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Serving as Laboratory Director at the Rockefeller University for 14 years (1975-1988), Yale University School of Medicine for one year (1974) and Harvard Medical School for 7 years (1988-1995) and utilizing the disciplines of physiology, cell biology, electron microscopy, molecular biology and protein chemistry, my coworkers and I were able to make the following seminal discoveries:
A method for preparing pancreatic tissue for structural (electron microscopy) and in-vitro functional studies in a manner that minimizes tissue damage and maximizes functional performance. This procedure is used beneficially with pancreatic tissue taken from guinea pig, rat or mouse. The procedure involves using a syringe and a needle to inject Krebs Ringer Bicarbonate (KRB) solution into the interstitial space thereby distending the tissue and revealing the smallest lobular structures perceived by the human eye. Then, using a pair of ophthalmic scissors, these lobular structures may be removed from the duct system and incubated in KRB under in vitro physiological conditions. Normally 5-10 lobules are incubated in a single Erlenmeyer flask at 37 degrees Celsius and in the presence of 95% oxygen and 5% carbon dioxide. In experiments that conduct pulse-chase studies that rely on rapid transfer of lobules from one flask to another or rapid washing conditions, a piece of nylon mesh cut into a 2 cm circle, may be used to attach the lobules to a single mesh disk in each incubation flask. The connective tissue surrounding the lobules attach spontaneously to the nylon mesh. Following attachment of the lobules to the nylon mesh, which occurs immediately during shaking in a temperature-controlled water-filled incubator, a pair of forceps may be used to pick up the group of lobules for washing or transferring manipulations without introducing injury to the tissue itself. Slide Presentation - Pancreatic Lobules Ultilizing enzymatic digestion with collagenase and vigorous shearing forces, pancreatic lobules can be further reduced to pancreatic acini. However, there is an important functional difference between lobules and acini. In pancreatic lobules the acinar lumen is sequestered from the incubation medium allowing use of ductal inhibitors to manipulate the fluid, electrolyte and pH conditions of the acinar lumen. In pancreatic acini the conditions of the acinar lumen can be directly controlled by the conditions of the incubation medium. These functional differences allowed Dr. Freedman and myself to discover the role of bicarbonate secretion from duct cells in regulating the dissolution of secretory enzymes during exocytic release and the cellular uptake of exocytic membranes (secretory granule membranes inserted into the APM) by activated endocytosis at the APM. Due to the fragile nature of tissue in the mouse pancreas, pancreatic acini obtained after treating with collagenase and shear forces, show relatively poor performance with high background levels of secretion. Thus, it is optimal to use pancreatic lobules when studying the mouse pancreas in vitro. Further, pancreatic lobules taken from mouse pancreas act more like acini in that the conditions of the acinar lumen appear to be directly controlled by the incubation medium. This observation suggests that small pancreatic ductile segments are severed during the tissue distension procedure. 2D GEL ELECTROPHORESIS & CHARACTERIZATION OF PANCREATIC ENZYMES Following my invention of 2D gel electrophoresis we determined that the exocrine pancreas synthesizes approximately twenty (20) enzymes and isoenzymes that are secreted into the intestinal tract where they are responsible for the digestion of food substrates in the small intestine. The functional families of enzymes characterized by the 2D procedure include proteinases (trypsin, chymotrypsin and elastase forms), lipases (lipase, colipase cholesterol ester lipase forms), glycosidases (amylase forms), nucleases (ribonuclease and deoxyribonuclease forms), trypsin inhibitors (pancreatic secretory trypsin inhibitor) and secretory stress proteins (pancreatic stone protein [PSP] and pancreatitis associated protein [PAP]). The 2D gel procedure allowed us to characterize the entire set of pancreatic enzymes in 5 species, including guinea pig, rat, rabbit, dog and human. Together, these studies are responsible for the characterization of approximately 100 proteins (secretory enzymes and isoenzymes). Slide Presentation - Characterization of Pancreatic Enzymes Slide Presentation - Human Pancreatic Proteins Utilizing double isotope labeling in pulse-chase experiments analyzed by cell fractionation and 2D gel electrophoresis, Alan Tartakoff, George Palade and I were able to define the extent of "leakage" and "absorption" artifacts that occur during tissue homogenization and cell fractionation. These carefully controlled studies led to the first demonstration, beyond any reasonable doubt, that secretory proteins are synthesized on ribosomes attached to the Rough Endoplasmic Reticulum (RER) membrane and then sequentially transferred to the Golgi Apparatus, condensing vacuoles and secretory granules before the process of exocytosis leads to the release of secretory enzymes into the pancreatic juice and the intestinal lumen, the site of digestive activity. Once successfully completed, these studies realized the ultimate dream of Albert Claude, the Belgian engineer who first tried to separate cell fractions in isotonic NaCl gradients and later shared the 1974 Nobel Prize in Medicine with Drs. George Palade and Christian DeDuve. Slide Presentation - Secretory Pathway Defined PARALLEL RELEASE OF PANCREATIC ENZYMES DURING BASAL & SECRETAGOGUE STIMULATION George Palade and I studied the kinetics of discharge of three enzymes (amylase, lipase and ribonuclease) and four protease zymogens (trypsinogen, chymotrypsinogen, procarboxypeptidase