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Background In addition to inhibition of muscle and glandular hyperactivity, botulinum neurotoxin (BoNT) type A also interferes with pain processing. Previously, in a rat model of paclitaxel-induced polyneuropathy, abobotulinumtoxinA (aboBoNT-A) elicited analgesic effects not only in the injected paw, but also in the contralateral, non-injected paw. Methods Here, we assessed bilateral analgesic effects of unilateral aboBoNT-A in several chronic pain models in Sprague-Dawley rats. Effects of aboBoNT-A on the paw withdrawal threshold in response to mechanical pressure was assessed in models of streptozotocin-induced diabetic polyneuropathy, chronic constriction injury (CCI)-associated mononeuropathy, and bilateral carrageenan-induced inflammatory pain. Results In diabetic polyneuropathy, aboBoNT-A (15, 20 U/kg) reversed hyperalgesia in the toxin-injected and non-injected paws. In unilateral CCI-exposed animals, 20 U/kg aboBoNT-A given ipsilateral to the injury reversed mechanical hyperalgesia, while 30 U/kg aboBoNT-A given contralateral to the injury had no effect. In carrageenan-induced bilateral inflammatory pain, aboBoNT-A (20, 30 U/kg) reversed hyperalgesia in both toxin-injected and non-injected paws. Discussion These results suggest that unilateral administration of aboBoNT-A results in bilateral reduction in mechanical hyperalgesia across neuropathic and inflammatory pain conditions, bilateral activation of sensory neurons being prerequisite for its expression. Future studies involving effects on other sensory modalities as well as those evaluating diffusion and migration of the toxin away from the injection site can shed light on mechanisms of this phenomenon. Significance The results expand evidence on bilateral analgesic effects of aboBoNT-A following unilateral administration across pain modalities, as the phenomenon is seen in more than one model of polyneuropathy as well as in a model of chronic inflammatory pain when the latter is rendered bilateral. The mechanism of bilateral analgesic effects of aboBoNT-A may require activation of the peripheral sensory neurons and involve retrograde axonal transport of the toxin into the spinal cord.

Intradetrusor injections of Botulinum toxin A—currently onabotulinumtoxinA—is registered as a second-line treatment to treat neurogenic detrusor overactivity (NDO). The common clinical practice is 30 × 1 mL injections in the detrusor, however, protocols remain variable and standardization is warranted. The effect of reducing the number of injection sites of Dysport® abobotulinumtoxinA (aboBoNTA) was assessed in the spinal cord-injured rat (SCI). Nineteen days post-spinalization, female rats received intradetrusor injections of saline or aboBoNTA 22.5 U distributed among four or eight sites. Two days after injection, continuous cystometry was performed in conscious rats. Efficacy of aboBoNTA 22.5 U was assessed versus aggregated saline groups on clinically-relevant parameters: maximal pressure, bladder capacity, compliance, voiding efficiency, as well as amplitude, frequency, and volume threshold for nonvoiding contractions (NVC). AboBoNTA 22.5 U significantly decreased maximal pressure, without affecting voiding efficiency. Injected in four sites, aboBoNTA significantly increased bladder capacity and compliance while only the latter when in eight sites. AboBoNTA significantly reduced NVC frequency and amplitude. This preclinical investigation showed similar inhibiting effects of aboBoNTA despite the number of sites reduction. Further studies are warranted to optimize dosing schemes to improve the risk-benefit ratio of BoNTA-based treatment modalities for NDO and further idiopathic overactive bladder.

In addition to inhibition of muscle and glandular hyperactivity, botulinum neurotoxin (BoNT) type A also interferes with pain processing. Previously, in a rat model of paclitaxel-induced polyneuropathy, abobotulinumtoxinA (aboBoNT-A) elicited analgesic effects not only in the injected paw, but also in the contralateral, non-injected paw.Here, we assessed bilateral analgesic effects of unilateral aboBoNT-A in several chronic pain models in Sprague-Dawley rats. Effects of aboBoNT-A on the paw withdrawal threshold in response to mechanical pressure was assessed in models of streptozotocin-induced diabetic polyneuropathy, chronic constriction injury (CCI)-associated mononeuropathy, and bilateral carrageenan-induced inflammatory pain.In diabetic polyneuropathy, aboBoNT-A (15, 20 U/kg) reversed hyperalgesia in the toxin-injected and non-injected paws. In unilateral CCI-exposed animals, 20 U/kg aboBoNT-A given ipsilateral to the injury reversed mechanical hyperalgesia, while 30 U/kg aboBoNT-A given contralateral to the injury had no effect. In carrageenan-induced bilateral inflammatory pain, aboBoNT-A (20, 30 U/kg) reversed hyperalgesia in both toxin-injected and non-injected paws.These results suggest that unilateral administration of aboBoNT-A results in bilateral reduction in mechanical hyperalgesia across neuropathic and inflammatory pain conditions, bilateral activation of sensory neurons being prerequisite for its expression. Future studies involving effects on other sensory modalities as well as those evaluating diffusion and migration of the toxin away from the injection site can shed light on mechanisms of this phenomenon.The results expand evidence on bilateral analgesic effects of aboBoNT-A following unilateral administration across pain modalities, as the phenomenon is seen in more than one model of polyneuropathy as well as in a model of chronic inflammatory pain when the latter is rendered bilateral. The mechanism of bilateral analgesic effects of aboBoNT-A may require activation of the peripheral sensory neurons and involve retrograde axonal transport of the toxin into the spinal cord.

The standardized extract of Ginkgo biloba EGb 761 has been used to reduce cognitive dysfunction. The present study was designed to evaluate the effect of postischemic oral treatment with EGb 761 in a model of vascular dementia in gerbils. Daily oral posttreatment with EGb 761 led to a significant recovery of spatial memory assessed by the object location test, inhibited the decrease in plasma SOD activity and protected the hippocampal CA1 neurons, even when administered after the insult. These data provide further evidence for the therapeutic potential of EGb 761 in the treatment of vascular dementia.

A number of experimental models of L-DOPA-induced dyskinesia have been proposed, but these models result in a low to medium rate of dyskinetic animals with mild to severe symptoms. The objective of this study was to combine a model of 6-OHDA-induced parkinsonism and of L-DOPA-induced dyskinesia in rats to establish a reliable preclinical model. Two stereotaxic injections of 6-OHDA were administered in the left striatum. This model led to 90-100% of rats with a marked contralateral circling behaviour, significant limb use asymmetry (20%), a decrease in ipsilateral striatal dopamine content (70%) and degeneration of dopamine neurons in the substantia nigra (70%). Chronic treatment with L-DOPA was administered for 35 days and consisted of three phases with incremental daily doses. The third phase resulted in 83-90% of rats developing severe abnormal involuntary movements (AIMs) which included limb and locomotive dyskinesia, axial dystonia and orolingual dyskinesia. Reproducibility of the model, criteria of strict blinding, placebo-controlled design, randomization of study subjects and pretrial determination of sample size were used to measure efficacy of amantadine and istradefylline and to validate the protocol design. Acute or subchronic post-treatment with amantadine reduced the severity of dyskinesia while istradefylline punctually attenuated AIMs. Our experimental conditions using gradual development of dyskinesia induced by increasing doses of L-DOPA resulted in a reliable model of L-DOPA-induced dyskinesia with a high rate of dyskinetic rats.

In addition to inhibition of acetylcholine release in the neuromuscular junction botulinum toxin type A (BoNT-A) also inhibits the release of mediators involved in pain perception. We have investigated the effect of two types of BoNT-A on mechanical hyperalgesia in the rat models of carrageenan-induced hyperalgesia and of paclitaxel-induced peripheral neuropathy. A subplantar (s.p.) injection of BoNT-A in the ipsilateral hindpaw 3 days before carrageenan administration reduced hypersensitivity. Dysport® and Botox® elicited comparable antihyperalgesic effects. Dysport® up to 30 U/kg and Botox® up to 20 U/kg did not impair the rat withdrawal nociceptive reflex or the locomotor performance as assessed by the rotarod test. Intraperitoneal administration of the skeletal muscle relaxant dantrolene produced, in contrast to BoNT-A, more motor impairment than analgesia. Paclitaxel treatment resulted in a peripheral neuropathy that affected the two hindpaws. Injection of 20 U/kg (s.p.) Dysport® produced a significant antihyperalgesic effect in the injected paw of neuropathic animals 3 days after administration. Unexpectedly, a similar analgesic effect was observed in the contralateral paw. The same results were also observed when Botox® was used instead of Dysport®. In contrast, a contralateral administration of Dysport® in the carrageenan test was ineffective. We conclude that BoNT-A elicits antinociceptive effects independent of the effects on muscular relaxation. Our results suggest that different mechanisms of action are responsible for the effect of BoNT-A in inflammatory and peripheral polyneuropathic rat models.

