Regulation of Local Hepatic Oxygen Delivery Following Stress

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Regulation of Local Hepatic Oxygen Delivery Following Stress

Mark G. Clemens, Markus Paxian, Walid Kamoun, Jean Ashburn, M. Nicole Kresge, and Sandra Merkel

Summary. Hepatic dysfunction is a common sequela of a wide variety of stresses such as ischemia/reperfusion, sepsis, chronic alcohol consumption and even remote trauma. Hypersensitivity to the constrictor activity of endothelin (ET)-1 is a common ﬁnding in the liver microcirculation in all of these stresses. The mechanism of this increased constrictor response appears to be dependent upon an increased expression of ETBreceptors but with concomitant uncoupling of the ETBreceptor from activation of endothelial nitric oxide synthase (eNOS). This uncoupling is at least in part the result of over- expression of caveolin-1 which binds to eNOS thus preventing activation by calmodulin. Increased expression of caveolin is associated with decreased basal eNOS activity and profound inhibition of endothelin-stimulated eNOS activity. Finally, uncoupling of ETB binding from eNOS activation leads to local tissue hypoxia and potentiation of injury. We propose that this mechanism is an important contributor to hepatic dysfunction following stress.

Key words. Endothelin, Caveolin-1, Microcirculation, Intravital microscopy

Introduction

A wide variety of stresses involving the liver, including inflammation such as following sepsis or oxidative stress associated with ischemia and reperfusion or hemorrhagic shock, lead to deficits in oxygen utilization by the liver. These deficits are caused by a combination of failure of the liver cells to effectively utilize available oxygen and changes in vascular regulation, resulting in areas

147 Department of Biology, University of North Carolina at Charlotte, 9201 University City Boulevard, Charlotte, NC 28223, USA

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of compromised perfusion. Although there is controversy regarding which of these mechanisms are the primary mechanism leading to hepatic injury in these conditions, work from our laboratory as well as others have demonstrated that the ability to effectively restore microvascular perfusion is a crit- ical determinant of the recovery of the liver [1]. Severe injury such as occurs after prolonged ischemia and reperfusion results in structural damage to the vasculature and rapid failure of the microcirculation. In addition, inflamma- tory stresses result in neutrophil accumulation in the liver, which can also result in parenchymal cell injury as well as vascular failure. However, deficits in oxygen extraction also occur in the absence of overt injury to the vasculature. In these cases data suggest that altered reactivity of the blood vessels to vasoconstrictors and vasodilators contributes to deficits in oxygen delivery [2,3].

Regulation of the liver microcirculation is complex. The liver has a dual circulation. While the hepatic artery circulation is regulated similar to most vascular beds, the volumetric ﬂow of the portal circulation is regulated largely by the upstream splanchnic viscera. These two circulations join to perfuse the sinusoids. Although the sinusoids, like capillaries in other vascular beds, do not contain vascular smooth muscle cells, it is now commonly accepted that they are subject to active constriction [4,5]. This constriction is mediated by the contraction of a specialized pericyte, the hepatic stellate cell (HSC) [5,6].

Under normal conditions, the HSC contracts in response to specific peptide mediators such as endothelin, but not a-adrenergic agonists such as phenyle- phrine [5]. They also relax in response to nitric oxide (NO) and carbon monoxide (CO). In response to long-term stresses leading to fibrosis, the HSCs become activated and take on a myofibroblast-like phenotype. In this activated state they become highly contractile [7].

Even following relatively short-term stresses that do not result in stellate cell transformation to myoﬁbroblasts, the microvasculature of the liver becomes hyper-responsive to the constrictor effect of endothelin (ET)-1 [8,9].

Endothelin expression is upregulated following endotoxemia, hypoxia, or ischemia [10]. In addition, the response to exogenous endothelin is increased following endotoxemia, ischemia reperfusion, blunt trauma, and chronic alcohol consumption [11–13]. This ubiquitous occurrence of hypersensitivity to endothelin associated with such conditions suggests that it is a common pathway to vascular dysregulation following most stresses that result in liver injury. The mechanisms for this increased sensitivity to endothelin and the functional signiﬁcance has, however, been unclear. The focus of this chapter will be to present the mechanisms related to altered hepatic vascular control following inﬂammatory or oxidative stress and the functional importance of this altered regulation related to oxygen delivery to the liver tissue.