A and procarboxypeptidase B) into the incubation medium under basal conditions and during stimulation with various secretagogues (CCK, cerulean and carbamylcholine chloride). For each experimental condition investigated, all seven activities, estimated to represent approximately 89% of the secretory output of the guinea pig pancreas, were discharged in constant proportions and were released from the tissue to the same proportional extent. The success of these studies depended on the careful determination of activation conditions (time, temperature, pH and ratio of activator to protein substrate) for each of the protease and lipase zymogens so that samples could be measured under concentration-response conditions that were strictly linear. Slide Presentation - Parallel Release of Pancreatic Enzymes MEMBRANE MODEL FOR PROTEIN TARGETING & RER SEQUESTRATION IN THE SIGNAL HYPOTHESIS Following my discovery that dog pancreatic microsomes are the membranes of choice for studying the translocation of nascent proteins across the RER membrane, Gunter Blobel and I first demonstrated that all of the pancreatic enzymes were initially synthesized as larger protein chains containing N-terminal "signal peptides" that are responsible for attachment of ribosomes to the membrane and translocation of the nascent enzyme across the membrane into a sequestered compartment, where, following protein folding, post-translational modifications and sequential transport through intracellular compartments (see above) the secretory enzymes were released into the acinar lumen contiguous with that of the pancreatic duct and intestinal lumen. Slide Presentation - Membrane Model for Protein Targeting DISCOVERY OF THE N-TERMINAL SIGNAL SEQUENCE FOR SECRETORY PROTEINS Based on my identification and characterization of pancreatic enzymes in the dog pancreas, Dr. Blobel and I then characterized the first N-terminal protein sequence for a secretory protein (cationic form of pancreatic trypsinogen in the dog pancreas), which provided the first supporting evidence for the signal hypothesis, which represented the major work honored by the 1999 Nobel Prize. Initially using N-terminal amino acid sequencing techniques and later using cDNA cloning techniques, we rapidly reported on the 15 N-terminal signal sequences associated with pancreatic presecretory proteins in functional enzyme families, including trypsinogen, chymotrypsinogen, proelastase, procarboxypeptidase, prophospholipase A2, lipase, amylase and ribonuclease forms in the rat and dog pancreas. Slide Presentation - Signal Peptides and RER Sequestration PURIFIED RIBONUCLEASE INHIBITOR In vitro translation and membrane transport studies were greatly assisted by the use of purified ribonuclease (RNase) inhibitor. The purification and characterization of RNase inhibitor from the human placenta, which represented a rich source, was accomplished by Peter Blackburn and Stanford Moore. Studies which demonstrated the utility of the RNase inhibitor in in-vitro translation studies were conducted in my laboratory in collaboration with Dr. Blackburn. Slide Presentation - Purified Ribonuclease Inhibitor IMPORTANCE OF REDOX POTENTIAL & CHAPERONES IN PROTEIN FOLDING FOLLOWING SEQUESTRATION IN THE ROUGH ENDOPLASMIC RETICULUM (RER) By utilizing 2D gel electrophoresis to monitor protein folding of nascent pancreatic enzymes, we could demonstrate that an optimal redox potential (ratio of oxidized glutathione [GSSG] to reduced glutathione [GSH]) was required along with protein disulfide isomerase to achieve the proper folding of proteins with authentic biological activity in secreted enzymes. Later studies revealed that chaperones were also required to fold proteins into their native configuration. Slide Presentation - Importance of Redox Potential for Protein Folding AUG INITIATION CODON CONSENSUS SEQUENCE DEFINED FOR PLANTS Together with Henrich Lutcke we defined the AUG initiation codon consensus sequence in plants. Slide Presentation - AUG Initiation Codon Consensus PANCREATIC ENZYMES CLONED IN THE DOG AND RAT PANCREAS In a collaboration with Professor Horst Kern at the Philipps Unversity in Marburg , Germany, we cloned, for the first time, the majority of genes that encode the pancreatic enzymes in both rat and dog pancreas. These cloning studies provided the amino acid sequences for all of the N-terminal signal peptides associated with presecretory proteins in the exocrine pancreas. Slide Presentation - Pancreatic Enzymes Cloned DIETARY ADAPTATION IN THE PANCREAS REGULATED BY DIFFERENTIAL GENE EXPRESSION Pavlov first recognized that the enzyme ferment in pancreatic juice was altered by changes in the nutritional substrates in the diet. The 2D gel electrophoresis technique proved to be optimal for measuring gene expression in the exocrine pancreas. Collaborating with the laboratory of Professor Horst Kern in the Philipps university, Marburg, Germany, we conducted detailed studies to determine the mechanisms that led to adaptation in the synthesis of pancreatic enzymes. DIETARY ADAPTATION IN THE EXOCRINE PANCREAS We studied the synthesis of 16 enzymes and isoenzymes in the rat pancreas in response to inverse changes in carbohydrate and protein in isocaloric diets. During adaptation to diets containing normal protein (22% of calories) or increased levels of protein (30%, 45%, 64%, 82% protein) and corresponding decreased levels of carbohydrate, the two amylase forms and 6 serine protease forms were synthesized in direct proportion to corresponding nutritional substrates in the diet. Similar changes were observed in adaptation of pancreatic lipase and colipase to inverse changes in lipid and carbohydrate in the diet. Changes in patterns of enzyme synthesis with cholecystokinin (CCK)-like hormones were similar to those obtained after administration of a high protein diet, suggesting that CCK mediates the effects of protein ingestion