AIM: To investigate serotonergic Ca²⁺ signaling and the expression of 5-hydroxytryptamine (5-HT) receptors, as well as Ca²⁺ transporting proteins, in hepatic stellate cells (HSCs).

METHODS: The intracellular Ca²⁺ concentration ([Ca²⁺]_i) of isolated rat HSCs was measured with a fluorescence microscopic imaging system. Quantitative PCR was performed to determine the transcriptional levels of 5-HT receptors and endoplasmic reticulum (ER) proteins involved in Ca²⁺ storage and release in cultured rat HSCs.

RESULTS: Distinct from quiescent cells, activated HSCs exhibited [Ca²⁺]_i transients following treatment with 5-HT, which was abolished by U-73122, a phospholipase C inhibitor. Upregulation of 5-HT_2A and 5-HT_2B receptors, but not 5-HT₃, was prominent during trans-differentiation of HSCs. Pretreatment with ritanserin, a 5-HT₂ antagonist, inhibited [Ca²⁺]_i changes upon application of 5-HT. Expression of type 1 inositol-5’-triphosphate receptor and type 2 sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase were also increased during activation of HSCs and serve as the major isotypes for ER Ca²⁺ storage and release in activated HSCs. Ca²⁺ binding chaperone proteins of the ER, including calreticulin, calnexin and calsequestrin, were up-regulated following activation of HSCs.

CONCLUSION: The appearance of 5-HT-induced [Ca²⁺]_i response accompanied by upregulation of metabotropic 5-HT₂ receptors and Ca²⁺ transporting/chaperone ER proteins may participate in the activating process of HSCs.

Hepatic stellate cells (HSCs), also known as “Ito cells” or “fat-storing cells”, localize between hepatocytes and sinusoids (space of Disse) in mammalian livers. In their healthy state, HSCs control retinoid homeostasis, sinusoidal blood flow, macromolecule transport, and potentially act as antigen-presenting cells in the liver[1,2]. However, in response to hepatic injury, HSCs undergo gross morphological and functional changes, transforming to a myofibroblast-like phenotype in a process called “activation” or “trans-differentiation”[3,4]. Manifestations of activated HSCs include: (1) the expression of contractile cytoskeletal proteins such as α-smooth muscle actin (α-SMA)[5,6]; (2) enhanced extracellular matrix synthesis[7,8]; (3) increased cell size and proliferation[9]; (4) decreased size of lipid droplets[8,10]; and (5) well developed endoplasmic reticulum (ER), Golgi bodies, and compacted microfilaments[11,12]. In particular, the deposition of cross-linked collagen during the activation process may result in cirrhotic changes accompanied by life-threatening hepatic dysfunction.

Serotonin [5-hydroxytryptamine (5-HT)] is a neurotransmitter that also acts as a multifunctional hormone in various tissues[13], where it modulates proliferation and differentiation of muscle, neurons, and mammary glands[14-16]. Serotonin released from platelets at sites of injury plays an important role in liver regeneration and fibrosis[17]. It has been reported that patients with cirrhosis of the liver and portal hypertension have increased plasma serotonin levels[18]. The expression levels of 5-HT_2A and 5-HT_2B are increased in the liver after hepatectomy as well as in activated HSCs[2,17]. Moreover, 5-HT₂ receptor antagonists suppress cell proliferation and expression of key fibrogenic factors in activated HSCs[2,19]. Among the mammalian 5-HT receptors (5-HT₁ to 5-HT₇), the 5-HT₂ receptor family is coupled to the G_q/11 protein and increases intracellular Ca²⁺ concentration ([Ca²⁺]_i) mobilized from ER reservoirs[20].