Alzheimer's disease (AD) is a human neurodegenerative disease characterized by two key histopathological hallmarks : beta amyloid plaques and neurofibrillary tangles. No animal species naturally develops AD. Lesions induced by toxic substances which modify neurotransmission have been used in rodents but are not suitable models for AD. More recently, transgenic mouse models reproducing physiopathologic aspects of AD have greatly contributed to our understanding of the disease and have provided models to evaluate new therapeutic approaches. While none of these models perfectly reproduces all the pathological characteristics of AD, they are extremely useful in the evaluation and development of novel therapeutic agents. Pharmacological evaluation should assess abnormal behavior, histopathologic lesions and biochemical or metabolic dysfunctions. New technologic tools such as neuroimaging and biological biomarkers have greatly facilitated these evaluations. Depending on the symptomatic or neuroprotective therapeutic objectives, these methods are becoming increasingly accurate and adaptable to the human patient. A multidisciplinary approach is going to optimize these models so that they can become more predictive and help bring forward new effective treatments for AD patients.

BIM-46187 (7-[2-amino-1-oxo-3-thio-propyl]-8-cyclohexylmethyl-2-phenyl-5,6,7,8-tetrahydro-imidazo-[1,2a]-pyrazine dimer, hydrochloride) is an inhibitor of the heterotrimeric G-protein complex signalling. Since many mediators of pain act through G-protein coupled receptors, the anti-hyperalgesic effects of BIM-46187 were assessed on experimental models of pain. In addition since opioids are widely used in pain management and act through specific G-protein-coupled receptors, the effects of BIM-46187 on the analgesic properties of morphine have also been investigated. BIM-46187 elicited a dose dependent analgesic effect in the models of carrageenan-induced hyperalgesia (0.1-1 mg/kg; i.v.) and chronic constriction injury (0.3-3 mg/kg; i.v.) in rats. BIM-46187, however, up to 10 mg/kg did not modify the paw oedema induced by carrageenan excluding an anti-inflammatory effect. In addition, at these doses, the compound was not sedative as shown by the lack of effect on the motor performance in the rotarod test. The combination of BIM-46187 and morphine (ratio 1/1) resulted in an unexpected synergistic effect in the model of carrageenan-induced hyperalgesia and in the chronic constriction injury model in rats when evaluated by isobolographic analysis. This synergy allowed a reduction of at least 20 fold in the dose of each compound. Conversely, the drug combination did not increase the side effects of morphine as assessed in the rotarod test. In conclusion, BIM-46187 elicits a potent anti-hyperalgesic effect and strongly synergizes with morphine. This work highlights the role of heterotrimeric G-protein complexes in pain and supports further investigations of the use of BIM-46187 alone, or in combination with low doses of morphine, in the management of pain.

BN 80933, a dual inhibitor of neuronal nitric oxide synthase and lipid peroxidation, prevents in vivo brain ischemic/reperfusion injury. In the present study, BN 80933 was shown to protect neurons from hypoxia-induced cell death in primary cultures of cortical neurons. BN 80933 prevented lactate dehydrogenase activity elevation induced by hypoxia, displaying an IC50 value of 0.15 +/- 0.05 microM. This effect was likely due to the antioxidant properties of BN 80933 because Trolox, but not NG-nitro-L-arginine, also elicited protection. The antioxidant property of BN 80933 was then further investigated on HT-22 cells subjected to buthionine sulfoximine- or glutamate-induced glutathione depletion. The relative order of potency of the various compounds to inhibit oxidative stress-induced neuronal death (BN 80933 > U104067 > butylated hydroxytoluene > 17beta-estradiol > Trolox > vitamin E) correlated with their ability to inhibit brain membrane lipid peroxidation (correlation coefficient = 0.939). BN 80933 afforded protection even when added 6 h after glutamate exposure. BN 80933 did not reverse intracellular glutathione depletion but prevented elevation of the level of beta-epiprostaglandin F2alpha (8-isoprostane), which appeared to be a delayed phenomenon. In conclusion, BN 80933 induces a potent cytoprotection that may be mediated by inhibition of delayed lipid peroxidation.

Histology is the gold standard to unveil microscopic brain structures and pathological alterations in humans and animal models of disease. However, due to tedious manual interventions, quantification of histopathological markers is classically performed on a few tissue sections, thus restricting measurements to limited portions of the brain. Recently developed 3D microscopic imaging techniques have allowed in-depth study of neuroanatomy. However, quantitative methods are still lacking for whole-brain analysis of cellular and pathological markers. Here, we propose a ready-to-use, automated and scalable method to thoroughly quantify histopathological markers in 3D in rodent whole brains. It relies on block-face photography, serial histology and 3D-HAPi (Three Dimensional Histology Analysis Pipeline), an open source image analysis software. We illustrate our method in studies involving mouse models of Alzheimer’s disease and show that it can be broadly applied to characterize animal models of brain diseases, to evaluate therapeutic interventions, to anatomically correlate cellular and pathological markers throughout the entire brain and to validate in vivo imaging techniques.

Resume La table ronde intitulee «Modeles de Partenariats Public-Prive en Europe, Comparaison France-pays europeens » reunissait des representants du secteur academique et industriel, de programmes scientifiques nationaux ou europeens dans le domaine de la Sante, d’acteurs regionaux et nationaux du partenariat public-prive et des biotechnologies, et bien entendu de representants d’institutions ou de programmes Europeens ou extraeuropeens dans le domaine de le recherche en Sante, avec pour objectifs: • le partage d’une vision sur les besoins, attentes et enjeux du Partenariat Public-Prive • a partir de l’analyse de cas concrets et originaux, ou d’initiatives nouvelles de partenariats Public-Prive • en tirer des enseignements - Facteurs clefs du succes. • identifier des axes de progres - Recommandations. L’actualite dans ce secteur est actuellement dominee par une initiative de partenariat public-prive a l’echelle de l’Union europeenne entre l’industrie du medicament (EFPIA) et la Commission europeenne (DG Recherche - Sciences de la Vie), sous la forme du projet de Plate-forme technologique Europeenne « Innovative Medicines Initiative » (IMI), dont l’architecture repose sur l’identification des principaux obstacles au developpement de medicaments innovants (pharmacologie et toxicologie predictives, identification et validation des biomarqueurs, recrutement de patients, evaluation du risque et cooperation avec les instances reglementaires) Simultanement, un certain nombre d’initiatives a l’echelle nationale ou regionale viennent complementer le maillage geographique du partenariat: la constitution de poles de competitivite a l’echelle regionale, ou l’appel d’offres du reseau Innovation Biotechnologie (RIB) de l’ANR sont autant d’incitations au partenariat public-prive, capables de structurer des consortia aptes a repondre secondairement a des appels d’offres de plus grande envergure a l’echelle europeenne, realisant un escalier du partenariat. En depit de mesures incitatives et de cadres regionaux et nationaux du partenariat, les difficultes decrites au niveau national reposent en partie sur la multiplicite des acteurs - multiples agences de valorisation, multiples organismes de recherche (operateur ou agence de moyens) qui peuvent compliquer la passation de contrats - rendant souhaitable une structuration plus lisible avec un referent unique par domaine (sante, technologie, etc.). Ces partenariats seront aussi facilites par un travail concernant les statuts, le recrutement et la mobilite, par un support aux scientifiques dans le pilotage des projets, et par un soutien permettant d’accompagner (sans les maintenu’ artificiellement en vie) des entreprises emergentes depuis le concept jusqu’au stade du developpement clinique. Concernant le developpement clinique, des pistes ont ete avancees quant a l’organisation de la recherche clinique en France et en Europe, necessairement structuree au sein du secteur public, et supposant un couplage entre infrastructures integrees a l’echelle de l’Europe et reseaux thematiques. Comme le souligne le volet « Efficacite » d’IMI, la qualite des infrastructures publiques europeennes sera un element cle de sa competitivite et de son attractivite pour le secteur academique comme industriel.

