Determination of pH by microfluorometry: intracellular and interstitial pH regulation in developing early-stage fish embryos (Danio rerio)

摘要

Microfluorometric techniques were applied in vivo for continuous monitoring of specific acid-base parameters in zebrafish (Danio rerio) embryos during early stages of ontogeny. Dextran-coupled pH-sensitive single-excitation/dual-emission dye SNARF-1 was pressure-injected into individual cells or the interstitial space of 16- to 256-cell embryos, and pH was continuously recorded during subsequent development for time periods of up to 8 h. A novel calibration technique was developed, essentially characterized by in vitro inorganic buffer calibration of the optical system and mathematical post-processing according to the effects of in vivo dye modifiers through a correlation established by direct comparison of optical techniques with pH microelectrodes. This approach results in high accuracy of microfluorometry, comparable with that of pH electrodes, and a recovery only limited by the physical stability of the utilized optical system. Intracellular pH (pHi) in Danio rerio embryos between 1k-cells stage and the end of epiboly was found to be well regulated to a mean value of 7.55+/-0.13 (+/-s.d.), a range distinctly more alkaline than typical values for adult fish but in accordance with embryonic pHi of a few non-fish species shortly after fertilization. Also, interstitial pH (pHint) was significantly higher (8.08+/-0.25) than values for extracellular pH in adult fish. Distributions of HCO3- across membranes and between interstitium and ambient fluid compared with respective potentials strongly suggest that pH in these early stages of ontogeny is already adjusted by active transfer processes. Non-respiratory changes in ambient pH between 7.7 and 8.5 did not significantly affect pHi, a result potentially attributable to low membrane leakage rate or to the potency of active transfer mechanisms. In order to assess the pH regulatory systems more quantitatively, embryos were exposed to ambient changes of carbon dioxide partial pressure (PCO2). The direct impact of PCO2 changes on cell pH was alleviated by cell non-bicarbonate buffering and subsequent rapid, almost complete, compensation by changes in cell [HCO3-] as an expression of transmembrane transfer of acid-base relevant ions. On the basis of these results, we conclude that the regulatory potency of embryonic cells is well developed, is active to resist extensive homoiostatic stress and is efficient to maintain critical metabolism in adverse conditions, even at early stages of ontogeny.