Cross-Atlantic Collaboration

Exocrine Pancreatic Secretion

100 Years of Scientific Discovery

19th Century

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.

1974 Nobel Prize:George PaladeChristian DeDuveAlbert Claude

King Gustaf, SwedenStanford Moore

Nobels

Alfred Nobel

A Collaboration into the 21st Century

Professor Horst Kern, Cell Biology, Philipps University, Marburg, GermanyProfessor George Scheele, Cell Biology, Rockefeller University, New York, NY

Pancreatic Lobules

• Secretory units intact - Acinar tissue – CCK stimulation - Ductules – Secretin stimulation

• Acinar lumen isolated from incubation medium

Lobule Advantages:

Pancreatic Acinar LumenApical Plasma Membrane

Exocytosis & Endocytosis

Hormone Stimulation

Accelerates Intracellular Transport

A Control (Resting tissue)

B Caerulein stimulation (CCK analogue)

2-D Gel Identifies Pancreatic EnzymesGP Dog Human

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.

Removal of Signal Peptide

Necessary 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

2-Dimensional Gel ElectrophoresisSecretory Enzymes & Isoenzymes

Separation of proteins by:- Charge (IEP)- Size (Mr in Daltons)

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

Hormones Regulate Pavlov’s Dietary Adaptation

CCK Stimulation:- Proteinase synthesis increased- Amylase synthesis decreased

Secretin stimulation:- lipase synthesis increased

Second Messenger Pathways

Regulate Protein Synthesis Patterns

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

1986

1993

Ex Libris

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 membranesTHP (Tamm Horsfall Protein/Uromodulin) – Membrane protein in kidney (TALH)

GP2 probe

THP probe

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

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

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

GP2 Release Correlates with HRP Uptake

Acinar lumen – GP2 Ab (Geuze) Alcian blue GP2 Ab

Sedimented PJ - Beaudoin

Exoplasmic Surface of ZGProtein Sorting in TGN

Role of GP2-Proteoglycan Matrix

Enzymatic Release of GP2 & Activation of Endocytosis

PH-dependent process

Association of GP2 & Proteoglycans

PH-Dependent Release

Cosedimentation

Cystic Fibrosis Bicarbonate Deficiency in Acinar Lumen

Ion Deficiencies

Location of CFTR

Dysfunction in Apical Membrane Trafficking

HYPOTHESIS

Flourescence Ab Localization of CFTRCFTR Ab – Low exposure

Control – Amylase Ab

CFTR Ab – High exposure

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

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

CFTR Knockout Mice (-/-)In-Vitro Correction of Membrane Trafficking Defect

HRP Uptake GP2 Release

• Alkaline pH

• Acidic pH + PI-PLC

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 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

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

Cystic Fibrosis

Conclusions:

Accomplishment

Joy

Satisfaction