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Though Turks and Afghans are located far off geographically yet it may not be perceived as a marker of friction between the two nations. The Islamic history in the middle Ages reveal that Islam has spread in the Turkish and Afghan regions amongst the indigenous tribes heralded a marked cultural and linguistic influence in the cited regions. The Turks' contact with the Islamic world started in the era of four Caliphs and the Umayyad dynasty reaching out to the western regions of the "Bab" region adjacent to Turkish territory in 22 AH. This contact resulted in the spread of Islam amongst the Turks and their men took part as part of Muslim army leading to propagation of Islam. Moreover, In the Abbasid era, the number of Turks assuming leadership positions had increased in the Muslim state. This contact sustained in the Islamic world to the East in the Turkish areas after the Muslims crossed the JIHUN River (Amu Darya) in 31 AH under the leadership of QUTAYBA Ibn Muslim. Thus Islam spread among the Turk Tribes under the rule of the SAMANI Governors where they spearheaded the extension of Muslim influence to Tirmiz, Bukhara, Samarkand and other Central Asian countries due to the simplicity of Islam. As for the Afghans' contact with Islam in the West, it began in the era of four Caliphs and the Umayyads eversince Herat was conquered in the year 23 AH; Jawzjan, Balkh and Takhar in the year 32 AH by Al-Ahnaf bin Qais from northern Afghanistan; Zabul in the year 43 AH and Kabul in 44 AH by Abd al-Rahman bin Samra. However the eastern areas of Afghanistan and the Khyber Pakhtunkhwa in Pakistan, although Islam reached there in the first century, did not enter the Islamic rule. They remained either under the tribal control or were ruled by the Indian kings until the Ghaznavids took over through Prince Subuktagin in Ghazni in the last decade of the 4th century. There has remained direct contact between the Turks and Afghans after the displacement of the Turkish tribes to the Gazani state in the south of the Jihun River.The state sovereignty of the Turkish majority was due to their leadership positions and a large part of the military formation included the Turks and Afghans. Hence the two nations lived with a clear linguistic and cultural influence with hundreds of basic words in common as for instance the Turkish names for Afghan men and women and dozens of Turkish names for villages and cities. There are even common tribal traditions and customs between the two peoples. This research addresses the historically direct contact between the Turkish and Afghan tribes with cultural and social impact between the Turks and Afghans.

Zinc oxide nanoparticles (ZnO NPs) exhibit diverse applications, including antimicrobial, UV-blocking, and catalytic properties, due to their unique structure and properties. This study focused on the characterization of zinc oxide nanoparticles (ZnO NPs) synthesized from Juglans regia leaves and their application in mitigating the impact of simultaneous infection by Meloidogyne arenaria (root-knot nematode) and Macrophomina phaseolina (root-rot fungus) in cowpea plants. The characterization of ZnO NPs was carried out through various analytical techniques, including UV–visible spectrophotometry, Powder-XRD analysis, FT-IR spectroscopy, and SEM-EDX analysis. The study confirmed the successful synthesis of ZnO NPs with a hexagonal wurtzite structure and exceptional purity. Under in vitro conditions, ZnO NPs exhibited significant nematicidal and antifungal activities. The mortality of M. arenaria juveniles increased with rising ZnO NP concentrations, and a similar trend was observed in the inhibition of M. phaseolina mycelial growth. SEM studies revealed physical damage to nematodes and structural distortions in fungal hyphae due to ZnO NP treatment. In infected cowpea plants, ZnO NPs significantly improved plant growth parameters, including plant length, fresh mass, and dry mass, especially at higher concentrations. Leghemoglobin content and the number of root nodules also increased after ZnO NP treatment. Additionally, ZnO NPs reduced gall formation and egg mass production by M. arenaria nematodes and effectively inhibited the growth of M. phaseolina in the roots. Furthermore, histochemical analyses demonstrated a reduction in oxidative stress, as indicated by decreased levels of reactive oxygen species (ROS) and lipid peroxidation in ZnO NP-treated plants. These findings highlight the potential of green-synthesized ZnO NPs as an eco-friendly and effective solution to manage disease complex in cowpea caused by simultaneous nematode and fungal infections.

The process of using dielectric fluid combined with different types of powders to improve the output of the machined surface is known as powder-mixed electrical discharge machining (PMEDM). In the electrical discharge machining (EDM) industry, this procedure is quickly gaining popularity. This investigation's goal is to ascertain whether tantalum carbide (TaC) powder-mixed dielectric fluid can reduce tool wear during the subsequent EDM machining of stainless-steel material. Two different EDM medium's tool wear rates and mathematical models were investigated during the machining. TaC powder at a concentration of 25.0 g/L in kerosene dielectric fluid was used for the machining process. The peak current, pulse on time, and pulse off time were the machining variables used. These variables' effects on the copper based EDMed electrode tools TWR were identified. The TWR for stainless steel (SUS 304) during electrical discharge machining was reduced by 37.9% when TaC powder additive was used, according to the results, demonstrating the effectiveness of this alternative method for reducing tool wear. The most influential factor, according to the tool wear ratio model for EDM with TaC powder additive (TWRPMEDM), is current, followed by pulse on-time and pulse off-time.

The Islamic conquests have expanded and Islam has spread across the globe from the beginning of Islam, while in The Land of India, the conquests of Muslims had started during the reign of the Guided Caliphs, including the “Muhallab” attack of the southern areas of Khyber Pakhtunkhwa, thus being considered the first of the Muslim invaders in India. The land of Sind was conquered by Muhammad bin Qasim, hence the rays of the light of Islam spread in Sindh and some areas of India in the first century, but most of the Indian territories were still under the Indian pagan majority and under Indian kings. However, the invaders did not exceed the borders of Kabul and Sindh, except the Ghaznavids, when Prince Subaktagin captured Ghazni, and other Afghan lands of Kabul, Laghman, etc in the late 4th century, thus he opened the gateway to India for those after him especially his son, Mahmud. The first conquest of the ruler of Ghazni was in Peshawar, Khyber Pakhtunkhwa in 1001 AD, and then followed by conquests in central and south India and lasted for more than twenty years. These conquests are famous and prominent, for their dynamic impacts. These conquests, in terms of their causes and objectives, are suspected by some Muslims and distorted by non-Muslims and have concealed their steps and features in terms of Islamic Da'wah. The questions raised here are: What are the causes and objectives of these conquests? Has it had a moral effect on spreading the call of Islam? And what steps have been taken for that? And what were its features? This paper aims to study these questions and to explain the causes of these conquests and uncover some doubts about them. And to highlight the milestones of the peaching of Islam and steps are taken in this regard.

Efficient access to clinical data by investigators is critical for data-driven biomedical research. Mediated query is commonly adopted to facilitate data access for investigators. However, the query mediation process remains a black-box. This study analyzed the email-based dialogues between investigators and clinical data query mediators from three institutions. We identify discussion topics and their frequencies, model task flows, and analyze user needs for query mediation support revealed from the dialogues. While the datasets between different institutions are distinct in some notable respects, we find that together they provide common insights for streamlining data access. From our findings, we conclude an intelligent dialogue-based query support model is feasible to automate human-mediated clinical data access for investigators and stakeholders.

