Lynn K. Hartzler (Committee Member), Robert W. Putnam (Committee Chair), Christopher N. Wyatt (Committee Member)
Master of Science (MS)
In this work, I have made attempts to clamp intracellular pH in the presence of hypercapnic acidosis (HA) in neurons from the locus coeruleus (LC) and nucleus of the solitary tract (NTS) in neonatal rat (ages P3 to P17) brainstem slices. Two approaches were used to minimize hypercapnia-induced ΔpHi: 1) an increase in intracellular buffering power with a high HEPES concentration using whole cell patching techniques in individual neurons, and 2) a weak acid diffusion technique that relies on an efflux of weak acid to counterbalance HA influx thereby clamping pHi in multiple neurons at once. pHi was measured using two pH-sensitive fluorescent dyes: membrane impermeable pyranine for the former approach, and the acetoxymethyl ester form of membrane permeable 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) for the latter. Blunting with HEPES buffer was performed in the NTS only, with a calculated average percent blunting of hypercapnia-induced acidification of 73.4%. Experiments blunting via weak acid diffusion utilized an inverted microscope and two weak acids: acetic acid and caproic (hexanoic) acid. In NTS neurons (n=56), acetic acid blunted acidification by only 33.1% and in LC neurons by only 19.6% (n=52). Caproic acid blunted hypercapnia-induced acidification by 50.7% (n=58) and 45.8% (n=47) in NTS and LC neurons, respectively. Experiments were also repeated using an upright microscope. In these experiments, acidification was blunted by 45.8% (n=56) and 52.6% (n=52) in NTS and LC neurons, respectively. Concurrent influx of weak base, trimethyl amine, with HA was also used to blunt ΔpHi, but the results showed no blunting and, in fact, a greater acidification in response to HA: -18.1% in the NTS (n=40) and -27.8% in the LC (n=28). These techniques are clearly insufficient to accomplish a complete clamping of hypercapnia-induced changes in pHi. However, we have determined that the ability to clamp pHi is highly affected by diffusion of the weak acid both up to and into the cell. Blunting could be improved with better superfusion of slices and the use of more permeable weak acids. Overall, the ability to blunt ΔpHi, especially in numerous cells simultaneously, would be valuable in studying systems where pHi may play a role in cellular function, such as the involvement of pHi in chemosensitive signaling, bicarbonate reabsorption in the proximal tubule, free fatty acid diffusion in adipocytes, and acid-sensing taste receptors.
Department or Program
Department of Neuroscience, Cell Biology & Physiology
Year Degree Awarded
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