Basic Science Aspects
Hydrogen Sulfide Differentially Affects The Hepatic Vasculature In Response To Phenylephrine And Endothelin 1 During Endotoxemia
Norris, Eric J.; Larion, Sebastian; Culberson, Catherine R.; Clemens, Mark G.
Department of Biology, University of North Carolina at Charlotte, Charlotte, North Carolina
Received 31 Jul 2012; first review completed 20 Aug 2012; accepted in final form 7 Sep 2012
Address reprint requests to Mark G. Clemens, PhD, Department of Biology, University of North Carolina at Charlotte, 9201 University City Blvd, Charlotte, NC 28223. E-mail: firstname.lastname@example.org.
Support was received through National Institutes of Health grant DK38201 and ARRA Supplement.
ABSTRACT: Despite being protective in many disease states, hydrogen sulfide (H2S) contributes to organ injury in sepsis. Like the other gasotransmitters, nitric oxide and carbon monoxide, H2S is a modulator of the microcirculation. Because microcirculatory dysfunction is a main cause of organ injury during sepsis, the present study was designed to test the effect of H2S on microvascular dysfunction in isolated perfused livers. In most microcirculatory beds, endotoxin activates the endothelium, resulting in hyporesponsiveness to catecholamines and a derangement in blood flow distribution. We demonstrate that H2S treatment attenuates the increase in portal pressure during infusion of the α1 adrenergic agonist, phenylephrine (PE) (P < 0.01). Hydrogen sulfide almost completely negated the increase in portal pressure in livers isolated from endotoxemic rats. Treatment with an inhibitor of endogenous H2S, DL-propargylglycine (PAG), reversed lipopolysaccharide-induced hyporesponsiveness to PE. Because hepatic microcirculatory dysfunction is associated with excessive sinusoidal vasoconstriction and not dilation, we investigated whether H2S affects endothelin 1 (ET-1)–induced vasoconstriction in isolated livers. Contrary to PE treatment, H2S did not affect the increase in portal pressure during infusion of ET-1, nor did it attenuate the hypersensitization of the liver to ET-1 during endotoxemia. Hepatic resistance in control rats was increased by PAG treatment during ET-1 infusion, but this increase was not exacerbated during endotoxemia. We monitored hepatic O2 consumption to assess the effect of vascular changes on oxygen consumption following ET-1 treatment. Low-dose ET-1 infusion caused an increase in hepatic O2 consumption, whereas low-dose ET-1 infusion decreased O2 consumption in endotoxemic livers. Interestingly, whereas we observed no effect of PAG on the vascular response to ET-1 infusion during endotoxemia, PAG treatment did maintain O2, suggesting a more complex effect of H2S inhibition. In summary, the discrepancies between the hepatic response to PE and ET-1 suggest that H2S differentially contributes to microcirculatory dysfunction in the systemic and hepatic microcirculations. We propose that this is due to H2S exerting a differential vasoactive function on presinusoidal and sinusoidal sites within the liver. Moreover, our findings suggest that H2S may contribute to the progression of sepsis by contributing to microvascular failure.
Organ failure is a common complication during sepsis and is associated with an increase in morbidity and mortality (1). Microcirculatory dysfunction is an important contributor to organ injury (2). In most vascular beds, sepsis attenuates vasoconstriction in response to catecholamine signaling. This contributes to intractable hypotension, inadequate tissue perfusion, and organ dysfunction. The portal venous circulation is also hyporesponsive to catecholamine signaling; however, depressed catecholamine signaling does not contribute to hepatic microvascular dysfunction (3). Instead, sinusoidal hyperconstriction, because of increased sensitivity to the vasopressor, endothelin 1 (ET-1), results in heterogeneous perfusion and focal hypoxia, which contributes to hepatic dysfunction and ultimately overall injury (4–6).
Hydrogen sulfide (H2S) is primarily produced as a byproduct of cysteine metabolism by two pyridoxal 5′ phosphate–dependent enzymes: cystathionine β synthase and cystathionine γ lyase (CSE) (7, 8). In addition, a third pyridoxal 5′ phosphate–independent enzyme, 3-mercaptopyruvate sulfurtransferase, synthesizes H2S (9). Cystathionine γ lyase is the predominant source of H2S in the cardiovascular system and liver (7). The liver is likely exposed to elevated levels of H2S during bacterial peritonitis due to an increase in hepatic CSE expression and the arrival of H2S in the portal circulation synthesized by bacteria of the gastrointestinal tract (10).
