The ecto-enzymes CD73 and adenosine deaminase modulate 5'-AMP-derived adenosine in myofibroblasts of the rat small intestine.

Adenosine is a versatile signaling molecule recognized to physiologically influence gut motor functions. Both the duration and magnitude of adenosine signaling in enteric neuromuscular function depend on its availability, which is regulated by the ecto-enzymes ecto-5'-nucleotidase (CD73), alkaline phosphatase (AP), and ecto-adenosine deaminase (ADA) and by dipyridamole-sensitive equilibrative transporters (ENTs). Our purpose was to assess the involvement of CD73, APs, ecto-ADA in the formation of AMP-derived adenosine in primary cultures of ileal myofibroblasts (IMFs). IMFs were isolated from rat ileum longitudinal muscle segments by means of primary explant technique and identified by immunofluorescence staining for vimentin and α-smooth muscle actin. IMFs confluent monolayers were exposed to exogenous 5'-AMP in the presence or absence of CD73, APs, ecto-ADA, or ENTs inhibitors. The formation of adenosine and its metabolites in the IMFs medium was monitored by high-performance liquid chromatography. The distribution of CD73 and ADA in IMFs was detected by confocal immunocytochemistry and qRT-PCR. Exogenous 5'-AMP was rapidly cleared being almost undetectable after 60-min incubation, while adenosine levels significantly increased. Treatment of IMFs with CD73 inhibitors markedly reduced 5'-AMP clearance whereas ADA blockade or inhibition of both ADA and ENTs prevented adenosine catabolism. By contrast, inhibition of APs did not affect 5'-AMP metabolism. Immunofluorescence staining and qRT-PCR analysis confirmed the expression of CD73 and ADA in IMFs. Overall, our data show that in IMFs an extracellular AMP-adenosine pathway is functionally active and among the different enzymatic pathways regulating extracellular adenosine levels, CD73 and ecto-ADA represent the critical catabolic pathway.

Molecular regulation of cholesterol metabolism: HDL-based intervention through drugs and diet.

The overloading of cholesterol in the arteries remains the principal cause of cardiovascular diseases. Since available anticholesterolemic drugs are not completely effective and have several severe adverse effects, the aim of this review is to analyze current research focused on the emerging, innovative therapeutic strategies based on both pharmacological and nutritional interventions to control cholesterol metabolism. Pharmacological interventions mainly involve the use of molecules capable of interfering with high-density lipoprotien (HDL) metabolism and the reverse cholesterol transport (RCT) through genetic control of apolipoprotein A-I (ApoA-I), agonism at liver X-receptor alpha (LXRalpha), or inhibition of cholesteryl ester transport protein (CETP), scavenger receptor BI(SR-BI), and ecto F0F1ATPase/synthase. Nutritional interventions are based on the use of fibres, phytosterols, and probiotics acting through interference with absorption and reabsorption of cholesterol by enterocyte and hepatocyte specific transporters, thus influencing RCT final step. The search for new drugs is still at the very beginning and new molecules are not yet ready to enter clinical use. However, several promising findings coming from innovative biotechnological research are expected shortly to produce probiotics, fibres, and phytosterols to be used as therapeutic tools. Among the most important advantages of natural products in respect to traditional drugs are the lack of severe adverse effects and their low cost.

The P2X7 purinergic receptor: from physiology to neurological disorders.

Purine nucleotides are well established as extracellular signaling molecules. P2X receptors are ATP-gated cation channels that mediate fast excitatory transmission in diverse regions of the brain and spinal cord. Several P2X receptor subtypes, including P2X(7), have the unusual property of changing their ion selectivity during prolonged exposure to ATP, which results in progressive dilation of the channel pore and the development of permeability to molecules as large as 900 Da. The P2X(7) receptor was originally described in cells of hematopoietic origin, including macrophages, microglia, and certain lymphocytes, and mediates the influx of Ca(2+) and Na(+) ions, as well as the release of proinflammatory cytokines. P2X(7) receptors may affect neuronal cell death through their ability to regulate the processing and release of interleukin-1beta, a key mediator in neurodegeneration, chronic inflammation, and chronic pain. Activation of P2X(7) receptors provides an inflammatory stimulus, and P2X(7) receptor-deficient mice have substantially attenuated inflammatory responses, including models of neuropathic and chronic inflammatory pain. Moreover, P2X(7) receptor activity, by regulating the release of proinflammatory cytokines, may be involved in the pathophysiology of depression. The P2X(7) receptor may thus represent a critical communication link between the nervous and immune systems, while providing a target for therapeutic exploitation. This review discusses the current biology and cellular signaling pathways of P2X(7) receptor function, as well as insights into the role for this receptor in neurological/psychiatric diseases, outstanding questions, and the therapeutic potential of P2X(7) receptor antagonism.