on enzyme synthesis in the pancreas. In contrast, changes in protein synthesis observed with secretin stimulation were similar to those observed with diets enriched in saturated or unsaturated fat. These findings suggest that secretin (and GIP) mediate the effects of triglyceride (fat) ingestion on synthesis of lipase and colipase in the pancreas. Taken together, the observed correlations suggest that differential regulation of functional groups of digestive enzymes (proteases, lipases and amylases) in the pancreas by changes in nutritional substrates in the diet (proteins, lipids and carbohydrates, respectively) is controlled largely through the effects of specific hormones. This hypothesis is supported by the following evidence. Protein has been shown to release CCK into the blood circulation from the intestinal mucosa and saturated or unsaturated lipids or fatty acids have been shown to release secretin in the same manner. Thus, positive feedback mechanisms appear to operate between nutritional substrates in the diet and regulation of the synthesis of pancreatic enzymes that are required for digestion of these substrates in the intestinal lumen. At least two of these feedback mechanisms appear to be mediated by specific hormones, CCK and secretin. We could also show that specific hormone-receptor interactions that lead to selective regulation of gene expression and protein synthesis can be correlated with specific second-messenger pathways within the pancreatic acinar cells. Taken together, these studies demonstrated that Pavlov's observations on dietary adaptation are mediated by specific gut hormones, involved in positive feedback mechanisms. ADAPTATION TO PROTEIN DEFICIENCY OR STARVATION During adaptation to diets containing decreased levels of protein (0-10%) and corresponding increased levels of carbohydrate, functional enzyme families were synthesized in inverse proportion to nutritional substrates in the diet. Under conditions of protein deficiency or starvation, changes in synthetic rates were related to isoelectric points of individual enzymes. For example, synthesis of anionic proteins, representing 45.6% of enzymes synthesized under normal dietary conditions, increased to 95% after 12 days of administration of a protein-free diet. Under conditions of protein starvation the pancreas maintains the synthesis and secretion of a select group of anionic proteinases, which allows for the digestion of newly found protein, a process that is necessary, at this point, for survival of the organism. Slide Presentation - Dietary Adaptation PATHOLOGICAL MECHANISMS BY WHICH PANCREATIC ENZYMES ARE RELEASED INTO THE BLOOD CIRCULATION Further collaborations with Professor Kern defined the route by which pancreatic enzymes, including amylase, enter the blood stream under conditions of acute experimental pancreatitis induced by supramaximal secretagogue stimulation. These studies demonstrated that pancreatic enzymes gain entry to the blood circulation under these pathological conditions through the process of exocytosis at the lateral plasma membrane. Slide Presentation - Lateral Exocytosis PANCREATIC SECRETORY STRESS PROTEINS DEFINED AND CHARACTERIZED Together with Drs. Rolf Graf and Daniel Bimmler at the University Hospital in Zurich we have defined a group of 16 kDa pancreatic secretory proteins, which have no digestive enzyme activities, but whose synthesis and secretion is dramatically increased in response to conditions of local or systemic stress. We have named this protein family "pancreatic secretory stress proteins". This group of proteins, synthesized and secreted by rat pancreatic acinar cells and composed of pancreatic stone protein (PSP/reg) and isoforms of pancreatic associated protein (PAP), show structural homologies, including conserved amino acid sequences, cysteine residues and highly sensitive N-terminal trypsin cleavage sites, as well as conserved functional responses in conditions of pancreatic or systemic stress. Trypsin activation of recombinant stress proteins or counterparts contained in rat pancreatic juice (PSP/reg, PAPI and PAP III) resulted in conversion of 16 kDA soluble proteins into 14 kDA soluble isoforms (pancreatic thread protein and Pancreatitis-associated thread protein, respectively that rapidly polymerize into insoluble sedimenting structures. Activating thread proteins show long-lived resistance to a wide spectrum of proteases contained in pancreatic juice, including serine proteases and metalloproteinases. Scanning and transmission electron microscopy indicate that activated thread proteins polymerize into highly organized fibrillar structures with helical configurations. Through bundling, branching and extension processes, these fibrillar structures form dense matrices that span large topological surfaces. These findings suggest that PSP/reg and PAPI and III isoforms consist of a family of highly regulated soluble secretory stress proteins, which, upon trypsin activation, convert into a family of insoluble helical thread proteins. We have cloned PSP/reg and the three PAP isoforms in the rat. Recent studies indicate that these pancreatic secretory stress proteins act as acute phase reactants in pancreatic juice under a variety of conditions including experimental acute pancreatitis (supramaximal caerulein stimulation and taurocholate-induced pancreatitis), chronic fibrosing pancreatitis in male WBN/Kob rats, during the perinatal post-weaning period and during simple surgical procedures and/or anesthesiology. These dense extracellular matrices, composed of helical thread proteins organized into higher ordered matrix structures, may serve adaptive physiological functions within luminal and/or interstitial compartments in the exocrine pancreas. Slide Presentation - Pancreatic Secretory Stress Proteins ROLE OF LUMINAL BICARBONATE AND PH DURING REGULATED SECRETION IN THE EXOCRINE PANCREAS The