The workshop entitled "Public-Private partnerships models in Europe-- comparison between France and European countries" brought together representatives of academia and industry, of national or European health research programs, of regional or national public-private partnership (PPP) initiatives, and of biotechnology with the following objectives: sharing a common vision on the needs, expectations and challenges of public-private partnership, based on the analysis of actual and original cases, and of new initiatives on public-private partnership, drawing conclusions and identifying key success factors, identifying trails for progress and drawing recommendations. The major event in this field is a European public-private partnership initiative between pharmaceutical industry (European Federation of Pharmaceultical Industry and Associations, EFPIA) and the European Commission (DG Research--health priority) resulting in the European Technology Platform project "Innovative Medicines Initiative" (IMI). Its architecture is based on the identification of the main bottlenecks to the development of innovative treatments (predictive pharmacology and toxicology, identification and validation of biomarkers, patients' recruitment, risk evaluation, and cooperation with the regulatory authorities). Simultaneously, initiatives both at the national and regional levels also foster PPP in the therapeutic field. Regional competitivity clusters acting in the biomedical sector, and national PPP calls such as the ANR (National Research Agency) RIB (Research and Innovation in Biotechnology) call are incentives for PPP projects. These regional and national PPP levels help public and private partners to further build consortia able to compete for EU-level calls, thus acting as incubators for EU PPP projects. In spite of incentives and of the regional and national structuring of PPP, weaknesses in the French system are linked to its fragmentation--multiple transfer agencies, multiple research organisations (operator or funding agency)--making contracts more difficult. This requires a simplified organisation, with a single referent per area (health, technology...). Improvement may also result from adaptation in the carreer, recruitment and mobility, from support to scientists in the management of projects, and from consistent support (without maintaining them artificially alive) to emerging companies from concept through clinical development. Pathways have been proposed to improve the efficiency of clinical research in France and Europe, involving the public hospital sector, and this requires the connection of disease-oriented networks and integrated infrastructures in Europe. As stated in the IMI strategic research agenda on efficacy, the quality of public infrastructures in Europe will be a key factor for its competitiveness and attractiveness for both academic and industry projects.

Nitric oxide (NO) and reactive oxygen species (ROS) act independently as well as cooperatively to induce neuronal death in acute neurological disorders. Inhibition of neuronal nitric oxide synthase (nNOS) and inhibition of lipid peroxidation induced by ROS have both been proposed as neuroprotective strategies in stroke and trauma. Recently, in our laboratory, the combination of the two strategies was found to be synergistic in reducing neuronal damage. Here, we report that BN 80933 [( S )- N -{4-[4-[(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2 H -1-benzopyran-2-yl)carbonyl]-1-piperazinyl]phenyl}-2-thiophenecarboximidamide], a compound that combines potent antioxidant and selective nNOS inhibitory properties in vitro , affords remarkable neuronal protection in vivo . Intravenous administration of BN 80933 significantly reduced brain damage induced by head trauma in mice, global ischemia in gerbils, and transient focal ischemia in rats. Treatment with BN 80933 (0.3–10 mg/kg) significantly reduced infarct volume (>60% protection) and enhanced behavioral recovery in rats subjected to transient (2-h) middle cerebral artery occlusion and 48-h or 7-day reperfusion. Furthermore, treatment with BN 80933 commencing up to 8 h after the onset of ischemia resulted in a significant improvement of neurological outcome. All these results indicate that BN 80933 represents a class of potentially useful therapeutic agents for the treatment of stroke or trauma and possibly neurodegenerative disorders that involve both NO and ROS.

1. The alpha 1-adrenoceptor subtypes mediating the regulation of arterial pressure (AP) and urethral perfusion pressure (UP) in the anaesthetized rat were characterized by using selective alpha 1-adrenoceptor agonists and antagonists. 2. Intravenous administration of selective alpha 1-adrenoceptor agonists elicited a dose-dependent increase in AP and UP. The rank order of agonist potency: oxymetazoline (ED50, 6.2 and 8.2 nmol kg-1 > phenylephrine (ED50, 32 and 27 nmol kg-1 > methoxamine (ED50, 300 and 296 nmol kg-1 was the same for AP and UP, respectively. 3. The effects of phenylephrine on AP and UP were antagonized, in a dose related-manner, by pretreatment with alfuzosin, BMY 7378, 5-methyl-urapidil, phentolamine, prazosin, spiperone and WB 4101.5-methyl-urapidil was the only alpha 1-adrenoceptor antagonist more potent on UP than on AP. 4. The potency of the different alpha 1-adrenoceptor antagonists tested on AP and UP was significantly correlated with their binding affinity for the expressed recombinant alpha 1a-, but not alpha 1b- or alpha 1d-, adrenoceptor subtype. 5. The results suggest that in the anaesthetized rat (1) both AP and UP are regulated by the alpha 1A-adrenoceptor subtype; and (2) the urogenital selectivity of 5-methyl-urapidil may be due to the existence of multiple forms of the alpha 1A-adrenoceptor.

A series of 2-alkyl-4-arylimidazoles were prepared and their binding affinities to the site-2 sodium (Na+) channel were determined. SAR studies led to highly potent Na+ channel blockers.

The development of dyskinesias following chronic L-DOPA replacement therapy remains a major problem in the long-term treatment of Parkinson's disease. This study aimed at evaluating the effect of IRC-082451 (base of BN82451), a novel multitargeting hybrid molecule, on L-DOPA-induced dyskinesias (LIDs) and hypolocomotor activity in a non-human primate model of PD. IRC-082451 displays multiple properties: it inhibits neuronal excitotoxicity (sodium channel blocker), oxidative stress (antioxidant) and neuroinflammation (cyclooxygenase inhibitor) and is endowed with mitochondrial protective properties. Animals received daily MPTP injections until stably parkinsonian. A daily treatment with increasing doses of L-DOPA was administered to parkinsonian primates until the appearance of dyskinesias. Then, different treatment regimens and doses of IRC-082451 were tested and compared to the benchmark molecule amantadine. Primates were regularly filmed and videos were analyzed with specialized software. A novel approach combining the analysis of dyskinesias and locomotor activity was used to determine efficacy. This analysis yielded the quantification of the total distance travelled and the incidence of dyskinesias in 7 different body parts. A dose-dependent efficacy of IRC-082451 against dyskinesias was observed. The 5 mg/kg dose was best at attenuating the severity of fully established LIDs. Its effect was significantly different from that of amantadine since it increased spontaneous locomotor activity while reducing LIDs. This dose was effective both acutely and in a 5-day sub-chronic treatment. Moreover, positron emission tomography scans using radiolabelled dopamine demonstrated that there was no direct interference between treatment with IRC-082451 and dopamine metabolism in the brain. Finally, post-mortem analysis indicated that this reduction in dyskinesias was associated with changes in cFOS, FosB and ARC mRNA expression levels in the putamen. The data demonstrates the antidyskinetic efficacy of IRC-082451 in a primate model of PD with motor complications and opens the way to the clinical application of this treatment for the management of LIDs.

This study investigated the distribution of platelet activating factor (PAF) binding sites in the brain and their involvement in global ischemia in a model of bilateral common carotid occlusion in the gerbil. In sagittal sections of gerbil brain, labeling with [3H]PAF was mainly located in the cortex, hippocampus and cerebellum. The corpus striatum, the superior and inferior colliculi showed lower binding, while the thalamus was only weakly labeled. Scatchard analysis of the data obtained from displacement curves with unlabeled PAF revealed the presence of one or two populations of binding sites with different affinity constant values depending on the brain structures. When the gerbils were submitted to 10 min ischemia, similar autoradiography with [3H]PAF demonstrated a dramatic reduction of labeling in all brain structures, particularly in the hippocampus. Immunoreactive endogenous PAF concentrations in brain tissues showed a marked increase in ischemic animals: (8977.3 +/- 1113 pg/g wet weight) as compared to sham-operated control: (997.7 +/- 77 pg/g wet weight). Endogenous PAF levels returned to basal values following 30 min reperfusion. These results indicate that PAF may be involved in the early stages of brain ischemia in the gerbil and suggest that endogenous PAF produced during ischemia may contribute to the down-regulation of [3H]PAF binding sites in the brain.

