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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.

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.

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.

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.

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.

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.

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.

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.

Inducible nitric oxide (NO) synthase in vascular smooth muscle cells (SMCs) appears to play a major role for the diminished responsiveness to vasoconstrictors observed in endotoxemia. However, cardiovascular dysfunctions associated with septic shock are also observed in the absence of endotoxin (LPS). Similar hemodynamic changes are produced either by a gram-negative bacteria (Escherichia coli) or by a gram-positive bacteria (Staphylococcus aureus), a microorganism without LPS, suggesting a common pathway leading to cardiovascular abnormalities. In the present study, we describe the induction of NO synthase in vascular SMCs by lipoteichoic acid (LTA), a component of the membrane of gram-positive bacteria. In cultured vascular SMCs, a 24-h incubation with LTA produced an increase in intracellular cyclic GMP. This effect was inhibited by methylene blue (MB), an inhibitor of guanylate cyclase. Incubation with a specific inhibitor of L-arginine, i.e., NG-nitro-L-arginine methyl ester (L-NAME), or depletion of L-arginine attenuated the LTA-induced cGMP production. A 5-h incubation of endothelium-free rings of rat aorta in the presence of LTA induced a loss of tonicity to the contractile response of phenylephrine. The contractions were restored by MB and by L-NAME. The effect of L-NAME was reversed by L-arginine. These results show that LTA, like LPS, expresses NO synthase in vascular SMCs.

Circular dichroism of endothelin-1 synthesized through a continuous flow process reveals, when dissolved in water, a strong concentration dependence of the spectrum. Furthermore, the general feature of the spectrum rules out the possibility of the existence of any alpha and beta structures. In addition, surface tension and conductivity measurements suggest that the peptide aggregates through formation of micelles.

It has been shown that in vitro angiotensin II (Ang II) potently downregulates endothelin-1 (ET-1) binding sites. In this study, we investigated in vivo the interactions between the renin-angiotensin system and ET-1. Sprague-Dawley rats were pithed, ventilated, and diastolic blood pressure (DBP) was recorded. ET-1 (1 nmol/kg) induced a biphasic response: a transient hypotension followed by a sustained increase of DBP. Captopril (5 mg/kg, i.v.) or Sar1-Ile8-Ang II (10 ng/kg/min) did not affect ET-1 responses. In other experiments, DBP was increased by infusion of methoxamine (MTX: 10, 20, 25, 32.5 micrograms/kg/min) or Ang II (50, 100, 150, 200 ng/kg/min). ET-1-induced hypotension correlated with the level of DBP (r = 0.94) for both agonists. The elevation of DBP in response to ET-1 was also related to the vascular tone but was dose-dependently attenuated by Ang II infusion when compared with MTX. Conversely, infusion of ET-1 (0.1 nmol/kg/min) blunted the pressor response to Ang II (0.1 micrograms/kg) but not to MTX (50 micrograms/kg). These doses induced the same increase of DBP in pithed control rats. Similarly, increased plasma renin activity induced by chronic salt depletion (0% NaCl) in pithed rats provoked a shift to the right of the dose-response curves to Ang II and ET-1 but not to MTX. These results suggest an in vivo cross-desensitization between ET-1 and Ang II.

The existence of distinct endothelin (ET) receptor subtypes has been reported in several tissues. In the present study, we investigated the binding characteristics of the three endothelin isoforms to cultured rat aortic smooth muscle cells. [125I]ET-1, [125I]ET-2, and [125I]ET-3 bound to an apparent single class of binding sites with apparent dissociation constants (Kd) of 111, 123, and 1410 pM and binding capacities (Bmax) of 54.1, 46.0, and 7.9 fmol/10(6) cells, respectively. The binding of the three radiolabeled endothelin isoforms was equally inhibited by ET-1 and ET-2. ET-3 was more effective in competing with [125I]ET-3 than with [125I]ET-1 or [125I]ET-2. In contrast to ET-1 and ET-2, the binding of ET-3 was reversible. Furthermore, 18 h of pre-exposure of the cells to 1 nM ET-1 or ET-2 decreased the ET-1 binding capacity, whereas ET-3 (10 nM) was ineffective. ET-3 binding characteristics were not affected by pretreatment of the cells with any of the endothelin isoforms. These results suggest the presence of two distinct endothelin receptor subtypes in rat aortic smooth muscle cells. The ET-1 and ET-2 preferring receptor (80-85%), sensitive to downregulation or internalization, elicits an irreversible binding. The second subtype (15-20%) binds the three endothelin isoforms with the same affinity in a reversible manner, and is insensitive to downregulation or internalization.