As the major intracellular calcium storage site, the ER possesses various kinds of calcium regulatory proteins that participate in: (1) pumping Ca²⁺ into the ER lumen, such as the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA); (2) releasing Ca²⁺ into the cytosol, such as IP₃ or ryanodine receptors; and (3) buffering Ca²⁺, such as calreticulin and calnexin, which are also known as chaperones. ER Ca²⁺ homeostasis is maintained by a balance between Ca²⁺ release and replenishment[21]. The free Ca²⁺ concentration in the ER ([Ca²⁺]_ER) ranges from 60-400 μmol/L, and disturbances in [Ca²⁺]_ER homeostasis can affect many of the functions of the ER including protein synthesis, secretion[22], protein folding[23], and sensitivity of cells to apoptosis[24]. Further, [Ca²⁺]_ER homeostasis might be critically required for the activation process of HSCs in order to keep up with accelerated protein synthesis. However, until now, the compensatory changes in ER protein expression involved in Ca²⁺ homeostasis and chaperone function have not been clearly elucidated.

[Ca²⁺]_i may be important for the activation of HSCs, primarily because [Ca²⁺]_i regulates the transcription of genes critical for cell function[25], and secondly because contractile elements such as α-SMA respond sensitively to [Ca²⁺]_i[26]. We hypothesized that serotonin, acting as an autocrine or paracrine mediator, can elicit a Ca²⁺ signal, and this signal might be involved in the activation of HSCs. Moreover, there may be an alteration in the ER function of HSCs such as Ca²⁺ release and protein folding. In this study, we isolated and cultured rat HSCs on plastic dishes in vitro, which has been widely accepted as an appropriate model for the study of activated HSCs[8,27]. Appearance of [Ca²⁺]_i transients induced by 5-HT and the upregulation of 5-HT₂ receptors and ER proteins were observed during HSC activation. These observed changes may participate in an activation signal as well as adaptive changes during the trans-differentiation of HSCs.

Trans-differentiation of HSCs is accompanied by marked increases in protein synthesis, including collagen, elastin, and glycoproteins[7]. It is well known that Ca²⁺ homeostasis in the ER is critical for the synthesis, folding, and secretion of protein[22,23]. In HSCs, the depletion of ER Ca²⁺ stores inhibits protein synthesis and increases intracellular degradation of collagen[34]. Maintaining a high Ca²⁺ gradient across the ER membrane (around 1000-fold) is accomplished by active Ca²⁺ transport by SERCAs. Among the three different isoforms of SERCAs, SERCA2 is considered to be a house-keeping protein expressed in the ER of most cell types, including HSCs[34]. We observed that SERCA2 was the main isotype in quiescent and activated HSCs (Figure 5A). During activation, the expression of SERCA2 (and also SERCA3) was increased, which likely helped to maintain appropriate ER Ca²⁺ concentrations.

Chaperone proteins in the ER facilitate the folding of newly synthesized proteins and glycoproteins. In particular, calreticulin and calnexin are important chaperones involved in a “quality control” system for protein synthesis[35]. In addition, these chaperones act as Ca²⁺ binding proteins in the ER. Overexpression of calreticulin increases the total amount of Ca²⁺ in intracellular stores, whereas calreticulin-deficient cells have reduced ER Ca²⁺ storage capacity[36]. Impaired collagen synthesis has been observed in cells derived from mice possessing genetic defects in ER chaperone proteins[37]. In this study, we observed for the first time that the expression of ER Ca²⁺ binding proteins was markedly increased during the activation process of HSCs, which might be an important adaptive change for trans-differentiation.

