Intracellular pH (pH
i) Measurement in Rat Duodenal Epithelium In Vivo Using SNARF-1
Tetsu Takeuchi, Lening Zhang, and Jonathan D. Kaunitz
Key words. SNARF-1, pHi, Duodenal epithelium, In vivo, Acid
Introduction
Our laboratory has previously used BCECF to measure the pHiof the duode- nal epithelium [1–3]. BCECF loads into epithelial cells and fluoresces well, but since BCECF is a dual excitation-single emission compound, a ratio pair cannot be obtained simultaneously, but requires sequential acquisition of image pairs. Measurement of pHi in vivo is impaired, due to the constant motion of epithelial cells which are affected by respiratory and cardiac move- ment. Exact registration of image pairs is not possible and ratiometric pHi
calculations must be therefore be performed manually. 5-(and 6)-Carboxy- seminaphthorhodafluor-1 (SNARF-1) is a single excitation dual emission fluorescent compound. The emission ratios calculated from the two peaks of 640 and 580 nm are a sensitive indicator of pH; furthermore, image pairs at two different wavelengths can be obtained simultaneously. It is thought that this specific feature can be used for more accurate determination of pHi
especially in vivo. In the present paper, we used SNARF-1 for measurement of pHiin rat duodenal epithelium in vivo.
Material and Methods
All studies were approved by the Animal Use Committee of the West Los Angeles Veterans Affairs Medical Center. An in vivo microfluorometric tech- nique was adapted from a technique originally developed by Tanaka et al. [1]
89 West Los Angeles VA Medical Center, Building 114, Suite 217, CURE: Digestive Diseases Research Center, and UCLA School of Medicine, Los Angeles, CA 90073, USA
to measure pHiin rat duodenal epithelial cells. Fluorescence of the duodenal mucosa observed microscopically was excited at 488 nm and divided into image pairs using the MicroImager with two emission filters (580/640 nm).
The entire duration of the experiment was 40 min and each image pair was captured every 2 min for 15 min from the beginning of acid exposure (every 5min for other observations). Three small areas of the duodenal epithelium from each recorded image pair were selected at random and each intensity of fluorescence was measured using image analyzer software (OpenLab, Improvision, Lexington, MA, USA).
Results
An examination of mucosa loaded with SNARF-1 in vivo produced image pairs such as depicted in Fig. 1. Duration of fluorescence and brightness was about 60 min. Note that fluorescence appears to be limited to the villous epithelial cells. Similar image pairs were obtained simultaneously at different wavelengths (580/640 nm). After baseline stabilization, pH 2.5 perfusion for 5 min rapidly reduced the pHi. After acid removal, pHi increased to baseline.
90 T. Takeuchi et al.
Fig. 1. In vivo image of chloromethyl SNARF-1-loaded duodenal mucosa. Identical image pairs are obtained simultaneously at two different wavelengths (580/640 nm). The dye localizes only in epithelial cells of villous tips
Discussion
pHimeasurement in vitro with SNARF-1 has been recently published [4,5].
Simultaneously acquired image pairs obtained with SNARF-1 enabled us to measure pHiin vivo more precisely. Image pairs obtained every 2 min showed more accurate changes of pHiwith acid exposure than measurements made using BCECF. Since our experiment needed at least 40 min to complete, we used chloromethyl SNARF-1 acetate. It is designed for long-term cellular retention via coupling to protein thiols. In summary, we demonstrated that SNARF-1 provides a useful alternative means for the measurement of duode- nal epithelial pHiin vivo and hereafter, as the disadvantages of SNARF-1 are overcome, this dye can be applied to other organs or species.
Acknowledgment. Supported by VA Merit Review funding and NIH/NIDDK RO1 54221.
References
1. Tanaka S, Guth PH, Engel E, et al (1998) A novel technique for the in vivo measurement of intracellular pH (pHi) of rat duodenal epithelial cells (abstract). Gas- troenterology 114:A306
2. Akiba Y, Kaunitz JD (1999) Regulation of intracellular pH and blood flow in rat duode- nal epithelium in vivo. Am J Physiol. 276(1 Pt 1):G293–G302
3. Akiba Y, Furukawa O, Guth PH, et al (2001) Cellular bicarbonate protects rat duodenal mucosa from acid-induced injury. J Clin Invest 108(12):1807–1816
4. Bassnett S, Reinisch L, Beebe DC (1990) Intracellular pH measurement using single exci- tation-dual emission fluorescence ratios. Am J Physiol 258(1 Pt 1):C171–C178 5. Blank PS, Silverman HS, Chung OY, et al (1992) Cytosolic pH measurements in single
cardiac myocytes using carboxy-seminaphthorhodafluor-1. Am J Physiol. 263(1 Pt 2):
H276–H284
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