Abstract
The effect of thrombin receptor activation on monocyte conformation was evaluated using the human monocyte cell line, THP-1, and the thrombin mimetic peptide, Trap-14. Treatment of THP-1 cells with Trap-14 induced rapid rounding of ameboid cells adherent to fibronectin-coated slides, whereas cell rounding was abrogated in the presence of the nitric oxide synthase inhibitor, NG-nitro-l-arginine or the endothelin B receptor antagonist, BQ-788. Endothelin-1 (ET-1) levels in the culture supernatant increased markedly within minutes of Trap-14 exposure with a concomitant loss in cellular ET-1 immunoreactivity. Importantly, loss of ET-1 immunoreactivity was blocked by pretreatment with the vesicle translocation inhibitor, nocodazole. Trap-14 potently induced the release of NO from THP-1 cells, whereas NO release was ablated by preincubation with BQ-788. These data demonstrate that thrombin receptor activation may inhibit cellular spreading as a result of autocrine ET-1 release and subsequent endothelin B receptor-dependent NO production, and suggest that initial exposure of inflammatory cells to thrombin may limit cellular activation and recruitment.
Inflammation involves the recruitment of leukocytes to an injured area regulated by cytokines and inflammatory mediators. Critical changes occur in the morphology and adhesivity of leukocytes that allow them to leave the circulation and collect at sites of injury. Aberrant modulation or insufficient regulation of this process has been implicated in the progression of inflammatory disease (1, 2). Thrombin is generated rapidly following injury, and increasing evidence suggests an important role for thrombin as an inflammatory mediator in addition to its classical role in coagulation (3, 4). Thrombin increases endothelial cell interactions with monocytes and neutrophils via up-regulation of cellular adhesion molecules (5, 6) and has been shown to be chemotactic for monocytes and neutrophils (7, 8). The capacity of thrombin to modulate leukocyte properties such as conformation and motility suggests that it may be of critical importance in the regulation of inflammation. Monocyte interactions with thrombin and other members of the coagulation cascade suggest connectivity between inflammatory and coagulative processes (9). Studies have demonstrated up-regulated cytokine expression and cytoskeletal changes in monocytes in response to thrombin or thrombin mimetic peptides (10, 11). Few studies, however, address the effect of thrombin on monocyte conformation. Change in cell conformation is crucial to the process of monocyte activation, and it is an accepted parameter for the designation of monocyte activity. Activated monocytes characteristically spread and extend pseudopodia to assume an ameboid conformation, whereas inactivated cells lack such processes and appear round. In this study, we have evaluated the effect of thrombin receptor activation on the conformation of the human monocytic cell line, THP-1. We demonstrate that thrombin receptor activation inhibits monocyte spreading on fibronectin-coated slides and induces the rapid withdrawal of pseudopodial processes, shifting the cellular conformation from ameboid to round. Cell rounding is abrogated in the presence of nitric oxide synthase (NOS)3 inhibitors, suggesting a critical role for NO in the modulation of cell conformation following thrombin receptor activation. These observations are consistent with previous reports that demonstrate the ability of NO to induce rounding of immune cells (12).
Methods and Materials
Reagents
The thrombin receptor agonist peptide, Trap-14 (SFLLRNPNDKYEPF-amide), was synthesized on an Advanced ChemTech Model 90 peptide synthesizer exactly as described (13). Peptides were cleaved with a mixture of 90% trifluoroacetic acid, 5% 1.2 ethanedithiol, 4% water, and 1% thioanisol, and purified by C18 column chromatography before use in assays. Endothelin receptor antagonists ETA selective, BQ-123, and ETB selective, BQ-788, were purchased from Peptides International (Louisville, KY). ET-1-specific enzyme immunoassay (EIA) kit was obtained from Peninsula Laboratories (Belmont, CA), whereas all other reagents were from Sigma (St. Louis, MO). Trap-14 and ET receptor antagonists were dissolved in physiologic salt solution (PSS) (130 mmol/L NaCl, 4.7 mmol/L KCl, 1.17 mmol/LMgSO4, 1.18 mmol/L KH2PO4, 14.9 mmol/L NaHCO3, 5.5 mmol/L dextrose, 0.03 mmol/L EDTA, 2.5 mmol/L CaCl2) or RPMI 1640 before use in assays.
Monoblastic cell line
The human monoblastic cell line, THP-1, was obtained from American Type Culture Collection (Rockville, MD) and cultured in RPMI 1640 (Sigma) supplemented with 10% FBS (Life Technologies, Grand Island, NY).
