ENTERIC GLIA EXHIBIT P-2U RECEPTORS THAT INCREASE CYTOSOLIC CALCIUM BY A PHOSPHOLIPASE C-DEPENDENT MECHANISM

Calcium signaling in fura-2 acetoxymethyl ester-loaded enteric glia wa s investigated in response to neuroligands; responses to ATP were stud ied in detail. Carbachol (1 mM), glutamate (100 mu M), norepinephrine (10 mu M), and substance P (1 mu M) did not increase the intracellular calcium concentration ([Ca2+](i)) in cultured enteric glia. An increa sing percentage of glia responded to serotonin (4%; 100 mu M), bradyki nin (11%; 10 mu M), and histamine (31%; 100 mu M), whereas 100% of gli a responded to ATP (100 mu M). ATP-evoked calcium signaling was concen tration dependent in terms of the percentage of glia responding and th e peak [Ca2+](i) achieved; responses were pertussis toxin insensitive, Based on responsiveness of enteric glia to purinergic agonists and pe ak [Ca2+](i) evoked, ATP = UTP > ADP > beta,gamma-methyleneadenosine 5 '-triphosphate much greater than 2-methylthioadenosine 5'-triphosphate = alpha,beta-methyleneadenosine 5'-triphosphate = AMP = adenosine, su ggesting a glial P-2U receptor. Depletion of D-myo-inositol 1,4,5-tris phosphate-sensitive calcium stores by thapsigargin (10 mu M) abolished glial responses to ATP. Similarly, calcium responses were decreased 9 2% by U-73122 (10 mu M), an inhibitor of phospholipase C, and 93% by t he phorbol ester phorbol 12-myristate 13-acetate (100 nM), an activato r of protein kinase C, Thus, cultured enteric glia can respond to neur otransmitters with increases in [Ca2+](i). Our data suggest that glial responses to ATP are mediated by a P-2U receptor coupled to activatio n of phospholipase C and release of intracellular calcium stores.