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"The Effect of PKC on Agonist-induced Cytosolic Calcium Signals in Primary Rat Hepatocytes"

by
Walson K. Metzger
M.D./Ph.D. Program

B.S. 2001, University of Florida


Thesis Advisor: Andrew P. Thomas, Ph.D.

Professor

Department of Pharmacology and Physiology

Tuesday, December 15, 2009
2:00 p.m., MSB H-609B


Abstract

The Protein Kinase C (PKC) family of serine-threonine kinases act as key elements in signaling pathways regulating a wide variety of essential cellular functions. Conventional and Novel PKCs are activated through the receptor-dependent phospholipase c (PLC)/ inositol triphosphate (IP3) / Ca2+ signaling pathway that leads to Ca2+ oscillations and waves (spatio-temporal organization) in hepatocytes. Since oscillatory PKC activation has been observed in parallel with the Ca2+ oscillations, and PKC has been shown to modulate aspects of the PLC/IP3/Ca2+ pathway, there is a strong possibility that this kinase family may participate in the feedback effects driving and modulating Ca2+ oscillations. Upon examining actions of broad specificity PKC activators and inhibitors on hormone-evoked Ca2+ oscillations in rat hepatocytes, we found that both PKC activation and inhibition led to similar responses: slowing of frequency or termination of Ca2+ oscillations, and broadening of individual Ca2+ spikes. A possible explanation for this observation is that PKC isoforms may have different targets that oppose one another, such that broad specificity activators and inhibitors yield a similar phenotype.
One tool often used in the study of PKC is down-regulation of phorbol-sensitive isoforms by chronic exposure to Phorbol-12-myristate-13-acetate (PMA). Western blot confirmed that PMA down-regulation (PMA-DR) depleted overnight cultured heptacytes of PMA-sensitive PKC isoforms (, , , ). When PMA-DR hepatocytes were exposed to 10M PE, a dose that normally causes cells to oscillate, the Ca2+ oscillations were markedly broader (slower rise and fall) and many cells displayed peak and plateau responses and were essentially non-oscillatory. With this dramatic shift in phenotype, we decided to investigate the Ca2+ signaling targets affected by PKC down-regulation. We tested the functions and activity of proteins and organelles with essential roles in the Ca2+ signaling pathway, including phospholipase C (PLC), IP3-Receptor, sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA), plasma membrane Ca2+ -ATPase (PMCA), store-operated Ca2+ entry (SOCE), mitochondrial Ca2+ handling, and IP3- metabolism. We concluded that the major targets of PKC feedback are IP3-metabolism and PLC activity, with PMA-DR hepatocytes displaying larger IP3 responses and greater PLC activity than control cells. The Ca2+ wave propagation velocity in PMA-DR cells was significantly reduced when compared to control cells. In order to investigate the role of individual PKC isoforms in Ca2+ signaling, we used selective peptide PKC modulators conjugated to HIV-TAT for plasma membrane permeability. These studies showed that PKC activation, and PKCI inhibition both increased the frequency of Ca2+ oscillations. Further analysis of the effect of these isoform-selective peptides on the targets we identified using PMA-DR, indicated that PKC activation increased cytosolic IP3 while PKCI inhibition increased PLC activity upon agonist stimulation. These results show that PKC feedback plays a major role in shaping and modulating Ca2+ oscillations and waves, and that individual PKC isoform activity has the potential to affect distinct cellular targets.


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