Effect of 5-HT7 receptor blockade on liver regeneration after 60-70% partial hepatectomy

Background Serotonin exhibits a vast repertoire of actions including cell proliferation and differentiation. The effect of serotonin, as an incomplete mitogen, on liver regeneration has recently been unveiled and is mediated through 5-HT2 receptor. The aim of the present study was to investigate the effect of 5-HT7 receptor blockade on liver regeneration after partial hepatectomy. Methods Male Wistar rats were subjected to 60-70% partial hepatectomy. 5-HT7 receptor blockade was applied by intraperitoneal administration of SB-269970 hydrochloride two hours prior to and sixteen hours after partial hepatectomy and by intraperitoneal administration of SB-258719 sixteen hours after partial hepatectomy. Animals were sacrificed at different time points until 72 h after partial hepatectomy. Liver regeneration was evaluated by [3H]-thymidine incorporation into hepatic DNA, the mitotic index in hematoxylin-eosin (HE) sections and by immunochemical detection of Ki67 nuclear antigen. Reversion of 5-HT7 blockade was performed by intraperitoneal administration of AS-19. Serum and liver tissue lipids were also quantified. Results Liver regeneration peaked at 24 h ([3H]-thymidine incorporation into hepatic DNA and mitotic index by immunochemical detection of Ki67) and at 32 h (mitotic index in HE sections) in the control group of rats. 5-HT7 receptor blockade had no effect on liver regeneration when applied 2 h prior to partial hepatectomy. Liver regeneration was greatly attenuated when blockade of 5-HT7 receptor was applied (by SB-258719 and SB-269970) at 16 h after partial hepatectomy and peaked at 32 h ([3H]-thymidine incorporation into hepatic DNA and mitotic index by immunochemical detection of Ki67) and 40 h (mitotic index in HE sections) after partial hepatectomy. AS-19 administration totally reversed the observed attenuation of liver regeneration. Conclusions In conclusion, 5-HT7 receptor is a novel type of serotonin receptor implicated in hepatocyte proliferation.


Background
Serotonin (5-HT) is an ancient chemical and neurotransmitter implicated in a vast variety of physiological and pathophysiological processes [1][2][3]. 5-HT mediates its actions through 14 distinct types of receptors encoded by a respective number of genes and its actions outnumber by far those of any other neurotransmitter. The majority of serotonin in the body (90%) is synthesized in the GI tract by enterochromafin cells and is known to control mood, behavior, memory, sleep and anxiety in the central nervous system (CNS). In the periphery, serotonin mediates vascular contraction and relaxation, GI tract smooth muscle cell tone (contraction and/or relaxation), platelet aggregation and is also acting as a growth factor for diverse cell types promoting survival, cell differentiation and proliferation as well as inhibition of apoptosis [1][2][3].
Reports regarding implication of serotonin in liver regeneration are dated back in the early 80s in non-English literature or even earlier [5,6]. A number of recent in vivo studies including studies from our laboratory have elucidated the role of serotonin in liver regeneration after partial hepatectomy [7][8][9] with platelets to be the major reservoir accounting for the increased hepatic concentrations of the monoamine during liver regeneration. From experiments with 5-HT 2 receptor blockade with ketanserin or ritanserin in our laboratory, it has become evident that serotonin exerts its actions mainly at the G1/S transition point and this suggests implication of the monoamine in the control of this major restrictive checkpoint of the cell cycle [8]. In cultured rat hepatocytes, in in vitro experiments, serotonin induces dose-dependent increase in DNA synthesis only in the presence of insulin and epidermal growth factor (EGF) [7] and recently serotonin has been shown to promote hepatocellular cancer growth in human hepatocellular cancer cell lines [10].
5-HT 7 receptor has been the last family of serotonin receptors to be discovered. It is a Gs coupled receptor with at least four different splice variants that differ in the length of the C termini and in the number of phosphorylation sites, and the above have significant biochemical consequences in the G protein coupling efficiency and the differential susceptibility to desensitization [11]. The distribution of the receptor has not been fully elucidated and its mRNA is most abundant in the thalamus, hippocampus and hypothalamus. In the central nervous system, 5-HT 7 receptor mediates thermoregulation, learning and memory, regulation of circadian rhythms and mood, and endocrine functions. In the periphery the receptor is localized mainly on smooth muscle cells in blood vessels in a variety of organs where it mediates relaxation of blood vessels as well as in the gastrointestinal tract where it regulates motility [2,3,12].
In the present study, we investigated the effect of 5-HT 7 receptor blockade on liver regeneration after partial hepatectomy.