There are conflicting reports in the literature about the vasoregulatory action of H2S (11–15). The first indication that H2S modulates the hepatic vasculature was reported by Fiorucci et al. (16). Using an isolated perfused liver system, they demonstrated that H2S attenuated the increase in portal pressure during infusion of norepinephrine in normal and cirrhotic livers. Therefore, H2S may be beneficial during cirrhosis and fibrosis by lowering intrahepatic resistance and reducing portal hypertension. Based on this finding, one would predict that elevated levels of H2S would prevent sinusoidal constriction during sepsis; however, there is ample evidence demonstrating that hepatic dysfunction during sepsis is the result of sinusoidal constriction resulting in tissue hypoxia (5, 6, 17, 18).
The hepatic microcirculation has multiple sites of blood flow regulation. Upstream of the sinusoids, terminal portal venules are surrounded by vascular smooth muscle cells (VSMCs) and respond to vasoactive molecules similarly to other vascular beds. Unlike the capillaries in other vascular beds, the hepatic sinusoids can modulate local tissue perfusion by changing their resistance, which makes them a second, functionally important site of hepatic perfusion (19). Unlike portal venules, the sinusoids lack VSMCs. Instead, they are surrounded by hepatic stellate cells (HSCs), which can modulate perfusion through individual sinusoids by contracting in response to vasoactive molecules (20, 21). This unique organization allows regulation of overall hepatic perfusion by presinusoidal regulatory and spatial distribution of hepatic blood flow within the liver lobule (22, 23). Previous work from our laboratory has demonstrated that the α1 adrenergic agonist, phenylephrine (PE), and ET-1 both increase hepatic vascular resistance (19). Importantly, PE acts only on presinusoidal sites of regulation, whereas ET-1 acts on presinusoidal and sinusoidal regulatory sites (19, 24, 25). Inflammatory stress primes the liver to the vasoconstrictive effect of ET-1, resulting in sinusoidal hyperconstriction and tissue hypoxia (5, 26).
Therefore, the present study was designed to investigate the effect of H2S on the different sites in hepatic microvasculature during endotoxemia. Using an isolated perfused liver system, we investigated the effect of H2S on total intrahepatic resistance during portal infusion of PE or ET-1 during endotoxemia. We hypothesize that H2S acts as a vasodilator in response to PE, but has no effect on ET-1–induced vasoconstriction, particularly during endotoxemia. This study is the first to provide evidence that H2S differentially regulates presinusoidal and sinusoidal sites of hepatic perfusion. Furthermore, our finding that H2S contributes to systemic vascular hyporesponsiveness but does not affect sinusoidal constriction adds to the current knowledge regarding the detrimental effects of H2S on organ injury during sepsis (10).
MATERIALS AND METHODS
Fifty male Sprague-Dawley rats (Charles River Laboratories, Fayetteville, NC) weighing 272 ± 13.2 g were used in this study. Rats remained quarantined for 4 days following arrival. Rats were maintained in pairs in standard cages with bedding with free access to standard rat chow and water in a temperature-controlled setting under 12-h light-dark cycles. Rats were removed from the vivarium on the morning of the experiment and transported to the laboratory where all surgical procedures were performed. Animals were randomly assigned to groups on the day of the experiment. All animal manipulation was in strict adherence with the National Institutes of Health guidelines, and experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of North Carolina at Charlotte.
Sodium sulfide (Na2S), L-PE, lipopolysaccharide (LPS, Escherichia coli O26:B6), and DL-propargylglycine (PAG) were purchased from Sigma-Aldrich Co (St Louis, Mo). Endothelin 1 was purchased from American Peptide Company (Sunnyvale, Calif).
