Activation of early events of the mitogenic response by a P2Y purinoceptor with covalently bound 3'-O-(4-benzoyl)-benzoyladenosine 5'-triphosphate.

Abstract

3'-O-(4-Benzoyl)benzoyl-ATP (BzATP), a photoaffinity analog of ATP, was used as a ligand for a P2Y purinoceptor (adenine nucleotide receptor) present in intact Swiss 3T3 and 3T6 cells and A-431 epidermoid carcinoma cells. Photolysis of serum-starved cells in the presence of 10-50 microM BzATP, followed by extensive washing to remove unincorporated BzATP, induced the release of arachidonic acid. A trace (less than 0.01%) of photoincorporated BzATP was as effective as when 50 microM BzATP or ATP was contained in the incubation medium during the assay. Photoincorporated BzATP also stimulated the production of prostaglandin E2 and the accumulation of cyclic AMP. In previous studies, we demonstrated that these three events are obligatory early steps in a pathway leading to DNA synthesis in the above cell lines. The evidence indicated that the purinoceptor activated by extracellular ATP or BzATP was linked to a pertussis toxin-sensitive GTP-binding protein. Consistent with these observations, we now find that pertussis toxin inhibits the effect of photoincorporated BzATP on arachidonic acid release. These results indicate that BzATP is an effective agonist for the P2Y purinoceptor concerned with stimulation of DNA synthesis in 3T3, 3T6, and A-431 cells. Furthermore, after photolysis it becomes irreversibly associated with intact cells and promotes the activation of early events required for DNA synthesis.

Physiological and pathological roles of purines: An update

“…For example, a role for ATP has been claimed in embryonic cell migration and during morphogenesis [Lohmann et al, 1991;Kubo, 1991;Rathbone et al, 1992al. Extracellular ATP has also been implicated as a mediator of programmed cell death or apoptosis [Di Virgilio et al, 1990;Zheng et al, 1991;Murgia et al, 19921; purine agonists have been shown to be involved in the trophic effects of sympathetic innervation [Albino-Teixeira et al, 19901; ATP has been implicated as a fusion signal for developing skeletal muscle cells [Keresztes et al, 19911; and purines can act synergistically with growth factors to influence growth mitogenesis and morphogenesis [Huang et al, 1989;Gonzalez et al, 1990;Rathbone et al, 1992bI. It has been suggested that extracellular ATP and ADP may play a physiological role in wound healing and as a mitogenic agent in the nervous system [Wang et al, 19901.…”

Tethered Benzophenone Reagents for the Synthesis of Photoactivatable Ligands

“…In particular, (i) BPs can be activated at wavelengths (> 320 nm) that are not associated with protein damage, (ii) BPs are reported to give highly efficient covalent labeling via insertion into unreactive C-H bonds, (iii) BP photolabeling shows no interference from water or bulk nucleophiles, and (iv) no special lighting conditions are necessary during synthesis and biochemical manipulations. Recently, the BP moiety has been used in polypeptides as 4-benzoylphenylalanine (ONeil & DeGrado, 1989;ONeil et al, 1989;Boyd et al, 1991;McNicoll et al, 1992;Shoelson et al, 1993;Williams & Shoelson, 1993) and as 4-benzoylbenzoyl esters and amides in nucleotides (Mahmood et al, 1989;Boyer et al, 1990;Chavan et al, 1990;Gonzalez et al, 1990;Pal & Coleman, 1990;Aloise et al, 1991;Bar-Zvi et al, 1992;Pal et al, 1992;Rajagopalan et al, 1993;Salvucci et al, 1993;Zarka & Shoshan-Barmatz, 19931, in sugars (Holman et al, 1988), in phospholipids (Ishidate et al, 1992), and in proteins (Leszyk et al, 1988;Agarwal et al, 1991;Rajasekharan et al, 1991;Combeau et al, 1992;Thiele & Fahrenholz, 1993). Interactions within lipid bilayers have also been probed using BP-lipid analogs (Yamamoto et al, 1993).…”

Homologous desensitization of ATP‐stimulated mitogenesis: Mechanism involves desensitization of arachidonic acid release and cAMP elevation but not the activation of protein kinase A

“…The production of PGE, and the accumulation of cyclic AMP were also stimulated, in a pertussis toxin sensitive manner (Gonzalez et al, 1990). Thus, a trace amount of BzATP, covalently linked to a Pzy purinoceptor, activated three "early events" as effectively as 50 pM ATP under ordinary conditions.…”

“…We previously observed that photolysis of quiescent 3T3 or 3T6 cells in the presence of BzATP, a photoaffinity analogue of ATP, followed by extensive washing to remove unincorporated BzATP, induced the release of arachidonic acid. The production of PGE, and the accumulation of cyclic AMP were also stimulated, in a pertussis toxin sensitive manner (Gonzalez et al, 1990). Thus, a trace amount of BzATP, covalently linked to a Pzy purinoceptor, activated three "early events" as effectively as 50 pM ATP under ordinary conditions.…”

“…Experiments were carried out as reported previously (Gonzalez et al, 1990). Confluent and quiescent 3T6 cells were washed two times with 1 ml HBS buffer (10 mM Hepes, pH 7.4; 140 mM NaC1, 5 mM KC1, 1 mM MgC12, 1 mM CaClz, 10 mM glucose) kept at 20°C and then 1 ml of the same buffer was added and the cell cultures were transferred to ice.…”