Role of the P2Y(13) Receptor in the Differentiation of Bone Marrow Stromal Cells into Osteoblasts and Adipocytes

Accumulating evidence indicates that extracellular nucleotides, signaling through purinergic receptors, play a significant role in bone remodeling. Mesenchymal stem cells (MSCs) express functional P2Y receptors whose expression level is regulated during osteoblast or adipocyte differentiation. P2Y13-deficient mice were previously shown to exhibit a decreased bone turnover associated with a reduction in the number of both osteoblasts and osteoclasts on the bone surfaces. We therefore examined whether P2Y13R activation was involved in the osteogenic differentiation of MSC. Our study demonstrated that ADP stimulation of P2Y13R+/+ (but not P2Y13R-/-) adherent bone marrow stromal cells (BMSCs) increased significantly the formation of alkaline phosphatase-colony-forming units (CFU-ALP) as well as the expression of osteoblastic markers (osterix, alkaline phosphatase, and collagen I) involved in the maturation of preosteoblasts into osteoblasts. The number of CFU-ALP obtained from P2Y13R-/- BMSC and the level of osteoblastic gene expression after osteogenic stimulation were strongly reduced compared to those obtained in wild-type cell cultures. In contrast, when P2Y13R-/- BMSCs were incubated in an adipogenic medium, the number of adipocytes generated and the level of adipogenic gene expression (PPARγ2 and Adipsin) were higher than those obtained in P2Y13R+/+ MSC. Interestingly, we observed a significant increase of the number of bone marrow adipocytes in tibia of P2Y13R-/- mice. In conclusion, our findings indicate that the P2Y13R plays an important role in the balance of osteoblast and adipocyte terminal differentiation of bone marrow progenitors. Therefore, the P2Y13 receptor can be considered as a new pharmacological target for the treatment of bone diseases like osteoporosis

Micronuclei to detect in vivo chemotherapy damage in a p53 mutated solid tumour

Apoptosis induction and micronuclei formation were compared following cytotoxic treatments in two rat glioma differing in p53 integrity. In vitro, micronuclei emergence but not apoptosis was linked to the p53 mutated status. In vivo, micronuclei assays were more sensitive to evaluate DNA damage induced by chemotherapy in a p53-mutated solid tumour.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Multiple P2Y receptors couple to calcium-dependent, chloride channels in smooth muscle cells of the rat pulmonary artery

BACKGROUND: Uridine 5'-triphosphate (UTP) and uridine 5'-diphosphate (UDP) act via P2Y receptors to evoke contraction of rat pulmonary arteries, whilst adenosine 5'-triphosphate (ATP) acts via P2X and P2Y receptors. Pharmacological characterisation of these receptors in intact arteries is complicated by release and extracellular metabolism of nucleotides, so the aim of this study was to characterise the P2Y receptors under conditions that minimise these problems. METHODS: The perforated-patch clamp technique was used to record the Ca(2+)-dependent, Cl(- )current (I(Cl,Ca)) activated by P2Y receptor agonists in acutely dissociated smooth muscle cells of rat small (SPA) and large (LPA) intrapulmonary arteries, held at -50 mV. Contractions to ATP were measured in isolated muscle rings. Data were compared by Student's t test or one way ANOVA. RESULTS: ATP, UTP and UDP (10(-4)M) evoked oscillating, inward currents (peak = 13–727 pA) in 71–93% of cells. The first current was usually the largest and in the SPA the response to ATP was significantly greater than those to UTP or UDP (P < 0.05). Subsequent currents tended to decrease in amplitude, with a variable time-course, to a level that was significantly smaller for ATP (P < 0.05), UTP (P < 0.001) and UDP (P < 0.05) in the SPA. The frequency of oscillations was similar for each agonist (mean≈6–11.min(-1)) and changed little during agonist application. The non-selective P2 receptor antagonist suramin (10(-4)M) abolished currents evoked by ATP in SPA (n = 4) and LPA (n = 4), but pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (10(-4)M), also a non-selective P2 antagonist, had no effect (n = 4, 5 respectively). Currents elicited by UTP (n = 37) or UDP (n = 14) were unaffected by either antagonist. Contractions of SPA evoked by ATP were partially inhibited by PPADS (n = 4) and abolished by suramin (n = 5). Both antagonists abolished the contractions in LPA. CONCLUSION: At least two P2Y subtypes couple to I(Cl,Ca )in smooth muscle cells of rat SPA and LPA, with no apparent regional variation in their distribution. The suramin-sensitive, PPADS-resistant site activated by ATP most resembles the P2Y(11 )receptor. However, the suramin- and PPADS-insensitive receptor activated by UTP and UDP does not correspond to any of the known P2Y subtypes. These receptors likely play a significant role in nucleotide-induced vasoconstriction

Ecto-phosphorylation on aortic endothelial cells. Exquisite sensitivity to staurosporine.