Triggering receptor expressed in myeloid cells (TREM2) is a member of the immunoglobulin superfamily and is expressed in macrophages, dendritic cells, microglia, and osteoclasts. TREM2 plays a role in phagocytosis, regulates release of cytokine, contributes to microglia maintenance, and its ectodomain is shed from the cell surface. Here, the question was addressed at which position sheddases cleave TREM2 and what are the proteases involved in this process. Using both pharmacological and genetic approaches we report that the main protease contributing to the release of TREM2 ectodomain is ADAM17, (a disintegrin and metalloproteinase domain containing protein, also called TACE, TNFα converting enzyme) while ADAM10 plays a minor role. Complementary biochemical experiments reveal that cleavage occurs between histidine 157 and serine 158. Shedding is not altered for the R47H-mutated TREM2 protein that confers an increased risk for the development of Alzheimers disease. These findings reveal a link between shedding of TREM2 and its regulation during inflammatory conditions or chronic neurodegenerative disease like AD in which activity or expression of sheddases might be altered., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: The fractalkine (CX3CR1) ligand is expressed in astrocytes and reported to be neuroprotective. When cleaved from the membrane, soluble fractalkine (sCX3CL1) activates the receptor CX3CR1. Although somewhat controversial, CX3CR1 is reported to be expressed in neurons and microglia. The membrane-bound form of CX3CL1 additionally acts as an adhesion molecule for microglia and infiltrating white blood cells. Much research has been done on the role of fractalkine in neuronal cells; however, little is known about the regulation of the CX3CL1 ligand in astrocytes., Methods: The mechanisms involved in the up-regulation and cleavage of CX3CL1 from human astrocytes were investigated using immunocytochemistry, Q-PCR and ELISA. All statistical analysis was performed using GraphPad Prism 5., Results: A combination of ADAM17 (TACE) and ADAM10 protease inhibitors was found to attenuate IL-1β-, TNF-α- and IFN-γ-induced sCX3CL1 levels in astrocytes. A specific ADAM10 (but not ADAM17) inhibitor also attenuated these effects, suggesting ADAM10 proteases induce release of sCX3CL1 from stimulated human astrocytes. A p38 MAPK inhibitor also attenuated the levels of sCX3CL1 upon treatment with IL-1β, TNF-α or IFN-γ. In addition, an IKKβ inhibitor significantly reduced the levels of sCX3CL1 induced by IL-1β or TNF-α in a concentration-dependent manner, suggesting a role for the NF-kB pathway., Conclusions: In conclusion, this study shows that the release of soluble astrocytic fractalkine is regulated by ADAM10 proteases with p38 MAPK also playing a role in the fractalkine shedding event. These findings are important for understanding the role of CX3CL1 in healthy and stimulated astrocytes and may benefit our understanding of this pathway in neuro-inflammatory and neurodegenerative diseases.

Widespread traumatic axonal injury (TAI) results in brain network dysfunction, which commonly leads to persisting cognitive and behavioural impairments following traumatic brain injury (TBI). TBI induces a complex neuroinflammatory response, frequently located at sites of axonal pathology. The role of the pro-inflammatory cytokine interleukin (IL)-1β has not been established in TAI. An IL-1β-neutralizing or a control antibody was administered intraperitoneally at 30 min following central fluid percussion injury (cFPI), a mouse model of widespread TAI. Mice subjected to moderate cFPI (n = 41) were compared with sham-injured controls (n = 20) and untreated, naive mice (n = 9). The anti-IL-1β antibody reached the target brain regions in adequate therapeutic concentrations (up to ~30 μg/brain tissue) at 24 h post-injury in both cFPI (n = 5) and sham-injured (n = 3) mice, with lower concentrations at 72 h post-injury (up to ~18 μg/g brain tissue in three cFPI mice). Functional outcome was analysed with the multivariate concentric square field (MCSF) test at 2 and 9 days post-injury, and the Morris water maze (MWM) at 14-21 days post-injury. Following TAI, the IL-1β-neutralizing antibody resulted in an improved behavioural outcome, including normalized behavioural profiles in the MCSF test. The performance in the MWM probe (memory) trial was improved, although not in the learning trials. The IL-1β-neutralizing treatment did not influence cerebral ventricle size or the number of microglia/macrophages. These findings support the hypothesis that IL-1β is an important contributor to the processes causing complex cognitive and behavioural disturbances following TAI., (© 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Background: BAF312 (Siponimod) is a dual agonist at the sphingosine-1 phosphate receptors, S1PR1 and S1PR5. This drug is currently undergoing clinical trials for the treatment of secondary progressive multiple sclerosis (MS). Here, we investigated the effects of BAF312 on isolated astrocyte and microglia cultures as well as in slice culture models of demyelination., Methods: Mouse and human astrocytes were treated with S1PR modulators and changes in the levels of pERK, pAkt, and calcium signalling as well as S1PR1 internalization and cytokine levels was investigated using Western blotting, immunochemistry, ELISA and confocal microscopy. Organotypic slice cultures were prepared from the cerebellum of 10-day-old mice and treated with lysophosphatidylcholine (LPC), psychosine and/or S1PR modulators, and changes in myelination states were measured by fluorescence of myelin basic protein and neurofilament H., Results: BAF312 treatment of human and mouse astrocytes activated pERK, pAKT and Ca(2+) signalling as well as inducing S1PR1 internalization. Notably, activation of S1PR1 increased pERK and pAKT in mouse astrocytes while both S1PR1 and S1PR3 equally increased pERK and pAKT in human astrocytes, suggesting that the coupling of S1PR1 and S1PR3 to pERK and pAKT differ in mouse and human astrocytes. We also observed that BAF312 moderately attenuated lipopolysaccharide (LPS)- or TNFα/IL17-induced levels of IL6 in both astrocyte and microglia cell cultures. In organotypic slice cultures, BAF312 reduced LPC-induced levels of IL6 and attenuated LPC-mediated demyelination. We have shown previously that the toxic lipid metabolite psychosine induces demyelination in organotypic slice cultures, without altering the levels of cytokines, such as IL6. Importantly, psychosine-induced demyelination was also attenuated by BAF312., Conclusions: Overall, this study suggests that BAF312 can modulate glial cell function and attenuate demyelination, highlighting this drug as a further potential therapy in demyelinating disorders, beyond MS.

The family of sphingosine-1-phosphate receptors (S1PRs) is G-protein-coupled, comprised of subtypes S1PR1-S1PR5 and activated by the endogenous ligand S1P. The phosphorylated version of Fingolimod (pFTY720), an oral therapy for multiple sclerosis (MS), induces S1PR1 internalisation in T cells, subsequent insensitivity to S1P gradients and sequestering of these cells within lymphoid organs, thus limiting immune response. S1PRs are also expressed in neuronal and glial cells where pFTY720 is suggested to directly protect against lysolecithin-induced deficits in myelination state in organotypic cerebellar slices. Of note, the effect of pFTY720 on immune cells already migrated into the CNS, prior to treatment, has not been well established. We have previously found that organotypic slice cultures do contain immune cells, which, in principle, could also be regulated by pFTY720 to maintain levels of myelin. Here, a mouse organotypic cerebellar slice and splenocyte co-culture model was thus used to investigate the effects of pFTY720 on splenocyte-induced demyelination. Spleen cells isolated from myelin oligodendrocyte glycoprotein immunised mice (MOG-splenocytes) or from 2D2 transgenic mice (2D2-splenocytes) both induced demyelination when co-cultured with mouse organotypic cerebellar slices, to a similar extent as lysolecithin. As expected, in vivo treatment of MOG-immunised mice with FTY720 inhibited demyelination induced by MOG-splenocytes. Importantly, in vitro treatment of MOG- and 2D2-splenocytes with pFTY720 also attenuated demyelination caused by these cells. In addition, while in vitro treatment of 2D2-splenocytes with pFTY720 did not alter cell phenotype, pFTY720 inhibited the release of the pro-inflammatory cytokines such as interferon gamma (IFNγ) and interleukin 6 (IL6) from these cells. This work suggests that treatment of splenocytes by pFTY720 attenuates demyelination and reduces pro-inflammatory cytokine release, which likely contributes to enhanced myelination state induced by pFTY720 in organotypic cerebellar slices.

