AbstractELECTRODE FABRICATION FOR in vitro MONITORING OF CATECHOLAMINE SECRETION: CONSEQUENCES AND EXPLANATIONS FOR DOCA-SALT SENSITIVE IMPAIRMENT OF ADRENAL CHROMAFFIN CELL SECRETIONByMatthew J. Fhaner Sympathetic activity is increased in multiple forms of hypertension. This observation led our group to hypothesize that increased sympathetic activity play an integral role in the on-set and long term maintenance of blood pressure in salt-sensitive hypertension (SSH). This form of hypertension is unique, as it is characterized by increased salt retention in the kidneys, leading to an increase in blood volume and in turn blood pressure. Studies were performed investigating the release of norepinephrine from sympathetic neurons innervating smooth muscle cells of mesenteric blood vessels, which determined that sympathetic neurons isolated from DOCA-salt rats released a higher amount of norepinephrine. This increase in overflow is partly attributed to impairment in the auto-regulatory α2-AR and the norepinephrine reuptake transporter. A gap remained in this work due to the spatial limitations of performing electrochemistry at individual neuronal junctions. To solve this, my work will use adrenal chromaffin cells to elucidate the affects of DOCA-salt hypertension on the frequency and kinetics of individual vesicle secretion. To accomplish this goal, electrode fabrication and optimization for single cell electrochemistry is required, alongside the isolation of single adrenal chromaffin cells. The results of this work revealed significant findings in both electrode fabrication processes and biological alterations in chromaffin cell signaling. 1) Applying a novel nucleation process to our published boron-doped diamond electrode fabrication process decreased the growth time needed to deposit a continuous diamond film in half. These electrodes will be useful for in vitro electrochemistry work for larger tissue such as mesenteric blood vessels and gastrointestinal neurotransmission; however carbon-fibers are superior for single cell work due to the ease at which they can be manipulated into a disk-shape. 2) Chromaffin cells isolated from DOCA-salt rats had a higher frequency of release events, with individual events occurring more rapidly, than Sham chromaffin cells. 3) These alterations appear to at least in part be caused by impairment in the function of BK and ATP-sensitive potassium channels in DOCA-salt cells. Further mechanistic investigation led to the conclusion that calcium influx, reactive oxygen species levels and catecholamines metabolism are not responsible for any alterations observed in DOCA-salt chromaffin cell secretion patterns. 4) Finally, fast-scan voltammetry determined that the ratio of norepinephrine:epinephrine secreting cells remains unchanged in DOCA-salt hypertension. These results suggest that increased release frequency along with more rapid secretion may also contribute to the alterations found in sympathetic neurons by previous group members. Also, potassium channels may provide a viable treatment option for managing salt-sensitive hypertension. Finally, for future work requiring BDD electrodes, application of a pre-growth carbon layer greatly enhances film deposition rates.
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