Generation of a alpha-synuclein-based rat model of Parkinson's disease.

Two missense mutations (A30P and A53T) in the gene for alpha-synuclein (alpha-syn) cause familial Parkinson's disease (PD) in a small cohort. There is increasing evidence to propose that abnormal metabolism and accumulation of alpha-syn in dopaminergic neurons play a role in the development of familial as well as sporadic PD. The complexity of the mechanisms underlying alpha-syn-induced neurotoxicity, however, has made difficult the development of animal models that faithfully reproduce human PD pathology. We now describe and characterize such a model, which is based on the stereotaxic injection into rat right substantia nigra pars compacta of the A30P mutated form of alpha-syn fused to a protein transduction domain (TAT). The TAT sequence allows diffusion of the fusion protein across the neuronal plasma membrane and results in a localized dopaminergic loss. Dopaminergic cell loss was evaluated both by tyrosine hydroxylase immunohistochemistry and by HPLC analysis of dopamine and its catabolite 3,4 dihydroxyphenylacetic acid. Infusion of TAT-alpha-synA30P induced a significant 26% loss in dopaminergic neurons. This dopaminergic loss was accompanied by a time-dependent impairment in motor function, evaluated utilizing the rotarod and footprint tests. In comparison to chemical neurotoxin-based (e.g. 6-hyroxydopamine, MPTP) animal models of PD, the alpha-syn-based PD animal model offers the advantage of mimicking the early stages and slow development of the human disease and should prove valuable in assessing specific aspects of PD pathogenesis in vivo and in developing new therapeutic strategies.

Antibiotic-induced dysbiosis of the microbiota impairs gut neuromuscular function in juvenile mice.

BACKGROUND AND PURPOSE: Gut microbiota is essential for the development of the gastrointestinal system, including the enteric nervous system (ENS). Perturbations of gut microbiota in early life have the potential to alter neurodevelopment leading to functional bowel disorders later in life. We examined the hypothesis that gut dysbiosis impairs the structural and functional integrity of the ENS, leading to gut dysmotility in juvenile mice. EXPERIMENTAL APPROACH: To induce gut dysbiosis, broad-spectrum antibiotics were administered by gavage to juvenile (3weeks old) male C57Bl/6 mice for 14 days. Bile acid composition in the intestinal lumen was analysed by liquid chromatography-mass spectrometry. Changes in intestinal motility were evaluated by stool frequency, transit of a fluorescent-labelled marker and isometric muscle responses of ileal full-thickness preparations to receptor and non-receptor-mediated stimuli. Alterations in ENS integrity were assessed by immunohistochemistry and Western blot analysis. KEY RESULTS: Antibiotic treatment altered gastrointestinal transit, luminal bile acid metabolism and bowel architecture. Gut dysbiosis resulted in distorted glial network, loss of myenteric plexus neurons, altered cholinergic, tachykininergic and nitrergic neurotransmission associated with reduced number of nNOS neurons and different ileal distribution of the toll-like receptor TLR2. Functional defects were partly reversed by activation of TLR2 signalling. CONCLUSIONS AND IMPLICATIONS: Gut dysbiosis caused complex morpho-functional neuromuscular rearrangements, characterized by structural defects of the ENS and increased tachykininergic neurotransmission. Altogether, our findings support the beneficial role of enteric microbiota for ENS homeostasis instrumental in ensuring proper gut neuromuscular function during critical stages of development.

Alpha-synuclein and Parkinson's disease.

