Takaharu Kondo

CFTR Functions as a Bicarbonate Channel in Pancreatic Duct Cells

Pancreatic duct epithelium secretes a HCO3−-rich fluid by a mechanism dependent on cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane. However, the exact role of CFTR remains unclear. One possibility is that the HCO3− permeability of CFTR provides a pathway for apical HCO3− efflux during maximal secretion. We have therefore attempted to measure electrodiffusive fluxes of HCO3− induced by changes in membrane potential across the apical membrane of interlobular ducts isolated from the guinea pig pancreas. This was done by recording the changes in intracellular pH (pHi) that occurred in luminally perfused ducts when membrane potential was altered by manipulation of bath K+ concentration. Apical HCO3− fluxes activated by cyclic AMP were independent of Cl− and luminal Na+, and substantially inhibited by the CFTR blocker, CFTRinh-172. Furthermore, comparable HCO3− fluxes observed in ducts isolated from wild-type mice were absent in ducts from cystic fibrosis (ΔF) mice. To estimate the HCO3− permeability of the apical membrane under physiological conditions, guinea pig ducts were luminally perfused with a solution containing 125 mM HCO3− and 24 mM Cl− in the presence of 5% CO2. From the changes in pHi, membrane potential, and buffering capacity, the flux and electrochemical gradient of HCO3− across the apical membrane were determined and used to calculate the HCO3− permeability. Our estimate of ∼0.1 µm sec−1 for the apical HCO3− permeability of guinea pig duct cells under these conditions is close to the value required to account for observed rates of HCO3− secretion. This suggests that CFTR functions as a HCO3− channel in pancreatic duct cells, and that it provides a significant pathway for HCO3− transport across the apical membrane.

Membrane potential and bicarbonate secretion in isolated interlobular ducts from guinea-pig pancreas

The interlobular duct cells of the guinea-pig pancreas secrete HCO3 − across their luminal membrane into a HCO3 −-rich (125 mM) luminal fluid against a sixfold concentration gradient. Since HCO3 − transport cannot be achieved by luminal Cl−/HCO3 − exchange under these conditions, we have investigated the possibility that it is mediated by an anion conductance. To determine whether the electrochemical potential gradient across the luminal membrane would favor HCO3 − efflux, we have measured the intracellular potential (Vm) in microperfused, interlobular duct segments under various physiological conditions. When the lumen was perfused with a 124 mM Cl−-25 mM HCO3 − solution, a condition similar to the basal state, the resting potential was approximately −60 mV. Stimulation with dbcAMP or secretin caused a transient hyperpolarization (∼5 mV) due to activation of electrogenic Na+-HCO3 − cotransport at the basolateral membrane. This was followed by depolarization to a steady-state value of approximately −50 mV as a result of anion efflux across the luminal membrane. Raising the luminal HCO3 − concentration to 125 mM caused a hyperpolarization (∼10 mV) in both stimulated and unstimulated ducts. These results can be explained by a model in which the depolarizing effect of Cl− efflux across the luminal membrane is minimized by the depletion of intracellular Cl− and offset by the hyperpolarizing effects of Na+-HCO3 − cotransport at the basolateral membrane. The net effect is a luminally directed electrochemical potential gradient for HCO3 − that is sustained during maximal stimulation. Our calculations indicate that the electrodiffusive efflux of HCO3 − to the lumen via CFTR, driven by this gradient, would be sufficient to fully account for the observed secretory flux of HCO3 −.

Chronic alcoholism and evolution of pain and prognosis in chronic pancreatitis.