The family of interleukin 17 receptors (IL17Rs), subtypes IL17RA-IL17RE, is targeted by the group of pro-inflammatory IL17 cytokines (IL17A-F) and moreover the newly developed anti-IL17A antibody secukinumab (AIN457) has shown promise in Phase II trials in multiple sclerosis. Here, we show that human astrocytes, isolated from a fetal cerebral cortex, express IL17RA and IL17RC and in vitro treatment with IL17A increases protein levels of IL6 in human astrocytes, which is enhanced in the presence of TNFα, as determined by homogeneous time resolved fluorescence. Studies on acutely isolated mouse astrocytes are comparable to human astrocytes although the protein levels of IL6 are lower in mouse astrocytes, which also show a lower response to IL17F and IL1β in promoting IL6 levels. In human astrocytes, IL17A and TNFα also induce mRNA expression of IL6, IL8 and the Th17 cytokines CXCL1, CXCL2, and CCL20, with little effect on Th1 cytokines CXCL9, CXCL10, and CXCL11. The effects of IL17A are associated with nuclear translocation of the NF-κB transcription factor, as determined by immunocytochemistry, where treatment of human astrocytes with the inhibitors of the NF-κB pathway and with secukinumab inhibits the IL17A and IL17A/TNFα-induced increase in nuclear translocation of NF-κB and levels of IL6. Taken together the data shows that IL17A signaling plays a key role in regulating the levels of cytokines, such as IL6, in human astrocytes via a mechanism that involves NF-κB signaling and that selective inhibition of IL17A signaling attenuates levels of pro-inflammatory molecules in astrocytes.

Blocking the function of the myelin protein Nogo-A or its signaling pathway is a promising method to overcome an important neurite growth inhibitory factor of the adult central nervous system (CNS), and to enhance axonal regeneration and plasticity after brain or spinal cord injuries. Several studies have shown increased axonal regeneration and enhanced compensatory sprouting, along with substantially improved functional recovery after treatment with anti-Nogo-A antibodies, Nogo-receptor antagonists, or inhibition of the downstream mediator RhoA/ROCK in adult rodents. Proof-of-concept studies in spinal cord-injured macaque monkeys with anti-Nogo-A antibodies have replicated these findings; recently, clinical trials in spinal cord-injured patients have begun. However, the optimal time window for successful Nogo-A function blocking treatments has not yet been determined. We studied the effect of acute as well as 1- or 2-weeks delayed intrathecal anti-Nogo-A antibody infusions on the regeneration of corticospinal tract (CST) axons and the recovery of motor function after large but anatomically incomplete thoracic spinal cord injuries in adult rats. We found that lesioned CST fibers regenerated over several millimeters after acute or 1-week-delayed treatments, but not when the antibody treatment was started with a delay of 2 weeks. Swimming and narrow beam crossing recovered well in rats treated acutely or with a 1-week delay with anti-Nogo-A antibodies, but not in the 2-week-delayed group. These results show that the time frame for treatment of spinal cord lesions with anti-Nogo-A antibodies is restricted to less than 2 weeks in adult rodents.

Increasing evidence suggests that interleukin-1β (IL-1β) is a key mediator of the inflammatory response following traumatic brain injury (TBI). Recently, we showed that intracerebroventricular administration of an IL-1β-neutralizing antibody was neuroprotective following TBI in mice. In the present study, an anti-IL-1β antibody or control antibody was administered intraperitoneally following controlled cortical injury (CCI) TBI or sham injury in 105 mice and we extended our histological, immunological and behavioral analysis. First, we demonstrated that the treatment antibody reached target brain regions of brain-injured animals in high concentrations (> 11 nm) remaining up to 8 days post-TBI. At 48 h post-injury, the anti-IL-1β treatment attenuated the TBI-induced hemispheric edema (P < 0.05) but not the memory deficits evaluated using the Morris water maze (MWM). Neutralization of IL-1β did not influence the TBI-induced increases (P < 0.05) in the gene expression of the Ccl3 and Ccr2 chemokines, IL-6 or Gfap. Up to 20 days post-injury, neutralization of IL-1β was associated with improved visuospatial learning in the MWM, reduced loss of hemispheric tissue and attenuation of the microglial activation caused by TBI (P < 0.05). Motor function using the rotarod and cylinder tests was not affected by the anti-IL-1β treatment. Our results suggest an important negative role for IL-1β in TBI. The improved histological and behavioral outcome following anti-IL-1β treatment also implies that further exploration of IL-1β-neutralizing compounds as a treatment option for TBI patients is warranted., (© 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Interleukin-1beta (IL-1beta) may play a central role in the inflammatory response following traumatic brain injury (TBI). We subjected 91 mice to controlled cortical impact (CCI) brain injury or sham injury. Beginning 5 min post-injury, the IL-1beta neutralizing antibody IgG2a/k (1.5 microg/mL) or control antibody was infused at a rate of 0.25 microL/h into the contralateral ventricle for up to 14 days using osmotic minipumps. Neutrophil and T-cell infiltration and microglial activation was evaluated at days 1-7 post-injury. Cognition was assessed using Morris water maze, and motor function using rotarod and cylinder tests. Lesion volume and hemispheric tissue loss were evaluated at 18 days post-injury. Using this treatment strategy, cortical and hippocampal tissue levels of IgG2a/k reached 50 ng/mL, sufficient to effectively inhibit IL-1betain vitro. IL-1beta neutralization attenuated the CCI-induced cortical and hippocampal microglial activation (P < 0.05 at post-injury days 3 and 7), and cortical infiltration of neutrophils (P < 0.05 at post-injury day 7). There was only a minimal cortical infiltration of activated T-cells, attenuated by IL-1beta neutralization (P < 0.05 at post-injury day 7). CCI induced a significant deficit in neurological motor and cognitive function, and caused a loss of hemispheric tissue (P < 0.05). In brain-injured animals, IL-1beta neutralizing treatment resulted in reduced lesion volume, hemispheric tissue loss and attenuated cognitive deficits (P < 0.05) without influencing neurological motor function. Our results indicate that IL-1beta is a central component in the post-injury inflammatory response that, in view of the observed positive neuroprotective and cognitive effects, may be a suitable pharmacological target for the treatment of TBI.

