Introduction

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The association between cocaine abuse and myocardial disease has been evolving in the literature for several years. The most prominent pharmacologic actions of cocaine are its ability to block sodium channels, which underlie its anesthetic properties (Cregler and Mark, 1986), and its sympathomimetic effects due to inhibition of catecholamine reuptake. In addition to its peripheral consequences, cocaine use has been associated with the development of myocardial ischemia and infarction (Smith et al., 1987; Nademanee et al., 1989). Direct cardiac effects attributed to cocaine exposure include arrhythmias (Laposta, 1991), decrease in contractility (Smikhovich et al., 1993), coronary vasoconstriction (Chokshi et al., 1990), mitochondrial dysfunction (Yuan and Acosta, 1996), initiation of neutrophil infiltration, and lipid peroxidation (Devi and Chan, 1999).

The present investigation examines the effects of acute cocaine exposure on complement synthesis by the isolated heart. Complement activation is largely responsible for tissue destruction in the reperfused myocardium (Homeister and Lucchesi, 1994). Previously, our laboratories determined that the heart increases complement mRNA expression and protein synthesis in response to ischemia/reperfusion (Yasojima et al., 1999). Because myocardial ischemia has been linked to cocaine abuse, it is possible that ischemia/reperfusion injury contributes to the detrimental effects of cocaine on the heart. Friedrichs et al. (1994) demonstrated that heparin reduces ischemia/reperfusion injury in the crystalloid-perfused isolated heart (Gralinski et al., 1996). Heparin inhibits the alternative (Weiler and Linhardt, 1989) and classical (Loos et al., 1976) pathways of plasma-derived complement, as well as interferes with assembly of the terminal components (Baker et al., 1975). To date, the effects of heparin on the activation of tissue-derived complement proteins remain unexplored.

Cocaine-mediated toxicity is thought to be due in part to the formation of free radicals arising from its metabolism by cytochrome P-450 (Boelsterli et al., 1993; Ndikum-Moffor et al., 1998). Free radicals participate in the inflammatory response and activate the transcription factor nuclear factor-B, which regulates several inflammatory genes (Abe and Berk, 1998). Oxygen radicals have been shown to initiate the transcription of a variety of genes, such as monocyte chemoattractant protein and colony-stimulating factor (Satriano et al., 1993). The complement system, an important component of many pathologic conditions involving inflammation, also is subject to activation by tissue-derived free radicals (Collard et al., 1997).

The present study introduces evidence that cocaine stimulates complement expression in the rabbit isolated heart via an antioxidant-sensitive mechanism. We also demonstrate that heparin inhibits formation of the membrane attack complex (MAC) despite cocaine-induced transcription of complement components in treated hearts. Our results suggest that increased complement expression may be one mechanism by which cocaine exerts its cardiotoxic effects.

	Materials and Methods

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Guidelines for Animal Research. The procedures used in this study were in accordance with the guidelines of the University of Michigan Committee on the Use and Care of Animals. Veterinary care was provided by the University of Michigan Unit for Laboratory Animal Medicine. The University of Michigan is accredited by the American Association of Accreditation of Laboratory Animal Health Care, and the animal care use program conforms to the standards in The Guide for the Care and Use of Laboratory Animals.

Langendorff Perfused Heart. Male New Zealand White rabbits (1.8-2.2 kg) were rendered unconscious by cervical dislocation. Hearts were removed, mounted, and perfused on a Langendorff apparatus with modified, oxygenated Krebs-Henseleit (K-H) buffer (pH 7.44; 37°C) through the aorta at a constant flow (20-24 ml/min). The K-H buffer was recirculated at 300 ml after the equilibration period. The perfusion medium was composed of 117 mM NaCl, 4.0 mM KCl, 1.2 mM MgCl₂  ·  6H₂O, 1.1 mM KH₂PO₄, 5.0 mM glucose, 5.0 mM L-glutamate, 2.0 mM pyruvic acid, 25.0 mM NaHCO₃, and 2.6 mM CaCl₂  ·  2H₂O. The buffer passed through a membranous "lung" composed of Silastic medical grade tubing (Dow Corning, Midland, MI). The membrane lung was gassed continuously with a mixture of 95% O₂/5% CO₂. The hearts were paced through the right atrium with electrodes attached to a laboratory stimulator (180 impulses/min, 2-ms duration, 4 V, Grass SD-5; Grass Instrument Co., Quincy, MA). A left ventricular drain, thermistor probe, and a latex balloon were placed via the left atrium and secured with a purse string suture at the atrial appendage. The latex balloon was expanded with water to achieve a left ventricular end-diastolic pressure of 5 mm Hg. Isolated hearts were stabilized under normoxic conditions for a 10- to 15-min equilibration period before beginning the protocol. The preparation has been described in detail previously (Yasojima et al., 1998b).

