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Department of Physiology, University of Saskatchewan, Saskatchewan, Canada, S7N 5E5
Abstract
Enhancement of the heme oxygenase/carbon monoxide (HO/CO) system has been shown to lower blood pressure (BP) in young (8 weeks), but not in adult (20 weeks) spontaneously hypertensive (SHR) rats. The reasons for this selective effect still remain puzzling. We investigated the effects of hemin on the HO/CO system of the pulmonary artery (PA) in SHR and Wistar-Kyoto (WKY) rats at different ages and evaluated the hemin-dependent changes in sGC and cGMP pathways. Hemin administration resulted in an evident reduction of BP (from 148.6 ± 3.2 to 125.8 ± 2.6 mmHg, P < 0.01) in young, but not in prehypertensive (4 weeks) or adult SHR or WKY rats at all ages. Coadministration of the HO inhibitor, chromium mesoporphyrin, with hemin, cancelled the BP-lowering effect of hemin. Remarkably, lower expression levels of HO-1, HO-2, and sGC paralleled with reduced HO activity and cGMP content were observed in PA from 8-week SHR rats, but not from adult SHR or WKY rats of all ages. Interestingly, hemin treatment restored these deficiencies, although the expression level of non-inducible HO-2 protein remained unchanged. We conclude that in young and prehypertensive SHR rats, an impaired HO/CO-sGC/cGMP system in the PA might be indicative of the pathogenesis and development of hypertension. In contrast, the HO/CO system in the PA of adult SHR rats was upregulated as a compensatory reaction to elevated BP and desensitization of the downstream targets of the sGC/cGMP pathway occurred.
Key Words: carbon monoxide • gasotransmitter • heme oxygenase • hypertension • pulmonary artery
One of the predominant sources of endogenous carbon monoxide (CO) is the heme oxygenase (HO)-catalyzed degradation of heme. In this process, the cleavage of the 
-methene bridge produces equimolar amounts of CO, biliverdin, and iron. CO is now widely accepted as one of the endogenous gasotransmitters (1) and elicits relaxation of vascular smooth muscle cells (SMCs) through several mechanisms comprising the opening of calcium-activated K+ channel, acting on cytochrome P450, or activating the soluble gyanylyl cyclase (sGC)(2, 3). The stimulation of sGC causes elevation of cGMP levels, which affects cellular functions through its effects on ion channels, phosphodiesterase, and protein kinases (4). Thus, the sGC/cGMP pathway constitutes an essential part of the HO/CO system in regards to the regulation of vascular tone.
Induction of the inducible isoform of HO (HO-1) generally occurs as an adaptive and protective response to alterations in homeostasis (5). The implication of HO-1 in hypertension has been reported in studies that demonstrated that acute administration of HO-1 inhibitors elicits peripheral vasoconstriction and induced a transient increase in blood pressure (BP) in Sprague–Dawley rats (6), whereas treatment with heme lowered BP in hypertensive rats via a HO-dependent mechanism (7). These studies showed the essential role of the HO/CO system in the regulation of arterial pressure and hypertension.
CO of vascular SMC origin has been shown to inhibit the expression of endothelin-1 in endothelial cells (8), evoking an antihypertensive effect. Further evidence of endogenous CO as a modulator of blood vessel tone is provided by the use of HO substrates or inhibitors to stimulate or block the activity of HO which, in turn, increases or decrease the vasodilatory response to CO (9). The co-dependent vasorelaxation is not consistent in all vascular beds and variable intensities of CO-induced dilatory response were attained in different vascular beds (10, 11). A significant vasorelaxation induced by CO has been observed in the pulmonary vein and pulmonary artery (PA; ref. 12). An age-dependent decrease in CO-induced relaxation of PA in piglets has also been reported (12). Defective contractility of PA was implicated not only in the pathogenesis of pulmonary hypertension but also in different forms of systemic hypertension with distinctive etiological origins including renin- and genetic-dependent hypertension (13, 14). Also, a positive correlation between the cardiac secretion of brain naturetic peptide in essential hypertension patients and elevated PA pressure has been established (15). As such, the pathophysiological significance of a defective contractile/dilatory response of the PA extends beyond the boundaries of pulmonary hypertension to encompass systemic hypertension. For this reason, the PA has been used for contractility studies in the spontaneously hypertensive rat spontaneously hypertensive (SHR) rat model (14) as well as in studies involving the regulation of pulmonary vascular tone in acute Pseudomonas pneumonia (16) in the Sprague-Dawley rat model.
The use of HO inducers to attenuate elevated BP in SHR has been reported to be effective in young animals but not in adults (17, 18). The reasons for this selective BP regulation in young SHR is complex and yet to be elucidated. The correlation between the expression and function of HO/CO-sGC/cGMP system in PA and BP levels of SHR at various ages (4, 8, and 20 weeks) has not been reported. In the present study, we assessed the importance of a functional HO/CO-sGC/cGMP system in PA to the development of genetic hypertension in SHR, an animal model of human essential hypertension.
