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J Appl Physiol 95: 2530-2536, 2003. First published August 22, 2003; doi:10.1152/japplphysiol.00681.2003
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Upregulation of hepatic prolactin receptor gene expression by 17{beta}-estradiol following trauma-hemorrhage

Yukihiro Yokoyama, Williams C. Kitchens, Balazs Toth, Martin G. Schwacha, Kirby I. Bland, and Irshad H. Chaudry

Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama 35294

Submitted 2 July 2003 ; accepted in final form 14 August 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Although studies show protective effects of 17{beta}-estradiol (E2) or prolactin (PRL) treatment in male rats after trauma-hemorrhage (TH), the mechanism of the salutary effects of these agents remains unknown. Because E2 modulates PRL receptor (PRL-R) expression in the liver, we examined whether E2 treatment after T-H has any effects on hepatic PLR-R gene expression. Male Sprague-Dawley rats were subjected to trauma (i.e., 5-cm midline laparotomy) and hemorrhage (35–40 mmHg for 90 min) followed by fluid resuscitation (Ringer lactate) or sham operation and then treated with E2 (50 µg/kg body wt sc) or vehicle immediately before resuscitation. Liver samples were collected at 3 h thereafter, and PRL-R mRNA expression was determined by PCR. Liver expression of PRL-R short-form gene was unaffected by T-H, whereas that of the long-form gene was suppressed. Treatment of T-H rats with E2 significantly increased PRL-R short-form gene expression and normalized PRL-R long-form gene expression to sham levels. In the isolated hepatocytes, PRL-R short-form gene expression was predominant compared with the long-form gene. In contrast, only the short form was detected in Kupffer cells. In vitro treatment by E2 demonstrated an increase in the PRL-R long-form gene in hepatocytes, but E2 had no effect on PRL-R short-form gene expression in either the Kupffer cells or hepatocytes. Thus E2 treatment after T-H in males appears to directly upregulate PRL-R long-form gene expression in hepatocytes. However, the upregulation of the PRL-R short form might involve the interaction of multiple cell types in the liver.

Kupffer cells; hepatocytes; liver; gender; shock

A BODY OF EVIDENCE HAS SHOWN that female sex hormones, i.e., estradiol (E2) and prolactin (PRL), have a protective role on immune and organ functions under stressful conditions such as trauma-hemorrhage (T-H) (8, 18, 20). Studies have also shown that proestrus female rats, which have high plasma E2 and PRL levels, do not demonstrate depression in immune functions (2, 3, 20) or cardiac and hepatic functions (13) after T-H. With regard to the liver, the release of proinflammatory cytokines by the Kupffer cells after T-H is significantly attenuated in proestrus females compared with males (19). Furthermore, the elevation in plasma alanine aminotransferase and decreased indocyanine green (ICG) clearance observed in males after T-H are attenuated in proestrus females under those conditions (13). Additionally, elevation of plasma E2 or PRL levels by pretreatment in male animals before T-H resulted in normalized immune functions and improved cardiac and hepatic functions (14, 18, 20, 22). In view of the above results, E2 and PRL have been proposed as novel adjuncts in the treatment of T-H. However, the precise mechanism by which female sex hormones mediate these observed beneficial effects remains poorly understood.

PRL, a single-chain 24-kDa protein, has multiple physiological functions, including lactation, reproduction, growth and development, water and electrolyte balance, and immunoregulation (5). PRL is promptly secreted from the anterior pituitary gland in response to a severe stress, and the activation of PRL receptor (PRL-R) leads to the modulation of gene expression in target organs (5). Two different PRL-R gene subtypes have been identified in the rats, i.e., short and long forms (30). Although the gene expression of PRL-R long form is predominant compared with short form in many organs (23), this expression pattern is reversed in the liver. Nevertheless, it remains unknown whether the altered expression of each receptor subtype occurs in the liver in response to T-H. Furthermore, the expression pattern of PRL-R subtype in different cell types of the liver has not been fully investigated under normal or pathophysiological conditions.