To evaluate the influence of chronic alcoholism on clinical features of chronic pancreatitis in Japan, pain evolution, pancreatic insufficiency, and long-term prognosis were studied by comparing chronic alcoholic pancreatitis (N=88)with idiopathic pancreatitis (N=67).The 155 patients with known course of the disease over three years were followed-up further for five more years, and pain evolution was evaluated once at the start and once at the end of the follow-up period. At the time of diagnosis, severe pain (59 vs 33%, P < 0.001), pancreatic calcification (63 vs 31%, P<0.001), advanced exocrine pancreatic insufficiency (72 vs 60%, NS),and overt diabetes (48 vs 17%, P<0.007)were more common in alcoholic than in idiopathic pancreatitis, respectively. Pain evolution was similar in both pancreatitis, and the pain decreased with time. The rate of abstinence was higher in groups with pain relief than without in alcoholic pancreatitis. Cumulative mortality rate during the five years was higher in alcoholic than idiopathic pancreatitis (26 vs 10%, P<0.01).These results suggest more favorable evolution of the disease can be expected by abstinence from alcohol.

Functional coupling of apical Cl−/HCO3− exchange with CFTR in stimulated HCO3− secretion by guinea pig interlobular pancreatic duct

Pancreatic ductal epithelium produces a HCO(3)(-)-rich fluid. HCO(3)(-) transport across ductal apical membranes has been proposed to be mediated by both SLC26-mediated Cl(-)/HCO(3)(-) exchange and CFTR-mediated HCO(3)(-) conductance, with proportional contributions determined in part by axial changes in gene expression and luminal anion composition. In this study we investigated the characteristics of apical Cl(-)/HCO(3)(-) exchange and its functional interaction with Cftr activity in isolated interlobular ducts of guinea pig pancreas. BCECF-loaded epithelial cells of luminally microperfused ducts were alkalinized by acetate prepulse or by luminal Cl(-) removal in the presence of HCO(3)(-)-CO(2). Intracellular pH recovery upon luminal Cl(-) restoration (nominal Cl(-)/HCO(3)(-) exchange) in cAMP-stimulated ducts was largely inhibited by luminal dihydro-DIDS (H(2)DIDS), accelerated by luminal CFTR inhibitor inh-172 (CFTRinh-172), and was insensitive to elevated bath K(+) concentration. Luminal introduction of CFTRinh-172 into sealed duct lumens containing BCECF-dextran in HCO(3)(-)-free, Cl(-)-rich solution enhanced cAMP-stimulated HCO(3)(-) secretion, as calculated from changes in luminal pH and volume. Luminal Cl(-) removal produced, after a transient small depolarization, sustained cell hyperpolarization of approximately 15 mV consistent with electrogenic Cl(-)/HCO(3)(-) exchange. The hyperpolarization was inhibited by H(2)DIDS and potentiated by CFTRinh-172. Interlobular ducts expressed mRNAs encoding CFTR, Slc26a6, and Slc26a3, as detected by RT-PCR. Thus Cl(-)-dependent apical HCO(3)(-) secretion in pancreatic duct is mediated predominantly by an Slc26a6-like Cl(-)/HCO(3)(-) exchanger and is accelerated by inhibition of CFTR. This study demonstrates functional coupling between Cftr and Slc26a6-like Cl(-)/HCO(3)(-) exchange activity in apical membrane of guinea pig pancreatic interlobular duct.

Effects of aging and gastric lipolysis on gastric emptying of lipid in liquid meal

Abstract: Lipid delays gastric emptying, and aging is associated with changes in gastric motor function and transit. However, little is known about the effect of lipid on gastric emptying time in the elderly. To determine the effect of aging on lipid gastric emptying, we used electrical impedance tomography (EIT) to study gastric emptying of liquid meals with or without lipid in five young (23.0 ± 0.6 years, mean ± SEM) and six elderly (73.3 ± 1.6 years) healthy male volunteers. These subjects drank 400 ml of non-lipid soup (triglycerides, 0 g) or lipid soup (triglycerides, 24.6 g) in liquid test meals. To study the effect of lipolysis in the stomach, a liquid test meal containing 240 mg of lipase in the lipid soup was also administered. Plasma cholecystokinin (CCK) concentration was measured by specific radioimmunoassay before and 30 min after the ingestion of a test meal. The gastric emptying time of the lipid soup was longer in the elderly than in the young subjects, and the time was significantly longer for lipid soup than for non-lipid soup (P < 0.05) in both the young and elderly subjects. Gastric emptying time for non-lipid soup was not significantly different between the elderly and young subjects. The administration of lipase shortened the gastric emptying time for lipid in both the elderly and the young subjects. Basal CCK concentration was significantly higher in the elderly than in the young subjects. However, there was no relationship between gastric emptying time and plasma CCK concentration after the ingestion of a test meal in the subjects overall. In conclusion, the delaying effect of lipid on gastric emptying is increased in the elderly, and the administration of lipase accelerates the emptying of lipid from the stomach.

