Functional activity of Pat-1 (Slc26a6) Cl(−)/HCO3(−) exchange in the lower villus epithelium of murine duodenum.

AIMS: The apical membrane anion exchanger putative anion transporter-1 (Pat-1) is expressed at significant levels in the lower villus epithelium of murine duodenum. However, previous studies of Cl(−)/HCO3(−) exchange in the lower villus have failed to demonstrate Pat-1 function. Those studies routinely included luminal glucose which induces Na(+) -coupled glucose transport and acidifies the villus epithelium. Since Pat-1 has been proposed to be an electrogenic 1Cl(−)/2HCO3(−) exchanger, membrane depolarization or cell acidification during glucose transport may obscure Pat-1 activity. Therefore, we investigated the effects of luminal glucose on Cl(−)(IN)/HCO3(−) (OUT) exchange activity in the lower villus epithelium. METHODS: Cl(−)(IN) /HCO (−) (OUT) exchange of villus epithelium in duodenal mucosa from Pat-1 knockout (KO), Slc26a3 [down-regulated in adenoma (Dra)] KO, cystic fibrosis transmembrane conductance regulator (Cftr) KO and wild-type (WT) littermate mice was measured using the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. Short-circuit current (I(sc) ) was measured in Ussing chambers. RESULTS: During glucose absorption, Cl(−)(IN)/HCO3(−) (OUT) exchange in the lower villus epithelium was abolished in the Dra KO and unaffected in the Pat-1 KO relative to WT. However, during electroneutral mannose absorption or electrogenic α-D-methyl glucoside absorption, Cl(−)(IN) /HCO3(−) (OUT) exchange was reduced in both Pat-1 KO and Dra KO villi. Exposure to high [K(+)] abolished Cl(−)(IN) /HCO3(−) (OUT) exchange in the Dra KO but not the Dra/Cftr double KO epithelium, suggesting that Pat-1 activity is little affected by membrane depolarization except in the presence of Cftr. CONCLUSIONS: The metabolic and electrogenic activity of glucose transport obscures Cl(−)(IN) /HCO3(−) (OUT) exchange activity of Pat-1 in the lower villus. The inhibitory effects of membrane depolarization on Pat-1 Cl(−)(IN) /HCO3(−) (OUT) exchange may require concurrent membrane association with Cftr.

Simultaneous imaging of multiple focal planes using a two-photon scanning microscope.

Despite all the advances in nonlinear microscopy, all existing instruments are constrained to obtain images of one focal plane at a time. In this Letter we demonstrate a two-photon absorption fluorescence scanning microscope capable of imaging two focal planes simultaneously. This is accomplished by temporally demultiplexing the signal coming from two focal volumes at different sample depths. The scheme can be extended to three or more focal planes.