Experimental animal model
Male Wistar rats, weighing 160-200 g, four to five months old (Hellenic Pasteur Institute, Athens, Greece) were used in this study. The animals were kept in a temperature-controlled room (22-25°C), under 12 h of light (08.00 h-20.00 h) and 12 h of darkness (20.00 h-08.00 h) and they had free access to a commercial pellet diet and tap water. The study protocol was approved by the Deontology Committee of the University of Peloponnese and animals were handled with humane care in accordance with the European Union Directive and adapted in the relevant Greek Presidential decree for the care and use of laboratory animals [13].
The experimental rats were randomly assigned to the following groups: Group A: rats submitted to 60-70% partial hepatectomy and intraperitoneal administration of normal saline 2 h prior and 16 h after partial hepatectomy. Group B: rats submitted to 60-70% partial hepatectomy and intraperitoneal administration of SB-269970 hydrochloride at the dose of 2 mg/kg bodyweight 2 h prior to partial hepatectomy. Group C: rats submitted to 60-70% partial hepatectomy and intraperitoneal administration of SB-269970 hydrochloride at the dose of 2 mg/kg bodyweight 16 h after partial hepatectomy. Group D: rats submitted to 60-70% partial hepatectomy and intraperitoneal administration of SB-269970 hydrochloride at the dose of 2 mg/kg bodyweight 2 h prior and 16 h after partial hepatectomy. Group E: rats submitted to 60-70% partial hepatectomy and intraperitoneal administration of SB-258719 at the dose of 4 mg/kg bodyweight 16 h after partial hepatectomy. Group F: rats submitted to 60-70% partial hepatectomy, intraperitoneal administration of SB-269970 16 h after partial hepatectomy at the dose of 2 mg/kg bodyweight followed by intraperitoneal administration of AS-19 at the dose of 10 mg/kg bodyweight. Group G: rats submitted to 60-70% partial hepatectomy, intraperitoneal administration of SB-258719 16 h after partial hepatectomy at the dose of 4 mg/kg bodyweight followed by intraperitoneal administration of AS-19 at the dose of 10 mg/kg bodyweight.
One hour prior to sacrifice the animals of all groups were injected with [ 3 H]-thymidine at the dose of 250 μCi/kg bodyweight intraperitoneally. A standard portion of the median liver lobe was used for histological evaluation and the rest was rapidly frozen in liquid nitrogen for further determinations. Liver weights were also tabulated for all groups of rats.

Histological evaluation
A standard portion of the median liver lobe was fixed in 4% buffered formalin for 24 hours. Sections 5-μm thick were processed routinely, stained with hematoxylin-eosin (HE) and analysed for mitoses. Mitoses were counted in 10 randomly selected high-power fields (HPF) and expressed as the mean number of mitoses/HPF. The mitotic index was also evaluated by the immunochemical detection of Ki67 nuclear antigen (Dako, MIB 5 clone, 1:50, with microwave pre-treatment).

Liver regeneration
The rate of liver regeneration was evaluated by the rate of [ 3 H]-thymidine incorporation into hepatic DNA, the mitotic index in HE sections and by immunochemical detection of Ki67 nuclear antigen.

Rate of [ 3 H]-thymidine Incorporation into Hepatic DNA
Animals of all groups were injected intraperitoneally with 250 μCi/kg bodyweight of [ 3 H]-thymidine 1 h prior to sacrifice. DNA was extracted from the tissue according to the method of Munro and Fleck [14] as modified by Kyprianidis et al. [15]. The content of tissue DNA was estimated by the method of Richards [16]. The rate of [ 3 H]thymidine incorporation into hepatic DNA was calculated from the radioactivity measured in a liquid scintillation counter (Wallac LKB 1211 Rackbeta, Sweden) and results were expressed as counts/min/μg of DNA.

Analysis of liver and serum lipid content
Frozen liver tissue (~100 mg) was homogenised in 1.6 ml phosphate-buffered saline and protein concentration was determined using the method of Lowry [17]. Lipids were extracted using chloroform: methanol (2:1) according to Folch et al. [18]. Phase separation was achieved with sulphuric acid 0.1% and the organic phase was solubilized in Triton X-100. Cholesterol, TG, FFA and phospholipid content were determined in liver tissue and plasma with the use of commercially available kits (Wako, Chemicals) and normalized to protein concentration of the homogenate. Free plasma glycerol levels were also determined in deproteinised serum samples as an indicator of lipolysis in adipose tissue [19].

Statistical analysis
Data were expressed as means ± SE. All observations were obtained from at least five animals. The statistical analysis of the results was performed by unpaired Student's t-test.