In Vitro Protocol. Eight experimental groups were studied, and all hearts were perfused with the following treatments for 70 min, unless otherwise indicated. Group A (control) consisted of hearts that were perfused for 70 min with K-H buffer only. Group B hearts were perfused with 1 µM cocaine hydrochloride. Group C hearts were perfused with 10 µM synthetic isomer (+)-cocaine hydrochloride. Group D hearts were perfused with 3 mM antioxidant 2-N-mercaptopropionyl glycine (MPG), for 10 min, before exposure to 1 µM cocaine hydrochloride. MPG was present throughout the duration of the protocol. Group E animals were injected with 300 U/kg heparin i.v., 2 h before removal of the heart and perfusion in the presence of 1 µM cocaine hydrochloride. Group F consisted of hearts treated with 10 µM of the sodium channel blocker procaine hydrochloride. Group G hearts were perfused with 30 µM procaine hydrochloride. Group H hearts underwent the exact same protocol as group E, but were perfused with 10 µM procaine hydrochloride instead of cocaine hydrochloride.

In Vivo Cocaine Administration. Two experimental groups were studied. Group I (control) rabbit tissue was obtained from drug-free rabbits (n = 3). Group J rabbits were injected with 1 mg/kg cocaine hydrochloride dissolved in sterile saline at approximately the same time of day for three consecutive days (n = 3). On the 4th day, rabbit hearts were removed and perfused on the Langendorff apparatus with saline for 5 min to remove residual plasma and cellular blood components. Rabbit liver tissue specimens were collected at the time of heart removal. The heart and liver were sectioned and the tissues snap frozen in liquid nitrogen.

Chemicals. ()-Cocaine hydrochloride and procaine hydrochloride were obtained from Sigma Chemical Co. (St. Louis, MO) and dissolved readily in K-H buffer. All other chemicals were purchased from Sigma Chemical Co. unless otherwise noted. (+)-Cocaine base was provided by the National Institute on Drug Abuse (Rockville, MD) and dissolved in a minimal amount of 1 N HCl before addition to the buffer reservoir.

Choice of Specific Primers. DNA sequences of rabbit C3, C8, C9, and cyclophilin were obtained from the GenBank database (accession nos. M32434, U20055, L26980, and YO0052, respectively). Cyclophilin mRNA was chosen as the internal standard because it is expressed at a relatively constant level in virtually all tissues. We previously reported on the primer sequences used to detect mRNAs for C3, C9, and cyclophilin (Yasojima et al., 1998b). The primer sequences chosen for amplifying C8 were forward 5'-TAAAAGACCGCACAAAAGGGACAC-3' and reverse 5'-ATGAAGACCAGCGAGACCAGCAACT-3'. Primers for human C1q and C1r (Yasojima et al., 1998a) were used to determine whether rabbit cDNA products could be obtained. Total RNA from rabbit heart was reverse-transcribed and the cDNAs amplified with the human C1q and C1r primers by the methods previously described in detail (Yasojima et al., 1998b). Single products close to the expected sizes were obtained on polyacrylamide gels. These products were subcloned into TGEM-T plasmid vector (Promega Biotec, Madison, WI) for sequencing. The sequences were determined by the cycle-sequencing method with T7- and sp6-sequencing primers on an autosequencer (NAPS Unit; University of British Columbia). The sequenced rabbit clone for C1q was 361 base pairs (bp) in length compared with 358 bp for the comparable human cDNA product. There was 82.6% homology overall, with 100% homology in the primer region. Primers for rabbit C1q were forward 5'- CCCAGGGATAAAAGGAGAGAAAGG-3' and reverse 5'-GGCGTGGTAGGTGAAGTAGTAGAG-3'. The assigned Genbank accession number is AF089083. The sequenced rabbit clone for C1r was 218 bp in length compared with 216 bp for the human product. There was 85.3% homology overall, with only 1-bp difference in the primer region. The assigned GenBank accession number is AF108768. New primers for rabbit C1r were designed forward 5'-GCCTCCCTGACAACGATACCTTCTA-3' and reverse 5'-TGTCCTGCTTTAGAGAGTGTCC-3'.

