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* Departments of Physiology and
Pharmacology, New York Medical College, Valhalla, New York 10595
Abstract
Heme is a co-factor required for the stimulation of soluble guanylate cyclase (sGC) by nitric oxide (NO) and carbon monoxide, and sGC activation by these agents is inhibited by superoxide. Because heme promotes oxidant generation, we examined the influence of rat pulmonary microvascular endothelial cells (PMECs) with a stable human heme oxygenase-1 (HO-1) transfection and heme on oxidant generation and cGMP. Culture of PMEC with low serum heme decreased cGMP and the detection of peroxide with 10 µM 2',7'-dichlorofluorescin diacetate and increased HO-1 further decreased cGMP without altering the peroxide detection under these conditions. Under conditions where heme (30 µM) has been shown to stimulate cGMP production in PMECsby mechanisms involving NO and CO, heme increased the detection of peroxide in a PMEC-dependent manner and HO-1 transfection did not markedly alter the effects heme on peroxide detection. The addition of 1 µM catalase markedly inhibited the effects of heme on peroxide detection whereas increasing (0.1 mM ebselen) or decreasing (depleting glutathione with 7 mM diethylmaleate) rates of intracellular peroxide metabolism or inhibiting the biosynthesis of oxidants (with 10 µM diphenyliodonium or 0.1 mM nitro-L-arginine) had only modest effects. The detection of superoxide by 10 µM dihydroethidium from PMECs was not increased by exposure to heme. These actions of oxidant probes suggest that intracellular oxidants have a minimal influence on the response to heme. Thus, exposure of PMECs to heme causes a complex response involving an extracellular generation of peroxide-derived oxidant species, which do not appear to originate from increases in intracellular superoxide or peroxide. This enables heme and HO to regulate sGC through mechanisms involving NO and CO, which are normally inhibited by superoxide.
Key Words: oxidative stress • NOS • lipid peroxidation
It is well established that heme oxygenase (HO) metabolizes heme in a manner that generates carbon monoxide (CO) in amounts that stimulate the soluble form of guanylate cyclase (sGC) and increase cellular levels of cGMP (1) and that it functions to prevent heme-mediated cellular injury (2). Our recent studies on the effects of heme and HO-1 transfection on cGMP generation by rat pulmonary microvascular endothelial cells (PMECs) have detected evidence that heme (30 µM) stimulates an increase in cGMP that is partially mediated through a nitric oxide (NO)-dependent mechanism and a mechanism associated with increased CO production (3). The HO-1-transfected PMECs show on exposure to heme further increases in CO production and cGMP levels, which are not dependent on NO biosynthesis. Concentrations of heme in the range of 30 µM appear to be just below the threshold of levels of heme that cause activation of oxidant-associated signaling mechanisms based on examination of the expression of endothelial adhesion proteins, a process which is inhibited by increased HO-1 expression (4). Because the stimulation of sGC by NO and CO is highly sensitive to oxidant species through mechanisms, which include inhibition by superoxide (5, 6), we investigated the effects of alterations in heme levels and HO-1 transfection on peroxide generation and intracellular superoxide levels in PMEC.
Materials and Methods
Cell Culture.
Rat pulmonary microvascular endothelium and PMECs containing a stable transfection with human HO-1 were cultured in 48-well tissue culture plates containing Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated bovine fetal calf serum (FCS), as previously described (3).
Fluorescent Detection of Oxidants.
The Dulbecco’s medium was removed and after washing was replaced with Hanks balanced salts solution. Mechanistic probes described in the Results section were then added, followed by 10 µM dihydroethidium (DHE) or 10 µM 2',7'-dichlorofluorescin diacetate (DCF) for the detection of superoxide or peroxide, respectively (7). After 30 min heme was then added and cells were incubated at 37°C over the period of 1 hr. A Bio-Tek florescent plate reader using excitation/emission filters of 485/620 nm and 485/528 nm were used for detection with DHE and DCF, respectively. DHE measurements were made immediately after removal of the incubation buffer. The background fluorescence from cell-free incubations was subtracted from data that is reported because heme increased the detection of peroxide in the absence of PMEC to levels that were 10% of the amounts observed in the presence of PMEC + heme. All probes employed were obtained from Sigma Chemical Company.
Measurements of cGMP.
The levels of cGMP in PMECs grown in 60-mm culture dishes were determined after removal of the medium containing FCS by using a 0.1 M HCl extraction and measurement with cGMP ELISA kits, as previously described (3).
Statistical Analysis.
Data are reported as mean ± SEM from four to eight separate experiments and were analyzed by Student t tests or ANOVA with a Bonferroni correction.
Results
Lowering Serum Heme Increases Peroxide Generation and Decreases cGMP Through a Mechanism Not Associated with Increased Intracellular Superoxide.
Incubation of PMECs with 5% of the serum heme levels of 0.5–1 µM that are normally present in the FCS-containing culture media for 24 hr caused a decrease in cGMP (Fig. 1
), which was associated with a decrease in the detection of peroxide (Fig. 2A), without alterations in the detection of intracellular superoxide (Fig. 2B). Increased levels of HO-1 decreased cGMP levels, without altering the detection of peroxide or intracellular superoxide, which suggests that changes in cGMP were not related to the levels of superoxide that were detected.