Results
In rats subjected to 60-70% partial hepatectomy (group A), liver regeneration as evaluated by [ 3 H]-thymidine incorporation into hepatic DNA, peaked at 24 and 32 h after partial hepatectomy and high rates were also observed at 40 h. The regenerative rates declined abruptly after 40 h and remained at low levels thereafter ( Figure 3).  In rats subjected to 60-70% partial hepatectomy and intraperitoneal administration of SB-269970 2 h prior to partial hepatectomy (group B), [ 3 H]-thymidine incorporation into hepatic DNA was maximal at 24 h and 32 h after partial hepatectomy with high rates also at 40 h ( Figure 3). The temporal pattern and values of regenerative rate were almost identical in groups A and B of rats ( Figure 3).
In group C of rats, intraperitoneal administration of SB-269970 16 h after partial hepatectomy greatly attenuated liver regeneration as evaluated by [ 3 H]-thymidine incorporation into hepatic DNA at 24 h after partial hepatectomy ( Figure 3). [ 3 H]-thymidine incorporation into hepatic DNA was maximal at 32 h after partial hepatectomy in group C of rats and sharply declined thereafter ( Figure 3). The maximal regenerative rate observed at 32 h in group C as well as the regenerative rates at all time points examined in this group were lower than the corresponding rates at the same time points for groups A and B (Figure 3).
In group D of rats [ 3 H]-thymidine incorporation into hepatic DNA peaked at 32 h after partial hepatectomy showing the same temporal pattern as in group C ( Figure 3). As in group C, liver regeneration was greatly attenuated at all time points examined.
In group E of rats [ 3 H]-thymidine incorporation into hepatic DNA peaked at 32 h after partial hepatectomy showing the same temporal pattern and similar values as in groups C and D ( Figure 4). As in group C, liver regeneration was greatly attenuated at all time points examined.
In groups F and G, AS-19 administration reversed the observed attenuation of liver regeneration and [ 3 H]thymidine incorporation into hepatic DNA peaked at 24 and 32 h after partial hepatectomy while it was also at high levels at 40 h. The time pattern and values of [ 3 H]thymidine incorporation into hepatic DNA in groups F and G were almost identical with that in group A ( Figures 5 and 6).   Mitotic index in HE sections was maximal at 32 h after partial hepatectomy in groups A and B with also relatively high levels at 24, 40 and 48 h and sharply declined thereafter (Figure 7). In groups C and D of rats, mitotic index was minimal until 32 h and two major peaks were observed at 40 and 60 h that were both lower than the corresponding peaks in groups A and B at 32 h (Figure 7). Mitotic index as evaluated by the immunochemical detection of Ki67 gradually increased between 8 and 24 h when it peaked in groups A and B of rats and remained at high levels until 40 h with abrupt decline thereafter (Figures 8 and 9). The index remained at low levels between 8 and 24 h after partial hepatectomy in groups C and D of rats with sharp increase at 32 h (Figures 8  and 10). The percentage of Ki67 nuclei remained at relatively high levels until 48 h after partial hepatectomy with gradual decrease afterwards in these groups of rats ( Figure 8). The regenerative rate as evaluated by Ki67 positive cells in groups C and D at 32 and 40 h was lower than that in groups A and B.
In group F intraperitoneal administration of AS-19 at the dose of 10 mg/kg of body weight totally reversed the observed attenuation of liver regeneration as evaluated by the percentage of Ki67 positive cells and regenerative rates were almost identical with these in group A (Figure 11). The observed effect of AS-19, as evaluated in initial pilot experiments was dose-dependent ( Figure 2). In group G of rats AS-19 administration also totally reversed the observed inhibition of liver regeneration and the time pattern   and values of Ki67 positive cells were also almost identical with these in groups A and F (data not shown).
Relative liver weight (liver weight in g/100 g bodyweight) sharply decreased, as expected after partial hepatectomy, with gradual increase thereafter in group A of rats. In groups C, D and E, relative liver weight remained at low levels without significant increases until 24 h after partial hepatectomy. In these groups a small increase was observed at 32 h with further increase at 40 and 48 h after partial hepatectomy. In groups F and G of rats, the relative liver weights showed the same gradual increases as in group A (Table 1).
Regarding lipid changes after partial hepatectomy, increase in liver triglyceride levels was observed at 18 h after partial hepatectomy with further increase at 24 h in group A of rats. Liver triglyceride content peaked at 40 h after partial hepatectomy and decreased thereafter but was still at high levels at 72 h after partial hepatectomy. Serum triglyceride concentration decreased at 18 and 24 h after partial hepatectomy and increased afterwards and these increases were still present at 72 h after partial hepatectomy. Serum FFA and free glycerol levels both increased at 18 h after partial hepatectomy and remained at high levels thereafter. The temporal patterns of liver and plasma lipid changes were similar in all groups of rats (Tables 2 and 3).