The pulmonary vascular reactivity to endothelin-1 (ET-1) was assessed in rats previously exposed to 11% O2 (hypoxic) or room air (controls) for 3 weeks. In isolated control lung preparations studied during conditions of increased tone by U46619 (50 pmol/min) and treated with meclofenamate (3 microM), low doses of ET-1 (30 and 100 pM) reduced the pressor response to U46619 by 58 +/- 5% (p less than 0.01). Vasodilation induced by ET-1 was not abolished by the antagonist of endothelium-dependent relaxing factor (EDRF) NG-monomethyl-L-arginine (5 x 10(-4) M), which suppressed vasodilator response to ionophore A23187 (10(-8)-10(-7) M). Higher doses of ET-1 (300 and 1,000 pM) induced vasoconstriction during conditions of basal tone, and the pressor response to 300 pM ET-1 was enhanced by EDRF antagonists. Administration of ET-1 to lungs from hypoxic rats failed to cause pulmonary vasodilation and instead induced a greater pulmonary pressor response (300 pM) than in control rat lungs (7 +/- 1.5 vs. 1.6 +/- 0.5 mm Hg, p less than 0.01), which was not further potentiated by EDRF antagonists. Infusion of 300 pM ET to conscious catheterized animals induced a sustained increase in pulmonary resistance only in the hypoxic group (from 305 +/- 37 to 389 +/- 55 mm Hg/L/min, p less than 0.01) (n = 7). The results suggest that depending on the dose, ET-1 can cause pulmonary vasodilation (independent of EDRF release) or vasoconstriction (opposed by EDRF). During chronic hypoxic pulmonary hypertension, ET-1 behaves only as a pulmonary vasoconstrictor.

Glutamate excitotoxicity, oxidative stress, and mitochondrial dysfunctions are common features leading to neuronal death in cerebral ischemia, traumatic brain injury, Parkinson's disease, Huntington's disease, Alzheimer's disease and amyotrophic lateral sclerosis. Nitric oxide (NO) alone or in cooperation with superoxide anion and peroxynitrite is emerging as a predominant effector of neurodegeneration The use of NO synthase (NOS) inhibitors and mutant mice lacking each NOS isoform have provided evidence for the injurious effects of NO derived from neuronal or inducible isoforms. New neuroprotective strategies have been proposed with selective NOS inhibitors for the neuronal (ARL17477) or the inducible (1400 W) isoforms or with compounds combining in one molecule selective nNOS inhibition and antioxidant properties (BN 80933), in experimental ischemia-induced acute neuronal damage. The efficacy of these new strategies is well established in acute neuronal injury but remains to be determined in more chronic neurological diseases.

BN 52021, a new specific PAF-acether receptor antagonist, was evaluated on several cardiovascular models. BN 52021 antagonized PAF-acether-induced extravasation in rats. Inhibition of the hypotensive action of PAF-acether was obtained by administration of the antagonist, given preventively or curatively. In isolated guinea-pig hearts, BN 52021 inhibited the vasoconstriction induced by PAF-acether whereas a small inhibition was observed with papaverine. On the other hand, phosphodiesterase inhibitors were very effective against coronary vasoconstriction induced by vasopressin while BN 52021 was without effect. PAF-acether increased the tonus of rat isolated portal vein; this effect was inhibited by BN 52021, without any reduction in basal myogenic activity. In this model Ca2+ antagonists (D 600, diltiazem) showed a small inhibitory effect but they strongly reduced basal myogenic activity. Neither PAF-acether nor BN 52021 modified phenylephrine-induced contraction of the isolated rabbit aorta with or without endothelium demonstrating that endothelium-dependent relaxing factor is not related to PAF-acether. Our results suggest that BN 52021 specifically block the cardiovascular effects of PAF-acether. This agent may thus be an useful tool for a better understanding of the role of PAF-acether in hemodynamic changes involved in anaphylaxis or shock.

Methyl-1 (S-methylphosphoryl-3) imidazolium (MSPI) was approximately equipotent with DFP in producing in mice a lethal intoxication and in inhibiting in vitro cholinesterases of electric-eel, brain and plasma mice. In vivo, a lethal dose of MSPI inhibited plasma but not brain ChE. The intoxication by MSPI was not cured by the combination of Atropine and TMB4 or of diazepam with Atropine and (or) TMB4 and the cholinesterase inhibition was not reversed by TMB4 in vivo nor in vitro in the conditions where DFP intoxication or ChE inhibition were.

The different methods used to explore the blood-brain barrier (made up of cerebral capillary vessels), and notably, at molecular level, isolated microvessel preparations, have greatly improved our knowledge in this particular field. Some of these methods could be used to evaluate the protective effects of therapeutic substances, such as Ginkgo biloba extract, on the blood-brain barrier.

Smooth muscle cells were cultured from guinea-pig aorta and labelled with 45Ca++ and 32Pi to investigate the possible effect of cicletanine, a new antihypertensive drug, on the release of intracellular Ca++ and the metabolism of phosphoinositide induced by histamine. In 45Ca++ labelled cells, histamine increased in a dose-dependent manner the 45Ca++ efflux in the first two minutes. Stimulation of 45Ca++ release was observed with H1-agonist [2-pyridylethylamine dihydrochloride (2-PEA)] but not with H2-agonist (dimaprit). In addition, histamine- or 2-PEA- induced 45Ca++ efflux was inhibited by the H1-antagonists (mepyramine and terfenadine) whereas the H2-antagonist (cimetidine) was without effect. Similar results were obtained in 32Pi labelled cells; both H1-agonists (histamine and 2-PEA) increased the labelling of phosphoinositides. This effect was completely blocked by mepyramine. These results demonstrate that the histamine-induced stimulation of 45Ca++ efflux and phosphoinositide metabolism are mediated through H1-receptors. In the above systems, cicletanine was as effective as the H1-antagonist (mepyramine) with an IC50 of 10(-6) M for both 45Ca++ efflux and phosphoinositide metabolism. Blockade of these systems by cicletanine may be part of the mechanism by which this drug produces relaxation of blood vessels and may account for its in vivo antihypertensive action.