E2 and PRL have a mutual endocrine relationship, and both hormones have multiple effects on immune and organ functions (23). E2 has been shown to stimulate pituitary PRL production and secretion (9, 10). E2 administration also increases the expression of PRL-R in the brain (21), mammary gland (7), ovary (33), and liver (30, 34). Because both PRL and E2 treatment elicit similar protective effects on immune and hepatic functions after T-H (14, 18, 20, 22), it is pertinent to determine whether E2 has any effects on the expression of PRL-R gene in this model. This might help to understand part of the mechanism of salutary effects of E2 on the hepatic damage after T-H. The aim of this study, therefore, was to investigate whether there are any alterations in the expression of PRL-R in the liver after T-H, with and without the administration of a single dose of E2. Additionally, hepatocytes and Kupffer cells were isolated after T-H to elucidate the different patterns of PRL-R subtype gene expression in each cell type. Furthermore, the direct effects of E2 on PRL-R gene expression in each cell type were determined by incubating the isolated cells with different concentrations of E2.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Animals. Experiments were performed by using male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 250–350 g at the time of operation. All procedures were performed in accordance with National Institutes of Health guidelines under a protocol approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham.

Experimental groups. The rats were randomly assigned to the following three treatment groups: sham-operated animals with vehicle treatment (SV), T-H animals with vehicle treatment (HV), and T-H animals with E2 treatment (HE). E2 ({beta}-estradiol 3-benzoate; Sigma Chemical, St. Louis, MO) was prepared by dissolving in corn oil (Sigma Chemical) and was injected subcutaneously (50 µg/kg body wt) just before the onset of resuscitation. An equal amount of corn oil (1 ml/kg) was used for the vehicle treatment. The dose of E2 used was based on our previous study, which revealed hepatoprotective effects of E2 after T-H (22). Furthermore, the time period of the study was 3 h after E2 or vehicle treatment. This time point was chosen to examine the early changes in gene expression after E2 treatment. In separate experiments, hepatocytes and Kupffer cells were isolated after T-H to examine the direct effects of E2 on the PRL-R gene expression in each cell type.

T-H procedure. The rats were fasted overnight but were allowed free access to water before the experiment. After anesthesia by isoflurane (Halocarbon Laboratories, River Edge, NJ) inhalation, soft tissue trauma (i.e., 5-cm midline laparotomy) was produced. After the abdominal wound was closed in layers, polyethylene tubes (PE-50, Becton Dickinson, Sparks, MD) were placed in both femoral arteries and the right femoral vein. The groin wounds were bathed in 1% lidocaine (Elkins-Sinn, Cherry Hill, NJ) throughout the procedure to minimize the wound pain. The rats were allowed to awaken, and afterward they were bled rapidly to a mean arterial pressure (MAP) of 35–40 mmHg. This hypotension was maintained until the animals could not maintain MAP of 35 mmHg unless extra fluid, in the form of Ringer lactate, was given. This time was defined as maximum bleed-out. After the maximum bleed-out, MAP was maintained between 35 and 40 mmHg until 40% of the maximum bleed-out volume was returned in the form of Ringer lactate. The total period of hypotension was 90 min. The animals were then treated by a subcutaneous injection of E2 or vehicle and were then resuscitated with four times the volume of the withdrawn blood over 60 min with Ringer lactate. After resuscitation, the catheters were removed and the wounds were sutured. Sham-operated animals underwent the same groin incision and vessel ligation without performance of the midline laparotomy and hemorrhage. Three hours after vehicle or E2 administration, the rats were anesthetized by isoflurane inhalation, and the blood samples were drawn from the abdominal aorta into a heparinized syringe. Plasma was then separated by centrifugation at 3,220 g for 12 min at 4°C and stored at -80°C until use. Hepatic tissue was harvested and quickly frozen in liquid nitrogen for the measurement of PRL-R gene expression.

Measurement of plasma E2 and PRL. Plasma E2 and PRL levels were measured by using commercially available ELISA kits (Cayman Chemical, Ann Arbor, MI) and preformed as recommended by the manufacturer.

