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Cocaethylene affects human microvascular endothelial cell p38 mitogen-activated protein kinase activation and nuclear factor-[kappa]B DNA-binding activity.

Cocaethylene (CE) [1] is formed from cocaine and ethanol in the liver by the human carboxylesterase-1 enzyme (1). Detectable amounts of CE are produced in vivo in more than 80% of cocaine abusers (2-4). Research has established that CE is dangerous to humans because of the shared molecular targets of cocaine and CE and the prolonged half-life of CE (2-4 h vs 30-45 min for cocaine) (5-9). Furthermore, the potential for pathologic outcomes over time in cocaine abusers is increased when CE is formed (3, 5). Thus, characterization of the mechanisms underlying CE-mediated toxicity is important to cocaine-related research.

Cocaine abuse leads to ischemic damage to tissues. The general pathologic mechanisms are fairly well characterized (3,10-14), with abundant case evidence demonstrating systemic disease in cocaine abusers (15-27). The high prevalence of vasculitis in such cases suggests that vascular pathology, initiated by an injurious effect of cocaine or CE, may contribute to such pathologies. Because most vascular tissue is microvascular and composed mostly of endothelial cells and connective tissue (28), the microvascular endothelium may play a central role in the pathogenic mechanisms underlying cocaine- and CE-associated systemic disease (29). Moreover, the persistence of CE in human serum suggests that it may be a prime affector of the microvascular endothelium.

The vascular endothelium responds to alterations in hemodynamics or injury by increasing vessel permeability and producing proinflammatory adhesion molecules and cytokines. These cellular changes are largely modulated by alterations in calcium, which regulates permeability via modulation of the actin-myosin cytoskeleton and affects autoregulatory (e.g., inositol trisphosphate and diacylglycerol) and signaling pathways associated with endothelial activation and survival. Such signaling pathways include the mitogen-activated protein kinase (MAPK) pathway, which when activated can go on to activate the nuclear factor-KB (NF-[kappa]B) pathway (30-32).

Typical tissue effects of increased endothelial permeability and proinflammatory signaling are inflammation, edema, and alterations in tissue-blood exchange of nutrients and waste. Such endothelial alterations could be implicated in the mechanisms of CE-associated vascular toxicity. We have demonstrated that exposure to 1 mmol/L CE alters the morphology of human microvascular endothelial cells (HMEC-1) without altering monolayer viability or inducing overt cytotoxicity (33). The morphologic change observed was associated with decreased monolayer electrical resistance, possibly modulated by an observed increase in HMEC-1 cellular calcium load and inositol trisphosphate generation. These findings suggest that CE is capable of significantly altering HMEC-1 cell signaling cascades.

We investigated whether CE exposure in the HMEC-1 cell model would lead to increased phosphorylation of p38 MAPK and would be associated with an alteration of the DNA-binding activity of NF-[kappa]B complexes.

Materials and Methods

REAGENT SOURCES

HMEC-1 cells were donated by the CDC, and CE fumarate was donated by the National Institute on Drug Abuse. Cell culture materials were from Corning. Fetal bovine serum, L-glutamine, phosphate-buffered saline (PBS; 1.06 mM K[H.sub.2]P[O.sub.4], 155.17 mM NaCL, 2.97 mM [Na.sub.2]HP[O.sub.4] - 7[H.sub.2]O, pH 7.4), and trypsin-EDTA were from Gibco-BRL. Human fibronectin was from Becton Dickinson. Sodium bicarbonate solution was from Cellgro. MCDB131 medium mix, water-soluble hydrocortisone, benzamidine, sodium fluoride, sodium azide, [beta]-mercaptoethanol, urea, protease inhibitor cocktails, phenylmethylsulfonyl fluoride, sulfuric acid (Hz504), lipopolysaccharide (LPS) from Salmonella typhosa, protease inhibitor cocktail for mammalian cells, and tetramethylbenzidine were from Sigma-Aldrich. Miscellaneous supplies and recombinant human epidermal growth factor were from Fisher Scientific. Phosphop38 MAPK assay kits were from R&D Systems. Core buffers, dithiothreitol (DTT), labeled Ig[kappa] oligonucleotide (Bio-Synthesis, Inc.), and materials for polyacrylamide gels and electrophoretic mobility shift assay (EMSA) imaging were donated by N. Herzog (Department of Pathology, University of Texas Medical Branch). The Micro BCA Protein Assay Reagent Kit for protein quantification was from Pierce. Phosphatase inhibitors were from Calbiochem.