One- and two-dimensional gel electrophoresis of proteins from bovine aortic endothelial cells (BAEC) incubated with [gamma-32P]ATP revealed the preferential labelling of a cell-associated 21 kDa substrate. The labelling of this band was detectable within 30 s, increased up to 30 min and was stable for at least 3 h following the wash-out of the ATP. This protein was also labelled after incubation of the cells with [gamma-35S]ATP. Incorporation of radioactivity into the 21 kDa band did not occur if the endothelial cells were treated with low concentrations of trypsin (0.01%) before or after the labelling period. The pattern of BAEC protein phosphorylation by [gamma-32P]ATP was completely different from that of the fetal calf serum used for the cell culture. The presence of serum during the incubation of BAEC with [gamma-32P]ATP did not modify qualitatively the labelling pattern and, in particular, did not enhance the phosphorylation of the 21 kDa substrate; this suggests that neither the kinase nor the 21 kDa substrate are adsorbed serum proteins. Staurosporine, a protein kinase inhibitor with low specificity, decreased the labelling of the 21 kDa protein with an IC50 of 2 nM. In contrast, at 100 nM, staurosporine did not decrease the accumulation of inositol phosphates induced by ATP via the activation of P2y receptors. These data indicate the presence of aortic endothelial cells of an ecto-kinase which uses extracellular ATP to produce the selective and long-lived phosphorylation of a 21 kDa endothelial substrate. Ecto-phosphorylation of this protein might play a role in the modulation of endothelial cell functions by ATP, in addition to the P2y receptors [Boeynaems & Pearson (1990) Trends Pharmacol. Sci. 11, 34-37]. The exquisite sensitivity of ecto-phosphorylation to inhibition by staurosporine and its specific inhibition by some isoquinolinesulphonamide compounds provide potential pharmacological tools to investigate this hypothesis

K(+) secretion activated by luminal P2Y(2) and P2Y(4) receptors in mouse colon

Extracellular nucleotides are important regulators of epithelial ion transport, frequently exerting their action from the luminal side. Luminal P2Y receptors have previously been identified in rat distal colonic mucosa. Their activation by UTP and ATP stimulates K(+) secretion. The aim of this study was to clarify which of the P2Y receptor subtypes are responsible for the stimulated K(+) secretion. To this end P2Y(2) and P2Y(4) knock-out mice were used to measure distal colonic ion transport in an Ussing chamber. In mouse (NMRI) distal colonic mucosa, luminal UTP and ATP with similar potency induced a rapid and transient increase of the transepithelial voltage (V(te)) (UTP: from −0.81 ± 0.23 to 3.11 ± 0.61 mV, n = 24), an increase of equivalent short circuit current (I(sc)) by 166.9 ± 22.8 μA cm(−2) and a decrease of transepithelial resistance (R(te)) from 29.4 ± 2.4 to 23.5 ± 2.0 Ω cm(2). This effect was completely inhibited by luminal Ba(2+) (5 mm, n = 5) and iberiotoxin (240 nm, n = 6), indicating UTP/ATP-stimulated K(+) secretion. RT-PCR analysis of isolated colonic crypts revealed P2Y(2), P2Y(4) and P2Y(6) specific transcripts. The luminal UTP-stimulated K(+) secretion was still present in P2Y(2) receptor knock-out mice, but significantly reduced (ΔV(te): 0.83 ± 0.26 mV) compared to wild-type littermates (ΔV(te): 2.08 ± 0.52 mV, n= 9). In P2Y(4) receptor knock-out mice the UTP-induced K(+) secretion was similarly reduced. Luminal UTP-stimulated K(+) secretion was completely absent in P2Y(2)/P2Y(4) double receptor KO mice. Basolateral UTP showed no effect. In summary, these results indicate that both the P2Y(2) and P2Y(4) receptors are present in the luminal membrane of mouse distal colonic mucosa, and stimulation of these receptors leads to K(+) secretion