Alpha 1-adrenergic receptor-mediated increase in the mass of phosphatidic acid and 1,2-diacylglycerol in ischemic rat heart.
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OBJECTIVE: 1,2-Diacylglycerol (1,2-DAG) and phosphatidic acid (PA) are produced by phospholipase C and D activity and play a key role as second messengers in receptor-mediated signal transduction. So far, little is known about alterations of endogenous 1,2-DAG and PA production during myocardial ischemia. METHODS: Rat isolated perfused hearts were subjected to global ischemia, total lipids were extracted, and separated by thin-layer chromatography. The mass of PA and 1,2-DAG were quantified using laserdensitometric analysis of visualized lipids. RESULTS: Compared to normoxic control values (1,2-DAG 713 +/- 45 ng/mg protein, PA 171 +/- 11 ng/mg protein), the myocardial content of 1,2-DAG and PA was unaltered after 10 min of ischemia. Prolonged myocardial ischemia (20 min), however, which was accompanied by marked overflow of endogenous norepinephrine, significantly increased the mass of both second messengers (1,2-DAG 1062 +/- 100 ng/mg protein, PA 340 +/- 29 ng/mg protein). The increase in PA and 1,2-DAG in response to ischemia was abolished by inhibition of ischemia-induced norepinephrine release as well as by alpha1-adrenergic blockade but unaffected by beta-adrenergic blockade. While inhibition of diacylglycerol kinase did not affect ischemia-induced increase in PA and 1,2-DAG, inhibition of phosphatidylinositol-specific phospholipase C activity significantly suppressed ischemia-induced increase in 1,2-DAG but did not affect endogenous production of PA indicating phospholipase C-independent formation of PA and activation of both, phospholipase C and D, in the ischemic heart. CONCLUSIONS: Ischemia elicits an alpha1-adrenergic receptor-mediated increase in the mass of myocardial PA and 1,2-DAG. The increase in endogenous PA is suggested to be due to the activation of myocardial phospholipase D, whereas 1,2-DAG is formed predominantly by activation of phospholipase C in the ischemic heart. (+info)
Nitric oxide inhibits angiotensin II-induced activation of the calcium-sensitive tyrosine kinase proline-rich tyrosine kinase 2 without affecting epidermal growth factor receptor transactivation.
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In a previous study, we showed that nitric oxide donors and N-acetylcysteine, either alone or in combination, inhibited the activation of several mitogen-activated protein kinases by angiotensin II in rat cardiac fibroblasts (Wang, D., Yu, X., and Brecher, P. (1998) J. Biol. Chem. 273, 33027-33034). In the present study, we have focused on the mechanism by which nitric oxide exerts this effect on the activation of extracellular signal-regulated kinase (ERK). We contrasted the effects of nitric oxide on ERK activation by angiotensin II and epidermal growth factor (EGF), since the transactivation of the EGF receptor has been implicated as a response to angiotensin II. We found that nitric oxide inhibited ERK activation by angiotensin II but did not inhibit the relatively slight but significant transactivation of the EGF receptor by angiotensin II. The tyrphostin AG1478, known to inhibit EGF receptor phosphorylation, also inhibited the angiotensin II and EGF-induced activation of ERK, the phosphorylation of the EGF receptor, and the subsequent association of Shc and Grb2. Nitric oxide did not affect either EGF receptor phosphorylation or Shc-Grb2 activation induced by either Ang II or EGF. However, the activation of the calcium-sensitive tyrosine kinase PYK2, which occurred in response to angiotensin II, but not EGF, was inhibited by nitric oxide. The data suggested that PYK2 activation may be an important inhibitory site in signaling pathways affected by nitric oxide. (+info)
Brief myocardial ischemia attenuates platelet thrombosis in remote, damaged, and stenotic carotid arteries.
