Expression and regulation of P2U-purinergic receptor in human granulosa-luteal cells

The P2U purinoceptor (P2UR) has been identified pharmacologically in the ov ary. However, the expression and regulation of the P2UR messenger RNA (mRNA ) in human ovarian cells are still poorly characterized. The present study was designed to examine the expression and regulation of the P2UR in human granulosa-luteal cells (hGLCs) by RT-PCR and Northern blot analysis. A PCR product corresponding to the expected 599-bp P2UR complementary DNA was obt ained from hGLCs. Molecular cloning and sequencing of the PCR product revea led an identical sequence to the reported P2UR complementary DNA. Two mRNA transcripts of 2.0 kb and 4.6 kb were identified in hGLCs using Northern bl ot analysis. The expression of the P2UR mRNA was down-regulated by human CG in a dose- and time-dependent manner. Treatment with 8-bromo-cAMP and fors kolin also attenuated P2UR mRNA levels. Calcium signaling following the act ivation of the PBUR in single hGLCs was studied using microspectrofluorimet ry. It revealed that, like ATP, uridine triphosphate (UTP) also induced cyt osolic calcium mobilization in a dose-dependent manner. These results demon strate for the first time that the P2UR mRNA is expressed in hGLCs and that PBUR mRNA is regulated by human CG, cAMP, and forskolin. The PBUR expresse d in hGLCs functional because activation of the P2UR by ATP or UTP resulted in rapid and transient mobilization of cytosolic calcium at the single cel l level. These findings further support a potential role of this neurotrans mitter receptor in the human ovary.