Background Two botulinum toxins A have been evaluated for the treatment of refractory neurogenic detrusor overactivity (NDO) in humans: Dysport (abobotulinumtoxinA) and Botox (onabotulinumtoxinA). However, these two distinct commercialized products have different potency units and are not interchangeable. Objective Assessment of the dose response and determination of minimal effective dose (MED) for Dysport and Botox in spinal cord–injured (SCI) rats with NDO. Design, setting, and participants Female, adult, Sprague-Dawley rats ( n =98) underwent T8-T9 spinal cord transection. Nineteen days after spinal cord injury, rats received intradetrusor injections (25μl injected, eight sites) of vehicle (V); Dysport 2, 5, 7.5, 10, and 12.5 U; and Botox 0.8, 2, 5, 7.5, and 10 U. Two days after injection, continuous cystometry was performed in conscious rats. Measurements Voiding contractions (VC) were assessed by duration of VC, intercontraction interval, voided volume, maximal pressure, pressure threshold change, and intravesical baseline pressure (BP), while nonvoiding contractions (NVC) were evaluated by amplitude, frequency, and volume threshold to elicit NVC. MEDs for Dysport and Botox were determined by analysis of variance step-down trend test. Results and limitations MEDs for Dysport and Botox were 10 U and 7.5 U, respectively. Regarding VC, only BP significantly decreased after 10 U Dysport and 7.5 U Botox compared to V (from 3.7±0.6 to 1.5±0.1 and 1.4±0.3mm Hg, respectively; p 0.01 and p 0.001, respectively). Dysport (10 U) and Botox (7.5 U) significantly inhibited NVC by decreasing their amplitude (from 7.4±1.1 to 5.8±0.5 and 5.4±0.6mm Hg, respectively; p 0.05); frequency (from 2.2±0.4 to 1.5±0.2 and 1.3±0.3 NVC per minute, respectively; p 0.01); and increasing volume threshold to elicit NVC (from 29.8±3.7 to 47.6±6.9 and 47.7±6.3%, respectively; p 0.05 and p 0.001, respectively). Conclusions This is the first preclinical dose-ranging study with Dysport and Botox under standardized conditions showing similar inhibiting effects on NDO, albeit at different MEDs. It highlights the importance of distinguishing each preparation for predicted outcomes and doses to be used. Further studies in patients with NDO are warranted to confirm these experimental results.

The capability of the methoxy-substituted 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3 or Edelfosine) and the two aza-alkylphospholipids BN 52205 and BN 52211 to bind to the PAF receptor was analysed in rabbit platelet membranes. Ether phospholipid concentrations were tested between 10(-5) M and 10(-11) M. The results indicate that ether phospholipids are not able to bind to the PAF receptor and do not prevent PAF binding to its receptor. Moreover, the cytotoxic effect of three potent PAF antagonists, BN 52021, BN 50730 and BN 50739, were analysed in HL60 promyelocytic cells. These cells were pre-and co-treated with PAF antagonists and ether phospholipids. The data show that the three PAF antagonists failed to counteract the activity of ET-18-OCH3, BN 52205 and BN 52211 thus demonstrating that the cytotoxic effect of these new anti-neoplastic drugs is not mediated by the PAF receptor.

Hypertension causes major structural vascular modifications including increase of wall:lumen ratio, vessel wall hypertrophy, and rarefaction of arterioles and small arteries. These structural alterations are accompanied with vascular dysfunctions. The stimuli responsible for this vascular remodeling are numerous and comprise physical, humoral, and locally synthetized factors. Among them, vasoactive substances such as vasoconstrictors (e.g., angiotensin II and endothelin) or vasodilating substances (e.g., prostacyclin and nitric oxide) may play a role as important as those of growth factors. The vascular protection afforded by antihypertensive drugs depends on their mode and their site of action and probably on their direct interference with the local hemodynamic and growth-affecting factors. This is well illustrated by cicletanine, a new antihypertensive agent that provides a good example of protection of the vasculature. This also emphasizes that vascular remodeling is important in hypertension and must be considered as an essential property of any new antihypertensive drug.

The development of l -dopa-induced dyskinesia (LID) remains a major problem in the long-term treatment of Parkinson’s disease (PD). This study aimed to assess the effect of the multitargeting molecule BN82451 on LID and to measure striatal mRNA expression of several genes in a rat model of PD. Rats were administered two unilateral injections of 6-OHDA in the striatum. After four weeks, the animals started a chronic daily treatment with increasing doses of l -dopa over a further four-week period. Over the course of l -dopa treatment, the rats developed abnormal involuntary movements (AIMs) classified as locomotive, axial, orolingual and forelimb dyskinesia. In animals rendered dyskinetic by l -dopa, administration of BN82451 at doses ranging from 1 to 10 mg/kg p.o. attenuated the severity of fully-established AIMs in a dose-related manner. This anti-dyskinetic effect could be achieved with lower doses of BN82451 administered sub chronically vs. acute single treatment. The improvement of AIMs is not due to a reduction in the general motor activity of dyskinetic rats. BN82451 treatment significantly reversed the overexpression of c-Fos, FosB and Arc mRNA associated with the dyskinesiogenic action of l -dopa. A significant correlation between the degree of overexpression of c-Fos, FosB and Arc mRNA and the dyskinesiogenic action of l -dopa was observed. The data demonstrate that BN82451 effectively attenuates LID and the associated molecular alterations in an animal model of PD and may represent a treatment option for managing dyskinesia.

[3H]Platelet-activating factor (PAF) binding to rabbit platelet membranes was examined before and after 20 min preincubation at 25 degrees C in the presence of PAF, lysoPAF, or of five different PAF receptor antagonists (L 652731, BN 52021, WEB 2086, BN 52111 and BN 52115). When platelet membranes were not washed after preincubation with PAF or PAF antagonists, no significant specific binding of [3H]PAF was observed, which suggests full occupancy of the binding sites. When membranes were extensively washed, full recovery of specific [3H]PAF binding was attained with L 652731 and partial recoveries (60%, 55% and 30%) were reached with BN 52021, WEB 2086 and PAF, respectively; no recovery was seen with the dioxolanes BN 52111 and BN 52115. Scatchard analysis of the binding data indicated that no significant change in the dissociation constant (Kd) and maximum number of binding sites (Bmax) occurred after preincubation of platelet membrane with L 652731, whereas a reduction of Bmax was observed when PAF and BN 52021 were measured. When platelet membranes were preincubated with WEB 2086, Bmax and Kd significantly increased. The data suggest differing binding properties for PAF and the PAF antagonists. Some of the PAF antagonists may tightly bind to the PAF receptor site(s) and/or irreversibly modify or downregulate PAF recognition sites. Our results also suggest that the interaction of PAF receptor antagonists with PAF receptor can be divided into at least two components, namely a reversible component and an irreversible one.

To assess the effects of endothelin 1 (ET) on the pulmonary and systemic vascular beds simultaneously, we examined the hemodynamic responses to ET in awake calves implanted with a Jarvik total artificial heart (TAH), a device that maintains constant cardiac output (CO). During basal conditions, successive incremental intravenous (i.v.) injections of 1, 3, and 10 micrograms ET caused a dose-dependent decrease in pulmonary arterial pressure (PAP), (from 24 +/- 3 to 15 +/- 1 mm Hg, p less than 0.05) while having no effect on systemic arterial (SAP), left atrial (LAP), and right atrial (RAP) pressures. Administration of 30 micrograms ET i.v. also decreased PAP, had no effect on LAP and RAP, but increased SAP from 100 +/- 6 to 118 +/- 4 mm Hg (p less than 0.05). The decrease in PAP was rapid, occurring within seconds and lasting 10 min, whereas the increase in SAP occurred after 2-5 min and was prolonged for greater than or equal to 20 min. As compared with injection in the right atrium, administration of 30 micrograms ET into the left atrium reduced PAP to a similar extent, but induced a greater increase in SAP (+32.5 +/- 4 vs +17.5 +/- 2 mm Hg, p less than 0.05). ET also dose-dependently reversed the acute pulmonary vasoconstriction induced by inhalation of an hypoxic gas mixture. In all cases, pulmonary vasodilation occurred without evidence of short-term tolerance. The results demonstrate that ET is a potent in vivo pulmonary vasodilator. In calves, the predominant hemodynamic response to ET is pulmonary vasodilation, with systemic vasoconstriction apparent only at higher concentrations of the peptide.

