No abstract

Leukotriene B4 (5S,12R-dihydroxy-6,14-cis,8,10-trans-eicosatetraenoic acid, LTB4) is released from neutrophils exposed to calcium ionophores. To determine whether LTB4 might be produced by ligand-receptor interactions at the plasmalemma, we treated human neutrophils with serum-treated zymosan (STZ), heat-aggregated IgG and fMet-Leu-Phe (fMLP), agonists at the C3b, Fc and fMLP receptors respectively. STZ (10 mg/ml) provoked the formation of barely detectable amounts of LTB4 (0.74 ng/10(7) cells); no omega-oxidized metabolites of LTB4 were found. Adding 10 microM-arachidonate did not significantly increase production of LTB4 or its metabolites. Addition of 50 microM-arachidonate (an amount which activates protein kinase C) before STZ caused a 40-fold increase in the quantity of LTB4 and its omega-oxidation products. Neither phorbol myristate acetate (PMA, 200 ng/ml) nor linoleic acid (50 microM), also activators of protein kinase C, augmented generation of LTB4 by cells stimulated with STZ. Neither fMLP (10(-6) M) nor aggregated IgG (0.3 mg/ml) induced LTB4 formation (less than 0.01 ng/10(7) cells). Moreover, cells exposed to STZ, fMLP, or IgG did not form all-trans-LTB4 or 5-hydroxyeicosatetraenoic acid; their failure to make LTB4 was therefore due to inactivity of neutrophil 5-lipoxygenase. However, adding 50 microM-arachidonate to neutrophil suspensions before fMLP or IgG triggered LTB4 production, the majority of which was metabolized to its omega-oxidized products (fMLP, 20.2 ng/10(7) cells; IgG, 17.1 ng/10(7) cells). The data show that neutrophils exposed to agonists at defined cell-surface receptors produce significant quantities of LTB4 only when treated with non-physiological concentrations of arachidonate.

Neutrophils respond to a variety of stimuli by generating superoxide anion, degranulating, and aggregating. Because it has been suggested that fusion of granules with the plasmalemma (degranulation) is necessary for aggregation and superoxide anion generation, we have tested whether these responses can be demonstrated in "neutrophilic cytoplasts" (granule-free vesicles of cytoplasm enclosed by plasmalemma). When examined by electron microscopy, cytoplasts were found to be approximately 4 ,um in diameter and essentially granule free. Cytoplasts exposed to fMet-Leu-Phe (0.1 FIM) generated superoxide anion after a lag of 16 sec but released no detectable 3glucuronidase, lysozyme, or elastase. Aggregation of cytoplasts, as measured by changes in light transmission, was also activated by fMet-Leu-Phe; no lag period was observed. Electron microscopy of the aggregates demonstrated clusters of cytoplasts with a scalloped appearance. Superoxide anion generation and aggregation of cytoplasts were also activated by phorbol 12-myristate 13-acetate, concanavalin A, and leukotriene B4. Exposure of cytoplasts to the dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] led to dye uptake and enhancement of fluorescence, implying that the vesicles were sealed and maintained a membrane potential across the plasmalemma. Exposure of DiOC6(3)-loaded cytoplasts to fMet-LeuPhe and PMA caused a rapid loss of dye fluorescence that was not inhibited by CN-, compatible with their lack of mitochondria. Exposure of dye-loaded cytoplasts to concanavalin A or leukotriene B4 caused an increase in fluorescence-i.e., a hyperpolarization. These results demonstrate that degranulation is not a prerequisite for aggregation or superoxide anion generation. (4), and cell-cell aggregation (5-8). There has also been controversy whether the 02 generating system is associated with the granules, and therefore dependent on degranulation (9, 10), or whether it is located in the plasmalemma.Stimuli that activate the neutrophil activate most or all of these responses, so that analysis of these discrete functions has proven difficult. To determine whether degranulation is essential to aggregation, we have prepared organelle-free "neutrophilic cytoplasts," according to the method of Roos et at (11). These cytoplasts are devoid of granules and are therefore incapable of degranulation. Cytoplasts have been shown to possess receptors for complement component C3b and phorbol 12-myristate 13-acetate (PMA) and to be capable of undergoing phagocytosis, 02 consumption, 02 generation, and H202 production (11). These particles are unique in having an intact receptor-response coupling mechanism for each of these functions.Cytoplasts also provide an opportunity to study the coupling process between receptor occupancy and the physiological responses of aggregation and 02 generation. Ionic movements have been shown to play an important role in stimulus-response coupling (12). Examination of ionic movements across the plasmalemma has been difficult due to interference by ...

No abstract

Percutaneous coronary intervention (PCI) has become a mainstay in the treatment of patients with coronary artery disease. Currently, more than one million coronary angioplasty and stent implantation procedures are performed annually. Although increasingly complex lesions and higher risk patients are being successfully treated percutaneously, restenosis and disease progression continue to cause significant morbidity. Restenosis occurs in approximately one-third of patients, one-half of who remain asymptomatic, while disease progression occurs at rates approaching 7% per year. Despite technological advances, unadjusted mortality rates have actually increased since the mid-1980s, and the current annual risk of a major adverse cardiac event following PCI is 5% to 7%. Although randomized clinical trials are needed to more definitively show a benefit, when performed six or more months following PCI, myocardial perfusion imaging reliably identifies patients most at risk of a poor long-term outcome. Directed reintervention can have a salutary impact on the prognosis of these patients. In view of recent data showing a positive impact of imaging and reintervention in patients after PCI, current guidelines should be reassessed.