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Laboratory for Research in Neonatal Physiology, Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163
Submitted 9 December 2002 ; accepted in final form 3 March 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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heme oxygenase; cerebrovascular; phosphorylation; calcium; carbon monoxide
Numerous questions remain relevant to CO and cerebral vasodilation. Of particular interest, and the subject of this study, is How is CO production controlled? Assuming that total cellular HO-2 does not change during the experimental time course, CO production could be controlled by regulation of substrate availability to HO-2 or the effective catalytic activity of HO-2. Catalytic activity includes the specific catalytic efficiency of the enzyme as well as intracellular localization to optimize substrate and cofactor proximity. CO production appears to be at least partially substrate dependent, because provision of exogenous substrate (heme) causes dilation that can be blocked by HO inhibitors (8). Whether cellular production of heme is used to regulate CO production by HO-2 remains an open question. HO-2 activity could be regulated at the catalytic level. Elevation of HO-2 catalytic activity by serine phosphorylation has been described (4). Whether alteration of cytosolic Ca concentration or tyrosine phosphorylation affects CO production is not known. Overall, considering the constitutive nature, cellular localization, and marked physiological effects of its products, the rudimentary level of our understanding of the mechanisms involved in control of HO-2 activity and cellular CO generation is astounding.
Therefore, this study, which uses freshly isolated piglet cerebral microvessels, was designed to address the hypothesis that CO production can be regulated by heme delivery and, at the catalytic level, be regulated by HO-2 catalytic activity. Whether cytosolic Ca signaling and/or protein phosphorylation could be involved is examined.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation of cerebral microvessels. Cerebral microvessels were isolated from the cerebral cortex as described before (2, 15, 17). The isolation was accomplished in cold Krebs solution [in mM: 120 NaCl, 5 KCl, 0.62 MgSO4, 1.8 CaCl2, 10 HEPES, and 6 glucose (pH 7.4)]. The dura mater and attached vessels were removed from the tissue, and the brain cortex tissue was washed three times with Krebs solution. The tissue was minced into tiny pieces using two scalpels in Krebs solution, transferred to a 40-ml Dounce homogenizer, and homogenized by 10 strokes of a loose-fitting pestle. The homogenate was passed through a 300-µm nylon mesh screen. The passage was refiltered over a 60-µm nylon mesh screen. The screen was removed and placed in a 50-ml centrifuge tube that contained Krebs solution. Microvessels (60–300 µm) were washed by agitation and scraping and were then centrifuged at 1,200 rpm for 5 min. Experimentation began immediately after vessel collection with resuspension of the microvessels in Krebs solution.
Experimental treatments. Treatments were started by replacement of the Krebs solution in the vial with fresh Krebs solution that contained the experimental treatment. Heme was prepared as heme-L-lysinate and was protected from light; aminolevulinate (ALA) was dissolved in Krebs solution; and ionomycin, PMA, tyrphostin-47, genestein, and phenyl arsine oxide (PAO) were dissolved in DMSO and diluted a minimum of 100-fold with Krebs solution. Ca-free Krebs solution was prepared by replacing CaCl2 with MgCl2 and including 4 mM EGTA and 5 x 10-6 M ionomycin. Orthovanadate and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) were dissolved in water and diluted with Krebs solution.
The apparent catalytic activity of HO in the intact cerebral microvessels was determined by providing exogenous substrate so that endogenous substrate delivery would not affect CO production. We assume that under the present experimental conditions, treatments used are unlikely to markedly alter O2 partial pressure or cellular reducing equivalents. Thus it seems reasonable to propose that catalytic activity (defined as the quantity of CO production per milligram of protein when substrate concentration is high and constant) includes HO-2 catalytic efficiency and fractional activation by intracellular relocation.
Measurement of CO production. For measurement of CO production, freshly isolated microvessels were placed inside amber vials (2.0 ml) that contained Krebs solution. All subsequent assay steps were carried out in the dark to prevent nonenzymatic photooxidative production of CO ex vivo due to the photodegradation of organic compounds. For the experiments in which the HO inhibitor zinc protoporphyrin (ZnPP, 2 x 10-5 M) was used, the vessels were pretreated with ZnPP for 30 min before the experiment was started and the inhibitor was maintained throughout. The internal standard (see below) was injected into the bottom of the Krebs solution in the vial, and the vial was immediately sealed with a rubberized Teflon-lined cap. Cerebral microvessels were incubated for 30 min at 37°C. Incubations were terminated by placing the samples in hot water (75°C).
