Shear stress modulates endothelial KLF2 through activation of P2X4.

Vascular endothelial cells that are in direct contact with blood flow are exposed to fluid shear stress and regulate vascular homeostasis. Studies report endothelial cells to release ATP in response to shear stress that in turn modulates cellular functions via P2 receptors with P2X4 mediating shear stress-induced calcium signaling and vasodilation. A recent study shows that a loss-of-function polymorphism in the human P2X4 resulting in a Tyr315>Cys variant is associated with increased pulse pressure and impaired endothelial vasodilation. Although the importance of shear stress-induced Krüppel-like factor 2 (KLF2) expression in atheroprotection is well studied, whether ATP regulates KLF2 remains unanswered and is the objective of this study. Using an in vitro model, we show that in human umbilical vein endothelial cells (HUVECs), apyrase decreased shear stress-induced KLF2, KLF4, and NOS3 expression but not that of NFE2L2. Exposure of HUVECs either to shear stress or ATPγS under static conditions increased KLF2 in a P2X4-dependent manner as was evident with both the receptor antagonist and siRNA knockdown. Furthermore, transient transfection of static cultures of human endothelial cells with the Tyr315>Cys mutant P2X4 construct blocked ATP-induced KLF2 expression. Also, P2X4 mediated the shear stress-induced phosphorylation of extracellular regulated kinase-5, a known regulator of KLF2. This study demonstrates a major physiological finding that the shear-induced effects on endothelial KLF2 axis are in part dependent on ATP release and P2X4, a previously unidentified mechanism.

Basic FGF-responsive telencephalic precursor cells express functional GABA(A) receptor/Cl-channels in vitro.

We have previously described the expression of specific gamma-aminobutyric acid (GABA)A receptor subunits and their transcripts in the cortical neuroepithelium (Ma and Barker, 1995, 1998). However, it is not clear whether neural precursor cells exposed to basic fibroblast growth factor (bFGF) in vitro reproduce the biological properties of neuroepithelial cells in vivo within the embryonic ventricular zone. In the present study, neural precursor cells were isolated from the telencephalic neuroepithelium of embryonic day 13-13.5 rats and exposed to bFGF in serum-free medium. Basic FGF-responsive cells were capable of dividing and differentiating into neurons and astrocytes. The rapidly dividing cells formed multicellular spheres and then a rosette-like formation in which a majority of cells expressed GABA(A) receptor alpha4, beta1, or gamma1 subunit proteins. We found in perforated patch-clamp recordings that GABA depolarized bromodeoxyundine (BrdU)+ precursor cells, and under voltage-clamp induced a bicuculline-sensitive current that reversed at the Cl- equilibrium potential. GABA also increased cytoplasmic Ca2+ in a significant number of BrdU+ cells that was blocked by bicuculline. The bicuculline sensitivity of these pharmacological effects implicates GABA(A) receptor/Cl- channels, since bicuculline is a competitive GABA(A) antagonist at these channels in well-differentiated cells. It is possible that the three GABA(A) receptor subunits (alpha4, beta1, and gamma1) found in precursor cells form the Cl- channels detected electrophysiologically. The functional GABA(A) receptor/Cl- channels and associated regulation of their cytoplasmic Ca2+ levels via bicuculline-sensitive mechanisms may play significant roles in the regulation of neural cell proliferation in this model neuroepithelium.

Neuronal and glial epitopes and transmitter-synthesizing enzymes appear in parallel with membrane excitability during neuroblastoma x glioma hybrid differentiation.

The membrane excitability and the presence of neural proteins, including neuronal and glial markers and neurotransmitter-synthesizing enzymes, were examined in parallel while the NG108-15 cell line was maintained in a serum-free medium. Whole-cell recordings in voltage-clamp or current-clamp configurations were used to evaluate the membrane excitability, and immunostaining was done with a panel of well-characterized antibodies against NSE, NF150, S-100 beta, GFAP, ChAT and TH. Culture for 4 to 10 days led to a striking rise in neurite outgrowth, electrical excitability and expression of neural proteins in type I neuron-like cells, which were of both neuronal and glial character, and expressed both cholinergic and adrenergic traits. After about 2 weeks, type II cells which lack neurite processes began to emerge. The type II cells proliferated, as revealed by BrdU uptake, and gradually overgrew differentiated cell types. They exhibited little or no membrane excitability and absence of immunoreactivity for the neuronal and glial specific proteins tested. These measurements indicate that the presence of these neural proteins at crucial stages of membrane excitability development is an important characteristics of NG108-15 cell differentiation, providing insights into the neural development and the reversible nature of neoplasia in the nervous system.