NO release
THP-1 cells were washed extensively with PSS just before use in assays. Approximately 106 THP-1 cells were placed in 5 ml of aerated (95% O2/5% CO2) PSS buffer and maintained at 24°C, followed by evaluation of NO release using an NO-specific amperometric probe, as described previously (14, 15). The probe tip was immersed into the cell suspension, and following establishment of baseline NO release (approximately 5 min), Trap-14 was introduced in the medium and the concentration of NO gas in solution was recorded by a computer-assisted data acquisition system at a sampling rate of 1/s.
ET-1 release
Approximately 106 THP-1 cells were placed in 500 μl PSS and maintained at 24°C, followed by addition of vehicle (PSS buffer) or Trap-14. ET-1 release observed following 5-min stimulation with Trap-14 or vehicle was evaluated using an ET-1-specific EIA kit (Peninsula Laboratories).
Immunodetection of ET-1 and NOS
THP-1 cells were washed and suspended in PSS and allowed to adhere to Vectabond (Vector Laboratories, Burlingame, CA)-treated glass slides. Cells were fixed with 3.7% formaldehyde for 10 min. After extensive washing, the cells were permeabilized with 0.1% saponin for 15 min for detection of ET-1 or with 0.1% Triton-X for 5 min for detection of constitutive NOS (cNOS). Cells were incubated with mouse anti-human Ab to ET-1 (Peninsula Laboratories), or rabbit anti-human cNOS (Signal Transduction Labs, Lexington, KY) for 1 h. After extensive washing with BSA-PSS, cells were incubated with rhodamine-labeled goat anti-mouse or goat anti-rabbit secondary Ab (Sigma) for 30 min. After washing, cells were postfixed with 3.7% formaldehyde for 10 min. Following extensive washing, cells were mounted with Fluoromount-G and coverslipped. Purified mouse or rabbit IgG was used as negative control. Immunofluorescence was evaluated by quantitative confocal microscopy (Meridian Ultima, Okemos, MI) using a ×40 objective and a pinhole of 1600 to evaluate total cellular immunofluorescence for ET-1. For evaluation of cNOS immunoreactivity, the cell was optically sectioned and cNOS immunofluorescence was determined using a ×100 objective and a pinhole setting of 40 μM to provide an axial resolution of approximately 0.3 μM.
Evaluation of cellular conformation
THP-1 cells were washed extensively with RPMI 1640 and diluted to a concentration of 106 cells/ml. Cell suspensions were added to glass slides coated with 5 mg/ml fibronectin, whereupon they were allowed to spread at 37°C for 1 h. After addition of agonist, changes in cell conformation were evaluated at 15-min intervals, as described previously (12, 16), using a ×40 objective and Nomarski optics (Nikon, TE-300). The degree of conformational change was based on measurements of cellular area and perimeter using image analysis software (Image Analytics, Hauppauge, NY). Changes in cellular conformation that ranged from inactive-rounded to active-ameboid were determined by measurement of cellular area and perimeter and were mathematically expressed by use of the form factor (FF) formula (4 × π A)/P2, in which A is the area of the cell and P is the perimeter of that given cell (12). The lower the FF value, the higher the cellular perimeter and the more ameboid the cellular shape. Changes in cell conformation induced by thrombin receptor activation were evaluated by addition of 100 μM Trap-14 or vehicle alone (RPMI 1640) to cells once they became ameboid, and changes in cell shape were followed for 1 h. For experiments in the presence of ET receptor antagonists or NOS inhibitors, these agents were added to the cell suspensions 15 min before addition of Trap-14 and cell conformation was followed as described above.
Statistics
Data were evaluated using analysis of variance in which a p value of <0.05 was considered to be statistically significant. Multiple comparisons were adjusted using the Bonferroni multirange test, with α set at 0.005.