Hepatocyte and Kupffer cell isolation. Hepatocytes and Kupffer cells were isolated by an in situ collagenase digestion method (35). Briefly, the liver was perfused with oxygenized Hanks' balanced salt solution (GIBCO-BRL, Gaithersburg, MD) for 10 min to wash out the blood and was perfused with 0.03% collagenase (Sigma Chemical) for 5 min. After the digestion, cell suspension was filtered through a sieve and centrifuged at 50 g for 3 min to separate parenchymal cells and nonparenchymal cells. Parenchymal cells were washed by repeating centrifugation at 50 g for 3 min. Nonparenchymal cells were further centrifuged over 16% metrizamide (Accurate Chemical, Westbury, NY) for 45 min to separate Kupffer cells (2,000 g, 45 min, 4°C). The cells at the interface were collected and washed twice by centrifugation with Hanks' balanced salt solution (450 g, 10 min, 4°C). The pellet was then resuspended in William's E medium (GIBCO-BRL) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO-BRL). After counting the cell numbers and assessing the cell viability by Trypan blue exclusion (average viability >95%), the concentration was adjusted to 1 x 106 cells/ml in William's E medium and plated on the 12-well culture dish. Hepatocytes were incubated in a collagen type I (Sigma) coated plate, whereas Kupffer cells were incubated in a noncoated plate. After 2-h incubation, the wells were washed to remove nonadherent cells. For both hepatocytes and Kupffer cells, TRIzol reagent (Invitrogen, Carlsbad, CA) was used to preserve mRNA. In a separate experiment, isolated hepatocytes and Kupffer cells were stimulated in vitro with different concentrations of E2 (0.5, 5, and 50 nM) for 16 h before mRNA were harvested. The incubation temperature of 36°C was used throughout the procedure.

RNA preparation. Total RNA was prepared from liver homogenate samples by using the RNeasy mini kit (QIAGEN, Valencia, CA), according to the manufacturer's instructions. Total RNA extraction from isolated hepatocytes and Kupffer cells was performed according to the manufacturer's instruction for TRIzol reagent (Life Technologies, Gaithersburg, MD). Briefly, 1 x 106 cells dissolved and frozen in 1 ml of Trizol reagent were thawed and incubated at room temperature for 5 min. Then 0.2 ml of chloroform was added, followed by vigorous shaking and incubation at room temperature for 2–3 min. The sample was then centrifuged for 10 min at 12,000 g at 4°C for phase separation. The aqueous phase was carefully withdrawn, and 0.5 ml of isopropyl alcohol was added. After incubation at room temperature for 10 min, samples were centrifuged at 12,000 g at 4°C to precipitate out the RNA. The RNA pellet was washed once with 1 ml of 80% (vol/vol) ethanol and centrifuged at 7,500 g for 5 min at 4°C. The RNA pellet was then air dried for 10 min at room temperature, dissolved in diethyl pyrocarbonate-treated distilled water, and stored at -80°C. RNA concentration and purity were determined on a spectrophotometer (SmartSpec 3000, Bio-Rad, Hercules, CA) by calculating the ratio of optical density at wavelengths of 260 and 280 nm.

Reverse transcription polymerase chain reaction. Reverse transcription of 1 µg of total RNA was performed by using the Advantage RT-for-PCR kit (Clontech Laboratory, Palo Alto, CA). The following components were combined: 1 µg of total RNA, 20 pmol of random hexamer primer, 1x reaction buffer (50 mM Tris · HCl, 75 mM KCl, 3 mM MgCl2, pH 8.3), dNTP mix (10 mM each), 1 U/µl of RNase inhibitor, Moloney-Murine leukemia virus reverse transcriptase, and 200 U/µg RNA. cDNA was amplified with each set of primers by using HotStartTaq Master Mix Kit (QIAGEN) in a total volume of 50 µl (the final concentrations: 1 µM sense and antisense primer, 1.5 mM MgCl2, 200 µM dNTPs; 0.5 unit Taq DNA polymerase). {beta}-Actin was used as an internal control. Primers used in this procedure are listed in Table 1. Common forward sequence was used for the amplification of a cDNA fragment derived from both PRL-R short and long form. The PCR reactions were carried out in a gradient Mastercycler (Eppendorf, Westbury, NY). The first cycle for PCR was carried out at 95°C for 15 min. The amplification was carried out with 35 cycles at 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min, followed by an additional 10 min of extension at 72°C. The number of reaction cycles was determined in a range that showed exponential amplification for PRL-R short, long, and {beta}-actin. The PCR products were electrophoresed on 1.5% agarose gel stained with ethidium bromide. The intensity of cDNA bands was measured in the 500 Fluorescence ChemiImager (San Leandro, CA).


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  		Table 1. Primer sequence used for RT-PCR

 

Statistical analysis. There were six to nine animals in each group. Results are presented as means ± SE. One-way ANOVA followed by the Student-Newman-Keuls test for multiple comparisons was used to determine significant differences among the experimental groups. When criteria for parametric testing were violated, the appropriate nonparametric (Mann-Whitney U-test) was used. P value <0.05 was considered to indicate a significant difference.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Plasma E2 and PRL levels. At 3 h after E2 treatment, plasma E2 levels were significantly increased in the T-H group (Fig. 1A). In contrast, plasma PRL levels did not show any significant difference in SV, HV, or HE, and the average plasma PRL concentrations of all groups were within the normal range of male rats (8–33 ng/ml; Fig. 1B).