Chloride transport in microperfused interlobular ducts isolated from guinea‐pig pancreas

Isolated interlobular ducts from the guinea‐pig pancreas secrete a HCO3−‐rich fluid in response to secretin. To determine the role of Cl− transporters in this process, intracellular Cl− concentration ([Cl−]i) was measured in ducts loaded with the Cl−‐sensitive fluoroprobe, 6‐methoxy‐N‐ethylquinolinium chloride (MEQ). [Cl−]i decreased when the luminal Cl− concentration was reduced. This effect was stimulated by forskolin, was not dependent on HCO3− and was not inhibited by application of the anion channel/transporter inhibitor H2DIDS to the luminal membrane. It is therefore attributed to a cAMP‐stimulated Cl− conductance, probably the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. [Cl−]i also decreased when the basolateral Cl− concentration was reduced. This effect was not stimulated by forskolin, was largely dependent on HCO3− and was inhibited by basolateral H2DIDS. It is therefore mediated mainly by Cl−/HCO3− exchange. With high Cl− and low HCO3− concentrations in the lumen, steady‐state [Cl−]i was 25‐35 mm in unstimulated cells. Stimulation with forskolin caused [Cl−]i to increase by approximately 4 mm due to activation of the luminal anion exchanger. With low Cl− and high HCO3− concentrations in the lumen to simulate physiological conditions, steady‐state [Cl−]i was 10–15 mm in unstimulated cells. Upon stimulation with forskolin, [Cl−]i fell to approximately 7 mm due to increased Cl− efflux via the luminal conductance. We conclude that, during stimulation under physiological conditions, [Cl−]i decreases to very low levels in guinea‐pig pancreatic duct cells, largely as a result of the limited capacity of the basolateral transporters for Cl− uptake. The resulting lack of competition from intracellular Cl− may therefore favour HCO3− secretion via anion conductances in the luminal membrane, possibly CFTR.

Genetic evidence for CFTR dysfunction in Japanese: background for chronic pancreatitis

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP regulated Cl− channel that is expressed in many epithelial tissues.1 In the pancreas CFTR plays a key role in the apical HCO3– transport in duct cells.2–5 Loss of its function due to mutations in the CFTR gene causes cystic fibrosis (CF) of the pancreas with exocrine insufficiency, chronic airway disease, and abnormally elevated sweat chloride concentration. Over 1000 mutations and 200 polymorphic loci in CFTR have now been identified.6 These mutations and polymorphisms confer quite variable phenotypes from classic CF to atypical CF with less severe pulmonary lesions, pancreatic sufficiency, and normal or borderline sweat chloride concentration.7,8 It is now well recognised that the spectrum of CFTR related disease is much broader than previously thought.9 Some individuals may exhibit an apparently single clinical feature or a monosymptomatic disease, such as chronic sinusitis, congenital bilateral absence of the vas deferens (CBAVD), and sweat chloride abnormalities. Recent evidence10–12 suggests that chronic pancreatitis, in at least a subset of the patients, belongs to this group of disease.13 Chronic pancreatitis is a progressive inflammatory disease of the pancreas that causes the loss of pancreatic acinar cells, ductal abnormalities often with intraductal stones, and irregular fibrosis. Alcohol is the most common cause of chronic pancreatitis in men but idiopathic pancreatitis is more common in women. The incidence of chronic pancreatitis in Western countries (about 4 per 100 000 population) is similar to that (5.4−5.9 per 100 000) in Japan.14 However, the incidence of CF in Japanese is very low (1 per 350 000) compared with whites (1 per 2500 live births).7,15 The apparent large difference of the incidence of CF and chronic pancreatitis may not support the presence of …

Sensitive serum markers for detecting pancreatic cancer.

