Proton electrochemical gradient and phosphate potential in submitochondrial particles.

The aerobic uptake of inorganic ions, such as 86Rb+ or 125I-, by submitochondrial particles, is about one order of magnitude lower than the uptake of organic ions, such as acridines or 8-anilino-1-naphthalene sulphonate. The values of deltapH, the transmembrane pH differential, and deltapsi, the transmembrane membrane potential are between 60 and 100 mV when calculated on the inorganic ions and between 150 and 240 mV when calculated on the organic ions. The discrepancy between the deltapH and deltapsi values from organic and inorganic ions is large at high but not at low ion/protein ratios. 2. In the absence of weak bases and strong acids the values of deltamuH, the proton electrochemical potential difference, are close to 100 mV and the magnitude of deltapH and deltapsi are similar. Weak bases decrease deltapH and enhance deltapsi. Strong acids decrease deltapsi and enhance deltapH. Interchangeability of deltapH with deltapsi occurs at low concentrations of weak bases and strong acids. High concentrations of weak bases and strong acids cause depression of deltamuH. 3. Concentrations of weak bases capable of abolishing deltapH, do not affect ATP synthesis. Concentrations of strong acids capable of abolishing deltapsi affect only slightly ATP synthesis. Concentrations of weak bases and strong acids capable of causing a decline of deltapH + deltapsi inhibit ATP synthesis. 4. Depression of deltamuH is paralleled by inhibition of ATP synthesis and decline of deltaGp, the phosphate potential. Abolition of ATP synthesis occurs only when deltamuH is below 20 mV. The deltaGp/deltamuH ratio increases hyperbolically with the decrease of deltamuH.     

Water channel proteins in bile formation and flow in health and disease: When immiscible becomes miscible

An essential function of the liver is the formation and secretion of bile, a complex aqueous solution of organic and inorganic compounds essential as route for the elimination of body cholesterol as unesterified cholesterol or as bile acids. In bile, a considerable amount of otherwise insoluble cholesterol is solubilized by carriers including two other classes of lipids, namely phospholipid and bile acids. Formation of bile and generation of bile flow are driven by the active secretion of bile acids, lipids and electrolytes into the canalicular and bile duct lumens followed by the parallel movement of water. Thus, water has to cross rapidly into and out of the cell interior driven by osmotic forces. Bile as a fluid, results from complicated interplay of hepatocyte and cholangiocyte uptake and secretion, concentration, by involving a number of transporters of lipids, anions, cations, and water. The discovery of the aquaporin water channels, has clarified the mechanisms by which water, the major component of bile (more than 95%), moves across the hepatobiliary epithelia. This review is focusing on novel acquisitions in liver membrane lipidic and water transport and functional participation of aquaporin water channels in multiple aspects of hepatobiliary fluid balance. Involvement of aquaporins in a series of clinically relevant hepatobiliary disorders are also discussed.     

The electrical and mechanical activity of the esophageal cell of Ascaris lumbricoides

The esophagus of Ascaris is a syncytial muscle organ of tubular shape in which the myofibrils are arranged radially between the lumen and the external surface. A resting potential of almost 40 mv (cytoplasm negative) is maintained by the extracellular organic anions (volatile fatty acids) found in the perienteric fluid. Replacement of these anions by Cl- ions results in a large depolarization. The resting potential is also decreased when the external pH is lowered. The leading phase of the action potential with a positive overshoot of about 18 mv elicits contraction of the myofibrils, development of negative pressure within the lumen, and suction of liquid and food particles. The mechanical energy stored in the elastic components of the cell is released when the myofibrils relax, thus injecting the contents of the lumen into the intestine. A fast and synchronous relaxation is elicited by a regenerative membrane polarization, a negative spike with a peak value of up to 108 mv produced by an increase in the permeability of the membrane to K+ ions. Cells completely depolarized in "chloride" saline are still able to generate such large potassium spikes.     