exocrine pancreas contains two types of epithelial cells with dissimilar but complementary functions: acinar cells, which secrete large amounts of protein (pancreatic digestive enzymes), and duct cells, which secrete fluid and bicarbonate ions. These two cell types are organized in an acinar-ductular complex that defines a common ductular space. Within this space the secretory products of these two cells are mixed and ultimately secreted, in response to peptidergic and cholinergic stimulation, into the digestive milieu of the intestinal lumen. Exocytosis, the last step in the regulated secretory pathway of pancreatic acinar cells, exteriorizes the secretory products (digestive enzymes) stored within zymogen granules through a process that involves insertion of granule membranes into the apical plasma membrane and release of the products into the acinar lumen, an extracellular fluid compartment that is contiguous with the pancreatic ductal system. Cholecystokinin (CCK) is the major hormone that elicits exocytosis in pancreatic acinar cells. Secretin is the major hormone that stimulates fluid and bicarbonate secretion from ductal cells. GP2/THP GENE FAMILY CLONED AND CHARACTERIZED Together with DrS. Shin-Ichi Fukuoka and Steven Freedman in my laboratory at Harvard Medical School in the 1990s, we cloned and characterized, for the first time, the GP2-THP gene family and elucidated the function of GP2 in the pancreas and Tamm-Horsfall Protein (THP) in the kidney. Glycoprotein-2 (GP2) is the most abundant glycoprotein in zymogen granule membranes accounting for 25-40% of membrane protein across species lines. Numerous investigations have been conducted on GP2 over the past 30 years. It was known to reside in the glycolipid-enriched exoplasmic leaflet of apical secretory compartments, predominantly in secretory granules. Earlier pulse-chase studies, conducted by others, indicated that GP2 is a membrane protein at early chase intervals (0-60 min.) and a soluble protein at later periods (1-6 hrs.) Immunocytochemical studies showed that small quantities of GP2 appear in ZG content and larger quantities in luminal and ductular secretions, the latter associated with linear branched aggregates. When isolated from pancreatic juice by ultracentrifugation, GP2 cosedimented with an extensive fibrillar meshwork stained with Alcian Blue, a cationic dye known to bind to repeating acidic groups (SO 4 ) on mucosubstances and proteoglycans. Although it was proposed that the GP2/proteoglycan matrix could provide, through sieving functions, a physiological barrier to bacterial invasion, the precise role of GP2 remained enigmatic. In 1990 Drs. Shin-Ichi Fukuoka, Steven Freedman and George Scheele provided the first successful cloning and description of the cDNA and amino acid sequence for rat and dog pancreatic GP2 and determined that pancreatic GP2 is homologous to Tamm-Horsfall protein (THP) produced by rat and human kidney. The C-terminal regions (474 amino acids) of rat GP2 and rat THP are highly conserved, demonstrating 53% identity and 86% similarity. A number of peptide domains, including an N-terminal signal sequence, a C-terminal transmembrane domain, one epithelial growth factor (EGF) motif, and eight N-linked carbohydrate attachments sites were conserved. Together, the structural studies suggested that GP2 and THP constitute a family of distinct but homologous genes (GP2/THP gene family). Northern blot studies indicated GP2 homologues in a variety of epithelial tissues, including parotid, submandibular gland, stomach, liver, and lung. Based on structural clues at the C-terminus of GP2, including a 20 residue hydrophobic transmembrane domain and potential phosphatidyl inositol glycan attachment sites, we used exogenous phospholipase C to demonstrate that GP2 was linked to the ZG membrane via a glycosyl phosphatidyl inositol (GPI) anchor. Kinetic analysis of GP2 release from ZGs permeabilized with low (sub-cmc) concentrations of saponin indicated a time-dependent release of GP2 at 37 o C, but not at 0 o C, findings that suggested the presence of a GP2-releasing enzyme in granule membranes. In order to better understand the paradox that GP2 remained attached to the ZG membrane during granule storage but was released following granule discharge at the APM we developed a novel working hypothesis that simple pH changes regulated these reciprocal processes. It had been known since the early 80s that the trans Golgi network (TGN), including zymogen granules are acidic (pH 5.5-6.0) due to the presence of a H + ATPase. It had been known since the 40s that pancreatic juice was alkaline (pH 8.0-8.5) and it was believed that the alkaline nature of pancreatic secretions was exclusively directed toward neutralization of acidic gastric secretions in the intestinal lumen. Yet the role of acid-base interactions in the secretory process and in the coupling of endocytosis to exocytosis had not been appreciated. Based on the hypothesis that mild pH changes exert major effects on exocrine products sequestered in regulated secretory compartments, our research efforts rapidly revealed major new insights into this area of basic and applied research. Slide Presentation - GP2/THP Gene Family BICARBONATE REGULATES THE ENZYMATIC CLEAVAGE OF GP2 FROM SECRETORY MEMBRANES & RETRIEVAL OF EXOCYTIC MEMBRANES FROM THE APICAL PLASMA MEMBRANE The success of this research depended on the development, by Dr. Steven Freedman and myself, of novel ways to manipulate the pH of the apical lumen in pancreatic tissue incubated under in vitro conditions. Direct measurements of pH in this compartment were precluded because of its small size. Instead, we exploited differences in medium access to luminal compartments in pancreatic lobules and acini. Pancreatic lobules with acinar units in contiguous attachment to ductal structures, contain luminal spaces that are protected from the incubation medium. We