Enteric Glia Exhibit P 2U Receptors that Increase Cytosolic Calcium by a Phospholipase C-Dependent Mechanism

Abstract

Calcium signaling in fura-2 acetoxymethyl ester-loaded enteric glia was investigated in response to neuroligands; responses to ATP were studied in detail. Carbachol (1 m M ), glutamate (100 µ M ), norepinephrine (10 µ M ), and substance P (1 µ M ) did not increase the intracellular calcium concentration ([Ca 2+ ] i ) in cultured enteric glia. An increasing percentage of glia responded to serotonin (4%; 100 µ M ), bradykinin (11%; 10 µ M ), and histamine (31%; 100 µ M ), whereas 100% of glia responded to ATP (100 µ M ). ATP-evoked calcium signaling was concentration dependent in terms of the percentage of glia responding and the peak [Ca 2+ ] i achieved; responses were pertussis toxin insensitive. Based on responsiveness of enteric glia to purinergic agonists and peak [Ca 2+ ] i evoked, ATP = UTP > ADP > Β,Γ-methyleneadenosine 5′-triphosphate > 2-methylthioadenosine 5′-triphosphate = Α,Β-methyleneadenosine 5′-triphosphate = AMP = adenosine, suggesting a glial P 2U receptor. Depletion of d- myo -inositol 1,4,5-trisphosphate-sensitive calcium stores by thapsigargin (10 µ M ) abolished glial responses to ATP. Similarly, calcium responses were decreased 92% by U-73122 (10 µ M ), an inhibitor of phospholipase C, and 93% by the phorbol ester phorbol 12-myristate 13-acetate (100 n M ), an activator of protein kinase C. Thus, cultured enteric glia can respond to neurotransmitters with increases in [Ca 2+ ] i . Our data suggest that glial responses to ATP are mediated by a P 2U receptor coupled to activation of phospholipase C and release of intracellular calcium stores.