Serum amylase, immunoreactive elastase (IRE), carcinoembryonic antigen (CEA), and carbohydrate antigen (CA 19‐9) were measured in 40 patients with pancreatic cancer. IRE and CA 19‐9 were further assayed in nonpancreatic malignancies (n = 98) and chronic nonmalignant disease (n = 194). In pancreatic cancer, elevated values were observed for amylase in 30% of the patients, for IRE in 70%, for CEA in 28%, and for CA 19‐9 in 68%. Elevation of IRE and/or CA 19‐9 was found in 95% of the 40 patients. Elevated serum IRE was observed more frequently in head cancer and resectable cancer, whereas elevation in CA 19‐9 occurred more often in body‐tail cancer and unresectable cancer. Elevation of serum IRE tends to occur at earlier stages of pancreatic cancer. No one test is adequate for the accurate diagnosis of pancreatic cancer. However, the two assays complement each other, and their combined use would provide a sensitive clue for tentative diagnosis of pancreatic cancer.

Evaluating exocrine function tests for diagnosing chronic pancreatitis

To evaluate the effectiveness of exocrine function tests in diagnosing chronic pancreatitis (CP), we compared the sensitivity and specificity of duodenal intubation with tubeless tests. While the secretin test (ST) was necessary to diagnose CP, especially in noncalcified CP, and tubeless tests demonstrated insufficient sensitivity to diagnose CP, the combination assay of tubeless tests was specific enough to diagnose severe exocrine dysfunction. Our studies found the sensitivity of secretin testing to diagnose definite CP to be 87%. In patients with probable CP, 60% had mild exocrine insufficiency and 40% had normal function. The false-positive rate of the ST results in nonpancreatic diseases, except diabetes mellitus, was 5%. The correlation between morphological changes in endoscopic retrograde pancreatography (ERP) and exocrine function evaluated by ST was 74%. In patients with calcified CP, 81 % had parallel results between ERP and the ST, but in non- calcified CP, 47% had parallel results. In patients with severe or moderate exocrine insufficiency demonstrated by ST, abnormally low levels were observed in 63% by N-benzoyl-L-tyro- syl-p-aminobenzoic acid (BT-PABA) test, 61% by fecal chymotrypsin test (FCT), and 44% by pancreatic amylase (PA). In patients with normal exocrine function demonstrated by ST, abnormally low levels were observed in 28% by BT-PABA test, 28% by FCT, and 10% by PA. A combination assay of BT-PABA test, FCT, and PA improved the specificity for diagnosing CP but not the sensitivity.

Serum immunoreactive elastase in diagnosis of pancreatic diseases

Diagnostic significance of serum immunoreactive elastase-1 was studied in 137 patients with pancreatic disease, 335 with various nonpancreatic diseases, and 416 healthy controls by using radioimmunoassay. Frequency of abnormally high serum elastase values exceeding 410 ng/dl was 100% in acute pancreatitis (N=14), 40% in chronic pancreatitis (N=80), and 72% in pancreatic cancer (N=43). In pancreatic cancer the mean value of serum elastase in resectable cancer (N=19) was significantly higher than that in unresectable cancer (N=24). Sensitivity, specificity, and efficiency of serum elastase in pancreatic cancer were 72.1%, 98.3%, and 95.9% against healthy controls; 72.1%, 85.9%, and 83.6% against nonpancreatic digestive diseases; and 72.1%, 60.0%, and 64.2% against chronic pancreatitis at a cutoff level of 410 ng/dl, respectively. High serum elastase could be a diagnostic clue to detect pancreatic duct obstruction due to pancreatic cancer, although further examination should be done by endoscopic retrograde pancreatography and other imaging studies.