Isolated perfused liver
Isolated perfused rat livers were used to investigate the effect of vasoactive modulators on hepatic portal resistance as described previously (27) with modification (28). Briefly, rats were anesthetized under isoflurane and subjected to laparotomy. Heparin (1 USP unit/rat mass [in grams]; Webster Veterinary, Devens, Mass) was injected into the inferior vena cava. The portal vein was exposed, and three loosely tied sutures were placed along the length of the vein. The portal vein was cannulated, and the sutures were tightened to secure the cannula in place. The inferior vena cava and abdominal aorta were severed to prevent hypervolemia due to perfusion and to allow for exsanguination. The pleural cavity was opened, and a cannula was inserted into the right atrium and directed downward in the superior vena cava. The cannula was secured in place just below the diaphragm. Lastly, the inferior vena cava was ligated above the renal veins to block perfusate flow. This isolates the liver in situ during perfusion with oxygenated Krebs-Henseleit buffer. Temperature of the buffer was maintained at 37°C and continuously oxygenated using 95% O2/5% CO2 gas. Following a 20-min stabilization period, increasing cumulative doses of PE (0.05–20 μM) or ET-1 (0.05–1.0 nM) were infused sequentially in 5-min intervals. Total intrahepatic resistance was determined by monitoring the pressure in the inflow tubing (portal pressure) using a Biopac Systems MP100 transducer (Goleta, Calif) during constant flow perfusion (16 mL/kg per minute). There were six different treatment groups to which rats were randomly assigned: (a) control, (b) LPS, (c) H2S, (d) H2S/LPS, (e) PAG, and (f) PAG/LPS. The H2S donor Na2S (50 μM) was administered in the perfusate during ET-1 or PE infusions for certain animal groups. In other experiments, the CSE inhibitor PAG (1 mg/kg) was injected intraperitoneally 30 min before liver isolation to inhibit endogenous H2S production. For the endotoxin model of sepsis, rats were given an injection of LPS (1 mg/kg, i.p.) 6 h before liver isolation and perfusion.
The amount of O2 extracted by the liver from the perfusate was calculated to assess hepatic oxygen consumption. The perfusate PO2 was monitored using an oxygen-sensitive Clark-type electrode connected to an oxygen monitor (YSI Life Sciences, Yellow Springs, Ohio). All measurements were recorded continuously using Biopac Systems MP 100 transducer and software (Goleta, Calif). Calibration of the electrode was performed daily using distilled water equilibrated with air and adjusted for temperature and elevation. The concentration of the inflow perfusate was measured immediately before and after the experiment. O2 extraction was calculated as the difference between the inflow perfusate PO2 and outflow perfusate PO2.
The data are presented as means ± SEM. Statistical significance was determined using two-way analysis of variance (ANOVA) analysis. Student-Newman-Keuls (SNK) post hoc test was used when ANOVA detected significance. Independent and repeated-measures analysis was used where appropriate. Statistical significance was P < 0.05.
Effect of endotoxin on hepatic resistance in response to PE and ET-1
An isolated perfused liver system was utilized to determine the effect of PE and ET-1 on the hepatic vasculature during endotoxemia. Baseline portal pressure in control rats was 3.2 ± 1.4 mmHg. Despite being slightly elevated, there was not a statistically significant effect of 6 h LPS treatment (4.5 ± 1.6 mmHg; P = 0.076). Phenylephrine infusion produced an increase in portal pressure with a maximal increase of 5.5 ± 0.8 mmHg with infusion of 20 μM PE in control animals (Fig. 1A). Lipopolysaccharide treatment significantly attenuated the increase in portal pressure due to PE infusion with a maximal increase in portal pressure of 4.1 ± 1.0 mmHg (P < 0.001, Fig. 1A). Lipopolysaccharide treatment had the opposite effect on the vasculature response to ET-infusion. Lipopolysaccharide potentiated the increase in portal pressure during ET-1 infusion (P < 0.001, Fig. 1B). In addition, LPS caused a significantly greater maximal increase in portal pressure (15.2 ± 1.1 mmHg in control vs. 18.7 ± 2.3 mmHg in LPS; P < 0.017).