The Mechanism of Enterohepatic Circulation in the Formation of Gallstone Disease

Bile acids entering into enterohepatic circulating are primary acids synthesized from cholesterol in hepatocyte. They are secreted actively across canalicular membrane and carried in bile to gallbladder, where they are concentrated during digestion. About 95 % BAs are actively taken up from the lumen of terminal ileum efficiently, leaving only approximately 5 % (or approximately 0.5 g/d) in colon, and a fraction of bile acids are passively reabsorbed after a series of modifications in the human large intestine including deconjugation and oxidation of hydroxy groups. Bile salts hydrolysis and hydroxy group dehydrogenation reactions are performed by a broad spectrum of intestinal anaerobic bacteria. Next, hepatocyte reabsorbs bile acids from sinusoidal blood, which are carried to liver through portal vein via a series of transporters. Bile acids (BAs) transporters are critical for maintenance of the enterohepatic BAs circulation, where BAs exert their multiple physiological functions including stimulation of bile flow, intestinal absorption of lipophilic nutrients, solubilization, and excretion of cholesterol. Tight regulation of BA transporters via nuclear receptors (NRs) is necessary to maintain proper BA homeostasis. In conclusion, disturbances of enterohepatic circulation may account for pathogenesis of gallstones diseases, including BAs transporters and their regulatory NRs and the metabolism of intestinal bacterias, etc.     

Effects of bile acids on actin polymerization in vitro

Bile acids are major determinants of canalicular bile secretion, and there are indications that choleretic bile acids increase bile canalicular contractions, in isolated rat hepatocytes. Therefore, we examined the influence of various bile acids on the rate of actin polymerization in vitro. The free forms of cholic acid, ursodeoxycholic acid, and chenodeoxycholic acid, as well as their taurine and glycine conjugates, were incubated with purified muscle actin, at a concentration of 100-300 nmoles/mg actin. The rate of actin polymerization was measured by viscometry and the fluorescence of the pyrene probe, linked to actin. Results showed that all bile acids slow the rate of polymerization, and that the effect was dose-dependent. However, the reduction by chenodeoxycholic acid was greater than that caused by the other bile acids. The results indicate that bile acids, particularly in high concentrations interact with actin, a finding that may be related to the increased bile canalicular contractility, and altered canalicular membrane morphology, induced by choleretic bile acids.     

Starch-primulin fluorescence

Summary Intact starch grains with primulin give initially a blue fluorescence that is weakly polarized. As more dye enters the grain, the fluorescence color gradually changes to a yellow-green fluorescence that is strongly polarized. Where the structure of the grain is disorganized, the primulin-starch complex fluoresces yellow. A bright yellow rim about all intact grains is interpreted as a Becke line.Fluorescence colors of primulin-starch are essentially unaffected by hydrogen ion concentration, by the presence of salts, by the solvent in which the primulin is dissolved, and by the dye fraction (of acetoneseparated primulin fractions) used as a solute. These colors have no relation to the type of starch in the primulin-starch complex.Absorption and fluorescence spectra of primulin and its fractions vary with the concentration of the dye, the nature of the solvent, and the nature of the fraction. Three types of primulin-solvent association are envisioned: primulin anion in water; primulin molecule hydrogen-bonded to solvent or substrate molecule in non-aqueous medium; primulin dimeric association-at high concenntrations-in non-aqueous medium.Primulin-starch fluorescence colors are explicable on the basis of dye concentration and pore size in the starch grain. Pore spaces that will accept dye molecules are estimated to have a diameter between 7 and 20°.     

Osservazioni sul rapporto fra accumulo protoplasmatico del fluorocromo uranina e intensità respiratoria in Oscillatoria irrigua Kütz

Abstract

OBSERVATIONS ON THE RELATION BETWEEN PROTOPLASMIC ACCUMULATION OF THE FLUOROCHROME URANIN AND RESPIRATORY INTENSITY IN OSCILLATORIA IRRIGUA Kütz. — Uranin (sodic fluorescein) is a vital dye of peculiar cytophysiological interest, since it is accumulated chiefly in the nucleus, cytoplasm and chondriosomes of living plants cell. The degree of accumulation of this dye is in evident relation with the cellular activity.

In the present paper the relation between respiration and secundary fluorescence induced by uranin in the cells of Oscillatoria irrigua is demonstrated. The A. has also observed that a reduction of the respiratory activity of so coloured cells through inhibitory substances as f. i. oxyquinoline results in a releasing of uranin from the cell. In this case the dye is no more detained in the cells and flows outwards.

The true nature of the accumulation of uranin and its relations with the cellular activity are not yet clear, but its value as research mean to appreciate the cellular activity of organisms is emphasized.     

