Yoshiko Iwasa

Gramicidin-perforated patch analysis on HCO3- secretion through a forskolin-activated anion channel in rat parotid intralobular duct cells.

Abstract. Forskolin-induced anion currents and depolarization were investigated to clarify the mechanism of HCO3− secretion in the intralobular duct cells of rat parotid glands. Anion currents of the cells were measured at the equilibrium potential of K+, using a gramicidin-perforated patch technique that negligibly affects intracellular anion concentration. The forskolin-induced anion current was sustained and significantly (54%) suppressed by glibenclamide (200 μm), a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. The anion current was markedly suppressed by addition of 1 mm methazolamide, a carbonic anhydrase inhibitor, and removal of external HCO3−. Forskolin depolarized the cells in the current-clamp mode. Addition of methazolamide and removal of external HCO3− significantly decreased the depolarizing level. These results suggest that activation of anion channels (mainly the CFTR Cl− channel located in luminal membranes) and production of cytosolic HCO3− induce the inward anion current and resulting depolarization. Inhibition of the Na+-K+-2Cl− cotransporter and the Cl−-HCO3− exchanger had no significant effect on the current or depolarization, indicating that the uptake of Cl− via the Na+-K+-2Cl− cotransporter or the Cl−-HCO3− exchanger is not involved in the responses. Taken together, we conclude that forskolin activates the outward movement (probably secretion) of HCO3− produced intracellularly, but not of Cl− due to lack of active Cl− transport in parotid duct cells, and that the gramicidin-perforated patch method is very useful to analyze anion transport.

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.

Suppression of carbachol-induced oscillatory Cl secretion by forskolin in rat parotid and submandibular acinar cells

Sympathetic stimulation induces weak salivation compared with parasympathetic stimulation. To clarify this phenomenon in salivary glands, we investigated cAMP-induced modulation of Ca(2+)-activated Cl(-) secretion from rat parotid and submandibular acinar cells because fluid secretion from salivary glands depends on the Cl(-) secretion. Carbachol (Cch), a Ca(2+)-increasing agent, induced hyperpolarization of the cells with oscillatory depolarization in the current clamp mode of the gramicidin-perforated patch recording. In the voltage clamp mode at -80 mV, Cch induced a bumetanide-sensitive oscillatory inward current, which was larger in rat submandibular acinar cells than in parotid acinar cells. Forskolin and IBMX, cAMP-increasing agents, did not induce any marked current, but they evoked a small nonoscillatory inward current in the presence of Cch and suppressed the Cch-induced oscillatory inward current in all parotid acinar cells and half (56%) of submandibular acinar cells. In the current clamp mode, forskolin + IBMX evoked a small nonoscillatory depolarization in the presence of Cch and reduced the amplitude of Cch-induced oscillatory depolarization in both acinar cells. The oscillatory inward current estimated at the depolarized membrane potential was suppressed by forskolin + IBMX. These results indicate that cAMP suppresses Ca(2+)-activated oscillatory Cl(-) secretion of parotid and submandibular acinar cells at -80 mV and possibly at the membrane potential during Cch stimulation. The suppression may result in the weak salivation induced by sympathetic stimulation.

Forskolin-Induced Clearance of the Fluorescent Dye Sulforhodamine from Rat Parotid Intralobular Duct Lumen: Visualization of the Secretory Function under a Confocal Laser Scanning Microscope

Cyclic AMP evokes fluid secretion with bicarbonate in exocrine ducts. Clearance of fluorescent dyes from rat parotid intralobular ducts by forskolin was visualized as a fluorescence change in the duct luminal space by optical sectioning under a confocal laser scanning microscope to clarify the secretory function in the ducts. When the isolated rat parotid intralobular duct segments were superfused with membrane-impermeable fluorescent dyes during the experimental period, fluorescent dyes were passively moved into the duct space. Forskolin and isobutylmethylxanthine decreased the fluorescence of anionic dye, sulforhodamine B, and neutral dye, dextran tetramethyl-rhodamine, in the duct space, suggesting that the forskolin-induced clearance of fluorescent dyes might be the result of fluid secretion in the ducts. Methazolamide inhibited a forskolin-induced sustained decrease in duct fluorescence and intracellular acidification. Low concentrations of external Cl?, DIDS, bumetanide and amiloride did not markedly inhibit a forskolin-induced decrease in duct fluorescence. These findings suggest that a major portion of the steady decrease in duct fluorescence by forskolin was related to intracellular HCO3? production, not the uptake mechanism of external Cl?. Glibenclamide, NPPB, DPC and DMA inhibited the forskolin-induced decrease. Forskolin evokes the clearance of fluorescent dyes from duct space possibly due to fluid secretion in rat parotid ducts, associated with secretion through CFTR and DPC-sensitive anion channels of carbonic anhydrase-dependent bicarbonate linked with the Na+/H+ exchange mechanism.

External Cl--Dependent Formation of Watery Vacuoles by Long-Term Hypotonic Shock in 3T3-L1 Cells

Osmotic shock transiently induces a volume change in the cells, followed by a restoration of the cell volume due to intracellular water regulation. Effect of long-term osmotic shock on the water regulation is not completely understood. Vacuole formation by long-term osmotic shock was investigated to clarify the water exclusion mechanism from cytoplasm into intracellular vacuoles in 3T3-L1 cells. Incubation of cells in hypotonic solution reversibly induced the vacuole formation. Staining of vacuoles with fluorescent dyes revealed that vacuoles were derived from endoplasmic reticulum and Golgi apparatus but not lysosomes. Membrane-impermeable fluorescent dyes were taken up into some vacuoles from cytoplasm and extracellular solution, suggesting that some vacuoles exhibit the dynamic changes for the connection of plasma membrane, and that transporter for membrane-impermeable dyes might be active in some vacuole membranes. External Cl-, but not Na+, was required for vacuole formation. DPC suppressed the vacuole formation and increased cell height, and further incubation with DPC increased the number of dead cells. Bumetanide, dimethylamiloride, and HgCl2 did not suppress the hypotonic stress-induced formation of water vacuoles. These findings suggest that 3T3-L1 cells regulate the intracellular water content through the DPC-sensitive external Cl--dependent vacuole formation during long-term osmotic stress.