adenosine-5--o-(3-thiotriphosphate) and diadenosine-tetraphosphate

The effects of diadenosine tetraphosphate (AP4A) diadenosine pentaphosphate (AP5A) and diadenosine hexaphosphate (AP6A) on the cytosolic-free calcium concentration ([Ca2+]i) were evaluated in cultured rat glomerular mesangial cells (MCs) using the fluorescent dye technique. The addition of 10 mumol L-1 AP4A, AP5A or AP6A significantly increased [Ca2+]i in MCs by 57 +/- 9 nmol L-1 n = 17; P < 0.01), 76 +/- 27 nmol L-1 (n = 9; P < 0.01) or 65 +/- 12 nmol L-1 (n = 18; P < 0.01) respectively. In the absence of extracellular calcium, there was no significant change in [Ca2+]i in MCs after administration of diadenosine polyphosphates, indicating that these agents induce transplasma membrane Ca2+ influx. AP6A significantly enhanced the angiotensin II-induced changes in [Ca2+]i in MCs. The AP5A-induced transplasma membrane Ca2+ influx was inhibited by the P2 purinoceptor blockers suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), but was not affected by the adenosine A1 receptor blocker 8-cyclopentyl-1.3-dipro-pylzanthine (CPDPX). Adenosine triphosphate (ATP) and adenosine 5'-O-(3-thio)triphosphate (ATP-gamma S) increased [Ca2+]i in MCs, whereas alpha, beta-methylene ATP had no effect on [Ca2+]i in MCs. Measurements of diacylglycerol and phosphatidic acid showed that AP5A and AP6A also stimulated phospholipase C, but had no effect on phospholipase D. The inhibition of phosphatidylcholine-specific phospholipase C significantly reduced the AP5A-induced [Ca2+]i increase. In summary, diadenosine polyphosphates induce Ca2+ influx through P2 purinoceptors and may be involved in the local regulation of vascular resistance evoked by the Ca(2+)-dependent contractile response of mesangial cells.

Topics: Adenosine Triphosphate; Affinity Labels; Angiotensin II; Animals; Calcium; Cell Membrane; Dinucleoside Phosphates; Dose-Response Relationship, Drug; Glomerular Mesangium; Male; Phospholipase D; Rats; Rats, Inbred WKY; Second Messenger Systems; Suramin; Type C Phospholipases; Vasoconstrictor Agents

Luciferase catalyzes the preferential synthesis of adenosine(5')tetraphospho(5')nucleoside (Ap4N) in the presence of luciferin (LH2), adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]) and NTP (other than ATP), with very low, or undetectable synthesis of Ap4A or Np4N, because ATP[gamma S] is a good adenylyl donor for the formation of the E-LH2-AMP complex, but a poor adenylyl acceptor from the complex, and NTP, other than ATP, are bad nucleotidyl donors, but good acceptors of the AMP moiety of the E-LH2-AMP complex. Synthesis of the corresponding Ap4N (or Ap5G in the case of p4G were obtained in the presence of ATP[gamma S] and GTP, UTP, CTP, XTP, dTTP, ITP, dGTP, dCTP, dITP, epsilon ATP (epsilon A, N6-ethenoadenosine) or p4G. The yield of synthesis of Ap4N was at least 50% of that theoretically expected. The process can be easily scaled-up, which allows synthesis of at least 1-5 mumol Ap4N. Further evidence for the synthesis of Ap4G from ATP[gamma S] and GTP was obtained by 1H-NMR and 31P-NMR spectroscopy. Synthesis of Ap4N, in yields lower than those above, can also be obtained in the presence of ADP and NTP; synthesis is due to the presence in commercial luciferase of enzymes (adenylate kinase and NDP kinase) that catalyze the synthesis of ATP from ADP and NTP. In the presence of ATP and polyphosphates, luciferase catalyzes the synthesis of a variety of compounds of adenosine 5'-polyphosphates (pnA; n = 3-20 and ApnA; n = 4-16). In the presence of P3 or P4, preferential synthesis of p4A and Ap5A or p5A and Ap6A were obtained, respectively, showing that both polyphosphates accept the adenylyl moiety of the E-LH2-AMP complex. Polyphosphates of chain length 5, 15 and 35 elicited the synthesis of a variety of PnA and ApnA. Ap4A is also split by luciferase in the presence of P3 or P4 (but not in the presence of P5) yielding preferential synthesis of p4A and Ap5A, or p5A and Ap6A, respectively.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Coleoptera; Dinucleoside Phosphates; Luciferases