guanosine-diphosphate and 5--adenylyl-(beta-gamma-methylene)diphosphonate

The role of nucleoside diphosphate kinase (NDKP), which converts GDP to GTP, in the coupling of mu-opioid receptors to G protein was investigated in membranes of Chinese hamster ovary cells stably transfected with the cloned rat mu-opioid receptor (rmor). Endogenous NDPK activity in membranes was determined to be 0.60+/-0.02 micromol/mg protein/30 min UDP (at 10 mM), a competitive substrate of NDPK for GDP with no effect on guanine nucleotide binding to G proteins, reduced basal [35S]GTPgammaS binding and unmasked morphine-stimulated [35S]GTPgammaS binding to pertussis toxin-sensitive G proteins, indicating that [35S]GTPgammaS binding to NDPK accounts for part of its high basal binding. UDP increased the extent of morphine-induced increase in [35S]GTPgammaS binding in the presence of GDP, most likely by reducing basal binding and inhibiting conversion of GDP to GTP. ATP greatly reduced morphine-induced increase in [35S]GTPgammaS binding, whereas AMP-PCP (adenylyl-(beta,gamma-methylene)-diphosphoate tetralithium salt), which cannot serve as the phosphate donor for NDPK, did not, demonstrating that effects of ATP is mediated by the NDPK product GTP. In addition, GDP and ATP increased the Kd and lowered the Bmax of the agonist [3H]DAMGO ([D-Ala2,N-Me-Phe4,Gly5ol]-Enkephalin) for the mu-opioid receptor and GDP alone increased Kd, most likely through their conversion to GTP by NDPK. Addition of exogenous NDPK enhanced the inhibitory effects of GDP and combined GDP and ATP on [3H]DAMGO binding. Thus, NDPK appears to play a role in modulating signal transduction of and agonist binding to mu-opioid receptors.

Topics: Adenosine Triphosphate; Analgesics, Opioid; Animals; Cell Membrane; Cells, Cultured; CHO Cells; Cloning, Molecular; Cricetinae; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Proteins; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Nucleoside-Diphosphate Kinase; Pertussis Toxin; Rats; Receptors, Opioid, mu; Uridine Diphosphate; Virulence Factors, Bordetella

Signal transduction via P2 purinergic receptors was investigated in HSG cells, a continuous cell line originally derived from an irradiated human salivary gland. Ligand specificity for nucleotide receptors in HSG cells was investigated with various nucleotides and their analogues. Inositol 1,4,5-trisphosphate (IP3) production was significantly increased by ATP, UTP and ATP gamma S. The ligand specificity of this effect agreed well with that of the P2U purinergic receptor. On the other hand, 45Ca2+ influx was stimulated by ATP, UTP > ATP gamma S, ADP, UDP > ADP beta S > AMPPNP, GTP, TTP > CTP, GDP, TDP, AMPPCP, AMPCPP. This ligand specificity of 45Ca2+ influx was much broader than IP3 production. Also pertussis and cholera toxin had no effect on both IP3 production and 45Ca2+ influx by ATP or UTP. 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (Bz-ATP) stimulates 45Ca2+ influx more effectively than IP3 formation. A 53-kDa membrane protein was photolabelled with [alpha-32P]Bz-ATP. This 53-kDa protein is a putative P2 purinergic receptor. In particular, the labelling was inhibited by a ligand profile that corresponded to that for 45Ca2+ influx. These findings suggest that nucleotides stimulate 45Ca2+ influx and IP3 formation by separate pathways via pertussis and cholera toxin-insensitive G proteins. Thus, in HSG cells, IP3 formation is coupled to the P2U subclass, while 45Ca2+ influx is coupled to another subclass, such as P2X, that regulates calcium channels.

Topics: Adenine Nucleotides; Adenosine Diphosphate; Adenosine Triphosphate; Adenylyl Imidodiphosphate; Affinity Labels; Calcium; Calcium Channels; Calcium Radioisotopes; Cell Line; Cholera Toxin; Cytidine Triphosphate; GTP-Binding Proteins; Guanosine Diphosphate; Humans; Inositol 1,4,5-Trisphosphate; Ligands; Membrane Proteins; Pertussis Toxin; Radiopharmaceuticals; Receptors, Purinergic; Salivary Ducts; Signal Transduction; Submandibular Gland; Substrate Specificity; Thionucleotides; Thymine Nucleotides; Uridine Triphosphate; Virulence Factors, Bordetella

We investigate the mechanisms underlying the intracellular calcium pulse that occurs in response to extracellular adenosine triphosphate (ATP) in osteoclasts. We find that pre-loading of GDP-beta-S abolishes the response in Ca(2+)-free medium, demonstrating an internal release of Ca2+ via a pathway that involves a G protein. GDP-beta-S does not block in normal Ca(2+)-containing medium, suggesting that ATP also induces a Ca2+ influx across the cell membrane. We confirmed this using the Mn2+ quenching technique, which shows significant opening of Ca2+ channels. We find a smaller response to adenosine diphosphate (ADP) and 2-methylthio-ATP (2-MeSATP), but no response to beta, gamma-methylene-ATP (AMP-PCP), adenosine monophosphate (AMP) or uridine triphosphate (UTP). Prior application of AMP and UTP, but not AMP-PCP, blocks the response to ATP. Our results indicate that the receptor is a P2 subtype that is not characteristic of any previously reported P2 receptor or combination of P2 receptors.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Calcium; Guanosine Diphosphate; Magnesium; Osteoclasts; Pertussis Toxin; Rabbits; Receptors, Purinergic P2; Signal Transduction; Thionucleotides; Uridine Triphosphate; Virulence Factors, Bordetella