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Endothelin Receptors

Endothelin acts through two major receptor subtypes, ETA and ETB. In the liver, the ETAreceptors are distributed primarily to the hepatocytes, stellate cells and vascular smooth muscle cells, while ETBreceptors are ubiquitously distributed over all cell types. The two receptor subtypes on different cells allow endothelin to exert both constrictor and dilator inﬂuences. Classically, ETAreceptors are considered to mediate constriction because of their distribution on vascular smooth muscle cells. ETBreceptors, conversely, mediate dilation by being coupled to activation of endothelial nitric oxide synthase (eNOS) (Fig. 1). Based on these functional characteristics, we originally hypothesized that an upregulation of ETA receptors could account for the hyperconstrictive response to endothelin. However, our previous studies have shown that although both endothelin itself and overall endothelin receptors are upregulated in response to stresses such as endotoxin or ischemia/reperfusion, the predominant receptor subtype that is responsible for the increase in total receptors is the ETB[6,14] receptor. This raised the question of how could an increase in the receptors that mediate dilation result in an enhance- ment of the constrictor response. Two major observations suggested a mechanism. First, although our original report did not ﬁnd any sinusoidal

Sinusoidal Endothelial cells

Endothelin-1

EtB Receptor

[Ca++]+_i Calmodulin eNOS L-arginine NO + L-citrulline

Smooth Muscle Cells

EtB /EtA Receptor

MLCK

vasodilation vasoconstriction

MLCKX

Vascular Effects of Endothelin-1

[Ca⁺⁺]i

Fig. 1. Schematic diagram of the interaction between ETAand ETBreceptors on endothelial cells and smooth muscle cells. In the liver, receptor distribution on hepatic stellate cells is similar to that of vascular smooth muscle cells. MLCK, myosin light chain kinase

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constrictor response to ETBreceptor activation, ETBagonists did have potent constrictor effects in the liver with the site of action being extrasinusoidal [5].

Second, it was possible to unmask the sinusoid constrictor response to an ETB

agonist by simultaneously inhibiting eNOS [6]. This observation demonstrated that there is a functional link between ETBreceptors and NOS activation in the liver, and suggested that interruption of this link may give rise to an increase in the functional constrictor response via inhibition of the com- pensatory dilation.

Caveolin-1 and Activation of eNOS

A possible mechanism for uncoupling of ETBreceptor binding and the activation of eNOS can be found in the putative role of caveolin-1 in eNOS regulation. Recent studies from several groups have provided evidence that increased constrictor tone in the hepatic portal circulation may be the result of decreased ability to activate eNOS. Rockey’s group showed, somewhat par- adoxically, that eNOS protein expression was normal but enzymatic activity was decreased in a cirrhosis model. A probable mechanism for this discrep- ancy was suggested by the work of Shah’s group which showed that the membrane scaffolding protein caveolin-1 was upregulated in cirrhosis and that eNOS was found to be associated with the caveolin [15,16].

Caveolin is a protein associated with membrane subdomains called caveolae. Caveolar domains are particularly rich in cholesterol and sphingolipids, and are a site of clustering of receptors and signal transduction proteins. Both ETBreceptors and eNOS have been shown to associate with caveolae. The role of caveolae in regulating eNOS is particularly relevant. It is thought that binding to caveolin is necessary to locate the eNOS molecule to this subdo- main where it is available for activation. On the other hand, binding to caveolin appears to inhibit binding to activated calmodulin and, thus, inhibits activation of eNOS.

These observations suggested that an increased expression of caveolin-1 in the liver might serve to uncouple ETBreceptor binding from activation of eNOS. We have examined this possibility is several models, including endo- toxemia and remote trauma. Injection of 1 mg/kg Escherichia coli lipopolysac- charide (LPS) results in an approximately twofold increase in caveolin protein in the liver. Immunohistochemistry studies indicated that the upregulation is largely associated with vascular tissue. To test whether the sinusoidal endothelial cells might be the site of the upregulation of caveolin-1, we treated isolated sinusoidal endothelial cells with LPS and measured induction of caveolin-1 protein by Western blot. Within 6 h, caveolin-1 levels were increased greater than twofold compared to vehicle controls. These results

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suggested that bacterial LPS upregulates caveolin-1 in sinusoidal endothelial cells in vivo. Moreover, this response was found not to be affected by the omis- sion of serum from the medium, suggesting that the mechanism is inde- pendent of CD14 activation. Since we also found that sinusoidal endothelial cells express toll like receptor 4 (TLR4), we have hypothesized that this pathway is important for the induction of caveolin in the liver.