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show that exposure of PMECs to 30 µM heme, conditions previously shown to be associated with marked increases in cGMP in PMEC (3), results in a marked increase in the detection of peroxide without increases in intracellular superoxide. The marked increases in peroxide caused by heme were not detectably altered by the presence of elevated levels of HO-1 (Fig. 2A). Because the presence of added catalase almost eliminates the heme-induced increase in peroxide, an extracellular source of this oxidant species is being detected (Fig. 3A). Ebselen is a probe that is thought to catalytically remove peroxide in a manner similar to glutathione peroxidase when intracellular reducing systems are present (8). This probe had only a minor inhibitory effect on the detection of heme-induced increases in peroxide (Fig. 3B), suggesting the source of peroxide generation was primarily extracellular.
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, the increased generation of peroxide did not alter the marked stimulation of peroxide detection by heme. Although exposure of PMECs to decreased serum heme lowered the levels of peroxide that were detected, the data in Figure 2A indicate that increased levels of peroxide were observed when the low serum exposed PMECs were subsequently treated with heme.
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), consistent with NOS (a flavoprotein inhibited by DPI) being a major contributor to the basal source of peroxide that was detected. Because NLA did not alter the levels of peroxide detected in the presence of heme (Fig. 5B), the production of peroxide by NOS in PMEC did not contribute to the increased levels of peroxide detected in the presence of heme. Based on the data in Figures 4 and 5, increased levels of HO-1 seemed to have minimal effects on the influence of methods of modulating basal cellular peroxide generation on the heme-stimulated production of peroxide.
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Exposure of PMECs to heme resulted in a marked increase in DCF fluorescence that was inhibited by the addition of catalase, consistent with detection of heme promoting extracellular hydrogen peroxide generation. Changes in endogenous cellular peroxide levels caused by stimulation of oxidase activity presumably through growth factors present in FCS (9) or by inhibition of peroxide biosynthesis with DPI and NLA did not alter the increases in peroxide caused by heme in a manner that could be associated with a role for these cellular sources of peroxide in the response to heme. For example, changing FCS concentration in the growth media from 0.05–10% increased basal peroxide generation and decreased the increase in peroxide caused by heme. Because enhancement of intracellular peroxide metabolism with ebselen and impairment of its metabolism by depletion of glutathione with diethylmaleate had only minor effects on the detection of heme-induced increases in peroxide, the observed increases in peroxide appear to be occurring primarily in the extracellular environment, through processes that do not appear to be associated with changes in the activities of intracellular peroxide generating systems.
The previously reported (3) effects of 30 µM heme and HO-1 on sGC activity and the absence of an increase in the detection of intracellular superoxide observed in PMECs under these conditions is consistent with this concentration of heme and HO-1 having a regulatory effects on sGC activity through mechanisms involving NO and CO, which are independent of changes in the actions of superoxide. Superoxide levels detected with DHE were not influenced by changes in heme or HO-1, indicating that this oxidant species was not responsible for the alterations in cGMP levels that were observed under these conditions. Because the basal levels of cGMP in PMEC grown in 10% FCS were not altered by inhibiting NOS (3), another mechanism sensitive to the lowering of heme with decreased FCS and increased HO-1 expression appears to be controlling the activity of sGC. The previously reported marked increase in cGMP caused by 30 µM heme seems to be partially dependent on the stimulation of sGC by CO and NO due to the marked increase in CO observed under these conditions and the partial attenuation of cGMP increases caused by inhibition of NOS (3). Since peroxide stimulates the activity of sGC (10) and NOS (11), it may also be a factor in the observed increase in cGMP caused by heme. However, the enhancement of increases in cGMP observed in HO-1 PMEC is associated with increased production of CO (3), and not with increased peroxide (Fig. 2), suggesting an enhanced role for CO in the stimulation of sGC through the metabolism of heme by HO-1. In contrast, peroxide generation by heme may be a contributing factor observed in previous studies examining the concentration-dependence of heme on adhesion protein expression in endothelium, an oxidant signaling-associated process that appeared to have a threshold of detection at 50 µM heme (4, 12). Thus, concentrations of heme in the range of up to 30 µM have signaling effects in endothelium influenced by HO-1 expression, which appear to be dominated by changes in NO, CO, heme levels, and the activity of sGC, and other systems regulated by these mediators that appear to be independent of changes in oxidant species.
Heme has previously been observed to generate extracellular oxidant species in the presence of biological molecules such as LDL, which are susceptible to oxidation (2). A novel observation in the present study is that heme can interact with endothelium or metabolites normally released from PMECs during brief incubations with glucose-containing saline buffer that results in the detection of peroxide. Interestingly, marked changes in intracellular peroxide did not have a detectable effect on the extracellular generation of peroxide by heme. Thus, extracellular oxidant species derived from the endothelium-heme interaction detected in the present study could have important effects on circulatory function under conditions of slightly elevated blood heme levels.
Footnotes
Supported by USPHS Grants HL31069, HL43023, HL55601, and HL66331 and AHA Grant 50948T.
1 To whom requests for reprints should be addressed at Department of Physiology, New York Medical College, Valhalla, NY 10595. E-mail: mike_wolin{at}nymc.edu 
References
This article has been cited by other articles:
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  S. V. Brodsky, F. Zhang, A. Nasjletti, and M. S. Goligorsky Endothelium-derived microparticles impair endothelial function in vitro Am J Physiol Heart Circ Physiol, May 1, 2004; 286(5): H1910 - H1915. [Abstract] [Full Text] [PDF]
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