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  		Fig. 1. Plasma estradiol (E2; A) and prolactin (PRL; B) levels measured by specific ELISA. Male Sprague-Dawley rats were subjected to sham (S) or trauma-hemorrhage (T-H; H). The rats were treated with either corn oil vehicle (V) or 50 µg 17{beta}-E2/kg body wt (E). At 3 h after E2 administration, blood was withdrawn from the aorta. Data are means ± SE of 8–9 animals in each group. *P < 0.05 vs. HV and #P < 0.05 vs. SV by ANOVA.

 

PRL-R gene expression in crude liver homogenate. Total hepatic mRNA expression for PRL-R short form did not change after T-H compared with sham in vehicle-treated rats. However, E2 treatment significantly increased the expression in the T-H group (Fig. 2A). In contrast, the expression of the PRL-R long-form gene was significantly depressed in the HV group compared with SV and was restored in the HE group (Fig. 2B).



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  		Fig. 2. Hepatic expression of PRL receptor short-(PRL-RS; A) and long-form (PRL-RL; B) mRNA. The expressions were analyzed by RT-PCR by using specific primers. Top: representative gels and {beta}-actin as the housekeeping gene are shown. Average intensities of the bands analyzed by densitometry are shown in the bar graphs. Data are the means ± SE of 6 separate experiments in SV, HV, and HE treatment. *P < 0.05 vs. SV and HV. #P < 0.05 vs. SV and HE by ANOVA.

 

PRL-R gene expression in isolated hepatocytes and Kupffer cells. PRL-R gene expression in purified hepatocytes and Kupffer cells stimulated by E2 in vitro after T-H was determined to elucidate which cell type was responsible for the altered expression of the PRL-R gene in the total liver homogenate. When the same amount of template and same number of amplification cycles were used for RT-PCR, differences in the amount of gene expression for each receptor type were observed in hepatocytes and Kupffer cells (Fig. 3). In hepatocytes, the mRNA expression for PRL-R short form was significantly higher than that for the long form, both in the sham and T-H groups (Fig. 3). Interestingly, the expression of the PRL-R short-form gene was detected in isolated Kupffer cells, but the level was significantly lower compared with the level observed in hepatocytes from both sham and T-H groups (Fig. 3). Furthermore, no PRL-R long-form gene expression was detected in Kupffer cells. Additionally, PRL-R long-form gene expression in hepatocytes was significantly lower in the T-H group compared with the sham group. These results are consistent with the observation using the liver homogenates.



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  		Fig. 3. The mRNA expression of PRL-RS and PRL-RL and {beta}-actin mRNA as the housekeeping gene in hepatocytes (HC) and Kupffer cells (KC). The expressions were analyzed by RT-PCR using specific primers. Top: 2 pairs of representative gels in sham and T-H are shown. Average intensities of the bands analyzed by densitometry are shown in the bar graphs. Values are means ± SE of 6 separate experiments in sham and T-H. *P < 0.05 vs. PRL-RS in the HC in sham group. **P < 0.05 vs. PRL-RS in the HC in T-H group. #P < 0.05 vs. PRL-RL in the HC in sham group by ANOVA. ^P < 0.05 vs. HC sham and T-H.

 

Effects of in vitro E2 treatment on the PRL-R expression. Hepatocytes and Kupffer cells were incubated with different concentrations of E2 (0, 0.5, 5, and 50 nM) to determine whether E2 directly alters the expression of the PRL-R gene. Stimulation with E2 did not alter the PRL-R short-form gene in either hepatocytes or Kupffer cells isolated after T-H (Fig. 4A). However, the downregulated expression of the PRL-R long-form gene after T-H in the hepatocytes was restored by the treatment of E2 (Fig. 4, B and C). No such change in the PRL-R long-form gene expression was observed in the cells isolated from sham animals.



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  		Fig. 4. The direct effects of E2 on PRL-R gene expression in isolated HC and KC. The cells were isolated after T-H and were incubated with different concentrations of E2 for 16 h before mRNA was harvested. Average intensities of the bands analyzed by densitometry are shown in the bar graphs (A: PRL-RS, B: PRL-RL). C: representative gel from the PCR products of HC PRL-RL and {beta}-actin. SH, HC from sham; HH, HC from T-H; SK, KC from sham; HK, KC from T-H. *P < 0.05 vs. SH.