Rho kinase, myosin-II, and p42/44 MAPK control extracellular matrix-mediated apical bile canalicular lumen morphogenesis in HepG2 cells

The molecular mechanisms that regulate multicellular architecture and the development of extended apical bile canalicular lumens in hepatocytes are poorly understood. Here, we show that hepatic HepG2 cells cultured on glass coverslips first develop intercellular apical lumens typically formed by a pair of cells. Prolonged cell culture results in extensive organizational changes, including cell clustering, multilayering, and apical lumen morphogenesis. The latter includes the development of large acinar structures and subsequent elongated canalicular lumens that span multiple cells. These morphological changes closely resemble the early organizational pattern during development, regeneration, and neoplasia of the liver and are rapidly induced when cells are cultured on predeposited extracellular matrix (ECM). Inhibition of Rho kinase or its target myosin-II ATPase in cells cultured on glass coverslips mimics the morphogenic response to ECM. Consistently, stimulation of Rho kinase and subsequent myosin-II ATPase activity by lipoxygenase-controlled eicosatetranoic acid metabolism inhibits ECM-mediated cell multilayering and apical lumen morphogenesis but not initial apical lumen formation. Furthermore, apical lumen remodeling but not cell multilayering requires basal p42/44 MAPK activity. Together, the data suggest a role for hepatocyte-derived ECM in the spatial organization of hepatocytes and apical lumen morphogenesis and identify Rho kinase, myosin-II, and MAPK as potentially important players in different aspects of bile canalicular lumen morphogenesis.     

植物乳杆菌DY6主要抑菌代谢物的分析和鉴定

【背景】被广泛应用于食品和饲料等多个行业的乳酸菌已成为制作生物防腐剂的研究热点。【目的】探究抑菌性能良好的植物乳杆菌DY6的抑菌物质,为其进一步应用提供参考依据。【方法】对植物乳杆菌发酵液中抑菌物质的理化特性进行研究,采用GC-MS分析发酵上清液代谢物,通过多元统计学分析方法推测主要抑菌物质,抑菌物质通过半制备进行初步分离后用GC-MS鉴定。【结果】植物乳杆菌DY6对金黄色葡萄球菌、大肠杆菌、沙门氏菌都有较强的抑制作用。采用不同发酵时间的发酵液作为研究对象,测定发酵上清液的抑菌能力,发酵0-4 h上清液无抑菌能力,发酵至8 h抑菌能力逐步上升,发酵24-48 h发酵上清液抑菌能力趋于稳定,在48 h时抑菌能力最佳,抑菌直径为15.28mm。通过多元统计学分析乳酸菌发酵液差异标志物,发现主要差异物为有机酸(如乳酸、乙酸、丙酸等)和脂肪酸(如辛酸、癸酸等)。经过半制备液相分离发酵上清液得到的抑菌组分,主要有有机酸(如乳酸、乙酸、3-苯基乳酸、苯丙酸等)和脂肪酸(如癸酸、辛酸、壬酸等),另外还有少量的醛类和醇类物质。【结论】确定了植物乳杆菌DY6的抑菌物质主要为有机酸和脂肪酸,为其进一步防腐应用提供了理论基础。     

Ionic transport mechanisms underlying fluid secretion by the pancreas

The pancreas is a 'leaky' epithelium and secretes a juice in which sodium and potassium have concentrations similar to those of plasma. The characteristic features of the secretion are its isosmolality and its high bicarbonate concentration. It is the latter that has attracted considerable attention. Secretion in the isolated cat pancreas is directly proportional to the bicarbonate concentration in the nutrient fluid. The ability of the gland to secrete weak acids has led to the view that because of the very different chemical nature of the anions, it is most likely that it is a component common to all buffers, the proton, that is subject to active transport. This is supported by the decrease in pH and the increase in rho CO2 of the venous effluent when secretion occurs and the sensitivity of secretion to the pH of the nutritional extracellular fluid. It is proposed that the cellular mechanisms are as follows: CO2 diffuses into the cell and is hydrated to carbonic acid under the influence of carbonic anhydrase. The bicarbonate ion so formed diffused into the ductular lumen and the proton is transported backwards through the epithelium with a proton pump (Mg2+ -ATPase) provisionally located in the luminal membrane and a hydrogen-sodium exchange carrier located in the basolateral membrane. Energy for the latter process is derived from the sodium gradient between extracellular fluid and cell. This gradient is maintained by a (Na+ + K+)-ATPase also located in the basolateral membrane. Chloride appears to be transported partly through a chloride-bicarbonate exchange mechanism but largely passively together with a large sodium and potassium component through the paracellular pathway. Osmotic equilibrium is likely to occur in the small ductules.     

