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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 1, 2003 as doi:10.1096/fj.03-0082fje. |
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* Unité de Recherche U.442, Institut National de la Santé et de la Recherche Médicale, Université Paris Sud, 91405 Orsay, France; and
Unité de Recherche U.469, Institut National de la Santé et de la Recherche Médicale and
UMR5101 Centre National de la Recherche Scientifique, 34094 Montpellier, France
3Correspondence: Unité de Recherche U.442, Institut National de la Santé et de la Recherche Médicale, Université Paris Sud, bât. 443, 91405 Orsay, France. E-mail: thierry.tordjmann{at}ibaic.u-psud.fr
SPECIFIC AIMS
After partial hepatectomy, liver mass is restored through a complex regulatory network of cytokines, growth factors, and hormones. In this study, we show that an interplay between hypothalamic vasopressin secretion and a remodeling of Ca2+ signals in the liver contributes significantly to rat liver compensatory growth and bile flow.
PRINCIPAL FINDINGS
1. Plasma arginine vasopressin (AVP) concentration highly increases through an activation of hypothalamic nuclei early after partial hepatectomy
Plasma AVP concentration increased sharply after partial hepatectomy, peaking 1 h after surgery (1.0±0.5 pg/mL, n=10 before surgery vs. 21.6±1.6 pg/mL, n=5 afterward), and remained higher than in the sham group for the following 24 h. A significant but smaller increase in AVP concentration was observed 1 h after sham operation (6.9±1.9 pg/mL, n=5).
We found that hypothalamic neurons of supraoptic (SON) and paraventricular (PVN) nuclei are stimulated after hepatectomy (and not sham surgery), using an antibody directed against the proto-oncogene c-fos protein product c-Fos, a well-established activation marker for neurons in response to a variety of stimuli. Only faint c-Fos immunostaining could be detected in the forebrains of sham-operated rats 1 h (Fig. 1
A, n=4) and 24 h (not shown) after surgery. In contrast, 1 h after hepatectomy, strong c-Fos immunostaining was associated with the nucleus of numerous neuron-like cells located within the SON (Fig. 1B
, n=4) and PVN (not shown). This labeling was also observed 2 h after hepatectomy but was absent 24 h later (data not shown). Examination of triple immunostained hypothalamus sections 1 h after hepatectomy indicated that most of the c-Fos-positive neurons located within the SON (and the PVN, not shown) were immunostained for AVP or ocytocin (OT) (Fig. 1C
). These data strongly support the idea that the increase in circulating AVP detected 1 h after hepatectomy originated in the hypothalamic AVP neurons. The amount of hypothalamic AVP mRNA, analyzed by semiquantitative RT-PCR, increased by 60% in the 8th hour after hepatectomy before returning to the basal level between the second and the third day (Fig. 1D
), suggesting that further AVP is synthesized to replenish neuronal stores, as previously reported after osmotic or hemorrhagic stress.
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2. Remodeling of AVP-induced Ca2+ signaling and V1a receptor expression after partial hepatectomy
We explored the responsiveness of hepatocytes to AVP by measuring the capacity of cells to mobilize intracellular Ca2+ in response to the hormone. Hepatocytes remained fully responsive to AVP (and to other Ca2+-mobilizing agonists) during the first 8 to 10 h and began to exhibit a lower sensitivity to AVP 12 h after hepatectomy. Then, two phases of deep hepatocyte desensitization occurred (at 20–24 h and 96–120 h). Almost basal cell sensitivity to hormones recovered 
6 days after hepatectomy. AVP-triggered [Ca2+]i responses exhibited significantly longer latencies in regenerating hepatocytes at 24 h (37±5 s vs. 19±4 s, P<0.05, n=5). Furthermore, a greater proportion of regenerating hepatocytes (57.5±3.5% vs. 23±7%, P<0.01, n=5) exhibited oscillating responses. Given that different sets of genes can be switched on or off by different [Ca2+]i kinetic patterns, hepatocyte Ca2+ signaling adaptation during liver regeneration may tune gene expression to leave the G0 quiescent state.