Results
Thrombin receptor activation triggers NO-dependent rounding of THP-1 cells
To evaluate the effects of thrombin receptor activation on cellular conformation, THP-1 cells adherent to fibronectin-coated slides were treated with vehicle (RPMI 1640) or Trap-14, and cell shape was visualized 1 h after exposure using computer-assisted phase-contrast imaging. Untreated control THP-1 cells (Fig. 1⇓A) were ameboid, whereas exposure to Trap-14 (Fig. 1⇓B) resulted in marked rounding of the cells. Cellular rounding induced by thrombin was impaired by pretreatment with the nonselective NOS inhibitor l-NNA (Fig. 1⇓C). Form factor analysis was employed to evaluate the extent of cell rounding. Form factor analysis describes the similarity of a cell to a circle, with rounded cells having a value of greater than 0.7 and ameboid cells having a value less than 0.5. The form factor values of THP-1 cells treated with 100 μM Trap-14 were increased significantly over a period of 1 h relative to that of vehicle-treated control cells, indicating a potent rounding of THP-1 cells in response to Trap-14 (Fig. 1⇓D). Cell rounding was inhibited significantly in the presence of l-NNA, suggesting that Trap-14-induced rounding of THP-1 cells may be mediated by NO.
Rounding of THP-1 cells in response to Trap-14. THP-1 cells were allowed to adhere and spread on fibronectin-coated glass slides for 1 h, after which the cells were exposed to RPMI 1640 (A) or 100 μM Trap-14 (B and C). Changes in cell conformation were evaluated over 1 h using Nomarski optics and computerized image analysis software (Image Analytics). Observed at 1 h after stimulation, Trap-14 induced significant rounding of ameboid THP-1 cells (B) when compared with control cells (A). This rounding was blocked by pretreatment with the NOS inhibitor, l-NNA (C). Numerical analysis of conformation over the course of the experiment (D) indicates that cell rounding in response to Trap-14 was rapid, with significant changes observed within minutes (∗, p < 0.05; ∗∗, p < 0.001).
THP-1 cells release NO in response to Trap-14 stimulation
To evaluate the role of NO release in Trap-14-induced cell rounding, NO release was measured using an amperometric probe. Stimulation of THP-1 cells with Trap-14 resulted in a marked increase in NO release, 43 ± 6 nM, that reached maximum within 5 min and returned to baseline levels within 10 min following stimulation. Trap-14 activation of NOS was potently abrogated by pretreatment with the NOS inhibitor l-NNA, 8 ± 3 nM (Fig. 2⇓, A and B). The transient nature of NO release induced by Trap-14 is consistent with that observed following activation of cNOS and is similar to that observed following Trap-14 or thrombin stimulation of vascular endothelium (14). We evaluated the presence of cNOS using a selective mAb. Unstimulated THP-1 cells exhibited significant cNOS immunofluorescence localized primarily to the cell periphery (Fig. 3⇓A). The expression of cNOS appeared to be clustered to areas of active cytoplasmic extension. These data are consistent with previous localization studies of cNOS in endothelial cells, in which cNOS was predominantly localized to plasmalemmal caveolae (17). THP-1 cells stimulated with Trap-14 for 30 min resulted in a marked alteration in cNOS localization with significant dispersion of cNOS throughout the cell (Fig. 3⇓B). The role of cNOS activation in Trap-14-induced NO release is further supported by the observation that rounding of THP-1 cells by Trap-14 was inhibited by pretreatment with the nonselective NOS inhibitor l-NNA, FF = 0.28 ± 0.06, whereas pretreatment with the iNOS-selective inhibitor aminoguanidine, FF = 0.74 ± 0.09, was without effect (Fig. 4⇓).
Real time evaluation of NO release from THP-1 cells. THP-1 cells were stimulated with 100 μM Trap-14, and NO release was evaluated in real time using an NO-specific amperometric probe (World Precision Instruments, Sarasota, FL). A rapid increase in NO levels was detected within 2 min of Trap-14 exposure to THP-1 cells, whereas Trap-14-stimulated NO release in the presence of 10 μM l-NNA was markedly attenuated (A). Analysis of peak values of NO release demonstrated that Trap-14-induced NO release was reduced significantly in the presence of l-NNA (B) (∗, p < 0.05, ANOVA).
Immunodetection of cNOS in THP-1 cells. THP-1 cells were fixed and permeabilized, whereupon the cells were treated with a mAb specific for cNOS and visualized with rhodamine-labeled secondary Ab. The localization of cNOS expression was performed by confocal sectioning using a ×100 objective and 40 μM pinhole. Cellular cNOS expression was evaluated by full reconstruction of all optical sections. Unstimulated THP-1 cells exhibited intense cNOS expression (A) that was localized primarily to regions at or in close proximity to the plasma membrane. Following treatment with Trap-14 for 30 min, that pattern of cNOS immunofluorescence was markedly altered (B) with dispersion of cNOS throughout the cell.
