P2 purinergic receptors potentiate parathyroid hormone receptor-mediated increases in intracellular calcium and inositol trisphosphate in UMR-106 rat osteoblasts.

The PTH receptor has been cloned and shown to activate both adenylate cyclase and phospholipase C. Evidence exists that both signaling pathways are important for mediating the net physiological effects of this hormone on bone remodeling. We have shown previously that UMR-106 osteoblastic sarcoma cells express two calcium-signaling P2 purinergic receptors, a P2U and a unique P2T receptor. Neither receptor modulates PTH receptor-mediated activation of adenylate cyclase. We now report that stimulation of either P2 receptor will, however, potentiate the magnitude of the calcium signal observed after subsequent addition of human (h) PTH-(1-34) to fluo-3-loaded UMR-106 cells. Results from experiments with staurosporine and phorbol 12-myristate 13-acetate argue against a role for protein kinase C as a mediator of this potentiating effect of P2 receptor ligands. The P2 receptor-mediated intracellular calcium elevation itself cannot account for the potentiating mechanism, because addition of ionomycin will not replicate the effect of P2 receptor ligands on hPTH-(1-34) signaling. Addition of EGTA after exposure to P2 ligands does not prevent the potentiation of hPTH-(1-34), indicating that P2 ligands potentiate the release of intracellular calcium after PTH receptor stimulation. Inositol trisphosphate production is potentiated in response to hPTH-(1-34) after first priming [3H]inositol-labeled cells with a P2 agonist. We conclude that UMR-106 cells express PTH receptors that are capable of activating adenylate cyclase, but may be unable to activate phospholipase C until cells receive a signal as a consequence of P2 receptor activation. The nature of the signal is unclear, but appears not to be mediated by either calcium or protein kinase C.

Separate P2T and P2U purinergic receptors with similar second messenger signaling pathways in UMR-106 osteoblasts.

UMR-106 rat osteogenic sarcoma cells express two calcium signaling P2 purinergic receptors. One is a P2U receptor with EC50's for adenosine triphosphate (ATP) and uridine triphosphate (UTP) of 2.6 and 2.4 microM, respectively. The other is a novel P2T receptor for adenosine diphosphate (ADP) (EC50 0.4 microM), adenosine 5'-O-(2-thiodiphosphate). (EC50 5 microM), 2-methylthio ATP (EC50 0.2 microM), and 2-methylthio ADP (EC50 0.04 microgram M). Responses to these ligands are desensitized by ADP but not by UTP. Responses to UTP and ATP are desensitized by UTP but not by ADP or adenosine 5'-O-(2-thiodiphosphate). 2-ChloroATP interacts with both receptors at high concentrations but with only the P2T receptor at low concentrations (EC50 0.05 microM). The weak platelet P2T receptor antagonist AMP blocks this P2T and not the P2U receptor. Addition of ATP after UTP desensitization of P2U receptors inhibits subsequent responsiveness to ADP but evidence for rapid conversion of ATP to ADP complicates interpretation of this apparent antagonism of P2T receptors by ATP. A subpassage of UMR-106.P135 cells lose P2U but retain P2T ligand responsiveness. Activation of either P2 receptor increases cellular IP3 concentrations in UMR-106 cells. Neither receptor can activate divalent cation entry as evidenced by their lack of effect on Mn++ quenching of fura-2 fluorescence. Neither receptor can modify parathyroid hormone receptor-mediated elevation of cellular cyclic AMP. This receptor for ADP demonstrates many of the same characteristics of the ADP receptor which we have previously reported as a P2T receptor expressed in K562 and Dami cells.

Evidence for separate calcium-signaling P2T and P2U purinoceptors in human megakaryocytic Dami cells.

Recently (J Pharmacol Exp Ther 261:580, 1992), we have shown that K562 leukemia cells express a calcium-signaling purinoceptor with characteristics of the P2T receptor subtype for adenosine diphosphate (ADP) previously found only in platelets. Because these results suggested that the P2T receptor may be an early marker for megakaryocytic differentiation, we studied whether this calcium-signaling receptor is also expressed in Dami cells, a human megakaryocytic leukemia cell line. Here we report evidence that Dami cells express a P2T receptor for ADP. The calcium response EC50 values for ADP, 2-methylthioadenosine diphosphate (2-MeS-ADP), and adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) in Dami cells are 0.4 mumol/L, 0.04 mumol/L, and 2 mumol/L, respectively, which approximate the potencies of these agonists in K562 cells and in platelets. The platelet P2T receptor antagonists 2-methylthioadenosine triphosphate (2-MeS-ATP), and 2-chloroadenosine triphosphate (2-Cl-ATP) were surprisingly potent agonists at the P2T receptor in both Dami and K562 cells. Dami cells, unlike K562 cells and platelets, also respond to adenosine triphosphate (ATP) and uridine triphosphate (UTP) with an increase in intracellular calcium. Adenosine monophosphate (AMP) is an effective antagonist of the response to ADP, 2-MeS-ADP, ADP beta S, 2-MeS-ATP, and 2-Cl-ATP, but not to ATP and UTP. The responses to maximal concentrations of UTP in combination with either ADP, 2-MeS-ADP, ADP beta S, or 2-MeS-ATP are additive. In contrast, ADP in combination with either 2-MeS-ADP, ADP beta S, 2-MeS-ATP, or 2-Cl-ATP are not additive. UTP desensitized Dami cells to ATP but not to ADP, 2-MeS-ADP, ADP beta S, or 2-MeS-ATP. Addition of ATP after UTP desensitization antagonized subsequent responsiveness to ADP. The data suggest that the receptor for ADP may be a unique P2T subtype, and the receptor for ATP and UTP is distinct from that of ADP and is most characteristic of the P2U (nucleotide) receptor subtype. Activation of either the P2T or P2U receptor causes a rapid generation of inositol trisphosphate in Dami cells.

Muscarinic m2 receptors in cerebellar granule cell cultures from rat: mechanism of short-term desensitization.

Cerebellar granule cell cultures of rat express only muscarinic m2 and m3 receptor subtypes and exhibit the pharmacological profile of muscarinic m2 receptors that couple to guanine nucleotide binding proteins to inhibit adenylyl cyclase. In vivo pretreatment with muscarinic agonists desensitizes the muscarinic m2 receptor with 50% complete desensitization within 15 to 20 min. After a 1-hr pretreatment with a maximal concentration of carbachol (short-term desensitization), m2 receptor responsiveness reappeared after a 1-hr treatment of cultures with atropine. However, after a 6-hr pretreatment with carbachol (long-term desensitization), m2 receptor responsiveness did not reappear after 1-hr treatment with atropine. Short-term desensitization was homologous for the m2 receptor because treatment of cultures with carbachol did not alter gamma-aminobutyric acidB receptor-mediated inhibition of adenylyl cyclase. Muscarinic m2 receptor desensitization was not mimicked by the addition of analogs of cyclic AMP, cyclic GMP or diacylglycerol to the cultures. The agonist-induced desensitization was not blocked by a cyclic AMP analog, 8-(4-chlorophenylthio)-cyclic AMP. Pretreatment with antisense oligodeoxynucleotides against the mRNA-encoding beta adrenergic receptor kinase attenuated the desensitization by carbachol (100 microM, 1 hr) of m2 receptors. Irreversible labeling of muscarinic m2 and m3 receptors with [3H]propylbenzilycholine mustard followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis produced a loss of the muscarinic m2 receptor (66-kDa protein), but not the muscarinic m3 receptor (92-kDa protein). We suspect that the short-term desensitization results from the phosphorylation of the muscarinic m2 receptor followed by loss of receptor from the plasma membrane.