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Myenteric plexus alterations hamper gastrointestinal motor function during intestinal inflammation. Hyaluronan (HA), an extracellular matrix glycosaminoglycan involved in inflammatory responses, may play a role in this process. In the colon of control rats, HA-binding protein (HABP), was detected in myenteric neuron soma, perineuronal space and ganglia surfaces. Prominent hyaluronan synthase 2 (HAS2) staining was found in myenteric neuron cytoplasm, suggesting that myenteric neurons produce HA. In the myenteric plexus of rats with 2, 4-dinitrobenzene sulfonic (DNBS)-induced colitis HABP staining was altered in the perineuronal space, while both HABP staining and HA levels increased in the muscularis propria. HAS2 immunopositive myenteric neurons and HAS2 mRNA and protein levels also increased. Overall, these observations suggest that inflammation alters HA distribution and levels in the gut neuromuscular compartment. Such changes may contribute to alterations in the myenteric plexus.

Antibiotic use during adolescence may result in dysbiosis-induced neuronal vulnerability both in the enteric nervous system (ENS) and central nervous system (CNS) contributing to the onset of chronic gastrointestinal disorders, such as irritable bowel syndrome (IBS), showing significant psychiatric comorbidity. Intestinal microbiota alterations during adolescence influence the expression of molecular factors involved in neuronal development in both the ENS and CNS. In this study, we have evaluated the expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tropomyosin-related kinase B (TrkB) in juvenile mice ENS and CNS, after a 2-week antibiotic (ABX) treatment. In both mucosa and mucosa-deprived whole-wall small intestine segments of ABX-treated animals, BDNF and TrKB mRNA and protein levels significantly increased. In longitudinal muscle-myenteric plexus preparations of ABX-treated mice the percentage of myenteric neurons staining for BDNF and TrkB was significantly higher than in controls. After ABX treatment, a consistent population of BDNF- and TrkB-immunoreactive neurons costained with SP and CGRP, suggesting up-regulation of BDNF signaling in both motor and sensory myenteric neurons. BDNF and TrkB protein levels were downregulated in the hippocampus and remained unchanged in the prefrontal cortex of ABX-treated animals. Immunostaining for BDNF and TrkB decreased in the hippocampus CA3 and dentate gyrus subregions, respectively, and remained unchanged in the prefrontal cortex. These data suggest that dysbiosis differentially influences the expression of BDNF-TrkB in the juvenile mice ENS and CNS. Such changes may potentially contribute later to the development of functional gut disorders, such as IBS, showing psychiatric comorbidity.

Several studies have been carried out in the last 30 years in the attempt to clarify the possible role of glutamate as a neurotransmitter/neuromodulator in the gastrointestinal tract. Such effort has provided immunohistochemical, biomolecular and functional data suggesting that the entire glutamatergic neurotransmitter machinery is present in the complex circuitries of the enteric nervous system (ENS), which participates to the local coordination of gastrointestinal functions. Glutamate is also involved in the regulation of the brain-gut axis, a bi-directional connection pathway between the central nervous system (CNS) and the gut. The neurotransmitter contributes to convey information, via afferent fibers, from the gut to the brain, and to send appropriate signals, via efferent fibers, from the brain to control gut secretion and motility. In analogy with the CNS, an increasing number of studies suggest that dysregulation of the enteric glutamatergic neurotransmitter machinery may lead to gastrointestinal dysfunctions. On the whole, this research field has opened the possibility to find new potential targets for development of drugs for the treatment of gastrointestinal diseases. The present review analyzes the more recent literature on enteric glutamatergic neurotransmission both in physiological and pathological conditions, such as gastroesophageal reflux, gastric acid hypersecretory diseases, inflammatory bowel disease, irritable bowel syndrome and intestinal ischemia/reperfusion injury.

Nitric oxide (NO) and glutamate, via N-methyl- d -aspartate (NMDA) receptors, participate to changes in neuromuscular responses after ischemic/reperfusion (I/R) injury in the gut. In the present study we investigated the existence of a possible interplay between nitrergic and NMDA receptor pathways in the guinea pig ileum after in vitro I/R injury, resorting to functional and biomolecular approaches. In normal metabolic conditions NMDA concentration-dependently enhanced both glutamate (analyzed by high performance liquid chromatography with fluorimetric detection) and NO (spectrophotometrically quantified as NO2− and NO3−) spontaneous overflow from isolated ileal segments. Both effects were reduced by the NMDA antagonists, (-)-AP5 (10 µM) and 5,7-diCl-kynurenic acid (10 µM, 5,7-diCl-KYN). Nω-propyl- l -arginine (1 µM, NPLA) and 1400W (10 µM), respectively, nNOS and iNOS inhibitors, reduced NMDA-stimulated glutamate overflow. After in vitro I/R, glutamate overflow increased, and returned to control values in the presence of NPLA and 1400W. NO2− and NO3− levels transiently increased during I/R and were reduced by both (-)-AP5 and 5,7-diCl-KYN. In longitudinal muscle myenteric plexus preparations, iNOS mRNA and protein levels increased after in vitro I/R; both parameters were reduced to control values by (-)-AP5 and 5,7-diCl-KYN. Both antagonists were also able to reduce ischemia-induced enhancement of nNOS mRNA levels. Protein levels of GluN1, the ubiquitary subunit of NMDA receptors, increased after I/R and were reduced by both NPLA and 1400W. On the whole, this data suggests the existence of a cross-talk between NMDA receptor and nitrergic pathways in guinea pig ileum myenteric plexus, which may participate to neuronal rearrangements occurring during I/R.

Neuronal and inducible nitric oxide synthase (nNOS and iNOS) play a protective and damaging role, respectively, on the intestinal neuromuscular function after ischemia-reperfusion (I/R) injury. To uncover the molecular pathways underlying this dichotomy we investigated their possible correlation with the orthodenticle homeobox proteins OTX1 and OTX2 in the rat small intestine myenteric plexus after in vivo I/R. Homeobox genes are fundamental for the regulation of the gut wall homeostasis both during development and in pathological conditions (inflammation, cancer). I/R injury was induced by temporary clamping the superior mesenteric artery under anesthesia, followed by 24 and 48 h of reperfusion. At 48 h after I/R intestinal transit decreased and was further reduced by Nω-propyl-l-arginine hydrochloride (NPLA), a nNOS-selective inhibitor. By contrast this parameter was restored to control values by 1400W, an iNOS-selective inhibitor. In longitudinal muscle myenteric plexus (LMMP) preparations, iNOS, OTX1, and OTX2 mRNA and protein levels increased at 24 and 48 h after I/R. At both time periods, the number of iNOS- and OTX-immunopositive myenteric neurons increased. nNOS mRNA, protein levels, and neurons were unchanged. In LMMPs, OTX1 and OTX2 mRNA and protein upregulation was reduced by 1400W and NPLA, respectively. In myenteric ganglia, OTX1 and OTX2 staining was superimposed with that of iNOS and nNOS, respectively. Thus in myenteric ganglia iNOS- and nNOS-derived NO may promote OTX1 and OTX2 upregulation, respectively. We hypothesize that the neurodamaging and neuroprotective roles of iNOS and nNOS during I/R injury in the gut may involve corresponding activation of molecular pathways downstream of OTX1 and OTX2.NEW & NOTEWORTHY Intestinal ischemia-reperfusion (I/R) injury induces relevant alterations in myenteric neurons leading to dismotility. Nitrergic neurons seem to be selectively involved. In the present study the inference that both neuronal and inducible nitric oxide synthase (nNOS and iNOS) expressing myenteric neurons may undergo important changes sustaining derangements of motor function is reinforced. In addition, we provide data to suggest that NO produced by iNOS and nNOS regulates the expression of the vital transcription factors orthodenticle homeobox protein 1 and 2 during an I/R damage.

The antiepileptic drug therapy of 1912 patients coming from various neurological clinics of Northern Italy has been evaluated in a collaborative survey. The following epidemiological data have been analysed: incidence of the various seizures types, according to the International Classification (Gastaut, 1969), in relation to the age; drug choice and therapeutic schedules in relation to the seizure type and to the age; number of drugs administered in various age and seizure groups; side effects in relation to type, number and plasma concentrations of the administered drugs. The analysis of the drug plasma level determinations, carried out either with gas-chromatographic methods or with the EMIT system, was effected in order to evaluate the number of patients who were under, in or over the suggested therapeutic ranges, the influence of age on drug disposition and the modifications of the drug plasma levels due to drug interactions.