Effect of H2S on hepatic resistance in response to PE and ET-1
To determine the effects of H2S on hepatic resistance, isolated livers were perfused with Krebs buffer or Krebs buffer containing the H2S donor Na2S (50 μM). Hydrogen sulfide did not significantly affect the resting portal pressure (4.1 ± 2.7 vs. 3.2 ± 1.4 mmHg in controls; not statistically significant). Hydrogen sulfide treatment significantly reduced the dose-dependent increases in portal pressure during PE infusions (Fig. 2A; P = 0.01). Hydrogen sulfide had no effect on the increase in portal pressure during infusion of ET-1 (Fig. 2B).
Effect of combined H2S/LPS treatment on hepatic resistance in response to PE and ET-1
Lipopolysaccharide treatment followed by perfusion with 50 μM H2S Krebs buffer did not affect the resting portal pressure (4.2 ± 0.5 mmHg). Infusion of 5 μM PE increased portal pressure 5.4 ± 0.5 mmHg (Fig. 3A). Lipopolysaccharide and H2S each reduced this response to 3.3 ± 0.5 and 3.1 ± 0.4 mmHg, respectively (P < 0.05). A synergistic interaction between LPS and H2S produced a nearly complete attenuation of the increase in portal pressure during 5 μM PE infusion (1.6 ± 0.5-mmHg increase in H2S/LPS; P < 0.05). Infusion of 0.5 nM ET-1 increased portal pressure by 13.6 ± 1.0 mmHg in control rats (Fig 3B). This effect was significantly increased in LPS-treated rats (19.3 ± 1.3-mmHg increase; P < 0.01). Hydrogen sulfide had no effect on the increase in portal pressure in either control or LPS-treated rats (15.0 ± 1.5 and 18.2 ± 1.5 mmHg, respectively).
Effect of inhibition of endogenous H2S synthesis on hepatic resistance during endotoxemia
Hepatic synthesis of H2S was inhibited by treating rats with DL-PAG (50 mg/kg i.p.) 30 min before liver isolation. Propargylglycine treatment alone had no effect on the resting portal pressure when compared with controls (3.6 ± 0.9 vs. 3.2 ± 1.4 mmHg). Propargylglycine treatment caused a greater increase in portal pressure due to infusion of PE (P < 0.001, Fig. 4A) and ET-1 (P < 0.001, Fig. 4B) when compared with control animals. To test the effect of endogenous H2S synthesis during endotoxemia, rats were treated for 6 h with LPS then given PAG 30 min before isolation. The combination of PAG and LPS had no effect on baseline portal pressure when compared with controls (4.8 ± 0.7 mmHg). Infusion of 5 μM PE resulted in an increase in portal pressure of 4.7 ± 0.5 mmHg in controls, which was reduced to 3.3 ± 0.6 mmHg in LPS-treated rats (P < 0.05, Fig. 5A). Propargylglycine treatment reversed the effect of LPS on PE-induced vasoconstriction during endotoxemia (6.1 ± 0.5 mmHg; P < 0.05). An 11.3 ± 0.8-mmHg increase in portal pressure was observed during infusion of 0.5 nM ET-1 in control rats (Fig. 5B). Lipopolysaccharide and PAG treatment both caused a greater increase in portal pressure to ET-1 (16.2 ± 1.1 and 15.4 ± 1.3 mmHg, respectively; P < 0.05). The combination of PAG and LPS was not significantly different from LPS or PAG alone (14.7 ± 1.3 mmHg).
Effect of H2S and PAG on O2 consumption during ET-1 infusion
There was no difference in resting hepatic O2 consumption between any of the treatment groups before ET-1 infusion. For livers in H2S treatment groups, the addition of 50 μM Na2S to the perfusate increased O2 consumption (7% over baseline; P < 0.05), which was used as a baseline for these groups (Fig. 6). In control livers, infusion of 0.05 nM ET-1 produced a modest, but repeatable increase in O2 consumption (6% increase over baseline; P < 0.05). In livers isolated from LPS-treated rats, infusion of 0.05 nM demonstrated a trend toward decreased oxygen consumption, but it was not statistically significant (100% vs. 92%; P = 0.058). However, hepatic O2 consumption during infusion of 0.05 nM ET-1 was significantly lower in the LPS group when compared with controls (90% vs. 106%; P < 0.05). The H2S group and H2S/LPS group were not significantly different from either the control or LPS group during infusion of 0.05 nM ET-1 (99% of baseline and 98% of baseline, respectively, Fig. 6A). Propargylglycine treatment was significantly lower than controls during 0.05 nM ET-1 infusion (P < 0.05). Whereas PAG appeared to restore O2 consumption in LPS-treated animals, the results were not statistically significant (P = 0.055, Fig. 6B). When the concentration of ET-1 was increased to 1 nM, hepatic O2 consumption was significantly reduced in all treatment groups (P < 0.001). Two-way ANOVA analysis demonstrated a significant effect of H2S on hepatic O2 consumption, which was increased over control and LPS treatment groups. Propargylglycine and PAG/LPS treatment groups were reduced during infusion of 1 nM ET-1 similar to control and LPS groups, and no significant differences were observed.