CELL CULTURE AND MAINTENANCE

HMEC-1 cells are immortalized human dermal microvascular endothelial cells that are representative of the human microvascular environment and have prolonged culture life that makes them suitable for in vitro model development (34). The cells were seeded at a density of 5 x [10.sup.6] cells in T150 flasks and incubated until confluent (72 h; 3 x [10.sup.7] cells). Medium composition (MCDB131) was as previously described (33). To decrease serum response, a change of cell media was performed 24 h before the beginning of each experiment.

CE FUMARATE STOCK PREPARATION

CE fumarate ([C.sup.18][H.sub.23]N[O.sub.4] x 1.5 [C.sub.4][H.sub.4][O.sub.4]) is more soluble than CE alone; fumarate does not affect HMEC-1 biochemistry (see Data 1 in the Data Supplement that accompanies the online version of this article at http://www. clinchem.org/content/vol52/issue10). CE fumarate stock (100 mmol/L) was prepared in MCDB131 medium (p38 MAPK) or calcium- and magnesium-free PBS (EMSA) in amber glass bottles. Stock was stored tightly sealed for up to 1 week at 4[degrees]C.

P38 MAPK PHOSPHORYLATION ASSAY

Monolayers were exposed to media containing no CE (negative control),1 mmol/L CE, or 0.1 mg/L of S. typhosa endotoxin/LPS (positive control) for 5 min, 1 h, 2 h, or 4 h. Cells were scraped from the plates, and lysis buffer (prepared as directed by the manufacturer) was added to each flask. Lysates were vortex-mixed, and aliquots were frozen at -20[degrees]C until analysis.

ELISA plates were coated with capture antibody overnight and rinsed according to manufacturer directions. Samples were centrifuged at 20008 for 5 min at ambient temperature, diluted according to manufacturer instructions, and vortex-mixed. Prepared samples were kept on ice.

Calibration curves of phospho-p38 MAPK were prepared in duplicate according to manufacturer instructions. Blanks, calibrators, and samples were added to ELISA plates at 100 [micro]L/well in duplicate. ELISA proceeded as directed by the manufacturer. Absorbance was measured at 450 nm (690 nm background correction), and the means of readings were found. Linear regressions of the calibration curves were used to determine concentrations of phospho-p38 in the experimental samples, reported here in pg/[10.sup.7] cells.

NF-[kappa]B ASSAYS

Extraction of Nuclear Protein from CE-Treated HMEC-1 Cells. We collected baseline samples from untreated cells. For time-point measurements, we harvested control HMEC-1 cells 1 or 4 h after adding PBS to the culture. Treated cultures were exposed to 1 mmol/L CE or 0.1 mg/L of S. typhosa endotoxin in PBS for 1 or 4 h. We then rinsed the flasks with Ca- and Mg-free PBS and placed them on ice. We used ice-cold PBS to harvest the cells. Cells were pelleted at 2008 for 10 min at 4[degrees]C. Pellets were resuspended in Ca- and Mg-free PBS and pelleted again at 2008 for 5 min at 4[degrees]C.

Nuclear extraction proceeded as described by Bassett et al. (35) and Dyer and Herzog (36), with modifications; all buffers mentioned were prepared according to their methods. Cell pellets were resuspended in ice-cold sucrose-based lysis buffer (containing DTT, phenylmethylsulfonyl fluoride, Nonidet P40, and phosphatase- and protease-inhibitor cocktails in a sucrose-salt buffer). Lysates were centrifuged at 5008 for 5 min at 4[degrees]C.

Pellets were resuspended in ice-cold low-salt buffer (containing DTT, Nonidet P40, phenylmethylsulfonyl fluoride, and phosphatase and protease inhibitor cocktails in a HEPES-EDTA-salt buffer). Suspended nuclei were lysed with cold high-salt buffer (same composition as the low-salt buffer, but with higher salt concentrations), added incrementally and with gentle mixing. Increased viscosity indicated nuclear lysis. Lysed nuclei were centrifuged at 136908 for 15 min at 4[degrees]C. Supernatants were stored in 25-F.i.L aliquots at -80[degrees]C.

Nuclear Extract Protein Quantification. We measured protein concentrations of the extracts with the Micro BCA Protein Assay Reagent Kit (Pierce), which uses the principles of the Lowry method of protein analysis (37), modified by Smith et al. (38), for quantification.

