Sodium, potassium-activated adenosine triphosphatase activity is impaired in the guinea pig pancreatic duct system in streptozotocin-induced diabetes.

In patients with type I diabetes mellitus, clinical studies have demonstrated decreased secretion of pancreatic juice by the pancreatic excretory duct system. The cause of this decrease is unknown, but could involve changes in initial signal transduction pathways or one or more of the electrolyte transport components that subserve regulated fluid secretion. We have compared responsiveness to secretin in pancreatic ducts isolated from healthy and diabetic Hartley guinea pigs and also have compared the expression of CFTR and Na+, K(+)-ATPase in these two groups, as the activities of these two proteins are essential for secretion of pancreatic juice. The increases in cyclic AMP levels evoked by exposure to either 0.1 nM or 0.1 microM secretin were not significantly different in pancreatic ducts isolated from healthy and diabetic guinea pigs nor were levels of CFTR or Na+, K(+)-ATPase expression. By contrast, Na+, K(+)-ATPase activity in pancreatic ducts isolated from diabetic guinea pigs was decreased by 70%, suggesting a change in the enzyme's catalytic properties in the diabetic tissues. The observed decrease would be expected to seriously compromise the production of pancreatic juice.

Confocal microscopic analysis of intracellular pH regulation in isolated guinea pig pancreatic ducts.

pH regulation in isolated guinea pig pancreatic interlobular duct segments loaded with the pH-sensitive fluorophore, 5-(6)-carboxy-SNARF-1-acetoxymethyl ester (SNARF-1), was characterized by laser-scanning confocal microscopy. In HCO3(-)-free medium, intracellular pH (pHi) of duct epithelial cells fell by 0.32 +/- 0.06 pH units in the presence of 0.5 mM amiloride and by 0.36 +/- 0.08 pH units in the absence of Na+. In the presence of extracellular HCO3-, pHi acidified in Na(-)-free medium but not in amiloride-containing medium. Superfusion with Cl(-)-free buffers or with buffers containing 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid produced a cytosolic alkalinization of 0.13-0.22 pH units. These observations demonstrate the presence of Na+/H+ exchange, Na(+)-HCO3- cotransport, and Cl-/HCO3- exchange in guinea pig pancreatic ducts. pHi recovered significantly from an NH4Cl pulse in HCO3(-)-free buffers containing amiloride and carbachol (50.4%) or amiloride and secretin (40.6%). This recovery was blocked by the H(+)-adenosinetriphosphatase (H(+)-ATPase) inhibitor bafilomycin A1 and by preincubation of ducts with nocodazole or cytochalasin D. These observations suggest that a vesicular H(+)-ATPase augments Na(+)-dependent H+ extrusion during agonist-stimulated bicarbonate secretion and that activation of this transport mechanism involves cytoskeletal elements.

Regulation of cyclic AMP levels in guinea pig pancreatic ducts and cultured duct epithelial monolayers.

Pancreatic duct bicarbonate secretion is mediated primarily by secretin-induced elevation of intracellular cyclic AMP, although little is known of the effects of other physiological regulators on pancreatic duct cyclic AMP metabolism. We investigated the effects of secretin and several other potential agonists on cyclic AMP levels in isolated guinea pig main and interlobular pancreatic duct segments and in cultured duct epithelial monolayers. Secretin (0.1 microM) caused a five- to eightfold elevation of cyclic AMP in both isolated ducts and cultured monolayers (EC50 = 0.15 nM). Main duct segments, while responsive, were less so than segments of interlobular duct. In isolated duct segments, carbachol, bombesin, cholecystokinin, substance P, calcitonin gene-related peptide, glucagon, insulin, isoproterenol, neurotensin, and prostaglandin E2 did not significantly alter resting or secretin-stimulated cyclic AMP levels. In contrast, 0.1 microM vasoactive intestinal peptide significantly increased cyclic AMP to a level comparable to that evoked by an equal concentration of secretin. Somatostatin significantly attenuated the effects of a submaximal (physiological) dose of secretin on duct cyclic AMP levels without altering resting cyclic AMP levels, suggesting that somatostatin's effects on pancreatic duct fluid secretion are mediated by inhibition of adenylyl cyclase activity.

Insulin, transforming growth factors, and substrates modulate growth of guinea pig pancreatic duct cells in vitro.

BACKGROUND & AIMS: Little is known of the physiological mechanisms that control cellular renewal in the pancreatic excretory duct system. This study investigated the effects of potential regulatory substances on the growth of cultured guinea pig pancreatic duct epithelial monolayers. METHODS: Pancreatic duct explants were cultured for 3 days on plastic and on permeable filters in the presence and absence of different substances. Growth of epithelial monolayers from these explants was measured by 5-bromo-2'-deoxyuridine incorporation and morphometric procedures. RESULTS: Epidermal growth factor and insulin both enhanced monolayer growth and together had an additive effect. Transforming growth factor alpha enhanced and transforming growth factor beta inhibited growth, whereas glucagon, somatostatin, pancreatic polypeptide, secretin, cerulein, bombesin, and dexamethasone had no significant effects. Monolayer growth on type 1 collagen-coated filters was enhanced when compared with that of monolayers grown on tissue culture plastic. Cell growth from explants on filters coated with type IV collagen and fibronectin was comparable with that on plastic, whereas growth on Matrigel- or laminin-coated filters was reduced. CONCLUSIONS: Insulin, transforming growth factors, and substrate components modulate growth of pancreatic duct epithelial cells in vitro, suggesting that they are important regulators of cell division in the excretory duct system of the intact pancreas.

Regulation of goblet cell degranulation in isolated pancreatic ducts.

Neurohumoral control of goblet cell degranulation in isolated segments of the guinea pig main pancreatic duct was examined using morphometric procedures. Goblet cells represent 25-30% of the epithelial cell population at the head of the main pancreatic duct, a percentage that decreases to 5-10% as the distance from the ampulla increases. Carbachol, bombesin, and vasoactive intestinal peptide (VIP) each stimulated degranulation of duct goblet cells, although cholecystokinin octapeptide, secretin, and histamine did not. The stimulatory effects of carbachol on goblet cell degranulation in isolated pancreatic ducts were blocked by atropine and enhanced by simultaneous exposure to VIP. These observations indicate that goblet cells in guinea pig pancreatic ducts express bombesin, VIP, and muscarinic cholinergic receptors and that multiple intracellular signaling pathways are involved in the regulation of goblet cell degranulation.

Intracellular mediators of goblet cell degranulation in isolated pancreatic ducts.

The involvement of particular intracellular signalling pathways in agonist-evoked degranulation of guinea pig pancreatic duct goblet cells was investigated. Carbachol, vasoactive intestinal peptide (VIP), calcium ionophore A23187, phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), cyclic AMP analogue Sp-5,6-DCl-cBIMPS and forskolin each caused degranulation of goblet cells in isolated ducts. Degranulation induced by carbachol was not inhibited by okadaic acid, cytochalasin-D or nocodazole. These results indicate that at least two major signalling pathways are involved in pancreatic duct goblet cell secretion.

Overview of pancreatic duct physiology and pathophysiology.

The epithelium of the excretory duct system of the exocrine pancreas secretes bicarbonate ions and mucins. Epithelial cells of the duct system also constitute primary sites of dysfunction in cystic fibrosis, pancreatitis and pancreatic cancer. The present work provides an overview of the current state of understanding of the physiology and pathophysiology of the pancreatic duct system and suggests approaches that will provide continued progress in exploration of the basic physiological processes operating in this tissue.