Stephen P. Halenda

Phospholipase D in cell signalling and its relationship to phospholipase C

Phospholipases C and D are phosphodiesterases which act on phospholipid head groups. Although the presence of these enzymes in living organisms has long been known, it is only recently that their role in cell signal transduction has been appreciated. The new developments on phospholipases D (PLD) are especially noteworthy, since these enzymes catalyze a novel pathway for second messenger generation. In a variety of mammalian cell systems, several biological or chemical agents have recently been shown to stimulate PLD activity. Depending on the system, activation of PLD has been suggested to be either dependent on, or independent of, Ca2+ and protein kinase C. PLD primarily hydrolyses phosphatidylcholine (PC) but phosphatidylinositol and phosphatidylethanolamine have also been reported as substrates. Different forms of endogenous PLD may also exist in cells. Exogenous addition of PLD causes alterations in cellular functions. In many instances, Ca2+ mobilizing agonists may stimulate both PLC and PLD pathways. Interestingly, several metabolites of these two enzymes are second messengers and are common to both pathways (e.g. phosphatidic acid, diglyceride). This has raised the issue of the interrelationship between these pathways. The regulation of either PLC or PLD by cellular components, e.g. guanine nucleotide binding proteins or protein kinases, is under intense investigation. These recent advances are providing novel information on the significance of phospholipase C and D mediated phospholipid turnover in cellular signalling. This review highlights some of these new discoveries and emerging issues, as well as challenges for future research on phospholipases.

ATP-stimulated smooth muscle cell proliferation requires independent ERK and PI3K signaling pathways

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.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.

Cell-Signaling Evidence for Adenosine Stimulation of Coronary Smooth Muscle Proliferation via the A1 Adenosine Receptor

For decades, it has been thought that adenosine is exclusively antimitogenic on vascular smooth muscles via the A2-type adenosine receptor. Recently, we have demonstrated that adenosine stimulates proliferation of porcine coronary artery smooth muscle cells (CASMC) through the A1 adenosine receptor. However, the cell-signaling mechanisms underlying A1 receptor–mediated CASMC proliferation in response to adenosine have not been defined. Here, we show that in cultured CASMC, adenosine stimulates phosphorylation of extracellular signal–regulated kinase (ERK), Jun N-terminal kinase (JNK), and AKT in a concentration- and time-dependent manner. This effect is fully mimicked by NECA (nonselective agonist), largely mimicked by CCPA (A1-selective agonist), weakly mimicked by 2-Cl-IB-MECA (A3-selective agonist), but not by CGS21680 (A2A-selective agonist), indicating that adenosine signals strongly via the A1 receptor to these mitogenic signaling pathways. This interpretation is supported by the finding that adenosine- and CCPA-induced phosphorylation of ERK, JNK, and AKT are inhibited by pertussis toxin (inactivator of Gi proteins) and by DPCPX (A1-selective antagonist), but not by SCH58261, MRS1706, and VUF5574 (A2A-, A2B-, and A3-selective antagonists, respectively). In addition, adenosine- and CCPA-induced phosphorylation of ERK, JNK, and AKT is inhibited, respectively, by U0126, PD98059 (mitogen-activated protein kinase kinase inhibitors), SP600125 (JNK kinase inhibitor), and wortmannin (phosphatidylinositol 3-kinase inhibitor). Furthermore, these kinase inhibitors abolish or diminish adenosine- and CCPA-induced increases in the rate of cellular DNA synthesis, bromodeoxyuridine incorporation, protein synthesis, and cell number. We conclude that adenosine activates the ERK, JNK, and phosphatidylinositol 3-kinase/AKT pathways primarily through the A1 receptor, leading to CASMC mitogenesis.

Novel Mitogenic Effect of Adenosine on Coronary Artery Smooth Muscle Cells Role for the A1 Adenosine Receptor

Adenosine is a vascular endothelial cell mitogen, but anti-mitogenic for aortic smooth muscle cells and fibroblasts when acting via the A2B 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 A1, A2A, A3, and barely detectable levels of A2B 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 A1 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 A1 receptor. When a cDNA encoding the porcine A1 receptor was cloned and expressed in COS-1 cells, A1 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 A1 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 A1 adenosine receptor, possibly contributing to the development of coronary artery disease.

PHOSPHOLIPASE D IN PLATELETS AND MEGAKARYOCYTIC CELLS

Phospholipase D (PLD) is stimulated in platelets by various agents. Phosphatidylcholine is the major substrate for PLD. This enzymatic pathway generates phosphatidic acid selectively. Guanine nucleotides also stimulate PLD in platelet membranes. Furthermore, tyrosine kinase may also be involved in platelet PLD regulation. It appears that multiple signals acting sequentially or in parallel converge on PLD. Among others, PLD has been proposed to play a role in platelet secretion and PLA2 regulation. PLD is also present in platelet percursor megakaryocytric cells and can be activated by platelet agonists. In these cells both PKC and G-proteins (e.g. Rho) may regulate PLD activity. The significance of PLD in megakaryocytes awaits investigation. These recent developments offer new avenues of research to further elucidate the biochemistry of platelet and megakaryocyte function.

Potentiation of arachidonic acid release by phorbol myristate acetate in platelets is not due to inhibition of arachidonic acid uptake or incorporation into phospholipids

Activators of protein kinase C, such as tumor-promoting phorbol esters (e.g., phorbol myristate acetate), mezerein, (-)-indolactam V and 1-oleoyl 2-acetoyl glycerol, potentiate arachidonic acid release caused by elevation of intracellular Ca2+ with ionophores. This action of protein kinase C-activators required protein phosphorylation, and was attributed to enhanced hydrolysis of phospholipids by phospholipase A2 (Halenda, et al. (1989) Biochemistry 28, 7356-7363). Recently Fuse et al. ((1989) J. Biol. Chem 264, 3890-3895) reported that the apparent enhanced release of arachidonate was actually due to inhibition of the processes of re-uptake and re-esterification of released arachidonic acid. They attributed this to loss of arachidonyl-CoA synthetase and arachidonyl-CoA lysophosphatide acyltransferase activities, which were measured in membranes obtained from phorbol myristate acetate-treated platelets. In this paper, we show that phorbol myristate acetate, at concentrations that strongly potentiate arachidonic acid release, does not inhibit either arachidonic acid uptake into platelets or its incorporation into specific phospholipids. Furthermore, the fatty acid 8,11,14-eicosatrienoic acid, a competitive substrate for arachidonyl-CoA synthetase, totally blocks arachidonic acid uptake into platelets, but, unlike phorbol myristate acetate, does not potentiate arachidonic acid release by Ca2+ ionophores. We conclude that the action of phorbol myristate acetate is to promote the process of arachidonic acid release by phospholipase A2.

Guanine nucleotides inhibit agonist-stimulated arachidonic acid release in both intact and saponin-permeabilized human platelets

The effects of guanine nucleotides on arachidonic acid (AA) release were studied in intact and saponin‐permeabilized human platelets. While GTP[S] itself caused a stimulation of AA release in permeabilized cells, GTP[S], GDP[S], GTP, ATP and other nucleotides inhibited AA release in response to thrombin and other agonists in intact, as well as permeabilized platelets. Inhibition of agonist‐stimulated AA release by nucleotides was partially attenuated by addition of ADP, and was abolished by prior stimulation of platelets to discharge the ADP‐containing dense granules. These results suggest: (i) that released ADP plays an important contributory role in agonist‐stimulated platelet AA release, and (ii) that guanine nucleotides can modulate platelet activation through an extracellular action which is distinct from their effects on G‐proteins.