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Am J Physiol Heart Circ Physiol 289: H1442-H1447, 2005. First published June 17, 2005; doi:10.1152/ajpheart.00464.2005
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Nitric oxide increases carbon monoxide production by piglet cerebral microvessels

Charles W. Leffler, Liliya Balabanova, Alexander L. Fedinec, and Helena Parfenova

Laboratory for Research in Neonatal Physiology, Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee

Submitted 6 May 2005 ; accepted in final form 9 June 2005


   ABSTRACT
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Carbon monoxide (CO) and nitric oxide (NO) can be involved in the regulation of cerebral circulation. Inhibition of production of either one of these gaseous intercellular messengers inhibits newborn pig cerebral arteriolar dilation to the excitatory amino acid glutamate. Glutamate can increase NO production. Therefore, the present study tests the hypothesis that NO, which is increased by glutamate, stimulates the production of CO by cerebral microvessels. Experiments used freshly isolated cerebral microvessels from piglets that express only heme oxygenase-2 (HO-2). CO production was measured by gas chromatography-mass spectrometry. Although inhibition of nitric oxide synthase (NOS) with N{omega}-nitro-L-arginine (L-NNA) did not alter basal HO-2 catalytic activity or CO production, L-NNA blocked glutamate stimulation of HO-2 activity and CO production. Furthermore, the NO donor sodium nitroprusside mimicked the actions of glutamate on HO-2 and CO production. The action of NO appears to be via cGMP because 8-bromo-cGMP mimics and 1H-[1,2,4]oxadiazole-[4,3-a]quinoxalin-1-one (ODQ) blocks glutamate stimulation of CO production and HO-2 catalytic activity. Inhibitors of neither casein kinase nor phosphotidylinositol 3-kinase altered HO-2 catalytic activity. Conversely, inhibition of calmodulin with calmidazolium chloride blocked glutamate stimulation of CO production and reduced HO-2 catalytic activity. These data suggest that glutamate may activate NOS producing NO that leads to CO synthesis via a cGMP-dependent elevation of HO-2 catalytic activity. These results are consistent with the findings in vivo that either HO or NOS inhibition blocks cerebrovascular dilation to glutamate in piglets.

heme oxygenase; guanosine 3'; 5'-cyclic monophosphate; nitric oxide synthase; glutamate

BOTH CARBON MONOXIDE (CO) and nitric oxide (NO) are endogenously produced, gaseous, intercellular messengers that can be involved in regulation of cerebral circulation. In neonatal pigs, CO regulation and modulation are involved in cerebrovascular circulatory control in response to neuronal activity, hypoxia, and changing blood pressure (12, 18, 26, 41). Whereas the contributions of NO to cerebral blood flow regulation increase with age (40, 47), NO is important in the newborn as a permissive factor enabling vascular responses to CO (15). In the piglet cerebrovascular circulation, glutamate-induced pial arteriolar dilation can be blocked by either inhibiting nitric oxide synthase (NOS) (14, 24), which produces NO, or heme oxygenase (HO) (18, 30), which produces CO. One possible explanation for these apparently conflicting data is that one gaseous messenger is necessary to allow dilation to the other. Indeed, as noted above, such a permissive contribution of NO to CO-induced dilation has been described. Another possibility is that glutamate receptor activation increases the production of one of the two gases and that gas in turn increases the production of the other, which is the final mediator of the dilatory response.

CO has been reported to directly affect NO production. In intestinal smooth muscle, CO increased NO that activated L-type Ca2+ channels (19). Conversely, CO dose dependently inhibited NO synthesis by rat renal arteries, although low concentrations of CO actually increased NO by causing release from a preformed pool (38).

Studies to date regarding the effects of NO on CO production have reported both increases and decreases of CO production caused by NO. Thus HO-2 expressed in Escherichia coli was inhibited by NO donors via binding of NO to a heme regulatory motif on HO-2 (5). Also, Rodriguez et al. (31) found that rats treated for 2 days with the NOS inhibitor NG-nitro-L-arginine methyl ester had increased renal CO production without any change in HO expression. Conversely, in isolated hearts (22) and porcine aortic endothelial cells (25) NO increased CO production.