EMSA. EMSA was performed as described by Bassett et al. (35) and Dyer and Herzog (39). We prepared a reaction cocktail containing 5 [micro]g of nuclear extract, 1 [micro]L of a 35 nmol/L stock of [sup.32]P-labeled Ig[kappa] oligonucleotide (which recognizes NF-[kappa]B proteins, sequence 5'-AGT TGA GGC GAC TTT CCC AGG C-3') and master mix buffer (5x band-shift buffer, 20 mmol/L DTT, poly(dI:dC). We mixed the band-shift buffer as described previously (35, 39), incubated the mixture at ambient temperature, and added 5 x loading buffer as described previously (35, 39). We then loaded samples onto a 6% polyacrylamide gel and conducted electrophoresis in 0.25 x TBE solution. After electrophoresis, we transferred the gel to Whatman paper and dried it at 75[degrees]C under reduced pressure.

Cold Competition EMSA. We used cold competition to distinguish bands specifically bound by the Ig[kappa] oligonucleotide from bands arising from proteins bound nonspecifically. The approach was the same as that for EMSA, except we added nonradiolabeled Ig[kappa] oligonucleotide to selected (positive controls and LPS-treated extracts) samples before adding the [sup.32]P-labeled Ig[kappa] oligonucleotide. Gel buffer addition, reactions, loading, and electrophoresis proceeded as described for EMSA.

Supershift Analysis. The approach for supershift analysis was nearly identical to EMSA as described above. However, we added specific antibodies for NF-[kappa]B-family proteins to the reaction cocktail for 30 min and then added the [sup.32]P-labeled Ig[kappa] oligonucleotide and poly(dI:dC) (5x band shift buffer contained 2 mmol/L DTT). Antibodies against p65 (H-286), p50 (nuclear localization signal), c-Rel (N466), and p52 (447) (donated by N. Herzog, purchased from Santa Cruz Biotechnology) were used at 2 [micro]g per reaction. Incubation with [sup.32]P-labeled Ig[kappa] oligonucleotide and poly(dI:dC) was followed by antibody incubation, and gel loading and processing continued as described above.

Gel Autoradiography and Imaging. We imaged cooled, dried gels for [greater than or equal to] 4 h with an InstantImager (Packard/ PerkinElmer). Band-radio intensities were reported in cpm/[mm.sup.2].

We performed autoradiography with Kodak X OMAT autoradiography film (Eastman Kodak) for 1 to 4 days. We scanned developed film with a UMAX PowerLook 1000 scanner (UMAX Technologies, Inc.). Shifted complexes on the resulting film were quantitated with Kodak Digital Science ID image analysis software.

Results

P38 MAPK PHOSPHORYLATION

In control HMEC-1 cells, mean (SD) baseline phospho-p38 MAPK [14.7 (8.6) pg/[10.sup.7] cells] showed a slight but statistically significant serum-induced increase over a 4-h period [84.0 (35.0) pg/[10.sup.7] cells; P <0.001] (Fig. 1). In LPS-treated cells, maximal phospho-p38 concentrations [395.1 (63.7) pg/[10.sup.7] cells; P <0.001 vs baseline] occurred within 2 h of exposure and persisted through the 4-h time point (Fig. 1). In HMEC-1 cells exposed to CE, significant, intermediate (between control and LPS values) increases in phospho-p38 concentrations occurred within 5 min and peaked at 4 h [162.2 (37.1) pg/[10.sup.7] cells (P <0.001 vs negative control) at 4 h]. No CE dose-response analysis was performed in this study, because previous results showed that lower concentrations of CE had little effect on HMEC-1 physiology (33).

[FIGURE 1 OMITTED]

NF-[kappa]B BAND IDENTIFICATION AND DENSITY

We conducted time-course measurements of NF-[kappa]B DNA binding activity with HMEC-1 cell nuclear extracts over a period of 4 h after exposure to LPS and CE. This experiment revealed 6 bands of potential interest (see Data 2 in the online Data Supplement). To determine which of the 6 bands contained specific NF-[kappa]B complexes, we performed a cold competition experiment for the same groups and time points (see Data 3 in the online Data Supplement). As a result of this experiment, we knew that 2 bands (named bands 1 and 2 here) contained specific NF-[kappa]B complexes.

LPS treatment led to significant increases in mean (SD) band 1 density at 4 h [Fig. 2A, 185% (9%) of control; P = 0.021] and significant modulation of band 2 density [Fig. 2B, 54% (3%) of control (P = 0.002) at 1 h; 167% (5%) of control (P = 0.005) at 4 h]. After CE treatment the densities of both bands decreased significantly at 1 h [Fig. 2, A and B; 31% (2%) of control (P = 0.021) and 43% (2%) of control (P <0.001), respectively], but at 4 h, band densities were similar to controls (P >0.05), demonstrating that LPS and CE have similar effects on HMEC-1 cell NF-[kappa]B DNA binding activity despite the difference in the magnitude of their effects (see Data 4 in the online Data Supplement).

