The Heme Oxygenase–Carbon Monoxide System as a Regulator of Microvascular Function

The Heme Oxygenase–Carbon

Monoxide System as a Regulator of Microvascular Function

Makoto Suematsu

Summary. Heme oxygenase (HO) catalyzes oxidative cleavage of protoheme IX to generate divalent iron, biliverdin, and carbon monoxide (CO). The inter- est in the HO–CO system has emerged in numerous disciplines among such as cardiovascular physiology, the central nervous and hepatic microvascular systems. Although for many years products of the HO reaction had been regarded as potentially toxic wastes, recent studies have implicated that these products play physiological roles. Both NO and CO share the ability to bind to the prosthetic group of heme proteins, structural changes and the func- tional outcomes of the proteins seem quite different between the gases. Dif- ferences in effects on soluble guanylate cyclase and hemoglobin between NO and CO led us to understand mechanisms as to how the proteins can distin- guish the gases to transducer signals in distinct ways. This chapter focuses on recent advances in both physiologic and pathophysiologic roles of CO and aims to provide updated information on these gas mediators as potential regulators of the organ function on the basis of data collected from the model of isolated perfused liver of rats.

Key words. Heme oxygenase, Carbon monoxide, Guanylate cyclase, Heme protein, Cytochrome P450

Introduction

Heme oxygenase (HO) degrades protoheme IX, giving rise to carbon monox- ide (CO), ferrous iron, and biliverdin, which is rapidly reduced to bilirubin [1]. Amongst the three products of HO, CO has been extensively studied as a

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Department of Biochemistry and Integrative Medical Biology, Keio University School of

Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan

potential neurotransmitter in the brain [2,3] and as a gaseous mediator in the liver [4,5]. The products of HO reaction including CO and bilirubin appear to modulate a variety of hepatobiliary functions in the liver. CO has been found to be the most important regulator of sinusoidal blood flow in normal liver [4]. This is based on our observation that elimination of endogenous CO by zinc protoporphyrin IX (ZnPP), a potent inhibitor of HO, caused to increase in the vascular resistance in the isolated perfused liver. The mechanisms for the gas reception and sinusoidal relaxation appear to involve soluble guany- late cyclase (sGC).

While CO shares the ability to activate sGC with NO, the two gases turned out to possess quite different properties to alter the function of heme pro- teins. We herein intend to compare and contrast roles of CO with those of NO, another gaseous monoxide that functions as a signal in diverse physiological processes. The parallelism exists between the two gaseous molecules as such the two monoxides are notable among signals for their rapid diffusion and ability to permeate cell membranes. Although CO and NO are structurally similar, there is a distinctive property in their chemical reactivity to the pros- thetic heme of the proteins. Considering the similarity and the difference, the present chapter attempts to address the possible mechanisms for mutual regulatory interactions between CO and NO.

Microvascular Actions of CO: Why Not NO in the Liver?

In mammals, HO exists in two forms: HO-1 and HO-2. HO-1 is induced by varied stressors such as cytokines, heavy metals, ROS and hypoxia. Excess NO could also cause the HO-1 induction. Microvascular actions of endogenously generated CO was first demonstrated in the liver [4–6]. We demonstrated intrahepatic distribution of two major HO isozymes immunohistochemically, with the finding that the two isozymes have distinct topographic patterns;

HO-1, the inducible form, is expressed prominently in Kupffer cells, while the

constitutive HO-2 is abundant in hepatocytes [6]. Carbon monoxide derived

from HO-2 is necessary to keep sinusoids in a relaxing state through mecha-

nisms involving sGC in hepatic stellate cells (HSC), also known as Ito cells

that constitute microvascular pericytes in this organ. Considering the

microanatomical orientation of the liver cells in and around sinusoids, HO-2

in parenchyma stands in the reasonable position for the gas reception by HSC

where CO released from hepatocytes can directly access to the cells and

thereby modulate their contractility without being captured by hemoglobin

in circulation. When exposed to disease conditions such as endotoxemia and

advanced cirrhosis, liver could upregulate HO-1 in Kupffer cells and hepato-

cytes as a result of cytokine responses [7]. In experimental models of endo- toxemia, such an induction of HO-1 expands the ability of liver to degrade heme and to trigger overproduction of CO. Under these circumstances, CO turned out to contribute to maintenance of blood perfusion as well as that of bile excretion.

