Candidate: Prentiss Jones, Jr.

Degree of: Doctor of Philosophy

Department: Biological Sciences

Title: Characterization of Membrane Potentials in Vascular Smooth Muscle of Hagfish, Lamprey and Trout

Committee: Dr. John Spitsbergen , Chair
Dr. William Jackson
Dr. Leonard Ginsberg
Dr. Kenneth Olson

Date: Monday, June 26, 2006 1:00 p.m. – 3:00 p.m.
1728 Wood Hall

Abstract: Characterization of membrane potentials in fish vasculature is without precedent in the literature. Previous studies have characterized membrane potentials in piscine tissue. However, these studies utilized non-vascular tissues such as cardiac muscle and skeletal muscle. Systemic arteries were harvested from Pacific hagfish, Eptatretus stouti and Sea lamprey Petromyzon marinus; the only surviving members of the most primitive class of true vertebrates (Agnatha) and the more advanced Rainbow trout, Onchorhyncus mykiss.

The hypoTheses of this study was that membrane potentials in fish vascular smooth muscle differ little between species when comparable vasculature is evaluated. A special feature of this study was the use of vascular smooth muscle from fish that inhabit chemically diverse environments and possess anatomical and morphological differences.

Histological examinations were conducted to assess the suitability of the selected fish model arteries for electrophysiological studies. Histologically prepared arteries examined via microscopy revealed vascular components (e.g., endothelial cells, connective tissue, and smooth muscle cells) similar to that reported in non-piscine animals. Osmometry and spectrophotometric techniques were used to prepare species specific physiological saline solutions necessary to maintain a “normal” environment during electrophysiological experiments.

Resting membrane potential and the response to changes in extracellular potassium was evaluated in hagfish and lamprey dorsal aortas and trout efferent branchial arteries. Membrane potentials were measured using sharp microelectrodes to impale continuously perfused arteries. Membrane potential measurements in unstimulated vessels showed similarities in response, -52.7 ±2.9 mV, n=15 (hagfish), -54.3 ±5.2 mV, n=32 (lamprey) and –48.3 ±1.6 mV, n=27 (trout) (p >0.05, ANOVA). Increasing the perfusate KCl concentration to 100 mM depolarized hagfish vascular smooth muscle cells 44.6 ±6.6 mV, n=5 and 80 mM KCl depolarized lamprey vascular smooth muscle cells 63 ±3 mV, n=7. Switching the KCl concentration back to normal re-polarized potassium depolarized cells.

Unexpectedly, the anesthetic used in this study (benzocaine) altered vascular tone. The effects of benzocaine on vascular tone were subsequently evaluated using a gas chromatography/mass spectrometry method developed for this study and via myography. In vitro exposure of lamprey and trout arteries to benzocaine concentrations measured in the plasma of these anesthetized animals induced contraction in lamprey aortas and relaxation in trout arteries. The present study suggests that membrane potentials are similar in comparable fish vasculature despite differences in vascular morphology and differences in the composition of intracellular and extracellular constituents. Additionally, this study suggests benzocaine possesses vasoactive properties in fish systemic arteries at anesthetic concentrations.

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