The existence of a functional interplay between alpha(2)-adrenoceptor and opioid receptor inhibitory pathways modulating neurotransmitter release has been demonstrated in the enteric nervous system by development of sensitivity changes to alpha(2)-adrenoceptor, mu- and kappa-opioid receptor agents on enteric cholinergic neurons after chronic sympathetic denervation. In the present study, to further examine this hypothesis we evaluated whether manipulation of alpha(2)-adrenoceptor pathways by chronic treatment with the antidepressant drug, desipramine (10 mg/kg i.p. daily, for 21 days), could entail changes in enteric mu- and kappa-opioid receptor pathways in the myenteric plexus of the guinea pig distal colon. In this region, subsensitivity to the inhibitory effect of both UK14,304 and U69,593, respectively alpha(2A)-adrenoceptor and kappa-opioid receptor agonist, on the peristaltic reflex developed after chronic desipramine treatment. On opposite, in these experimental conditions, supersensitivity developed to the inhibitory effect of [D-Ala, N-Me-Phe4-Gly-ol5]-enkephalin (DAMGO), mu-opioid receptor agonist, on propulsion velocity. Immunoreactive expression levels of alpha(2A)-adrenoceptors, mu- and kappa-opioid receptors significantly decreased in the myenteric plexus of the guinea pig colon after chronic desipramine treatment. In these experimental conditions, mRNA levels of alpha(2A)-adrenoceptors, mu- and kappa-opioid receptors significantly increased, excluding a direct involvement of transcription mechanisms in the regulation of receptor expression. Levels of G protein-coupled receptor kinase 2/3 and of inhibitory G(i/o) proteins were significantly reduced in the myenteric plexus after chronic treatment with desipramine. Such changes might represent possible molecular mechanisms involved in the development of subsensitivity to UK14,304 and U69,593 on the efficiency of peristalsis. Alternative molecular mechanisms, including a higher efficiency in the coupling between receptor activation and downstream intracellular effector systems, possibly independent from inhibitory G(i/o) proteins, may be accounted for the development of supersensitivity to DAMGO. Increased sensitivity to the mu-opioid agonist might compensate for the development of alpha(2A)-adrenoceptor and kappa-opioid receptor subsensitivity. On the whole, the present data further strengthen the concept that, manipulation of alpha(2)-adrenergic inhibitory receptor pathways in the enteric nervous system entails changes in opioid inhibitory receptor pathways, which might be involved in maintaining homeostasis as suggested for mu-opioid, but not for kappa-opioid receptors.

The actions of purine and pyrimidine compounds on isolated segments of the mouse intestine were investigated during postnatal development. The localization of P2Y(1), P2Y(2), P2Y(4), P2X(1,) P2X(2) and P2X(3) receptors were examined immunohistochemically, and levels of expression of P2Y(1), P2X(1) and P2X(2) were studied by Western immunoblot. From day 12 onwards, the order of potency for relaxation of longitudinal muscle of all regions was 2-MeSADPor=alpha,beta-meATPor=ATP=UTP=adenosine, suggesting P2Y(1) receptors. This was supported by the sensitivity of responses to 2-MeSADP to the selective antagonist MRS 2179 and P2Y(1) receptor immunoreactivity on longitudinal muscle and a subpopulation of myenteric neurons. A further alpha,beta-meATP-sensitive P2Y receptor subtype was also indicated. ATP and UTP were equipotent suggesting a P2Y(2) and/or P2Y(4) receptor. Adenosine relaxed the longitudinal muscle in all regions via P1 receptors. The efficacy of all agonists to induce relaxation of raised tone preparations increased with age, being comparable to adult by day 20, the weaning age. During postnatal development the contractile response of the ileum and colon was via P2Y(1) receptors, while the relaxant response mediated by P2Y(1) receptors gradually appeared along the mouse gastrointestinal tract, being detectable in the stomach from day 3 and in the duodenum from day 6. In the ileum and colon relaxant responses to 2-MeSADP were not detected until days 8 and 12, respectively. 2-MeSADP induced contractions on basal tone preparations from day 3, but decreased significantly at day 12 and disappeared by day 20. At day 8, contractions of colonic longitudinal muscle to ATP showed no desensitisation suggesting the involvement of P2X(2) receptors. Immunoreactivity to P2X(2) receptors only was observed on the longitudinal muscle of the colon and ileum from day 1 and on a subpopulation of myenteric neurons from day 3. These data suggest that P2Y(1) receptors undergo postnatal developmental changes in the mouse gut, with a shift from contraction to relaxation. Such changes occur 1 week before weaning and may contribute to the changes that take place in the gut when the food composition changes from maternal milk to solid food.

Interferon (IFN)-gamma plays a pivotal role in the pathogenesis of multiple sclerosis (MS), while IFN-beta may be able to modify the clinical course of the disease, eventually also by counterbalancing IFN-gamma-mediated effects. Catecholamines (CA) exert important effects on the immune response, both as transmitters between the nervous and the immune system, as well as autocrine/paracrine mediators in immune cells, and several lines of evidence support their involvement in MS. In particular, dysregulated production of CA seems to occur in peripheral blood mononuclear cells (PBMCs) of MS patients. We assessed the effects of IFN-beta and IFN-gamma on endogenous CA in PBMCs. In cultured PBMCs stimulated with phytohaemagglutinin (PHA), IFN-beta increased CA production and induced CA release in the culture medium, while IFN-gamma decreased both CA production and the expression of mRNA for the CA-synthesizing enzyme tyrosine hydroxylase. Coincubation with both IFNs prevented the inhibitory effect of IFN-gamma, as well as the stimulatory effect of IFN-beta. IFNs are the first physiological compounds shown to affect endogenous CA in PBMCs: in view of the role of CA-dependent mechanisms in the immune response, these findings may help to better understand the mechanisms of action of IFN-beta as an immunomodulatory drug in MS.

Dopamine (DA) modulates apoptosis in neuronal and non-neuronal cells, and dopaminergic pathways contribute to neurodegenerative disease. Human lymphocytes express dopaminergic receptors and DA transporters, and synthesize endogenous catecholamines, which may modulate apoptosis in these cells. In the present study, dopaminergic modulation of apoptosis was investigated in human peripheral blood mononuclear cells (PBMCs) obtained from healthy donors. Twenty-four-hour DA reduced at 0.1-5 x 3 10(-6) M and enhanced at 1-5 x 310(-4) M spontaneous apoptosis. DA 1 x 310(-6) M was inhibited by the D1-like receptor antagonist SCH 23390 1 x 310(-6) M, but not by the D2-like receptor antagonists domperidone 1 x 3 10(-6) M or haloperidol 1 x 3 10(-6) M, while the effect of DA 5 x 3 10(-4) M was prevented by the antioxidants glutathione 5-10 mM or N-acetyl-l-cysteine 1-10 mM. Intracellular reactive oxygen species were respectively reduced and increased by 1-3 h incubation with DA 0.1-10 x 3 10(-6) M and 1-5x310(-4) M. Twenty-four-hour DA 1 x 3 10(-6) M or 5 x 3 10(-4) M had no effect on PBMC expression of Cu/Zn superoxide dismutase or Bcl-2; however, DA 5 x 3 10(-4) M decreased caspase-3 activity. In human PBMCs, DA seems to promote apoptosis through oxidative mechanisms but may also result in cell rescue from apoptotic death possibly through activation of D1-like receptors. The dual effect of DA on human PBMCs closely resembles that on striatal neurons. Lymphocytes of patients with Parkinson's disease may show reduced DA content and impaired DA transporter immunoreactivity. Human PBMCs may thus represent a simple and readily accessible model to study DA-related mechanisms relevant for neurodegenerative disease.