Alpha-synuclein (alpha-syn) is a small soluble protein expressed primarily at presynaptic terminals in the central nervous system. Interest in alpha-syn has increased dramatically after the discovery of a relationship between its dysfunction and several neurodegenerative diseases, including Parkinson's disease (PD). The physiological functions of alpha-syn remain to be fully defined, although recent data suggest a role in regulating membrane stability and neuronal plasticity. Various trigger factors, either environmental or genetic, can lead to a cascade of events involving misfolding or loss of normal function of alpha-syn. In dopaminergic neurons, this may promote a vicious cycle in which elevation in cytoplasmic dopamine, oxidative stress, alpha-syn dysfunction, and disruption of vesicle function lead to dopaminergic cell loss and PD. Alpha-syn dysfunction appears to be a common feature of all forms of PD. The mechanism by which alpha-syn induces neuronal cell toxicity may invoke multiple pathways, such as aggregation or interaction with other proteins and molecules, including synphilin-1, chaperone 14-3-3 protein, and dopamine itself. This complexity has hindered the development of models to study PD. The available animal models of PD, each present distinct advantages and limits. Findings to date suggest that alpha-syn-based models represent a paradigm, which is closest to the human pathology.

Isolation and culture of neural progenitor cells from rat postnatal cerebellum.

The mammalian brain contains undifferentiated, mitotically active, and multipotent neural stem/progenitor cells that in vivo contribute new neurons and glia to specific areas of the mature brain. When isolated under the appropriate conditions, these cells maintain in vitro the ability to proliferate and differentiate into cells that express neuronal and glial markers. Neural stem/progenitor cells have been identified and isolated from many regions of the embryonic, postnatal, and adult central nervous system, including cerebellum. This chapter details techniques to isolate and culture neural progenitor cells from rat postnatal cerebellum, which can be used as an in vitro model to study the molecular mechanisms underlying proliferation and differentiation into mature neural cells induced by various stimuli including pharmacological agents.

alpha-Synuclein- and MPTP-generated rodent models of Parkinson's disease and the study of extracellular striatal dopamine dynamics: a microdialysis approach.

The classical animal models of Parkinson's disease (PD) rely on the use of neurotoxins, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine and, more recently, the agricultural chemicals paraquat and rotenone, to deplete dopamine (DA). These neurotoxins elicit motor deficits in different animal species although MPTP fails to induce a significant dopaminergic neurodegeneration in rats. In the attempt to better reproduce the key features of PD, in particular the progressive nature of neurodegeneration, alternative PD models have been developed, based on the genetic and neuropathological links between -synuclein ( -syn) and PD. In vivo microdialysis was used to investigate extracellular striatal DA dynamics in MPTP- and -syn-generated rodent models of PD. Acute and sub-acute MPTP intoxication of mice both induce prolonged release of striatal DA. Such DA release may be considered the first step in MPTP-induced striatal DA depletion and nigral neuron death, mainly through reactive oxygen species generation. Although MPTP induces DA reduction, neurochemical and motor recovery starts immediately after the end of treatment, suggesting that compensatory mechanisms are activated. Thus, the MPTP mouse model of PD may be unsuitable for closely reproducing the features of the human disease and predicting potential long-term therapeutic effects, in terms of both striatal extracellular DA and behavioral outcome. In contrast, the -syn-generated rat model of PD does not suffer from a massive release of striatal DA during induction of the nigral lesion, but rather is characterized by a prolonged reduction in baseline DA and nicotine-induced increases in dialysate DA levels. These results are suggestive of a stable nigrostriatal lesion with a lack of dopaminergic neurochemical recovery. The -syn rat model thus reproduces the initial stage and slow development of PD, with a time-dependent impairment in motor function. This article will describe the above experimental PD models and demonstrate the utility of microdialysis for their characterization.

P2X(7) Receptors in Neurological and Cardiovascular Disorders.