The role of H2S in disease and inflammation is the source of considerable debate. Hydrogen sulfide prevents myocardial and hepatic ischemia-reperfusion injury, which is partially the result of an increase in cellular antioxidant capacity via induction of antioxidant gene expression (29, 30). On the contrary, inhibition of endogenous H2S synthesis significantly reduces organ injury and improves survival in septic mice by attenuating the inflammatory response (10, 31). In the earliest stages of sepsis, hepatocellular injury is initially the result of excessive cytokine release from Kupffer cells (32, 33); however, it is now well established that continued hepatocellular dysfunction is primarily the result of hepatic microvascular failure, particularly in the sinusoids (4–6, 18). As a vasoregulatory molecule that is elevated in the liver during sepsis (10), it is possible that H2S contributes to the dysregulation of hepatic sinusoidal perfusion.
Fiorucci et al. (16) were the first to demonstrate a vasoregulatory role of H2S in the hepatic microcirculation. In their study, H2S attenuated the increase in intrahepatic resistance to norepinephrine in normal and cirrhotic rats. This vasodilatory effect would likely be beneficial during chronic conditions, like cirrhosis, by decreasing intrahepatic resistance and attenuating portal hypertension (31). If H2S acts as a vasodilator in the hepatic microcirculation, one would predict that H2S could also be beneficial during sepsis via improved tissue perfusion. The hepatic microcirculation is regulated at presinusoidal and sinusoidal sites (34). Therefore, we tested the effect of H2S on the vascular response to PE and ET-1 that act at these sites, respectively, in an endotoxin model of sepsis. Our study demonstrates that H2S differentially modulates the response of the hepatic microcirculation to vasopressor, which suggests that H2S may exert a different vasoregulatory function at different sites within the liver. Moreover, our results highlight the complex role H2S serves in modulating the microcirculation during endotoxemia.
Several factors contribute to systemic microcirculatory dysfunction during sepsis, including activation of procoagulant pathways, recruitment of leucocytes, and excess production of vasoactive agents (2). In healthy individuals, the activation of α1 adrenergic receptors by circulating catecholamines causes VSMC contraction and reduces blood flow through the vascular bed, which allows for the spatial distribution of cardiac output. During sepsis, the excessive release of vasodilators suppresses the response of the endothelium to catecholamines (3). The diffuse peripheral vasodilation contributes to systemic hypotension and organ dysfunction (35).
In this study, we first investigated the effect of H2S on presinusoidal portal venules, which respond to catecholamines in a similar manner to systemic resistance vessels. Evidence to support this was provided by studies demonstrating decreased vascular responsiveness to PE in livers isolated from endotoxemic rats. Moreover, administration of an inhibitor of NO synthase reversed the hyporesponsiveness of the hepatic vasculature, suggesting the importance of increased production of vasodilatory gas nitric oxide (NO) in vascular hyporesponsiveness (3). Like NO, H2S acts as a vasodilator, which suggests that a similar effect of H2S should occur during endotoxemia (12). Infusion of increasing doses of PE produced a dose-dependent increase in portal pressure in isolated livers, which was significantly diminished in livers isolated from LPS-treated rats. The addition of exogenous H2S (50 μM) to the perfusate almost completely abrogated the increase in portal pressure in response to PE in livers isolated from endotoxemic rats, suggesting a synergistic effect of LPS and H2S.