A saturated solution of the isotopically labeled CO (13C16O; isotopic purity >99%) was used as the internal standard for quantitative measurements by gas chromatography-mass spectrometry (GC-MS; Refs. 8 and 17). GC-MS analysis of the headspace gas was performed using a Hewlett-Packard 5970 mass-selective ion detector interfaced to a Hewlett-Packard 5890A gas chromatograph. The separation of CO from other gases was carried out on a Varian-5A mole-sieve capillary column (30 m height; 0.32 mm ID) with a linear temperature gradient from 35 to 65°C at 5°/min. Helium was the carrier gas at a column-head pressure of 4.0 psi. Immediately after termination of incubation, aliquots (100 µl) of the headspace gas were injected using a gas-tight syringe into the splitless injector, which had a temperature of 120°C. Ions with massto-charge ratios (m/z) of 28 and 29, which correspond to 12C16O and 13C16O, respectively, were recorded via selective ion monitoring. The amount of CO in samples was calculated from the ratio of peak areas of m/z values of 28 and 29. The results are expressed as picomoles of CO released into the headspace gas per 100 µg of protein in 30 min. Protein was measured by the Bradford method.
Statistical analysis. Values are presented as means ± SE. The results were subjected to ANOVA for repeated measures with Tukey's post hoc test to isolate differences between groups. A level of P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ZnPP was used solely to show that a known inhibitor of HO could prevent CO from being produced in response to exogenous substrate. The dose of ZnPP was selected because when we used cranial windows in intact piglets, we found that 2 x 10-5 M ZnPP, similar to the chromium mesoporphyrin concentration (8), blocked dilation of pial arterioles caused by heme but not dilation caused by CO. The data shown in the presence of ZnPP (see Figs. 1 and 2) are for ZnPP combined with the highest level of substrate (heme or ALA) used. No attempts were made to evaluate whether inhibition was complete, i.e., repeatedly rinsing vessels and replacing medium after incubation with ZnPP, allowing longer preincubation, etc. Clearly, even in the presence of excess substrate, ZnPP lowered CO production to below the basal production level. Additional control experiments were performed in which all of the treatments were incubated in the vials without microvessels, and no CO production was detected. In mammalian cells, the only known source of CO is heme metabolism by HO, so we assume that the CO measured when ZnPP was present was due to incomplete inhibition or residual CO from before addition of the inhibitor.
Elevations of cytosolic Ca could affect heme delivery and/or HO-2 catalytic activity. Therefore, we administered treatments that affect cytosolic Ca and measured CO production. To elevate cytosolic Ca, cerebral microvessels were treated with ionomycin in Ca-replete media. Ionomycin dose dependently increased CO production from endogenous substrate (Fig. 3). In contrast, the catalytic activity of HO did not appear to be affected by changes in cytosolic Ca concentration. Ionomycin did not change the rate of CO production from exogenous heme in the presence of normal extracellular Ca (Fig. 4). Furthermore, when microvessels were incubated in Ca-free medium with ionomycin to reduce cytosolic Ca, CO production from exogenous heme was equal to production when extracellular Ca was normal and ionomycin was not present.
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Protein phosphorylation is a common cellular mechanism for altering enzyme activity posttranscriptionally. Therefore, we determined whether the PKC activator PMA increased CO production. PMA did dose dependently increase CO production from endogenous substrate in cerebral microvessels (Fig. 5). However, PMA did not alter the catalytic activity of HO-2 as assessed by conversion of exogenous heme to CO (Fig. 5). Additionally, the PKC inhibitor H-7 did not affect the catalytic activity of HO-2 (Fig. 6). These data suggest that heme availability for HO-2-mediated catabolism may be increased via activation of PKC but do not suggest that PKC-mediated phosphorylation alters the catalytic activity of HO-2 in piglet cerebral microvessels.
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Tyrosine phosphorylation does appear to contribute to regulation of HO-2 catalytic activity. The PTK inhibitors genistein and tyrphostin-47 reduced CO production from endogenous substrate (Fig. 7). In addition, PTK inhibition decreased the catalytic activity of HO-2 as measured by generation of CO from exogenous heme (Fig. 7). Furthermore, the protein tyrosine phosphatase (PTP) inhibitors sodium orthovanadate and PAO increased CO production from endogenous and exogenous heme (Fig. 8). Therefore, it appears that tyrosine phosphorylation is important for HO-2 activity. Whether tyrosine kinases and/or phosphatases affect heme availability is less certain but may be suggested by the greater effect of PTP inhibitors when exogenous heme is not provided.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present experiments, freshly isolated cerebral microvessels were used. This is important for HO-2 activity regulation experiments, because freshly isolated microvessels only express HO-2 (8). Conversely, the mitogens required for cell proliferation and maintenance in culture induce expression of the inducible HO isoform HO-1.
HO-2 is expressed constitutively. HO-2 generates CO from cellular heme.
Heme is produced in cells from glycine with the rate-limiting enzyme in
porphyrin biosynthesis being 
-ALA synthase (ALAS; Refs.
6 and
9). Cellular mechanisms of
regulation of HO-2 activity and processes involved in control of heme delivery
are poorly understood. The constitutive nature of HO-2 in the brain and brain
vasculature would seem to dictate mechanisms for regulation of CO production
either by control of HO-2 catalytic activity or substrate delivery. CO
production by HO-2 could be controlled by delivery of electrons from NADPH via
cytochrome P-450 reductase, by delivery of heme to the enzyme (all
tissues make heme), and negatively by biliverdin inhibition of HO-2
(9,
10,
19). It has been demonstrated
that heme catabolism via HO-1 can be limited by the availability of heme
(1). The present results
suggest that the catabolism of heme physiologically by HO-2 is substrate
limited, because either ALA or, more directly, heme increases CO production.