This study investigated the pulmonary vascular response to endothelin (ET) in rats. In conscious rats, an incremental intravenous bolus of ET-1 (100-1,000 pM) caused, after an initial drop in systemic arterial pressure (Psa), a secondary dose-dependent increase of Psa concomitant with a decrease of cardiac output (CO) and heart rate (HR). Pulmonary arterial pressure (Ppa) remained unchanged, and pulmonary vascular resistance (PVR) increased significantly only after 1,000 pM (+ 40.0 +/- 10.4 at 15 min). Meclofenamate (6 mg/kg iv) did not alter hemodynamic response to ET (300 pM). After autonomic blockade with hexamethonium (6 mg/kg iv) plus atropine (0.75 mg/kg iv), bradycardia response to ET (300 pM) was blocked, but CO decreased, systemic vascular resistance increased, and PVR remained unchanged as in controls. In anesthetized ventilated rats, bolus injections of ET (10-1,000 pM) induced a transient dose-related decrease in compliance (-10.9 +/- 1.8% after 1,000 pM) but no change of conductance. In isolated lungs, Ppa increased at doses greater than 100 pM, and edema developed in response to 1,000 pM ET. The rise of Ppa in response to 300 pM was not altered by meclofenamate (3.2 x 10(-6) M) but was potentiated by inhibitors of endothelium-derived relaxing factor(s) (EDRF), methylene blue (10(-4) M), pyrogallol (3 x 10(-5) M), and NG-monomethyl-L-arginine (6 x 10(-4) M) (3.9 +/- 0.3, 4.6 +/- 0.5, and 5.9 +/- 0.3 mmHg, respectively, compared with 1.5 +/- 0.5 mmHg in control lungs). These results suggest that circulating ET is a more potent constrictor of the systemic circulation than of the pulmonary vascular bed.(ABSTRACT TRUNCATED AT 250 WORDS)

Alzheimer's disease (AD) is a human neurodegenerative disease characterized by two key histopathological hallmarks : beta amyloid plaques and neurofibrillary tangles. No animal species naturally develops AD. Lesions induced by toxic substances which modify neurotransmission have been used in rodents but are not suitable models for AD. More recently, transgenic mouse models reproducing physiopathologic aspects of AD have greatly contributed to our understanding of the disease and have provided models to evaluate new therapeutic approaches. While none of these models perfectly reproduces all the pathological characteristics of AD, they are extremely useful in the evaluation and development of novel therapeutic agents. Pharmacological evaluation should assess abnormal behavior, histopathologic lesions and biochemical or metabolic dysfunctions. New technologic tools such as neuroimaging and biological biomarkers have greatly facilitated these evaluations. Depending on the symptomatic or neuroprotective therapeutic objectives, these methods are becoming increasingly accurate and adaptable to the human patient. A multidisciplinary approach is going to optimize these models so that they can become more predictive and help bring forward new effective treatments for AD patients.

This study examined the effect of various doses of endothelin (from 0.2 to 2 nmol/kg body wt) on regional hemodynamics in conscious unrestrained rats. Normal rats were instrumented chronically with femoral artery and vein catheters and pulsed Doppler flow probes simultaneously on the renal and superior mesenteric arteries and the abdominal aorta. Endothelin induced a biphasic response of mean arterial pressure. First, endothelin provoked a sharp hypotension with tachycardia, vasodilation of the hindquarter, and a pronounced decrease in renal and mesenteric blood flows. After this initial response, endothelin induced a dose-dependent increase of mean arterial pressure. Changes in the hindquarter vascular resistance were less pronounced than those in renal and mesenteric vascular resistances. Endothelin (2 nmol/kg) reduced renal flow (-86%) resulting from a vasoconstriction (+1,818%) significantly more pronounced than for the mesenteric vascular bed. In another set of experiments, endothelin (2 nmol/kg) induced an increase in proteinuria, characterized by an increase in excreted albumin and by the appearance of proteins with molecular weights of 20,000-280,000. Renal vascular bed exhibited a pronounced sensitivity to the vasoconstrictive effect of endothelin associated with changes in renal function.

Endothelin (ET) belongs to a family of 21 amino acid peptides comprising at least three isoforms in man. Originally identified as an endothelium-derived vasoconstrictive substance, ET arises from a precursor peptide which is cleaved and released by a specific ET-converting enzyme. ET exerts multiple pharmacological effects through its own receptors. Among them, ET evokes the sustained and poorly reversible contraction of isolated vascular preparations and induces in vivo a long-lasting pressor effect preceded by transient hypotension. The mechanism of action of ET is not yet clarified although ET has been shown to stimulate phospholipase C and protein kinase C. It seems probable that this peptide acts more as a paracrine or autocrine signal than a circulating hormone. The availability of specific receptor antagonists and inhibitors of its biosynthesis will allow to understand the biological relevance of this novel family of peptides.

Free radical and calcium buffering mechanisms are implicated in cochlear cell damage that has been induced by sound trauma. Thus in this study we evaluated the therapeutic effect of a novel dual inhibitor of calpains and of lipid peroxidation (BN 82270) on the permanent hearing and hair cell loss induced by sound trauma. Perfusion of BN 82270 into the scala tympani of the guinea pig cochlea prevented the formation of calpain-cleaved fodrin, translocation of cytochrome c, DNA fragmentation and hair cell degeneration caused by sound trauma. This was confirmed by functional tests in vivo, showing a clear dose-dependent reduction of permanent hearing loss (ED50 = 4.07 microM) with almost complete protection at 100 microM. Furthermore, BN82270 still remained effective even when applied onto the round window membrane after sound trauma had occurred, within a therapeutic window of 24 h. This indicates that BN 82270 may be of potential therapeutic value in treating the cochlea after sound trauma.

A series of 4-arylimidazole carbamates was synthesized and their binding affinities to the site-2 sodium (Na+) channel were determined. SAR studies led to the identification of compound 10, a potent Na+ channel blocker which was efficacious in pain models in vivo.

After extensive necrosis, progressive diaphragm muscle weakness in the mdx mouse is thought to reflect progressive replacement of contractile tissue by fibrosis. However, little has been documented on diaphragm muscle performance at the stage at which necrosis and fibrosis are limited. Diaphragm morphometric characteristics, muscle performance, and cross-bridge (CB) properties were investigated in 6-wk-old control (C) and mdx mice. Compared with C, maximum tetanic tension and shortening velocity were 37 and 32% lower, respectively, in mdx mice (each P < 0.05). The total number of active CB per millimeter squared (13.0 ± 1.2 vs. 18.4 ± 1.7 × 109/mm2, P < 0.05) and the CB elementary force (8.0 ± 0.2 vs. 9.0 ± 0.1 pN, P < 0.01) were lower in mdx than in C. The time cycle duration was lower in mdx than in C (127 ± 18 vs. 267 ± 61 ms, P < 0.05). Percentages of fiber necrosis represented 2.8 ± 0.6% of the total muscle fibers, and collagen surface area occupied 3.6 ± 0.7% in mdx diaphragm. Our results pointed to severe muscular dysfunction in mdx mouse diaphragm, despite limited necrotic and fibrotic lesions.

Spinal cord injury (SCI) induces plasticity within neural pathways innervating the lower urinary tract (LUT), with the recruitment of nociceptive “silent” C-fibers leading to the development of an abnormal autonomic micturition reflex and emergence of neurogenic detrusor overactivity (NDO), thereby greatly compromising bladder filling during the micturition cycle. In SCI patients, botulinum toxin A has been evaluated for the treatment of refractory NDO and have been reported to decrease urinary incontinence frequency and maximum intravesical pressure while increasing bladder capacity and compliance Onabotulinum (Ona) toxin A intradetrusor injections is registered as a second-line treatment to treat NDO in humans. Injection protocol remains variable among clinical studies. Hovever approved Ona label recommends 30 injection points in the bladder wall. The main objective of this study was to determine the effect of reducing the number of injection sites by comparing the effect of 4 versus 8 injection sites with abobotulinum toxin A (aboBoNTA) in the SCI rat, a relevant model of NDO METHODS Experimental design Nineteen days post-SCI, rats received intradetrusor injections in 4 or 8 sites of saline or aboBoNTA 22.5U. Two days after injections, effect of aboBoNTA on urodynamic clinically relevant parameters was determined by analysis of variance test versus aggregated saline groups (ASG). Four experimental groups were considered: saline (NaCl 0.9%) 4 sites (12.5µl total volume, n=12; S-4sites), saline 8 sites (25µl total volume, n=11; S-8sites), aboBoNTA 4 sites (12.5µl total volume, 5.6U per site, n=20; aboBoNTA-4sites) and aboBoNTA 8 sites (25µl total volume, 2.8U per site, n=20; aboBoNTA-8sites).