Materials and Methods
Animal Preparation.
The experimental protocol is in accordance with the principles and guidelines of the Canadian Council on Animal Care and the University of Saskatchewan Standing Committee on Animal Care and Supply approved the protocol. Male SHR and Wistar-Kyoto (WKY) rats were obtained from Charles River Laboratories (Willington, MA). Hemin (15 mg/kg/day) and chromium mesoporphyrin IX (CrMP; 4 µmol/kg) (Porphyrin Products, Logan, UT) were dissolved in 0.1 M NaOH, titrated to pH 7.4 with 0.1 M HCl, diluted 1:10 with phosphate buffer, and administered intraperitoneally for 4 consecutive days (18). Systolic BP was determined without anesthesia using a standard tail-cuff noninvasive BP measurement system (Model 29-SSP, Harvard Apparatus, Montreal, Canada) after acclimatization before and 23 hr after the last hemin or CrMP injection (18).
Carbon Monoxide Preparation and Treatment.
A saturated solution of CO was prepared as we previously described (2). Briefly, pure CO (99.99%; Canadian Liquid Air Ltd., Oshawa, ON, Canada) was bubbled into a 20 ml solution of phosphate-buffered saline in a sealed glass tube for 20 min under a pressure of 100 kilopascals at 37°C. One microliter of this CO-saturated solution contained 30 ng of the gas. The desired concentration (1 µM) was obtained by diluting the CO-saturated solution with phosphate-buffered saline. Samples were then incubated with 1 µM CO in a shaking water bath for 30 min at 37°C.
Western Immunoblotting for HO-1, HO-2, and sGC Proteins and Measurement of HO Activity and cGMP Content.
Isolated and frozen PA tissue was homogenized in 10 mM Tris-buffered saline [20 mM Tris-HCl, pH 7.4, 0.25 M sucrose, and 1 mM EDTA] (1:10, w:v) in the presence of a freshly added cocktail of protease inhibitors. Western blot experiments were performed as described previously (19).
HO activity was measured as bilirubin production as reported previously (20). PA from animals treated in vivo with either hemin or a combination of hemin and CrMP (21) were used in one set of experiments. In another set of experiments, the isolated PA tissues were incubated for 30 min at 37°C in vitro with one of the following substances: CO (1 µM), biliverdin (1 µM), and ferrous ammonium sulphate (1 µM; ref. 22). The following groups of PA samples were also used: oxyhemoglobin (HbO2; 1 µM) for 30 min followed by hemin (1 µM) for 4 hr, CrMP (1 µM) for 30 min plus hemin (1 µM) for 4 hr, CO (1 µM), and CO plus HbO2 (1 µM) for 30 min at 37°C in vitro as previously described (23). The cGMP content was determined by a radioimmunoassay kit (125I-cGMP-RIA, Amersham International plc, Amersham, UK), as we previously described (20).
Statistical Analysis.
All data were expressed as mean ± SEM from at least three independent experiments performed in duplicate except otherwise stated. Statistical analyses were done using unpaired Student t test, analyses of variance in conjunction with Newman-Keuls test, and analyses of variance for repeated measures where appropriate. Group differences at the level of P < 0.05 were considered statistically significant.
Results
Effects of Hemin on BP.
Systolic BP was significantly higher in young (8 weeks SHR; 148.6 ± 3.2 mmHg) than in age-matched WKY rats (127.4 ± 1.5 mmHg; P < 0.01) but was significantly lower than in adult (20 weeks) SHR rats (182.5 ± 4.6 mmHg; P < 0.05). After completion of the hemin treatment, the higher systolic BP of young SHR rats was reduced to 125.8 ± 2.6 mmHg (n = 10, P < 0.01). There was no change in BP of young WKY rats after hemin treatment (125.7 ± 2.5 mmHg, n = 10). The hemin-induced BP lowering in young SHR rats was completely abolished by the coadministration of CrMP with hemin (n = 5). BP remained elevated in 20-week SHR rats (198.1 ± 2.7 mmHg, n = 10) or remained unaffected in age-matched WKY rats (137.5 ± 2.1 vs 138.2 ± 4 mmHg, n = 10) after hemin treatment. Hemin administration did not alter the BP of the juvenile (4 weeks) SHR and age-matched WKY (not shown).
HO-1 and HO-2 Protein Expression in the PA of SHR and WKY Rats.
Lower HO-1 and HO-2 protein expression in the PA of young SHR were detected by Western immunoblotting (Figs. 1
and 2). Hemin treatment enhanced HO-1 protein expression, restoring it to levels comparable to age-matched WKY rats. Hemin also upregulated HO-1 expression in both young and adult WKY rats, although to a lesser amount as compared to young SHR rats (Fig. 1). The basal expression level of HO-1 (Fig. 1), but not HO-2 (Fig. 2), was significantly higher in the PA of adult SHR than in age-matched WKY rats. Neither HO-1 nor HO-2 expression in the PA from adult SHR rats was altered by hemin treatment.