Because both inhibition of NOS and HO block glutamate-induced dilation, and glutamate has been reported to increase NO production (6, 8), we wondered whether NO could stimulate CO production, adding another form of interaction to the permissive action of NO in CO-mediated dilation to glutamate. Therefore, the present experiments were designed to test the hypothesis that NO stimulates CO production by piglet cerebral microvessels. We also examined potential mechanisms by which NO could stimulate the production of CO.


   MATERIALS AND METHODS
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RESULTS
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Experiments using animals were reviewed and approved by the Animal Care and Use Committee of the University of Tennessee Health Science Center. Brains were removed from 1- to 3-day-old piglets under ketamine (33 mg/kg) and acepromazine (3.3 mg/kg) anesthesia.

Isolation of cerebral microvessels. Cerebral microvessels were isolated from the brains as described before (15, 16). 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 tissue was washed three times with the isolation solution. The tissue was minced into tiny pieces using two scalpels in isolation solution and then transferred to a 40-ml Dounce homogenizer and homogenized with 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 containing Krebs solution. Microvessels that passed through the 300-µm but not 60-µm mesh screen were washed off by agitation and scraping and then centrifuged at 1,200 rpm for 5 min. Experimentation began immediately after vessel isolation (<30 min from brain removal) with resuspension of the microvessels in Krebs solution.

Experimental treatments. Treatments were begun by replacement of Krebs solution in the vial with fresh Krebs containing the experimental treatment. Glutamate (10–4 M), N{omega}-nitro-L-arginine (L-NNA)(10–3 M), sodium nitroprusside (SNP) (10–7-10–5 M), 8-bromo-cGMP (10–5 M), 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB) (2 x 10–5 M), LY-294002 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY-294002) (2.5 x 10–5 M), and calmidizolium chloride (CzCl) (2 x 10–5 M) were dissolved in Krebs solution. Heme (5 x 10–6 M) was prepared as heme-L-lysinate or hemin in basic Krebs and protected from light. Guanylyl cyclase was inhibited with 1H-[1,2,4]-oxadiazolo-[4,3-a] quinoxaline-1-one (ODQ). ODQ (5 x 10–4 M) was initially dissolved in DMSO to a concentration of 4 x 10–2 M and diluted ~100 times with Krebs. DMSO at double this concentration did not effect CO production or HO-2 catalytic activity.

The concentrations of treatments used were selected based on one or more of three sources. First, where data were available, inhibitor concentrations were those that had been found to be effective in vivo from topical application to the piglet cerebral cortex using cranial windows [L-NNA (17) and ODQ (14)]. Second, other inhibitor concentrations were selected from literature sources of use in vitro [TBB (1), LY-294002 (7), and CzCl (2)]. Finally, agonist concentrations were in all cases selected from submaximal dilator concentrations in piglet cranial window experiments [heme (18), glutamate (14), SNP (17), and 8-bromo-cGMP (14)].

The apparent catalytic activity of HO-2 in the intact cerebral microvessels was determined by providing exogenous substrate so endogenous substrate availability 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 CO production per milligram protein when substrate concentration is high and constant, includes HO 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) containing Krebs solution. All subsequent assay steps were carried out in the dark to prevent nonenzymatic photo-oxidative production of CO ex vivo due to the photodegradation of organic compounds. Krebs buffer in each vial was replaced with fresh Krebs or fresh Krebs solution containing the experimental treatment to begin incubation. The internal standard (see below) was injected into the bottom of 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 ice water (0°C), and CO production was determined immediately.

A saturated solution of the isotopically labeled CO (13C16O) (isotopic purity >99%) was used as an internal standard for quantitative measurements by gas chromatography-mass spectrometry (GC-MS) (15, 16).