RNA Preparation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Total RNA from ~500 mg of each tissue sample was extracted by the acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987). The extracted RNA was quantified by scanning spectrophotometry. The A260/280 ratio of all preparations was >1.8. The RNA was then reverse transcribed and specific cDNAs amplified by the PCR technique as described previously (Yasojima et al., 1998a,b).

Restriction Digest Analysis. The PCR products were purified by the ethanol precipitation procedure (Yasojima et al., 1998a). Unique restriction sites and restriction enzymes were selected with the DNA strider computer program. The restriction enzymes chosen were as follows: SacI for C1q, Sau3AI for C1r, HincII for C3, MseI for C8, and BamHI for C9. The restriction digestion reaction was carried out for 2 h at 37°C. The digested PCR products were analyzed by electrophoresis on a 6% nondenaturing polyacrylamide gel.

Western Blot Analysis. Western blots were performed on the cytosolic fraction of homogenates of rabbit heart. Heart samples were homogenized in 5× vol/protein extraction buffer (0.02 M Tris-HCl; pH 7.5) containing the protease inhibitors phenylmethylsulfonyl fluoride (10 µg/ml) and aprotinin (10 µg/ml), and 1 mM EDTA. Homogenates were centrifuged at 18,000g at 4°C for 30 min. The protein content of the supernatants was determined, and the samples were diluted in SDS sample buffer (60 mM Tris, pH 6.8; 2.5% SDS, 5% -mercaptoethanol) to a final protein content of 1 mg/ml and boiled for 3 min. Due to the high molecular weight of MAC, modifications of the electrophoresis and protein transfer steps were required. Samples containing 10 µg of protein were loaded onto a 3% polyacrylamide gel and separation was carried out for 2.5 h at 100 V in a cold room with the apparatus surrounded by ice. The transfer to the membranes was then carried out at 100 V for 5 h in the cold. Membranes were blocked in 5% low-fat milk for 2 h. Immunoblots were treated for 4 h at room temperature with a chicken anti-rabbit MAC antibody (1:500 dilution). The anti-rabbit MAC antibody was developed in conjunction with Lampire Biological Laboratories (Pipersville, PA) with rabbit C5b-9 antigen supplied by Dr. S. Bhakdi (Institute of Medical Microbiology and Hygiene, Johannes Gutenberg University, Mainz, Germany). The membranes were washed and treated for 3 h with a goat anti-chicken IgG (Sera Lab, 1:8000). Immunoreactivity was visualized by incubation with Supersignal CL-HRO chemiluminescent substrate (Pierce Chemical Co., Rockford, IL) After draining, the membranes were covered in clear plastic wrapping and exposed to X-ray film (Hyperfilm ECL; Amersham, Arlington Height, IL) for 20 s.

Statistical Analysis. Data are expressed as means ± S.E. Differences between control and experimental groups were checked for statistical significance (P < .05) by ANOVA followed by the Student's t test for unpaired observations. Dunnet's t tests with Holm's (Holm, 1979) stepdown correction for multiple comparisons was used for determining significant differences.

	Results

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Hemodynamic Parameters. Cocaine and procaine treatment only slightly modified the hemodynamic parameters of coronary perfusion pressure (CPP) and percentage of baseline left ventricular-developed pressure (%LVDP) after 70 min of perfusion (Tables 1 and 2) compared with control. However, 10 µM (+)-cocaine, 3 mM MPG/1 µM cocaine, 300 mg/kg heparin/1 µM cocaine, and 3 mM MPG/10 µM procaine hearts preserved %LVDP significantly better after 70 min of perfusion compared with control (P < .05; all groups versus control) (Table 2). MPG/cocaine, MPG/procaine, and heparin/cocaine hearts maintained essentially normal CPP values throughout the protocol (P < .05; all groups versus control) (Table 1).