Discussion
The ability of the liver to regenerate after surgical resection or any short of hepatic injury has been known from long and has drawn immense scientific interest. 60-70% partial hepatectomy is the most commonly applied stimulus for the study of liver regeneration mainly due to the fact that the mitotic stimulus is accurately applied in time and not associated with necrotic or inflammatory processes [20]. A great number of substances influence the regenerative process and traditionally they are classified as complete and incomplete (auxiliary) mitogens [20]. The autonomic nervous system, both sympathetic and parasympathetic, is implicated in liver regeneration although the exact mechanisms of its effects still remain obscure [21][22][23]. Among neurotransmitters norepinephrine, mainly through α 1 -adrenergic receptor [23][24][25] (actions through β-adrenergic receptors have also been reported) [26], and serotonin, mainly through 5-HT 2 receptor, are considered auxiliary mitogens [7][8][9].   Serotonin is an important neurotransmitter of the autonomic nervous system and in the liver serotonergic nerve fibres are localized in the tunica media of branches of the hepatic artery, portal vein, bile ducts and the connective tissue of the interlobular septae in humans and rats [27,28]. 5-HT receptors are expressed in various liver cell types, apart from hepatocytes, as hepatic stelate cells and sinusoidal endothelial cells [4,29,30]. From experiments on differential 5-HT receptor subtype expression and blockade experiments with various receptor antagonists of other research groups it has become evident that 5-HT 2α and 5-HT 2β receptors mediate liver regeneration [31] and molecular pathways have been elucidated in the case of 5-HT 2β receptor [32][33][34].
In our study, 5-HT 7 receptor blockade greatly attenuated liver regeneration when applied close to the G 1/ S transition point of the cell cycle and this is the first study to reveal implication of the 5-HT 7 receptor in liver regeneration and more specifically in this major restrictive cell cycle check point. In the central nervous system, blockade of 5-HT 7 receptor has been reported to increase hippocampal cell proliferation [35] and the receptor is also implicated at least in the initial stages of T-cell activation and possibly in T-cell proliferation [36]. Additionally, 5-ΗΤ 7 receptor has been recently found to be expressed in hepatocytes although the full repertoire of its actions in the liver still remains obscure [37].
SB-269970 used in our study is considered a highly selective ligand for 5-HT 7 receptors (pKi= 8.9 ± 0.1) with at least 100-fold greater affinity in relation to other types of 5-HT receptor subtypes but some researchers have also reported that it is also a potent α 2 -adrenergic receptor blocker [38][39][40][41]. Although only α 1 -adrenoreceptors have been reported to participate in liver regeneration, the observed inhibitory effect by SB-269970 could also be attributed to α 2 -receptor blockade especially since α 2adrenoreceptors are expressed in hepatocytes [42,43]. Activation of α 2 -adrenergic receptors has been reported to induce cell proliferation in different cell types [44][45][46], whereas competitive inhibition of these receptors attenuates cell proliferation and/or induces apoptosis [44,45,47]. However, there are reports that connect α 2receptor stimulation with inhibition of cell growth [48]. In order to elucidate the above, another series of experiments has been conducted in our laboratory with intraperitoneal administration of SB-258719 (pKi= 7.5) at the dose of 4 mg/kg bodyweight at 16 h after partial hepatectomy [38,49,50]. SB-258719 is a known weak inverse agonist of 5-HT 7 receptor without any known actions on other type of serotonin receptors and its administration had the same effect on liver regeneration as SB-269970 administration and the above clearly suggests that the observed inhibitory effect must be attributed to 5-HT 7 receptor blockade.
In order to verify that the observed effect on liver regeneration is due to blockade of 5-HT 7 receptor we conducted another series of experiments with the selective 5-HT 7 receptor agonist AS-19 [51][52][53]. AS-19 is considered a selective 5 HT 7 agonist (Ki = 0.6 nM, IC 50 = 0.83nM) [54]. AS-19 administration reversed the observed attenuation of liver regeneration caused by administration of SB-269970 and SB-258719 and this verifies the implication of 5-HT 7 in liver regeneration.

Conclusions
The results of this study indicate that 5-HT 7 receptor is implicated in liver regeneration after partial hepatectomy. Serotonin through 5-HT 7 receptor seems to exert its auxiliary proliferative effect close to G1/S transition point and during the S phase. Therefore, the results identify a novel type of 5-HT receptor that mediates the proliferative effect of the monoamine in the liver.