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; 0.32 mm ID) with a linear temperature gradient from 35°C to 65°C at 5° per minute. Helium was the carrier gas at a column head pressure of 4.0 psi. Aliquots (100 µl) of the headspace gas were injected using a gas-tight syringe into the splitless injector having a temperature of 120°C. Ions at m/z 28 and 29 corresponding 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 28 and m/z 29. The results are expressed as picomoles of CO released into the headspace gas per 100 µg 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 post hoc to isolate differences between groups. A level of P < 0.05 was considered significant.


   RESULTS
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Acute NOS inhibition did not affect either basal CO production or HO-2 catalytic activity. Thus, 30–60 min after treatment, cerebral microvessels produced 19 ± 3 pmol CO/100 µg·30 min without and 24 ± 4 pmol CO/10 µg·30 min with L-NNA treatment (n = 4 separate microvessel preparations). Furthermore, HO-2 catalytic activity, detected as heme-stimulated CO production, was 51 ± 11 pmol CO/100 µg·30 min before and 51 ± 8 pmol CO/100 µg·30 min after L-NNA treatment (n = 4).

In contrast, NOS inhibition markedly attenuated glutamate stimulation of CO production (Fig. 1). Furthermore, augmentation of HO-2 catalytic activity by glutamate was attenuated when microvessels were treated with L-NNA (Fig. 1). Thus, in L-NNA-treated microvessels, exogenous heme-stimulated CO production was no different whether or not glutamate was applied (Fig. 1). These data suggest that NO may be involved in the mechanism by which glutamate stimulates CO production.



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  		Fig. 1. Effect of glutamate (10–4 M) on carbon monoxide (CO) production by piglet cerebral microvessels from endogenous and exogenous heme (5 x 10–6 M) in the absence and presence of N{omega}-nitro-L-arginine (L-NNA; 10–3 M). Values are means ± SE. *P < 0.05 compared with preceding bar. n = 4 animals.

 
Because the data above suggest that NO may increase CO production, we addressed this hypothesis directly by using the NO donor SNP. SNP strongly stimulated CO production (Fig. 2). In addition, similarly to glutamate, SNP augmented HO-2 catalytic activity (Fig. 2). These data are consistent with the hypothesis that glutamate increases NO that increases CO production by elevating HO-2 catalytic activity.



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  		Fig. 2. Effect of sodium nitroprusside (SNP) on CO production and heme oxygenase-2 (HO-2) catalytic activity (exogenous heme, 5 x 10–6 M) by piglet cerebral microvessels. Values are means ± SE. *P < 0.05 compared with no sodium nitroprusside (SNP). {dagger}P < 0.05 compared with exogenous heme with no SNP. n = 4 animals.

 
Because NO is a strong activator of guanylyl cyclase, we addressed the hypothesis that the mechanism by which NO increases HO-2 activity and thus CO production is by increasing cGMP. Initially, guanylyl cyclase was blocked with ODQ (14). As anticipated, ODQ decreased CO production by cerebral microvessels and strongly attenuated the increase CO caused by exogenous heme (catalytic activity) (Fig. 3). Furthermore, as would be expected if glutamate increases NO that elevates cGMP that then augments CO production, ODQ blocked glutamate-induced CO production (Fig. 4) and glutamate-induced stimulation of HO-2 activity (93 vs. 40 pmol CO/10 µg·30 min without and with ODQ, respectively). In addition, the stable cGMP analog 8-bromo-cGMP stimulated CO production (Fig. 5). In the presence of sufficient 8-bromo-cGMP to increase CO production similarly to glutamate, glutamate did not increase and L-NNA did not decrease CO production (Fig. 5). These data suggest that glutamate may increase CO production by stimulating NO production by the microvessels. NO activates guanylyl cyclase producing cGMP that increases the catalytic activity of HO-2. Data in Figs. 2 and 5 suggest 60–70 pmol CO/100 µg protein·30 min is approximately maximal CO production without provision of additional substrate (as in Figs. 1 and 3). The fact that glutamate did not further increase CO production when coadministered with 10–5 M 8-bromo-cGMP suggests glutamate may not increase cellular heme.



