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- Hit 213
- Writer 이태화
Genetic Bases for Ion Transporting Defects and Salivary
- Date/Time : Fri July 4., 2003
- Speaker : 박기랑 교수
- 주성대 바이오공학과
- Place : Life Science Bldg. #104
- For inquires : Professor Young sook Lee Dept. of Life Science
생명과학과 이영숙 교수 (☎279-2296)
Saliva formation is thought to involve a two-stage process. Initially, acinar cells secrete an isotonic plasma-like fluid, the generation of which depends on the concerted activity of a number of membrane transporter proteins to drive transepithelial Cl- movement and significant HCO3- efflux. During the second stage of secretion, ductal cells modify acinar secretions primarily by conserving NaCl in a flow rate dependent fashion. According to the current model, Na+/K+/2Cl- cotransport and coupled Na+/H+ and Cl-/HCO3- exchange utilize the inwardly directed Na+ chemical gradient generated by the Na+ pump to mediate Cl- uptake across the basolateral membrane of acinar cells. The initial acidification of the cytosol resulting from HCO3- efflux into the lumen is buffered by an increase in Na+/H+ exchanger activity, which by alkalinizing the cytosol, promotes both the secretion of HCO3- via the apical anion channel and the uptake of Cl- mediated by the basolateral Cl-/HCO3- exchanger. To directly test this possibility and to confirm molecular identity of transporter proteins involved in saliva secretion we studied the in vivo and in vitro functioning of acinar cells from the parotid glands of mice with targeted disruption of Na+/K+/2Cl- cotransporter isoform 1 (Nkcc1), Na+/H+ exchanger isoform 1, isoform 2, or isoform 3 (Nhe1, Nhe2, or Nhe3, respectively). Immunohistochemistry indicated that NHE1 was localized to the basolateral and NHE2 to apical membranes of both acinar and duct cells, whereas NHE3 was restricted to the apical region of duct cells. Na+/H+ exchange activity was reduced more than 95% in acinar cells of NHE1-deficient mice (Nhe1-/-). Salivation in response to a muscarinic agonist (pilocarpine), the primary in situ salivation signal was reduced significantly in both Nhe1-/-and Nhe2-/- mice, particularly during prolonged stimulation, whereas the loss of NHE3 had no effect on secretion. Expression of Na+/K+/2Cl- cotransporter mRNA increased dramatically in Nhe1-/- parotid glands but not in those of Nhe2-/- or Nhe3-/- mice, suggesting that compensation occurs for the loss of NHE1. The sodium content, chloride activity and osmolality of saliva in Nhe2-/- or Nhe3-/- mice were comparable with those of wild-type mice. In contrast, Nhe1-/- mice displayed impaired NaCl absorption. These data suggest that in parotid duct cells apical NHE2 and NHE3 do not play a major role in Na+ absorption. These results also demonstrate that basolateral NHE1 and apical NHE2 modulate saliva secretion in vivo, especially during sustained stimulation when secretion depends less on Na+/K+/2Cl- cotransporter activity. In wild-type mice Na+/K+/2Cl- cotransporter isoform 1 (NKCC1) was localized to the basolateral membranes of parotid acinar cells, whereas expression was not detected in duct cells. The lack of functional NKCC1 resulted in a dramatic reduction (>60%) in the volume of saliva secreted in response to pilocarpine. Consistent with defective Cl- uptake, a loss of bumetanide-sensitive Cl- influx was observed in parotid acinar cells from Nkcc1-/- mice. Cl-/HCO3- exchanger activity was increased in parotid acinar cells isolated from Nkcc1-/- mice suggesting that the residual saliva secreted by mice lacking NKCC1 is associated with anion exchanger-dependent Cl- uptake. Indeed, expression of the Cl-/HCO3- exchanger AE2 was enhanced suggesting that this transporter compensates for the loss of functional Na+/K+/2Cl- cotransporter. Furthermore, the ability of the parotid glands to conserve NaCl was abolished in Nkcc1-/- mice. This deficit was not associated with changes in the morphology of the ducts, but transcripts levels for the a-, b-, and g-subunits of epithelial Na+ channel were reduced. These data directly demonstrate that NKCC1 is the major Cl- uptake mechanism across the basolateral membrane of acinar cells and is critical for driving saliva secretion in vivo.