To investigate the role of atrial natriuretic factor (ANF) in renal responses to a decrease in central blood volume, we examined the effects of ANF infusion on renal function and hormones during prolonged lower-body negative pressure (LBNP). Ten healthy volunteers participated in two experimental sequences, each comprising a 120-min baseline period followed by the application of -20 mm Hg LBNP for 90 min. During one of the two sequences, ANF was infused throughout LBNP application at the constant rate of 2.5 ng/kg/min. Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were measured by using inulin and p-aminohippuric acid clearance techniques. LBNP induced a significant decrease in ERPF (534 +/- 28 to 457 +/- 26 ml/min; p < 0.05), GFR (120 +/- 2.5 to 112 +/- 2.5 ml/min; p < or = 0.01), in urine excretion (12 +/- 0.9 to 5.6 +/- 0.5 ml/min; p < 0.001), in sodium excretion (0.36 +/- 0.03 to 0.30 +/- 0.02 mmol/min; p < 0.05), and in plasma ANF (19 +/- 3 to 11 +/- 2 pg/ml; p = 0.001) concomitant with an increase in plasma renin activity (PRA; 0.48 +/- 0.09 to 0.87 +/- 0.16 ng/ml/h; p = 0.01) and of forearm vascular resistance (FVR; p < 0.05). The combination of ANF infusion with LBNP led to a slight increase in plasma ANF from baseline (from 20 +/- 2 to 28 +/- 3 pg/ml; p < 0.05). Compared with values obtained during LBNP with saline vehicle infusion, values obtained during LBNP with ANF infusion were similar for ERPF (463 +/- 23 vs. 457 +/- 26 ml/min), for GFR (111 +/- 2 vs. 112 +/- 2 ml/min), and for urine excretion (7 +/- 0.6 vs. 5.6 +/- 0.5 ml/min; p = 0.07), but greater for fractional excretion of sodium (2.38 +/- 0.25% vs. 1.91 +/- 0.11%; p < 0.05) and FVR (p < 0.05), and smaller for PRA (0.49 +/- 0.1 vs. 0.87 +/- 0.16 ng/ml/h; p < 0.01). These data show that ANF infusion attenuates the antinatriuretic effect of low-level LBNP and its PRA-increasing effects without altering renal hemodynamic responses to LBNP, although there is a decrease in the LBNP-induced forearm vasoconstriction. These results were obtained with plasma ANF levels slightly higher than those in baseline. They support the hypothesis that a decrease in ANF secretion might contribute to the antinatriuretic effect of LBNP.

s Toxins 2011 / Toxicon 68 (2013) 60–123 82 Purpose of study: The work was designed to localize the regions employed by botulinum neurotoxin type B (BoNT/B) for binding to mouse brain synaptosomes (snp). Methods used: Sixty 19-residue overlapping peptides (peptide C31 was 24 residues) encompassing BoNT/B H chain (residues 442-1291) were synthesized chemically and used to inhibit 125I-labeled-BoNT/B binding to mouse brain snp. Summary of results: Synaptosome-binding regions were noncompeting and existed on both HN and HC BoNT domains. At 37oC, inhibitory activities on HN resided, in decreasing order, in peptides 638–656 (26.7%), 596–614 (18.2%), 512–530 (13.9%), 778–796 (13.8%) and 526–544 (11.6%). On HC, peptide 1170-1188 displayed highest activity (44.6%) followed by peptides 1128-1146 (21.6%), 1184-1202 (18.6%), 1156-1174 (13.0%), 946-964 (11.8%), 1114-1132 (11.2%), 1100-1118 (6.2%), 876-894 (6.1%), 1268-1291 (4.6%) and 1226-1244 (4.3%). The remaining 45 HN and HC peptides had no activity. At 4oC, peptide C24 (1170-1188) retained high activity (inhibiting, 31.2%), while activities of each peptides N15, C21 and C25 fell to slightly under 10%. The snp-binding regions overlapped with sites that bind synaptotagmin II and gangliosides. Despite low sequence homology, BoNT/B and BoNT/A snp-binding regions display significant structural homology. In the BoNT/B 3D structure, the greater part of HC, one HN face and part of the belt on the same side participate in interaction with snp. Conclusions: BoNT/B binds to the cell membrane through the HC head and employs regions on one HN face and the belt, reserving flexibility for the belt's unbound part to release the light chain. Most snp-binding regions coincide or overlap with blocking-antibody-binding regions, explaining how such antibodies prevent BoNT/B toxicity. Supported by theWelch Foundation and an unrestricted grant from Allergan. http://dx.doi.org/10.1016/j.toxicon.2012.07.070 Effects of botulinum toxin type A on digit abduction score and running wheel assays and assessment of diffusion using calcium-activated potassium channel expression M. Auguet, C. Favre-Guilmard, S. Cornet, D. Carre, M.N. Rocher, B. Pignol, B. Pham, P.E. Chabrier Ipsen Innovation, Les Ulis, France. E-mail address: michel.auguet@ipsen.com (M. Auguet). Purpose of study: Injection of BoNT/A in the mouse hind limb produces local weakening which can be evaluated by the Digit Abduction Score (DAS assay) or by continuous measurement in the running wheel. The purpose of the study was to compare the effect of BoNT/A on these two models. In addition expression of a small conductance calcium-activated potassium channel, SK3, has been used as a tool to assess BoNT/A diffusion. Methods used: Male CD1 mice were used in the two models. abobotulinumtoxinA (Dysport , Ipsen Biopharm, Wrexham, UK) was injected into the right gastrocnemiussoleus complex of mice under light isoflurane anesthesia. DAS assay consisted of evaluating the abduction of the digits of the hind paw, whereas the running wheel assay measured the distances covered during a night cycle period of 12 hr over a period of one month. Protocol and analysis were designed to allow determination of different parameters such as onset, time of maximal effect, recovery and global efficacy. The level of the expression of SK3 in gastrocnemius and tibialis anterior muscles was quantified by Western blot analysis 14 days after treatment with 2 U/mouse in satellite mice from the running wheels assay. Summary of results: The two models resulted in a dose-related effect/duration with comparable sensitivity in term of ED50 (0.3-0.4 U/mouse). However, maximal score in the DAS assay was reached at a lower dose than the one inducing maximal inhibition of the running distance. The DAS assay is more qualified to define the onset of effect, which is also dose-related, whereas the running wheel is convenient to assess the global efficacy and recovery. Injection of a dose (2 U/mouse) that abolishes the covered distances on activity wheels results in a two-fold increase in expression of SK3 in the gastrocnemius, but not in the tibialis anterior, indicating a lack of diffusion of the toxin. Conclusions: The two models are complementary to characterize the activity of a BoNT/A. http://dx.doi.org/10.1016/j.toxicon.2012.07.071 Molecular pharmacophores against BoNT/B C. Chambers, G.E. Garcia Physiology and Immunology Branch, Research Division, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD, USA E-mail address: carolyn.chambers1@us.army.mil (C. Chambers). Purpose of study: To design pharmacophores by molecular modeling to identify inhibitory compounds against botulinum neurotoxin type B (BoNT/B) binding to neuronal cells. Blocking the binding of toxin to the target cell would render BoNT harmless. Methods used: Computer-based surface analyses were conducted against three pockets on the surface of the heavy chain binding domain of BoNT/B using the Molecular Operating Environment (MOE), with virtual screening of commercially available compounds and small molecule databases from the National Cancer Institute Developmental Therapeutics Program. Thirty compounds per pocket were evaluated as potential inhibitors that would prevent binding of the binding domain to its ganglioside and/or protein receptor. Summary of results: The combined screening of more than 12 million compounds using the computer-based surface MOE virtual screening analysis yielded 60 small molecule compound potential inhibitors from simulations that used a five-point pharmacophore docked into pocket1, the ganglioside GT1b binding site, and yielded 30 small molecule potential inhibitors from simulations that used a four-point pharmacophore docked into pocket-4, the protein receptor synaptotagmin binding site. We tested

