P2U Receptor Stimulation Increases Intracellular Ca²⁺ in Hybrid N18TG2 X Mesencephalon (MES-23.5) Cells Via Two Distinct Mechanisms

Himanshu N. Parikh, Li-Xin Liu and Louis A Chiodo, Department of Pharmacology,
Texas Tech University Health Sciences Center, Lubbock, Texas

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Himanshu Parikh
Department of Pharmacology
Texas Tech University Health Science Center
3601, 4th Street
Lubbock, TX 79430
phrhp@ttuhsc.edu
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KEY WORDS: Keywords: fura-2, intracellular calcium, purinergic receptors, MES-23.5 cells

Abstract
(Neuroscience-Net, Volume 2, Article #10014; August ?, 1997)

In the present study, we have characterized the signal transduction pathway associated with activation of P₂ receptors endogenously present in a cultured hybrid N18TG2 X mesencephalon (MES-23.5) cells and responsible for increases in intracellular Ca²⁺ ( [Ca²⁺]_i ) in response to extracellular adenosine triphosphate (ATP) application. The agonist potency profile for this receptor was UTP > ATP >> 2-methylthio-adenosine 5'- triphosphate (2-MeSATP) > a, b-methylene adenosine 5'-triphosphate (a,b-MeATP), typical of P_2U purinergic receptor type. Extracellular application of UTP or ATP (1-100 micromolar) to MES-23.5 cells resulted in a rapid, transient increase in [Ca²⁺]_i that was inhibited by >50% when extracellular Ca²⁺ was removed. These findings demonstrated that P_2U receptor activation increased [Ca²⁺]_i by two mechanisms: mobilization of Ca²⁺ from internal stores and increased Ca²⁺ entry into the cell. Enhanced entry of Ca²⁺ as a result of P_2U receptor-induced intracellular Ca²⁺ mobilization suggested the presence of Ca²⁺ release-activated Ca²⁺ channels (CRAC) in MES-23.5 cells. In direct support of this hypothesis, whole-cell patch recordings demonstrated a sustained inward current in response to bath application of thapsigargin (which is known significantly increase [Ca²⁺]_i in these cells) or ATP. Ca²⁺ entry and mobilization in MES-23.5 cells was pertussis toxin sensitive in that pretreatment of cells completely blocked the increase in [Ca²⁺]_i, In addition, U-73122, a phospholipase C inhibitor, also inhibited P_2U receptor mediated mobilization of [Ca²⁺]_i. These results demonstrate the presence of functional P_2U purinergic receptors on MES-23.5 cells. These receptors are coupled to phospholipase C by a pertussis toxin sensitive G-protein and their activation results in the mobilization of Ca²⁺ from internal stores and the subsequent activation of CRAC Ca²⁺ entry.