Skeletal effects of serotonin (5-hydroxytryptamine) transporter inhibition: evidence from in vitro and animal-based studies

The regulation of bone metabolism continues to be an area of intense investigation, with recent evidence indicating a potential contribution from the neural system. In particular, the neurotransmitter serotonin (5-hydroxytryptamine [5-HT]) has been hypothesized to play a role in skeletal metabolism via its transporter (5-HTT). The 5-HTT is a plasma membrane transporter that is highly specific for the uptake of extracellular 5-HT, thereby facilitating the intracellular storage and/or degradation of 5-HT. The 5-HTT is clinically important as it is the key target of pharmaceutical agents aimed at treating affective disorders, such as major depressive disorder. By antagonizing the 5-HTT, selective serotonin reuptake inhibitors (SSRIs) potentiate 5-HT activity and effectively relieve the symptoms of depression. However, questions have been raised regarding the potential skeletal effects of SSRIs given the recent identification of a functional 5-HTT and functional 5-HT receptors in bone cells. This paper discusses the preclinical evidence for the skeletal effects of 5-HT and the inhibition of the 5-HTT. In particular, it discusses the: (1) role of 5-HT and the function of the 5-HTT; (2) presence of functional 5-HTTs in bone; (3) potential sources and response mechanisms for 5-HT in bone, and; (4) in vitro and in vivo skeletal effects of 5-HT and 5-HTT inhibition.     

Norepinephrine-mediated Regulation of 5HT<Subscript>1</Subscript> Receptor Functioning in Human Platelets

Adaptive changes in serotonergic 5HT1 receptor signalling are believed to underlie the therapeutic effectiveness of antidepressant drugs. Since cells are continuously exposed to neurotransmitters/neuromodulators, spatially and temporally integrated, the responsiveness of a receptor system is dependent upon the physio-pathological state of the cell and the interaction between different neurotransmitters. In the present work, we investigated heterologous regulation of 5HT1 receptors induced by norepinephrine (NE) in human platelets. NE platelet treatment induced a time and concentration dependent 5HT1 receptor desensitisation mediated by both alpha and beta receptors through activation of intracellular protein kinases. In particular NE, through PKC activation, regulated 5HT1 receptor phosphorylation on threonine residues, causing in turn serotonin receptor-G protein uncoupling and functional responsiveness drop. These results suggest that high NE levels (released i.e. during stress disorders) may play an important role in regulating the 5HT1 responsiveness and in controlling effectiveness of drugs acting on these neurotransmitter systems.     

The role of peripheral nerve fibers and their neurotransmitters in cartilage and bone physiology and pathophysiology

The peripheral nervous system is critically involved in bone metabolism, osteogenesis, and bone remodeling. Nerve fibers of sympathetic and sensory origin innervate synovial tissue and subchondral bone of diathrodial joints. They modulate vascularization and matrix differentiation during endochondral ossification in embryonic limb development, indicating a distinct role in skeletal growth and limb regeneration processes. In pathophysiological situations, the innervation pattern of sympathetic and sensory nerve fibers is altered in adult joint tissues and bone. Various resident cell types of the musculoskeletal system express receptors for sensory and sympathetic neurotransmitters. Osteoblasts, osteoclasts, mesenchymal stem cells, synovial fibroblasts, and different types of chondrocytes produce distinct subtypes of adrenoceptors, receptors for vasointestinal peptide, for substance P and calcitonin gene-related peptide. Many of these cells even synthesize neuropeptides such as substance P and calcitonin gene-related peptide and are positive for tyrosine-hydroxylase, the rate-limiting enzyme for biosynthesis of catecholamines. Sensory and sympathetic neurotransmitters modulate osteo-chondrogenic differentiation of mesenchymal progenitor cells during endochondral ossification in limb development. In adults, sensory and sympathetic neurotransmitters are critical for bone regeneration after fracture and are involved in the pathology of inflammatory diseases as rheumatoid arthritis which manifests mainly in joints. Possibly, they might also play a role in pathogenesis of degenerative joint disorders, such as osteoarthritis. All together, accumulating data imply that sensory and sympathetic neurotransmitters have crucial trophic effects which are critical for proper limb formation during embryonic skeletal growth. In adults, they modulate bone regeneration, bone remodeling, and articular cartilage homeostasis in addition to their classic neurological actions.     

