Characterization of the P2Y-purinoceptor involved in the ATP-induced rise in cytosolic Ca2+ concentration in rat ileal myocytes.
1. The P2-purinoceptor subtype and the intracellular signalling mechanism(s) involved in the rise in the free cytosolic Ca2+ concentration ([Ca2+]i) induced by ATP and analogues were analyzed in myocytes isolated from the longitudinal muscle layer of rat ileum by means of molecular and physiological techniques. 2. The P2-purinoceptor expressed by ileal smooth muscle cells shared 100% amino acid identity with the rat P2Y1-receptor. 3. Short applications of the purinoceptor agonists induced a transient rise in [Ca2+]i in an all-or-nothing manner. The rank order of potency of the analogues of ATP and ADP, determined by measuring the percentage of responding cells was 2-methylthioATP = 2-chloro-ATP > ADP > ATP, with concentrations giving [Ca2+]i response in 50% of cells ranging between 3 nM and 0.6 microM. The concentration-response curves to ADP and ATP were shifted to the right by 10 microM pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). 4. Although the rise in [Ca2+]i induced by stimulation of the ileal P2v-purinoceptor was inhibited by heparin (5 mg ml-1), we were not able to detect stimulation of phospholipase C under conditions (37 degrees C) where muscarinic cholinoceptor activation markedly increased inositol phosphate (InsP) accumulation. However, the carbachol (CCh)-induced increase in InsP accumulation was suppressed when the agonist was applied at 20 degrees C while a CCh-induced [Ca2+]i rise similar to that obtained in response to the P2-purinoceptor agonist was still observed. 5. Our results indicate that the rat ileal myocytes express a PPADS-sensitive P2-purinoceptor similar to the P2Y1-receptor subtype. Although there is no detectable increase in InsP production, stimulation of these receptors leads to a rise in [Ca2+]i by activation of the inositol 1,4,5-trisphosphate receptor-channel of the intracellular Ca2+ store, indicating that they couple to phospholipase C.
Pacaud
P
P
Institut de Pharmacologie Moléculaire et Cellulaire CNRS UPR 411, Universitè de Nice Sophia-Antipolis, Valbonne, France.
Feolde
E
E
Frelin
C
C
Loirand
G
G
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D007082:Ileum / Q000187:drug effects* / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects*; D051381:Rats; D017208:Rats, Wistar; D011983:Receptors, Purinergic / Q000187:drug effects*
D011983:Receptors, Purinergic; D000255:Adenosine Triphosphate; D002118:Calcium