Diego Fornasari

Human neuronal nicotinic receptors

Nicotine is a very widely used drug of abuse, which exerts a number of neurovegetative, behavioural and psychological effects by interacting with neuronal nicotinic acetylcholine receptors (NAChRs). These receptors are distributed widely in human brain and ganglia, and form a family of ACh-gated ion channels of different subtypes, each of which has a specific pharmacology and physiology. As human NAChRs have been implicated in a number of human central nervous system disorders (including the neurodegenerative Alzheimers disease, schizophrenia and epilepsy), they are suitable potential targets for rational drug therapy. Much of our current knowledge about the structure and function of NAChRs comes from studies carried out in other species, such as rodents and chicks, and information concerning human nicotinic receptors is still incomplete and scattered in the literature. Nevertheless, it is already evident that there are a number of differences in the anatomical distribution, physiology, pharmacology, and expression regulation of certain subtypes between the nicotinic systems of humans and other species. This review will attempt to survey the major achievements reached in the study of the structure and function of NAChRs by examining the molecular basis of their functional diversity viewed mainly from pharmacological and biochemical perspectives. It will also summarize our current knowledge concerning the structure and function of the NAChRs expressed by other species, and the newly discovered drugs used to classify their numerous subtypes. Finally, the role of NAChRs in behaviour and pathology will be considered.

Neuronal nicotinic receptors, important new players in brain function.

Acetylcholine receptors are cationic channels whose opening is controlled by acetylcholine. They are key molecules in the cholinergic nicotinic transmission in a number of areas of the central and peripheral nervous system. Because of the structural complexity, given by the numerous subunits that forms these receptors, they have different pharmacological and biophysical properties. Here we give a brief account of the known and consolidated data regarding neuronal nicotinic receptors, as as an introduction to the articles reported in this issue, in order to allow readers who are not familiar with the field to place the detailed information in the right context.

Adverse effects associated with non-opioid and opioid treatment in patients with chronic pain.

Chronic pain is a debilitating condition that is associated with many common diseases; this places a major burden on the healthcare system. There are currently numerous analgesic agents available for the treatment of chronic pain. In general, the oral non-opioid analgesic, paracetamol, is recommended for the initial treatment of mild to moderate pain. Therapeutic doses of paracetamol do not appear to result in hepatotoxicity, although overdose may lead to acute liver failure. Current data suggest that paracetamol has acceptable gastrointestinal tolerability. Another class of non-opioid analgesic with confirmed efficacy for the treatment of chronic mild to moderate pain are non-steroidal anti-inflammatory drugs (NSAIDs), although this efficacy is offset by the potential of adverse gastrointestinal events. In particular, non-selective NSAIDs, also known as cyclooxygenase (COX) inhibitors, carry an increased risk of serious upper gastrointestinal complications, including ulcers, perforation and bleeding. The introduction of COX-2 inhibitors provided a NSAID-based option with improved gastrointestinal safety, but increased risk of cardiovascular effects. Opioids are powerful analgesic agents used to treat moderate to severe chronic pain. However, treatment with opioids is associated with a number of common adverse effects, including constipation, nausea or vomiting, pruritus, somnolence or cognitive impairment, dry mouth, tolerance or dependence and urinary retention. Although there are multiple strategies in place to manage adverse events that arise from both non-opioid and opioid analgesic therapy, a better understanding of the mechanisms involved in the development of specific drug-related adverse effects is required along with proper prescribing practices and adequate physician/patient education. Balanced against the adverse effects of pain management medications, there is a need to be mindful of the widespread, often serious, adverse consequences of poorly managed pain itself.Chronic pain is a debilitating condition that is associated with many common diseases; this places a major burden on the healthcare system. There are currently numerous analgesic agents available for the treatment of chronic pain. In general, the oral non-opioid analgesic, paracetamol, is recommended for the initial treatment of mild to moderate pain. Therapeutic doses of paracetamol do not appear to result in hepatotoxicity, although overdose may lead to acute liver failure. Current data suggest that paracetamol has acceptable gastrointestinal tolerability. Another class of non-opioid analgesic with confirmed efficacy for the treatment of chronic mild to moderate pain are non-steroidal anti-inflammatory drugs (NSAIDs), although this efficacy is offset by the potential of adverse gastrointestinal events. In particular, non-selective NSAIDs, also known as cyclooxygenase (COX) inhibitors, carry an increased risk of serious upper gastrointestinal complications, including ulcers, perforation and bleeding. The introduction of COX-2 inhibitors provided a NSAID-based option with improved gastrointestinal safety, but increased risk of cardiovascular effects. Opioids are powerful analgesic agents used to treat moderate to severe chronic pain. However, treatment with opioids is associated with a number of common adverse effects, including constipation, nausea or vomiting, pruritus, somnolence or cognitive impairment, dry mouth, tolerance or dependence and urinary retention. Although there are multiple strategies in place to manage adverse events that arise from both non-opioid and opioid analgesic therapy, a better understanding of the mechanisms involved in the development of specific drug-related adverse effects is required along with proper prescribing practices and adequate physician/patient education. Balanced against the adverse effects of pain management medications, there is a need to be mindful of the widespread, often serious, adverse consequences of poorly managed pain itself.