The 5-HT1B/D receptor agonist sumatriptan has been proposed to treat dyspeptic symptoms, because it facilitates gastric accommodation. It is unknown whether stimulation of 5-HT1B/D receptors is involved. Thus, in four conscious dogs, we compared the effects of sumatriptan alone or combined with N-[4-methoxy-3-(4-methyl-1-piperazinyl) phenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)-[1,1-biphenyl]-4-carboxamide hydrocloride (GR-127935), N-[3-[3 (dimethylamino)-ethoxy]-4-methoxyphenyl]-2′-[methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)]-[1,1-biphenyl]-4-carboxamide hydrocloride (SB-216641 hydrochloride), or 3-[4-(4-chloro-phenyl)piperazin-1-yl]-1,1-diphenyl-2-propanol hydrochloride (BRL-15572 hydrochloride) (respectively, nonselective 5-HT1B/D, selective 5-HT1B, and selective 5-HT1D receptor antagonists) on gastric accommodation to isobaric distensions performed with a barostat. An exponential and a linear model were used to fit the pressure-volume relationship. An exponential equation fitted the data better than a linear equation. Sumatriptan (800 nmol/kg iv) induced an immediate gastric relaxation (Δvolume: 112 ± 44 ml, P < 0.05). After sumatriptan, the pressure-volume curve was shifted toward significantly higher volumes. This effect was fully reversed by GR-127935 or SB-216641 but not by BRL-15572. In conclusion, 5-HT1B receptors seem to play an important role in modulating gastric accommodation to a distending stimulus. An exponential model for pressure-volume curves fits well with the concept of gastric adaptive relaxation.

Chronic sympathetic denervation entails subsensitivity to alpha(2)-adrenoceptor agonists and supersensitivity to kappa- and mu-opioid receptor agonists modulating cholinergic neurons in the guinea pig colon. A possible role for signal transduction G proteins in contributing to development of these sensitivity changes was investigated. Pertussis toxin (PTX), a blocker of the G(i/o)-type family of G proteins significantly reduced the inhibitory effects of UK14,304 (alpha(2)-adrenoceptor agonist), U69593 (kappa-opioid receptor agonist) and DAMGO (mu-opioid receptor agonist) on acetylcholine (ACh) overflow in preparations obtained from normal animals, but not in those obtained from sympathetically denervated animals. In this experimental condition, immunoblot analysis revealed reduced levels of G(alphao), G(alphai2), G(alphai3) and G(beta) in myenteric plexus synaptosomes. On reverse, synaptosomal levels of G(alphai1) and G(alphaz), a PTX-insensitive G-protein, increased after chronic ablation of the sympathetic pathways. These data suggest that changes in the function and expression of inhibitory G proteins coupled to alpha(2)-adrenoceptors, kappa- and mu-opioid receptors occur in the myenteric plexus of the guinea pig colon after chronic sympathetic denervation. The possibility that regulation of G proteins represents one of the biochemical mechanisms at the basis of the changes in sensitivity of enteric cholinergic neurons to alpha(2)-adrenoceptor, kappa- and mu-opioid receptor agonists is discussed.

Mechanisms underlying the gastric toxicity of nonsteroidal anti-inflammatory drugs (NSAIDs) have been extensively investigated, whereas those leading to intestinal damage are not completely understood. Several hypotheses have been put forward on the pathophysiology of intestinal damage by NSAIDs: enhanced intestinal permeability, inhibition of cyclooxygenase (COX), enterohepatic recirculation, and formation of adducts. The effects of COX-2 selective inhibitors, which appear to have better gastric tolerability when compared to nonselective NSAIDs, on normal and inflamed intestinal mucosa (as in Crohn's disease or ulcerative colitis) are still largely unexplored. If COX-2 inhibition plays a key role in suppressing the inflammatory process, recent evidence suggests that COX-2 products are involved in maintaining the integrity of intestinal mucosa, in the healing of gastrointestinal ulcers and in the modulation of inflammatory bowel disease (IBD). Animal models of intestinal inflammation have so far yielded conflicting results on the effects of COX-2 selective inhibitors on the intestinal mucosa. It is now clear that NSAIDs do not act through cyclooxygenase inhibition, but also have different targets such as nuclear factor-kappaB and/or peroxisome proliferator-activated receptors gamma. The peculiar pharmacological profile of each compound may help to explain the different impact of each NSAID on the inflammatory process and on IBD. Notably, the salicylic acid derivative 5-ASA is widely used in the treatment of IBD and is believed to act through nuclear factor-kappaB inhibition. Although the use of COX-2 selective inhibitors remains contraindicated in patients with IBD, studying their effects on intestinal mucosa may offer new insights into their subcellulars mechanisms of action and open new avenues for the development of novel therapies for IBD.

Acetylcholine and tachykinins act as co-transmitters along excitatory pathways at different gut levels. Since cholinergic pathways are involved in maintaining gastric tone during fasting, our aim was to study the possible role of tachykininergic pathways in modulating canine gastric tone and compliance in vivo by using selective tachykinin receptor antagonists. In four fasting, conscious dogs, we characterized the pressure-volume relationship in the proximal stomach by using a barostat. We increased the pressure of the intragastric bag by 2 mmHg increments every 3 min, starting from a baseline value of 2 up to 12 mmHg. Drug effects were investigated by studying pressure-volume relationships before and 15 min after intravenous (i.v.) administration of SR140333, SR48968, or SR142801 (respectively, NK (1)-, NK (2)-, and NK (3)-receptor antagonist, each at the dose of 1 mg kg (-1)) or atropine (100 microg kg (-1)). Pressure-volume curves were fitted by nonlinear regression analysis. Before drug administration, the curve that best fitted the pressure-volume relationship was exponential. SR140333, SR48968 and SR142801 did not affect baseline gastric tone or the gastric pressure-volume curve at any distension level. At a distending pressure of 6 mmHg, the Delta volumes obtained after administration of SR140333, SR48968 or SR142801 vs control were 65 +/- 28, 27 +/- 26, 14 +/- 20 ml, respectively. The same was true even when all three antagonists were administered together to achieve simultaneous blockade of all three tachykinin receptor subtypes. Atropine increased baseline gastric volume (Delta volume = 237 +/- 15 ml; P0.01) and significantly (P0.0001) shifted the pressure-volume curve to the left. After atropine, a linear equation best fitted the pressure-volume curve. We conclude that tachykininergic pathways are not involved in modulating canine gastric tone and compliance during fasting, whereas cholinergic pathways play a major role not only in maintaining gastric tone, but also in modulating the compliance of the proximal stomach to a distending stimulus.

Alterations of the enteric glutamatergic transmission may underlay changes in the function of myenteric neurons following intestinal ischemia and reperfusion (I/R) contributing to impairment of gastrointestinal motility occurring in these pathological conditions. The aim of the present study was to evaluate whether glutamate receptors of the NMDA and AMPA/kainate type are involved in myenteric neuron cell damage induced by I/R. Primary cultured rat myenteric ganglia were exposed to sodium azide and glucose deprivation (in vitro chemical ischemia). After 6 days of culture, immunoreactivity for NMDA, AMPA and kainate receptors subunits, GluN(1) and GluA(1-3), GluK(1-3) respectively, was found in myenteric neurons. In myenteric cultured ganglia, in normal metabolic conditions, -AP5, an NMDA antagonist, decreased myenteric neuron number and viability, determined by calcein AM/ethidium homodimer-1 assay, and increased reactive oxygen species (ROS) levels, measured with hydroxyphenyl fluorescein. CNQX, an AMPA/kainate antagonist exerted an opposite action on the same parameters. The total number and viability of myenteric neurons significantly decreased after I/R. In these conditions, the number of neurons staining for GluN1 and GluA(1-3) subunits remained unchanged, while, the number of GluK(1-3)-immunopositive neurons increased. After I/R, -AP5 and CNQX, concentration-dependently increased myenteric neuron number and significantly increased the number of living neurons. Both -AP5 and CNQX (100-500 µM) decreased I/R-induced increase of ROS levels in myenteric ganglia. On the whole, the present data provide evidence that, under normal metabolic conditions, the enteric glutamatergic system exerts a dualistic effect on cultured myenteric ganglia, either by improving or reducing neuron survival via NMDA or AMPA/kainate receptor activation, respectively. However, blockade of both receptor pathways may exert a protective role on myenteric neurons following and I/R damage. The neuroprotective effect may depend, at least in part, on the ability of both receptors to increase intraneuronal ROS production.