Neglect is a complex human cognitive spatial disorder typically induced by damage to prefrontal or posterior parietal association cortices. Behavioral treatments for neglect rarely generalize outside of the therapeutic context or across tasks within the same therapeutic context. Recovery, when it occurs, is spontaneous over the course of weeks to months, but often it is incomplete. A number of studies have indicated that anti-Nogo-A antibodies can be used to enhance plasticity and behavioral recovery following damage to motor cortex, and spinal cord. In the present studies the anti-Nogo-A antibodies IN-1, 7B12, or 11C7 were applied intraventricularly to adult rats demonstrating severe neglect produced by unilateral medial agranular cortex lesions in rats. The three separate anti-Nogo-A antibody groups were treated immediately following the medial agranular cortex lesions. Each of the three antibodies induced dramatic significant behavioral recovery from neglect relative to controls. Severing the corpus callosum to destroy inputs from the contralesional hemisphere resulted in reinstatement of severe neglect, pointing to a possible role of interhemispheric mechanisms in behavioral recovery from neglect.

Object: Central nervous system axons regenerate poorly after traumatic brain injury (TBI), partly due to inhibitors such as the protein Nogo-A present in myelin. The authors evaluated the efficacy of anti-Nogo-A monoclonal antibody (mAb) 7B12 administration on the neurobehavioral and cognitive outcome of rats following lateral fluid-percussion brain injury, characterized the penetration of the 7B12 or control antibodies into target brain regions, and evaluated the effects of Nogo-A inhibition on hemispheric tissue loss and sprouting of uninjured motor tracts in the cervical cord. To elucidate a potential molecular response to Nogo-A inhibition, we evaluated the effects of 7B12 on hippocampal GAP-43 expression., Methods: Beginning 24 hours after lateral fluid-percussion brain injury or sham injury in rats, the mAb 7B12 or control antibody was infused intracerebroventricularly over 14 days, and behavior was assessed over 4 weeks., Results: Immunoreactivity for 7B12 or immunoglobulin G was detected in widespread brain regions at 1 and 3 weeks postinjury. The brain-injured animals treated with 7B12 showed improvement in cognitive function (p < 0.05) at 4 weeks but no improvement in neurological motor function from 1 to 4 weeks postinjury compared with brain-injured, vehicle-treated controls. The enhanced cognitive function following inhibition of Nogo-A was correlated with an attenuated postinjury downregulation of hippocampal GAP-43 expression (p < 0.05)., Conclusions: Increased GAP-43 expression may be a novel molecular mechanism of the enhanced cognitive recovery mediated by Nogo-A inhibition after TBI in rats.

Stroke often results in devastating neurological disabilities with no specific treatment available to improve functional recovery. Neurite growth inhibitory proteins such as Nogo-A play a critical role in impeding regain of function after stroke. We have reported that treatment with anti-Nogo-A antibody using the intracerebroventricular route resulted in improvement of function and neuroplasticity in adult or aged rats after stroke. This present study tested a more clinically accessible route for applying anti-Nogo-A antibodies, the intrathecal route. Anti-Nogo-A or control antibody was administered intrathecally at lower lumbar levels 1 week after middle cerebral artery occlusion in adult rats. Our results show that anti-Nogo-A antibody delivered by this intrathecal route for 2 weeks penetrated into brain parenchyma and bound to myelin-enriched structures such as the corpus callosum and striatal white matter. Animals receiving anti-Nogo-A antibody treatment significantly improved recovery of function on the skilled forelimb reaching task as compared to stroke only and stroke/control antibody animals. These findings show that anti-Nogo-A antibody delivered through the intrathecal route is as effective in restoring lost functions after stroke as the intracerebroventricular route. This is of great importance for the future application of anti-Nogo-A immunotherapy for ischemic stroke treatment.

Functional recovery following acute CNS injury in humans, such as spinal cord injury and stroke, is exceptionally limited, leaving the affected individual with life-long neurological deficits such as loss of limb movement and sensation leading to a compromised quality of life. As yet, there is no effective treatment on the market for such injuries. This lack of functional recovery can at least in part be attributed to the restriction of axonal regeneration and neuroplasticity by several CNS myelin proteins that have been shown to be potent inhibitors of neurite outgrowth in vitro, namely myelin-associated glycoprotein (MAG), Nogo-A and oligodendrocyte myelin glycoprotein (OMgp). Nogo-A contains multiple neurite outgrowth inhibitory domains exposed on the surface of myelinating oligodendrocytes located within its amino-terminal region (amino-Nogo-A) and C-terminal region (Nogo-66). Although structurally dissimilar; Nogo-66, MAG and OMgp exert their inhibitory effects by binding the GPI-linked neuronal Nogo-66 receptor (NgR) that transduces the inhibitory signal to the cell interior via transmembrane co-receptors LINGO-1 and p75(NTR)or TROY. Although the receptor(s) for amino-Nogo-A are unknown, amino-Nogo-A and NgR ligands mutually activate the small GTPase RhoA. Consistent with their neurite outgrowth inhibitory function, approaches counter-acting Nogo-A using function-blocking antibodies, NgR using peptide antagonists and receptor bodies or RhoA using deactivating enzymes have been shown to significantly enhance axonal regeneration and neuroplasticity leading to improved functional recovery in animal models of acute CNS injury. These in vivo findings thus provide a sound basis for the development of an effective treatment for acute CNS injuries in humans.

Stroke is a prevalent and devastating disorder, and no treatment is currently available to restore lost neuronal function after stroke. One unique therapy that improves recovery after stroke is neutralization of the neurite inhibitory protein Nogo-A. Here, we show, in a clinically relevant model, improved functional recovery and brain reorganization in the aged and adult rat when delayed anti-Nogo-A therapy is given after ischemic injury. These results support the efficacy of Nogo-A neutralization as treatment for ischemic stroke, even in the aged animal and after a 1-week delay, and implicate neuronal plasticity from unlesioned areas of the central nervous system as a mechanism for recovery.

The Nogo-66 receptor (NgR) plays a pivotal role in the inhibition of neuroregeneration as the receptor for multiple neurite outgrowth inhibitors such as Nogo-A. We have previously shown that NgR undergoes zinc metalloproteinase-mediated ectodomain shedding in neuroblastoma cells. Here, we demonstrate that the NgR-related protein NgR homologue-1 is released from neuroblastoma cells as a full-length ectodomain (NgRH1-ecto) and an N-terminal fragment (NTF-NgRH1) containing the leucine-rich repeat region of the protein. Inhibitors of the major protease classes failed to block the release of NgRH1-ecto, suggesting that this occurs via a protease-independent mechanism, presumably by a phospholipase-like enzyme. The release of NTF-NgRH1 was blocked by a hydroxamate-based zinc metalloproteinase inhibitor and tissue inhibitor of metalloproteinases-2 and -3, but not -1, implicating the involvement of membrane-type matrix metalloproteinases in this process. Our findings thus highlight the parallels between the ectodomain shedding of NgRH1 and that previously described for NgR.

