Gramicidin-perforated patch recording revealed the oscillatory nature of secretory Cl- movements in salivary acinar cells.
Elevations of cytoplasmic free calcium concentrations ([Ca(2+)](i)) evoked by cholinergic agonists stimulate isotonic fluid secretion in salivary acinar cells. This process is driven by the apical exit of Cl(-) through Ca(2+)-activated Cl(-) channels, while Cl(-) enters the cytoplasm against its electrochemical gradient via a loop diuretic-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC) and/or parallel operations of Cl(-)-HCO(3)(-) and Na(+)-H(+) exchangers, located in the basolateral membrane. To characterize the contributions of those activities to net Cl(-) secretion, we analyzed carbachol (CCh)-activated Cl(-) currents in submandibular acinar cells using the "gramicidin-perforated patch recording configuration." Since the linear polypeptide antibiotic gramicidin creates monovalent cation-selective pores, CCh-activated Cl(-) currents in the gramicidin-perforated patch recording were carried by Cl(-) efflux via Cl(-) channels, dependent upon Cl(-) entry through Cl(-) transporters expressed in the acinar cells. CCh-evoked oscillatory Cl(-) currents were associated with oscillations of membrane potential. Bumetanide, a loop diuretic, decreased the CCh-activated Cl(-) currents and hyperpolarized the membrane potential. In contrast, neither methazolamide, a carbonic anhydrase inhibitor, nor elimination of external HCO(3)(-) had significant effects, suggesting that the cotransporter rather than parallel operations of Cl(-)-HCO(3)(-) and Na(+)-H(+) exchangers is the primary Cl(-) uptake pathway. Pharmacological manipulation of the activities of the Ca(2+)-activated Cl(-) channel and the NKCC revealed that the NKCC plays a substantial role in determining the amplitude of oscillatory Cl(-) currents, while adjusting to the rate imposed by the Ca(2+)-activated Cl(-) channel, in the gramicidin-perforated patch configuration. By concerting with and being controlled by the cation steps, the oscillatory form of secretory Cl(-) movements may effectively provide a driving force for fluid secretion in intact acinar cells.
Sugita
Makoto
M
Department of Oral Physiology, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553, Japan. sugisan@hiroshima-u.ac.jp
Hirono
Chikara
C
Shiba
Yoshiki
Y
D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002034:Bumetanide / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D018118:Chloride Channels / Q000378:metabolism; D027963:Chloride-Bicarbonate Antiporters / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D018679:Cholinergic Agonists / Q000494:pharmacology; D004232:Diuretics / Q000494:pharmacology; D006096:Gramicidin / Q000494:pharmacology*; D008297:Male; D008704:Methazolamide / Q000494:pharmacology; D009579:Nitrobenzoates / Q000494:pharmacology; D018408:Patch-Clamp Techniques / Q000379:methods*; D010507:Periodicity; D051381:Rats; D017208:Rats, Wistar; D012469:Salivary Glands / Q000378:metabolism* / Q000502:physiology*; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism
D020533:Angiogenesis Inhibitors; D000900:Anti-Bacterial Agents; D018118:Chloride Channels; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides; D018679:Cholinergic Agonists; D004232:Diuretics; D009579:Nitrobenzoates; D017923:Sodium-Hydrogen Exchangers; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D006096:Gramicidin; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D002217:Carbachol; D002118:Calcium; D008704:Methazolamide