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BACKGROUND: Brief antecedent periods of coronary artery occlusion improve subsequent vessel patency in damaged and stenotic coronary arteries via release of adenosine from ischemic/reperfused myocardium and resultant adenosine receptor stimulation. However, the site of receptor stimulation-circulating blood-borne elements (ie, platelets) versus vessel-wall components of the culprit artery-remains unclear. If platelet adenosine receptors are involved, then the benefits of brief coronary occlusion (1) should be manifested systemically and improve patency at a remote site and (2) should be inhibited by an antagonist of adenosine A(2) receptors, whereas, in contrast, (3) brief vascular occlusion not associated with appreciable adenosine release should be ineffective in improving vessel patency. METHODS AND RESULTS: In Protocol 1, anesthetized rabbits received 5 minutes of transient coronary occlusion, 5 minutes of transient bilateral carotid occlusion (purported to cause negligible adenosine release from the brain), or no intervention. All rabbits then underwent injury plus stenosis of the left carotid artery, resulting in repeated cyclic variations in carotid blood flow (CFVs). Carotid patency during the initial 2 hours after stenosis (assessed by quantifying the nadir of the CFVs and area of the flow-time profile) was significantly enhanced with antecedent coronary-but not carotid-occlusion versus controls. In Protocol 2, improvement in carotid patency after brief coronary occlusion was corroborated in anesthetized dogs. However, the benefits of brief coronary occlusion were abrogated by the A(2)/A(1) antagonist CGS 15943. CONCLUSIONS: Brief antecedent coronary artery occlusion enhanced vessel patency in remote, damaged, and stenotic carotid arteries, largely due to adenosine receptor stimulation on circulating elements. (+info)
Intracellular movement of green fluorescent protein-tagged phosphatidylinositol 3-kinase in response to growth factor receptor signaling.
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Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase which has been implicated in mitogenesis, protein trafficking, inhibition of apoptosis, and integrin and actin functions. Here we show using a green fluorescent protein-tagged p85 subunit that phosphatidylinositol 3-kinase is distributed throughout the cytoplasm and is localized to focal adhesion complexes in resting NIH-3T3, A431, and MCF-7 cells. Ligand stimulation of an epidermal growth factor receptor/c-erbB-3 chimera expressed in these cells results in a redistribution of p85 to the cell membrane which is independent of the catalytic activity of the enzyme and the integrity of the actin cytoskeleton. The movement is, however, dependent on the phosphorylation status of the erbB-3 chimera. Using rhodamine-labeled epidermal growth factor we show that the phosphatidylinositol 3-kinase and the receptors colocalize in discrete patches on the cell surface. Low concentrations of ligand cause patching only at the periphery of the cells, whereas at high concentrations patches were seen over the whole cell surface. Using green fluorescent protein-tagged fragments of p85 we show that binding to the receptor requires the NH(2)-terminal part of the protein as well as its SH2 domains. (+info)
N-acetylation of the heterocyclic amine batracylin by human liver.
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Batracylin (8-aminoisoindolo[1,2-b]quinazolin-12(10 H)-one; BAT) is a heterocyclic amine that exhibits antitumor activity in a number of in vivo and in vitro models. The acetyl product has been implicated in BAT toxicity in animals, cells, and bacteria. The ability of human N-acetyltransferase (NAT) to form this product was investigated. Nine human liver samples were analyzed for NAT1 and NAT2 genotypes. Seven of the samples possessed at least one NAT1*4 allele. Three samples contained one or more NAT2*4 allele and were classified as rapid acetylators. The remaining six had two alleles associated with the slow phenotype. NAT activities were evaluated with BAT, sulfamethazine (SMZ), a preferential substrate for human NAT2, and p-aminobenzoic acid, a substrate for NAT1. BAT activities in the nine donor samples ranged from 14.9 to 0.56 nmol/min/mg. The mean apparent K(m) values in rapid acetylators for BAT, SMZ, and p-aminobenzoic acid were 6.59 +/- 3.21, 278 +/- 69.4, and 31.2 +/- 12.5 microM, respectively. The apparent K(m) values for slow acetylators did not differ from the rapid acetylator phenotype. However, a significant difference in the apparent V(max) for BAT and SMZ was observed between rapid and slow acetylators. Comparing the apparent intrinsic clearance (V(max)/K(m)) for BAT and SMZ, a significant correlation (r(2) = 0.97, p <.001) was observed. These data demonstrate that BAT N-acetylation is similar to SMZ, and suggests that BAT is a preferential substrate for human NAT2. Thus, rapid acetylators would be more likely to develop toxicity when exposed to this drug. (+info)
Inhibition of bladder carcinoma angiogenesis, stromal support, and tumor growth by halofuginone.
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We have previously demonstrated that halofuginone, a widely used alkaloid coccidiostat, is a potent inhibitor of collagen alpha1(I) and matrix metalloproteinase 2 gene expression. Halofuginone also suppresses extracellular matrix deposition and cell proliferation. We investigated the effect of halofuginone on transplantable and chemically induced mouse bladder carcinoma. In both systems, oral administration of halofuginone resulted in a profound anticancerous effect, even when the treatment was initiated at advanced stages of tumor development. Although halofuginone failed to prevent proliferative preneoplastic alterations in the bladder epithelium, it inhibited further progression of the chemically induced tumor into a malignant invasive stage. Histological examination and in situ analysis of the tumor tissue revealed a marked decrease in blood vessel density and in both collagen alpha1(I) and H19 gene expression. H19 is regarded as an early marker of bladder carcinoma. The antiangiogenic effect of halofuginone was also demonstrated by inhibition of microvessel formation in vitro. We attribute the profound antitumoral effect of halofuginone to its combined inhibition of the tumor stromal support, vascularization, invasiveness, and cell proliferation. (+info)
Potentiation of the cytotoxicity of thymidylate synthase (TS) inhibitors by dipyridamole analogues with reduced alpha1-acid glycoprotein binding.