The central nervous system myelin components oligodendrocyte-myelin glycoprotein, myelin-associated glycoprotein and the Nogo-66 domain of Nogo-A inhibit neurite outgrowth by binding the neuronal glycosyl-phosphatidylinositol-anchored Nogo-66 receptor (NgR) that transduces the inhibitory signal to the cell interior via a transmembrane co-receptor, p75NTR. Here, we demonstrate that human NgR expressed in human neuroblastoma cells is constitutively cleaved in a post-ER compartment to generate a lipid-raft associated C-terminal fragment that is present on the cell surface and a soluble N-terminal fragment that is released into the medium. Mass spectrometric analysis demonstrated that the N-terminal fragment terminated just after the C-terminus of the ligand-binding domain of NgR. In common with other shedding mechanisms, the release of this fragment was blocked by a hydroxamate-based inhibitor of zinc metalloproteinases, but not by inhibitors of other protease classes and up-regulated by treatment with the cellular cholesterol depleting agent methyl-beta-cyclodextrin. The N-terminal fragment bound Nogo-66 and blocked Nogo-66 binding to cell surface NgR but failed to associate with p75NTR, indicative of a role as a Nogo-66 antagonist. Furthermore, the N- and C-terminal fragments of NgR were detectable in human brain cortex and the N-terminal fragment was also present in human cerebrospinal fluid, demonstrating that NgR proteolysis occurs within the human nervous system. Our findings thus identify a potential cellular mechanism for the regulation of NgR function at the level of the receptor.

Axon injury following cerebral ischemia has received little scientific attention compared to the abundance of information dealing with the pathophysiology of grey matter ischemia. There are differences in the initial response of grey and white matter to ischemia in vitro. In this study we investigate whether the vasoactive peptide, endothelin-1, can generate a focal ischemic lesion in the white matter and compare the findings with endothelin-1-induced lesions in the grey matter. Using a minimally invasive technique to microinject endothelin-1 into selected brain regions, we observed an acute reduction in local MRI perfusion in the injected hemisphere after 1 hour. Twenty-four hours after microinjection of 10 pmoles of endothelin-1, we observed a loss of neurons in the grey matter. At 72 hours, neutrophils were absent and a macrophage/microglia response and astrocyte gliosis were detected. No breakdown in the blood-brain barrier was detected. After injection of 10 pmoles endothelin-1 into the cortical white matter, we observed prolific amyloid precursor protein-positive immunostaining (indicative of axonal disruption) and an increase in tau-1 immunostaining in oligodendrocytes at 6 hours. Similar to the grey matter lesions, no neutrophils were present, a macrophage/microglia response did not occur until 72 hours and there was no disruption in the blood-brain barrier. Focal injections of endothelin-1 into specific areas of the rat CNS represent a model to investigate therapeutic approaches to white matter ischemia.

Nogo-66 receptor (NgR) has recently been identified as the neuronal receptor of the myelin-associated proteins Nogo-A, oligodendrocyte protein (OMgp) and myelin-associated glycoprotein (MAG), and mediates inhibition of axonal regeneration both in vitro and in vivo. Through database searches, we have identified two novel proteins (NgRH1 and NgRH2) that turned out to be homologous in their primary structures, biochemical properties and expression patterns to NgR. Like NgR, the homologues contain eight leucine-rich repeats (LRR) flanked by a leucine-rich repeat C-terminus (LRRCT) and a leucine-rich repeat N-terminus (LRRNT), and also have a C-terminal GPI signal sequence. Northern blot analysis showed predominant expression of NgRH1 and NgRH2 mRNA in the brain. In situ hybridization and immunohistochemistry on rat brain slices revealed neuronal expression of the genes. NgRH1 and NgRH2 were detected on the cell surface of recombinant cell lines as N-glycosylated GPI anchored proteins and, consistent with other GPI anchored proteins, were localized within the lipid rafts of cellular membranes. In addition, an N-terminal proteolytic fragment of NgR comprising the majority of the ectodomain was found to be constitutively secreted from cells. Our data indicate that NgR, NgRH1 and NgRH2 constitute a novel receptor protein family, which may play related roles within the CNS.

Monocyte chemoattractant protein-1 (MCP-1) has been implicated in many inflammatory and autoimmune diseases. The G-protein-coupled receptor CCR-2B is probably the most important MCP-1 receptor in vivo, and loss of MCP-1 effector function alone is sufficient to impair monocytic trafficking in inflammation models. MCP-1 signalling appears to be a relevant target, especially in rheumatoid arthritis (RA). In RA patients, MCP-1 is produced by synovial cells and infiltrating monocytes, plasma MCP-1 concentrations correlate with swollen joint count, and elevated serum MCP-1 concentrations were found in juvenile RA in patients with active disease. Modulation of MCP-1 signalling in experimental RA showed beneficial effects on inflammation and joint destruction. With respect to chronic neuroinflammation, a critical role for MCP-1 has been established in animal models for multiple sclerosis. In acute neuroinflammation, experimental evidence for a detrimental role of MCP-1 in stroke and excitotoxic injury has been found. Several selective small molecular weight CCR-2B antagonists and MCP-1-blocking antibodies have been described. The proof for the validity of targeting MCP-1 signalling in disease, however, has yet to be established in clinical trials.

Nogo-A is a myelin-associated neurite outgrowth inhibitory protein limiting recovery and plasticity after central nervous system injury. In this study, a purified monoclonal anti-Nogo-A antibody (7B12) was evaluated in two rat stroke models with a time-to-treatment of 24 hours after injury. After photothrombotic cortical injury (PCI) and intraventricular infusion of a control mouse immunoglobulin G for 2 weeks, long-term contralateral forepaw function was reduced to about 55% of prelesion performance until the latest time point investigated (9 weeks). Forepaw function was significantly better in the 7B12-treated group 6 to 9 weeks after PCI, and reached about 70% of prelesion levels. Cortical infarcts were also produced in spontaneously hypertensive rats (SHR) by permanent middle cerebral artery occlusion (MCAO). In the control group, forepaw function remained between 40% and 50% of prelesion levels 4 to 12 weeks after MCAO. In contrast, 7B12-treated groups showed significant improvement between 4 and 7 weeks after MCAO from around 40% of prelesion levels at week 4 to about 60% to 70% at 7 to 12 weeks after MCAO. Treatment in both models was efficacious without influencing infarct volume or brain atrophy. Neuroanatomically in the spinal cord, a significant increase of midline crossing corticospinal fibers originating in the unlesioned sensorimotor cortex was found in 7B12-treated groups, reaching 2.3 +/- 1.5% after PCI (control group: 1.1 +/- 0.5%) and 4.5 +/- 2.2% after MCAO in SHR rats (control group: 1.8 +/- 0.8%). Behavioral outcome and the presence of midline crossing fibers in the cervical spinal cord correlated significantly, suggesting a possible contribution of the crossing fibers for forepaw function after PCI and MCAO. The results suggest that specific anti-Nogo-A antibodies bear potential as a new rehabilitative treatment approach for ischemic stroke with a prolonged time-to-treatment window.

The present study investigated the activation of extracellular-signal-regulated kinase (ERK) and the potential role of interleukin-1 beta (IL-1beta) in the brain's response to focal brain ischemia in the permanent middle cerebral artery occlusion (pMCAO) model. Phosphorylated ERK p44 and p42 were increased time-dependently and significantly 18- and 28-fold, respectively, at 24-h post-pMCAO. Similarly, IL-1beta protein levels were significantly increased with the peak at 24 h in the lesioned core of the ischemic hemisphere compared to the contralateral side. Previous studies using various stimuli have shown ERK-dependent IL-1 induction. The results from our study suggest that this relation may also exist in vivo in ischemic brain tissue. Based on the progressive nature of IL-1 induction, we hypothetized that inhibition of interleukin-converting enzyme (ICE) could provide an extended time-window for neuroprotection. Therefore, we applied N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD x fmk), an ICE blocker 3 or 6 h after pMCAO. Reductions of infarct volume, however, were not observed. Taken together with previous results, where we showed protective activity of zVAD x fmk when given immediately after pMCAO, we conclude that the time window for zVAD x fmk is less than 3 h.

