Induction and Role of NO Synthase in Hypotensive Hepatic Failure
Abstract Nitric oxide (NO) plays an important role in the physiological and pathophysiological control of the vascular system. NO is synthesized by isoforms of the enzyme NO synthase (NOS). Hepatic failure is complicated by hypotension, low systemic vascular resistance, and resistance to vasoconstrictor drugs. The potential role of NO in these abnormalities was investigated by using in vitro pharmacological interventions on hepatic arteries obtained from both donor and recipient patients at the time of liver transplantation. The presence of NOS mRNA was investigated by reverse transcription polymerase chain reaction (RT-PCR) with primers designed from human endothelial NOS (eNOS) and inducible NOS (iNOS) cDNA sequences. Arteries from patients with hepatic failure had an impaired constrictor response to phenylephrine compared with those of donor arteries. The constrictor effect of phenylephrine was potentiated by NG-monomethyl-l-arginine, an inhibitor of NOS, which had no effect in donor control arteries. RT-PCR identified human eNOS mRNA in donor and recipient arteries and human iNOS mRNA in recipient arteries only. Induction of NOS in the vasculature with subsequent NO-induced vasodilatation may therefore contribute to the hemodynamic abnormalities observed in hepatic failure and potentially in other pathologies associated with endotoxemia. Whether selective inhibitors of iNOS will improve hemodynamic control or clinical outcome in these conditions requires further study.
- Received October 20, 1995.
- Accepted March 20, 1996.
NO is known to play an important role in cell-to-cell communication.1 NO is synthesized from l-arginine by isoforms of the enzyme NOS, which can be divided into two functional classes. The constitutive, calcium-dependent enzymes are localized in endothelial (eNOS) and neuronal (nNOS) tissue and produce NO in short bursts for physiological purposes. In the endothelium eNOS plays a crucial role in the regulation of blood pressure and regional blood flow. The endotoxin- or cytokine-“inducible” isoform (iNOS) is calcium independent at physiological calcium concentrations and produces NO for as long as the enzyme is induced. Cloning studies have identified the DNA sequence for human eNOS,2 nNOS,3 and iNOS.4
Hepatic failure is often complicated by hypotension, low systemic vascular resistance, and reduced sensitivity to vasoconstrictor drugs.5 6 This condition is similar to the clinical syndrome in septic or endotoxin shock7 and has been reproduced in humans by injection of endotoxin.8 Plasma concentrations of endotoxins are known to be elevated in cirrhosis,9 a common cause of hepatic failure. It has thus been proposed that endotoxin could play a role in NOS induction in the vessel wall, with the consequent enhanced synthesis of NO causing the pathological vasodilatation observed in hepatic failure.10 A role for NO in the vasodilatation of endotoxin shock is supported by the demonstration that L-NMMA, an inhibitor of NOS, increases vascular resistance.11 Similarly, in animal models of endotoxemia, L-NMMA has been shown to improve the hemodynamics of this condition.12 13
Using hepatic arteries from patients with hepatic failure who were undergoing liver transplantation and arteries from donor patients as controls, we studied vascular reactivity in vitro and used RT-PCR to examine the expression of human eNOS and NOS mRNA.
Hepatic arteries were obtained from donor (n=7) and recipient (n=10) patients at the time of liver transplantation. Recipients had fulminant or Child-Pugh grade B or C hepatic failure, and all donors had been found to be healthy. Pathological conditions in the recipients included hepatitis C, non-A/non-B hepatitis, chronic active hepatitis, cryptogenic cirrhosis, and sclerosing cholangitis. Donor tissue had been harvested 10.5±0.9 (mean±SEM) hours earlier by the same surgical team and stored in transport medium (University of Wisconsin solution). Each recipient artery was studied immediately. At the time of transplantation a short segment of hepatic artery was collected into Krebs’ buffer if surplus artery was available. Arteries from recipients were obtained more distally than those from donors. Arteries were dissected free of connective tissue, cut into 2- to 3-mm rings, and suspended in 20-mL organ baths containing Krebs’ buffer at 37°C under 2 g resting tension. Some tissue was frozen at −70°C for subsequent RT-PCR. After a 1-hour wash and relaxation, a concentration-response curve to PhE was constructed. Subsequently some of the rings were exposed to 100 μmol/L L-NMMA for 20 minutes, and a second concentration-response curve to PhE was constructed. In some rings not exposed to L-NMMA, the effects of ACh or GTN were studied after preconstriction with PhE. When more than one ring per patient was studied, responses were averaged and the mean±SEM then calculated for n patients.