NF-[kappa]B BAND COMPOSITION

The results of the supershift assays, which demonstrate the specific NF-[kappa]B proteins present in each band and how treatment with LPS and CE affected band composition, are shown in Fig. 3. The 70Z/3 pre-B-cell extract was used as a positive control for NF-[kappa]B banding patterns, because the compositions of 70Z/3 bands have been characterized (36, 39). Fig. 3A shows that all HMEC-1 cell extracts tested contained RelA(p65) and that RelA is present in both bands at all time points. LPS treatment decreased RelA at 4 h, as evidenced by an absence of band elimination relative to controls at the 4-h time point. CE treatment did not appear to affect the RelA content of either band.

[FIGURE 2 OMITTED]

Both bands appeared to contain p50, as evidenced by the supershifting and strong band elimination observed at all time points (Fig. 3B). At both time points, LPS treatment increased the p50 content of band 1 and decreased the p50 content of band 2. CE treatment showed the same pattern over time as LPS, increasing band 1 p50 content and decreasing band 2 p50 content.

No supershifting occurred, so band elimination was the indicator for the presence of c-Rel in the HMEC-1 extracts (Fig. 3C). Band 1 appeared to contain some c-Rel in all groups. Band 2 showed significant elimination at baseline. LPS decreased the elimination of band 2 over time, but CE did not affect band 2 density.

At baseline t, p52 was present in the HMEC-1 cell extracts, as indicated by the presence of a faint supershift near the loading wells (Fig. 3D). However, because the densities of bands 1 and 2 did not appear to change after treatment with anti-p52 antibody, it is possible that the supershifted band originated from one of the other band shifts on the gel. LPS treatment appeared to decrease any p52 that may have been present in band 2. However, CE treatment appeared to have no effect on the p52 content-related densities of bands 1 and 2.

Given the known band composition of NF-[kappa]B in the 70Z/3 extracts, and confirmation by supershifts, we identified the bands seen in the HMEC-1 extracts. The results of the supershift and band density analyses are summarized in Table 1 and indicate that band 1 was probably composed of ReIA/p50 heterodimers, in agreement with the banding pattern of the 70Z/3 controls. Band 2 composition, appears to be more complex, however, with RelA, p50, and c-Rel present at all time points. The p52 content of band 2 is inconclusive, because in all groups, little change in band 2 density was observed at baseline. LPS treatment led to increased band 1 density at 4 h and decreased band 2 density at 1 h, and CE treatment led to decreased densities of both bands at 1 h.

[FIGURE 3 OMITTED]

Discussion

Cocaine abuse causes systemic disease in addition to the well-known neurologic and cardiovascular disturbances. The mechanisms) of cocaine-related diseases are poorly characterized despite their potential lethality. We postulate that CE affects the vascular endothelium, and the resulting systemic disturbances in vascular function commonly lead to tissue ischemia and systemic diseases. Previously, we demonstrated that a lethal concentration of CE leads to vascular endothelial dysfunction via alterations in monolayer permeability (33). We began elucidating the mechanism by demonstrating that the same concentration of CE alters endothelial monolayer resistance, intracellular calcium ion flux, and the generation of inositol 1,4,5-trisphosphate (data not shown). In the current study, we hypothesized that because of the altered calcium flux and its known influence on endothelial signaling, p38 MAPK would show greater activation (evidenced by phosphorylation) and be associated with altered nuclear localization of NF-[kappa]B in HMEC-1. The resulting evidence supports this hypothesis, enabling us to further characterize alterations in endothelial signaling associated with CE exposure and demonstrate the continuum of the effects of CE.

The first major finding of this study was an increase in p38 MAPK phosphorylation in CE-exposed HMEC-1 cells. This increase was significantly greater than the effects of serum stimulation but intermediate compared with maximal stimulation of p38 MAPK phosphorylation in HMEC-1 by LPS from S. typhosa. This finding is largely substantiated by our previous studies (33) and by other reports of observed endothelial permeability increases associated with p38 MAPK phosphorylation (31, 40, 41).

The paucity of data in this area of cocaine and CE research means that there are few results with which ours can be compared. One cardiac study of cocaine exposure (0.01-1 [micro]mol/L) in a rat cardiomyocyte model reported no change in p38 MAPK phosphorylation (42). Because of this disparity of data, we suggest that the cellular response to cocaine and/or CE exposure involves potential dose-, drug-, and model-dependent variables that require characterization in each experimental setting and must be further tested in these and other models.