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  		Fig. 3. Effect of 1H-[1,2,4]oxadiazole-[4,3-a]quinoxalin-1-one (ODQ, 5 x 10–4 M) on CO production from endogenous and exogenous heme (5 x 10–6 M) by piglet cerebral microvessels. Values are means ± SE. *P < 0.05 compared with control. {dagger}P < 0.05 compared with heme without ODQ. n = 6 animals.

 


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  		Fig. 4. Effect of ODQ (5 x 10–4 M) on glutamate (10–4 M) stimulation of CO production by piglet cerebral microvessels. Values are means ± SE. *P < 0.05 compared with control. {dagger}P < 0.05 compared with ODQ without glutamate. n = 6 animals.

 


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  		Fig. 5. Effect of 8-bromo-cGMP (8-BrcGMP, 10–5 M) on CO production by piglet cerebral microvessels without and with glutamate (10–4 M) and/or L-NNA (10–3 M). Values are means ± SE. *P < 0.05 compared with L-NNA alone. n = 8 animals.

 
The present results and past data from others and us (see DISCUSSION) have shown that phosphorylation can increase HO-2 catalytic activity. Therefore, we examined the possibility that two kinases that have been shown to alter HO-2 catalytic activity in other tissues of rats might do the same in piglet cerebral microvessels. The casein kinase inhibitor TBB (20 µM) did not change HO-2 catalytic activity detected by conversion of exogenous heme (10–6 M) to CO (106 ± 29 and 104 ± 25 pmol/100 µg protein·30 min without and with TBB, respectively, n = 8), and glutamate (1 mM) still increased HO-2 catalytic activity (160 ± 31 and 220 ± 45 pmol/100 µg protein·30 min without and with TBB, respectively. n = 8). Similarly, the phosphotidylinositol 3-kinase inhibitor LY-294002 (25 µM) did not alter catalytic activity in piglet microvessels (at 10–4 M heme: 76 ± 21 and 90 ± 26 pmol/100 µg protein·30 min without and with LY-294002, respectively, n = 8).

Because glutamate may increase cytosolic Ca2+ (37) and Ca2+/calmodulin (CaM)-dependent endothelium NOS (eNOS) increases NO in endothelial cells in response to increased cytosolic Ca2+ (20, 32), we examined the effect of CaM inhibition with CzCl on glutamate-induced CO production (Fig. 6). Whereas CzCl had no effect on basal CO production, it completely blocked glutamate stimulation of CO production. Furthermore, CzCl reduced HO-2 catalytic activity [CO production from exogenous heme (10–5 M)] from 53 ± 7 to 36 ± 5 pmol CO/100 µg protein·30 min (P < 0.05, n = 4).



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  		Fig. 6. Effect of calmidizolium Cl (CzCl) (2 x 10–5 M) on glutamate (10–4 M) stimulation of CO production by piglet cerebral microvessels. Values are means ± SE. *P < 0.05 compared with control. n = 7 animals.

 

   DISCUSSION
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The new findings on newborn pig cerebral microvessels are the following: 1) basal NO production does not appear to be involved in either controlling HO-2 catalytic activity or basal CO production; 2) increasing NO and cGMP elevates HO-2 catalytic activity and stimulates CO production; 3) glutamate elevates NO and, thereby, cGMP, which enhances HO-2 catalytic activity, causing CO production to increase; and 4) CaM is required for glutamate-induced stimulation of HO-2 activity and CO production.

Cellular CO production results from metabolism of heme by HO. In freshly isolated cerebral microvessels from newborn pigs, as in the intact brain, in vivo, of the two known, highly catalytically active isoforms of HO, only HO-2 expression is detectable (28). HO-2 is constitutively expressed and induced by few stimuli (21), so expression of HO in the present experiments can be considered invariant.