RT-PCR. RT-PCR amplification from total RNA extracts was used to establish the presence and relative values of the mRNAs for C1q, C1r, C3, C8, and C9 in all heart samples. These are the only complement genes for which the sequences have been determined for rabbit, and therefore the only ones that could be amplified. Identification of PCR products from typical RT-PCR experiments is illustrated in Fig. 1. The primers chosen to amplify each cDNA yielded a single product corresponding to the anticipated size based on the known sequences. The C1q primers generated a product of 361 bp, which gave the expected fragments of 214 and 147 bp when treated with the restriction enzyme SacI. The C1r primers generated a product of 218 bp, which was cleaved by Sau3AI to yield the predicted digestion fragments of 65 and 153 bp. The C3 primer generated a product of 298 bp, and treatment with HincII yielded fragments of 253 and 45 bp. The C8 primers yielded a product of 441 bp and treatment with MseI gave fragments of 160 and 281 bp. The C9 primers generated a product of 202 bp and treatment with BamHI resulted in fragments of 135 and 67 bp. The cyclophilin primers yielded a product corresponding to the calculated size of 206 bp (data not shown). These results establish that unique reaction products were being amplified that correspond to each complement mRNA being analyzed.

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Representative polaroid photographs showing gel electrophoresis of the RT-PCR products for C1q, C1r, C3, C8, and C9 obtained from total RNA extracts of perfusion control and 1 µM cocaine-treated rabbit hearts. A single band of the expected size (arrows) was obtained for each product, i.e., 361 bp for C1q, 218 bp for C1r, 298 bp for C3, 441 bp for C8, and 202 bp for C9. In each case, lane 1 is control and lane 2 is cocaine treated. Notice the more intense bands in lane 2, indicating up-regulated mRNA levels after cocaine perfusion. Bands in the lower right illustrate representative Western blot results for MAC of complement from a cocaine-perfused heart extract (lane 1), a heparin-pretreated cocaine-perfused heart extract (lane 2), and a buffer-perfused heart extract (lane 3). Presence of the bands indicates full activation of the complement pathway. Notice the more intense band for the cocaine-perfused heart than the heparin-cocaine-treated heart, and the faint band in the control. See Materials and Methods for details.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Bar graphs (means ± S.E.) showing relative C1q, C1r, C3, C8, and C9 mRNA levels in perfusion controls and cocaine-treated hearts (groups A-E). Compared with buffer perfused controls, 1 and 10 µM cocaine significantly increased all the mRNA levels. Perfusion with 10 µM (+)-cocaine did not significantly alter complement transcription. MPG (3 mM) suppressed the cocaine-induced (1 µM) complement transcription and significantly reduced expression of the mRNA for C3. Pretreatment with heparin (300 U/kg i.v.) 2 h before removal of the hearts did not attenuate up-regulation of any of the complement mRNAs except for C3 (n = 6 in each group).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Bar graphs (means ± S.E.) showing relative C1q, C1r, C3, C8, and C9 mRNA levels in perfusion controls and procaine-treated hearts (groups A, F, G, H). Treatment with 10 µM procaine significantly increased transcription of C1q, C1r, and C8, whereas treatment with 30 µM procaine significantly up-regulated C1q, C1r, C8, and C9 mRNAs. MPG (3 mM) blocked complement gene expression stimulated by 10 µM procaine and significantly reduced C3 mRNA expression (n = 6 in each group).

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Bar graphs (means ± S.E.) showing relative C1q, C1r, C3, C8, and C9 mRNA levels in hearts and livers of rabbits administered cocaine (1 mg/kg) for three consecutive days before organ removal. Hearts were then perfused with buffer for 5 min to remove blood. Cocaine treatment significantly increased all the mRNA levels in rabbit heart and liver compared with controls. Note the higher up-regulation in heart compared with liver (n = 3 in each group)

Western Blot Analysis. The bottom right bands in Fig. 1 are representative of Western blot data for the MAC from a heart perfused with 1 µM cocaine only (lane 1), a heart similarly perfused with 1 µM cocaine after in vivo heparin pretreatment 2 h before heart removal (lane 2), and a heart perfused with buffer only (lane 3). An intense MAC band is observed after cocaine perfusion, which is reduced considerably by pretreatment with heparin. Heparin administration appeared to attenuate assembly of the MAC, even though it did not affect cocaine-induced C1q, C1r, C3, C8, and C9 transcription. The control heart shows a faint band consistent with the modest complement mRNA expression detected with RT-PCR.