P2X receptors are ATP-gated cation channels that mediate fast excitatory transmission in diverse regions of the brain and spinal cord. Several P2X receptor subtypes, including P2X(7), have the unusual property of changing their ion selectivity during prolonged exposure to ATP, which results in a channel pore permeable to molecules as large as 900 daltons. The P2X(7) receptor was originally described in cells of hematopoietic origin, and mediates the influx of Ca(2+) and Na(+) and Ca(2+) and Na(+) ions as well as the release of proinflammatory cytokines. P2X(7) receptors may affect neuronal cell death through their ability to regulate the processing and release of interleukin-1beta, a key mediator in neurodegeneration, chronic inflammation, and chronic pain. Activation of P2X(7), a key mediator in neurodegeneration, chronic inflammation, and chronic pain. Activation of P2X(7) receptors provides an inflammatory stimulus, and P2X(7) receptor-deficient mice have substantially attenuated inflammatory responses, including models of neuropathic and chronic inflammatory pain. Moreover, P2X(7) receptor activity, by regulating the release of proinflammatory cytokines, may be involved in the pathophysiology of depression. Apoptotic cell death occurs in a number of vascular diseases, including atherosclerosis, restenosis, and hypertension, and may be linked to the release of ATP from endothelial cells, P2X(7) receptor activation, proinflammatory cytokine production, and endothelial cell apoptosis. In this context, the P2X(7) receptor may be viewed as a gateway of communication between the nervous, immune, and cardiovascular systems.

Fluoxetine-induced proliferation and differentiation of neural progenitor cells isolated from rat postnatal cerebellum.

Previous studies have shown that the serotonin-reuptake inhibitor (SSRI) fluoxetine affects neural progenitors derived from postnatal cerebellum or adult hippocampus and stimulates their proliferation. In the human cerebellum, the proliferation of cerebellar granule cells (CGC) continues until the 11th postnatal month and could be influenced in infants by breastfeeding-delivered SSRIs. Current information about fluoxetine effects on postnatal cerebellar neural progenitors is limited. Here we report the characterization of fluoxetine actions on rat postnatal cerebellar neural progenitors. RT-PCR and immunostaining revealed the expression of serotonin transporter (SERT), 5HT(1A) receptors, tryptophan hydroxylase (TPH), and serotonin (5HT). Protracted in vitro fluoxetine treatment increased cell proliferation and differentiation. The proliferative effects of fluoxetine, 5HT, and the selective agonist of 5HT(1A) receptors trans-8-hydroxy-2-(N-n-propyl-N-3'-iodo-2'-propenyl)aminotetralin (8-OH-PIPAT) were abolished by the selective antagonist of 5HT(1A) receptors, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY-100635). Furthermore, fluoxetine-induced activation of both the cAMP-response element-binding (CREB) protein and extracellular signal-regulated protein kinases (ERK1/2), which was abolished by the selective inhibitor of MAP kinase kinase (MEK) 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), and increased cyclin D1 expression. All these effects were prevented by WAY-100635. Collectively, our results demonstrate that rat postnatal cerebellum contains neural progenitors capable of proliferating and differentiating in response to fluoxetine exposure, possibly through the activation of 5HT(1A) receptors. The relevance of these findings for possible SSRI effects on the developing postnatal/infant human cerebellum should be explored.

Evaluation of the prescription and utilization patterns of statins in an Italian local health unit during the period 1994-2003.

OBJECTIVES: The prescription pattern of statins in the Local Health Unit (LHU) of Treviso (northern Italy) over a 10-year period was evaluated, with the aim of evaluating the persistence with and adherence to therapy. METHODS: Data on 21,393 subjects who received at least one prescription for statins during the period between January 1, 1994 and December 31, 2003 were retrieved from the LHU database in order to track the pharmacological history of individual patients. The data included age, sex, drug formulation, strength, number of drug packages prescribed, and prescription date. The adopted indicators for drug utilization included the Defined Daily Dose (DDD), the Received Daily Dose (RDD), and a surrogated Prescribed Daily Dose (sPDD), extrapolated from available prescription data. An Adherence to Therapy Index (ATI) was calculated from the ratio between the amount of drug actually prescribed and the amount of sPDD. Based on the ATI, patients were grouped into non-adherent, poor-adherent, and good-adherent groups. The distribution of adherence level among patient-age classes and statin-prescribed patients in primary or secondary prevention was evaluated. RESULTS: All drug-utilization indicators showed an increase in statin use over the study period in terms of both the number of prescribed patients and the sPDD. Persistence with and adherence to therapy remained low, with a 50% discontinuation rate in the first year, and persistent patients did not follow the therapy regularly. Patients in secondary prevention were the most adherent to their drug regimen, although only 41% of these had a good compliance. CONCLUSIONS: Our findings suggest an increase in statin use which is, however, accompanied by poor patient persistence with and adherence to statin therapy.

