Extracellular ATP in concentrations of 5-50 gLM displayed very little mitogenic activity by itself but it caused synergistic stimulation of [3H]thymidine incorporation in the presence of phorbol 12-tetradecanoate 13-acetate, epidermal growth factor, platelet-derived growth factor, insulin, adenosine, or 5'-(N-ethyl) These findings raise the possibility that exogenous ATP acts as a growth factor, and this was implied in two recent papers (23, 24), where ATP was compared to "more conventional mitogens." However, it is premature to call ATP a mitogen without more evidence than has been available. One must be careful to rule out the possibility that ATP is converted to adenosine by ubiquitous hydrolytic enzymes on the cell surface (ectoenzymes) (32). It was demonstrated some years ago in Rozengurt's laboratory (11-13) and by others (33) that adenosine is a potent mitogen for Swiss 3T3 cells, and this is also true for certain adenosine analogues such as NECA.In this paper, we show that exogenous ATP is a mitogen for 3T3, 3T6, A431, HFF (34), and DDT1-MF2 (35) cells. We confirm that adenosine is also mitogenic, but several lines of evidence show that ATP acts independently, in its own right, and not by being first converted to adenosine by ectoenzymes. MATERIALS AND METHODS Materials. [3H]Thymidine was purchased from Amersham.Culture media and fetal bovine serum (FBS) were from GIBCO. ATP, adenosine, epidermal growth factor (EGF), adenosine 5'-[8,y-imido]triphosphate (AdoPP[NH]P), and all other nucleotides were from Boehringer Mannheim. Bovine insulin, NECA, and phorbol 12-tetradecanoate 13-acetate (PTA) were from Sigma. Platelet-derived growth factor (PDGF) was obtained from Amgen Biologicals. All other chemicals were of the highest purity available.Cell Culture. Swiss 3T3 and 3T6 mouse fibroblasts, human A431 epidermoid carcinoma cells (14), and DDT1-MF2 cells (35), derived from a vas deferens tumor of the hamster, were maintained in Dulbecco's modified Eagle's medium (DMEM; GIBCO) containing 10 mM Hepes, 44 mM NaHCO3, penicillin (110 units/ml), streptomycin (100 ,ug/ml), and 0.5% (vol/vol) (3T6), 5% (A431, DDT1-MF2), or 10% (3T3) FBS.Cells were grown at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. Two days after seeding, the 3T3 cells were given fresh medium supplemented as before and then allowed to become confluent and quiescent for 5 days. The A431, DDT1-MF2, and 3T6 cultures were used 4, 5, or 7904The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The mitogenic effect of extracellular ATP on porcine aortic smooth muscle cells (SMC) was examined. Stimulation of [3H]thymidine incorporation by ATP was dose-dependent; the maximal effect was obtained at 100 microM. ATP acted synergistically with insulin, IGF-1, EGF, PDGF, and various other mitogens. Incorporation of [3H]thymidine was correlated with the fraction of [3H]thymidine-labeled nuclei and changes in cell counts. The stimulation of proliferation was also determined by measurement of cellular DNA using bisbenzamide and by following the increase of mitochondrial dehydrogenase protein. The effect of ATP was not due to hydrolysis to adenosine, which shows synergism with ATP. ATP acted as a competence factor. The mitogenic effect of ATP, but not adenosine, was further increased by lysophosphatidate, phosphatidic acid, or norepinephrine. The inhibitor of adenosine deaminase, EHNA, stimulated the effect of adenosine but not ATP. The adenosine receptor antagonist theophylline depressed adenosine-induced mitogenesis. ADP and the non-hydrolyzable analogue adenosine 5'-[beta, gamma-imido]triphosphate (AMP-PNP) were equally mitogenic. Thus extracellular ATP stimulated mitogenesis of SMC via P2Y purinoceptors. The mechanism of ATP acting as a mitogen in SMC was further explored. Extracellular ATP stimulated the release of [3H]arachidonic acid (AA) and prostaglandin E2 (PGE2) into the medium, and enhanced cAMP accumulation in a dose-dependent fashion similar to ATP-induced [3H]thymidine incorporation. Inhibitors of the arachidonic acid metabolism pathway, quinacrine and indomethacin, partially inhibited the mitogenic effect of ATP but not of adenosine. Pertussis toxin inhibited ATP-stimulated DNA synthesis, AA release, PGE2 formation, and cAMP accumulation. Down-regulation of protein kinase C (PKC) by long-term exposure to phorbol dibutyrate (PDBu) partially prevented stimulation of DNA synthesis and activation of the AA pathway by ATP. The PKC inhibitor, staurosporine, antagonized mitogenesis stimulated by ATP. No synergistic effect was found when PDBu and ATP were added together. Therefore, a dual mechanism, including both arachidonic acid metabolism and PKC, is involved in ATP-mediated mitogenesis in SMC. In addition, ATP acted synergistically with angiotensin II, phospholipase C, serotonin, or carbachol to stimulate DNA synthesis. Finally, the possible physiological significance of ATP as a mitogen in SMC was further studied. The effect of endothelin and heparin, which are released from endothelial cells, on ATP-dependent mitogenesis was investigated. Extracellular ATP acted synergistically with endothelin to stimulate a greater extent of [3H]thymidine incorporation than was seen with PDGF plus endothelin. Heparin, believed to have a regulatory role, partially inhibited the stimulation of DNA synthesis caused both by ATP and PDGF.(ABSTRACT TRUNCATED AT 400 WORDS)

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A receptor can be activated either by specific ligand-directed changes in conformation or by intrinsic, spontaneous conformational change. In the beta(2)-adrenergic receptor (AR) overexpression transgenic (TG4) murine heart, spontaneously activated beta(2)AR (beta(2)-R*) in the absence of ligands has been evidenced by elevated basal adenylyl cyclase activity and cardiac function. In the present study, we determined whether the signaling mediated by beta(2)-R* differs from that of a ligand-elicited beta(2)AR activation (beta(2)-LR*). In ventricular myocytes from TG4 mice, the properties of L-type Ca(2+) current (I(Ca)), a major effector of beta(2)-LR* signaling, was unaltered, despite a 2.5-fold increase in the basal cAMP level and a 1.9-fold increase in baseline contraction amplitude as compared with that of wild-type (WT) cells. Although the contractile response to beta(2)-R* in TG4 cells was abolished by a beta(2)AR inverse agonist, ICI118,551 (5 x 10(-7) M), or an inhibitory cAMP analog, Rp-CPT-cAMPS (10(-4) M), no change was detected in the simultaneously recorded I(Ca). These results suggest that the increase in basal cAMP due to beta(2)-R*, while increasing contraction amplitude, does not affect I(Ca) characteristics. In contrast, the beta(2)AR agonist, zinterol elicited a substantial augmentation of I(Ca) in both TG4 and WT cells (pertussis toxin-treated), indicating that L-type Ca(2+) channel in these cells can respond to ligand-directed signaling. Furthermore, forskolin, an adenylyl cyclase activator, elicited similar dose-dependent increase in I(Ca) amplitude in WT and TG4 cells, suggesting that the sensitivity of L-type Ca(2+) channel to cAMP-dependent modulation remains intact in TG4 cells. Thus, we conclude that beta(2)-R* bypasses I(Ca) to modulate contraction, and that beta(2)-LR* and beta(2)-R* exhibit different intracellular signaling and target protein specificity.