A rapid and simple HPLC-ED method is described to identify and measure catecholamines (CTs) and their major metabolites in immune cells. Using this method, intracellular CTs were quantified in human peripheral blood mononuclear cells (PBMCs), T and B lymphocytes, monocytes and granulocytes. Immune cell subsets were separated by density gradient centrifugation and immunomagnetic cell sorting. CTs were also found in the human hematopoietic cell lines NALM-6 (pre-B) and (in smaller amounts) in Jurkat (T lymphoblastoid) and U937 (promonocytic). In cultured PBMCs, intracellular CTs were reduced by both the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine and the chromaffin granule depletant reserpine. In NALM-6 cells, both alpha-methyl-p-tyrosine and the dopamine-beta-hydroxylase inhibitor disulfiram reduced intracellular CTs, supporting the presence of active synthetic pathways in these cells. Since sympathoadrenergic mechanisms play a key role in the interactions between the immune system and the nervous system, these findings may be relevant for a better understanding of the neuro-immune network.

To assess the role of AMPA and kainate receptors in modulating neurotransmitter release from the myenteric plexus, the effect of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainic acid on endogenous acetylcholine (ACh) and noradrenaline (NA) overflow from the guinea-pig isolated colon was studied. AMPA inhibited spontaneous ACh overflow and increased electrically-evoked NA overflow. Kainic acid did not influence both ACh and NA overflow. AMPA-mediated effects on ACh and NA overflow were significantly reduced by the AMPA/kainate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, CNQX. The inhibitory effect of AMPA on ACh overflow could be due, at least in part, to the AMPA-induced NA overflow as it was greatly reduced after adrenoceptor blockade and virtually abolished in sympathetically-denervated animals. The possible functional significance of these findings was studied by measuring the efficiency of the peristaltic reflex in the presence of the different agonists. The efficiency of peristalsis was enhanced by AMPA, whereas it was not modified by kainic acid. In conclusion, AMPA receptors, but not kainate receptors, may play a role in the modulation of ACh and NA release and of peristalsis in the guinea-pig colon.

Enteric ganglia can maintain integrated functions, such as the peristaltic reflex, in the absence of input from the central nervous system, which has a modulatory role. Several clinical and experimental observations suggest that homeostatic control of gut function in a changing environment may be achieved through adaptive changes occurring in the enteric ganglia. A distinctive feature of enteric ganglia, which may be crucial during the development of adaptive responses, is the vicinity of the final effector cells, which are an important source of mediators regulating cell growth. The aim of this review is to focus on the possible mechanisms underlying neuronal plasticity in the enteric nervous system and to consider approaches to the study of plasticity in this model. These include investigations of neuronal connectivity during development, adaptive mechanisms that maintain function after suppression of a specific neural input, and the possible occurrence of activity-dependent modifications of synaptic efficacy, which are thought to be important in storage of information in the brain. One of the applied aspects of the study of plasticity in the enteric nervous system is that knowledge of the underlying mechanisms may eventually enable us to develop strategies to correct neuronal alterations described in several diseases. GASTROENTEROLOGY 1999;117:1438-1458

In the guinea-pig colon, acetylcholine (ACh) release from intrinsic cholinergic motor neurons is inhibited by adrenoceptors, opioid and muscarinic receptors. Chronic sympathetic denervation resulted in supersensitivity to the inhibitory effect of DAMGO (μ-opioid agonist) on ACh release and on the peristaltic reflex. After chronic treatment with naltrexone (NTX) supersensitivity to DAMGO and subsensitivity to UK14,304 (α2-adrenoceptor agonist) developed for both functional parameters. The facilitatory effect of scopolamine on ACh release remained unchanged after chronic NTX treatment, whereas it was potentiated after chronic sympathetic denervation. These data suggest the existence of a functional interaction between different inhibitory pathways modulating cholinergic motor neurons in the guinea-pig coloN. Namely, chronic manipulation of an inhibitory pathway may entail adaptive sensitivity changes in another inhibitory pathway so that homeostasis can be maintained.

We have studied the effect of [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO, opioid mu-receptor agonist) and ICI-204,448 (kappa-receptor agonist) on endogenous noradrenaline release in the guinea-pig isolated distal colon. DAMGO enhances noradrenaline over-flow and this effect is antagonized by naloxone (pIC50 = 10.27) and nor-binaltorphimine (pIC50 = 7.97), and concentration-dependently turned into inhibition by yohimbine. ICI-204,448 inhibits noradrenaline overflow and is antagonized by naloxone (pIC50 = 9.38) and nor-binaltorphimine (pIC50 = 10.48), but is not affected by yohimbine. Evidence is thus given that mu- and kappa-opioid receptors modulate noradrenaline release in the guinea-pig colon. Modifications by yohimbine of the effect of DAMGO indicate the existence of a functional relationship between mu-receptors and alpha(2)-autoreceptors in this model.

Drug prescribing habits and adverse drug reaction occurrence were investigated in a psychiatric hospital ward for chronic resident patients, and the usefulness of a drug prescription and adverse event (AE) routine monitoring programme in this setting was assessed. A computerized data base was established and updated bimonthly with clinical data concerning each patient. In particular, information about drug prescriptions was routinely obtained from the clinical records, stored in the data base and periodically retrieved and analysed. The ward doctors were required to report on a form the AEs, which were subsequently examined by a panel of clinical pharmacologists and hospital consultants, to assess their relationship with drug treatments. Moreover, the data base was periodically scrutinized, to search for further AEs. Drug prescription monitoring, besides providing valuable information about drug utilization, allowed identification of some questionable issues (e.g. polypharmacy, low frequency of treatment revisions). The AE monitoring system proved to be effective and the participation of the ward doctors was satisfactory. It is concluded that in this psychiatric hospital setting a drug prescription and AE routine monitoring programme can provide useful information and promote improvement of the quality of care.

In the guinea pig colon, chronic sympathetic denervation entails supersensitivity to inhibitory mu-opioid agents modulating cholinergic neurons. The mechanism underlying such adaptive change has not yet been unravelled, although protein kinase C (PKC) may be involved. A previous study indirectly demonstrated that activation of mu-opioid receptors on myenteric neurons facilitates PKC activity. Such coupling may counteract the inhibitory action of mu-opioid agents on acetylcholine overflow, since PKC, per se, increases this parameter. After chronic sympathetic denervation such restraint abates, representing a possible mechanism for development of supersensitivity to mu-opioid agents. In the present study, this hypothesis was further investigated. After chronic sympathetic denervation, Ca(2+)-dependent PKC activity was reduced in colonic myenteric plexus synaptosomes. The mu-opioid agent, DAMGO, increased Ca(2+)-dependent PKC activity in synaptosomes obtained from normal, but not from denervated animals. In myenteric synaptosomes obtained from this experimental group, protein levels of Ca(2+)-dependent PKC isoforms betaI, betaII and gamma decreased, whereas alpha levels increased. In whole-mount preparations, the four Ca(2+)-dependent PKC isoforms co-localized with mu-opioid receptors on subpopulations of colonic myenteric neurons. The percentage of neurons staining for PKCbetaII, as well as the number of mu-opioid receptor-positive neurons staining for PKCbetaII, decreased in denervated preparations. The same parameters related to PKCalpha, betaI or gamma remained unchanged. Overall, the present data strengthen the concept that mu-opioid receptors located on myenteric neurons are coupled to Ca(2+)-dependent PKCs. After chronic sympathetic denervation, a reduced efficiency of this coupling may predominantly involve PKCbetaII, although also PKCbetaI and gamma, but not PKCalpha, may be implicated.

The existence of a close relation between presynaptic inhibitory alpha(2)-adrenoceptor and mu-opioid receptor pathways is well established. Such interplay may occur during chronic conditions that give rise to neuroadaptive changes involving both receptor systems. The aim of this study was to examine the effect of chronic treatment with the tricyclic antidepressant drug, desipramine, on alpha(2)-adrenoceptors and mu-opioid receptors in the guinea pig brain. Guinea pigs were treated with 10 mg/kg desipramine, injected i.p. for 21 days, every 24 h. The levels of expression of alpha(2)-adrenoceptors and mu-opioid receptors, the G protein receptor regulatory kinase, GRK2/3 and signal transduction inhibitory G proteins in synaptosomes of the guinea pig hippocampus and cortex were evaluated by immunoblotting. Quantitative analysis of alpha(2)-adrenoceptor and mu-opioid receptor mRNA levels has been carried out by competitive reverse transcriptase polymerase chain reaction. The expression levels of alpha(2)-adrenoceptors and mu-opioid receptors and the respective mRNAs were found unchanged in the cortex, after chronic desipramine treatment. In these experimental conditions alpha(2)-adrenoceptor and mu-opioid receptor levels decreased, while the relevant transcripts increased, in the hippocampus. GRK2/3 levels remained unchanged and increased, respectively, in the cortex and the hippocampus, after chronic exposure to desipramine. In the same experimental conditions, Galpha(i1), Galpha(i2), Galpha(o) and Galpha(z) levels remained unchanged, while Galpha(i3) levels decreased, in the cortex; whereas, Galpha(i1), Galpha(i2) and Galpha(i3) levels significantly increased, and Galpha(o) and Galpha(z) levels remained unchanged, in the hippocampus. On the whole, the present data suggest that alpha(2)-adrenoceptor and mu-opioid receptor expression and transcription are similarly influenced by chronic treatment with desipramine, in the guinea pig cortex and hippocampus. Furthermore, alterations in the levels of regulatory GRK2/3 and of inhibitory signal transduction G proteins, relevant to activation of both receptor pathways, have been documented. The distinct pattern of adaptations of the different protein studied in response to chronic desipramine treatment in both regions is discussed.

