Polymorphonuclear leukocytes (PMNL) possess relatively large amounts of 5-lipoxygenase, the enzyme catalyzing the sequential conversion of arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and LTA 4 (9). Upon cell activation, significant amounts of LTB 4 and its -oxidized metabolites 20-hydroxy-and 20-carboxy-LTB 4 are released into the extracellular milieu together with nonenzymatic breakdown products of LTA 4 , namely ⌬ 6 -trans-LTB 4 isomers and 5,6-dihydroxyeicosatetraenoic acids (5,6-diHETEs) (10 -12). Recent studies in complex organ systems (13-18) showed that perfusion of PMNL in the isolated lung or heart of the rabbit only caused a significant increase in the production of cysteinyl leukotrienes when PMNL were activated during the perfusion process. These data suggest that transcellular biosynthesis of cysteinyl leukotrienes might indeed be of physiopathological relevance when tight cell-cell interactions occur, such as during adhesion and diapedesis of PMNL through the microvascular endothelium of a functioning organ system.In light of these observations it was of interest to assess the relative amount of LTA 4 released from PMNL and therefore available for transcellular biosynthesis of cysteinyl leukotrienes, with respect to total LTA 4 synthesized. The release of LTA 4 into the extracellular milieu would remove this intermediate from intracellular LTA 4 hydrolase that catalyzes conversion of LTA 4 into LTB 4 . Intracellular LTB 4 can be further metabolized by a specific cytochrome P-450 to 20-hydroxy-LTB 4 and 20-carboxy-LTB 4 (19). The extracellular (released) LTA 4 will react with water with a half-life lower than 30 s (20) to yield the nonenzymatic products, ⌬ 6 -trans-LTB 4 , ⌬ 6 -trans-12-epi-LTB 4 , and 5,6-dihydroxyeicosatetraenoic acid isomers. But PMNL are able to take up exogenously added LTA 4 (21) and metabolize it into LTB 4 , thus reducing the fraction of released LTA 4 that is actually available for transcellular metabolism (or nonenzymatic hydrolysis).In the present study experiments were designed to test the effect of dilution on the quantitative profile of LTA 4 metabolites produced after challenge with the Ca 2ϩ ionophore A23187. The hypothesis that in diluted cell preparations LTA 4 would have less chance of being reabsorbed and metabolized by vicinal PMNL has been tested. In a previous study, Cluzel et al. (22) showed that the use of diluted cell suspensions provided important information concerning the amount of platelet activating factor and LTB 4 released by PMNL. Using a similar approach, we provide evidence that significant transcellular * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.§ To whom correspondence should be addressed. Tel.: 39-2-20488312; Fax 39-2-29404961. 1 The abbreviations used are: 5-LO, 5-lipoxygenase; PMNL, polymorphonuclear leukocytes; LTA, leukotriene A; RP-HP...

Abstract-Recent studies have suggested that part of the vasorelaxation caused by nifedipine, a 1,4-dihydropyridine Ca 2ϩ antagonist, depends on the endothelium. To study the effect of endothelium-dependent vasorelaxation, the release of NO and superoxide (O 2 Ϫ ) in the presence of nifedipine in isolated cultured rabbit endothelial cells was measured. Highly sensitive electrochemical microsensors were placed onto the cell membrane, and the kinetics of NO and O 2 Ϫ were measured simultaneously with time resolutions of 0.1 and 0.05 ms, respectively. Nifedipine at its therapeutical concentrations stimulated NO release and scavenged O 2 Ϫ in endothelial cells. The linear relationship between NO concentration and nifedipine concentration was observed in the range between 0.01 and 1 nmol/L. NO concentration reached a maximum of 200Ϯ10 nmol/L at 1.2 nmol/L of nifedipine. The NO concentration was Ϸ50% and 30% of the concentration measured in the presence of receptor-dependent (acetylcholine) and the receptor-independent (Ca 2ϩ ionophore A23187) NO synthase (eNOS) agonists, respectively. NO release stimulated by eNOS agonists was followed by the generation of the NO scavenger superoxide. The concentration of O 2 Ϫ was significantly lower after stimulation with nifedipine (peak 5Ϯ0.5 nmol/L) than after stimulation with acetylcholine (15Ϯ1 nmol/L) and Ca 2ϩ ionophore (25Ϯ1 nmol/L). The average rate of NO release by nifedipine is relatively slow (17 nmol/L per second). This is in sharp contrast to the fast rate of NO release by acetylcholine and Ca 2ϩ ionophore (40 and 300 nmol/L per second, respectively). These experiments show that nifedipine, apart from its well-known Ca 2ϩ antagonistic properties in vascular smooth muscle cells, stimulates the release of significant concentration of NO in endothelium and also preserves NO concentration. Both these effects may be beneficial in the treatment of hypertension. Key Words: nifedipine Ⅲ endothelium Ⅲ nitric oxide Ⅲ superoxide T he 1,4-dihydropyridine known as nifedipine is a commonly used cardiovascular drug that is applied for the control of angina, hypertension, and other vascular diseases. 1 Nifedipine can be viewed as a prototypical drug for several generations of 1,4-dihydropyridine antagonists and activators, which potently activate L-type Ca 2ϩ channels, thus modulating the vascular tone via the Ca 2ϩ influx into smooth muscle cells.It has been also shown that NO is the endothelium-derived relaxing factor. 2 NO is normally generated in the circulation via stimulation of vascular endothelium NO synthase (eNOS) by endothelial mechanochemical receptors. A variety of stimuli, including increased vascular flow, and pharmacological agents, such as acetylcholine (ACh), bradykinin, substance P, ATP, and histamine, produce vascular relaxation by the release of NO. 2 Many vasodilator drugs are thought to act predominantly through either endothelium-dependent or -independent pathways. There is conflicting evidence as to whether 1,4-dihydropyridines may alter the synthesis or relea...

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