UTP and ATP increase extracellular signal-regulated kinase 1/2 phosphorylation in bovine chromaffin cells through epidermal growth factor receptor transactivation.

Adenosine triphosphate (ATP) is coreleased with catecholamines from adrenal medullary chromaffin cells in response to sympathetic nervous system stimulation and may regulate these cells in an autocrine or paracrine manner. Increases in extracellular signal-regulated kinase (ERK) 1/2 phosphorylation were observed in response to ATP stimulation of bovine chromaffin cells. The signaling pathway involved in ATP-mediated ERK1/2 phosphorylation was investigated via Western blot analysis. ATP and uridine 5'-triphosphate (UTP) increased ERK1/2 phosphorylation potently, peaking between 5 and 15 min. The mitogen-activated protein kinase (MAPK/ERK)-activating kinase (MEK) inhibitor PD98059 blocked this response. UTP, which is selective for G-protein-coupled P2Y receptors, was the most potent agonist among several nucleotides tested. Adenosine 5'-O-(3-thio) triphosphate (ATPgammaS) and ATP were also potent agonists, characteristic of the P2Y(2) or P2Y(4) receptor subtypes, whereas agonists selective for P2X receptors or other P2Y receptor subtypes were weakly effective. The receptor involved was further characterized by the nonspecific P2 antagonists suramin and reactive blue 2, which each partially inhibited ATP-mediated ERK1/2 phosphorylation. Inhibitors of protein kinase C (PKC), protein kinase A (PKA), Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), and phosphoinositide-3 kinase (PI3K) had no effect on ATP-mediated ERK1/2 phosphorylation. The Src inhibitor PP2, epidermal growth factor receptor (EGFR) inhibitor AG1478, and metalloproteinase inhibitor GM6001 decreased ATP-mediated ERK1/2 phosphorylation. These results suggest nucleotide-mediated ERK1/2 phosphorylation is mediated by a P2Y(2) or P2Y(4) receptor, which stimulates metalloproteinase-dependent transactivation of the EGFR.

Tyrosine hydroxylase phosphorylation increases in response to ATP and neuropeptide Y co-stimulation of ERK2 phosphorylation.

ATP and neuropeptide Y (NPY) are examples of agents co-secreted with catecholamines from neuronal and neuroendocrine cells which may regulate the function of the cells from which they are released. For example, ATP and NPY could influence chromaffin cell activity in an autocrine or paracrine manner. The primary recognized function of chromaffin cells is the synthesis and secretion of catecholamines; therefore, we hypothesize that ATP and NPY can regulate catecholamine synthesis in chromaffin cells. ATP increases phosphorylation of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, at Ser31 with a potency similar to that for ERK1/2 phosphorylation, the kinase responsible for TH phosphorylation at this site. Moreover, NPY co-stimulation increases the potency of ATP for both ERK1/2 and TH phosphorylation, while having no effect on these parameters alone. ATP and NPY had no effect on TH phosphorylation at Ser40, the primary site responsible for acute activation of the enzyme. Correspondingly, ATP and NPY did not increase TH activity. Additionally, ATP or ATP and NPY had no effect on TH expression. TH phosphorylation at Ser31 may be responsible for stabilization of the enzyme or may increase the rate and extent of phosphorylation of Ser40. We propose that ATP and NPY may serve to enhance the stimulatory effects of other agents on TH activity.

Glutamate production by HIV-1 infected human macrophage is blocked by the inhibition of glutaminase.

Mononuclear phagocyte (macrophages and microglia) dysfunction plays a significant role in the pathogenesis of human immunodeficiency virus (HIV) associated dementia (HAD) through the production and release of soluble neurotoxic factors including glutamate. The mechanism of glutamate regulation by HIV-1 infection remains unclear. In this report, we investigated whether the enzyme glutaminase is responsible for glutamate generation by HIV-1 infected monocyte-derived macrophages. We tested the functionality of novel small molecule inhibitors designed to specifically block the activity of glutaminase. Glutaminase inhibitors were first characterized in a kinetic assay with crude glutaminase from rat brain revealing an uncompetitive mechanism of inhibition. The inhibitors were then tested in vitro for their ability to prevent glutamate generation by HIV-infected macrophages, their effect upon macrophage viability, and HIV infection. To validate these findings, glutaminase specific siRNA was tested for its ability to prevent glutamate increase during infection. Our results show that both glutaminase specific small molecule inhibitors and glutaminase specific siRNA were effective at preventing increases in glutamate by HIV-1 infected macrophage. These findings support glutaminase as a potential component of the HAD pathogenic process and identify a possible therapeutic avenue for the treatment of neuroinflammatory states such as HAD.

Betagamma subunits mediate the NPY enhancement of ATP-stimulated inositol phosphate formation.

Neuropeptide Y (NPY) enhances ATP-stimulated inositol phosphate (InsP) formation in bovine chromaffin cells through an unknown mechanism. Chromaffin cells were transduced with the carboxyl terminus of beta-adrenergic receptor kinase 1 (betaARK1CT), a Gbetagamma subunits scavenger, using a recombinant adenovirus system. The adenovirus also expresses a green fluorescent protein (GFP) which serves as an index of transduction. Flow cytometry showed that up to 80% of chromaffin cells were transduced by the virus. There was a direct correlation between the betaARK1CT inhibition of the NPY enhancement of ATP-stimulated InsP formation and the percent of cells expressing GFP ( r2=0.9993 ). These results demonstrate that Gbetagamma subunits are required for the NPY enhancement of ATP-stimulated InsP formation in bovine chromaffin cells.

Effect of the ecto-ATPase inhibitor, ARL 67156, on the bovine chromaffin cell response to ATP.

Bovine chromaffin cells contain an ecto-ATPase (K(m)=1.57 +/- 0.27 x 10(-4) M) which can hydrolyze ATP present in the culture media. ARL 67156 is a competitive inhibitor of this ATPase (K(i)=2.55 +/- 1.36 x 10(-7) M). A small increase in potency (threefold) is seen when ARL 67156 is included during measurement of ATP-stimulated inositol phosphate formation. ARL 67156 also acts on chromaffin cell P2Y receptors to increase inositol phosphate formation (EC(50)=4.9 x 10(-5) M). It is useful as an ecto-ATPase inhibitor in studies with bovine chromaffin cells since it exhibits a 300-fold selectivity for the ecto-ATPase versus the P2Y receptor.

Transduction of bovine adrenal chromaffin cells using a recombinant adenovirus expressing GFP.

Insertion of genetic material into bovine chromaffin cells employing various techniques have produced low to moderate transduction rates. Recent technology using adenoviral gene transfer has become one of the most powerful methods for introducing genes into mammalian cells. We examined whether a recombinant adenovirus could provide a convenient vector to transfer genes of interest for mechanistic studies on chromaffin cells. Our results show that 100% transduction of chromaffin cells was accomplished within 18 h with a recombinant adenovirus as revealed by the expression of green fluorescent protein (GFP) in chromaffin cells. Transduction was dependent on time and viral titer but independent of cell age and density in culture. There was no effect of the recombinant adenovirus on the secretory function [3H]-norepinephrine ([3H]-NE release) of the chromaffin cells. The results demonstrate that the recombinant adenovirus provides an effective process for the complete transfection of bovine chromaffin cells with a selected gene.