Muscarinic pathways are involved in maintaining gastric tone during fasting and atropine is known to decrease gastric tone via blockade of a tonic vagal cholinergic input. Our aim was to assess the role of different muscarinic receptors in modulating canine gastric tone and compliance in vivo by using "selective" muscarinic receptor antagonists (telenzepine, AF-DX 116 and 4-DAMP for M1, M2, and M3 receptors, respectively) and the non-selective muscarinic receptor antagonist atropine. In four fasting, conscious dogs, we characterized the pressure-volume relationship in the proximal stomach by using a barostat. Drug effects were investigated by studying pressure-volume relationships before and 15 min after intravenous administration telenzepine, AF-DX 116, 4-DAMP or atropine. Pressure-volume curves were fitted by non-linear regression analysis. Before drug administration, the curve that best fitted the pressure-volume relationship was exponential. Atropine (100 microg kg-1) immediately decreased baseline gastric tone, i.e. relaxed the stomach (Deltavolume at 2 mmHg=236+/-15 ml; P0.05), and significantly (P0.01) shifted the pressure-volume curve to the left. Telenzepine, at the lowest dose (3 microg kg-1), shifted the pressure-volume curve to the right (P0.01). AF-DX 116 at the lower dose (422 microg kg-1) had no effect on baseline gastric tone or the gastric pressure-volume curve, whereas the higher dose (2532 microg kg-1) significantly shifted the pressure-volume curve to the left (P0.01), but did not increase baseline gastric volume. Finally, 4-DAMP (13.5, 45, 135 microg kg-1) immediately decreased baseline gastric tone (Deltavolume at 2 mmHg=97+/-29 ml, 110+/-35 ml and 155+/-21 ml, respectively) and significantly shifted the pressure-volume curve to the left (P0.01). We conclude that muscarinic pathways are involved in modulating canine gastric tone and compliance during fasting: M3 receptors seem to play a key role in excitatory pathways, whereas the shift of pressure-volume curve to the right observed with the lowest dose of telenzepine is consistent with the existence of M1 receptors on inhibitory pathways.

Activation of human peripheral blood mononuclear cells (PBMC) triggers endogenous production of catecholamines (CA) through protein kinase (PK) C-dependent induction of tyrosine hydroxylase (TH; EC 1.14.16.2), the first and rate-limiting enzyme in the synthesis of CA. Since CA themselves are major mediators of the neural input to the immune system, we have examined their ability to affect PKC-induced TH mRNA expression and CA production in human isolated PBMC. In T- and B-lymphocytes (but not in monocytes) the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) (but not its inactive analogue 4alpha-phorbol-12,13-didecanoate) induced TH mRNA expression which was followed by an increase in the amount of intracellular CA. Coincubation of human PBMC with dopamine (DA) (but not with norepinephrine or epinephrine) inhibited TPA-induced TH mRNA expression. The effect of DA was concentration-dependent and was mimicked by the dopaminergic D1-like receptor agonist SKF-38393 but not by the D2-like receptor agonist bromocriptine. The D1-like antagonist SCH-23390 shifted to the right the concentration-response curves of both DA and SKF-38393, while neither the D2-like antagonist domperidone, nor the alpha1-adrenoceptor antagonist prazosin, the alpha2-adrenoceptor antagonist yohimbine, or the beta-adrenoceptor antagonist propranolol affected to any significant extent the inhibitory effect of DA. SKF-38393 also significantly reduced TPA-induced increase of intracellular CA, an effect which was antagonized by SCH-23390. It is thus suggested that in human T- and B-lymphocytes PKC activation leads to TH mRNA expression and subsequent increase of intracellular CA, which can be inhibited by D1-like receptor activation. Inhibition of intracellular CA production in human PBMC promotes cell survival through reduction of activation-induced apoptosis, and dopaminergic modulation of TH expression and intracellular CA content may thus represent a novel mechanism in the cross-talk between the nervous and the immune system as well as among immune system cells.

Dopamine (DA) is a neurotransmitter in the central and peripheral nervous system, which can be either cytotoxic or cytoprotective under selected conditions. Such effects involve oxidative mechanisms and are likely to play a role in neurodegenerative disorders. Because increasing evidence points to peripheral blood lymphocytes (PBL) as a feasible model for studying DA-related mechanisms of cell death and survival, we have explored in these cells the effects of DA on oxidative metabolism and apoptosis. Our results show that, whereas DA 100-500 microM resulted in increased intracellular reactive oxygen species (ROS) levels and apoptotic cell death through oxidative stress, DA 0.1-5 microM decreased ROS levels and apoptosis. DA (both 1 and 500 microM) partially counteracted the decrease in Cu/Zn superoxide dismutase levels observed in untreated PBL. However, whereas the effect of the low dose lasted for the whole incubation period (24 h), the effect of DA 500 microM was transient. DA-dependent reduction of ROS levels and apoptosis was prevented by D1-like (but not D2-like) receptor antagonism. The present findings add knowledge about the sensitivity of PBL to DA and strengthen the rationale for exploiting these cells as an easily accessible peripheral model for the ex vivo investigation of oxidative stress-related dopaminergic mechanisms underlying human neurodegenerative diseases.

Supersensitivity to muscarinic, kappa- and mu-opioid agents modulating cholinergic neurons in the guinea pig colon develops after chronic sympathetic denervation. A possible role for protein kinase C (PKC) in contributing to development of these sensitivity changes was investigated. The PKC activator, phorbol-12-myristate-13-acetate (PMA), enhanced acetylcholine (ACh) overflow in preparations obtained from normal animals. The facilitatory effect of PMA was significantly reduced after prolonged exposure to the phorbol ester and by the PKC inhibitors, chelerythrine and calphostin C. Subsensitivity to the facilitatory effect of PMA developed after chronic sympathetic denervation. In this experimental condition, immunoblot analysis revealed reduced levels of PKC in myenteric plexus synaptosomes. The facilitatory effect of the muscarininc antagonist, scopolamine, on ACh overflow was significantly reduced by the phospolipase C (PLC) inhibitor, U73122, chelerythrine and calphostin C, both in normal and denervated animals. However, in both experimental groups, PLC antagonists and PKC antagonists did not affect the inhibitory effect of the muscarinic agonist, oxotremorine-M on ACh overflow. The inhibitory effects of U69593 (kappa-opioid receptor agonist) and DAMGO (mu-opioid receptor agonist) on ACh overflow significantly increased in the presence of U73122, chelerythrine and calphostin C in preparations obtained from normal animals, but not in those obtained from sympathetically denervated animals. These results indicate that activation of PKC enhances ACh release in the myenteric plexus of the guinea pig colon. At this level, chronic sympathetic denervation entails a reduced efficiency of the enzyme. In addition, PKC is involved in the inhibitory modulation of ACh release mediated by muscarinic-, kappa- and mu-opioid receptors, although with different modalities. Muscarinic receptors inhibit PKC activity, whereas kappa- and mu-opioid receptors increase PKC activity. Both the inhibitory and the facilitatory effect on PKC involve modulation of PLC activity. The possibility that the change in PKC activity represents one of the biochemical mechanisms at the basis of development of sensitivity changes to opioid and muscarinic agents after chronic sympathetic denervation is discussed.