The second major finding of this study involves the characterization of HMEC-1 NF-[kappa]B production and how its DNA-binding pattern changes when HMEC-1 cells are exposed to CE. We have confirmed that at baseline HMEC-1 cells produce primarily the p50 homodimer, which likely serves to suppress activation of genes regulated by NF-[kappa]B. Small concentrations of ReIA/p50 complexes are also present at baseline and likely modulate the promotion of transcription of NF-[kappa]B-regulated genes (43, 44). In addition, after 1 h of CE exposure in the HMEC-1 cell model, we observed a decrease in both the activating (ReIA/p50) and suppressing (p50/p50) NF-[kappa]B complexes, with little or no alteration of other NF-[kappa]B protein content in the nuclear complexes. Thus, it is likely that in HMEC-1 cells exposed to CE, the nuclear localization of different NF-[kappa]B complexes and other transcription factors not described in this study are altered. CE exposure led to transient changes in the tested proteins, in contrast to LPS exposure, which altered the DNA binding of NF-[kappa]B dimers at both time-points. In other endothelial models, such changes in NF-[kappa]B are known to be associated with increased transcription of NF-[kappa]B-regulated genes, such as surface adhesion molecules and cytokines (43, 44).

Lee et al. (45), in the only study we identified as comparable to this one, showed that human brain microvascular endothelial cells exposed to cocaine (up to 200 [micro]mol/L) for 2 h exhibited increased DNA binding of RelA and p50. Lee et al. (45) identified 2 NF-[kappa]B-specific bands that were similarly identified and contained the same complexes that we observed in this study. DNA binding, however, differed vastly between our study and theirs. This difference is likely related to the drug and concentration used and the specialized nature of the brain vs dermal endothelium. Also, in signaling studies, the times of sampling can make a large difference in the results observed--a difference of as little as 1 h could affect differentiation of upregulation from suppression. Thus, future studies of NF-[kappa]B DNA binding conducted in our laboratory are likely to include additional, lower CE concentrations, more time points of sampling, and potentially cocaine, so that we may test these theories.

In summary, we have demonstrated that exposure of HMEC-1 monolayers to a lethal dose of CE alters p38 MAPK phosphorylation and NF-[kappa]B DNA binding activity. These findings suggest that adhesion molecules and cytokine production are potentially affected by exposure of endothelial cells to CE. Considering the number of signaling pathways that have yet to be characterized in CE-exposed endothelium, we strongly encourage further mechanistic research in this direction. Knowledge of such pathways aids in the general characterization of healthy and pathologic occurrences in the endothelium and could direct efforts to develop therapeutic agents that will abrogate or ameliorate pathology observed in cocaine abusers with systemic and vascular disease.

We thank B. Elsom and 5. Fennewald for technical assistance. This study was supported by NIH Grant F31 DA15580 and by the Department of Pathology, University of Texas Medical Branch.

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DANYEL HERMES TALKER, NORBERT K. HERZOG, and ANTHONY O. OKORODUDU *

Department of Pathology, University of Texas Medical Branch, Galveston, TX.

[1] Nonstandard abbreviations: CE, Cocaethylene; MAPK, mitogen-activated protein kinase; NF-[kappa]B, nuclear factor-[kappa]B; HMEC, human microvascular endothelial cell; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; DTT, dithiothreitol; EMSA, electrophorefic mobility shift assay.

* Address correspondence to this author at: University of Texas Medical Branch, 301 University Blvd., Rte. 0551, Galveston, TX 77555-0551.

Received December 16, 2005; accepted July 10, 2006. Fax 409-772-9231; e-mail aookorod@utmb.edu.

Previously published online at DOI: 10.1373/clinchem.2005.065250
Table 1. Summary, NF-[kappa]B DNA binding activity in
HMEC-1 cells. (a)

Band RelA (p65) p50 c-Rel p52

1 [check] [check] X X
2 [check] [check] [check] ?

 LPS

Band 1 h 4 h

1 [left and right arrow] *
2 [down arrow] *

 CE

Band 1 h 4 h

1 [down arrow] * [left and right arrow]
2 [down arrow] * [left and right arrow]

(a) The results of the supershift assays and band density analysis are
shown. The presence of a specific NF-[kappa]B protein in a band
(labeled on the left) is marked with [check], and the absence of an
NF-[kappa]B protein is marked with X; inconclusive tests are marked
with ?. Changes in band density after LPS or CE treatment are shown as
increase (none found), decrease ([down arrow]), or no change ([left
and right arrow]). Asterisk (*) indicates a statistically significant
change (P <0.05) from baseline within each group, as calculated with
Student 2-tailed t-test.
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Title Annotation:Drug Monitoring and Toxicology
Author:Tacker, Danyel Hermes; Herzog, Norbert K.; Okorodudu, Anthony O.
Publication:Clinical Chemistry
Date:Oct 1, 2006
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