Therefore, CO production can be regulated by delivery of substrate (heme) and catalytic activity of HO-2. HO-2 catalytic activity may be altered by cofactor availability, cellular localization, and/or posttranslational modifications of the enzyme. Under the experimental conditions used, it is unlikely that oxygen, NADPH, or NADPH-cytochrome c reductase would be limiting. The rate-limiting step in heme synthesis is the production in mitochondria of {delta}-aminolevulinic acid from succinyl CoA and glycine catalyzed by the tightly regulated enzyme {delta}-aminolevulinic acid synthase (13, 23). However, we did not find evidence of alteration of heme provision contributing to either glutamate- or NO-induced CO production. This conclusion is particularly supported by the finding that glutamate did not increase CO production when catalytic activity of HO-2 was already increased by 8-bromo-cGMP. If glutamate increased heme delivery, one would expect augmented CO production in the context of elevated HO-2 activity rather than no change. Therefore, it appears that glutamate, NO, and cGMP stimulate CO production by increasing HO-2 catalytic activity. Indeed, glutamate and SNP increased conversion of exogenous heme to CO.

HO-2 catalytic activity control mechanisms may be cell type and tissue specific. In neurons HO-2 activity can be stimulated by casein kinase 2 (CK2)-catalyzed phosphorylation of serine-79 (1). Glutamatergic activation of HO-2 results from metabotropic glutamate receptor-induced Ca2+ release, activation of protein kinase C (PKC), and CK2 phosphorylation (3). Conversely, in freshly isolated piglet cerebral microvessels and microvascular endothelial cells in culture, CO production is increased by ionotropic, but not metabotropic, glutamate receptor stimulation (27). In addition, protein tyrosine kinase inhibition decreased and tyrosine phosphatase inhibition increased basal CO production and glutamate stimulated CO production (15). Neither treatment of the cerebral microvessels with phorbol ester to activate PKC, H-7 to inhibit PKC, nor TBB to inhibit CK2 increased HO-2 catalytic activity (16 and present study).

In contrast to the present study, in preparations of HO-2 expressed in E. coli, NO donors inhibited HO-2 (5), possibly suggesting that NO can have a direct inhibitory effect on HO-2 that is masked in the intact system by cGMP-induced stimulation. Such a direct inhibitory effect of NO has been reported in HO-1-rich aortic endothelial cell microsomes where nitrosylation of heme prevented catabolism by HO (11). Interestingly, in contrast to the present studies of the effects of acute exposure (minutes) to NO, chronic exposure to NO consistently inhibits HO (11, 31).

CzCl, which inhibits CaM, decreased HO-2 catalytic activity and blocked glutamate stimulation of CO production. Also, Boehning et al. (2) showed Ca2+-CaM regulation of HO-2 catalytic activity. Similarly to the present experiments in piglet microvessels, glutamate increased HO-2 activity, and that increase was blocked by CzCl in rat cortical neurons. These results were surprising to us because we had found previously that the Ca2+ ionophore ionomycin in Ca2+-replete media increased CO production but did not increase HO-2 catalytic activity (15). Furthermore, ionomycin and Ca2+-free media to deplete cellular Ca2+ did not decrease basal CO production nor prevent glutamate-induced stimulation of CO production. Thus the results of our earlier study that elevations of cytosolic Ca2+ increased CO production but did not detectably increase HO-2 catalytic activity appear inconsistent with the present findings and those of Boehning et al. (2) However, in our previous study we either flooded the cell with Ca2+ with Ca2+ ionophore or eliminated extracellular Ca2+ and attempted to empty intracellular stores. In the present study, Ca2+ was not manipulated but instead Ca2+-CaM signaling was eliminated by blocking CaM. Interestingly, Ca2+-independent, CaM-dependent regulation of enzyme activity has been described (9, 35, 42) but not for HO-2. However, in rat cortical neurons, ionomycin did increase bilirubin production from exogenous heme (2). Explanations for the difference between rat cortical neurons and newborn pig microvascular endothelium in the effects of ionomycin on HO-2 catalytic activity are not immediately apparent.