Remodeling of hormone receptor density and lobular distribution were found during liver regeneration. Autoradiographs ([125I]HO-LVA) of liver sections from sham-operated and hepatectomized rats showed that regenerating livers contained 2.48 ± 0.3-fold (P<0.001, n=5) less V1a binding sites than sham livers 24 h after surgery. The amount of V1a mRNA analyzed with RT-PCR was lower in hepatectomized rats than in sham-operated rats 8 and 12 h after surgery (36.9±4.6 and 48.7±6% of sham values, respectively; P<0.001, n=3) and was similar in both rat groups at 24 h, remaining stable thereafter. These kinetics suggest that the observed drop in V1a density 24 h after hepatectomy is at least partly due to transcriptional regulation. The V1a receptor distribution was highly heterogeneous, perivenous (PV) areas being richer in this receptor than periportal (PP) regions, as reported in control non-operated rats. At 24 h, the PV/PP gradient in V1a density was significantly (P<0.001) higher (3.16±0.13, n=5) in hepatectomized rats than in sham-operated rats (2.18±0.09, n=5) because of a more pronounced loss of V1a receptors in the PP than in the PV area.
3. AVP contributes to liver growth after partial hepatectomy
The specific V1a receptor antagonist SR49059 was used to study the in vivo effect of AVP in the rat after hepatectomy. V1a antagonism strongly reduced liver mass restoration, mainly in the first 24 (P<0.001, n=6) to 48 (P=0.05, n=6) h after partial hepatectomy (Fig. 2
A). The liver mass was subsequently restored similarly in control and SR49059-treated rats. The peak DNA synthesis 24 h after partial hepatectomy was also significantly damped in SR49059-treated rats compared with controls, as measured by the percentage of hepatocytes incorporating BrdU (P=0.001, n=4) (Fig. 2B
) and by assessing thymidine kinase activity (P=0.05, n=4) (Fig. 2C
). Thus, AVP may help quiescent G0 hepatocytes to enter the cell cycle in G1 and to progress to the S phase, thus restoring liver mass. In line with these results, significantly lower amounts of cyclin D1 and cyclin A proteins were detected in treated vs. untreated rats 24 and 48 h (P<0.05, n=4) after hepatectomy. Also, NF-
B, a crucial transcription factor implicated in the priming of hepatocytes leading to immediate early gene expression, was significantly less activated in rats treated with the V1a antagonist than in vehicle-treated rats, mainly at 30 min (P<0.01, n=4) after partial hepatectomy. Given that NF-B is submitted to Ca2+-dependent regulation, it is tempting to suggest that AVP-elicited [Ca2+]i oscillations in hepatocytes contribute to the activation of this transcription factor.
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4. AVP contributes to bile flow after partial hepatectomy
AVP is a regulator of bile flow in the rat due to its capacity to generate intercellular calcium waves, which are spatially oriented along the acinus axis by a V1a receptor gradient. Given that soon after hepatectomy and later, the AVP concentration is high in the plasma and that the V1a receptor gradient is enhanced in the liver lobule, we examined the regulation of bile flow by AVP (10–300 pM) in the regenerating liver. We calculated that the choleretic effect of AVP was significantly enhanced (P<0.001, n=5) and the cholestatic effect was largely dampened (P<0.001, n=5) in hepatectomized rats at 24 h compared with sham-operated rats. These results suggest that AVP is an in vivo regulator of bile flow when the functional mass of hepatocytes is reduced. Based on our previous work, the observed amplification of the V1a receptor lobular gradient may account for the enhanced impact of AVP on bile flow during liver regeneration. Further studies are required to determine the consequences of a steeper V1a receptor density gradient along hepatocyte plates on intercellular Ca2+ wave kinetics during liver regeneration.
CONCLUSIONS AND SIGNIFICANCE
In conclusion, our study provides new insights into the physiological impact of AVP on liver function (Fig. 3
). A very early hypothalamic release of AVP after partial hepatectomy helps the liver to initiate its mass recovery. On the other hand, AVP appears to stimulate bile flow during liver regeneration. The remodeling of hepatocyte AVP-induced Ca2+ signaling after partial hepatectomy is suggested to underlie these effects, a lower number of V1a receptors together with a steeper lobular receptor gradient appearing to promote AVP efficiency during liver regeneration.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0082fje; doi: 10.1096/fj.03-0082fje 
2 A.N. and V.S. contributed equally to this work.
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