We used a suicide inhibitor of CSE to block hepatic H2S synthesis during endotoxemia to determine if endogenous H2S production contributes to vascular hyporesponsiveness. Propargylglycine treatment alone significantly potentiated the increase in intrahepatic resistance in response to PE, suggesting that H2S is involved in the constant regulation of hepatic blood flow. Importantly, PAG treatment reversed the hyporesponsiveness of the presinusoidal venules to PE during endotoxemia.
Given that the only target of PE in the hepatic microcirculation are presinusoidal resistance vessels (19), the previous results suggest that H2S is involved in the modulation of hepatic microvasculature at the level of portal terminal venules. Because the liver is the largest internal organ and a major recipient of cardiac output, excessive hepatic vasodilation could potentially contribute to systemic hypotension during sepsis. Hydrogen sulfide derived from CSE is an important regulator of systemic blood pressure (15). Because portal venules and systemic arterioles are regulated in a similar manner by VSMCs, it is likely that H2S contributes to hyporesponsiveness of resistance vessels in most tissues. In support of this hypothesis, circulating H2S levels in septic rats have been shown to be negatively correlated to blood pressure (36).
Based on our results, PAG treatment may provide a protective effect during sepsis by attenuating systemic vascular hyporesponsiveness to catecholamines; however, its effect in the hepatic sinusoid remains unclear. Previously, we showed that sinusoidal tone is modulated by HSCs, which contract in response to ET-1 (19). Hepatic stellate cells are activated following inflammatory stress, which enhances their contractility to ET-1 (26). The priming effect of LPS on the HSCs results in sinusoidal hyperconstriction, which is a main cause of tissue hypoxia and cell death (5). In support of the importance of ET-1 in hepatic dysfunction, elevated ET-1 levels are highly correlated to disease severity in cirrhosis (37). Thus, the sinusoid is a critical regulatory site for hepatic perfusion during sepsis. Previous work from our laboratory and others demonstrated that impaired synthesis of NO contributes to sinusoidal dysfunction (38–40). The vasoregulatory effect of H2S in the hepatic sinusoids remains unclear. Therefore, we investigated the effect of H2S on the sinusoids during endotoxemia by assessing its effect on ET-1 infusion.
Infusion of increasing concentrations of ET-1 resulted in progressive increase in portal pressure. In agreement with our previous reports, the effect of ET-1 was significantly potentiated by endotoxin treatment. Based on its vasodilatory action, we hypothesized that H2S would attenuate the hypersensitization of the hepatic sinusoid to ET-1 during endotoxemia. Surprisingly, we observed no effect of H2S on ET-1–induced vasocontriction in either control or endotoxin-treated rats. Interestingly, PAG treatment increased the vascular response to ET-1 in control livers but had no effect in the endotoxin group. These results suggest that H2S differentially modulates the vascular response to ET-1 when compared with PE.
This differential effect may be due to the cells responsible for modulating lumenal diameter. Portal terminal venules, as well as arterioles, are modulated by VSMCs. The vasodilatory effect of H2S is primarily the result of VSMC hyperpolarization via activation of KATP channels (11, 41, 42). Hepatic sinusoidal resistance is regulated by HSCs, which may be differentially regulated by H2S. It has been suggested that the H2S donor (NaHS) can prevent HSC contraction (43). However, that study was performed in isolated HSCs over the course of 18 h with NaHS. The spontaneous activation of HSCs following isolation may not reflect actual in vivo conditions (44). Furthermore, NaHS rapidly releases H2S, which if not contained in a closed system rapidly escapes culture media and enters the atmosphere in a short period during incubation (45). Although the finding of that study is promising, more conclusive research is needed to assess the effect of H2S on HSCs.
One drawback of our isolated perfused liver system is that we are assessing total intrahepatic resistance by monitoring changes in pressure during constant flow perfusion. An anticipated consequence of this is that the vasoconstriction at one location can be counteracted by vasodilation at another location. The suppression of a response during PE clearly demonstrates a vasodilatory effect of H2S; however, the lack of an effect during ET-1 infusion may be due to a set of more complex vascular events. It is possible that H2S can induce vasoconstriction in the hepatic sinusoid that which lacks VSMCs and vasodilation at presinusoidal sites.