Whether HO-2 catalytic activity is Ca dependent had not been reported
previously. Regulation of enzyme activity by phosphorylation also has been
little explored. Two other enzymes that produce key autocrine and/or paracrine
mediators, cyclooxygenase-2
(13) and nitric oxide synthase
(5), can be rapidly altered
posttranscriptionally by tyrosine phosphorylation; this now appears to be the
case with HO-2.
The heme that is the substrate for HO-2 is intracellular heme, which is continually being produced, assimilated into enzymes, and degraded. Clearly, there are marked intracellular localizations of heme with higher amounts in mitochondria, which is where the first as well as the last three reactions in heme synthesis occur. ALAS, the rate-limiting and most regulated enzyme in heme synthesis, is inhibited in a negative-feedback manner by mitochondrial heme, and ALAS gene expression is regulated by cellular heme. Because the heme (and ALA) in this experiment was topically applied extracellularly, the cytoplasmic and mitochondrian concentrations are not known. It appears that the intracellular heme concentration that we achieved is in excess of that obtained by endogenous production, because the CO production was greater than can be achieved by known agonist stimulation. HO-2 is an endoplasmic reticulum enzyme, so cytoplasmic heme concentration would be relevant to substrate delivery. If one concedes that Bach 1 is a transcription factor by which intracellular heme can regulate gene expression, it is logical to assume that cellular heme concentration is within the binding range for Bach 1. The Kd for hemin binding to Bach 1 is on the order of 10-7 M (12). Furthermore, inhibition of Bach 1 DNA binding by hemin occurred over the range of 3 x 10-8 to 3 x 10-6 M. Thus the significant elevation of CO production produced when heme was applied at 10-6 M and the strong tendency for an increase at 10-7 M suggest that the heme applied exogenously readily entered the cell and produced cytoplasmic heme concentrations near the conceivable physiological range.
These data are the first to indicate that increased cellular tyrosine phosphorylation increases HO-2 catalytic activity. It is unlikely that the effects of the inhibitors are unrelated to the desired action, because we used two different PTK inhibitors and two different PTP inhibitors and the results were virtually identical. A previous report indicated that HO-2 catalytic activity in rat and mice neuronal cultures could be increased by serine phosphorylation (4), but we could not detect an effect of either PMA or H-7 on conversion of exogenous heme to CO in piglet cerebral microvessels. It remains possible that if a PKC species that is resistant or inaccessible to H-7 was involved, PKC could play a role in regulating CO production. The present data open the field of HO-2 regulation by tyrosine phosphorylation, but the mechanisms involved have not been addressed. Questions related to whether the tyrosine phosphorylation effect on activity is direct or via another enzyme that alters HO-2 catalytic activity, the phosphorylation sites, and the kinases and/or phosphatases involved, must be pursued in future experiments. Whether increased tyrosine phosphorylation changes the catalytic activity of the enzyme intrinsically or causes intracellular relocation is likewise not addressed.
Either elevation of cytosolic Ca with ionomycin or activation of PKC with PMA stimulates CO production by cerebral microvessels. Although cytosolic Ca was not measured in the present experiments, we are confident that the treatments altered cytosolic Ca as desired. First, ionomycin treatment with Ca-replete medium did increase CO production. Second, we previously measured cerebral microvascular smooth muscle (data not shown) and endothelial (14) Ca changes caused by ionomycin and Ca-replete and Ca-free media. Of course, ionomycin and PMA could share a common mechanism, because activation of PKC can produce an increase in cytosolic Ca. However, whatever mechanisms are involved in ionomycin- and phorbol ester-induced CO production must result from alterations of heme delivery, because conversion of exogenous heme to CO, i.e., catalytic activity of HO-2, is not changed.
CO is a physiologically significant messenger molecule in the brain. The enzyme responsible for CO production, HO-2, is found in highest concentrations in the brain with localization in neurons and vascular endothelium (9, 18, 20). Endogenously produced CO may contribute to regulation of vascular tone. Endogenously produced CO and exogenous CO can cause dilation of arteries and arterioles (3, 7). CO is a very potent dilator of the neonatal cerebral circulation (8). Exogenous heme can produce dilation that is blocked by chromium mesoporphyrin, HO-2 is strongly expressed in neonatal cerebral microvessels, and CO production levels by piglet cerebral microvessels are the highest of the tissues we examined (8) and rival production by olfactory neurons (6).
In conclusion, these data suggest that regulation of CO production in the newborn cerebral microvasculature could be accomplished by control of heme availability and posttranslational modification of HO-2 catalytic activity.
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ACKNOWLEDGMENTS |
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This research was supported by the National Heart, Lung, and Blood Institute; the American Heart Association, Southeast Affiliate; and the Vascular Biology Center of Excellence at the University of Tennessee Health Science Center. C. D. Sullivan was supported by a medical student summer-training grant from the National Institutes of Health.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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