found that the composition of the acinar lumen in lobules could be altered by addition of ion substitutes and inhibitors of ion transport to the incubation medium. In contrast, pancreatic acini, prepared with collagenase digestion and vigorous shearing forces, contain acinar units devoid of ductular elements and associated luminal compartments are accessible to the incubation medium. Accordingly, we found that the composition of the acinar lumen in acini could be easily altered by changing the composition of the incubation medium. Utilizing rat pancreatic lobules, Freedman and I could show that alkalinization of the acinar lumen regulates enzymatic cleavage of the GPI anchor associated with GP2 and that release of GP2 from secretory (exocytic) membranes regulates retrieval (endocytosis) of ZG membranes from the APM. In lobules five lines of evidence have been provided that GP2 released from the APM depends on bicarbonate secreted from ductal elements:
Using rat pancreatic acini, which are devoid of ductal elements, we directly demonstrated that enzyme-mediated release of GP2 from the APM is regulated according to the pH of acinar lumen. In medium with pH values of 6.0 pancreatic acini released GP2 poorly. In medium adjusted to physiological pH (7.4) acini released GP2 in the intermediate range. In contrast, GP2 release from acini was maximal at pH 8.3, conditions that mimic ductal juice pH during secretin stimulation. To correlate GP2 release with membrane trafficking at the APM, horseradish peroxidase (HRP) uptake was used to measure endocytosis directly at the APM as a function of luminal pH. HRP uptake was quantified by biochemical assay and further visualized in the electron microscope by Professor Horst F. Kern. At pH 6.0, apical endocytosis was abolished. Acinar lumina showed marked dilatation and numerous "arrested" exocytic complexes, which in several cases formed extensive protrusions of the luminal space into the supranuclear Golgi regions of the cell forming "cavea". Dilated luminal membranes showed a marked decrease in density of microvillae, which appeared to be truncated in length. HRP penetrated into the luminal space and into arrested exocytic complexes. Notable, however, was the absence of HRP uptake into vesicular compartments in the cytosol. Over the pH range from 7.0-8.3 endocytosis increased in a progressive fashion to robust levels. In the electron microscope acinar lumina appeared contracted with normal density of microvilli. HRP, which appeared in luminal spaces, was also abundantly present within vesicular compartments at the apical pole of the cell and in a variety of vesicular compartments in the apical cytoplasm, particularly in the region of the Golgi Complex. Taken together, these findings clearly indicate that luminal pH regulates retrieval of exocytic membranes from the apical plasma membrane and suggest that GP2 release plays a regulatory role in this process. Slide Presentation - Bicarbonate Regulates Membrane Trafficking at the APM BICARBONATE COUPLES ENDOCYTOSIS TO EXOCYTOSIS AT THE APICAL PLASMA MEMBRANE OF PANCREATIC ACINAR CELLS The success of these studies depended upon the development of a two-step protocol that allowed us to define the chemical factors responsible for coupling endocytosis to exocytosis. CCK-8 stimulation during the first hour led to marked exocytosis and dilatation of the acinar lumen and provided a model for the study of apical exocytosis (step 1). Removal of CCK-8 by rapid washes followed by incubation of pancreatic acini during the second hour led to cellular retrieval of secretory (exocytic) membranes from the APM and provided an in vitro model of apical endocytosis (step 2). When hormone-stimulated secretion was conducted in the presence of incubation medium at pH 6.0, exocytosis and endocytosis could be completely dissociated. Because of the fine precision by which pH manipulations dissociate endocytosis/exocytosis and further regulate molecular and cellular processes involved in endocytosis, we believe that the 2-step exocytosis/endocytosis model greatly facilitates the study of membrane events at the APM for both exocytosis and endocytosis. Under the time conditions of our study (2 hrs.) mild changes in pH of the incubation medium (6.0-8.3) had little or no effect on cellular integrity as judged by the absence of changes in the cellular release of enzymes in response to hormonal stimulation, cellular ATP levels, lactate dehydrogenase release, or binding of HRP to acinar cells. ENZYMATIC RELEASE OF GP2 ACTIVATES ENDOCYTOSIS AND RETRIEVAL OF EXOCYTIC MEMBRANES FROM THE APICAL PLASMA MEMBRANE We have used this new and powerful model of membrane trafficking to directly determine whether cleavage of GPI-anchored proteins from the APM of rat acinar cells is responsible for activation of endocytosis. Under conditions of incubation of pancreatic acini at pH 6.0 where GP2 release and apical endocytosis are abolished, we added PI-specific, phospholipase C to the incubation medium. PI-PLC treatment immediately reversed the inhibition and restored endocytosis to levels observed under physiological conditions. Control studies indicated that endocytosis was not activated through second messenger pathways (incubation of tissue at pH 6.0 had no effects on intracellular pH or intracellular calcium concentrations; administration of phorbol esters or diacyl glyceride had no effect on apical endocytosis). Two GPI-linked proteins were released from the luminal compartment, GP2 and alkaline phosphatase (AP). GP2, but not AP, release correlated with endocytosis observed under physiological conditions at pH 7.4 or 8.3. By electron microscopic evaluation, incubation of cells at pH 6.0 with PI-PLC led to induction of HRP uptake into vesicles at the apical pole of the cell, a reduction in apical plasma membranes, and a concomitant contraction of the acinar lumen space. Internalized HRP