The alpha 1 adrenoceptor subtypes mediating contraction of rabbit prostate and hypogastric artery were pharmacologically characterized using an isolated organ bath technique. The prostate had the same sensitivity to the contractile action of methoxamine and phenylephrine, whereas the hypogastric artery was five times less sensitive to the action of methoxamine in comparison with phenylephrine. Clonidine elicited contraction in the hypogastric artery but not in the prostate. BMY7378 was about 70-fold more potent to antagonize the phenylephrine-induced contraction in the hypogastric artery (pA2 8.14) than in the prostate (pA2 6.28), and 5-methyl-urapidil was about three-fold more potent on prostrate than on hypogastric artery. The potency of different alpha 1-adrenoceptor antagonists tested in the rabbit prostate was significantly correlated with their binding affinity for the expressed recombinant alpha 1A-, but not alpha 1B- or alpha 1D-, adrenoceptor subtype, whereas, the potency of the alpha 1-adrenoceptor antagonists tested in the rabbit hypogastric artery was better correlated with the defined alpha 1D-adrenoceptor. Chloroethylclonidine produced a 10-fold rightward shift in the phenylephrine concentration-response curve in the hypogastric artery but only had a weak effect in the prostate. The results indicate that significant heterogeneity exists among alpha1-adrenoceptor in the rabbit hypogastric artery (alpha 1D-adrenoceptor) and the prostate (alpha 1A-adrenoceptor).

We investigated the constrictor effects of Angiotensin I (Ang I) and Angiotensin II (Ang II) on rabbit peripheral (aorta, carotid artery, mesenteric artery, saphenous artery) and cerebral (basilar artery) vessels and in rat aorta in functional organ bath studies. The effect of angiotensin converting enzyme (ACE) inhibition by captopril was also assessed in these preparations. Ang II elicited concentration-dependent contractions with comparable potency in rabbit and rat endothelium-free vascular rings (pD2 about 8.5) which indicates a lack of species and regional variation in the contractile responses to Ang II. The responses to Ang II were reduced by the presence of a functional endothelium in rabbit mesenteric artery and in rat aorta. Since ACE determines the plasma and tissue conversion of Ang I to active Ang II, we calculated the ratio R (EC50 Ang I-induced contraction: EC50 Ang II-induced contraction) as an indicator of the tissue ACE effectiveness. In the aorta without endothelium, Ang I was found to be much less potent than Ang II in the rabbit (R = 44) compared with the rat (R = 3.5). This species difference in the aortic conversion of Ang I to Ang II was confirmed by the use of captopril. Captopril (10(-6) M) shifted the Ang I concentration/ response curve by 2- and 14-fold to the right in rabbit and rat respectively. In other rabbit blood vessels, the rank order of potency to Ang I in endothelium denuded rings was basilar artery > > carotid artery > or = aorta > or = saphenous artery. In addition, the R value was the lowest for the basilar artery (R = 2.5). This is in agreement with the highest rightward shift (78-fold) of the Ang I concentration/response curve by captopril for basilar artery in comparison with only 3-, 8- and 3-fold shifts observed in carotid artery, saphenous artery and aorta respectively. In conclusion, our data provide evidence for a greater influence of ACE in rabbit basilar artery than in peripheral vessels.

The reactivity and the structure as well as the growth of the vascular wall depend on a variety of locally synthetised factors in a process of a permanent structure-function adaptation. These substances exert their inhibitory or stimulatory growth effects by paracrine or autocrine mechanisms. These factors command the reorganisation of the structure of existing blood vessel or the creation of new vessels. They are synthetised and secreted form either endothelial and smooth muscle cells or circulating cells (in particular macrophages, platelets). The growth factors are multiple and interactive insuring a role of physiological vascular modeling in normal conditions but they may participate and even induce dramatic structural dysfunctions that are observed in pathologies such as venous diseases, atherosclerosis or hypertension. Among them, the polypeptides PDGF (platelet derived growth factor), FGF (fibroblast growth factor) and TGF beta (transforming growth factor beta) play a major role. Other factors like cytokines, IGFs (insulin like growth factors), PAF (platelet activating factor) endothelins or nitric oxide have also to be considered. Thus, the vascular wall structure is under the influence of a complex group of growth factors which become to be identified and may be the targets of new therapies of the vessels.

In recent years, endothelial dysfunction and arterial remodelling in various cardiovascular diseases have emerged as two key concepts, with numerous interrelationships. Both endothelial dysfunction and arterial remodelling occur in various pathologies including heart failure, atherosclerosis, restenosis after angioplasty, and pulmonary hypertension, and have modified the therapeutic approach by offering new pharmacological targets: specific receptors not only at the site of the vascular smooth muscle cells but also on the endothelial cells, growth factors that stimulate proliferation of smooth muscle, and receptors and enzymes of the extracellular matrix. Among the various substances under research, the present review will discuss angiotensin II receptor antagonists, endothelin receptor antagonists, nitrates-NO donors, potassium channel activators, and substances interfering with proteoglycans and other components of the extracellular matrix.

In the central nervous system, several cellular types are able to produce nitric oxide (NO). In particular in neuronal cells, the excitatory amino-acid receptor activation induces NO synthesis and release. Since excessive activation of these receptors is responsible of neuronal death in excitotoxicity or cerebral ischemia, the hypothesis of a NO role in neuronal damage has been proposed. The use of NO synthesis inhibitors in excitotoxicity or cerebral focal ischemia models has provided contradictory data. Here we attempt to present the main bibliographic data concerning this controversial research area in order to a better comprehension of NO role in neuronal death.

Dysfunctions of EDRF/L-arginine-NO pathway have been demonstrated in genetic and experimental hypertension. NO is produced through the conversion of L-arginine to L-citruline by NO synthases (NOS) which exist at least in two isoforms. The first termed constitutive (NOSc) and located in the endothelium of the vascular wall results in the basal and stimulated NO production. The second termed inducible (NOSi), which produces large amounts of NO, can be expressed in both smooth muscle and endothelial cells. The aim of the study was to examine and compare in isolated aortic rings of WKY and SHR rats, the activity of the two isoforms of endothelial NO synthases and their influence on the constrictor response induced by angiotensin II. On phenylephrine preconstricted endothelium intact aortic rings (10(-6) M, WKY = 1.2 +/- 0.04 g; SHR = 1.2 +/- 0.07 g; n = 7), carbachol (10(-5) M) induced a greater relaxation in WKY (84 +/- 2.5%) than in SHR (63 +/- 8.5%) rat. This suggests the presence of a low NOSc stimulated activity in the hypertensive strain. When the incubation period was limited to 30 min after equilibration period, L-arginine (3.10(-4) M) did not induce relaxation. When the incubation period was prolonged (180 min), L-arginine induced a relaxation (WKY = 75 +/- 8%; SHR = 58 +/- 10%; n = 7). This relaxation was not observed in a medium containing actinomycin D (10(-6) M) or after endothelium removal, indicating the induction of an endothelial NOSi in the two strains.(ABSTRACT TRUNCATED AT 250 WORDS)

The potent pulmonary vasodilating property of atrial natriuretic factor (ANF) may alter gas exchange in patients with COPD. We examined the hemodynamic and gas exchange responses to intravenous infusion of ANF (0.01 and 0.03 ng/min/kg body weight) in eight stable patients with COPD studied during spontaneous breathing, using the inert gas elimination technique. When compared with baseline, ANF infusion was associated with a dose-dependent decrease in pulmonary artery pressure (from 27.3 +/- 2.5 to 23.9 +/- 1.8 and 20.2 +/- 1.7 mm Hg, respectively) and a dose-dependent increase in blood flow perfusing poorly ventilated and unventilated units (VA/Q0.1: from 5.80 +/- 2.05 to 7.25 +/- 2.5 and 12.0 +/- 5.4 percent of total blood flow, respectively; p = 0.02). However, PaO2 remained unchanged (70.2 +/- 3.6, 68.1 +/- 3.8 65.4 +/- 3.5 mm Hg, respectively) because of a significant increase in minute ventilation (VE) from 8.6 +/- 0.8 to 9.6 +/- 0.8 and 10.3 +/- 0.7 L/min (p0.002). Six additional COPD patients receiving intravenously administered ANF at the same dosages were studied during controlled mechanical ventilation using right heart catheterization. In these patients, pulmonary vasodilation was associated with a significant increase in venous admixture (from 12.7 +/- 2.4 to 14.4 +/- 2.9 and 17.5 +/- 3.5 percent of total blood flow, respectively; p0.02), and a dose-dependent reduction in arterial PO2 (from 117 +/- 17 to 110 +/- 15 and 96.4 +/- 8.8 mm Hg, respectively; p0.05). The present results show that ANF infusion is associated with alterations in the VA/Q relationship in patients with COPD. However, a decrease in arterial oxygenation may be prevented by an increase in VE.