Effect of NOS inhibitors on Trap-14-induced cell rounding. Trap-14-induced cell rounding was evaluated in the presence of 1 μM NOS inhibitor l-NNA or 10 μM iNOS-selective inhibitor, aminoguanidine. Cells were allowed to adhere to fibronectin-coated slides for 1 h, and cell conformation was evaluated before addition of Trap-14 (open bars) and 1 h after Trap-14 treatment (closed bars). Trap-14 induced cell rounding in the absence of NOS inhibitors (None), while l-NNA significantly abrogated Trap-14-induced cell rounding (∗, p < 0.05, ANOVA). Aminoguanidine had no effect, suggesting that the activity was not mediated by activation of inducible NOS.
Trap-14-induced NO release is mediated by ET-1
Our laboratory has demonstrated previously that Trap-14-induced NO release in rat aorta is mediated by autocrine ET-1 release with subsequent activation of the ETB receptor (14). In this study, we demonstrate the presence of ET-1 in THP-1 cells that is released in response to Trap-14 exposure and a requirement of monocyte ETB receptor activation for the induction of NO release. ET-1 immunoreactivity was detected in THP-1 cells using fluorescent confocal microscopy. Unstimulated THP-1 cells exhibited intense ET-1-specific immunofluorescence that was reduced markedly following 5-min exposure to 100 μM Trap-14. The reduction in fluorescence observed following Trap-14 exposure was abrogated by pretreatment of THP-1 cells with the microtubular assembly inhibitor, nocodazole (Fig. 5⇓, A–D). Consistent with the mobilization of ET-1 following Trap-14 stimulation, release of ET-1 was markedly increased, 177 ± 31 pM, following 5-min exposure to Trap-14 relative to vehicle-treated control cells, 9 ± 12 pM (Fig. 6⇓). These data are consistent with our previous observations in which rapid release of ET-1 was observed following thrombin or Trap-14 stimulation of rat aorta (13, 14), and suggest that thrombin receptor activation may trigger the mobilization of ET-1 from vesicular storage sites.
ET-1 mobilization. For immunodetection of ET-1, THP-1 cells were formaldehyde fixed and saponin permeabilized, whereupon the cells were evaluated for the presence of immunoreactive ET-1 using a mAb specific for ET-1 (Bioaffinity Labs) and rhodamine-labeled secondary Ab. Fluorescent images were obtained by confocal microscopy, and fluorescence values (in pixels) were obtained by computer analysis (Meridian Instruments, Lansing, MI). Intense ET-1 immunofluorescence was detected in untreated THP-1 cells (A), whereas a marked reduction in fluorescence was observed in THP-1 cells exposed to Trap-14 (B). ET-1 immunofluorescence was not reduced by Trap-14 in the presence of 1 nM nocodazole (C). Analysis of fluorescence values for the above experiment (D) indicates a significant loss in ET-1 immunoreactivity in response to Trap-14 (∗, p < 0.05, ANOVA), suggesting the release of ET-1. The loss was abrogated by pretreatment with nocodazole, suggesting that rapid release of ET-1 is dependent upon microtubular assembly.
ET-1 release. THP-1 cells were treated with 100 μM Trap-14 for 5 min, whereupon the culture supernatant was assayed for the presence of ET-1 by ET-1-specific immunoassay (Peninsula Laboratories). Treatment of THP-1 cells with Trap-14, but not vehicle alone, resulted in a significant increase in ET-1 levels detected in the culture supernatant (∗, p < 0.05, ANOVA).
To evaluate the role of ET receptor activation in thrombin-induced NO release from monocytes, THP-1 cells were pretreated with an antagonist specific for the ETA (BQ-123) or ETB (BQ-788) receptor subtype, and NO release following thrombin receptor activation was evaluated. Trap-14-induced release of NO was abrogated by pretreatment with the ETB, 9 ± 4 nM, but not the ETA receptor antagonist, 49 ± 5 nM (Fig. 7⇓). These data coupled to our previous demonstration of thrombin-induced NO release in vascular endothelium by an ETB receptor-dependent mechanism suggest that thrombin receptor activation-induced NO release from monocytes may be indirect and requires ET-1 release and subsequent ETB receptor activation.
Contribution of ET-1 to Trap-14-induced NO release. NO release from THP-1 cells induced by 100 μM Trap-14 was evaluated in the presence and absence of 0.1 μM ET receptor antagonists, ETA-selective BQ-123 or ETB-selective BQ-788, and peak values for NO release were determined. Maximal production of NO induced by Trap-14 was markedly attenuated in the presence of BQ-788, but not BQ-123 (∗, p < 0.05, ANOVA).