 


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
To the best of our knowledge, this is the first study to show that the hepatic expression of the PRL-R gene is altered after T-H and can be modulated by a single administration of E2. Second, our findings demonstrate a unique expression pattern of PRL-R genes in hepatocytes and Kupffer cells after injury, which has not previously been reported. The distribution of PRL-R immunoreactivity detected by anti-PRL-R antibodies has been shown to be similar to the expression of the PRL-R gene, although these were not entirely parallel (4, 14, 26). Furthermore, during ontogeny or in response to estrogen treatment, the rat liver increases both mRNA levels and binding levels of PRL-R (14). These results, therefore, collectively suggest that PRL-R gene expression in rat liver is regulated similarly at the transcriptional and translational levels. In view of this, we examined the expression of the PRL-R gene to understand and extrapolate the functional regulation of the PRL-R system after T-H, with or without E2 treatment.

There is a delicate regulation and mutual interaction between E2 and PRL. Studies have shown that E2 upregulates the expression of the PRL-R gene in various organs (17, 29, 31). In the liver, PRL-R gene expression has been reported to be upregulated in response to E2 administration (1, 12, 32). However, neither form of PRL-R gene was found to be upregulated by E2 administration in the sham group in this study. The difference between our results and the previous reports showing upregulation of PRL-R gene by E2 is likely due to the method of drug administration and time course of the study. Our protocol used a single administration of E2 as a therapeutic dose, and the time point for the measurement of mRNA expression was relatively short (3 h posttreatment). In contrast, previous studies used chronic treatment with E2 (1, 32). Interestingly, in the T-H group, the expression pattern of PRL-R subtype gene was altered at that time period. Although the expression of the short-form gene did not show any change, the message for the long form was significantly downregulated. Moreover, E2 treatment increased the expression of both PRL-R short- and long-form gene in the liver in the in vivo study. A restored level of the PRL-R gene implies that E2 can indirectly enhance the effects of PRL on hepatic function after T-H.

Unexpectedly, plasma PRL levels did not show any change after E2 treatment in the T-H group, despite the increased plasma E2 levels in this group. Previous study showed that either E2 administration (11) or hemorrhagic insult (16) temporarily induces PRL release from the pituitary gland. The reason why our experiment showed unchanged levels of plasma PRL, either by hemorrhagic insult or E2 treatment, is unknown. However, because we measured plasma PRL levels only at 3 h after T-H and E2 treatment, it is possible that plasma PRL levels may have increased at an earlier or later time point. The other time points were, however, not measured in the present study. It is also possible that E2 administration increases the binding affinity of PRL-R to its ligand in the liver, as shown by Guillaumot et al. (12). However, this notion remains controversial because other studies showed that E2 does not change the binding affinity of PRL-R, even though it significantly increases the number of PRL-R (4, 32). Additional studies to elucidate the change in binding affinity of PRL-R in the liver after T-H with and without E2 treatment should clarify this aspect.

To further elucidate which hepatic cell type was responsible for the altered PRL-R gene expression, hepatocytes and Kupffer cells were isolated, and the expression pattern of each receptor subtype was elucidated. Hepatocytes demonstrated predominant expression of the PRL-R short-form gene and lesser expression of PRL-R long-form gene in concordance with the previous report using cells from liver homogenates (23). It is noteworthy to mention that Kupffer cells displayed significantly lower levels of PRL-R short-form mRNA expression compared with the hepatocytes by using equal quantities of template and PCR cycle amplifications. More interestingly, there was no expression of PRL-R long-form gene in the isolated Kupffer cells. To our knowledge, this is the first report demonstrating the expression pattern of the PRL-R gene in isolated Kupffer cells in rats.