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This study was designed to evaluate the developmental stage-dependent status of the HO/CO system and the sGC/cGMP metabolic pathway in the PA of SHR rats and to delineate the mechanisms by which hemin selectively lowers BP in young SHR rats. Our studies reveal insufficiency of HO-1, HO-2, and sGC in the PA of 4- and 8-week-old SHR rats, which correlated well with reduced cGMP level. Interestingly, hemin treatment restored the deficits in HO-1 and sGC expression as well as the cGMP level, which were accompanied by a concomitant reduction in BP in 8-week-old SHR rats. The hemin-dependent augmentation of HO-1 resulted in an increased CO production, and the latter activated sGC and increased cGMP content. Increased cGMP lowers [Ca2+]i, alters the phosphorylation of the contractile apparatus, and inhibits voltage-dependent calcium channels in vascular SMCs (2). The final effect is vasorelaxation and reduction of elevated BP level in young SHR rats. Although HO-2 remained low after hemin treatment, nonetheless, the upregulated HO-1 expression would compensate this deficit. Despite the low sGC and cGMP levels in 4-week-old SHR rats, the BP of these juvenile animals was still within normal range. Moreover, similar upregulation of sGC and cGMP levels in the PA was observed after hemin treatment. The suppressed sGC/cGMP pathway in 4-week-old SHR rats may represent a genetic defect that is only manifested physiologically at a later stage of life. Accordingly, beyond a certain age (>4 weeks), the impact of the impaired sGC/cGMP pathway may outplay other intrinsic hypertensive-restraining metabolic pathways, that prior to 4 weeks of age, were effective in keeping BP at normotensive setting. Consequently, the impaired sGC/cGMP pathway emerges and manifests itself as a cardiovascular-related pathology, for example, hypertension. However, we do not exclude the involvement of other metabolic pathways. Although hemin treatment enhanced the sGC/cGMP pathway in the PA of 4- and 8-week-old WKY rats, a greater magnitude of increase was registered in the corresponding age-matched SHR rats. Thus, the hemin-elicited increase in the sGC/cGMP pathway may fall below the threshold necessary to trigger a BP-lowering response. The ability of hemin to enhance both the expression and activity of sGC in the PA is a novel observation that could be accounted for by the direct effect of hemin on the transcription and/or translation of sGC, although CO produced from the HO-driven breakdown of heme might also affect the expression of sGC. Importantly, the regulation of sGC by CO and/or hemin may constitute an alternative reaction through which CO can selectively control the expression and activity of sGC, particularly in pathophysiological conditions characterized by deficient sGC expression, as exemplified by our studies. Another important finding of this study is the higher basal levels of HO-1, sGC, and cGMP in the PA of adult SHR rats in comparison with age-matched WKY rats, although the former had hypertension but the latter were normotensive. The expressions of HO-1 and sGC proteins, as well as cGMP level and BP, were unaffected by hemin in adult SHR rats. One explanation for these observations could be that the basal levels of HO-1 and sGC proteins in adult SHR rats have been greatly increased as a compensatory reaction to the elevated BP. As such, they could not be further enhanced by hemin. In addition, fully developed hypertension in adult SHR rats may cause desensitization of the downstream targets of cGMP. Interestingly, impaired vasorelaxant response (data not shown) to 1-Benzyl-3-(5'-hydroxymethyl-2'-furyl)indazole (YC-1) in adult SHR rats with a remarkably higher EC50 in comparison to young SHR or WKY rats of all ages was detected. In this light, a defective and desensitized sGC/cGMP pathway in adult SHR rats could explain the ineffectiveness of the sGC/cGMP pathway to lower BP in adult SHR rats.
Taken together, our results indicate that the functional status of the HO/CO-sGC/cGMP system in the PA is closely related to the developmental stages of genetic hypertension, and insufficiency of this system might contribute to the genesis of hypertension. However, the activation of a functional HO/CO-sGC/cGMP system serves to lower BP only when the cGMP targets remain normally sensitive to cGMP, and the failure of this would compromise tissue contractility. Moreover, hemin also regulates cytochrome P-450 production and thus the formation of vasoconstrictive factors like 20-HETE (24) in addition to detoxifying heme (25). Therefore, the benefits of an upregulated HO/CO system by hemin span beyond the recognized therapeutic potentials to even include an emerging detoxifying capacity.
Acknowledgments
The authors are grateful for the technical assistance from Ginger Bearl, Koleen Safinuik, and Deryk Meszaros.
Footnotes
This work was supported by Canadian Institutes of Health Research. J.F. Ndisang is supported by a postdoctoral fellowship from Health Services Utilization and Research Commission (HSURC); and R. Wang by an Investigator award from Canadian Institutes of Health Research/Regional Partnership Program.
1 To whom requests for reprints should be addressed at Department of Physiology College of Medicine University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, Canada S7N 5E5. E-mail: wangrui{at}duke.usask.ca 
References
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P < 0.01) and was not affected by hemin. The ages of the animals in weeks are designated as 20 and 8 in (A). n = 4.