As mentioned, HO-1 is an inducible enzyme, and with exposure to stress stimuli such as cytokine, hypoxia and superoxide, HO-1 increases not only in Kupffer cells but also in parenchymal cells [6,7]. It should be noted that under the normal condition, nitric oxide synthase (NOS) is expressed in the sinu- soidal endothelium; however, under the pathophysiological condition, an inducible form of NOS can be expressed in hepatocytes. This lack of NO pro- duction in the hepatocytes in normal liver may be of a physiological impor- tance in some ways. The first importance may arise when we consider the fact that the hepatocyte is the locus of the urea cycle where arginine (a substrate of NOS) and citrulline (by-product of NOS reaction) are the playing partners.

Under the circumstance that hepatocytes start expressing inducible NOS in response to cytokine, the efficiency of urea cycle may be reduced due to a possible shunt between arginine and citrulline created by catalytic action of inducible NOS. The shunt of this kind is undesirable for the efficient elimi- nation of ammonia. In addition, it has been reported that NO binds to the heme moiety of cytochrome c oxidase, and consequently it causes mitochon- drial dysfunction. This could represent the second importance of not having much NO in hepatocytes.

The role of CO in sinusoidal relaxation was clearly shown by experiments using varied forms of hemoglobin; namely oxyhemoglobin (HbO

, a ferro- heme compound that traps both CO and NO) and methemoglobin (metHb, a ferriheme form that traps NO alone but not CO). Of the two, only the HbO

was able to reproduce the vasoconstrictor effect of ZnPP, a potent HO inhibitor [4–6]. Furthermore, oxyhemoglobin that was encapsulated in SIM!250 nm-diameter liposome (HBV-O

) so that it was restricted to the sinu- soids, failed to induce vasoconstriction, suggesting that the locus of action of CO is extrasinusoidal [6]. Considering the fact that free hemoglobin origi- nated from senescent erythrocytes is immediately oxidized to metHb and is metabolized either in Kupffer cells or in the hepatocytes, one might realize a problematic design if NO were the dominant mediator to relax vascular tone.

If so, the delivered metHb would bind to NO before it could act on a recep-

tor protein(s), most likely to soluble guanylate cyclase, and this elimination of

NO would make it impossible to maintain low vascular resistance in the

hepatic microcirculation. In other words, using CO not NO appears to be a

clever design to maintain low vascular tone in the hepatic microcirculation

under physiological conditions.

Carbon Monoxide Effects on Heme Proteins:

Analogy and Difference with NO

Both NO and CO share the binding ability to heme proteins, functional outcome occurring on the proteins seems quite different between the two gases. In case of hemoglobin, CO stabilizes the six-coordinated form of the prosthetic heme and increases the affinity of molecular oxygen in other sub- units, whereas NO binds to the a subunit of the heme and breaks the proxi- mal histidine-Fe bond, forming a five-coordinated nitrosyl heme complex to decrease the affinity of oxygen in b subunits. Likewise the case of hemoglo- bin, differences between NO and CO in the heme structure in the b subunit of sGC appear to cause distinct activation states of the catalytic a subunit of the enzyme. Because of such a structural difference in the heme coordination between NO and CO, the interaction of the two gases on the prosthetic heme of the enzyme leads to a unique regulatory response of the enzyme: low tissue NO makes CO a modestly stimulatory modulator of the enzyme, whereas high tissue NO makes CO an inhibitory one. Observation that vascular smooth muscle cell-specific heme oxygenase-1 transgenic mice exhibit systemic hypertension rather than hypotension supports such a possibility [8]. This notion was also confirmed by our recent studies by showing that the interac- tions between the two gases cause fine-tuning of the sGC function in vivo [9].

In this study, we applied the newly developed monoclonal antibody (mAb) 3221 against sGC that can recognize the specific structure produced by the enzyme activation. Immunohistochemical analyses of rat retina where the background NO-generating activities appear heterogeneous among different neuronal layers revealed that light-induced upregulation of HO-1 activates sGC in retinal pigment epithelia (low NO), while suppressing the enzyme in inner plexiform layer (high NO). Physiologic roles of CO in this particular organ have not fully been investigated. However, distinct from NO, retina could benefit from the non-radical CO to maintain housekeeping cyclic guanosine monophosphate without a risk of potential degradation of retin- oids. The detail description on difference between NO- and CO-mediated signaling events is also available in another review article published from our laboratory [10]. Such a way to use CO is likely to be the case in relaxation of hepatic stellate cells to guarantee sinusoidal patency or in apoptotic control of spermatogenesis, where NO-breakable DNA or vitamin A is abundantly stored, respectively [10].

Acknowledgments. The authors acknowledge support by the 21st Century

Center-of-Exellence Program and by the Leading Project for Biosimulation

from the Ministry of Education, Sciences, and Technology of Japan. A portion

of the Project was also supported by a Grant-in-Aid for Creative Scientific Research by the Japan Society for the Promotion of Sciences 16GS0015.

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