accumulated in the Golgi region of the cell. These results indicate that cleavage of GPI-anchored proteins, particularly GP2 from the APM, serves as the molecular signal that activates apical endocytosis. These studies also provided the first rationale for the physiological importance of GPI anchor cleavage in the control of membrane homeostasis. Under conditions in which exocrine cells conserve membranes during secretory events, endocytic vesicles move from the APM to the Golgi Complex where they are reutilized in the secretory process. Slide Presentation - Enzymatic Release of GP2 Activates Endocytosis BICARBONATE REGULATES SOLUBILIZATION OF SECRETORY ENZYMES IN THE EXOCRINE PANCREAS We also demonstrated, for the first time, that proteins targeted to the regulated exocrine path show intrinsic properties for reversible, pH-dependent aggregation, under conditions of mild acidification. In the absence of ZG membranes we could demonstrate that 14 well defined canine pancreatic proteins aggregate under pH conditions (5.5) that mimic the TGN. Upon reversal of pH to neutrality protein aggregates dissociated and enzymes achieved complete solubility in the surrounding medium. Of considerable interest, two proteins representative of constitutive secretory processes, bovine serum albumin (IEP = 6.0) and cationic immunoglobulin (IEP = 8.6) were excluded from this pH-dependent and reversible aggregation process. Taken together, these studies favor intrinsic pH-dependent aggregation events over sorting receptors for assembly of secretory granules in regulated exocrine cells. These studies elucidated the role of acidic pH in the aggregation/packaging of secretory enzymes into condensing vacuoles and zymogen granules. Slide Presentation - Bicarbonate Solubilizes Secreted Proteins HYPOTHESIS: GP2 FUNCTIONS IN MEMBRANE SORTING, ASSEMBLY AND TRAFFICKING OF ZYMOGEN GRANULE PROTEINS & MEMBRANES In parallel, we conducted extensive studies on the effects of acidic pH, which mimic those present in the TGN, on GP2 structure. We demonstrated that the globular domain, purified from ZG membranes after phospholipase cleavage, shows pH-and calcium-dependent self association into sedimenting complexes. The reaction is reversible as a function of pH and abolished at low temperatures (0-10 o C) and in the presence of nonionic detergents. At pH 5.5 in the presence of 20 mM calcium, oligomeric structures (~300 kDa) consistent with tetrameric complexes were observed by gel filtration chromatography and elliptical structures (14-18 nm) were observed in the electron microscope by negative staining techniques. These observations provided the first demonstration that the luminal component of a membrane protein associated with the regulated exocytic pathway is capable of homophilic binding/oligomerization reactions under conditions similar to those believed to exist in the TGN. Recent investigations in our laboratory suggest that a GP2/proteoglycan (PG) matrix is initially formed on the luminal leaflet of condensing vacuoles and ZG membranes (Freedman, S., Lu, L. and Scheele, G., unpublished findings). Isolation of zymogen granules from 35 SO 4 labeled pancreatic lobules revealed that 92.8% of the label was associated with ZG membranes. A wash with Na 2 CO 3 at pH 11.2 could release 70.7% of labeled proteoglycans. GP2 remained attached via its GPI anchor. However, release of GP2 from the membrane with PI-PLC also released significant quantities of radiolabeled proteoglycans. Co-association of GP2 and proteoglycans with PI-PLC treatment argues that a GP2/PG matrix associates tightly with the luminal aspect of ZG membranes via the GPI anchor of GP2. We believe that the complex between membrane-associated GP2 and proteoglycans is first assembled on the luminal aspects of condensing vacuole membranes. PH-dependent formation of tetrameric GP2 complexes in the TGN would allow for multivalent association of proteoglycan chains, resulting in the formation of a fibrillar meshwork. Based on our knowledge of the tensile properties of proteoglycans we hypothesize that the GP2/PG matrix serves as a submembranous skeletal structure with the following functions during ZG assembly and storage: COLOCALIZES DURING ASSEMBLY OF MEMBRANE MICRODOMAINS WITHIN CONDENSING VACUOLES (CV) AND ZYMOGEN GRANULES (ZG) We hypothesize that the GP2/PG matrix serves important sorting functions during granule assembly as follows: • Glycolipid-rich membrane microdomains containing GPI-anchored membrane proteins (GP2) assemble in the TGN. • Progressive decreases in pH achieved in the TGN lead to the formation of GP2 tetramers (homophilic binding reactions) tethered to the membrane via GPI anchors. Decreased pH in the TGN also allows sulfation of glycosaminoglycan chains associated with proteoglycans. • Heterophilic interactions between tetrameric GP2 complexes and sulfated proteoglycans in the TGN form a submembranous skeletal meshwork on the luminal aspect of the ZG membrane. • Glycolipid-rich membranes containing a submembranous GP2/PG matrix are retained in condensing vacuoles and ZGs. Phospholipid-rich membranes, devoid of GP2 and associated proteoglycans may vesiculate and depart from CVs in order to be distributed to endolysosomal compartments. • Continued remodeling of CV membranes combined with progressive condensation (pH- and ion-induced aggregation) of exocrine products lead to the formation of mature ZGs where, during the storage phase of secretion, the limiting membrane remains quiescent. DETERMINES, IN PART, THE PHYSICAL CHARACTERISTICS OF ZYMOGEN GRANULES The GP2/PG matrix may determine the size, shape, and stability of ZGs. Granules formed in the presence of GP2 are remarkably uniform at 1 ± 0.1 um diameter. Granules formed in the absence of GP2 are variegated in shape and range in size from 0.1 - 2.5 um. The turgid properties of proteoglycans tethered to the ZG membrane via multivalent