Vascular smooth muscle cells respond to the purinergic agonist ATP by increasing intracellular calcium concentration and increasing the rate of cell proliferation. In many cells the extracellular signal-regulated kinase (ERK) cascade plays an important role in cellular proliferation. We have studied the effect of extracellular ATP on ERK activation and cell proliferation. ATP binding to a UTP-sensitive P2Y nucleotide receptor activates ERK1/ERK2 in a time- and dose-dependent manner in coronary artery smooth muscle cells (CASMC). ATP-induced activation of ERK1/ERK2 is dependent on the dual-specificity kinase mitogen-activated protein kinase/ERK kinase (i.e., MEK) but independent of phosphatidylinositol 3-kinase (PI3K) activity. We provide evidence that both ERK1/ERK2 and PI3K activities are required for CASMC proliferation. Thus ATP-stimulation of CASMC proliferation requires independent activation of both the ERK and PI3K signaling pathways.

The tumor-promoting phorbol diester 4,8-phorbol 12-myristate 13-acetate (PMA) inhibited mobilization of intracellular Ca2' in platelets by thrombin (also trypsin and 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine). PMA was effective over the same concentration range that activates protein kinase C in intact platelets; ICse vs. thrombin = 2 ng/ml, 3.4 nM: >90% inhibition at 10-20 ng/ml. Suppression of thrombin-induced Ca2' mobilization was evident within 30 sec of pretreatment with PMA and was essentially complete by 6-10 min at 10-20 ng of PMA per ml. Thrombin-induced secretion was initially accelerated in the presence of PMA, but after 1 min it was progressively inhibited when Ca2+ mobilization was depressed by >60%. PMA did not inhibit Ca2+ mobilization or secretion caused by A23187. Thrombininduced phosphatidylinositol 4,5-[32P]bisphosphate breakdown and [32Plphosphatidic acid production were also initially increased by PMA and then progressively depressed. Inhibition of thrombin-induced lipid metabolism required higher concentrations of PMA (IC5s = 10 ng/ml), and it was not overcome by A23187. 4a-Phorbol 12,13-didecanoate, which lacks the ability to activate protein kinase C, did not inhibit any responses to thrombin. These results suggest that activation of protein kinase C, which initially fosters secretion and aggregation, may subsequently exert negative feedback on the receptor-mediated mobilization of intracellular Ca2+ and the hydrolysis of phosphatidylinositol 4,5-bisphosphate.The stimulation of platelets to secrete and aggregate by agonists, such as thrombin and 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine (PAF-acether), is associated with a rapid increase of the cytoplasmic free Ca2l concentration ([Ca2+]i) (1,2) and the hydrolysis of the phosphodiester bond of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) (3, 4). The latter reaction is closely correlated with stimulusresponse coupling in many cell types, including platelets (5). The products of this reaction, 1,2-diacylglycerol (acyl2Gro) and myo-inositol 1,4,5-trisphosphate (Ins-1,4,5-P3), appear to play important roles in various cellular activation processes.Acyl2Gro acts as a second messenger to stimulate Ca2+/ phosphatidylserine-dependent protein kinase C by increasing the affinity of the enzyme-lipid complex for Ca2+, so that the enzyme can be activated even at resting levels of [Ca2+]i (6).Acyl2Gro is subsequently metabolized by further hydrolysis to release arachidonic acid and by phosphorylation to form phosphatidic acid (PtdOH). The action ofacyl2Gro on protein kinase C can be duplicated by tumor-promoting phorbol esters (7). Ins-1,4,5-P3, on the other hand, has been impli- (17) is mediated by cyclic AMP. In this paper we report that PMA inhibits Ca2+ mobilization, breakdown of Ptdlns-4,5-P2, formation of PA, and secretion caused by thrombin. This suggests that the protein kinase C pathway can exert feedback inhibition on receptor-mediated platelet activation subsequent to its initial role in promoting secretion. METH...

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Abstract-Adenosine is a vascular endothelial cell mitogen, but anti-mitogenic for aortic smooth muscle cells and fibroblasts when acting via the A 2B adenosine receptor. However, we show that adenosine increases porcine coronary artery smooth muscle cell (CASMC) number, cellular DNA content, protein synthesis, and PCNA staining. RT-PCR analysis indicates that porcine CASMC express A 1 , A 2A , A 3 , and barely detectable levels of A 2B receptor mRNAs. The mitogenic effect of adenosine is mimicked by NECA, CCPA, and R-PIA, but not by CGS21680 and 2-Cl-IB-MECA, and is inhibited by DPCPX, indicating a prominent role for the A 1 receptor. This interpretation is supported by the finding that adenosine-and CCPA-induced DNA synthesis is significantly inhibited by pertussis toxin, but substantially potentiated by PD81723, an allosteric enhancer of the A 1 receptor. When a cDNA encoding the porcine A 1 receptor was cloned and expressed in COS-1 cells, A 1 receptor pharmacology is confirmed. Anti-sense oligonucleotides to the cloned sequence dramatically suppress the mitogenic effect of adenosine and CCPA. Conversely, over-expression of the cloned A 1 receptor in CASMC increases adenosine-and CCPA-induced DNA synthesis. Furthermore, stimulation with adenosine or CCPA of intact coronary arteries in an organ culture model of vascular disease increases cellular DNA synthesis, which was abolished by DPCPX. We conclude that adenosine acts as a novel mitogen in porcine CASMC that express the A 1 adenosine receptor, possibly contributing to the development of coronary artery disease. Key Words: adenosine receptors Ⅲ coronary artery smooth muscle cells Ⅲ proliferation Ⅲ molecular cloning Ⅲ porcine T he diverse cellular actions of adenosine are mediated by a family of adenosine receptors (ARs), of which 4 subtypes (A 1 R, A 2A R, A 2B R, and A 3 R) have been cloned and pharmacologically characterized. 1,2 In general, the A 1 R and A 3 R are coupled with G i/o proteins, whose activation causes a decrease of intracellular cAMP. In contrast, activation of the G s -coupled A 2A R and A 2B R increases intracellular cAMP. 1,2 Thus, the end biological action of adenosine in a particular organ or cell population may depend on the relative expression level and signaling efficiency of the individual AR subtypes. 3 Adenosine, like many other vasodilators, historically has been thought to act as an inhibitor of the proliferation of vascular smooth muscle cells (VSMCs). This contention was supported by recent studies in cultured aortic VSMCs, showing that adenosine was antimitogenic through its activation of the A 2B R. 4 -8 To the best of our knowledge, however, current studies of the long-term effects of adenosine on VSMC proliferation have been limited to aortic smooth muscle.Given the heterogeneity of VSMCs 9 and the diversity of the expression profile of ARs in cells from different blood vessels, 10 it remains to be shown whether the antiproliferative action of adenosine can be extended to other VSMCs, particularly coronary artery smo...