PhE and ACh were obtained from Sigma Chemical Co, GTN from American Hospital Supplies, and L-NMMA from Wellcome Research Laboratories.
RNA Isolation and RT-PCR
Tissue was homogenized and Poly(A)+ mRNA isolated by the Micro-FastTrack procedure (Invitrogen). RNA PCR was performed with the GeneAMP RNA-PCR reaction kit (Perkin-Elmer). Human eNOS was amplified with the following primers: 5′-CAGTGTCCAACATGCTGCTGGAAATTG-3′ and 5′-TAAAGGTCTTCTTCCTGGTGATGCC-3′, bases 1003 to 1029 (sense) and 1464 to 1488 (antisense), respectively, of the coding sequence2 that amplify a 485-bp product. Human iNOS was amplified with the following primers: 5′-GGCCTCGCTCTGGAAAGA-3′ and 5′-TCCATGCAGACAACCTT-3′, bases 1218 to 1235 (sense) and 1701 to 1717 (antisense), respectively, of the coding sequence4 that amplify a 499-bp product. Human eNOS was amplified in the presence of 1 mmol/L MgCl2 by 35 cycles of the following sequence: 96°C for 35 seconds, 62°C for 2 minutes, and 72°C for 2 minutes. Human iNOS was amplified in the presence of 1 mmol/L MgCl2 by 35 cycles of the following sequence: 96°C for 35 seconds, 56°C for 2 minutes, and 72°C for 2 minutes. PCR products were then sequenced directly after their elution from agarose gels. Sequencing was performed with the primers used in the PCR, dye-labeled terminators, and Taq cycle sequencing. Reaction products were analyzed on a 373A sequencing machine (Applied Biosystems).
The 10 patients with hepatic failure (39.4±4.7 years old) were vasodilated, with a systemic vascular resistance index of 806.6±143.0 dyn · s/cm3 · m2. Mean arterial blood pressure was 70.0±3.3 mm Hg and cardiac output 10.8±0.8 L/min. Donor age (44.6±4.5 years) was similar to that of the recipients. All recipient patients were maintained on low-dose (renal) dopamine and 2 were on inotropic support with epinephrine infusion. Four donor patients were maintained on low-dose dopamine and 2 on dobutamine. Sepsis was excluded in recipient patients before transplantation, but early chest sepsis was possible in 1. All recipient patients were taking prophylactic antibiotics, with negative blood cultures and a white cell count of 6.5±1.6. Two recipients had experienced recent gastrointestinal bleeding. There was no evidence of sepsis in donor patients.
The hepatic artery from recipients had a smaller diameter (2.7±0.3 mm, 29 rings) than the donor artery (4.1±0.4 mm, 19 rings) and was resistant to PhE-induced contraction compared with that of the donor artery (Fig 1⇓). At 3×10−5 mol/L PhE, the donor artery increased tension by 5.6±0.9 g and the recipient artery increase tension by 2.1±0.5 g (P<.05, unpaired t test). L-NMMA potentiated the response to PhE in the recipient artery (by 106±32% at 3×10−5 mol/L PhE, P<.05 paired t test) but had no effect on the donor artery (Fig 2⇓). Arteries from patients who were receiving inotropic support did not respond differently from those of patients who were not receiving such drugs. The effect was also independent of hepatic pathology. ACh (10−8 to 10−6 mol/L) either had no effect or induced a small contraction at the highest concentration studied, and GTN (10−8 to 10−4 mol/L) relaxed rings in both groups similarly (Fig 3⇓).