We have also demonstrated that caveolin association with eNOS increases following stresses. This ﬁnding is consistent with the hypothesis that increased caveolin expression serves to inhibit the activation of eNOS by calmodulin. In support of this hypothesis, we have also found that eNOS activity (but not protein expression) is decreased following remote trauma in the whole liver, and endothelin-stimulated eNOS activity is decreased following treatment of isolated sinusoidal endothelial cells with LPS. Taken together, these results support the hypothesis that increased caveolin expression inhibits activation of eNOS by ETB receptor activation, leading to a hyper- constriction response (see Fig. 2). This then raised the question of whether uncoupling of ETBreceptor binding from eNOS activation leads to functional alterations in tissue oxygen delivery.

LPS Increases Cav-1 and Sequesters eNOS

Ca²⁺

CaM

/

NO

ETBR ET-1

eNOS- Inactive

Ca²⁺/CaM

eNOS Active

LPS

Ca²⁺/CaM

?

cytokines

eNOS- Inactive

Endothelial Cell

HSC SMC

Cav-1

Fig. 2. Proposed schema for regulation of response to endothelin B (ETB) receptor binding following endotoxemia. LPS, lipopolysaccharide; Cav-1, caveolin-1; ETBR, endothelin B receptor; eNOS, endothelial nitric oxide synthase; SMC, smooth muscle cells; HSC, hepatic stellate cells

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Effect of Endothelins on Oxygen Delivery

To examine the effect of uncoupling ETBreceptors from NOS activation on tissue oxygenation, we used an intravital microscopy technique to examine tissue oxygenation and redox potential. Figure 3 shows the redox potential response of the liver (as indicated by nicotinamide adenine dinucleotide (NADH) fluorescence) to ET-1 which stimulates both ETAand ETBreceptors, versus IRL1620, which is a specific ETBagonist. The results showed that IRL 1620exerted modest effects on redox potential compared to ET-1. This is consistent with the ETAactivation by ET-1 causing sinusoidal constriction, while the response to IRL 1620 is balanced by NO-mediated dilation. In contrast, when NOS activity was inhibited by treatment with L-nitroarginine, IRL 1620 provoked a significantly enhanced response, similar to that produced by ET- 1. Similar responses were observed regarding tissue PO2(Fig. 4). These results are also consistent with our observation that even though both ET-1 and IRL 1620 stimulate increased oxygen demand in isolated hepatocytes, ET-1, but

IRL1620

ET-1

baseline

NADH fluorescence

ET or IRL ET or IRL + L-NA

Fig. 3. Nicotinamide adenine dinucleotide (NADH) fluorescence in response to ET-1 or IRL 1620 without or with simultaneous treatment with L-nitroarginine (L-NA). L- Nitroarginine treatment alone did not cause significant change in NADH fluorescence. Rat livers were observed by intravital fluorescence microscopy in vivo. Endothelin-1 or IRL 1620were infused via the portal vein

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not IRL 1620, actually produces a decrease in oxygen consumption in isolated perfused liver, suggesting inadequate oxygen delivery to the tissue. This notion is further substantiated by the observation that inhibition of eNOS combined with either IRL 1620 or ET-1 potentiates cell injury, as indicated by increased enzyme release.

Conclusion

Hypersensitivity to endothelin-1 is a common ﬁnding in the liver microcirculation following a wide variety of stresses. The mechanism of this increased constrictor response appears to be dependent upon an increased expression of ETBreceptors but with concomitant uncoupling of the ETBreceptor from activation of eNOS. This uncoupling is at least in part the result of overex- pression of caveolin-1, which binds to eNOS thus preventing activation by calmodulin. Increased expression of caveolin is associated with decreased basal eNOS activity and profound inhibition of endothelin-stimulated eNOS activity. Finally, uncoupling of ETB binding from eNOS activation leads to

IRL1620

ET-1

baseline ET or IRL ET or IRL + L-NA

RuPhen fluorescence (tissue PO

₂

)

Fig. 4. Tissue PO2in response to ET-1 or IRL 1620. The experiment was the same as in Fig. 3 except that tris(1,10-phenanthroline) ruthenium(II) chloride hydrate (RuPhen) was infused intravenously as an indicator of tissue PO2. Increased ﬂuorescence indicates hypoxia

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local tissue hypoxia and potentiation of injury. We propose that this mechanism is an important contributor to hepatic dysfunction following stress.

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