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Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...

Des Moines University

Mercy College of Health Sciences

The high requirement of O2 in the renal proximal tubule stems from a high rate of Na(+) transport. Adenosine A1 receptor (A1R) activation regulates Na(+) transport in this nephron segment. Thus, the effect of the acute activation and the mechanisms of A1R on the rate of O2 consumption were evaluated. The A1R-antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPX) and adenosine deaminase (ADA), which metabolize endogenous adenosine, reduced O2 consumption (40-50%). Replacing Na(+) in the buffer reversed the ADA- or CPX-mediated reduction of O2 consumption. Blocking the Na/H-exchanger activity, which decreases O2 usage per se, did not enhance the ADA- or CPX-induced inhibition of O2 consumption. These data indicate that endogenous adenosine increases O2 usage via the activation of Na(+) transport. In the presence of endogenous adenosine, A1R was further activated by the A1R-agonist N(6)-cyclopentyladenosine (CPA); CPA inhibited O2 usage (30%) and this effect also depended on Na(+) transport. Moreover, a low concentration of CPA activated O2 usage in tissue pretreated with ADA, whereas a high concentration of CPA inhibited O2 usage; both effects depended on Na(+). Protein kinase C signaling mediated the inhibitory effect of A1R, while adenylyl cyclase mediated its stimulatory effect on O2 consumption. In summary, increasing the local concentrations of adenosine can either activate or inhibit O2 consumption via A1R, and this mechanism depends on Na(+) transport. The inhibition of O2 usage by A1R activation might restore the compromised balance between energy supply and demand under pathophysiological conditions, such as renal ischemia, which results in high adenosine production.

Dual Effect of Adenosine A1 Receptor Activation on Renal O2 Consumption.

Na+/H+ exchanger-3 (NHE3) plays an essential role in maintaining sodium and fluid homeostasis in the intestine and kidney epithelium. Thus, NHE3 is highly regulated and its function depends on binding to multiple regulatory proteins. Ezrin complexed with NHE3 affects its activity via not well-defined mechanisms. This study investigates mechanisms by which ezrin regulates NHE3 activity in epithelial Opossum Kidney cells. Ezrin is activated sequentially by phosphatidylinositol-4,5-bisphosphate (PIP2) binding and phosphorylation of threonine 567. Expression of ezrin lacking PIP2 binding sites inhibited NHE3 activity (-40%) indicating that ezrin binding to PIP2 is required for preserving NHE3 activity. Expression of a phosphomimetic ezrin mutated at the PIP2 binding region was sufficient not only to reverse NHE3 activity to control levels but also to increase its activity (+80%) similar to that of the expression of ezrin carrying the phosphomimetic mutation alone. Calcineurin Homologous Protein-1 (CHP1) is part, with ezrin, of the NHE3 regulatory complex. CHP1-mediated activation of NHE3 activity was blocked by expression of an ezrin variant that could not be phosphorylated but not by an ezrin variant unable to bind PIP2. Thus, for NHE3 activity under baseline conditions not only ezrin phosphorylation, but also ezrin spatial-temporal targeting on the plasma membrane via PIP2 binding is required; however, phosphorylation of ezrin appears to overcome the control of NHE3 transport. CHP1 action on NHE3 activity is not contingent on ezrin binding to PIP2 but rather on ezrin phosphorylation. These findings are important in understanding the interrelation and dynamics of a CHP1-ezrin-NHE3 regulatory complex.

Victor Babich

Physiology and Pharmacology Department

Des Moines University

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

Victor Babich

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Physiology and Pharmacology Department

Des Moines University

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

Physiology and Pharmacology Department