Current perspectives on NMDA-type glutamate signalling in bone

Bone is a complex, evolving tissue, architecturally defined by the activities of osteoclasts and osteoblasts that continually resorb and replace the mineralised matrix. Numerous regulatory mechanisms exist to control bone remodelling and the maintenance of bone mass. The consequences of inappropriate or uncoupled bone resorption and formation are significant and invariably lead to different disease states, the most prevalent being osteoporosis. In recent years, much attention has focused on unravelling the systemic and local signalling interactions that influence the differentiation and function of bone cells with a view to developing our understanding of bone biology and identifying potential new targets for therapeutic intervention. Several lines of evidence indicate that neurotransmitters and neuromodulators have influential roles to play in the regulation of bone remodelling and much of this research has involved analysis of the excitatory amino acid glutamate. This review will summarise current understanding of glutamate signalling in bone cells, addressing specifically the function of N-methyl-D-aspartate (NMDA)-type glutamate receptor signalling mechanisms, and will address the functional significance and future prospects for this area of research.     

Pathophysiology of astroglial purinergic signalling

Astrocytes are fundamental for central nervous system (CNS) physiology and are the fulcrum of neurological diseases. Astroglial cells control development of the nervous system, regulate synaptogenesis, maturation, maintenance and plasticity of synapses and are central for nervous system homeostasis. Astroglial reactions determine progression and outcome of many neuropathologies and are critical for regeneration and remodelling of neural circuits following trauma, stroke, ischaemia or neurodegenerative disorders. They secrete multiple neurotransmitters and neurohormones to communicate with neurones, microglia and the vascular walls of capillaries. Signalling through release of ATP is the most widespread mean of communication between astrocytes and other types of neural cells. ATP serves as a fast excitatory neurotransmitter and has pronounced long-term (trophic) roles in cell proliferation, growth, and development. During pathology, ATP is released from damaged cells and acts both as a cytotoxic factor and a proinflammatory mediator, being a universal "danger" signal. In this review, we summarise contemporary knowledge on the role of purinergic receptors (P2Rs) in a variety of diseases in relation to changes of astrocytic functions and nucleotide signalling. We have focussed on the role of the ionotropic P2X and metabotropic P2YRs working alone or in concert to modify the release of neurotransmitters, to activate signalling cascades and to change the expression levels of ion channels and protein kinases. All these effects are of great importance for the initiation, progression and maintenance of astrogliosis-the conserved and ubiquitous glial defensive reaction to CNS pathologies. We highlighted specific aspects of reactive astrogliosis, especially with respect to the involvement of the P2X(7) and P2Y(1)R subtypes. Reactive astrogliosis exerts both beneficial and detrimental effects in a context-specific manner determined by distinct molecular signalling cascades. Understanding the role of purinergic signalling in astrocytes is critical to identifying new therapeutic principles to treat acute and chronic neurological diseases.     

Purinergic signalling in the musculoskeletal system

It is now widely recognised that extracellular nucleotides, signalling via purinergic receptors, participate in numerous biological processes in most tissues. It has become evident that extracellular nucleotides have significant regulatory effects in the musculoskeletal system. In early development, ATP released from motor nerves along with acetylcholine acts as a cotransmitter in neuromuscular transmission; in mature animals, ATP functions as a neuromodulator. Purinergic receptors expressed by skeletal muscle and satellite cells play important pathophysiological roles in their development or repair. In many cell types, expression of purinergic receptors is often dependent on differentiation. For example, sequential expression of P2X5, P2Y₁ and P2X2 receptors occurs during muscle regeneration in the mdx model of muscular dystrophy. In bone and cartilage cells, the functional effects of purinergic signalling appear to be largely negative. ATP stimulates the formation and activation of osteoclasts, the bone-destroying cells. Another role appears to be as a potent local inhibitor of mineralisation. In osteoblasts, the bone-forming cells, ATP acts via P2 receptors to limit bone mineralisation by inhibiting alkaline phosphatase expression and activity. Extracellular ATP additionally exerts significant effects on mineralisation via its hydrolysis product, pyrophosphate. Evidence now suggests that purinergic signalling is potentially important in several bone and joint disorders including osteoporosis, rheumatoid arthritis and cancers. Strategies for future musculoskeletal therapies might involve modulation of purinergic receptor function or of the ecto-nucleotidases responsible for ATP breakdown or ATP transport inhibitors.     