Vasoactive intestinal peptide in the normal and inflamed feline gallbladder

Intravenous infusion of vasoactive intestinal peptide (VIP) causes gallbladder mucosal fluid secretion by an action on epithelial cell receptors in the cat. Gallbladder fluid secretion is observed also in experimental cholecystitis and this secretion is abolished when the intramural gallbladder nerves are blocked. In the present study, immunoreactive VIP was detected in the gallbladder contents (29 +/- 5 (S.E.M.) pM) in the obstructed lumen of the gallbladder in cats with experimental cholecystitis and gallbladder mucosal fluid secretion, but not in the normal feline gallbladder. During luminal perfusion of the gallbladder in vivo, the calculated secretion of VIP into the gallbladder lumen in animals with experimental cholecystitis was significantly higher (0.31 +/- 0.08 (S.E.M.), pmol/h) than in controls (0.11 +/- 0.02 (S.E.M.), pmol/h) while plasma levels of VIP were similar. Recovery of exogenously administered VIP was similar in normal and inflamed gallbladders. The present results support the hypothesis that intramural VIP-releasing nerve fibers may be activated in cholecystitis.     

Prebiotic phosphate: a problem insoluble in water ? ]

It is well-known that in water phosphate readily reacts with calcium, precipitating as insoluble apatite. How phosphorus could have been available for prebiotic reactions is still an open problem. We suggest that phosphorus-containing compounds might have accumulated in a hydrophobic medium, since the absence of calcium ions would have prevented them from precipitating as apatite. Hydrophobic compounds may have been synthesized on the early Earth through the polymerization of methane or through Fischer-Tropsch-type reactions. Moreover, hydrophobic compounds would have been delivered to the early Earth by extraterrestrial infall. In previous articles (Morchio and Traverso [1999], Morchio et al. [2001]) we suggested that such hydrophobic material would have formed a hydrophobic layer on the surface of the sea, which would have provided an environment thermodynamically more suitable than water for the concentration and polymerization of organic molecules fundamental to life, particularly amino acids and (pyrimidine) bases. It may be hypothesized that elemental phosphorus or phosphorus-containing compounds (such as phosphite) deriving from volcanic eruptions would have ended up raining down into the hydrophobic layer, accumulating due to the absence of calcium ions, in an environment protected against hydrolysis. Phosphorus-containing compounds might have interacted with hydrophobic molecules in the layer giving rise to polymers. In particular, phosphite might have reacted with the hydrophobic amino acids, giving rise to phosphoamino acids, which, in turn, might have interacted with pyrimidine bases (relatively abundant in the layer) giving rise to peptides and oligonucleotide-like polymers. Indeed, it has been experimentally shown (Zhou et al. [1996]) that, in an anhydrous organic medium (pyridine), dialkilphosphite reacts with amino acids to form phosphoamino acids, which interact with pyrimidine nucleosides to give nucleotides, short oligonucleotides and phosphoryl peptides.     

The mechanism of biliary secretion of reduced glutathione. Analysis of transport process in isolated rat-liver canalicular membrane vesicles

Transport of reduced glutathione (GSH) was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibit uptake of [2-3H]glycine--labeled GSH into an osmotically reactive intravesicular space. Although the canalicular membrane vesicles possess gamma-glutamyltransferase and aminopeptidase M, enzymes that hydrolyze glutathione into component amino acids, inactivation of the vesicle-associated transferase by affinity labeling with L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) had no effect on the initial rate of GSH transport. Chemical analysis revealed that intact GSH accounted for most of vesicle-associated radioactivity. The initial rate of transport followed saturation kinetics with respect to GSH concentration; an apparent Km of 0.33 mM and V of 1.47 nmol/mg protein in 20 s were calculated. These results indicate that transport of GSH across the canalicular membranes is a carrier-mediated process. Replacement of NaCl in the transport medium by KCl, LiCl or choline chloride had no effect on the transport activity of the vesicles. The rate of GSH uptake by the vesicles was enhanced by valinomycin-induced K+-diffusion potential (vesicle inside-positive) and was inhibited by probenecid, indicating that GSH transport across the canalicular membranes is electrogenic and involves the transfer of negative charge. The transport of GSH was inhibited by oxidized glutathione or S-benzyl-glutathione. This transport system in canalicular plasma membranes may function in biliary secretion of GSH and its derivatives which are synthesized in hepatocytes by oxidative processes or glutathione S-transferase.     