Upon stimulation from the extracellular space, ER Ca²⁺ is the main source for releasing Ca²⁺ and is responsible for enabling biologic signaling mediated by Ca²⁺. In addition, Ca²⁺ release from the ER stimulates store-operated Ca²⁺ entry into the cytosol, which eventually increases the refilling of the ER Ca²⁺ reservoir. It has been shown that cytosolic Ca²⁺ signaling is important for proliferation and differentiation of HSCs[25]. Similar to myofibroblast-like cells, activated HSCs can have a contractile response to [Ca²⁺]_i changes, which may increase vascular resistance leading to portal hypertension in vivo[32]. During trans-differentiation, the expression of L-type calcium channels increases, which may contribute to cytosolic Ca²⁺ signaling in HSCs[26,38]. In the present study, we observed that 5-HT increased [Ca²⁺]_i only in activated HSCs via a serotonergic receptor. Until now, 5-HT-induced [Ca²⁺]_i changes have not been reported in HSCs. Physiologic concentrations of 5-HT in plasma are known to be less than 100 nmol/L, but those in cirrhotic patients are significantly elevated (3-4 fold) compared to controls[39]. Moreover, intrahepatic neighboring cells secrete 5-HT to act as an autocrine/paracrine regulator[40]. Thus, we hypothesize that local 5-HT concentration close to the releasing cells might be higher than the plasma level and repetitive exposure may have additive effects on [Ca²⁺]_i-mediated changes in the process of HSC activation.

We observed that 5-HT elicited a [Ca²⁺]_i response via the metabotropic 5-HT₂ receptor in activated HSCs. This was demonstrated by the findings that 5-HT-induced [Ca²⁺]_i transients were (1) completely blocked by a PLC inhibitor; (2) not altered by nominally Ca²⁺ free conditions; and (3) reduced by a 5-HT₂ blocker. 5-HT_2A is known to mediate mitogenic effects in fibroblasts[41], while 5-HT_2B is involved in the development of the heart and enteric nervous system[42]. However, we did not discriminate whether the 5-HT_2A and/or 5-HT_2B receptor mediated the serotonergic Ca²⁺ signaling in activated HSCs. We also observed that the type I IP₃ receptor (IP₃R 1) is the main isoform expressed in activated HSCs, which is consistent with a recent report by Kruglov et al[32]. The expression level of IP₃R 1 was increased during the activation process (Figure 5B).

Various ligands for G_q/11-coupled metabotropic receptors could be important extracellular stimuli, as they generate IP₃ by activating phospholipase-C. Interestingly, it has been reported that the expression of the P2Y metabotropic purinoceptor (P2Y6) is rapidly upregulated following activation of HSCs, with a similar increase in ATP-induced [Ca²⁺]_i transients[33]. The same study also reported that extracellular UDP increases the transcription of procollagen in activated HSCs via activation of the P2Y receptor, and this effect is partially inhibited by a P2Y receptor blocker. These results add further support to the hypothesis that Ca²⁺ signaling released from ER stores is associated with HSCs undergoing the process of activation. We also observed that ATP increased [Ca²⁺]_i, which might be mediated by the metabotropic P2Y receptor (Figure 3). However, acetylcholine did not induce calcium changes, indicating that muscarinic acetylcholine receptors do not functionally exist in activated HSCs, even in the presence of machinery for ER Ca²⁺ release.

In this study, we observed the pronounced increase in serotonergic [Ca²⁺]_i response related to the upregulation of metabotropic 5-HT₂ receptors, type 1 inositol-5’-triphosphate receptor, type 2 sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase, and Ca²⁺ binding ER chaperone proteins following trans-differentiation of HSCs. These changes may be involved in the pathophysiologic (profibrotic) process of rat HSCs as well as being a compensatory mechanism for maintaining ER Ca²⁺ homeostasis and protein synthesis/maturation. Switching on and off of the serotonergic signaling pathway might be implicated in potential treatment for portal hypertension. Yet, the biological relevance of a 5-HT-induced [Ca²⁺]_i transient in HSCs remains to be clarified. Moreover, it is not obvious whether simply switching-off this serotonergic signaling is an ideal target for developing treatments for liver cirrhosis. While there is evidence to suggest that 5-HT₂ antagonists reduce proliferation and increase cell death of isolated HSCs[2,19], a recent study found that fibrotic changes induced by CCl₄ are not ameliorated by a 5-HT₂ antagonist[29,43]. Further studies to elucidate the detailed role of serotonergic signaling in HSCs are needed in order to develop therapeutic approaches to hepatic fibrosis.