Although both microvascular smooth muscle and endothelial cells are stimulated to generate CO by glutamate, CO production and the response to glutamate are more pronounced in endothelial cells (15). Piglet cerebrovascular endothelial cells express ionotropic and, to a lesser extent, metabotropic glutamate receptors, as well as glutamate transporters. Ionotropic receptor stimulation causes increased CO production (27). Ionotropic glutamate receptor stimulation can increase reactive oxygen species in endothelial cells (33). However, reactive oxygen species would be expected to decrease NO (10) and thus work against stimulation of CO production. Glutamate increases cytosolic Ca2+ via metabotropic and ionotropic receptor mechanisms in neurons (44) and glia (34, 36). Glutamate-induced elevations in Ca2+ also occur in endothelial cells (29). Because eNOS (see below) activity can be regulated by Ca2+-CaM and elevation of CO production by glutamate was blocked by CzCl, ionotropic glutamate receptor-induced elevation of cytosolic Ca2+ could be involved in glutamate-induced CO production.

The present data suggest NO is an intermediary signal between glutamate receptor stimulation and CO production in cerebral microvessels. The cell types included in the microvessel preparation are primarily endothelial cells and microvascular smooth muscle, with adhering pericytes, astrocytes, and perivascular nerve endings. Because the vessels are used immediately on collection, inducible NOS (iNOS) could not be induced. The predominant NOS is probably eNOS, but the potential inclusion of neuronal NOS (nNOS) cannot be excluded. Because eNOS can be activated to produce NO by an elevation of cytosolic Ca2+ (20, 32, 39, 46), glutamate may increase endothelial cell Ca2+ that would activate eNOS. Conversely, eNOS activity can be increased by elevations of eNOS sensitivity to CaM (reviewed in Ref. 43) so glutamate could increase NO without increasing cytosolic Ca2+ concentration.

These data on isolated vessels and those from intact cerebrovascular circulation in vivo are not entirely consistent, suggesting potential additional sources of CO in vivo may contribute to pial arteriolar dilation to glutamate. Thus in the present study L-NNA totally abolished glutamate-induced CO production. However, in vivo, whereas L-NNA blocked glutamate-induced dilation, a constant background amount of SNP completely restored dilation to glutamate (13). If CO causes dilation to glutamate and NO causes the increase in CO, how could glutamate cause dilation if NO is held constant? In vivo cerebral microvessels are accompanied by astrocytes and neurons, in particular, that also have glutamate receptors, HO-2, and nNOS. In fact, involvement of nNOS in glutamate-induced cerebrovascular dilation in the mouse cerebellum has been demonstrated (45). Furthermore, the inclusion of HO-2 and glutamate receptors in astrocytes and neurons provides the possibility of glutamate-induced stimulation of CO independent of NO in either of these cell types. Because the vascular smooth muscle appears to require a permissive level of cGMP that can be provided by eNOS-derived NO (15), a constant level of NO, or cGMP, could allow arteriolar dilation to occur in response to increases CO from neurons and/or glia.

cGMP is the predominant mechanism by which NO increases CO production in microvessels. Inhibition of guanylyl cyclase completely blocked the CO increases caused by glutamate and SNP and reduced HO-2 catalytic activity. 8-Bromo-cGMP mimicked the stimulatory effects of glutamate and SNP. Therefore, the present data are consistent with the hypothesis that NO increases HO-2 catalytic activity and glutamate-induced CO production by increasing cGMP.


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This study was supported by National Heart, Lung, and Blood Institute and National Institute of Neurological Disorders and Stroke.


   ACKNOWLEDGMENTS
 
We thank G. Short for preparing the figures and M. Lester for clerical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. W. Leffler, Dept. of Physiology, 894 Union Ave., Memphis, TN 38163 (e-mail: cleffler{at}physio1.utmem.edu)

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.


   REFERENCES
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