Enhancement of Cellular Adenosine Triphosphate Levels in PC12 Cells by Extracellular Adenosine.

Abstract

Extracellular adenosine (Ado), a metabolite of adenosine triphosphate (ATP), has been shown to function as an intercellular signaling molecule by interacting with specific P1 receptors.1,2) Extracellular ATP released from nerve cells, endothelial cells, chromaffin cells, or platelets can act as a transmitter or modulator via P2 receptors. 1,3) A number of cellular responses induced by Ado [4][5][6] and ATP [7][8][9][10][11] in rat pheochromocytoma PC12 cells, a neuronal model, have been demonstrated to be mediated mainly through P1 and P2 receptors. PC12 cells are known to have A2a and A2b but not A1 or A3 receptors among the P1 receptor subtypes, and P2X 2 , P2Y 1 , and P2Y 2 among the P2 receptor subtypes.2)The common responses induced by Ado and ATP in PC12 cells are to increase the cellular levels of cAMP and the activity of cAMP-regulated tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis. 4,12) It is of interest to determine whether cellular levels of ATP in PC12 cells would be affected by the addition of Ado, ATP, and related compounds.In general, extracellular ATP is known to be metabolized rapidly to Ado, and then converted to inosine and other metabolites by ecto-enyzmes on cell surfaces or soluble enzymes in extracellular fluids. 3,13,14) Extracellular ATP was rapidly degraded to adenosine 5Ј-monophosphate (AMP) by PC12 cells, but the formation of Ado from AMP was slow, 4,15) despite having ecto-5Ј-nucleotidase, which catalyzes the conversion of AMP to Ado.16) It is of interest to examine whether PC12 cells have the ability to metabolize Ado, and to know whether extracellular Ado influences the cellular contents of adenine compounds, especially ATP.In this investigation, we examined the effects of Ado and adenine nucleotides on cellular levels of adenine compounds, especially ATP, in PC12 cells. We showed that extracellular Ado enhanced the cellular ATP level, probably via the salvage pathway of adenine nucleotides, but not P1 and P2 receptors of Ado and ATP, respectively. MATERIALS AND METHODSMaterials Ado, AMP, adenosine 5Ј-diphosphate (ADP), and ATP were purchased from Yamasa Shouyu (Japan). 2-Chloro-N 6 -cyclopentyladenosine (CCPA), N 6 -cyclohexyladenosine (CHA), 5Ј-N-ethylcarboxaminoadenosine (NECA), dipyridamole, and reactive blue 2 were obtained from Sigma Chemical (U.S.A.). 2[p-(2-Carboxyethyl)phenethylamino]-5Ј-N-ethylcarboxamidoadenosine (CGS 21680) and coformycin were from Funakoshi Chemical (Japan) and Nakarai Tesque (Japan), respectively. Theophylline and chloroacetaldehyde were from Wako Chemicals (Japan). Fetal calf serum and inactivated horse serum were from IS Japan (Japan) and Gibco BRL (U.S.A.), respectively. Other chemicals of reagent grade were commercially obtained.Cell Culture Rat pheochromocytoma PC12 cells were kindly supplied by Professor K. Miura (Wako University, Tokyo, Japan). Cells were grown at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 5% heat-inactivated horse serum, glutamine 30 mg/ ml, and kanamycin 60 ...

Enhancement of Cellular Adenosine Triphosphate Levels in PC12 Cells by 2,5-Dideoxyadenosine, a P-Site Inhibitor of Adenylate Cyclase

“…14,15) As previously reported, 4) theophylline had no effect on the Ado-induced ATP levels in PC12 cells without pretreatment. On the contrary, pretreatment of PC12 cells with theophylline suppressed the Ado-induced ATP-enhancing effect, suggesting that theophylline might inhibit HGPT activity to produce ATP through Ado.…”

“…4) Ribose-modified analogues of Ado such as DDA and deoxyAdo were found to increase cellular ATP levels (Tables 1-3). However, adenine ringmodified analogues of Ado such as methylAdo and cyclohexylAdo had no effect on ATP levels in PC12 cells (Table 1).…”

“…These results are agreement with the previous results that various P1 receptor agonists, Ado analogues with chemical modification of adenine ring, did not affect cellular ATP levels. 4) DDA and deoxyAdo are neither proper substrates of HGPT for the synthesis of adenine nucleotides nor precursors of ATP because of the lack of a hydroxyl group at positions 2 and 5. The addition of Ado was found to enhance additionally the DDA-induced cellular ATP levels ( Table 2).…”

“…4) In brief, PC12 cells were seeded in 35-mm Petri dishes at approximately 2ϫ10 6 cells and cultured for 3 d. PC12 cells were incubated with 1.5 ml of 100 mM adenine-related compound in Locke's solution {5.6 mM glucose, 154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO 3 , 5 mM 2-[4-(2-hydroyethyl)-1-piperazinyl]-ethanesulfonic acid (Hepes), pH 7.3} at 37°C for 2 h. After washing twice with Locke's solution, the cells were homogenized in perchloric acid. The perchloric acid-soluble compounds extracted from PC12 cells were treated at 85°C for 15 min with chloroacetaldehyde for fluorescence derivatization as described previously.…”

“…Recently, Fujimori et al 4) reported that extracellular Ado enhances cellular ATP levels in PC12 cells, a neuronal model, and speculated that the Ado-evoked ATP level enhancement might result in stimulation of ATP synthesis through a salvage pathway requiring hypoxanthine-guanine phosphoribosyltransferase (HGPT). Changes in cellular levels of cAMP released by adenylate cyclase are known to involve cellular glucose metabolism.…”