In their recent Review, Gordon and Barnes [1] discuss the possible role and functional relevance of monoamine transporters in lymphocytes. They first present available evidence concerning the occurrence of serotonin and dopamine transporters in lymphocytes, subsequently provide an exhaustive analysis of the effects of monoamine transporter ligands on the immune response, and conclude by asking whether the observed effects could be mediated at least in part through direct targeting of lymphocyte transporters. To answer the question of Gordon and Barnes, a direct link should be traced between the effect of monoamine transporter ligands on monoamine trafficking across the lymphocyte plasma membrane and the regulation of lymphocyte function. At present, however, possibly with the notable exception of our own studies on serotonininduced modulation of apoptosis in lymphoma cells [2], evidence is unfortunately still indirect and circumstantial. At least in the case of catecholamines (CAs), thepaucityof data could be explained by the complexity of their physiopharmacology in lymphocytes. Indeed, although dopamine, norepinephrine and epinephrine can be detected inhumanperipheralbloodmononuclearcells (PBMCs),even in the absence of activating stimuli [3], it is only after mitogen stimulation that cultured PBMCs express the mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in CA synthesis, and subsequently increase the intracellular content of CA through protein kinase C (PKC)dependent mechanisms, up to 20–40-fold the basal values [4]. This finding is in line with the reported upregulation of adrenoceptors [5,6] which occurs in lymphocytes following mitogen, glucocorticoid or proinflammatory cytokine treatment, and which is taken as indirect evidence that CAs are preferentially involved in the functional regulation of activated rather than resting lymphocytes. This idea is strengthened by the observation that pharmacological inhibition of TH does not affect spontaneous apoptosis in unstimulated PBMCs, but leads to a significant reduction of the percentage of apoptotic cells following mitogen stimulation [7], suggesting a role for endogenous CA in the regulation of activation-induced cell death. CAs newly synthesized on activation are not immediately released, but rather accumulate into the cells, possibly resulting in receptor-independent, oxidative stress-induced apoptosis (by analogy, e.g. with dopaminergic neurons [8]). Intracellular CAs accumulating in activated lymphocytes are nevertheless also likely to provide these cells with a supply of mediators to be released on appropriate stimulation. In preliminary experiments, we observed that elevated extracellular Kþ concentrations ([K]e) leads to dramatic CA release from mitogen-activated lymphocytes (R. Bombelli et al., unpublished), thus increasing extracellular CA from pico–nanomolar up to submicromolar concentrations (and possibly to even higher values in the vicinity of the cells). Elevated [K]e is characteristic of various pathological conditions and is per se a sufficient stimulus to activate integrin-mediated adhesion and migration of T cells (reviewed in Ref. [9]). An excess [K]e could thusbothassist therecruitmentof lymphocytes toaninjured tissue and lead to local increase of CAs, which in turn might act on lymphocytes themselvesand/oronneighbouring cells. Available evidence allows us to hypothesize that CAs in lymphocytes subserve several functions, thus representing an attractive target for pharmacological intervention. Much study is, however, needed to unravel the physiopharmacology of CA production, storage and release in these cells. In particular, the effects of drugs acting on monoamine transporters should be examined taking into account that, on activation, lymphocytes upregulate their endogenous CA system, including CA production, intracellular storage and CA receptor expression. In addition, distinct lymphocyte subsets might produce and/or use CAs to a different extent. The expression and activity of CA transporters themselves should therefore be investigated in the various subsets, before and after stimulation. Figure 1 shows a hypothetical scheme of the endogenous CA system in lymphocytes, highlighting various issues, which await clarification. Nonetheless, as awareness increases that immune and nervous system cells use the same (or similar) molecules and mechanisms, we cannot but join the proposal of Gordon and Barnes [1] that the discipline of neuroimmunopharmacotherapy is really in its dawn. As a minor point, we would like to mention that even if a literature search for the occurrence of norepinephrine transporters in lymphocytes was unsuccessful [1], at least indirect evidence is available in this regard. More than two decades ago, Audus and Gordon [10] described in murine lymphocytes a single population of desipramine-binding sites with an apparent dissociation constant (Kd) of ,0.4 nM.Morerecently,weprovidedexperimentalevidence that incubation of human PBMCs with desipramine or with the dopamine transporter ligand GBR12909 increased the extracellular levels of both dopamine and norepinephrine [3], an observation that is compatible with the occurrence of both transporters on the human lymphocyte membrane. Corresponding author: Marco Cosentino (marco.cosentino@uninsubria.it). Update TRENDS in Immunology Vol.24 No.11 November 2003 581

Several reports suggest that enteric cholinergic neurons are subject to a tonic inhibitory modulation, whereas few studies are available concerning the role of facilitatory pathways. Glutamate, the main excitatory neurotransmitter in the central nervous system (CNS), has recently been described as an excitatory neurotransmitter also in the guinea-pig enteric nervous system (ENS). The present study aimed at investigating the presence of glutamatergic neurons in the ENS of the human colon. At this level, the presence of ionotropic glutamate receptors of the NMDA type, and their possible interaction with the enteric cholinergic function was also studied. In the human colon, l -glutamate and NMDA concentration dependently enhance spontaneous endogenous acetylcholine overflow in Mg 2+ -free buffer, both effects being significantly reduced by the antagonists, (±)-2-amino-5-phosphonopentanoic acid (±AP5) and 5,7-diCl-kynurenic acid. In the presence of Mg 2+ , the facilitatory effect of l -glutamate changes to inhibition, while the effect of NMDA is significantly reduced. In addition, morphological investigations reveal that glutamate- and NR1-immunoreactivities are present in enteric cholinergic neurons and glial cells in both myenteric and submucosal plexus. These findings suggest that, as described for the guinea-pig ileum, glutamatergic neurons are present in enteric plexuses of the human colon. Modulation of the cholinergic function can be accomplished through NMDA receptors.

Sympathoadrenergic mechanisms may play a role in multiple sclerosis (MS). We examined catecholamine (CA) levels and production and tyrosine hydroxylase (TH) expression in peripheral blood mononuclear cells (PBMCs) from MS patients, and the correlation between CA production and apoptosis in PBMCs. PBMCs from MS patients had increased norepinephrine (NE) levels. However, phytohaemagglutinin (PHA)-stimulated PBMCs from MS patients with active disease synthesized less dopamine (DA) than cells from both healthy controls and patients with inactive disease. PBMCs from patients with inactive disease showed lower expression of TH. Pharmacological inhibition of TH in cultured PBMCs stimulated with PHA reduced the percentage of apoptotic cells. Since a failure of activation-induced apoptosis in immune cells may be involved in MS, it is suggested that altered CA production by PBMCs may be implicated in such dysregulation.

Using immunocytochemistry coupled to fluorescence and electron microscopy, we investigated the expression and ultrastructural localization of tyrosine hydroxylase (TH, EC 1.14.16.2), the rate-limiting enzyme in the biosynthesis of catecholamines, in human peripheral blood mononuclear cells (PBMCs), with PC12 cells as positive controls. In unstimulated PBMCs, TH-specific immunoreactivity was localized to the plasma membrane. However, after stimulation with the polyclonal mitogen phytohaemagglutinin (PHA), TH immunoreactivity was almost completely localized to electron-dense cytoplasmic granules, which resembled those found in PC12. TH-positive granules, however, were larger (300–500 nm) than in PC12 cells (100–200 nm). Flow cytometry analysis of TH expression showed about 46–50% positive cells in unstimulated PBMCs and in PHA-stimulated PBMCs in the G0/G1 phase of the cell cycle, but more than 80% positive cells in PHA-stimulated PBMCs in the S+G2/M phase. In agreement with previous observations, PHA stimulation also induced de novo expression of TH mRNA as well as increased intracellular catecholamine content, suggesting the occurrence of TH upregulation at the level of both gene expression and enzyme activity. The ultrastructural localization of TH in human PBMCs seems therefore regulated by cell stimulation and related to the functional activity of the enzyme.

Although it is now established that immunocompetent cells produce catecholamines (CA), which in turn may act as autocrine/paracrine mediators, little is known about the mechanisms regulating CA production in these cells. In the present study, evidence is provided that stimulation of human cultured peripheral blood mononuclear cells (PBMCs) with phytohaemagglutinin (PHA) induces the expression of tyrosine hydroxylase (TH) mRNA and subsequently increases intracellular CA levels through protein kinase C (PKC) activation and the contribution of intracellular Ca(++)-dependent mechanisms. Increased production of CA in PHA-stimulated PBMCs suggests a preferential involvement of catecholaminergic pathways in the functional modulation of activated cells. These findings may help to better define the role of immunocompetent cell-derived CA in the neuroimmune network.