Inflammatory processes have been implicated in the pathogenesis of brain damage after stroke. In rodent stroke models, focal ischemia induces several proinflammatory chemokines, including monocyte chemoattractant protein-1 (MCP-1). The individual contribution to ischemic tissue damage, however, is largely unknown. To address this question, the authors subjected MCP-1-deficient mice (MCP-1-/-) to permanent middle cerebral artery occlusion (MCAO). Measurement of basal blood pressure, cerebral blood flow, and blood volume revealed no differences between wild-type (wt) and MCP-1-/- mice. MCAO led to similar cerebral perfusion deficits in wt and MCP-1-/- mice, excluding differences in the MCA supply territory and collaterals. However, compared with wt mice, the mean infarct volume was 29% smaller in MCP-1-/- mice 24 hours after MCAO (P = 0.022). Immunostaining showed a reduction of phagocytic macrophage accumulation within infarcts and the infarct border in MCP-1-/- mice 2 weeks after MCAO. At the same time point, the authors found an attenuation of astrocytic hypertrophy in the infarct border and thalamus in MCP-1-/- mice. However, these effects on macrophages and astrocytes in MCP-1-/- mice occurred too late to suggest a protective role in acute infarct growth. Of note: at 6 hours after MCAO, MCP-1-/- mice produced significantly less interleukin-1beta in ischemic tissue; this might be related to tissue protection. The results of this study indicate that inhibition of MCP-1 signaling could be a new acute treatment approach to limit infarct size after stroke.

1. Intravenous administration of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 150 micrograms kg-1) into conscious sham-operated rats caused significant increases in basal glycaemia with minor changes in basal insulinaemia. Glucose-stimulated (intravenous glucose tolerance test) plasma insulin levels were significantly inhibited in 8-OH-DPAT-treated sham-operated animals. These metabolic changes were associated with significant and sustained falls in blood pressure (BP) and heart rate (HR) preceded by transient (less than 5 min) increases only in BP. 2. In adrenodemedullated animals, 8-OH-DPAT failed to cause an initial vasoconstriction, hyperglycaemia, or inhibition of glucose-stimulated plasma insulin despite eliciting falls in BP and HR that were comparable to those observed in sham-operated animals. 3. Noradrenaline, adrenaline and dopamine levels in the adrenal tissue were reduced by about 95% in adrenodemedullated rats as compared to sham-operated rats. A functionally intact adrenal cortex was indicated by the presence of corticosterone in the plasma of both adrenodemedullated and sham-operated rats. 4. The present data demonstrate that 8-OH-DPAT mediates an initial increase in BP and changes in metabolic parameters via intact adrenal medulla and may thus be consequential to the release of adrenaline, whereas the sustained cardiovascular effects of 8-OH-DPAT are not.

In the present study, the role of the adrenal gland in the hypoinsulinaemic and hyperglycaemic effects of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) vis-à-vis the cardiovascular effects were examined using adrenalectomized and sham-operated (with intact adrenals) Sprague-Dawley rats. Intravenous administration of 8-OH-DPAT (150 micrograms/kg) into conscious sham-operated animals caused significant increases in basal plasma glucose levels without any change in basal plasma insulin levels whereas glucose-stimulated plasma insulin levels were markedly inhibited. These changes were associated with significant and sustained falls in blood pressure (BP) and heart rate (HR) preceded by transient (less than 5 min) increases only in BP. In adrenalectomized animals, 8-OH-DPAT-mediated initial vasoconstriction, hyperglycaemia and inhibition of evoked plasma insulin levels were abolished. However, in these animals, 8-OH-DPAT produced falls in BP and HR that were comparable to those observed in sham-operated animals. Plasma corticosterone was increased 15 min after 8-OH-DPAT in sham-operated animals whereas it was undetectable in adrenalectomized animals. The data indicate that unlike the effects of 8-OH-DPAT to decrease BP and HR, the initial increase in BP and its effects on the metabolic parameters are clearly dependent on intact adrenals.

1. Effects of the prototype selective 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-dipropylamino)tetralin (8-OH-DPAT), were studied on the glycaemia and insulinaemia in conscious spontaneously hypertensive (SH) rats concurrently with blood pressure (BP) and heart rate (HR); underlying mechanism(s) were investigated in anaesthetized and pithed SH rats and in the perfused rat pancreas. 2. Intravenous (i.v.) injections of 8-OH-DPAT (150 micrograms kg-1, i.v.) into fasted conscious but not anesthetized SH rats increased glycaemia; glucose-stimulated (i.v. glucose tolerance test) plasma insulin levels were significantly inhibited in both cases without significant changes in glucose tolerance. Metabolic changes were associated with prominent decreases in BP and HR. 3. No inhibitory effect of 8-OH-DPAT, 150 micrograms kg-1 i.v., on glucose-stimulated plasma insulin was observed in pithed SH rats; in contrast, clonidine (8 micrograms kg-1 i.v.), produced marked inhibition of insulin levels in association with glucose intolerance. Neither compound decreased BP; rather, pronounced vasopressor effects were observed. 4. In the isolated perfused pancreas of the rat, 8-OH-DPAT, at 10(-8) and 10(-7) M, concentrations known to activate 5-HT1A receptors in vitro, failed to modify glucose-stimulated insulin release. Inhibition (39 +/- 7%) was seen only at a high concentration of 10(-6) M. 5. The present data suggest that like the cardiovascular effects of 8-OH-DPAT, the inhibition of glucose-stimulated insulin release is mediated via the central nervous system. However, it is suggested that different mechanisms are involved in the cardiovascular actions and metabolic effects of 8-OH-DPAT in the SH rat; the latter are likely to reflect a consequence of activation of the hypothalamic-adrenal axis.

1. With radioligand binding techniques, MDL 73005 EF (8-[2-(2,3-dihydro-1,4-benzodioxin-2-yl-methylamino)ethyl]-8-az aspiro[4, 5]decane-7,9-dione methyl sulphonate) shows high affinity (pIC50 8.6) and selectivity (greater than 100 fold compared to other monoamine and benzodiazepine receptor sites) for the 5-hydroxytryptamine (5-HT)1A recognition site; it was both more potent and more selective than buspirone in this respect. 2. In rats pretreated with reserpine, 8-hydroxy-2-(di-n-propyl-amino) tetralin (8-OH-DPAT) induced forepaw treading and flat body posture; in the same model, MDL 73005EF and buspirone showed minimal agonist activity and at high doses MDL 73005EF inhibited responses to 8-OH-DPAT. 3. In rats trained to discriminate 8-OH-DPAT from saline in a drug discrimination paradigm, both MDL 73005EF and buspirone generalized dose-dependently and completely to the 8-OH-DPAT cue. 4. To define the anxiolytic potential of MDL 73005EF, it was examined in the elevated plus-maze test and in the water-lick conflict test in comparison with diazepam and buspirone. In both tests MDL 73005EF induced effects similar to those seen following diazepam. Buspirone had similar effects to both MDL 73005EF and diazepam in the water-lick conflict test but opposite effects in the elevated plus-maze. 8-OH-DPAT also had opposite effects in the elevated plus-maze test to MDL 73005EF and diazepam. 5. The anti-conflict effects of MDL 73005EF were reversed by low doses of the 5-HT1A receptor agonist, 8-OH-DPAT; those of buspirone were neither antagonised nor mimicked by 8-OH-DPAT. 6. These results suggest that an interaction with 5-HTIA receptors is the basis of the anxiolytic-like activity of MDL 73005EF. However, its mechanism of action is clearly different from that of buspirone, possibly reflecting a greater selectivity for the 5-HTlA receptors located presynaptically on central 5- hydroxytryptaminergic neurones.

