adenosine-5--o-(3-thiotriphosphate) and 8-(4-sulfophenyl)theophylline

We examined the effects of purines and the pyrimidine UTP on cellular proliferation in the human astrocytoma cell line 1321N1. Treatment of cultured cells with 100 microM ATP or 2-chloroadenosine (2-CA) resulted in significant reductions in cell numbers after 2 days, whereas adenosine (ADO) exhibited a slower time course of inhibition of cell growth. Treatment with 100 microM UTP had no effect on cell numbers. 2-Chloroadenosine but neither ATP nor ADO resulted in an increase in cell death rates. A significant portion of the inhibitory response to ATP, ADO, or 2-CA was sensitive to the purine nucleoside transport inhibitor S-(p-nitrobenzyl)-6-thioguanosine, suggesting that uptake into cells was required for the inhibitory response. At least the majority of the observed responses to purines was not mediated by P1 (adenosine) receptors, because effects of ATP, ADO, or 2-CA were not affected by treatment of cells with the P1 receptor antagonist 8-(p-sulfophenyl)-theophylline. The absence of any known P2 (nucleotide) receptors in 1321N1 cells, coupled with the failure of the relatively stable ATP analog adenosine 5'-O-(3-thiotriphosphate) to alter cell growth rates, suggests that ATP acts indirectly to inhibit proliferation via one or more metabolic products. Although intracellular effects of purine nucleosides should be taken into account in future studies using 1321N1 cells, our findings also suggest 1321N1 cells as an excellent model for intracellular actions of nucleosides.

Topics: 2-Chloroadenosine; Adenosine; Adenosine Triphosphate; Astrocytoma; Brain Neoplasms; Cell Death; Cell Division; Humans; Purine Nucleosides; Purinergic P1 Receptor Antagonists; Theophylline; Tumor Cells, Cultured; Uridine Triphosphate

1. ATP has previously been shown to act as a sympathetic cotransmitter in the rat kidney. The present study analyses the question of whether postganglionic sympathetic nerve endings in the kidney possess P2-receptors which modulate noradrenaline release. Rat kidneys were perfused with Krebs-Henseleit solution containing the noradrenaline uptake blockers cocaine and corticosterone and the alpha2-adrenoceptor antagonist rauwolscine. The renal nerves were electrically stimulated, in most experiments by 30 pulses applied at 1 Hz. The outflow of endogenous noradrenaline (or, in some experiments, of ATP and lactate dehydrogenase) as well as the perfusion pressure were measured simultaneously. 2. The P2-receptor agonist adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS, 3-30 microM) reduced the renal nerve stimulation (RNS)-induced outflow of noradrenaline (estimated EC50 =8 microM). The P2-receptor antagonist cibacron blue 3GA (30 microM) shifted the concentration-inhibition curve for ATPgammaS to the right (apparent pKB value 4.7). 3. Cibacron blue 3GA (3-30 microM) and its isomer reactive blue 2 (3-30 microM) significantly increased RNS-induced outflow of noradrenaline in the presence of the P1-receptor antagonist 8-(p-sulphophenyl)theophylline (8-SPT, 100 microM) by about 70% and 90%, respectively. The P2-receptor antagonist suramin (30-300 microM) only tended to enhance RNS-induced outflow of noradrenaline. When the nerves were stimulated by short pulse trains consisting of 6 pulses applied at 100 Hz (conditions under which autoinhibition is inoperative), reactive blue 2 did not affect the RNS-induced outflow of noradrenaline. 4. RNS (120 pulses applied at 4 Hz) induced the outflow of ATP but not of the cytoplasmatic enzyme lactate dehydrogenase. 5. ATPgammaS (3-30 microM) concentration-dependently reduced pressor responses to RNS at 1 Hz. Cibacron blue 3GA, reactive blue 2 as well as suramin also reduced pressor responses to RNS (maximally by 50 to 70%). 6. This study in rat isolated kidney, in which the release of endogenous noradrenaline was measured, demonstrates that renal sympathetic nerves possess prejunctional P2-receptors that mediate inhibition of transmitter release. These prejunctional P2-receptors are activated by endogenous ligands, most likely ATP, released upon nerve activity. Both, P2-receptor agonists and P2-receptor antagonists reduced pressor responses to RNS either by inhibiting transmitter release or by blocking postjunctional vasoconstri

Topics: Adenosine Triphosphate; Animals; Blood Pressure; Kidney; L-Lactate Dehydrogenase; Male; Norepinephrine; Rats; Rats, Wistar; Receptors, Purinergic P2; Theophylline; Triazines