Purpose Drug treatment of idiopathic Parkinson's disease (IPD) is a difficult task, and comorbidity and comedication add to its complexity. Since in IPD there is little information about drug use, this study investigated drug prescribing and indications in IPD patients. Methods From June 1997 to April 1998, a cross-sectional survey of IPD outpatients was performed and demographic and clinical data and information about drug treatments was collected and analysed. Results In the 130 IPD patients included in the study, anti-Parkinson drug (APD) prescriptions increased with disease duration and severity. Levodopa was most frequently used, followed by dopamine agonists and anticholinergic agents. Levodopa with other APDs was given to older patients with later IPD onset. Prescriptions of drugs for other indications (non-APDs) were given to 80.8% of the patients and their number increased with patient age. Non-APD prescriptions concerned mainly the circulatory system, mental disorders, the musculo-skeletal system and the digestive system. Prescriptions for indications corresponding to secondary symptoms that often complicate IPD increased with patient age and also with IPD duration. Conclusion In IPD patients, disease duration and severity and patient age seem to be major determinants of drug use. Indications for drug prescription suggest that main comorbidity includes neuropsychiatric, circulatory, musculo-skeletal and digestive disorders. Analysis of prescribing patterns in IPD can provide a readily accessible indirect indicator of patient health status for both health services and epidemiologic research purposes. Copyright © 2002 John Wiley & Sons, Ltd.

The actions of adenosine, adenosine 5'-triphosphate (ATP), 2-methylthio adenosine diphosphate ADP (2-MeSADP), 2-methylthio ATP (2-MeSATP), alpha,beta-methylene ATP (alpha,beta-meATP) and uridine triphosphate (UTP) on isolated segments of mouse stomach (fundus), duodenum, ileum and colon were investigated. The localization of P2Y(1), P2Y(2), P2Y(4), P2X(1) and P2X(2) receptors and neuronal nitric oxide synthase (NOS) were examined immunohistochemically, and P2Y(1) mRNA was examined with in situ hybridization. The order of potency for relaxation of longitudinal muscle of all regions was: 2-MeSADP>/=2-MeSATP>alpha,beta-meATP>ATP=UTP=adenosine. This is suggestive of P2Y(1)-mediated relaxation and perhaps a further P2Y receptor subtype sensitive to alpha,beta-meATP. As ATP and UTP are equipotent, the presence of a P2Y(2) receptor is indicated. ATP responses were inhibited by the P2Y(1)-selective antagonist MRS 2179, and suramin. P2Y(1) receptors were visualized immunohistochemically in the smooth muscle of the ileum and in a subpopulation for myenteric neurones, which also stained for NOS. P2Y(1) mRNA was localized in neurones in both myenteric and submucosal ganglia in the ileum. Taken together, these results suggest that ATP was acting on non-adrenergic, non-cholinergic inhibitory neurons, which release both nitric oxide (NO) and ATP. Reduced relaxations to 2-MeSADP by tetrodotoxin and N(omega)-nitro-L-arginine methyl ester, are consistent with this possibility. Adenosine acts via P1 receptors to relax smooth muscle of the mouse gut. Segments of mouse colon (in contrast to the stomach and small intestine) were contracted by nucleotides with the potency order: 2-MeSATP>alpha,betameATP>ATP; the contractions showed no desensitization and were antagonized by suramin and PPADS, consistent with responses mediated by P2X(2) receptors. Immunoreactivity to P2X(2) receptors was demonstrated on both longitudinal and circular muscle of the colon, but not in the other regions of the gut, except for a small subpopulation of myenteric neurones. In summary, neuronal P2Y(1) receptors appear to mediate relaxation, largely through NO in all regions of the mouse gut, and to a lesser extent by P2Y(1), P2Y(2) and a novel P2Y receptor subtype responsive to alpha,beta-meATP in smooth muscle, while P2X(2) receptors mediate contraction of colonic smooth muscle.

Sumatriptan is a 5-HT1B/D receptor agonist of documented efficacy in relieving migraine and associated symptoms such as nausea and vomiting. In the past decade, several studies reported an important delay of gastric emptying induced by sumatriptan in healthy humans. The impact of this gastric motor effect of sumatriptan in migraineurs is difficult to predict: a further delay in gastric emptying could be detrimental (i.e. increased nausea and epigastric symptoms) in patients already having delayed gastric emptying. However, in patients with functional dyspepsia, sumatriptan is also reported to improve gastric accommodation to a meal and reduce perception of gastric distention, hence relieving epigastric symptoms. Thus, reduced visceral perception could be a mechanism involved in reducing nausea during a migraine attack. Paradoxically, sumatriptan is reported both to relieve the nausea of a migraine attack and to have nausea as a side effect. Although careful analysis of the time of onset of nausea may offer a clue as to the origin of this symptom, available data do not support definite conclusions, all the more so because the gastric motor effect of second-generation triptans are still unexplored. Taken together, the available evidence warrants further studies to clarify the following issues: first, the mechanism responsible for the gastric motor effect of sumatriptan [receptor subtype(s) involved; central vs peripheral mechanism]; secondly, the effects on gastric motility/visceral sensitivity of second-generation triptans (which are 5-HT1B/D receptor agonists) and more recent selective 5-HT1D receptor agonists (proposed as investigational antimigraine agents with less potential to induce coronary vasoconstriction through 5-HT1B receptors); finally, the possible use of drugs improving gastric accommodation to a meal in the management of those dyspeptic patients with impaired fundic relaxation/altered visceral sensitivity.

The aim of the present functional study was to assess the role of beta3-adrenoceptors in the light of recent findings suggesting the existence of a putative fourth beta-adrenoceptor in adipose and heart tissue. The effect of the non-conventional beta3-adrenoceptor partial agonist CGP12177A is resistant to the effect of the beta3-adrenoceptor antagonist SR59230A. Under isotonic conditions in circular muscle strips of human distal colon, the concentration-effect relationship of CGP12177A and SR59104A (beta3-adrenoceptor agonists), alone and in the presence of CGP20712A (beta1-adrenoceptor antagonist) ICI118551 (beta2-adrenoceptor antagonist) and SR59230A, all 0.1 microm was studied. CGP12177A concentration-dependently relaxed circular muscle strips (pEC50=6.16+/-0.05). This effect was left unchanged by beta1-/beta2-adrenoceptor blockade, but antagonised by SR59230A (pA2=8.12+/-0.02). SR59104A concentration-dependently relaxed circular muscle strips (pEC50=5.43+/-0.01), an effect that was not significantly affected by pretreatment with CGP20712A and ICI118551, but competitively antagonised by SR59230A (p KB=7.89). Isoprenaline-induced relaxations were antagonised by propranolol with a low pA2value (7.76+/-0.16). These results provide further evidence for the presence of functional beta3-adrenoceptors in the human colon, but do not support a role for an atypical beta-adrenoceptor distinct from the beta3-subtype.

Evidence is presented that human neutrophils contain catecholamines and several of their metabolites. In vitro, incubation with alpha-methyl-p-tyrosine or pargyline affects intracellular dopamine, norepinephrine and their metabolites, suggesting catecholamine synthesis and degradation by these cells. Reserpine reduces intracellular dopamine and norepinephrine and desipramine reduces intracellular norepinephrine, suggesting the presence of storage and uptake mechanism. In view of the ability of catecholamines to affect neutrophil function, the present results support the hypothesis that autoregulatory adrenergic mechanisms may exist in these cells.

Evidence has been obtained that peripheral blood mononuclear cells contain dopamine, norepinephrine, epinephrine, and their metabolites. Pharmacologic inhibition of tyrosine hydroxylase or monoamine oxidase profoundly affected intracellular catecholamines (CTs) and their metabolites, indicating that these cells are able to synthesize and breakdown CTs. The sensitivity of intracellular CTs to reserpine and the presence of CTs in the extracellular medium suggest that CTs are stored and released. Moreover, the increase of extracellular CTs in the presence of monoamine uptake blockers point to the presence of functional uptake mechanisms. Altogether, these results indicate the existence of a CT lifecycle in human mononuclear cells and warrant further studies to investigate the role of adrenergic autoregulatory mechanisms in modulation of the immune response and in the pathogenesis of diseases involving the immune system.