Interestingly, the expression of the PRL-R long-form gene in hepatocytes is significantly downregulated after T-H compared with sham. Furthermore, this depressed expression was restored with E2 treatment in the in vitro study. These observations are similar to the pattern of PRL-R long-form gene modification obtained from the liver homogenates in the in vivo study. The beneficial effects of PRL for the treatment of T-H-induced hepatic damage have been shown in our laboratory's previous reports (14). Increased plasma PRL levels by metoclopramide treatment significantly improved the depressed hepatocellular function after T-H, which was assessed by the ICG clearance technique (14). Interestingly, a similar beneficial effect on ICG clearance was observed by E2 treatment within the same model (22). We, therefore, propose that the beneficial effects of E2 are partly mediated by its effect on PRL-R modulation. PRL has other effects on the functions of the hepatocytes. Reports have shown that PRL stimulates the regeneration in hepatocytes after partial hepatectomy via the activation of protein kinase C (6). It could, therefore, be postulated that PRL contributes to the regeneration processes of the damaged liver because hepatocyte damage does occur after T-H (6). Therefore, the restored expression of the PRL-R long-form gene in the hepatocytes after E2 treatment might have protective effects in preventing the hepatocyte damage after T-H.

In contrast to the PRL-R long form, no effect of E2 on the PRL-R short-form gene expression in hepatocytes was observed in the in vitro study, despite its increase in the in vivo studies using liver homogenates. Although studies have shown the upregulation of PRL-R short-form gene expression after E2 treatment (30, 34), those studies used total hepatic cells and not isolated liver cells specifically. To date, there is no available information regarding the modulation of PRL-R short-form gene expression in different cell types of the liver. Furthermore, studies examining the interaction of each liver cell type in the regulation of PRL-R short-form gene expression have not been conducted, and thus it remains unclear which cell type(s) is (are) involved in the modulation of the PRL-R short form. Based on our study, it can be speculated that, for the modulation of the PRL-R short form, the interaction between hepatocytes and other cells, such as the Kupffer cell, endothelial cell, and hepatic stellate cell, might be required.

Another important finding in this study is the evidence that isolated Kupffer cells only express the PRL-R short-form gene, although its expression was not modified by either T-H insult or E2 treatment. Kupffer cells have been shown to be a major source of inflammatory cytokine released after T-H (25). In this regard, the existence of PRL-mediated processes has been demonstrated in isolated Kupffer cells in T-H models (18, 36). Although T-H markedly increases the level of mRNA for IL-1{beta}, IL-6, and TNF in Kupffer cells, in vivo treatment of animals with PRL after hemorrhage significantly decreased the expression of these cytokines (36). Our previous studies have also demonstrated that PRL directly suppresses the proinflammatory cytokine release from isolated Kupffer cells in vitro after T-H (18). These findings suggest that PRL directly acts on Kupffer cells and contributes to blunting the systemic inflammatory response associated with T-H. Taken together with our present results, we propose that any receptor-dependent effects of PRL on Kupffer cells are mediated through the activation of the PRL-R short form but not through the long form. Further studies will help to elucidate the cellular mechanisms involving activation of a specific signal transduction pathways and gene expression after stimulation of the PRL-R short form in Kupffer cells.

The expression pattern of each receptor subtype in other hepatic cells, such as endothelial cells and hepatic stellate cells, was not evaluated in the present study. These cells are important in regulating the hepatic microcirculation by producing and reacting to vasoregulators in a paracrine or autocrine fashion (26). It is, therefore, important to determine the PRL-R expression on hepatic stellate cells and endothelial cells in normal and patholophysiological conditions in future studies.

In addition to the cells from the liver, distinct patterns of PRL-R gene expression and PRL-R-positive cells in the thymus of mice have been reported after T-H that are modulated by age and gender (24). Whether or not the thymic PRL-R expression is also increased after T-H or E2 treatment in the rat liver remains to be determined. Our previous studies have also demonstrated the presence of androgen and estrogen receptors in T cells (28). Whether or not E2 treatment upregulates estrogen receptor and downregulates androgen receptors in the hepatocytes and Kupffer cells also remains to be determined.

In summary, our results demonstrate decreased PRL-R long-form gene expression in the liver after T-H, in both liver homogenates and isolated hepatocytes. Furthermore, this decreased expression of PRL-R long-form gene was restored by treatment of animals with a single dose of E2 after T-H. These results indicate that E2 may modulate hepatic PRL and PRL-R system through its long-form receptor after T-H.


   DISCLOSURES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This work was supported by National Institute of General Medical Sciences Grant R37 GM-39519 (to I. H. Chaudry).


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. H. Chaudry, Center for Surgical Research, Univ. of Alabama at Birmingham, Volker Hall G094, 1670 Univ. Boulevard, Birmingham, Alabama 35294–0019 (E-mail: Irshad.Chaudry{at}ccc.uab.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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