Journal
CELLULAR PHYSIOLOGY AND BIOCHEMISTRY
Volume 31, Issue 6, Pages 883-891Publisher
KARGER
DOI: 10.1159/000350106
Keywords
Red blood cells; Ca2+ content; Phosphatidylserine; Protein kinase C; P-type Ca2+ channel; Flow cytometry; Fluorescence imaging
Categories
Ask authors/readers for more resources
Background/Aims: Protein kinase C alpha (PKC alpha) is activated by an increase in cytosolic Ca2+ in red blood cells (RBCs). Previous work has suggested that PKC alpha directly stimulates the Ca(V)2.1 channel, whereas other studies revealed that Ca(V)2.1 is insensitive to activation by PKC. The aim of this study was to resolve this discrepancy. Methods: We performed experiments based on a single cell read-out of the intracellular Ca2+ concentration in terms of Fluo-4 fluorescence intensity and phosphatidylserine exposure to the external membrane leaflet. Measurement modalities included flow cytometry and live cell imaging. Results: Treatment of RBCs with phorbol 12-myristate 13-acetate (PMA) led to two distinct populations of cells with an increase in intracellular Ca2+: a weak-responding and a strong-responding population. The EC50 of PMA for the number of cells with Ca2+ elevation was 2.7 +/- 1.2 mu M; for phosphatidylserine exposure to the external membrane surface, it was 2.8 +/- 0.5 mu M; and for RBC haemolysis, it was 2.9 +/- 0.5 mu M. Using pharmacological manipulation with the Ca(V)2.1 inhibitor omega-agatoxin TK and the broad protein kinase C inhibitor GO6983, we are able to show that there are two independent PMA-activated Ca2+ entry processes: the first is independent of Ca(V)2.1 and directly PKC alpha-activated, while the second is associated with a likely indirect activation of Ca(V)2.1. Further studies using lysophosphatidic acid (LPA) as a stimulation agent have provided additional evidence that PKC alpha and Ca(V)2.1 are not directly interconnected in a signalling chain. Conclusion: Although we provide evidence for a lack of interaction between PKC alpha and Ca(V)2.1 in RBCs, further studies are required to decipher the signalling relationship between LPA, PKC alpha and Ca(V)2.1. Copyright (C) 2013 S. Karger AG, Basel
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available