Developmental Expression of Endothelial Nitric Oxide Synthase (eNOS) in the Rat Liver

Abstract

Transition from fetal to postnatal life requires significant changes in cardiac, pulmonary, and hepatic blood flow. As such, there must be changes in vascular control in these vascular systems. Vascular resistance, a major contributor to blood flow, is mediated in the ductus arteriosus and pulmonary vasculature by endothelial nitric oxide synthase (eNOS). This study was conducted to determine the ontogeny of hepatic eNOS expression and activity. Additionally, the expression and activity of inducible nitric oxide synthase (iNOS) was measured to determine whether perinatal hypoxia resulted in detectable levels. NOS mRNA and proteins were determined by reverse transcription PCR assay and semiquantitative Western blot analysis, respectively. NOS activity was measured by the formation of [ 14 C]-citrulline from [14 C]-arginine. Localization of eNOS within the liver was determined by immunohistochemistry. eNOS mRNA was detectable at low levels at 18-d gestation and increased after birth, reaching a maximum level (4.5-fold increase) at 20 d of life. Parallel patterns for eNOS protein and activity were seen, with 6.9-fold and 16.1-fold increases, respectively. In the prenatal rat, eNOS was localized to areas of extramedullary hematopoiesis, with little signal in the sinusoids. Postnatally, there was a decrease in staining in the hematopoietic cells and a gradual increase in the staining of the endothelium of the sinusoids and central veins. iNOS mRNA and protein could not be detected at any age. eNOS expression and activity are developmentally regulated, increasing after birth coincident with an initial relative hypoxia and an increase in shear forces upon closure of the ductus venosus. NO is a short-lived free radical that influences physiologic processes in essentially every organ and tissue. Some of the roles of NO include neuromodulator, prevention of clotting, mediation of bactericidal and tumoricidal activity of macrophages, and regulation of blood pressure. NO is enzymatically synthesized from L-arginine by three known NOS isoforms: constitutively expressed eNOS (or NOS-3), nNOS (or NOS-1), iNOS (or NOS-2) (1-3). NO production by eNOS is primarily regulated by fluctuations in intracellular calcium levels. In the liver under normal conditions, only eNOS is present, produced by the sinusoidal endothelial cells (4, 5). The resulting low level of NO production is a major contributor of the basal vascular tone in the normal liver (4, 5).eNOS plays a role in control of vascular tone in most vascular beds (6). eNOS participates in the control of vascular tone during the transition from fetal circulation to postnatal circulation in the ductus arteriosus (7) and lung (8). The role of eNOS in control of the ductus arteriosus is age dependent. The effects of eNOS are more pronounced in determining vascular tone of the ductus arteriosus in the preterm animal than in the near-term or adult animal (7, 9). During the transition period, blood flow to the lungs is increased by a number of factors, including closure of the ductus ...

Nitric Oxide Production and its Contribution to Hepatocyte Proliferation in Normal Juvenile Rats

“…Obolenskaya et al [14] used methods similar to those reported here and demonstrated that NO production is very low in adult rats. In addition, NOS 3 (eNOS) protein in the liver reaches a maximum 20 days after birth, and these levels persist in adults [13]. These reports were in good agreement with the present study, where minimal NO production was observed in the livers of normal 5-week-old rats, when NOS 3 protein expression is almost at the same level as in adults.…”

“…Meanwhile, NOS 1 and NOS 2 mRNA were not detected in the liver during juvenile stages. Previous report has suggested that NOS 3 is the only NOS isoform expressed in the liver of newborn rats, and its expression continues into adulthood [13]. Thus, it is suggested that the NO production in the liver was derived from NOS 3.…”

“…On the other hand, in juvenile rats, the hepatocytes have high proliferative ability and are still undifferentiated with respect to their functions, which appear sequentially during the postnatal developmental stages [2,7,12]. Under normal physiological conditions, NOS 3 is constitutively expressed in the endothelial cells of the adult liver, while NOS 1 and NOS 2 are undetectable [10,13,22]. On the other hand, NO-derived NOS 2 is one of the key mediators of liver regeneration [16,26,27] wherein NOS 2 is expressed inductively in hepatocytes and kupffer cells [21].…”

Developmental changes in arginase expression and activity in the lung

“…This cannot be supported by our findings of eNOS expression in lung tissue. The developmental pattern of eNOS expression is thought to be such that it peaks after the neonatal period as shown in porcine lung (2) and also rat liver (28). In the present study, we documented that eNOS expression in rat lung was similar across ages with a tendency toward higher values in the fetus and newborn compared with adult lung.…”

Selective inhibition of iNOS attenuates trauma-hemorrhage/resuscitation-induced hepatic injury

“…At least two isoforms of NOS are found in the liver: endothelial NOS (eNOS) and inducible NOS (iNOS). NO generated by eNOS plays a vital physiological role in maintaining appropriate microvascular tone and blood flow and exhibits protective effects (30). Studies have shown that nonspecific NOS inhibition or genetic deletion of eNOS results in reduced microvascular perfusion and aggravated liver injury (10,17).…”