The venous endothelium, acting as an interface between the blood and the layer of smooth muscle cells, plays a major role in many essential functions of vessel physiology. The endothelium prevents and inhibits pathological activation of the coagulation system and plays an active role in the regulation of venous tone. Mediators include the prostacyclin which has vasodilating and antiagregating properties and nitric oxide (NO), a vasodilator and inhibitor of thrombosis, adhesion and cellular proliferation. To maintain endothelial integrity and tissue cohesion, endothelial cells express adhesion factors which regulate cell to cell and cell to extracellular matri interactions. Finally, the endothelium plays a role in vasotrophism by synthesizing, or activating growth factors which act directly on cell renewal of the endothelium itself and proliferation of adjacent tissues. Thus, the venous endothelium cannot be considered simply as a biologically inert barrier but rather as an active cell layer capable of intercell signalling and interactions constantly modified by the pathophysiological environment.

The two endothelin (ET) receptor subtypes (ETA and ETB) have been characterized in rat kidney from normal rats and rats with acute renal failure induced by hypertonic glycerol administration. In control rats, the total number of ET receptors in kidney cortex and medulla was 155 and 386 fmol/mg of protein, respectively. The ratio of ETA to ETB receptors was 54:46 in renal cortex and 35:65 in renal medulla. Treatment of rats with 10 ml/kg glycerol (50%, w/v) intramuscularly resulted in severe renal dysfunction; the serum urea concentration increased from 0.46 to 2.65 g/liter and the creatinine clearance decreased from 1.06 to 0.30 ml/min. Ligand binding studies showed that glycerol-induced acute renal failure was associated with a marked up-regulation of ETA and ETB receptor subtypes in both cortex and medulla. In glycerol-treated rats, the total ET receptor density in kidney cortex and medulla was increased to 294 and 1172 fmol/mg of protein, with ETA/ETB ratios of 52:48 and 31:69, respectively. The upregulatory effect of glycerol treatment was significantly more pronounced in renal medulla than renal cortex and affected ETB receptors preferentially, compared with ETA receptors. Subsequently, ETA and ETB receptor mRNA levels were markedly increased by glycerol administration in both kidney cortex and medulla, as assessed by polymerase chain reaction coupled to reverse transcription. These results suggest that up-regulation of renal ET receptors, particularly ETB receptors in kidney medulla, may account for or contribute to renal function impairment induced by glycerol, and they support a pathophysiological role for ET in acute renal failure.

Endothelins (ETs) are a family of vasoactive peptides occurring in three isoforms (ET-1, ET-2, ET-3) encoded by three distinct genes in the human genome. ETs arise from precursor peptides (big-ETs) that are cleaved and released by an endothelin-converting enzyme. ET-1 secretion, which can be stimulated by various agents, is preferentially directed towards the abluminal site of endothelial cells, suggesting a local paracrine action of the peptide. ETs exert their actions through the activation of at least two receptor subtypes: ET-A receptors, which mediate the proliferative and vasoconstrictive effects, and ET-B receptors, which mediate vasorelaxation. Although, the potential roles of ETs are mostly hypothetical, considering their potent cardiovascular effects, it has been suggested that maintenance of a basal vascular tone and regulation of vascular growth and haemostasis may well represent the biological functions of this family of peptides. The recent discovery of specific receptor antagonists will provide a means to assess their physiological and pathophysiological roles.

Binding and internalization of the synthetic double-stranded complex poly(A).poly(U) were studied on a murine monocyte/macrophage cell line J774A1. Poly(A).poly(U) increased in a dose-dependent fashion the oligo-2',5'-adenylate synthetase demonstrating that those cells were responsive to this agonist. Binding of [32P]poly(A).[32P]poly(U) to the cells reached an apparent kinetic equilibrium within 4 h and was saturable (apparent Kd = 9.99 +/- 0.09.10(-2) g/l and Bmax 13.3 +/- 5.3.10(-3) g/l per 10(6) cells) and temperature-dependent. The binding of poly(A).poly(U) was competitively inhibited by various polynucleotides but not by other structurally unrelated compounds. Analysis of cell-associated [32P]poly(A).[32P]poly(U) demonstrated a minimal degradation of this polyribonucleotide over a 4-h incubation period. Autoradiography of cells incubated with [3H]poly(A).[3H]poly(U) revealed that poly(A).poly(U) was internalized and migrated to cell nuclei. These results suggest that poly(A).poly(U) is internalized in J774A1 cells via an endocytotic process.

To study the involvement of endogenous endothelin (ET) in the development of cerebral ischemia, we measured by radioimmunoassay brain tissue content of immunoreactive (ir)-ET-1 in a model of focal cerebral ischemia in the rat. Permanent occlusion of the middle cerebral artery (OMCA) was accompanied after 24 h by a progressive but marked elevation of ir-ET-1 in the ipsilateral compared with the contralateral hemisphere (119% after 24 h; 184% after 48 h and 459% after 72 h). The pial vessels and the arteries of the circle of Willis did not respond with ir-ET-1 production. The increase in ir-ET-1 content in tissues was first observed in the caudate nucleus (after 24 h) and later in the cortex (after 48 h), which was more variably injured. Transient ischemia followed by recirculation led to a slight increase of ir-ET-1, which also appeared after 24 h of recirculation. This study demonstrates that during permanent OMCA, the tissue content of ir-ET-1 markedly and progressively increases, whereas less severe ischemia (transient) is accompanied by a modest elevation of ir-ET-1 levels. These results suggest that endogenous ir-ET-1 production is involved in the development and the severity of ischemic injury.

Acute renal failure was induced in rat with a hypertonic glycerol solution and endothelin-1 (ET-1) binding was measured in kidney membrane preparations. In control animals, [125I]-ET-1 bound to specific recognition sites in kidney cortex (Bmax = 134 +/- 11 fmol/mg protein) and medulla (Bmax = 300 +/- 9 fmol/mg protein) with an apparent dissociation constant (Kd) of 0.16 +/- 0.06 nM and 0.39 +/- 0.07 nM for cortex and medulla, respectively. A single i.m. dose of 10 ml/kg glycerol (50% w/v) resulted in alterations of renal function that were maximal 48 h after glycerol administration. After this 48-h period, serum urea was increased from 0.20 +/- 0.01 g/L to 1.16 +/- 0.20 g/L (p0.001) and creatinine clearance was reduced from 1.04 +/- 0.15 ml/min to 0.23 +/- 0.06 ml/min (p0.001). Renal ET-1 receptor density was significantly increased in glycerol-treated rats to 255 +/- 14 fmol/mg protein in renal cortex (p0.01), and 576 +/- 55 fmol/mg protein in renal medulla (p0.01), with no significant modification of the Kd values. These results suggest that upregulation of ET-1 receptors is involved in renal hemodynamic impairment induced by glycerol.

Nitric oxide (NO) is a free radical molecule which has been described to play a role as a messenger molecule in at least three systems: white blood cells, blood vessels and most recently in the nervous system. In the brain, NO is produced enzymatically in postsynaptic structures in response to activation of excitatory amino acid receptors. A major action of NO is to activate soluble guanylate cyclase and to raise cGMP level in target cells. The role of NO as a messenger in long-term potentiation and in long-term depression has been established and recent studies have directly implicated NO in neuronal damage associated with vascular strokes. Concerning the role of NO in the excitatory amino acid neurotoxicity, more studies will be necessary to elucidate the implication of NO mediating neuronal damage. Whatever the exact function of NO, it is sure that this substance play an important role in the brain and that pharmacological manipulations of NO pathway will constitute a novel approach for therapeutical applications in the future.