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

2. 19 th Century

3. 20 th 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. 1974 Nobel Prize: George Palade Christian DeDuve Albert Claude King Gustaf, Sweden Stanford Moore Nobels Alfred Nobel

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

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

7. Pancreatic Acinar Lumen Apical Plasma Membrane

8. Exocytosis & Endocytosis

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

10. 2-D Gel Identifies Pancreatic Enzymes GP Dog 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 Peptide Necessary for correct Folding of Enzymes A Unfolded precursors aggregate B-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 Electrophoresis Secretory 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 Pathways Regulate Protein Synthesis Patterns

17. Experimental Pancreatitis Supramaximal Secretagogue Stimulation In vivo Model – Marburg in vitro model – New York Intracellular granule fusion Lateral Exocytosis – amylase secretion into the interstitial space and blood stream

18. 1986 1993 Ex Libris

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

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

21. Apical Membrane Trafficking Acinar Lumen PH Regulates Endocytosis Prestimulation, 1 nM CCK, pH 7, 1 hr: a 2 nd hr, HRP, pH 7.4 b 2 nd hr, HRP, pH 7.4 (higher magnif) c 2 nd hr, HRP, pH 6.0 d 2 nd 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 2 nd hr, HRP, pH 6.0 b 2 nd hr, HRP+PLC, pH 6.0, Apical lumen c 2 nd hr, HRP+PLC, pH 6.0, Golgi region d 2 nd hr, HRP+PLC, pH 6.0, Apical lumen, increased magnification

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

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

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

26. Cystic Fibrosis Bicarbonate Deficiency in Acinar Lumen Ion Deficiencies Location of CFTR Dysfunction in Apical Membrane Trafficking HYPOTHESIS

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

28. CFTR Knockout Mice (-/-) Membrane Trafficking defects HRP Uptake Impaired 1-Step Protocol Lobules prestim., 0.5 nM CCK-8, 0.5 hr A Wild-type mice: CFTR +/+ B Heterozygous mice: CFTR +/- C Homozygous mice: CFTR -/- D Homozygous mice: CFTR -/- 2-step Protocol: E 2 nd period, pH 8.0 F 2 nd 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 HRP UptakeGP2 Release Alkaline pH Acidic pH + PI-PLC

31. Membrane Trafficking & Protein Sorting 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 exocytosis 2. 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 milieu 2. Secretin stimulates alkaline fluid secretion from ductal cells 3. Reveals acid-base interplay between acinar and ductal cells Conclusions:

32. Pancreatic Defects in Cystic Fibrosis: 1. Genetic defect in the CFTR – Deficiency in chloride secretion by duct cells 2. Associated defect in bicarbonate secretion 3. Progressive acidification of the acinar and duct lumen 4. Persistent aggregation of secretory enzymes released into the duct lumen 5. Massive dilatation of the acinar lumen 6. Marked decrease of ZGs 7. 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 lumen leading 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 Cystic Fibrosis Conclusions:

33. Accomplishment Joy Satisfaction