Effect of 5-HT₇receptor blockade on liver regeneration after 60-70% partial hepatectomy

Serotonin (5-HT) is an ancient chemical and neurotransmitter implicated in a vast variety of physiological and pathophysiological processes [1]-[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]-[3].

In the liver, serotonin is implicated in the regulation of blood flow at the level of portal vein and sinusoids through activation of 5-HT₂ subtype of receptors [1], in biliary tree growth (5-HT_1α and 5-HT_1β receptors), in the development of liver cirrhosis through activation and proliferation of HSC cells (5-HT_2α and 5-HT_2β) and hepatocyte proliferation (mainly 5-HT_2α/β) [4]. Hepatocytes express SERT, 5-HT_2α and 5-HT_2β and possibly other types of serotonin receptors and HSC cells express 5-HT_1β, 5-ΗΤ_1F, 5-HT_2α, 5-HT_2β, 5-HT₇ and SERT [1].

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]-[9] with platelets to be the major reservoir accounting for the increased hepatic concentrations of the monoamine during liver regeneration. From experiments with 5-HT₂ 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-HT₇ 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-HT₇ 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-HT₇ receptor blockade on liver regeneration after partial hepatectomy.

In rats subjected to 60-70% partial hepatectomy (group A), liver regeneration as evaluated by [³H]-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), [³H]-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 [³H]-thymidine incorporation into hepatic DNA at 24 h after partial hepatectomy (Figure 3). [³H]-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 [³H]-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 [³H]-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 [³H]-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 [³H]-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).

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]-[23]. Among neurotransmitters norepinephrine, mainly through α₁-adrenergic receptor [23]-[25] (actions through β-adrenergic receptors have also been reported) [26], and serotonin, mainly through 5-HT₂ receptor, are considered auxiliary mitogens [7]-[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]-[34].

In our study, 5-HT₇ 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-HT₇ receptor in liver regeneration and more specifically in this major restrictive cell cycle check point. In the central nervous system, blockade of 5-HT₇ 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-ΗΤ₇ 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-HT₇ 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 α₂-adrenergic receptor blocker [38]-[41]. Although only α₁-adrenoreceptors have been reported to participate in liver regeneration, the observed inhibitory effect by SB-269970 could also be attributed to α₂-receptor blockade especially since α₂-adrenoreceptors are expressed in hepatocytes [42],[43]. Activation of α₂-adrenergic receptors has been reported to induce cell proliferation in different cell types [44]-[46], whereas competitive inhibition of these receptors attenuates cell proliferation and/or induces apoptosis [44],[45],[47]. However, there are reports that connect α₂-receptor 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-HT₇ 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-HT₇ receptor blockade.

In order to verify that the observed effect on liver regeneration is due to blockade of 5-HT₇ receptor we conducted another series of experiments with the selective 5-HT₇ receptor agonist AS-19 [51]-[53]. AS-19 is considered a selective 5 HT₇ agonist (Ki = 0.6 nM, IC₅₀ = 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-HT₇ in liver regeneration.

It is known from long that liver regeneration is accompanied by transient hepatic steatosis and intracellular accumulation of triglycerides in hepatocytes through increased lipolysis in the adipose tissue and increased hepatic lipogenesis [55],[56]. Serotonin induces lipolysis in adipocytes and promotes gluconeogenesis in hepatocytes through 5-HT_2b receptor during fasting adaptation [57]. Additionally serotonin is also implicated in the regulation of lipid metabolism through 5-HT_2c receptors by altering sympathetic outflow at the brain level [58]. In our experiments no significant differences have been observed in serum and liver lipids during liver regeneration after 5-HT₇ receptor blockade and consequently 5-HT₇ receptor does not seem to be implicated in the adaptive changes of lipid metabolism during liver regeneration.

5-HT₇ receptors have been reported to activate MAPK [59],[60] and this activation has also been reported to be RAS-dependent [61]. The above seems to represent a more general pattern of MAPK activation from Gs- coupled receptors with RAS independent pathways to have also been described [62],[63]. Both 5-HT_2α and 5-HT_2β receptors have also been reported to activate MAPK through similar pathways [33],[64] and this hints at a possible role of 5-HT₇ receptor in mitogenesis and cell-cycle progression although further research is needed at this point.

The results of this study indicate that 5-HT₇ receptor is implicated in liver regeneration after partial hepatectomy. Serotonin through 5-HT₇ 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.