Trap-14-induced conformational changes require ETB receptor activation
The contribution of ET-1 activity to cell rounding was evaluated using phase-contrast microscopy and image analysis software, as described above. Trap-14-induced cell rounding (Fig. 8⇓A) was inhibited by pretreatment with the ETB-selective antagonist BQ-788 (Fig. 8⇓B), but not by the ETA-selective antagonist BQ-123 (Fig. 8⇓C). Numerical analysis of data collected over the course of the experiment (Fig. 8⇓D) demonstrates the significant attenuation of Trap-14-induced cell rounding by the ETB-selective antagonist BQ-788, suggesting a requirement for ETB receptor activation. Taken together, these data demonstrated that acute exposure of THP-1 cells to the thrombin mimetic peptide Trap-14 induces cell rounding that is mediated by cNOS-coupled release of NO, and this activity requires ET-1 release and subsequent activation of the ETB receptor.
Contribution of ET-1 to Trap-14-induced rounding of THP-1 cells. THP-1 cells were allowed to adhere and spread on fibronectin-coated glass slides in the presence or absence of 0.1 μM ETA or ETB receptor antagonists for 1 h. Cells were then exposed to 100 μM Trap-14 (A–C), and changes in cell conformation were evaluated over 1 h, as described earlier. Observed at 1 h after stimulation, Trap-14 induced significant rounding of ameboid THP-1 cells (A) that was attenuated in the presence of the ETB receptor antagonist BQ-788 (B), but not the ETA antagonist BQ-123 (C). Numerical analysis of conformation over the course of the experiment (D) indicates that Trap-14-induced cell rounding (♦) was abrogated significantly by pretreatment with BQ-788 (▪), but not BQ-123 (▴), suggesting a requirement for ETB receptor activation.
Discussion
Recent reports have suggested an important role for thrombin in inflammation in addition to its established function as a procoagulant. While its ability to augment endothelial adhesivity toward leukocytes attributed to up-regulation of endothelial adhesion molecules is well known, leukocyte responses to thrombin are as yet poorly defined. Previous reports have demonstrated that thrombin is chemotactic for monocytes (8) and that it induces up-regulated release of monocyte-chemotactic protein-1 and LPS-induced cytokines (18). Few studies have evaluated the response of monocytes to thrombin in terms of cellular conformation, a classical indicator of monocyte activation. Activated monocytes characteristically spread and extend pseudopodia to assume an ameboid conformation (12). This report studies the effect of thrombin receptor activation on the conformation of the monocytic cell line THP-1 and demonstrates that acute exposure of adherent THP-1 cells to the thrombin mimetic peptide Trap-14 induces rapid cell rounding. Cell rounding is inhibited by pretreatment with the NOS inhibitor l-NNA, suggesting that the activity is mediated by NO. Consistent with a critical role for NO in Trap-14-induced cell rounding, we observed the release of nanomolar quantities of NO from THP-1 cells in response to Trap-14. The ability of NO to affect cell conformation has only recently been appreciated, and recent evidence suggests that this activity is mediated by alteration of cytoskeletal actin (19, 20). The rapid and transient profile of NO release and inability of the iNOS-selective inhibitor aminoguanidine to inhibit subsequent cell rounding implicate the involvement of the constitutive pathway of NO synthesis in Trap-14-induced cell rounding. While cNOS activity in monocytic cells has been an issue of some debate, the presence of cNOS in THP-1 cells has been demonstrated recently by Reiling et al. (21), and cNOS activity in monocytes has been demonstrated by others (22). Immunofluorescence studies described in this study demonstrate expression of cNOS in unstimulated THP-1 cells with predominant localization at or in close proximity to the plasma membrane. Following stimulation with Trap-14, a rapid dispersion of cNOS was observed, suggesting cNOS activation in THP-1 cells treated with Trap-14. Optical sectioning of THP-1 cells by confocal microscopy revealed significant cNOS immunofluorescence 2.4 μM below the plasma membrane in Trap-14-treated, but not control THP-1 cells (not shown). These data are consistent with the demonstration by others of predominant localization of cNOS in caveolae of endothelial cells with rapid release into the cytosol following activation (17, 23).