interactions with tetrameric GP2 may exert a tensile force that determines, in part, the radius of curvature of the granule membrane and its regular shape. STABILIZES ZG MEMBRANES DURING THE GRANULE STORAGE PHASE OF SECRETION A submembranous skeletal structure composed of a GP2/PG matrix may serve to retard or prevent vesicular budding from the ZG membrane. Membranes stabilized through their interaction with submembranous structures may retard clathrin-mediated processes on the cytosolic aspect of the granule membrane. Inhibition of clathrin processes would prevent vesicular budding in granule membranes. This hypothesis is consistent with the observation that limiting membranes of mature ZGs, during storage, are completely inactive with regard to vesicular budding. Slide Presentation - Hypothetical Roles for GP2 LUMINAL EVENTS DURING COUPLED EXOCYTOSIS AND ENDOCYTOSIS AT THE APICAL PLASMA MEMBRANE Since the GP2/PG matrix appears to function as a skeleton to stabilize membrane structure and inhibit vesicular budding mechanisms as described above, it follows that once exocytosis occurs at the APM, the skeletal elements must be removed to allow membrane retrieval by vesicular processes. We envision the transformation process at the APM as follows: • Exocytosis : Insertion of ZG membranes into the APM - Exocytosis, the last step in the regulated secretory pathway, exteriorizes the secretory products (digestive enzymes) stored within ZGs through a process that involves insertion of granule membranes into the APM. This transport step is highly regulated by second messenger pathways located within the cytosolic space that respond to hormone-receptor interactions at the basolateral plasma membrane of acinar cells. Cholecystokinin (CCK) is the major hormone that elicits exocytosis in pancreatic acinar cells. • Neutralization Of The Acidic PH Of Exocytic Contents - The primary secretory product released from acinar cells into the acinar lumen by exocytosis represents secretory (pro)enzymes highly aggregated in an acidic milieu (pH 5.5-6.5). Bicarbonate secreted from ductal elements is required to neutralize the pH of exocytic contents. Because pH induced aggregation of exocrine products is reversible, neutralization of exocytic contents leads to dissociation of protein aggregates and solubilization of (pro)enzymes within the acinar lumen. Secretin is the major hormone that stimulates fluid and bicarbonate secretion from ductal cells. • Solubilization Of Aggregated Proteins - The initial phases of dissociation of protein aggregates can be expected to greatly increase osmotic forces that enhance the influx of fluid and bicarbonate into these complexes. The swelling effects associated with the dissociation/solubilization process requires that the acinar lumen dilates during regulated exocytosis. • PH- And Enzyme-Induced Release Of The GP2/PG Matrix From The Apical Plasma Membrane - Following neutralization of the acidic milieu of exocytic contents and solubilization of secretory proteins in neutral or alkaline medium, increased pH at the APM are required for the enzymatic cleavage of the GPI anchor of GP2. The pH-dependent nature of this process would ensure that protein aggregates are dissociated and secretory enzymes are solubilized before the GP2/PG matrix is released from the luminal aspects of ZG membranes inserted into the APM by exocytic processes. • Clathrin-Mediated Recycling Of ZG Membranes To The Golgi Complex - Following removal of the GP2/PG skeletal matrix from their luminal surfaces, ZG membranes are free to be retrieved by the cell through clathrin-mediated processes. These cytosol-mediated processes are responsible for vesiculation of granule membranes and their return to the Golgi Complex where they may participate in another secretory round. This conservation process allows the acinar cell to deliver large quantities of digestive enzymes to the intestinal lumen without engaging in increased membrane biosynthesis. Identification of biochemical coupling reactions within the acinar lumen and recognition that these pH-dependent reactions are coordinated through the actions of different hormones (CCK and secretin) on divergent epithelial cells (acinar and ductal cells, respectively) provides a new appreciation for the importance of combined CCK and secretin stimulation during pancreatic secretion in response to a meal. These concepts further suggest that disruption of biochemical coupling processes in the acinar lumen plays a critical role in the development of chronic pancreatic disease in cystic fibrosis. Back To Top Slide Presentation - Pancreatic Dysfunction in Cystic Fibrosis CLAUDE BERNARD: ON THE SCIENTIFIC METHOD Quotations From CHAPTER I - OBSERVATION AND EXPERIMENT • "These may be called 'Experiments to see', because they are intended to make a first observation emerge, unforeseen and undetermined in advance, but whose appearance may suggest an experimental idea and open a path for research." • "It is impossible to devise an experiment without a preconceived idea." • "An experimenter. reflects, tries out, gropes, compares, contrives so as to find the experimental conditions best suited to gain the end which he sets before him." • "In scientific investigation, minutiae of method are of the highest importance." • "The true scientist is one whose work includes both experimental theory and experimental practice. He notes a fact. A propos of this fact, an idea is born in his mind. In the light of this idea, he reasons, devises an experiment, imagines and brings to pass its material conditions. From this experiment new phenomena result which must be observed, and so on and so forth." • "If the facts used as a basis for reasoning are ill-established or erroneous, everthing will crumble or be falsified." CHAPTER II - THE A PRIORI IDEA AND DOUBT IN EXPERIMENTAL REASONING EXPERIMENTAL TRUTHS ARE OBJECTIVE (p28) • "The experimental