Intravital study of the hydrophobic interactions in protein fibrils by a polarization-fluorescence method. I. Collagen fibrils

It has been shown earlier that anisotropy of extrinsic fluorescence (AEF) of ordered structures in living cell may serve as a measure of the free structure energy. The experimental study of AEF changes of myelin at varying temperatures (0-30 degrees C) revealed cold denaturation. This phenomenon occurs only in cases when the structure in question is stabilized through hydrophobic interactions. The purpose of this work was to show that not only membranes but also some native ordered protein structures might be analysed by fluorescence polarization to detect hydrophobic interactions. The fluorescence anisotropy of primulin adsorbed by collagen fibrils from rat tail has been investigated. The fluorophore orientation decreases with temperature, i.e. here the phenomenon of cold denaturation is observed. When the medium humidity falls down to 80%, no cold denaturation occurs. AEF of collagen fibrils depends to a considerable extent on organic substances (ethanol, ethylene glycol) added in small concentrations to the incubation medium. It is concluded that the dependence of the whole collagen molecular structure in fibrils on the content and structure of the solvent shell is of critical nature.     

Bacteria-degraders as the base of an amperometric biosensor for detection of anionic surfactants

Several strains belonging to genera Pseudomonas and Achromobacter and characterized by the ability to degrade anionic surfactants were tested as potential bases of microbial biosensors for surfactant detection. For each strain the substrate specificity and stability of sensor signals were studied. The total amount of the substrates tested (including carbohydrates, alcohols, aromatics, organic acids, etc.) was equal to 60; the maximal signals were observed towards the anionic surfactants. The lower limit of detection for sodium dodecyl sulfate used as a model surfactant was in the field of 1 microM for all the strains. The created microbial biosensor model can extend the practical possibilities for rapid evaluation of surfactants in water media.     

白细胞介素-6在工程菌分批培养中的高效表达及其纯化

在确定了培养基及pH值的基础上,进一步观察了升温诱导过程中有机酸的产生及其对工程菌E.coli DH5α(pHV-hIL-6)生长和rIL-6表达的影响。当有机酸浓度低于70mmol/L以下时,菌密度达到干重2～3.5g/L之间收菌,rIL-6的表达水平为25％～32％;当有机酸浓度达到70mmol/L以上时,工程菌的生长不受影响,而rIL-6的表达明显受抑制。产生的有机酸以乙酸为主。收集菌体后,经过破菌,分离提纯的包涵体,其rIL-6的纯度可达到70％。用GuHCl缓冲液溶解包涵体,样品稀释后经过Q Sepharose F F柱纯化,可得到纯度达95％以上的rIL-6。采用依赖IL-6的小鼠杂交瘤细胞系7TD1及MTT比色法测定生物活性,rIL-6的比活性为2×10~8U/mg。     

Spatial and temporal distribution of solutes in the developing carrot taproot measured at single-cell resolution

The time-course and spatial distribution of sugars and ions in carrot (Daucus carota L.) was studied at fine resolution using single cell (SiCSA) and tissue analysis. Four phases of osmolyte accumulation in the taproot were identified: an amino acid (germination) phase, when internal sources of amino acids provide seedlings with osmotica; an ion phase, when inorganic and organic ions were the main solutes; a hexose phase, when concentrations of glucose and fructose sharply increased and reached their maximum; and a sucrose phase, when sucrose became the major solute. Spatial distribution of sugar in taproot cells showed a general trend of highest concentration on both sides of the vascular cambium (some 200 mM sucrose, 150 mM glucose) and a minimum in the pith (some 100 mM sucrose, 60 mM glucose) and in periderm. Electrolytes (e.g. potassium) followed a distribution generally reciprocal to that of sugars; minimum in the tissue adjacent to the cambium (some 10 mM) and maximum in the pith and periderm (some 60-100 mM). The cambial cells contained unexpectedly low concentrations of sugars and potassium. These spatial and temporal patterns indicate that amino acids, other electrolytes and sugars are interchangeable in the tissue osmotic balance. The nature of the solute is developmentally determined both temporally and spatially. During the accumulation of electrolytes following the initial amino acid phase, osmotic pressure to 420 mosmol kg-1 rises and then remains constant despite large changes in the concentration of individual solutes. This indicates that osmotic pressure is regulated independently of the individual concentrations of solutes.     

