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Cross-Atlantic Collaboration Exocrine Pancreatic Secretion

Cross-Atlantic Collaboration Exocrine Pancreatic Secretion. 100 Years of Scientific Discovery. 19 th Century. 20 th Century.

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Cross-Atlantic Collaboration Exocrine Pancreatic Secretion

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  1. Cross-Atlantic CollaborationExocrine Pancreatic Secretion 100 Years of Scientific Discovery

  2. 19th Century

  3. 20th Century In Subcellular Particles: F. Hayashi, edit., The Ronald Press, New York, 1959….. Dr. Philip Siekevitz and I decided to undertake such a project, which, as can be seen, combines new techniques with old and simple experimental approaches. It is, one could say, a collaboration over almost a century between Rudolf Heidenhain, Philip Siekevitz and myself. George E. Palade, Rockefeller Institute for Medical Research, New York, N.Y.

  4. Nobels Alfred Nobel 1974 Nobel Prize:George PaladeChristian DeDuveAlbert Claude King Gustaf, SwedenStanford Moore

  5. A Collaboration into the 21st Century Professor Horst Kern, Cell Biology, Philipps University, Marburg, GermanyProfessor George Scheele, Cell Biology, Rockefeller University, New York, NY

  6. Pancreatic Lobules Lobule Advantages: • Secretory units intact - Acinar tissue – CCK stimulation - Ductules – Secretin stimulation • Acinar lumen isolated from incubation medium

  7. Pancreatic Acinar LumenApical Plasma Membrane

  8. Exocytosis & Endocytosis

  9. Hormone StimulationAccelerates Intracellular Transport A Control (Resting tissue) B Caerulein stimulation (CCK analogue)

  10. 2-D Gel Identifies Pancreatic Enzymes Dog GP Human

  11. Signal Hypothesis – Translocation of Secretory Proteins into the RER N-terminal signal sequence targets proteins for translocation into the RER – Explains absence of initiator Met in mature enzymes.

  12. Removal of Signal PeptideNecessary for correct Folding of Enzymes A Unfolded precursors aggregateB-D Correctly folded proteins migrate to correct position in 2-D gel Demonstrated importance of (i) membrane translocation, (ii) optimal redox potential and (iii) protein disulfide isomerase

  13. 2-Dimensional Gel ElectrophoresisSecretory Enzymes & Isoenzymes Separation of proteins by:- Charge (IEP)- Size (Mr in Daltons)

  14. Dietary Adaptation A. Protein-Sufficient Diet (Abundance Mechanism): - Enzymes synthesized in direct proportion to nutritional substrates in diet B. Protein-Deficiency (Survival Mechanism): - Enzymes synthesized according to isoelectric point - 95% of synthesis channeled into acidic proteinases

  15. Hormones Regulate Pavlov’s Dietary Adaptation CCK Stimulation:- Proteinase synthesis increased- Amylase synthesis decreased Secretin stimulation:- lipase synthesis increased

  16. Second Messenger PathwaysRegulate Protein Synthesis Patterns

  17. Experimental PancreatitisSupramaximal Secretagogue Stimulation In vivo Model – Marburgin vitro model – New York • Intracellular granule fusion • Lateral Exocytosis – amylase secretion into the interstitial space and blood stream

  18. Ex Libris 1993 1986

  19. GP2-THP Gene Family GP2 – The major protein in pancreatic ZG membranesTHP (Tamm Horsfall Protein/Uromodulin) – Membrane protein in kidney (TALH) Widespread tissue distribution: GP2 probe THP probe Unique N-terminal THP sequence: (Four EGF motifs, each defined as 1-6)

  20. Apical Membrane TraffickingAcinar Lumen pH Regulates Endocytosis, not Exocytosis Prestimulation, CCK, 1 hr, pH 7.4:A Dilated acinar lumen, pH 6.0B Contracted acinar lumen, pH 8.3Controls:C Resting pancreas, pH 6, 1 hrD CCK stimulation, pH 6, 1 hr