Dopamine D2-receptors were directly identified in receptor binding assays with washed particulate preparations of rabbit carotid body using the selective ligand, [3H]domperidone. High affinity, saturable specific binding of [3H]domperidone was clearly demonstrable and chronic section of the sinus nerve resulted in a 32% decrease in the labelling of the dopamine D2-sites. Adenylate cyclase activity was also detected in rabbit carotid body homogenates and although this enzyme was stimulated 4-fold by 10 mM sodium fluoride, neither dopamine nor isoprenaline significantly altered basal activity. On the other hand, in the intact carotid body incubated in vitro, 10(-5) M isoprenaline increased the basal cyclic AMP content 6-fold, though dopamine was again ineffective. The effect of various selective dopamine receptor antagonists and agonists was also studied on chemoreceptor afferent discharge. The results confirm that depression of 'spontaneous' chemosensory discharge is the predominant effect of dopamine (0.01-100 micrograms) in rabbits. The 'selective' D2-agonist, LY 141865, proved very effective (ID50 3.3 nmol) and was equipotent with dopamine (ID50 4.2), whereas, the D1-agonist, SK & F 38393, was very ineffective (ID50 150). The D2-antagonists domperidone and (-)-sulpiride produced a dose-related decrease in the chemodepressant responses to dopamine and LY 141865. However, there was no evidence for any appreciable excitatory action of either of these agonists after blockade of their chemo-depressant effects. The D2-antagonists variably affected the spontaneous activity, there being an increase in discharge on average, whereas responses to hypoxia, cyanide and CO2 were reduced. The present results from biochemical and neuropharmacological studies, provide strong evidence for the presence of functional dopamine D2-receptors in the rabbit carotid body, and suggest that the receptor involved in dopamine-induced depression of chemosensory discharge is of D2-type.

The catecholamine content of the carotid body of several mammalian species has been assayed using high performance liquid chromatography coupled to electrochemical detection and radioenzymatic assays. Although there were strain differences in the content of catecholamines in the carotid body of the rat, noradrenaline was equal to or exceeded the dopamine level in this species. No apparent differences were found in carotid bodies of animals killed by cervical dislocation or those dissected from anaesthetized animals. Noradrenaline concentrations were found to be substantially higher than those of dopamine in the cat and guinea-pig carotid body, though dopamine was the predominant amine in the rabbit and ferret. Unilateral superior cervical ganglionectomy or chemical sympathectomy with 6-hydroxydopamine substantially depleted noradrenaline without influencing dopamine in the rat carotid body. A marked selective reduction in noradrenaline was also observed in the rabbit and guinea-pig following ganglionectomy, though similar procedures in the cat failed to alter the levels of either catecholamine in the carotid body. The present data highlights the marked species variation in catecholamine content and the contribution to the latter by sympathetic innervation to this organ. This information will be useful in determining the species specificity regarding the relative roles of dopamine and noradrenaline in the modulation of chemoreceptor afferent discharge.

N-Acetyltyramine, N-acetyldopamine and N-acetyloctopamine were the major products when either L-[3H]tyrosine or [3H]tyramine were incubated with thoracic ganglia of the desert locust, Schistocerca gregaria. No label was incorporated into L-DOPA under these conditions, although 2-3% of the radioactivity could be recovered in dopamine and octopamine. Addition of the aromatic amino acid decarboxylase inhibitor, 3-hydroxybenzylhydrazine (NSD 1015), prevented the formation of N-acetylcompounds from L-[3H]tyrosine, without resulting in an accumulation of label in L-DOPA. In contrast, incubation of samples of haemolymph with L-[3H]tyrosine resulted in the recovery of 7% of label in L-DOPA, which was increased to 17% in the presence of NSD 1015. These results provide evidence that the initial step in the synthesis of dopamine and octopamine by S. gregaria nervous tissue is the conversion of L-tyrosine to tyramine, which is subsequently metabolised to N-acetyltyramine, N-acetyldopamine or N-acetyloctopamine.

Trans-diclofurime has been shown to be a potent calcium antagonist which resembles verapamil in vitro and in vivo. Trans-diclofurime was a potent antagonist of Ca2+-induced contractions in K+-depolarized taenia preparations from the guinea pig caecum (pA2 = 8.3 +/- 0.2), whereas the cis isomer was 50 times less active (pA2 = 6.6 +/- 0.1); the inhibitory effects of trans-diclofurime were reversed noncompetitively by the Ca2+ channel activator Bay K 8644. Trans-diclofurime and verapamil were equipotent inhibitors of electrically evoked contractions of guinea pig left atria preparations; the inhibitory effects were frequency dependent and cis-diclofurime was 10 times less effective. Both diclofurime isomers prolonged the effective refractory period at high concentrations, indicating that they also possess local anaesthetic properties. Trans-diclofurime and verapamil reduced blood pressure in pithed rats infused with angiotensin II. Hypotensive effects were accompanied by bradycardia and prolongation of PR intervals, leading to second-degree atrioventricular block. The cis isomer was less potent. Diclofurime is thus a very potent calcium antagonist in heart and smooth muscle and has some additional membrane-stabilizing properties.

MDL 72567 (2,6 dimethyl,3 methoxycarbonyl,4-(2-nitrophenyl), 5-(2-furoyl)1,4 dihydropyridine) was a potent antagonist of Ca2+-induced contractions in K+-depolarized taenia preparations from the guinea pig caecum (pA2 8.8 +/- 0.1). MDL 72567 was a potent displacer of [3H]nitrendipine binding from rat cortical membrane preparations (Ki 3.99 nM), indicating an effect at the dihydropyridine binding site, which is consistent with the finding that the inhibitory effects of MDL 72567 in smooth muscle were prevented by the dihydropyridine Ca2+ channel activator Bay K 8644. MDL 72567 slowed spontaneously beating rat atria preparations to a greater extent than did nifedipine, however, for a given negative inotropic effect. Furthermore, in pithed rat preparations infused with angiotensin II to elevate blood pressure, the hypotensive effects of MDL 72567 (3 nmol/kg-3 mumol/kg, intravenously, i.v.) were accompanied by bradycardia, whereas nifedipine, PY 108-068, and nicardipine lowered blood pressure without affecting heart rate. When compared with nifedipine, MDL 72567 caused less reflex tachycardia for a given fall in blood pressure, in anesthetized beagles and in conscious renal hypertensive dogs. In anesthetized dogs, MDL 72567 increased cardiac contractility at all hypotensive doses tested (30-3,000 nmol/kg, i.v.), whereas nifedipine caused profound myocardial depression at higher doses (1,000-3,000 nmol/kg, i.v.) even though the compounds had equivalent vasodilator effects. Thus, although MDL 72567 appears to cause a direct myocardial slowing that can partially offset reflex tachycardia, the compound has negligible negative inotropic effects and may therefore be useful in angina pectoris or even in congestive heart failure.