Although H2S is generally regarded as a vasodilator, there are several reports that it can function as a vasoconstrictor. In VSMCs, H2S has been shown to lower cAMP levels and inhibit contraction (14). Hydrogen sulfide could increase the sensitivity to ET-1 by lowering cAMP levels in HSCs. cAMP has been shown to desensitize ETA receptors on HSCs to ET-1 (46). A second mechanism, reported by Ali et al. (13), demonstrated that high doses of exogenous H2S produce hypotension, whereas low-dose intravenous infusion of the H2S donor, NaSH, produced a transient increase in mean arterial pressure in rats. Interestingly, no increase in MAP was observed following treatment with L-NAME, a NO synthase inhibitor suggesting an interaction between the two gasses. It has been proposed that H2S may interact with NO to form a vasoinactive nitrosthiol, thereby quenching the NO signal (47). This could be of particular interest in endotoxemia as ET-1–stimulated NO synthesis in sinusoidal endothelial cells (SECs) is diminished (38). It is possible in our study that the addition of exogenous H2S may quench NO availability, causing constriction in the sinusoids while also hyperpolarizing VSMCs in the terminal portal venules, resulting in no net change in intrahepatic resistance.
Previously, we reported that H2S potentiates the increase in O2 consumption induced by PE infusion, which may contribute to hypoxic stress (28). Endothelin 1 stimulates glycogenolysis and O2 consumption in isolated rat hepatocytes (19, 48). Therefore, we investigated the effect of H2S and PAG on ET-1–stimulated O2 consumption. Infusion of low concentrations of ET-1, which did not produce a vascular response in control livers, increased hepatic O2 consumption. At higher doses of ET-1, O2 consumption was significantly reduced to the excessive vasoconstriction of the sinusoids. Hepatic O2 consumption was significantly reduced by LPS treatment, most likely due to enhanced sinusoidal vasoconstriction. This finding highlights the importance of sinusoidal integrity in maintaining proper O2 delivery during endotoxemia. The addition of H2S to the perfusate caused an initial increase in O2 consumption due to hepatic oxidation of H2S. Although H2S had no effect in control or LPS livers during 0.5 nM ET-1 infusion, it was associated with a significant increase in O2 consumption at the highest dose of ET-1. Throughout the experiment, H2S was undetectable in outflow perfusate, indicating complete hepatic H2S oxidation. Despite the decreased O2 consumption during sinusoidal hyperconstriction, the liver still utilized O2 for H2S oxidation, providing further evidence that H2S oxidation may contribute to hypoxic stress during endotoxemia. We observed that PAG potentiated the vascular effect of ET-1; in agreement with this, hepatic O2 consumption was significantly reduced compared with controls during low-dose infusion of ET-1. Although the PAG results in LPS-treated animals were not statistically significant (P = 0.055), there was a trend toward restoring hepatic O2 consumption to baseline levels. Because the reduction in O2 consumption in LPS-treated animals is the result of sinusoidal constriction, it is tempting to speculate that PAG treatment may improve sinusoidal perfusion during endotoxemia.
The present study sought to investigate the effect of H2S on different regulatory sites in the hepatic microcirculation. Using an isolated perfused organ system, we demonstrated a differential effect of H2S on presinusoidal resistance vessels that respond to PE and the sinusoids, which are modulated primarily by ET-1. We conclude that H2S contributes to the loss of vasomotor control in resistance vessels subject to regulation via catecholamine signaling. Although we cannot conclude that H2S causes vasoconstriction in the sinusoids, we do demonstrate that there is no attenuation of ET-1 hypersensitization, suggesting differential regulation in the sinusoids. In addition, we show that hepatic oxidation occurs despite limited O2 availability due to sinusoidal hyperconstriction. This finding combined with our previous report that H2S lowers hepatic oxygen availability in vivo supports the hypothesis that elevated H2S levels may exacerbate hepatic tissue hypoxia. The discrepancy between the effects of H2S on PE and ET-1 demonstrates the need for a better understanding of H2S as a vascular modulator. Overall, our study demonstrates that the regulation of hepatic vascular tone by H2S is complex and likely an important modulator in microvascular dysfunction during endotoxemia.
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