P-2-PURINOCEPTOR-MEDIATED FORMATION OF INOSITOL PHOSPHATES AND INTRACELLULAR CA2-ARTERY SMOOTH-MUSCLE CELLS( TRANSIENTS IN HUMAN CORONARY)

1 The effects of extracellular adenosine 5'-triphosphate (ATP) on smoo th muscles are mediated by a variety of purinoceptors. In this study w e addressed the identity of the purinoceptors on smooth muscle cells ( SMC) cultured from human large coronary arteries. Purinoceptor-mediate d increases in [Ca2+](i) were measured in single fura-2 loaded cells b y applying a digital imaging technique, and the formation of inositol phosphate compounds was quantified after separation on an anion exchan ge column. 2 Stimulation of the human coronary artery SMC (HCASMC) wit h extracellular ATP at concentrations of 0.1-100 mu M induced a transi ent increase in [Ca2+](i) from a resting level of 49+/-21 nM to a maxi mum of 436+/-19 nM. The effect was dose-dependent with an EC(50) value for ATP of 2.2 mu M. 3 The rise in [Ca2+](i) was independent of the p resence of external Ca2+, but was abolished after depletion of intrace llular stores by incubation with 100 nM thapsigargin. 4 [Ca2+](i) was measured upon stimulation of the cells with 0.1-100 mu M of the more s pecific P-2-purinoceptor agonists alpha,beta-methyleneadenosine 5'-tri phosphate (alpha,beta-MeATP), 2-methylthioadenosine 5'-triphosphate (2 MeSATP) and uridine 5'-triphosphate (UTP). alpha,beta-MeATP was withou t effect, whereas 2MeSATP and UTP induced release of Ca2+ from interna l stores with 2MeSATP being the most potent agonist (EC(50)=0.17 mu M) , and UTP having a potency similar to ATP. The P-1 purinoceptor agonis t adenosine (100 mu M) did not induce any changes in [Ca2+](i). 5 Stim ulation with a submaximal concentration of UTP (10 mu M) abolished a s ubsequent ATP-induced increase in [Ca2+](i), whereas an increase was i nduced by ATP after stimulation with 10 mu M 2MeSATP. 6 The phospholip ase C (PLC) inhibitor U73122 (5 mu M) abolished the purinoceptor-activ ated rise in [Ca2+](i), whereas pretreatment with the G(i) protein inh ibitor pertussis toxin (PTX, 500 ng ml(-1)) was without effect on ATP- evoked [Ca2+](i) increases. 7 Receptor activation with UTP and ATP res ulted in formation of inositol phosphates with peak levels of inositol 1,4,5-trisphosphate (Ins(1,4,5)P-3) observed 5-20 s after stimulation . 8 These findings show, that cultured HCASMC express G protein-couple d purinoceptors, which upon stimulation activate PLC to induce enhance d Ins(1,4,5)P-3 production causing release of Ca2+ from internal store s. Since a release of Ca2+ was induced by 2MeSATP as well as by UTP, t he data indicate that P-2Y- as well as P-2U-purinoceptors are expresse d by the HCASMC.