Pain Mechanisms in Patients with Chronic Pain

The mechanisms involved in the development of chronic pain are varied and complex. Pain processes are plastic and unrelieved pain may lead to changes in the neural structure involved in pain generation. Nociceptive pain announces the presence of a potentially damaging stimulus that occurs when noxious stimuli activate primary afferent neurons. Neuropathic pain is initiated or caused by a primary lesion or dysfunction in the nervous system resulting from trauma, infection, ischaemia, cancer or other causes such as chemotherapy. The exact mechanisms involved in the pathophysiology of chronic pain are not well understood, but rapid and long-term changes are thought to occur in parts of the central nervous system that are involved in the transmission and modulation of pain following injury. Peripheral and central sensitization of sensory nerve fibres are the primary reasons for hypersensitivity to pain after injury, and mainly occur in inflammatory and neuropathic pain. During these processes the sensation of pain is enhanced as a result of changes in the environment, the nerve fibres and modifications of the functional properties and the genetic programme of primary and secondary afferent neurons. Non-steroidal anti-inflammatory drugs and opioid analgesics are two of the most common classes of drugs used for the treatment of pain. Response to drug treatment shows significant interindividual variability and can lead to side effects. The neurobiological mechanisms that cause pain may account for the different types of pain observed. Identification of these mechanisms may allow us to move from an empirical therapeutic approach to one that it is specifically targeted at the particular mechanisms of the type of pain experienced by an individual patient.The mechanisms involved in the development of chronic pain are varied and complex. Pain processes are plastic and unrelieved pain may lead to changes in the neural structure involved in pain generation. Nociceptive pain announces the presence of a potentially damaging stimulus that occurs when noxious stimuli activate primary afferent neurons. Neuropathic pain is initiated or caused by a primary lesion or dysfunction in the nervous system resulting from trauma, infection, ischaemia, cancer or other causes such as chemotherapy. The exact mechanisms involved in the pathophysiology of chronic pain are not well understood, but rapid and long-term changes are thought to occur in parts of the central nervous system that are involved in the transmission and modulation of pain following injury. Peripheral and central sensitization of sensory nerve fibres are the primary reasons for hypersensitivity to pain after injury, and mainly occur in inflammatory and neuropathic pain. During these processes the sensation of pain is enhanced as a result of changes in the environment, the nerve fibres and modifications of the functional properties and the genetic programme of primary and secondary afferent neurons. Non-steroidal anti-inflammatory drugs and opioid analgesics are two of the most common classes of drugs used for the treatment of pain. Response to drug treatment shows significant interindividual variability and can lead to side effects. The neurobiological mechanisms that cause pain may account for the different types of pain observed. Identification of these mechanisms may allow us to move from an empirical therapeutic approach to one that it is specifically targeted at the particular mechanisms of the type of pain experienced by an individual patient.

Expression of the α3 nicotinic receptor subunit mRNA in aging and Alzheimer's disease

Changes in the number of high-affinity nicotine binding sites have been widely reported in specific regions of the human brain during aging and in degenerative neurological diseases associated with aging, such as Alzheimers disease. Nicotinic receptors are highly diverse and a description of the molecular subtypes affected in such conditions has not been achieved to date. To investigate the status of the α3 subunit-containing subtypes in such conditions, we assessed by in situ hybridisation the α3 mRNA density in the hippocampus, entorhinal cortex and thalamus of Alzheimers patients and age-matched controls. No significant difference in the expression of the α3 mRNA, either qualitative or quantitative, was found between Alzheimers individuals and controls in any of the analysed areas. This result suggests that the nicotine binding changes occurring in these areas in Alzheimers patients are not correlated to a variation of the α3 mRNA in the same regions. Nevertheless, a negative correlation between the α3 mRNA density and the age was observed in the entorhinal cortex of both the Alzheimers and the normal subjects, suggesting a potentially extensive decay of the α3-expressing neurons or loss of α3-containing receptors in intact neurons of the entorhinal cortex in the late elderly.

Expression and Transcriptional Regulation of the Human α3 Neuronal Nicotinic Receptor Subunit in T Lymphocyte Cell Lines

Abstract: The expression of neurotransmitter receptors on the surface of immunocompetent cells is generally accepted as evidence that the nervous system can influence immune responses, even though many aspects of these interactions remain to be elucidated. In this article, we analyzed the expression of the α3 nicotinic receptor subunit in human cell lines of myeloid and lymphoid origin and show that the α3 mRNA and the receptor molecules containing this subunit are specifically expressed in T lymphocyte cell lines. We have previously characterized the structural properties of the human α3 nicotinic subunit gene promoter and defined its functional profile in neuronal cells; in this study, we analyzed the activity of the α3 promoter in T lymphocytes and found that the same minimal promoter located in the 0.16‐kb BglII‐AccIII fragment is responsible for the expression of the α3 mRNA in both neuronal and T lymphocyte cell lines. However, the α3 transcription initiation patterns in the two cell types were both qualitatively and quantitatively different, and the minimal promoter was differentially modulated by downstream and upstream regulatory elements. These findings suggest that distinct transcriptional mechanisms allow the same promoter to be regulated in a tissue‐specific fashion, according to the different functional needs of the two cell types.