DYNAMICS OF THREE ORGANIC ACIDS (MALIC,ACETIC AND SUCCINIC ACID) IN SUNFLOWER EXPOSED TO CADMIUM AND LEAD

Sunflower (Helianthus annuus L.) has been considered as a good candidate for bioaccumulation of heavy metals. In the present study, sunflower was used to enrich the cadmium and lead in sand culture during 90 days. Biomass, Cd and Pb uptake, three organic acids and pH in cultures were investigated. Results showed that the existence of Cd and Pb showed different interactions on the organic acids exudation. In single Cd treatments, malic and acetic acids in Cd10 showed an incremental tendency with time. In the mixed treatments of Cd and Pb, malic acids increased when 10 and 40 mg?L^–1 Cd were added into Pb50, but acetic acids in Pb50 were inhibited by Cd addition. The Cd10 supplied in Pb100 stimulated the secretion of malic and succinic acids. Moreover, the Cd or Pb uptake in sunflower showed various correlations with pH and some organic acids, which might be due to the fact that the Cd and Pb interfere with the organic acids secretion in rhizosphere of sunflower, and the changes of organic acids altered the form and bioavailability of Cd and Pb in cultures conversely.     

Effect of fatty acids on the synthesis and secretion of apolipoprotein B by rat hepatocytes

The modulation of apolipoprotein B synthesis and secretion by fatty acids in rat hepatocytes was studied. Maximum apolipoprotein B production was obtained in the case of oleic acid followed by linoleic, stearic and palmitic/linolenic acid when compared to control which was not supplemented with any fatty acids. Oleic acid was found to exert a concentration dependent increase in the secretion of [³H] apolipoprotein B into the medium while that associated with the cell layer was not affected. Pulse chase experiments in the presence of oleic acid showed that it caused an increase in the secretion of apolipoprotein B into the medium.¹⁴C-acetate incorporation into cholesterol and cholesteryl ester associated with the cell layer and secreted very low density lipoproteins also showed an increase in the presence of oleic acid indicating an increase in cholesterogenesis. The effect of oleic acid on [³H] apolipoprotein B and very low density lipoproteins secretion appeared to be mediated through cholesterol as (i) ketoconazole, an inhibitor of cholesterol synthesis caused significant reduction in the stimulatory effect of oleic acid on apolipoprotein secretion and (ii) mevinolin, another inhibitor of cholesterol synthesis also reversed the stimulatory effect of oleic acid on apolipoprotein B secretion. These results indicated that oleic acid may influence apolipoprotein B synthesis and secretion in hepatocytes probably by affecting cholesterol/cholesteryl ester formation which may be a critical component in the secretion of apolipoprotein B as lipoproteins     

Carbachol-induced fluid movement through methazolamide-sensitive bicarbonate production in rat parotid intralobular ducts: quantitative analysis of fluorescence images using fluorescent dye sulforhodamine under a confocal laser scanning microscope

Fluid secretion is observed at the openings of ducts in the exocrine gland. It remains unclear whether the ducts are involved in fluid secretion in the salivary glands. In the present study, we investigated the exclusion of fluorescent dye from the duct lumen by carbachol (CCh) in isolated parotid intralobular duct segments to clarify the ability of the ducts for the fluid secretion. When the membrane-impermeable fluorescent dye, sulforhodamine, was added to the superfused extracellular solution, quantitative fluorescence images of the duct lumen were obtained under the optical sectioning at the level of the duct lumen using a confocal laser scanning microscope. CCh decreased the fluorescent intensity in the duct lumen during the superfusion of the fluorescent dye, and CCh flushed out small viscous substances stained with the fluorescent dye from isolated duct lumen, suggesting that CCh might induce fluid secretion in the duct, leading to the clearance of the dye and small stained clumps from the duct lumen. CCh-induced clearance of the fluorescent dye was divided into two phases by the sensitivity to external Ca2+ and methazolamide, an inhibitor for carbonic anhydrase. The initial phase was insensitive to these, and the subsequent late phase was sensitive to these. A major portion in the late phase was inhibited by removal of bicarbonate in the superfusion solution and DPC, but not low concentration of external Cl-, bumetanide or DIDS, suggesting that methazolamide-sensitive production of HCO3-, but not the Cl- uptake mechanism, might contribute to the CCh-induced clearance of the dye from the duct lumen. These results represent the first measurements of fluid movement in isolated duct segments, and suggest that carbachol might evoke fluid secretion possibly through Ca2+-activated, DPC-sensitive anion channels with HCO3- secretion in the rat parotid intralobular ducts.