骨再生的力学生物学问题

生物力学主要探讨力学刺激与细胞的形态、结构和功能之间的关系。骨组织改变其形态和结构以适应力学刺激,表现为骨的适应性重建。骨的生长是骨塑形和骨重建两个过程协同作用的结果,以调整骨的形状、大小和组成,适应其所处的力学环境。骨组织工程的目的就是修复骨组织的正常生物力学功能。近年来,骨组织工程的研究主要集中于模拟骨生长的在体生理条件,从而刺激细胞形成有功能的骨组织。生物反应器能够模拟体内生理状态,为种子细胞在生物支架材料上生长提供一个适宜的力学环境。     

Acetylcholine promotes the proliferation and collagen gene expression of myofibroblastic hepatic stellate cells

The mechanisms that initiate and perpetuate the fibrogenic response, during liver injury, are unclear. Animal studies, however, strongly support a role for the autonomic nervous system (ANS) in wound healing. Therefore, the ANS may also mediate the development of cirrhosis. Hepatic stellate cells (HSC), the liver's major matrix-producing cells, are activated by injury to become proliferative, fibrogenic myofibroblasts. HSC respond to sympathetic neurotransmitters by changing phenotype, suggesting that HSC may be the cellular effectors of ANS signals that modulate hepatic fibrogenesis during recovery from liver damage. We show here that the parasympathetic neurotransmitter acetylcholine markedly stimulates the proliferation of myofibroblastic HSC and induces HSC collagen gene expression in these cells. By extending evidence that HSC are direct targets of the ANS, these results support the proposed neuroglial role of HSC in the liver and suggest that interrupting ANS signalling may be useful in constraining the fibrogenic response to liver injury.     

Neuropeptidergic regulation of bone resorption and bone formation

Immunohistochemical studies have revealed an extensive network of nerve fibers in the vicinity and within the skeleton, not only in the periosteum of bone but also in cortical and trabecular bone as well as in the bone marrow. Phenotyping of the skeletal nerve fibers have demonstrated the expression of a restrictive panel of different signalling molecules including neuropeptides, neurotransmitters and neurotrophins. In this review, the presence of receptors for the neuropeptides vasoactive intestinal peptide, calcitonin gene-related peptide and substance P on osteoblasts and osteoclasts and the capacity of these receptors to regulate bone formation, osteoclast formation and activity are described. These findings, together with data obtained by chemically and surgically targeted nerve deletion and observations made in paraplegic patients, strongly suggest that neuro-osteogenic interactions play an important role in skeletal function.     

Regulation of nicotinic acetylcholine receptors by protein phosphorylation

Neurotransmitter receptors and ion channels play a critical role in the transduction of signals at chemical synapses. The modulation of neurotransmitter receptor and ion channel function by protein phosphorylation is one of the major regulatory mechanisms in the control of synaptic transmission. The nicotinic acetylcholine receptor (nAcChR) has provided an excellent model system in which to study the modulation of neurotransmitter receptors and ion channels by protein phosphorylation since the structure and function of this receptor have been so extensively characterized. In this article, the structure of the nAcChR from the electric organ of electric fish, skeletal muscle, and the central and peripheral nervous system will be briefly reviewed. Emphasis will be placed on the regulation of the phosphorylation of nAcChR by second messengers and by neurotransmitters and hormones. In addition, recent studies on the functional modulation of nicotinic receptors by protein phosphorylation will be reviewed.     

Neurotransmitters as early signals for central nervous system development

During brain ontogenesis, the temporal and spatial generation of the different types of neuronal and glial cells from precursors occurs as a sequence of successive progenitor stages whose proliferation, survival and cell-fate choice are controlled by environmental and cellular regulatory molecules. Neurotransmitters belong to the chemical microenvironment of neural cells, even at the earliest stages of brain development. It is now established that specific neurotransmitter receptors are present on progenitor cells of the developing central nervous system and could play, during neural development, a role that has remained unsuspected until recently. The present review focuses on the occurrence of neurotransmitters and their corresponding ligand-gated ion channel receptors in immature cells, including neural stem cells of specific embryonic and neonatal brain regions. We summarize in vitro and in vivo data arguing that neurotransmitters could regulate morphogenetic events such as proliferation, growth, migration, differentiation and survival of neural precursor cells. The understanding of neurotransmitter function during early neural maturation could lead to the development of pharmacological tools aimed at improving adult brain repair strategies.     

