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

P2 nucleotide receptors on C2C12 satellite cells

In developing muscle cells environmental stimuli transmitted by purines binding to the specific receptors are crucial proliferation regulators. C2C12 myoblasts express numerous purinergic receptors representing both main classes: P2X and P2Y. Among P2Y receptors we have found the expression of P2Y1, P2Y2, P2Y4, P2Y6 and P2Y12 family members while among P2X receptors P2X4, P2X5 and P2X7 were discovered. We have been able to show that activation of those receptors is responsible for ERK class kinase activity, responsible for regulation of cell proliferation pathway. We have also demonstrated that this activity is calcium dependent suggesting Ca2+ ions as secondary messenger between receptor and kinase regulatory system. More specifically, we do suspect that in C2C12 myoblasts calcium channels of P2X receptors, particularly P2X5 play the main role in proliferation regulation. In further development of myoblasts into myotubes, when proliferation is gradually inhibited, the pattern of P2 receptors is changed. This phenomenon is followed by diminishing of the P2Y2-dependent Ca2+ signaling, while the mRNA expression of P2Y2 receptor reminds still on the high level. Moreover, P2X2 receptor mRNA, absent in myoblasts appears in myotubes. These data show that differentiation of C2C12 cell line satellite myoblasts is accompanied by changes in P2 receptors expression pattern

ADP stimulation of inositol phosphates in hepatoctyes: role of conversion to ATP and stimulation of P2Y2 receptors.

1. Accumulation of inositol (poly)phosphates (InsP(x)) has been studied in rat hepatocytes labelled with [(3)H]inositol. Stimulation with ADP resulted in a significant increase in total [(3)H]InsP(x), whereas 2-MeSADP had only a small effect and ADPβS was ineffective. UTP and ITP also stimulated substantial increases in [(3)H]InsP(x). 2. The dose–response curve to ADP was largely unaltered by the presence of the P2Y(1) antagonist, adenosine-3′-phosphate-5′-phosphate (A3P5P). Similarly, inclusion of MRS 2179, a more selective P2Y(1) antagonist, had no effect on the dose–response curve to ADP. 3. The inclusion of hexokinase in the assay reduced, but did not abolish, the response to ADP. 4. HPLC analysis revealed that ADP in the medium was rapidly converted to AMP and ATP. The inclusion of hexokinase removed ATP, but exacerbated the decline in ADP concentration, leading to increased levels of AMP. 2-MeSADP was stable in the medium and ATP was largely unaffected. 5. The addition of the adenylate kinase inhibitor, diadenosine pentaphosphate (Ap(5)A) significantly reduced the ADP response. HPLC analysis conducted in parallel demonstrated that this treatment inhibited conversion of ADP to ATP and AMP. 6. Inclusion of the P1 antagonist CGS 15943 had no effect on the dose–response curve to ADP. 7. These observations indicate that hepatocytes respond to ADP with an increase in inositol (poly)phosphates following conversion to ATP. P2Y(1) activation in hepatocytes does not appear to be coupled to inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) production