Date of Award

January 2015

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Peter S. Aronson

Subject Area(s)

Physiology, Medicine

Abstract

NHE3, the predominant proximal tubule sodium-hydrogen exchanger, is regulated by a variety of conditions and physiologic factors, including dopamine, a critical natriuretic hormone. Alterations in NHE3 phosphorylation comprise an important mechanism by which NHE3 activity is controlled.

We transfected rat NHE3 into opossum kidney (OK) cells and used a panel of phosphoantibodies against the S552, S605, S661, and S791 NHE3 phosphorylation sites to analyze the effects of forskolin and IBMX (F/I)--cyclic adenosine monophosphate (cAMP) activators--and dopamine (DA) on patterns of NHE3 phosphorylation.

At S552, F/I induced a 139% increase in phosphorylation while DA, a 67% increase. At S605, F/I caused a 953% increase in phosphorylation whereas DA, a 349% increase. F/I resulted in a 117% increase in phosphorylation at S661 while DA induced a 221% increase. Lastly, at S791, F/I decreased phosphorylation by 84% while DA increased phosphorylation by 31%. All densitometry analyses were statistically significant with p<0.05 except for the anti-PS791 signal in response to DA.

F/I increased NHE3 phosphorylation at S552 and S605 to a greater extent than DA did as demonstrated in previously published studies. Novel findings presented here include greater phosphorylation at S661 in response to DA than to F/I, and decreased phosphorylation at S791 in response to F/I with no apparent decrease in response to DA.

This study suggests that both PKA and PKC could phosphorylate NHE3 at S661 and that one or more phosphatases likely dephosphorylate NHE3 at S791 in response to PKA activation. While largely hypothesis-generating, this study demonstrates the complexity of NHE3 regulation by multi-site patterns of phosphorylation.

Comments

This thesis is restricted to Yale network users only. This thesis is permanently embargoed from public release.

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