Retrospective analysis of opioid prescriptions in cancer patients in a northern Italian region.

We evaluated patterns of use of opioids in palliative care across one region in Italy by cross-referencing a cancer registry with unique patient identifiers, with prescription databases. There were 90 803 patients in the registry, of whom 39 597 died during the study period. Only 8539 (21%) of these were prescribed opioids at the time of their death. Prescribed daily doses of oral morphine used (45 mg) and of buprenorphine (0.71 mg) were low compared with injected morphine (28.6 mg, equivalent to approximately 90 mg of oral morphine) and especially with doses of transdermal fentanyl (1.13 mg, equivalent to approximately 180 mg morphine). The reasons for this acceptance of transdermal fentanyl and reluctance to use oral morphine are unclear, but it seems that more effort in educating healthcare professionals and patients about the use of morphine would be useful. The use of more detailed prescribing data such as prescribed or received daily doses can add to our understanding of headline prescribing data.

Opioid prescription for terminally ill outpatients in a district of northern Italy: a retrospective survey.

A retrospective survey of the opioid prescriptions issued for cancer outpatients (2125) of the Treviso district (Veneto Region, northern Italy) during the time period 1993-2000 was carried out with the specific aims to establish the rate of opioid prescription and verify whether terminally ill outpatients (1697) who had died by the end of December 2000 received adequate opioid prescription, as compared with the Defined Daily Doses (DDDs) of opioids suggested by the World Health Organization (WHO) for a standard population. For both women and men, the maximum rate of opioid prescription was at the age of beyond 90 years. Men were more prescribed than women between 60 and 79 years of age, whereas women were more prescribed than men beyond 90 years. Opioid prescriptions concerned only morphine, buprenorphine, and pentazocine. The Anatomical Therapeutic Chemical (ATC)/DDDs analysis of opioid prescriptions indicated that total opioid use increased about 1.7-fold between 1993 and 1996, mainly because of an increase (55.4%) in morphine prescriptions. Afterwards, total opioid use remained stable, with an estimated mean annual value of 108.2+/-6.4 DDDs/million inhabitants/day. Considering terminally ill outpatients who had died by the end of December 2000, oral morphine turned out to be the most commonly prescribed opioid (64% of patients) and, among the three opioids, pentazocine was more prescribed to older patients. From the comparison between the number of "expected opioid DDDs" (i.e. days for which patients should have been prescribed opioids at the WHO recommended DDDs) and the number of prescribed opioid DDDs (i.e. days for which patients had been offered adequate opioid treatment) for individual patients, it could be estimated that only 38.1% of opioid prescriptions were adequate and a mean of 55.8 DDDs of opioids per patient were not prescribed. The opioid prescription inadequacy increased with the length of time from first prescription to patient death. In addition, a questionnaire investigation, conducted in 2001 among general practitioners of the Treviso district to evaluate their attitudes toward opioid prescribing, evidenced insufficient knowledge of general practitioners in theory and use of opioid analgesics in cancer pain management. A total of 104 (32.5%) general practitioners responded and most of them feared opioid side effects, such as respiratory depression (49.6%), constipation (41.7%), and addiction (8.7%). Furthermore, many of the respondents considered opioids capable of reducing the patient length of life (22.2%) and inappropriate to treat pediatric patients (50.6%). About 44% of the respondents experienced external pressure by relatives of patients against opioid prescription and a majority of them (58.2%) considered the recently revised Italian legislation on opioid prescription ineffective for improving their prescribing pattern. In conclusion, present data show that the vast majority of terminally ill cancer outpatients in the Treviso district received inadequate opioid prescriptions in relation to either drug daily dosage or therapy duration. Misconceptions of general practitioners of the district about opioids could contribute to the inappropriate use of these analgesics in cancer pain management. As far as we know, the ATC/DDD methodology for the opioid prescription analysis used in this survey has not been applied before.