The osseous tissue is a complex structure composed of two major cell types: the osteoclasts that are responsible for bone resorption and the osteoblasts involved in bone remodeling. Functionally related to bone cells are the chondrocytes which produce and maintain the extracellular matrix of cartilage [1]. All these cell components play individual roles in osteoporosis, a metabolic disease of osseous tissue characterized with a decrease in bone density resulting in fragility of bones and their fractures [2]. The development of osteoporosis is orchestrated by parathyroid hormone, 1α,25-dihydroxyvitamin D3, estrogen, calcitonin, and a moiety of local factors such as the insulin-like growth factors, the transforming growth factors, prostaglandins, osteoclast differentiation factor, bone morphogenetic proteins, etc. with an important role in control over the osseous metabolism played by the nervous system [3] presented in the bones with its sympathetic and parasympathetic branches [4].

There are data on important role of serotonin (5-hydroxytryptamine, 5-HT) in the control of bone mineral density [5]. Serotonin is a small molecule C10H12N2O with molecular weight of 176.215, which is produced from L-tryptophan by aromatic amino acid decarboxylase; it can be oxidized by monoamine oxidase. In addition to the abovementioned enzymes and indoleamine, the serotoninergic system is comprised of serotonin transporter SERT and the serotonin receptors, which are subdivided into 7 classes (5-HT1 to 5-HT7). In humans and animals, the serotonin receptors are located in the membranes of osseous, nervous, and other cells where they mediate the effects of 5-HT and endogenous ligands. The rat osteoblasts incorporate 6 subtypes of 5-HT receptors: 5-HT1A, 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2B, and 5-HT2C. Of them, 5-HT2A and 5-HT1B demonstrated the greatest expression level both at the early and late differentiation stages [17].

Most part of 5-HT molecules (90-95%) are synthesized by enterochromaffin cells located in mucous coat of antral and pyloric segments of stomach, duodenum, and ileum; in addition, they are stored by the platelets. 5-HT is a messenger both in CNS and in the periphery, osseous tissues included. Application of serotonin can regulate proliferation of osteoblasts and their functions in vitro [17].

Serotonin augments the stimulatory effect of parathyroid hormone on activity of c-Jun (AP-1) transcription factor in osteoblasts of UMR 106-H5 rats [6]. In addition, serotonin promotes proliferation of mesenchymal stem cells and primary osteoblasts [7, 8]. The strength of osseous tissue is maintained by estrogens (the antagonists of parathyroid hormone) which stimulates enhancement of calcium concentration in the blood. The parathyroid hormone inhibits reabsorption of phosphates in proximal renal tubules and up-regulates their secretion [9]. In contrast, the endogenous renal serotonin up-regulates reabsorption of phosphates in vivo.

One of the drugs employed to correct osteoporosis is alfacalcidol (1α, 3β, 5Z, 7E)-9,10-secocholesta-5,7,10(19)-triene-1,3-diol, a regulator of calcium and phosphorus metabolism and a potent prodrug for active vitamin D [11, 12]. It had been developed in Japanese Chugai Pharmaceutical Co., Ltd. and Teijin Institute for Bio-Medical Research [8] to treat osteoporosis by increasing bone mineral density (BMD) [5-7]. Alfacalcidol is a dose-dependent drug which 1) improves bone strength and density [9] and 2) diminishes the risk of vertebral and extra-vertebral fractures [10, 11]. In addition, the treatment with analogs of active vitamin D decreases the fall-related injury rate. Meta-analysis showed that alfacalcidol (an analog of vitamin D) is more effective in preventing the loss of bone mass, fractures, and falls in comparison with native vitamin D [10, 12].

There was no spectacular progress in modeling systemic osteoporosis and assessment of its severity, which impedes approbation and the use of novel treatment technologies and drugs in clinics [13, 14]. Among the hints that suggest the novel steps in solving old tasks is the dual role of serotonin in BMD control. Really, low concentrations of 5-HT can inhibit proliferation, differentiation, and mineralization of osteoblasts, but these down-regulatory effects are somewhat blended at rather high serotonin concentrations. Thus, here we used 5-HT only at minimal doses, which were sufficient to model osteoporosis and to test efficacy of serotonin in provoking this disease.

