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Use of ion-selective microelectrodes to determine the contribution of H(+), K(+), and HCO(-) towards the asymmetric current in Xenopus laevis oocytes
The asymmetric current previously identified using voltage probe techniques in maturing Xenopus laevis oocytes has been further investigated using non-invasive ionselective microelectrodes. Three-dimensional fluxes of hydrogen (H+), potassium (K+), and bicarbonate ([special characters omitted]), of varying magnitudes, were found to be hidden within this net 10 pmol current. These fluxes were seen to vary with respect to developmental stage and presence of surrounding follicular tissue. The mechanisms of H + and K+ transport were also examined with P and V-type ATPase pump inhibitors, ortho-vanadate and N-ethyl maleimide (NEM), respectively. A representative flux pattern of H+, K+, and [special characters omitted] ions around fully developed Xenopus oocytes was obtained from this research. H+ effluxes, as well as K+ influxes and effluxes, were detected at the animal, vegetal, and equatorial regions of the membrane. The transport of these two ions was seen to be highly correlated in defolliculated oocytes, with large H+ effluxes accompanied by large K+ influxes. Small effluxes of [special characters omitted] were measured at the animal and vegetal hemispheres. The use of ATPase pump inhibitors also provided information regarding possible transport mechanisms. Vanadate (0.01 mM) and NEM (1 mM) significantly reduced the H+ flux in defolliculated and follicle-enclosed oocytes, respectively. However, the observed K+ fluxes were unaffected by these inhibitors. The combination of these results suggests that ATPase pumps are important only in the transport of H+ ions and more specifically, the presence of a P-type ATPase on the surface of the oocyte membrane and a V-type ATPase in the follicular tissue. The technique of measuring extracellular fluxes from developing cells with ion-selective electrodes was also examined. Construction of more efficient sampling rules, determination of electrode efficiencies using these sampling rules, and calibration of the carbonate electrode were some of the advances resulting from this research. In addition, the implementation of a multiple head ion probe provided simultaneous data collection of more than one ion type from the same oocyte, allowing for the study of ion-ion interactions and the determination of bicarbonate flux Patterns with H+ and carbonate electrodes.
Cellular biology|Anatomy & physiology|Animals
Faszewski, Ellen Evelyn, "Use of ion-selective microelectrodes to determine the contribution of H(+), K(+), and HCO(-) towards the asymmetric current in Xenopus laevis oocytes" (1999). Doctoral Dissertations Available from Proquest. AAI9920597.