Neurotransmitters and Integration in Neuronal-Astroglial Networks

Two major neural cell types, glia, astrocytes in particular, and neurones can release chemical transmitters that act as soluble signalling compounds for intercellular communication. Exocytosis, a process which depends on an increase in cytosolic Ca²⁺ levels, represents a common denominator for release of neurotransmitters, stored in secretory vesicles, from these neural cells. While neurones rely predominately on the immediate entry of Ca²⁺ from the extracellular space to the cytosol in this process, astrocytes support their cytosolic Ca²⁺ increases by appropriating this ion from the intracellular endoplasmic reticulum store and extracellular space. Additionally, astrocytes can release neurotransmitters using a variety of non-vesicular pathways which are mediated by an assortment of plasmalemmal channels and transporters. Once a neuronal and/or astrocytic neurotransmitter is released into the extracellular space, it can activate plasma membrane neurotransmitter receptors on neural cells, causing autocrine and/or paracrine signalling. Moreover, chemical transmission is essential not only for homocellular, but also for heterocellular bi-directional communication in the brain. Further detailed understanding of chemical transmission will aid our comprehension of the brain (dys)function in heath and disease.     

The role of semaphorin3A in myogenic regeneration and the formation of functional neuromuscular junctions on new fibres

Current research on skeletal muscle injury and regeneration highlights the crucial role of nerve–muscle interaction in the restoration of innervation during that process. Activities of muscle satellite or stem cells, recognized as the ‘currency’ of myogenic repair, have a pivotal role in these events, as shown by ongoing research. More recent investigation of myogenic signalling events reveals intriguing roles for semaphorin3A (Sema3A), secreted by activated satellite cells, in the muscle environment during development and regeneration. For example, Sema3A makes important contributions to regulating the formation of blood vessels, balancing bone formation and bone remodelling, and inflammation, and was recently implicated in the establishment of fibre‐type distribution through effects on myosin heavy chain gene expression. This review highlights the active or potential contributions of satellite‐cell‐derived Sema3A to regulation of the processes of motor neurite ingrowth into a regenerating muscle bed. Successful restoration of functional innervation during muscle repair is essential; this review emphasizes the integrative role of satellite‐cell biology in the progressive coordination of adaptive cellular and tissue responses during the injury‐repair process in voluntary muscle.     

Glial cells

The nervous system is built from two broad categories of cells, neurones and glial cells. The glial cells outnumber the neurones and the two cell types occupy a comparable amount of space in nervous tissue. The main glial cell types are, in the central nervous system, astrocytes and oligodendrocytes and, in the peripheral nervous system, Schwann cells, enteric glial cells and satellite cells. In the embryo, glial cells form a cellular framework that permits the development of the rest of the nervous system, and regulate neuronal survival and differentiation. The best known function of glia in the adult is the formation of myelin sheaths around axons thus allowing the fast conduction of signalling essential for nervous system function. Glia also maintain appropriate concentrations of ions and neurotransmitters in the neuronal environment. Increasing body of evidence indicates that glial cells are essential regulators of the formation, maintenance and function of synapses, the key functional unit of the nervous system.     

The role of glutamate in the regulation of bone mass and architecture

Communication between the cells in bone underlies the way that the tissue functions physiologically, and in nearly all pathologies, the pathogenesis of skeletal diseases. The number of molecules involved in intercellular signalling in bone grows constantly and it is perhaps unsurprising that the list includes many with functions in other tissues. In recent years, evidence has accumulated to show that molecules involved in neurotransmission have paracrine roles in the skeleton. The focus of this review is the excitatory amino acid glutamate and its role in regulating bone formation and resorption. Specifically, this article will concentrate on the functional role of the system, and the reasons why mechanisms like synaptic transmission are relevant to what might appear to be a slow responding tissue, as the sites of expression of glutamate signalling components in bone have been reviewed already. While there is strong evidence for a regulatory role for glutamate in osteoblast and osteoclast differentiation and function in vitro, in vivo data is less advanced. Preliminary data from in vivo systems does however suggest that glutamate has a physiological function in the skeleton.     

