ABSTRACT
BACKGROUND AND PURPOSE: The Gi -coupled, ADP-activated P2Y12 receptor is well characterized as playing a key role in platelet activation via crosstalk with the P2Y1 receptor in ADP-evoked intracellular Ca2+ responses. However, there is limited knowledge on the role of P2Y12 receptors in ADP-evoked Ca2+ responses in other blood cells. Here, we investigated the role of P2Y12 receptor activation in the modulation of ADP-evoked Ca2+ responses in human THP-1 monocytic cells. EXPERIMENTAL APPROACH: A combination of intracellular Ca2+ measurements, RT-PCR, immunocytochemistry, leukocyte isolation and siRNA-mediated gene knockdown were used to identify the role of P2Y12 receptor activation. KEY RESULTS: ADP-evoked intracellular Ca2+ responses (EC50 2.7 µM) in THP-1 cells were abolished by inhibition of PLC (U73122) or sarco/endoplasmic reticulum Ca2+ -ATPase (thapsigargin). Loss of ADP-evoked Ca2+ responses following treatment with MRS2578 (IC50 200 nM) revealed a major role for P2Y6 receptors in mediating ADP-evoked Ca2+ responses. ADP-evoked responses were attenuated either with pertussis toxin treatment, or P2Y12 receptor inhibition with two chemically distinct antagonists (ticagrelor, IC50 5.3 µM; PSB-0739, IC50 5.6 µM). ADP-evoked responses were suppressed following siRNA-mediated P2Y12 gene knockdown. The inhibitory effects of P2Y12 antagonists were fully reversed following adenylate cyclase inhibition (SQ22536). P2Y12 receptor expression was confirmed in freshly isolated human CD14+ monocytes. CONCLUSIONS AND IMPLICATIONS: Taken together, these data suggest that P2Y12 receptor activation positively regulates P2Y6 receptor-mediated intracellular Ca2+ signalling through suppression of adenylate cyclase activity in human monocytic cells.
Subject(s)
Adenosine Diphosphate/metabolism , Calcium Signaling , Calcium/metabolism , Monocytes/metabolism , Receptors, Purinergic P2Y12/metabolism , Calcium/analysis , Dose-Response Relationship, Drug , Humans , RNA, Small Interfering/pharmacology , Receptors, Purinergic P2Y12/genetics , Structure-Activity Relationship , THP-1 CellsABSTRACT
This study focused on the hypothesis that KCNA genes (which encode K(V)alpha1 voltage-gated K(+) channels) have enhanced functional expression in smooth muscle cells of a primary determinant of peripheral resistance - the small mesenteric artery. Real-time PCR methodology was developed to measure cell type-specific in situ gene expression. Profiles were determined for arterial myocyte expression of RNA species encoding K(V)alpha1 subunits as well as K(V)beta1, K(V)alpha2.1, K(V)gamma9.3, BK(Ca)alpha1 and BK(Ca)beta1. The seven major KCNA genes were expressed and more readily detected in endothelium-denuded mesenteric resistance artery compared with thoracic aorta; quantification revealed dramatic differential expression of one to two orders of magnitude. There was also four times more RNA encoding K(V)alpha2.1 but less or similar amounts encoding K(V)beta1, K(V)gamma9.3, BK(Ca)alpha1 and BK(Cabeta)1. Patch-clamp recordings from freshly isolated smooth muscle cells revealed dominant K(V)alpha1 K(+) current and current density twice as large in mesenteric cells. Therefore, we suggest the increased RNA production of the resistance artery impacts on physiological function, although there is quantitatively less K(+) current than might be expected. The mechanism conferring up-regulated expression of KCNA genes may be common to all the gene family and play a functional role in the physiological control of blood pressure.
Subject(s)
Mesenteric Arteries/physiology , Multigene Family , Muscle, Smooth, Vascular/physiology , Potassium Channels/genetics , Potassium Channels/metabolism , Vascular Resistance , Animals , Aorta, Thoracic/metabolism , Electric Conductivity , Gene Expression , Male , Mesenteric Arteries/metabolism , Mice , Mice, Inbred C57BL , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Patch-Clamp Techniques , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA/metabolism , Up-RegulationABSTRACT
Ductal branching within the mammary gland is stimulated by prolactin (PRL) and progesterone (P) acting through their receptors (PRLR and PR). Analysis of mammary gland PRLR expression revealed increasing expression of the long form (L-PRLR) and two of the three short forms (S1- and S3-PRLR) during puberty that became maximal late in pubescence and early gestation, then declined during gestation. By contrast, S2-PRLR mRNA levels remained constant. Examination of stromal PRLR revealed the consistent expression of L-PRLR mRNA. By contrast, S1-PRLR was present only in the mammary fat pad of neonates, whereas high neonatal expression of S2-PRLR became undetectable during puberty. Stromal expression of S3-PRLR decreased to low levels during puberty and was undetectable during lactation and involution. Exogenous PRL stimulated DNA synthesis in both epithelial and adjacent stromal cells in vivo. Distribution of PRLR mRNA in mammary epithelium was homogeneous before puberty and heterogeneous during puberty, gestation, and early lactation. A mutual role for PRLR and PR was suggested wherein PR mRNA increased beyond 6 weeks to maximal levels during puberty and gestation then became undetectable during lactation. In situ hybridization revealed that PR mRNA distribution is homogeneous in the ductal epithelium before 6 weeks and heterogenous during puberty and gestation and that PRLR and PR are similarly distributed in the ductal epithelium. Neither hormone stimulated DNA synthesis in mammary glands of ovariectomized females while their effects interacted markedly. These results demonstrate differential PRLR transcription by epithelial and stromal cells and a similar distribution of PRLR and PR that may facilitate the interaction between P and PRL during ductal branching in the mammary gland.