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Dipyridamole has been shown to enhance the in vitro activity of antimetabolite anticancer drugs through the inhibition of nucleoside transport. However, the clinical potential of dipyridamole has not been realized because of the avid binding of the drug to the plasma protein alpha1-acid glycoprotein (AGP). Dipyridamole analogues that retain potent nucleoside transport inhibitory activity in the presence of AGP are described and their ability to enhance the growth inhibitory and cytotoxic effects of thymidylate synthase (TS) inhibitors has been evaluated. Three dipyridamole analogues (NU3026, NU3059 and NU3060) were shown to enhance the growth inhibitory activity of the TS inhibitor CB3717 and block thymidine rescue in L1210 cells. The extent of potentiation at a fixed analogue concentration (10 microM) was related to the potency of inhibition of thymidine uptake. A further analogue, NU3076, was identified, which was more potent than dipyridamole with a Ki value for inhibition of thymidine uptake of 0.1 microM compared to 0.28 microM for dipyridamole. In marked contrast to dipyridamole, inhibition of thymidine uptake by NU3076 was not significantly affected by the presence of AGP (5 mg ml(-1)). NU3076 and dipyridamole produced equivalent potentiation of the cytotoxicity of the non-classical antifolate TS inhibitor, nolatrexed, in L1210 cells with both compounds significantly reducing the LC90, by > threefold in the absence of salvageable thymidine. Thymidine rescue of L1210 cells from nolatrexed cytotoxicity was partially blocked by both 1 microM NU3076 and 1 microM dipyridamole. NU3076 also caused a significant potentiation of FU cytotoxicity in L1210 cells. These studies demonstrate that nucleoside transport inhibition can be maintained in the absence of AGP binding with the dipyridamole pharmacophore and that such analogues can enhance the cytotoxicity of TS inhibitors. (+info)
Inhibition of matrix metalloproteinase-2 expression and bladder carcinoma metastasis by halofuginone.
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Matrix metalloproteinase-2 (MMP-2) plays a critical role in tumor cell invasion and metastasis. Inhibitors of this enzyme effectively suppress tumor metastasis in experimental animals and are currently being tested in clinical trials. MMP-2 transcriptional regulation is a part of a delicate balance between the expression of various extracellular matrix (ECM) constituents and ECM degrading enzymes. Halofuginone, a low-molecular-weight quinazolinone alkaloid, is a potent inhibitor of collagen type alpha1 (I) gene expression and ECM deposition. We now report that expression of the MMP-2 gene by murine (MBT2-t50) and human (5637) bladder carcinoma cells is highly susceptible to inhibition by halofuginone. Fifty percent inhibition was obtained in the presence of as little as 50 ng/ml halofuginone. This inhibition is due to an effect of halofuginone on the activity of the MMP-2 promoter, as indicated by a pronounced suppression of chloramphenicol acetyltransferase activity driven by the MMP-2 promoter in transfected MBT2 cells. There was no effect on chloramphenicol acetyltransferase activity driven by SV40 promoter in these cells. Halofuginone-treated cells failed to invade through reconstituted basement-membrane (Matrigel) coated filters, in accordance with the inhibition of MMP-2 gene expression. A marked reduction (80-90%) in the lung colonization of MBT2 bladder carcinoma cells was obtained after the i.v. inoculation of halofuginone-treated cells as compared with the high metastatic activity exhibited by control untreated cells. Under the same conditions, there was almost no effect of halofuginone on the rate of MBT2 cell proliferation. These results indicate that the potent antimetastatic activity of halofuginone is due primarily to a transcriptional suppression of the MMP-2 gene, which results in a decreased enzymatic activity, matrix degradation, and tumor cell extravasation. This is the first description, to our knowledge, of a drug that inhibits experimental metastasis through the inhibition of MMP-2 at the transcriptional level. Combined with its known inhibitory effect on collagen synthesis and ECM deposition, halofuginone is expected to exert a profound anticancerous effect by inhibiting both the primary tumor stromal support and metastatic spread. (+info)