Attitudes to adverse drug reaction (ADR) spontaneous reporting were investigated among all the National Health Service (NHS) doctors operating in the territory of the Area Health Authority n.1 of Varese (Italy), to assess their awareness of the reporting system and to identify reasons for under reporting. Three hundred and fifty doctors were sent questionnaires and 207 (59.1%) were returned completed. More than 77% of the responders stated to have noticed ADRs, which were mainly reported to the pharmaceutical manufacturers and, in a minority of cases, to the NHS. Fifty per cent did not report ADRs to anyone. Important factors for deciding to report were unusualness and severity of the reaction, and involvement of a new drug. The main reason for not reporting was the clinical negligibility of the reaction. There was little knowledge about the types of reactions to be preferentially reported and the purposes of ADR reporting systems. Nevertheless, nearly everyone asked for feed-back information about reported ADRs. NHS doctors in this district have little information concerning ADR reporting systems. Some effective measures to improve the situation could be: inclusion of pharmacovigilance into pre- and post-graduated continuing education programs, provision of guidelines for ADR spontaneous reporting and of feed-back information to reporters, implementation of regional pharmacovigilance units.

1. Lymphocyte subsets and neutrophil function were studied in chronic psychiatric patients on long-term multiple drug therapy, and the correlation with disease and drug treatments was also investigated. 2. Patients showed decreased CD4+ percentage, CD4+/CD8+ ratio, B lymphocyte percentage, increased absolute and percentage NK, and impaired neutrophil chemotaxis, phagocytosis, and oxidative metabolism. Correlations were found between: patient status and lower CD4+ percentage, CD4+/CD8+ ratio, neutrophil chemotaxis, phagocytosis, and oxidative metabolism; schizophrenia and higher absolute and percentage NK, and neutrophil random migration; benzodiazepines and higher CD8+ absolute number and lower NBT reduction frequency; carbamazepine and higher NK percentage, neutrophil chemotaxis, phagocytosis, stimulated O2-production, and lower microbicidal activity. 3. The present study provides direct evidence for the existence of immunologic abnormalities in a population of chronic psychiatric patients on long-term drug therapy. Although a possible role by other factors could not be excluded, such abnormalities are strongly associated with chronic administration of benzodiazepines and carbamazepine. Further investigations are warranted to confirm these findings and disclose the possible mechanism(s) involved.

Over the years, a vast literature has accumulated on the adrenergic mechanisms controlling gut motility, blood flow, and mucosal transport. The present review is intended as a survey of key information on the relevance of adrenergic mechanisms modulating gut motility and will provide an outline of our knowledge on the distribution and functional role of adrenoceptor subtypes mediating motor responses. alpha1-Adrenoceptors are located postsynaptically on smooth muscle cells and, to a lesser extent, on intrinsic neurons; alpha2-adrenoceptors may be present both pre- and postsynaptically, with presynaptic auto- and hetero-receptors playing an important role in the modulation of neurotransmitter release; beta-adrenoceptors are found mainly on smooth muscle cells. From a clinical standpoint, adrenoceptor agonists/antagonists have been investigated as potential motility inhibiting (antidiarrheal/antispasmodic) or prokinetic agents, although at present their field of application is limited to select patient groups.

The role of adrenergic mechanisms in the modulation of the immune response is well established [1], and several lines of evidence point to their involvement in immunemediated diseases of the nervous system such as multiple sclerosis (MS) [2]. Adrenoceptor ligands such as the b-adrenoceptor agonists terbutaline and isoproterenol [3] and the a-adrenoceptor antagonist prazosin [4] produce beneficial results in rats with experimental allergic encephalomyelitis (EAE), an established animal model of MS. Nonetheless, the rational basis for a therapeutic role of adrenoceptor ligands in MS has been hampered by the limited comprehension of the contribution of such mechanisms to human disease. The interesting article by De Keyser and co-workers, published recently inTiPS [5], discussed the involvement of b2-adrenoceptors expressed on astrocytes in inflammatory demyelinationandaxonaldegeneration inMS,andprovides novel insights into this intriguing topic. In this regard, we would like to add further points for consideration. First, noradrenaline acting on b2-adrenoceptors on astrocytes might be derived not only from noradrenergic nerve endings but also from activated lymphocytes infiltrating the CNS. Human peripheral lymphocytes express, following their activation, tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines, and subsequently increase their intracellular content of dopamine, noradrenaline and adrenaline by up to 20–40-fold basal levels [6]. Intracellular catecholamines that accumulate in activated lymphocytes appear to be involved in the modulation of activationinduced apoptosis [7]. In addition, they provide these cells with a supply of mediators to be released following appropriate stimulation. Interestingly, interferon b (IFN-b), which is at least partially beneficial in MS, appears to be a strong inducer of catecholamine production and release from activated lymphocytes [8]. Although the relevance of this in vitro effect for the in vivo activity of IFN-b remains to be established, it cannot be excluded that this might contribute to a ‘restorative’ effect by IFN-b of the astrocyte–lymphocyte crosstalk. It would therefore be of interest to investigate the effects of IFN-b on b-adrenoceptor expression on astrocytes. Second, human lymphocytes express adrenoceptors, which might represent additional targets for noradrenaline, either released by nerve endings or derived from immune cells. In patients with MS, b2-adrenoceptor expression on lymphocytes: (i) is increased; (ii) correlates withmagnetic resonance imaging (MRI) disease activity in primary progressive MS; and (iii) also occurs in the secondary progressive form of MS (although no relationship withMRI disease activity has been demonstrated) [9]. Increased production of cAMP has been detected in resting, but not activated, lymphocytes from MS patients following stimulation in vitro with b2-adrenoceptor agonists [10]. However, despite this phenomenon, reduced immunological responses to b2-adrenoceptor agonists appear to characterize lymphocytes from MS patients and from mice affected by EAE [11,12]. Biochemical deficiencies downstream of cAMP might therefore account for the aberrant signalling through b2-adrenoceptors in MS and EAE diseases, although these remain to be clearly elucidated [12]. The observation that IFN-b counteracts activation-induced reduction of b-adrenoceptor agonistinduced cAMP accumulation in human lymphocytes [13] might have interesting implications. It can therefore be proposed that in MS b2-adrenoceptor-dependent cAMP signalling might play a role not only in astrocytes, as pointed out by De Keyser and co-workers [5], but also in lymphocytes. In addition to b2-adrenoceptors, lymphocytes express a wide array of other G-proteincoupled receptors that, following stimulation, might Corresponding author: Marco Cosentino (marco.cosentino@uninsubria.it). Available online 8 May 2004 Update TRENDS in Pharmacological Sciences Vol.25 No.7 July 2004 350

1. In order to clarify whether atypical or beta 3-adrenoceptors can modulate canine colonic motility in vivo, we studied the effects of SR 58611A (a selective agonist for atypical beta-adrenoceptors) alone and after pretreatment with beta-adrenoceptor antagonists on colonic motility in the conscious dog. The gastrocolonic response (postprandial increase in motility) was monitored by means of electrodes and strain-gauge force transducers chronically implanted along the distal colon. In some experiments, heart rate was also measured. The possible role of beta 3-adrenoceptors in mediating the effects of SR 58611A was also tested in vitro in circular muscle strips taken from the canine distal colon. 2. Intravenous infusion of SR 58611A, ritodrine or isoprenaline at doses inducing the same degree of tachycardia inhibited the gastrocolonic response to a different extent, with SR 58611A and ritodrine being more effective than isoprenaline. 3. In a dose-response study, SR 58611A was more potent in inhibiting colonic motility than in inducing tachycardia: the ED35 values for inhibition of colonic motility and induction of tachycardia were 23 and 156 micrograms kg-1, i.v., respectively. 4. The inhibitory effect of SR 58611A 100 micrograms kg-1, i.v., on the gastrocolonic response was reversed by alprenolol (non-selective beta-adrenoceptor antagonist), but resistant to CGP 20712A (beta 1-adrenoceptor antagonist) or ICI 118551 (beta 2-adrenoceptor antagonist). 5. In vitro, SR 58611A concentration-dependently relaxed circular muscle strips, an effect that was competitively antagonized by alprenolol with a pA2 value of 7.1, but resistant to CGP 20712A (100 nM), ICI 118551 (100 nM) or tetrodotoxin (1 microM). 6. The present study provides strong functional evidence for a role of atypical or beta 3-adrenoceptors in the modulation of canine colonic motility both in vivo and in vitro by an inhibitory effect most likely at the smooth muscle level.