Experiments were carried out on female Wistar rats from MCSPC. The study was approved by the Ethics Committee of MCSPC in strict adherence to humanistic principles specified in European Economic Community Directive 86/609/EEC and Helsinki Declaration on Protection of Vertebral Animals.

The rats (n=20) were randomized into equal control and 3 experimental groups. All rats were maintained on the standard granulated food. For 6 months, group 1 experimental rats received weekly intramuscular serotonin (50-100 µg/kg) dissolved in physiological saline. Group 2 experimental rats received serotonin as in group 1 rats, but it was supplemented with alfacalcidol (0.0025-0.0030 g/kg per os). Group 3 experimental rats received weekly alfacalcidol (0.0025-0.0030 g/kg per os) for 6 months.

The rats were narcotized with sodium etaminal (40 mg/kg intraperitoneally) known to produce a minimum effect on autonomic nervous system. The upper and middle thirds of the inner thigh were cut longitudinally, thereupon the femoral bone was isolated to obtain the specimens with the length of 0.8-1.9 cm. These specimens were treated with 0.9% hydrochloric acid for 24 h, fixed in 9% formalin, dehydrated in alcohols of increasing concentrations from 70° to 96°, embedded in paraffin, and stained with hematoxylin and eosin.

The routine kits were used to assay for phosphorus, calcium, and alkaline phosphatase in blood serum. The blood (1 ml) was drawn from the tail vein. Olympus AU680 Immunochemical Analyzer (Beckman Coulter) and the standard kits were employed to measure phosphorus, calcium, magnesium, and iron in bone specimens

The study used following chemicals: sodium etaminal (sodium 5-ethyl-5-barbiturate C11H19N2NaO3, Sigma-Aldrich); serotonin adipate (5-hydoxytryptamine adipate C16H21N2O4, Lorr, Russia); alfacalcidol (1-hydroxycholecalciferol), (1α, 3β, 5Z, 7E)-9,10-secocholesta-5,7,10(19)-triene-1,3-diol (Tocris Bioscience).

In all groups, morphological examination of the osseous tissue was carried out at ×240 magnification. Particular attention was focused on the osteoblasts, osteons, and the collagen fibers.

The data were analyzed statistically using Statistica-6 software, χ2-test, and Student’s t test at р<0.05. The results are summarized as m±SEM.

In group 1 rats, chronic injections of serotonin affected phosphorus and calcium metabolism: in comparison with the control values, it increased serum phosphorus and calcium by 118.7 % and 34.2%, correspondingly (Table 1). In parallel, the level of alkaline phosphatase increased by 30.4% compared to the normal value characteristic of the rats with stable renal excretion and hepatic protein-synthesizing functions.

Table 2 shows that serotonin also changed the mineral composition and metabolism in the bones: the concentrations of examined elements dropped in osseous tissue by 30% (calcium), 55% (phosphorus), 89% (magnesium), and 83% (iron). Thus, chronic administration of serotonin provoked moderate osteoporosis.

To correct the state of osseous tissue during osteoporosis, we employed alfacalcidol known to normalize absorption and balance of calcium ions, down-regulate secretion of parathyroid hormone, and stimulate osteogenesis. The combined effect of serotonin and alfacalcidol were examined in the corresponding experimental group (Table 2). This table shows that simultaneous chronic administration of serotonin and alfacalcidol inhibited the drop of calcium and phosphorus in osseous tissue, their ratio being 1.12:1. At this, osseous magnesium dropped dramatically due to disturbed balance between iron and magnesium indicating incomplete mineralization of osseous tissue.

Individual administration of alfacalcidol produced no effect on serum mineral elements: the content of phosphorus and calcium remained stable (Table 1). According to the level of albumin, alfacalcidol virtually did not change the protein synthesis in the liver. In contrast, this drug inhibited the renal excretory function by 20% as indicated by creatine (Table 1).