Effects of chemotherapy on bone metabolism and skeletal growth

In recent years there has been a significant increase in both acute and chronic toxicity associated with the more successful but now highly intensive chemotherapy (CT) regimens used to treat childhood cancers. The incidence of childhood cancers coincides with periods of rapid skeletal development. Consequently, short stature and osteoporosis are important long-term effects in adult survivors. Clinical data indicate that the effects of CT, including glucocorticoids, on final height are due to direct effects of these drugs on the skeleton. The multiple modes of action of CT drugs suggest a complex and diverse influence on chondrocytes, extracellular matrix and bone cells. However, only limited data demonstrate these direct effects on the proliferative capacity of growth plate chondrocytes and on key steps of endochondral ossification, the multistep process that determines rate and extent of long bone growth. Endochondral ossification requires coordinated maturation, proliferation and differentiation of growth plate chondrocytes leading to hypertrophic cells which eventually undergo apoptosis to leave a cartilaginous scaffold that is mineralized prior to the laying down of new bone. Disruption of the physiological cellular activity of growth plate chondrocytes and/or bone cells result in skeletal growth disturbances. Thus, CT drugs which disrupt normal cell division may manifest their effects on the growth plate as either a reduction in cell number and/or the loss of functional integrity of extracellular matrix. Histological and cell kinetic studies, using in vivo and in vitro models of long bone growth, are essential to increase our understanding of the cellular mechanisms involved and to finally determine how the individual growth potential might be maintained during treatment for childhood cancers.     

Colocalization of neurotransmitter receptors on astrocytes in explant cultures of rat CNS

In recent years evidence has accumulated that astrocytes express functional receptors for a variety of neurotransmitters/neuromodulators. By means of electrophysiological and combined autoradiographic and immunohistochemical methods we have demonstrated the colocalization of cholinergic, adrenergic and peptidergic receptors on astrocytes in explant cultures from various regions of rat central nervous system. A great number of biochemical and electrophysiological studies from other laboratories have shown that most of the neurotransmitters exert their effects on second messenger systems and on Ca2+-activated K+-channels. Furthermore, certain neurotransmitters are involved in the regulation of energy metabolism by stimulating enzymatic breakdown of glycogen in astrocytes. It was suggested that there is a cross-talk between the various neurotransmitter receptors on the glial membrane and that these receptors act in a synergistic or antagonistic way. The coexistence of cholinergic and peptidergic receptors on astrocytes is of great interest since both neurotransmitter systems are involved in cognitive functions and are impaired in patients with Alzheimer's dementia. The question is therefore raised whether not only neurones but also astrocytes might be involved in neurodegenerative disorders such as Alzheimer's disease.     

Beyond the role of glutamate as a neurotransmitter

Glutamate is the principal excitatory neurotransmitter of the central nervous system, but many studies have expanded its functional repertoire by showing that glutamate receptors are present in a variety of non-excitable cells. How does glutamate receptor activation modulate their activity? Do non-excitable cells release glutamate, and, if so, how? These questions remain enigmatic. Here, we review the current knowledge on glutamatergic signalling in non-neuronal cells, with a special emphasis on astrocytes.     

Role of nonsynaptic communication in regulating the immune response

The discovery of nonsynaptic communication in the 1960s and 1970s was an important milestone in investigating the function of the nervous system, and it revolutionized our view about information transmission between neurons. In addition, nonsynaptic communication has a practical importance not only within the nervous system, but in the communication between the peripheral nervous system and other organ systems. Nonsynaptic communication takes place in different immune organs, which are innervated by sympathetic nerve terminals. In addition, the function of microglia, one of the immunocompetent cell types of the brain, can also be affected by neurotransmitters released from axon varicosities. The various functions of immune cells are modulated by released neurotransmitters without any direct synaptic contact between nerve endings and targeted immune cells requiring only functional neurotransmitter receptors on immune cells. Here, we briefly overview the role of the various receptor subtypes mediating nonsynaptic modulation of the function of immunocompetent cells both in the periphery and in the central nervous system.