Use of RT-PCR with the human eNOS-specific primer generated a 485-bp product in both donor (n=3) and recipient (n=3) arteries (Fig 4⇓). The human iNOS-specific primer generated a 499-bp product in recipient artery but not in any donor artery (Fig 5⇓). After amplification with the iNOS-specific primers, the PCR fragment was subsequently isolated, subjected to DNA sequencing, and found to be identical to the published human iNOS cDNA sequence.4
Hepatic arteries from patients with hepatic failure were found to be resistant to constriction with PhE compared with control arteries from donor patients. This finding is comparable to the resistance to conventional vasoconstrictor drugs that can be observed clinically. The response to PhE was enhanced by the inhibition of NO synthesis by L-NMMA in hepatic arteries from patients with hepatic failure but not in control arteries. This potentiation restored the response of recipient arteries toward that of donor arteries. This effect of L-NMMA suggests basal synthesis of NO by recipient arteries, which is not detected in donor arteries. Differences in the peak effect of PhE may be partly accounted for by different vessel diameters, since recipient arteries were obtained more distally than were donor arteries. However, differences in vessel diameter alone would not explain the differences in response to L-NMMA. If basal NO synthesis were a widespread phenomenon, then it could account for the vasodilatation recorded preoperatively in the hepatic failure patients. The hepatic artery is a branch of the celiac axis and so is likely to be representative of systemic arteries, although a regional response cannot be excluded.
Arteries from neither recipients nor donors relaxed to ACh, which implies defective endothelial function in both. This may be attributable to the surgical preparation of both sets of arteries as well as to the disease process in recipient arteries. Comparable responses of both sets of tissues to GTN confirm that the underlying vascular smooth muscle function was intact. Whether transport of donor tissue altered the results is unclear. Because donor tissue had similar relaxation responses and greater contraction than recipient tissue, it seems unlikely that transport had a significant effect; however, this possibility cannot be excluded. Use of vasoactive drugs also seems unlikely to account for the effect, because arteries from patients who were taking such agents responded similarly to those who were not. Furthermore, the effect was independent of hepatic pathology, so specific viral induction is unlikely.
Synthesis of NO by recipient arteries was confirmed by the identification (by RT-PCR) of mRNA for human iNOS, which was absent in control donor arteries. Via the production of NO, the iNOS synthesized could account for the in vitro and in vivo abnormalities observed. All tissues under study expressed human eNOS mRNA, as identified by RT-PCR. The lack of effect of ACh or L-NMMA on the activity of this enzyme may be due to the presence of only small amounts of eNOS, since RT-PCR can detect very low levels of mRNA. The eNOS mRNA may reside in the remaining endothelium or within smooth muscle itself, which would contain endothelium in small vessels. iNOS is known to produce greater amounts of NO than does eNOS over a given period of time,1 which could explain the profound effects of iNOS induction.
The proposed mechanism for the effects we observed would be that liver disease, via endotoxemia and cytokine production, leads to induction of iNOS in the vessel wall. The NO subsequently produced would account for the hypotension and other hemodynamic abnormalities observed in this condition. This confirms the hypothesis previously proposed by Vallance and Moncada.10 It may also explain the hemodynamic abnormalities that are commonly observed in other causes of endotoxemia, including septic shock. The role of L-NMMA or other selective iNOS inhibitors in treating these conditions requires investigation. Preliminary data (unpublished observations) have demonstrated that L-NMMA is an effective agent in increasing blood pressure and systemic vascular resistance in fulminant hepatic failure, but further studies are needed.
Selected Abbreviations and Acronyms
|(NO)S||=||(nitric oxide) synthase|
|RT-PCR||=||reverse transcription polymerase chain reaction|
Prof Martin is a British Heart Foundation Professor of Cardiovascular Science. Prof Robinson is a British Heart Foundation Junior Research Fellow. We thank Marcus Oxer for assistance with the DNA sequencing and Annie Higgs for help with the manuscript.
Charles IG, Palmer RMJ, Hickery MS, Baylis MT, Chubb AP, Hall VS, Moss DW, Moncada S. Cloning, characterisation and expression of a cDNA encoding an inducible nitric oxide synthase from the human chondrocyte. Proc Natl Acad Sci U S A.. 1993;90:11419-11423.
Schrier RW, Caramelo C. Hemodynamic and hormonal alterations in hepatic cirrhosis. In: Epstein M, ed. The Kidney in Liver Disease. Baltimore, Md: Williams & Wilkins Co; 1988;265-285.
Parillo JE. Septic shock in humans. Ann Intern Med.. 1990;113:227-242.
Wright CE, Rees DD, Moncada S. Protective and pathological roles of nitric oxide in endotoxin shock. Cardiovasc Res.. 1992;26:48-57.