Degree Name

PhD (Doctor of Philosophy)

Program

Biomedical Sciences

Date of Award

December 1994

Abstract

Using conventional microelectrodes, I studied the electrical basis for determining the resting V$\sb{\rm m}$ in intact EC's from hamsters. The resting V$\sb{\rm m}$ were found to be $-$40 mV for aortic EC's and $-$43 mV for vena caval EC's. The contributions of ions to the resting V$\sb{\rm m}$ of aortic EC's were compared in terms of the transference number (t$\sb{\rm ion}$). To develop a technique for in situ monitoring changes in V$\sb{\rm m}$ of postcapillary venular EC's in the hamster mesentery, a voltage-sensitive fluorescent probe, bisoxonol, was used to load the cells and the fluorescence signals were analyzed under an intravital microscope by recording the fluorescence intensity (I$\sb{\rm f}$) and processing fluorescent images of the bisoxonol-loaded cells. Calibrations were conducted by simultaneously measuring changes in V$\sb{\rm m}$ with microelectrodes and bisoxonol from aortic EC's and by varying extracellular Na$\sp{+}$ in microvessels. Both calibrations yielded the linear relationship between V$\sb{\rm m}$ and bisoxonol I$\sb{\rm f}$, showing the slope of 5.7%/mV for aortic EC's and 5.2%/mV for microvascular EC's. Altering extracellular K$\sp{+}$ to 25, 50, and 100 mM in the suffusate depolarized microvascular EC's by 5, 8, and 10 mV; whereas, the same alterations via both suffusion and perfusion induced the depolarization by 18, 30, and 42 mV, indicating that the K$\sp{+}$ conductance has an asymmetric distribution. Ba$\sp{2+}$ (1 mM) produced a depolarization by 70 mV, suggesting that the activity of K$\sp{+}$ channels dominates the resting V$\sb{\rm m}$. To correlate the bradykinin-induced increase in microvascular permeability to the changes in V$\sb{\rm m}$, the albumin flux (J$\sb{\rm A}$) was measured using TRITC-albumin along with monitoring V$\sb{\rm m}$. Bradykinin(l $\mu$M) induced a hyperpolarization of EC's by 8 mV and a biphasic increase in J$\sb{\rm A}$ from the basal level of 1.00 x 10$\sp{-6}$ to a transient peak of 9.17 x 10$\sp{-6}$ followed by a sustained level of 3.05 x 10$\sp{-6}$ cm/s. The linear correlations of net increases in both the peak and the sustained values of J$\sb{\rm A}$ to changes in V$\sb{\rm m}$ indicate that the hyperpolarization determines the peak in part and the sustained level in all. Under high K$\sp{+}$ (50 mM), bradykinin produced a repolarization from a depolarized V$\sb{\rm m}$ of -54 mV to -66 mV and a smaller increase in J$\sb{\rm A}$ from the basal level of 0.38 x 10$\sp{-6}$ to the peak of 5.51 x 10$\sp{-6}$ followed by a significantly lowered, sustained level of 1.11 x 10$\sp{-6}$ cm/s. The repolarization under high K$\sp{+}$ indicates that besides the activation of Ca$\sp{2+}$-dependent K$\sp{+}$ channels, other electrical events may be implicated. The correlation between the repolarization and the lowered value of J$\sb{\rm A}$ at the peak implies that this variation in V$\sb{\rm m}$ also mediates the bradykinin-induced increase in J$\sb{\rm A}$ under high K$\sp{+}$ condition. (Abstract shortened by UMI.)

Document Type

Dissertation - unrestricted

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