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Ouabain-sensitive rubidium transport in cultured OK cells expressing rodent Na+, K+-ATPase (Na-K pump) á1 or á3 isoforms were used to measure intracellular signaling and structural variations involved in transport responses. Since kinetic differences between isozymes seem to be of little functional consequence, more insight into Na-K pump subunit signaling diversity and its physiological relevance was sought by determining their interaction with regulatory proteins such as protein kinase C (PKC) which also exists in multiple forms. The evaluations began with comparing the effects of phorbol ester as a PKC agonist and a conventional PKC inhibitor on rodent á1 and á3. Cytoskeletal framework integrity was then examined as a requirement for the possible secretion or removal of functional Na-K pump units through intracellular vesicle trafficking. This study found evidence in favor of the conventional PKC subfamily being responsible for ouabain-inhibitable transport increases in cells expressing rodent á3 Na-K pump. Also, á3 appeared to have a greater sensitivity to the conventional PKC signaling pathway than á1. As for the cytoskeletal examination, the pharmacological approach used lead to noisy and unreliable signals. Biotinylation, real-time visualization, such as GFP labeling, or immunolabeling are alternative methods recommended to provide more conclusive evidence for rodent á3* Na-K pump trafficking.

According to the American Stroke Association, about 700,000 people suffer a new or recurrent stroke each year in the United States. Of these people, approximately 163,000 die, making stroke the third leading cause of death in the U.S., only behind heart disease and cancer. Depending on the area of the brain affected by the stroke, functions such as motor activity, speech, behavior, and/or memory can be hampered. The hippocampus is a bilateral structure that is highly susceptible to hypoxic and/or ischemic insult. One of the early responses to ischemia is the transient and reversible inhibition of synaptic activity mediated by endogenous adenosine acting on neuronal A1 receptors. Increase in adenosine during ischemia is thought to play a key prosurvival role by attenuating excitotoxic damage through inhibiting glutamate release and activating Akt. Akt is activated by PI3K-dependent and PI3K-independent mechanisms. Akt, also known as PKB, has been shown to be both necessary and sufficient to promote cell survival by growth factors in vitro. Akt directly phosphorylates multiple proteins resulting in the inhibition of apoptotic and/or necrotic cell death. Bcl-2 and Bcl-xL are two proteins that are disinhibited by the direct Akt phosphorylation of Bad. These two proteins function to maintain mitochondrial integrity during ischemia, thus inhibiting the release of cytochrome c which is a strong inducer of the apoptotic pathway. This thesis explores the activation mediated by PI3K and the significance of this activation in neuronal survival mechanisms.