	Discussion

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The present study demonstrates that cocaine (1 µM) increases C1q, C1r, C3, C8, and C9 mRNA and MAC protein expression in the rabbit isolated heart. In addition, i.v. administration of cocaine to the rabbit augments hepatic and myocardial complement synthesis in treated animals. The anesthetic, procaine hydrochloride (10 and 30 µM), also augments myocardial complement generation. The antioxidant MPG (3 mM) abolishes cocaine and procaine-stimulated complement transcription, suggesting a possible role for reactive oxygen species in up-regulation of complement gene expression. The actions of cocaine appear to be stereospecific because 10 µM (+)-cocaine failed to induce complement expression. The present study also shows that heparin inhibits MAC formation, yet does not prevent cocaine-induced transcription of C1q, C1r, C3, C8, and C9 mRNA. The latter observation is in keeping with our previous findings that heparin pretreatment of the rabbit subsequently protected the isolated perfused heart from the deleterious functional and cytotoxic effects associated with global ischemia and reperfusion (Friedrichs et al., 1994).

The concentrations of cocaine and procaine used (1 and 10 µM, respectively) only modestly altered CPP and %LVDP compared with controls, lessening the possibility that modulation of complement expression was due to compromised function. These changes in the hemodynamic parameters only slightly affected complement expression because buffer-perfused hearts contained relatively low levels of complement mRNA (except for C3, which is constitutive) and MAC protein. Hearts treated with the synthetic isomer (+)-cocaine, MPG/cocaine, MPG/procaine, or heparin/cocaine preserved %LVDP significantly (P < .05) better than buffer-perfused controls. MPG/cocaine, MPG/procaine, and heparin/cocaine hearts also sustained significantly lower CPP values throughout the protocols (P < .05). Because the hearts are perfused in a recirculating system, tissue components released from the myocardium over time may cause the observed increase in CPP and decrease in %LVDP. The lack of similar observations in the hearts perfused with (+)-cocaine, MPG, or heparin implicates that the potential effects of cellular components or tissue metabolites appear to be negated by these agents.

Cocaine modifies immune cell function and expression of cytokines and adhesion molecules (Pellegrino and Bayer, 1998; Gan et al., 1999). It has been demonstrated that cocaine increases blood-brain barrier permeability, thereby promoting neuroinvasion by HIV-1 (Zhang et al., 1998). Application of the results from this study alludes to a role for local generation of complement and formation of MAC in facilitating HIV-1 entry. The MAC forms channels 9 to 12 nm in diameter, large enough to allow access of viral particles into the cell. Cocaine alters immune cell activation and proliferation, as well as cytokine production (Pellegrino and Bayer, 1998). Gan et al. (1999) recently demonstrated that cocaine up-regulates the expression of adhesion molecules in vitro, in turn promoting leukocyte migration. Cocaine administration is reported to result in platelet activation in vivo (Kugelmass et al., 1995). These actions, individually or acting in concert, may partially account for cocaine-associated myocardial tissue injury. Aside from its noted peripheral and central nervous system effects, cocaine abuse also has been associated with direct cardiotoxicity to myocytes. Myocardial ischemia and infarction are common features observed in chronic cocaine users (Smith et al., 1987; Nademanee et al., 1989). Our investigation presents another facet of the immune system affected by cocaine exposure. Previous work reporting that myocardial complement generation increases during ischemia/reperfusion implicates a role for local complement production under certain pathologic conditions (Yasojima et al., 1999a). In light of this evidence, we propose that up-regulation of complement expression may account for the deleterious effects of cocaine on myocardial tissue.

Although cocaine is primarily hydrolyzed by plasma esterases, a fraction undergoes metabolic conversion by cytochrome P-450 (Ndikum-Moffor et al., 1998). Cytochrome P-450 metabolism of cocaine generates free radicals, and the P-450 metabolite norcocaine nitroxide causes hepatotoxicity in vivo (Boelsterli et al., 1993; Ndikum-Moffor et al., 1998). Various P-450s have been detected in myocardial tissue (McCallum et al., 1993; Wu et al., 1997). Although the importance and extent of cocaine metabolism by myocardial P-450s has yet to be established, it is feasible that a portion of the cocaine perfused through the hearts was subject to the P-450 metabolic pathway, possibly forming radical species. Free radicals initiate the transcription of a variety of genes and activate transcription factors (Abe and Berk, 1998; Satriano et al., 1993). The complement system, a major contributor to the inflammatory response, also can be activated by free radicals (Collard et al., 1997).

