stainless steel cannulas and gastric, duodenal and colonic silicone tubing that were exteriorized to the interscapular region of the animal's neck. Three to four days postoperatively synthetic PACAP 1-38 (0.1-10 l~nol) or 0.15 M NaCl as a control were supplied icy in 10 pl 20 minutes before injection of intestinal markers: stomach (phenol red 0.75 mg in 1.5 ml 0.15 M NaCI), proximal d u o d e n u m (51Cr 0.4 pCi in 0.2 ml of 0.15 M NaCI) and proximal colon (51Cr 0.4 ~Ci in 0.2 ml of xanthum gum). Thirty minutes later, gastric emptying was determined by standard methods and small and large bowel transits were assessed by calculation of the geometric center of the markers. RESULTS: PACAP 1-38 administered into the lateral cerebral ventricle resulted in a dose-dependent and significant inhibition of large bowel transit. Small bowel transit was depressed slightly. Up to a dose of 10 nmol injected icv, PACAP 1-38 had no influence on gastric emptying of a liquid meal. CONCLUSIONS: PACAP 1-38 may participate in the central control of gastrointestinal motility.
Abstracts fi'om the 11 th hzternational Symposium on Regulatory Peptides
TRAFFICKING OF THE RECEPTOR FOR GASTRIN RELEASING PEPT1DE IN SWISS 3T3 CELLS AFTER EXPOSURE TO AGONIST. N.W. Bunnett, G. Dulai, L. Slice, J.H. Walsh, E.F. Grady, University of California, San Francisco, and University of California, Los Angeles. Endocytosis of receptors may contribute to desensitization and resensitization of cellular responses. Internalization of the receptor for gastrin releasing peptide (GRP-R) has been directly observed in transfected cell lines. Here we extend these studies to Swiss 3T3 cells, which naturally express the GRP-R and which respond to GRP by growth. Swiss 3T3 cells were enriched for high expression of GRP-R by FACS using cyanine 3-GRP (cy3-GRP). There were -10,000 binding sites/cell prior to sorting, and ~80,000 binding sites/cell in the sorted population, as determined by Scatchard analysis using 125I-GRP. Cells were incubated with 200 nM cy3-GRP or 50 nM GRP for 2 h at 4"C and warmed to 37°C for 0-120 rain. They were washed, fixed in 4% paraformaldehyde, and observed by confocal microscopy. Some cells were processed for immunofluorescence using an antiserum to the C-terminus of the mouse GRP-R, and a monoclonal antibody to the human transferrin receptor. At 40C, cy3-GRP was detected at the cell-surface. After 5 min at 37°C, numerous small vesicles containing cy3-GRP were observed immediately beneath the plasma membrane. By 10 min, prominent vesicles with cy3-GRP were evenly distributed throughout the cell. After 30 min, in some cells, a perinuclear staining pattern occurred which was more prominent after 60 min, and was present in most cells after 90 min. We then examined the distribution of the GRP-R. At 4°C, weak surface GRP-R immunoreactivity was evident. After 30s at 37°C, GRP-R was visible in very small vesicles. By 5 rain, the GRP-R was present in small and larger vesicles. By 10-15 min, GRP-R immunoreactivity was intense, and present in vesicles of even size distributed randomly throughout the cells. After 30 min, the GRP-R was in a small perinuclear sorting region in some cells. By 60 min, many fine vesicles as well as a perinuclear staining for GRP-R were apparent. At 90-120 min, most cells had some GRP-R immunoreactivity by the nucleus~ in very fine vesicles, and on the cell surface. After 2, 5 and 10 rain at 37*C, the GRP-R was colocalized with the transferrin receptor, thus it is present in a recffcling compartment. We measured the rate of GRP-R internalization in binding experiments with 125I-GRP, using an acid wash to separate surface from internalized peptide. Binding of 125I-GRP to Swiss 3T3 cells at 40C, followed by an acid wash showed 90% of surface counts were internalized after 20 min at 37°C. Sucrose and phenylarsine oxide inhibited internalization. Thus, GRP induces rapid internalization of the GRP-R into a recycling compartment in Swiss 3T3 cells. Unlike transfected rat kidney epithelial cells, which recover surface GRP-R 30 min after internalization, Swiss 3T3 cells process internalized GRP-R at a much slower rate. Supported by NSF IBN9510314.
CENTRAL NERVOUS SYSTEMEFFECTSOF PACAP 1-38 ON GASTROINTESTINALTRANSIT IN THE RAT
M. Burlage, U.R. F61sch, W.E. Schmidt. 1st Department of Medicine, Christian Albrechts University 24105 Kiel, Germany Pituitary Adenylate Cyclase Activating Polypeptide (PACAP 1-38) is a novel member of the glucagon/secretin/VIP peptide family and acts as neurotransmitter. PACAP is expressed in neurons of the esophagus, stomach, small and large bowel of sheep, rat and man. In vitro PACAP contracts guinea pig ileum and relaxes human longitudinal sigmoidal muscle strips. The purpose of this study was to examine if intracerebroventricularly (icv) injected PACAP affects gastrointestinal motility in awake rats. METHODS: Male Sprague-Dawley rats (250 - 300 g) were equipped with intracerebroventricular stainless steel cannulas and gastric, duodenal and colonic silicone tubing that were exteriorized to the interscapular region of the animal's neck. Three to four days postoperatively synthetic PACAP 1-38 (0.1-10 l~nol) or 0.15 M NaCl as a control were supplied icy in 10 pl 20 minutes before injection of intestinal markers: stomach (phenol red 0.75 mg in 1.5 ml 0.15 M NaCI), proximal d u o d e n u m (51Cr 0.4 pCi in 0.2 ml of 0.15 M NaCI) and proximal colon (51Cr 0.4 ~Ci in 0.2 ml of xanthum gum). Thirty minutes later, gastric emptying was determined by standard methods and small and large bowel transits were assessed by calculation of the geometric center of the markers. RESULTS: PACAP 1-38 administered into the lateral cerebral ventricle resulted in a dose-dependent and significant inhibition of large bowel transit. Small bowel transit was depressed slightly. Up to a dose of 10 nmol injected icv, PACAP 1-38 had no influence on gastric emptying of a liquid meal. CONCLUSIONS: PACAP 1-38 may participate in the central control of gastrointestinal motility.