When used alone, alfacalcidol produced the diverse effects on osseous mineral metabolism: it doubled the calcium content, halved the content of iron, and produced no significant effect on osseous phosphorus and magnesium (Table 2). Thus, individual administration of alfacalcidol and its combined use with serotonin showed that alfacalcidol can correct the disturbances in osseous mineral metabolism characteristic of serotonin-provoked osteoporosis.

Morphological examination showed that serotonin-provoked osteoporosis was characterized with a decreased number of osteoblasts in the visual field indicating the development of moderate osteoporosis. In contrast, individual injections of alfacalcidol induced formation of numerous osteoblasts, the osteons with greater optical dense, and the collagen fibers. When the rats were simultaneously treated with serotonin and alfacalcidol, the osseous tissue was characterized with increased number of osteoblasts indicating stimulation of bone regeneration processes.

The experimental animal models play important role in the studies of etiology, pathophysiology, and diagnostics of osteoporosis. Moreover, they pave the way to the development of novel approaches to prevention and therapy of this disease [19]. At present, the basic laboratory animals used to examine osteoporosis are the rats, because they are widely available, rapidly grow, have a short life span, and inexpensive in maintenance [20]. There are various ways to develop the standard osteoporosis models – for instance, by ovariectomy [20], which reliably simulates various features of osteoporosis [21]. Here we developed a pharmacological model of osteoporosis produced by chronic (6 month) injections of serotonin in small doses. It is based on the fact that functional serotoninergic pathways can mediate the effects of 5-HT on the osseous tissues [22, 23]. Specifically, the experimental data attest to the effect of serotonin on bone remodeling [17].

Examination of primary cultures of rat osteoblasts (ROB) and various cell lines of osteoblastic clones (including ROS 17/2.8, UMR 106-Н5, and Py1a) with reverse transcriptase-polymerase chain reaction revealed the presence of mRNA serotonin transporter 5-HTT as well as the family of serotonin receptors 5-НТ1А, 5-HT1D, 5-HT2A, and 5-HT2B [24]. Immunoblotting detected expression of the proteins related to 5-HT1A, 5-HT2A, and 5-HT2B receptors. The binding loci of 5-HTT were revealed in ROS 17/2.8 and UMR 106-H5 cell lines. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, showed the highest potency to antagonize [125I]RTI-55 binding in ROS and UMR cells. GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5-HT uptake rate in ROS cells was 110 pmol/10 min per well, with a Km value of 1.13 µmol. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC50 values within the nanomolar range. In normal differentiating ROB cultures, 5-HTT functional activity was observed initially at day 25, and activity increased almost eightfold by day 31. In mature ROB cultures, the estimated density of [125I]RTI-55 binding sites was 600 fM/mg protein. Functional down-regulation of transporter activity was assessed after treatment with phorbol 12-myristate 13-acetate (PMA), which caused a pronounced (40%) reduction in the maximal uptake rate of [3H]5-HT, an effect that was prevented by pretreatment with staurosporine. The affinity of 5-HT for the transporter increased significantly after PMA treatment [24].

Injections of serotonin at low doses resulted in a dose-dependent drop in the proliferation rate of rat osteoblasts, which agrees with the reported data [25]. In that study, proliferation of the native osteoblasts decreased after exposure to serotonin for 24 h.

Our study focused on the development of the model with serotonin-induced low BMD, which is a characteristic feature of osteoporosis. By testing this osteoporosis model with alfacalcidol, we obtained the corroborative data on its validity. Really, alfacalcidol corrected the development of full-scale serotonin-induced osteoporosis by up-regulating osteoblastic activity. It should be stressed that despite a wide use of this drug in the treatment of osteoporosis, its mode of action in the osseous tissue is little known [9]. Correction of serotonin-induced osteoporosis with alfacalcidol attests to imperfect osteogenesis manifested by anomalous value of Mg:Fe ratio. Further studies are needed to reveal the mechanism of imbalanced saturation of the bones with calcium observed during individual administration of alfacalcidol.

The authors declare that they have no competing interests.

All authors mentioned in the above have contributed to this article from data collection to final drafting work. All authors also declare to have read and approved the final manuscript.

S.V. Revenko, Department of Physiology, Russian Cardiology Research-and-Development Center, Ministry of Health of the Russian Federation, Moscow, Russia edited this article for English.