The present study demonstrates that the free radical scavenger MPG blocks cocaine-induced complement transcription. MPG scavenges several types of free radicals and can gain entry into the cell (Mitsos et al., 1986). MPG also inhibits complement activation (Kilgore et al., 1994); however, its effects in this study appear to be at the transcriptional level. Melchert et al. (1992) determined that cytochrome P-450 preferentially metabolizes ()-cocaine (cocaine) over the synthetic isomer (+)-cocaine, which is mainly hydrolyzed by esterases. Our data show that cocaine increases myocardial complement generation, whereas (+)-cocaine does not. Together, these observations implicate free radicals as mediators of cocaine-induced complement protein synthesis. However, it cannot be excluded that MPG is working through mechanisms other than acting as an antioxidant. Cocaine also may affect complement synthesis through a receptor-ligand interaction; however, the validity of this hypothesis cannot be determined from the current data. MPG also inhibited up-regulation of C1q, C1r, C3, C8, and C9 mRNA transcription stimulated by procaine exposure. Although it has not been established whether the metabolism of procaine generates free radicals, the attenuation of complement gene expression in the MPG/procaine-treated hearts indicates a role for free radicals in procaine's mechanism of action.

Both cocaine and procaine block sodium channels, which accounts for their anesthetic properties. Inhibition of sodium channels may be another mechanism by which these drugs increase myocardial complement expression. We observed that the ATP-sensitive potassium channel (KATP) channel opener pinacidil decreases complement expression in response to ischemia/reperfusion (Tanhehco et al., 1999). The KATP channel blocker glyburide reverses the effect of pinacidil, suggesting that modulation of KATP channel may regulate complement gene expression. However, activation of the KATP channel has been shown to preserve myocardial function against hydrogen peroxide-mediated oxidative stress (Gan et al., 1998). These data suggest that inhibition of complement production by ion channel modulators may be secondary to, or completely dissociated from, their direct effects on ion channel function.

Pretreatment in vivo with heparin 2 h before removing the heart inhibited MAC expression in cocaine-treated hearts, but did not prevent C1q, C1r, C8, and C9 transcription. Administration of heparin over a similar time course before an ischemic event also ameliorates ischemia/reperfusion injury in vitro and in vivo (Friedrichs et al., 1994; Gralinski et al., 1996). Heparin binds to 13 complement components, including C6, C8, and C9, all of which are essential for formation of MAC (Sahu and Pangburn, 1993). Heparin disables both the alternative (Weiler and Linhardt, 1989) and classical (Loos et al., 1976) pathways, as well as blocks organization of the terminal components (Baker et al., 1975). In addition, the function of C1 inhibitor is potentiated by heparin (Caldwell et al., 1999). C1 inhibitor, as its name implies, regulates C1 esterase activity (Caldwell et al., 1999). The up-regulation of C1q and C1r mRNA implicates a role for the classical pathway in the isolated heart model. Any of the aforementioned actions of heparin may explain how the glycosaminoglycan interferes with MAC assembly in the cocaine-treated hearts. Inhibition of local myocardial tissue complement activation may be one mechanism by which heparin protects the ischemic heart. Heparin could be exerting its effects at an extracellular or intracellular site because it has been shown to bind avidly to the cell surface and be taken up by vascular smooth muscle cells (Castellot et al., 1985).

In summary, we present evidence that cocaine up-regulates C1q, C1r, C3, C8, and C9 mRNA expression and MAC formation in the myocardium. Western blot analysis for MAC indicates that the isolated heart is capable of generating all of the complement components necessary for activation of the cascade and formation of MAC. The antioxidant MPG attenuates cocaine and procaine-induced complement expression, suggesting that cocaine and procaine increase complement transcription through a free radical-mediated mechanism. Heparin does not reduce C1q, C1r, C8, and C9 mRNA up-regulation by cocaine, but does prevent MAC assembly. This may explain the previously reported ability of heparin to reduce ischemia/reperfusion injury. Our study suggests that activation of tissue complement may contribute to the cardiotoxicity of cocaine.