method is concerned only with the search for objective truths, not with any search for subjective truths." INTUITION OR FEELING BEGETS THE EXPERIMENTAL IDEA (p32) EXPERIMENTERS MUST DOUBT, AVOID FIXED IDEAS AND MAINTAIN THEIR FREEDOM OF MIND (p35) • "The great experimental principle, then, is doubt, that philosophic doubt which leaves to the mind its freedom and initiative and from which the virtues most valuable to investigators in physiology and medicine are derived." • "Men who have excessive faith in their theories or ideas are not only ill prepared for making discoveries, they also make very poor observations." • "The truly scientific spirit, then should make us modest and kindly. We really know very little, and we are all fallible when facing the immense difficulties presented by investigation of natural phenomenon." • "But as these theories and ideas are by no means immutable truth, one must always be ready to abandon them, to alter them or to exchange them as soon as they cease to represent the truth. In a word, we must alter theory to adapt it to nature, but not nature to adapt it to theory." • "To sum up, two things must be considered in experimental science: method and idea. The object of method is to direct the idea . in the search for truth. But everywhere the idea must be submitted to a criterion. In science the criterion is the experimental method or experiment; this criterion is indispensable and we must apply it to our own ideas as well as to those of others." INDEPENDENT CHARACTER OF EXPERIMENTAL METHOD (p40) • "Genius is revealed in a delicate feeling which correctly foresees the laws of natural phenomenon. But this we must never forget, that correctness of feeling and fertility of idea can be established and proved only by experiment." • "Man can learn nothing except by going from the known to the unknown." • "In my opinion there is only one way of reasoning for the mind, just as there is only one way of walking for the body. But when a man goes ahead on solid flat ground, by a straight road whose whole extent he knows and sees, he advances toward his goal at an assured and rapid pace. On the contrary, when a man follows a winding road in the dark and over unknown hilly ground, he dreads precipices and goes forward cautiously, step by step. Before taking a second step, he must make sure that he has placed his first foot on a spot that is firm, then go forward in the same way verifying experimentally, moment by moment, the solidity of the ground and always changing the direction of his advance according to what he encounters." DOUBT IN EXPERIMENTAL REASONING (p48) • "In all natural phenomena the principles from which we start, like the conclusions which we reach, embody only relative truths. The experimenter's stumbling block, then, consists in thinking that he knows what he does not know, and in taking for absolute, truths that are only relative. Hence, the unique and fundamental rule of scientific investigation is reduced to doubt." • "Experimental reasoning is precisely the reverse of scholastic reasoning." • "Scholasticism must always have a fixed and indubitable starting point. Experimenters, on the contrary, never accept an immutable starting point. Experimenters, then, always doubt even their starting point. Of necessity they keep a supple and modest mind and accept contradiction, on the one condition that it be proven." • "Systematic or scholastic reasoning is natural to inexperienced, proud minds. Of those who think they are following the experimental path in physiology and in medicine, many, as we shall see later, are still scholastics." • "The experimenter, on the contrary, who always doubts and who does not believe that he possesses absolute certainty about anything, succeeds in mastering the phenomena that surround him and in extending his power over nature. Man can do, then, more than he knows and true experimental science gives him power only in showing him his ignorance." • "Finally, we may say that in all experimental reasoning there are two possibilities: either the experiment's hypothesis will be disproved or it will be proved by experiment." PRINCIPLE OF EXPERIMENTAL CRITERION (p52) • "A fact is nothing in itself, it has value only through the idea connected with it or through the proof it supplies .. This relationship is the scientific truth." • "In spite of our efforts, we are still very far from the absolute truth and it is probable, especially in the biological sciences, that it will never be given us to see it in its nakedness. But this need not discourage us, for we are constantly nearing it. And moreover, with the help of our experiments, we grasp relations between phenomena which, though partial and relative, allow us more and more to extend our power over nature." PROOF AND COUNTERPROOF (p55) • "Indeed, proof that a given condition always preceded or accompanies a phenomenon does not warrant concluding with certainty that a given condition is the immediate cause of that phenomenon. It must still be established that, when this condition is removed, the phenomenon will no longer appear. If we limited ourselves to the proof of presence alone, we might fall into error at any moment and believe in relations of cause and effect where there was nothing but simple coincidence." • "Coincidences form one of the most dangerous stumbling blocks encountered by experimental scientists in complex sciences like biology." • "'Experimentum crucis' provides the counterproof." • "Some physicians fear and avoid counterproof. As soon as they make observations in the direction of their ideas, they refuse to look for contradictory facts, for fear of seeing their hypothesis vanish." • "If we mean to find truth, we can solidly settle our ideas only by trying to destroy our own conclusions by counter experiments." Quotations Selected by Dr. George A. Scheele |
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