NO release from Trap-14-stimulated THP-1 cells and subsequent cell rounding are blocked by pretreatment with an ETB receptor antagonist and are consistent with the mechanism for Trap-14 induction of NO previously described in rat aortic rings (13). In this study, we demonstrate that THP-1 cells release ET-1 within minutes of Trap-14 stimulation. Although up-regulated ET-1 release from monocytes 24 h after stimulation with thrombin has been described previously (24), the rapid nature of ET-1 release demonstrated in this work is novel. Mobilization of ET-1 was observed within minutes of thrombin receptor activation, and was inhibited by nocodazole, an inhibitor of microtubular assembly. Taken together, these observations suggest that a pool of ET-1 is present in vesicular storage sites in monocytes. While the presence of ET-1 vesicles in vascular endothelium has been suggested by others (25, 26), this is the first demonstration of ET-1 storage in monocytes and it provides evidence to suggest a role for autocrine ET-1 release in the modulation of monocyte reactivity.
Rounding of THP-1 cells in response to Trap-14 suggests that activation of the thrombin receptor may paradoxically inhibit monocytic spreading. This observation is seemingly at odds with the prevailing reputation of thrombin as a proinflammatory mediator. It must be noted, however, that studies investigating the role of thrombin as an inflammatory mediator primarily evaluate the effect of thrombin on the endothelium. While a few reports have addressed cytokine release and chemotaxis by leukocytes in response to thrombin, little is known about thrombin-induced changes in leukocyte reactivity and the contribution of such changes to leukocyte recruitment. From the time of initial mobilization of leukocytes from the circulation, until their ultimate extravasation to the primary site of injury, extensive interaction exists between leukocytes and endothelium to ensure strict spatial and temporal regulation of inflammation. Current models proposed to explain the process of leukocyte recruitment have suggested the necessity of negative regulation of leukocyte reactivity (27). NO has been implicated as an agent that inhibits leukocyte activity (28, 29), and excessive accumulation of leukocytes after inhibition of NO synthesis is well known (30, 31). We propose that thrombin receptor activation triggers the rapid release of ET-1 from vesicles, and subsequent binding of ET-1 to ETB receptors results in rapid and transient cNOS-coupled NO production (Fig. 9⇓). Functional coupling of ETB receptor activation to NO release has been shown previously (13, 14), and this pathway is further supported by independent demonstration of cNOS and ET receptor localization in caveolae by others (17, 32). Cytoskeletal actin may be disrupted following ADP-ribosylation induced by NO, resulting in destabilization of pseudopodial extensions and cellular rounding. The negative regulation of leukocyte reactivity by thrombin would be relieved by down-regulation of ETB receptor signaling or cNOS activity. Such modulation may be instrumental in restricting inappropriate leukocyte reactivity, thereby ensuring strict regulation of inflammatory response progression.
Inhibition of monocyte spreading by thrombin. We propose a model in which thrombin receptor activation triggers the rapid exocytosis of ET-1 from preformed vesicles with subsequent binding of ET-1 to monocyte ETB receptors. An influx of extracellular Ca2+ triggered by ETB receptor activation may activate cNOS, resulting in the rapid and transient production of NO. The concentration of ETB receptors and cNOS in caveolae may potentiate and localize the effects of thrombin-induced NO release. ADP-ribosylation of actin mediated by NO may result in depolymerization of actin in pseudopodial extensions, resulting in inhibition of cell spreading and alteration of cell conformation from ameboid to round.
Acknowledgments
We thank Jeanette Schaeffer and Queens College Image Core Facility for technical assistance. Graphic output was supported by a generous gift from Queens College Biology Department, Alumni Fund.
Footnotes
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↵1 This work was supported by a grant from the American Heart Association, 9750211N, and the City University of New York Internal Awards Program, PSC-City University of New York.
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↵2 Address correspondence and reprint requests to Dr. Harold I. Magazine, Department of Biology, Queens College, 65-30 Kissena Blvd., Flushing, NY 11367. E-mail address: Magazine{at}QC.EDU
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↵3 Abbreviations used in this paper: NO, nitric oxide; cNOS, constitutive nitric oxide synthase; EIA, enzyme-linked immunoassay; ET, endothelin; FF, form factor; iNOS, inducible NOS; l-NNA, NG-nitro-l-arginine; NOS, nitric oxide synthase; PSS, physiologic salt solution.
- Received March 9, 1998.
- Accepted July 1, 1998.
- Copyright © 1998 by The American Association of Immunologists
References
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