  21. Apical Membrane TraffickingAcinar Lumen PH Regulates Endocytosis Prestimulation, 1 nM CCK, pH 7, 1 hr:a 2nd hr, HRP, pH 7.4b 2nd hr, HRP, pH 7.4 (higher magnif)c 2nd hr, HRP, pH 6.0d 2nd hr, HRP, pH 6.0 (higher magnif) Note “arrested” exocytic image

  22. PI-PLC Releases GP2 and Activates Endocystosis at pH 6.0 Acini prestimulated, 1 nM CCK, pH 7.4, 1 hr:a 2nd hr, HRP, pH 6.0b 2nd hr, HRP+PLC, pH 6.0, Apical lumenc 2nd hr, HRP+PLC, pH 6.0, Golgi regiond 2nd hr, HRP+PLC, pH 6.0, Apical lumen, increased magnification

  23. GP2 Release Correlates with HRP Uptake Sedimented PJ - Beaudoin Alcian blue Acinar lumen – GP2 Ab (Geuze) GP2 Ab

  24. Role of GP2-Proteoglycan Matrix Protein Sorting in TGN Exoplasmic Surface of ZG Enzymatic Release of GP2 & Activation of Endocytosis PH-dependent process

  25. Association of GP2 & Proteoglycans Cosedimentation PH-Dependent Release

  26. Cystic FibrosisBicarbonate Deficiency in Acinar Lumen Ion Deficiencies Dysfunction in ApicalMembrane Trafficking Location of CFTR HYPOTHESIS

  27. Flourescence Ab Localization of CFTR CFTR Ab – Low exposure CFTR Ab – High exposure Control – Amylase Ab

  28. CFTR Knockout Mice (-/-)Membrane Trafficking defects HRP Uptake Impaired 1-Step ProtocolLobules prestim., 0.5 nM CCK-8, 0.5 hrA Wild-type mice: CFTR +/+B Heterozygous mice: CFTR +/-C Homozygous mice: CFTR -/-D Homozygous mice: CFTR -/-2-step Protocol:E 2nd period, pH 8.0F 2nd period, pH 8.0

  29. CFTR Knockout Mice (-/-)PH Defect in Secretin-Stimulated Pancreatic Juice

  30. CFTR Knockout Mice (-/-)In-Vitro Correction of Membrane Trafficking Defect • Alkaline pH • Acidic pH + PI-PLC HRP Uptake GP2 Release

  31. Membrane Trafficking & Protein Sorting Conclusions: Role of GP2-Proteoglycan Matrix in:1. Sorting of membranes in the Trans-Golgi Network (TGN)2. Role of TGN acidification in sorting of soluble proteins - Soluble proteins translocate to endo-lysosomal compartments - Precipitating proteins retained in condensing vacuoles and ZGs Bicarbonate Secretion by Ductal Cells Regulates:1. PH of the acinar lumen – Neutralizes the acid pH associated with exocytosis2. Solubilization of secretory (pro)enzymes 3. Membrane trafficking at the apical plasma membrane - Role of GP2 release in activating endocytosis & membrane recycling at the APM Acinar Lumen Couples Acinar and Duct Cell Function:1. CCK stimulates release of secretory enzymes from ZGs in an acidic milieu2. Secretin stimulates alkaline fluid secretion from ductal cells3. Reveals acid-base interplay between acinar and ductal cells

  32. Cystic Fibrosis Conclusions: Pancreatic Defects in Cystic Fibrosis:1. Genetic defect in the CFTR – Deficiency in chloride secretion by duct cells2. Associated defect in bicarbonate secretion3. Progressive acidification of the acinar and duct lumen4. Persistent aggregation of secretory enzymes released into the duct lumen5. Massive dilatation of the acinar lumen6. Marked decrease of ZGs7. Loss of the apical pole of the acinar cell 8. Progressive loss of pancreatic exocrine function Biochemical Defect in Cystic Fibrosis: Progressive deficiency of chloride & bicarbonate in the acinar and duct lumenleading to inappropriate acidification of pancreatic luminal compartments Potential Treatment Modality:Restoration or administration of bicarbonate to the duct lumen with correction of the pH defect

  33. Accomplishment Satisfaction Joy

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