Neuronal and Extraneuronal Expression and Regulation of the Human α5 Nicotinic Receptor Subunit Gene

Abstract : The mRNA encoding the human α5 nicotinic subunit was detected in several structures of the nervous system but appeared to be mainly expressed in cerebellum, thalamus, and the autonomic ganglia. For the first time, the α5 transcript was also detected in several non‐neuronal tissues, with maximal expressions being found throughout the gastrointestinal tract, thymus, and testis. Many other extraneuronal sites expressed α5, but there were also nonexpressing organs, such as the liver, spleen, and kidney. To understand the transcriptional mechanisms controlling such a diversified expression of α5 in neuronal and nonneuronal cells, we isolated the 5′‐regulatory region of the human gene and characterized its properties. Here we identify the α5 core promoter and demonstrate that the DNA regions surrounding it contain elements (with positive or negative activities) that work in a tissue‐specific fashion. In particular, the segment specifying the 5′‐untranslated region in neuronal cells has most of the properties of an enhancer because it activates a heterologous promoter in a position‐ and orientation‐independent fashion. We therefore conclude that the expression of α5 relies on a highly complex promoter that uses distinct regulatory elements to comply with the different functional and developmental requirements of the various tissues and organs.

Molecular cloning of human neuronal nicotinic receptor α3-subunit

Neuronal nicotinic receptors (nAchRs) have been isolated or cloned in insect, bird and mammalian neurons, but no information exists on the primary structure of human neuronal nAchRs. By screening a cDNA library from the human neuroblastoma cell line IMR 32 with a cDNA probe corresponding to the full length of rat alpha 3-nicotinic subunit, we have identified an open reading frame encoding a protein of 502 amino acids. This protein shows all the features of members of the ligand-gated receptor superfamily and has two cysteine residues at positions 192, 193 which are typical of the nicotinic alpha-subunits. Because of its high homology to rat alpha 3 (93% amino acid identity), we conclude that we have cloned the human alpha 3-nicotinic subunit.

The Appropriate Treatment of Chronic Pain

Chronic pain is a common healthcare problem worldwide that ranks as a predominant reason for consulting a physician, yet effective management of chronic pain remains suboptimal, often resulting in unnecessary suffering and decreased quality of life, lost productivity and excessive healthcare costs. To overcome the challenges associated with the management of chronic pain, increased awareness and both patient and physician education are required. Improving physician knowledge of pain assessment and management guided by recommendations for a comprehensive, multifactorial, personalised treatment approach involving pharmacological and non-pharmacological approaches is key to achieving effective pain relief. Guidelines for the management of non-cancer and cancer pain recommend thorough patient assessment before individualized therapy based on the type and intensity of pain. The availability of mechanism-specific analgesics has facilitated improvements in the treatment of chronic non-cancer pain, which may be of neuropathic, muscle, inflammatory, mechanical/compressive or mixed origin. Stepwise escalation of analgesic therapy (paracetamol, non-steroidal anti-inflammatory drugs, mild to strong opioids) according to the World Health Organization’s three-step pain ladder remains the standard approach for the selection of treatment for chronic cancer pain, although there is now a greater awareness of the requirements for effective administration of opioids including dose titration, use of short versus long-acting opioids, opioid rotation, management of adverse effects, and ongoing monitoring. Selection of an effective, appropriate, personalized analgesic regimen for patients with chronic pain is achievable and is expected to enhance compliance, overall functioning and quality of life.

Sp proteins and Phox2b regulate the expression of the human Phox2a gene.

Phox2a is a vertebrate homeodomain transcription factor that is involved in the specification of the autonomic nervous system. We have isolated the 5′ regulatory region of the human Phox2agene and studied the transcriptional mechanisms underlying its expression. We first identified the minimal gene promoter by means of molecular and functional criteria and demonstrated that its activity relies on a degenerate TATA box and a canonical Sp1 site. We then concentrated on the region immediately upstream of the promoter and found that it stimulates transcription in a neurospecific manner because its deletion caused a substantial decline in reporter gene expression only in neuronal cells. This DNA region contains a putative binding site for homeodomain transcription factors, and its mutation severely affects the transcriptional activity of the entire 5′ regulatory region, thus indicating that this site is necessary for the expression of Phox2a in this cellular context. The use of the electrophoretic mobility shift assay showed that Phox2b/PMX2b is capable of specifically interacting with this site, and cotransfection experiments demonstrated that it is capable of transactivating the human Phox2a promoter. Many data obtained from knock-out mice support the hypothesis that Phox2a acts downstream of Phox2b during the development of most of the autonomic nervous system. We have provided the first molecular evidence that Phox2b can regulate the expression of Phox2a by directly binding to its 5′ regulatory region.