An increase in specific dopamine D2 receptor binding sites was observed in membranes prepared from the carotid bodies of rabbits treated for 8 weeks and then withdrawn for 4-9 days from the D2 antagonist domperidone (2-5 mg/kg per day). Recordings of chemoreceptor afferent discharge from the carotid body also revealed that this change in receptor density was accompanied by an increased sensitivity to the chemodepressant effects of exogenous dopamine. The chemoreceptor responsiveness of the carotid body to hypoxia is blunted in rabbits treated chronically with domperidone, but this can be restored to normal by an acute dose of the D2 antagonist. These experiments provide evidence that is compatible with a chemo-inhibitory role for endogenous dopamine in the rabbit's carotid body. Furthermore, these results suggest that the carotid body provides a useful model for the functional studies of dopamine D2 receptors.

1. The effects of the dihydropyridine calcium channel blocker nifedipine and the activator Bay K 8644 were investigated in different behavioural tests involving dopaminergic systems. These were the discriminative stimulus induced by amphetamine, rotational behaviour in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions and apomorphine-induced yawning in rats. 2. The yawning induced by apomorphine (40 micrograms kg-1 s.c.) was significantly potentiated by nifedipine (5-10 mgkg-1 i.p.). Bay K 8644 (0.05-0.5 mgkg-1 i.p.) dose-dependently inhibited yawning induced by apomorphine (80 micrograms kg-1 s.c.) and, at 0.4 mgkg-1, inhibited the nifedipine potentiation of apomorphine-induced yawning. In contrast to their effects on apomorphine-induced yawning, nifedipine and Bay K 8644 had no effect on apomorphine-induced penile erection. 3. Bay K 8644 (0.06-0.5 mgkg-1 i.p.) and nifedipine (5-20 mgkg-1 i.p.) had no dose-related effect on the discrimination performance of rats trained to discriminate amphetamine from saline. However, nifedipine dose-dependently reduced the response rate of amphetamine-treated rats. Bay K 8644 had no effect on this measure except at high doses that also caused disruption. 4. Neither nifedipine (5-10 mgkg-1 i.p.) nor Bay K 8644 (0.06-0.5 mgkg-1 i.p.) affected the turning behaviour induced by amphetamine (1 mgkg-1 i.p.) in rats with unilateral 6-OHDA lesion of the medial forebrain bundle, and did not induce turning themselves. 5. As the dihydropyridine compounds affected apomorphine-induced yawning but not penile erection, and did not affect amphetamine-induced rotation or drug discrimination, it seems unlikely that they are affecting dopamine release in vivo.

In receptor binding assays (+/-)MDL 72832, 8-[4-(1,4-benzodioxan-2-ylmethylamino)butyl]-8-azaspiro++ +[4,5] decane-7,9-dione, was a potent (pIC50 9.1), selective and stereospecific ligand for central 5-HT1A recognition sites. In functional tests, (+/-)MDL 72832 and its S(-) and R(+) enantiomers blocked stereoselectively the 8-OH-DPAT-induced neuronal inhibition of the transmurally stimulated guinea-pig ileum and the cardiovascular effects of 8-OH-DPAT in anaesthetized rats. In contrast, (+/-)MDL 72832 and its enantiomers were exclusively '8-OH-DPAT-like' in their ability to fully and stereoselectively generalize to the 8-OH-DPAT discriminative stimulus and, in reserpinised rats, to induce forepaw treading and flat body posture. These results characterize (+/-)MDL 72832 as a potent, stereoselective ligand with mixed agonist and antagonist properties at central and peripheral 5-HT1A receptors. The similar stereoselective requirements for the recognition site and functional effects provides compelling evidence that the 5-HT1A recognition site is indeed a functional receptor.

1. Trans-diclofurime has been shown to be a potent group II calcium antagonist in in vitro and in vivo test systems. In contrast to the dihydropyridines, group II calcium antagonists have a reduced propensity to cause reflex tachycardia due to well-balanced inhibitory effects in smooth muscle and heart. Since effects on autonomic reflexes are more reliably assessed in conscious animals, the cardiovascular effects of trans-diclofurime have been examined and compared to those of nifedipine, verapamil and diltiazem in the conscious spontaneously hypertensive rat (SHR). 2. Each SHR had an indwelling catheter in the femoral artery to record mean arterial pressure (MAP) and heart rate (HR) and a cannula in the femoral vein for drug infusion over 1 min. 3. Nifedipine (0.1-3.0 mumol kg-1 i.v.) caused dose-related falls in MAP accompanied by dose-related increases in HR. Trans-diclofurime and verapamil (0.3-3.0 mumol kg-1 i.v.) also caused dose-related decreases in MAP, but significant tachycardia was only seen at 1.0 and 3.0 mumol kg-1. Trans-diclofurime (0.3 mumol kg-1) induced a significant fall in HR. Diltiazem (1.0-10.0 mumol kg-1 i.v.) induced dose-related falls in MAP, significant bradycardia was evident with 1.0 mumol kg-1 and tachycardia with 10 mumol kg-1. Trans-diclofurime and diltiazem induced less tachycardia than nifedipine and verapamil for equivalent falls in MAP. 4. These results suggest that trans-diclofurime is a potent antihypertensive agent in conscious SHR and, like diltiazem, the hypotensive effects are associated with less tachycardia than is usually apparent with calcium antagonists such as nifedipine or verapamil. S. The cardiovascular effects of trans-diclofurime in conscious SHR are those expected of a class II calcium antagonist and are consistent with its proposed mode of interaction with the diltiazem site in the calcium channel.

Trans-diclofurime has been shown to be a very potent class II calcium antagonist (see preceding report), and we have examined its molecular interactions with the different receptor sites at the Ca2+ channel. Trans-diclofurime did not affect [3H]nitrendipine binding to rat cortical membranes at 37 degrees C and showed weak inhibitory effects at 25 degrees C, whereas at 0 degrees C 80% of the binding was inhibited noncompetitively (IC50, 13 nM); cis-diclofurime was 22-fold less potent. Trans-diclofurime, like diltiazem, blocked the inhibitory effects of verapamil on [3H]nitrendipine binding. Trans-diclofurime is a potent displacer of [3H]diltiazem binding (IC50, 15 nM; IC50 for diltiazem, 55 nM); the diclofurime isomers showed high stereoselectivity, with high Hill coefficients (0.85-1.0). In contrast, the stereoselectivity of the isomers was lower as inhibitors of [3H]verapamil binding, as were the Hill coefficients (0.55-0.65). It is proposed that the functional potency of the diclofurime isomers as calcium antagonists can be explained on the basis of their relative affinities for the diltiazem site and that this site is coupled to the dihydropyridine site in a positive heterotropic allosteric manner. A model for the interaction of group II calcium antagonists with the Ca2+ channel is proposed.

The present experiments were carried out to investigate the cardiovascular effects of endothelin 1 (ET) in pithed spontaneously hypertensive (SH) rats and to evaluate its mechanism of action. The results show that ET (0.1-3 nmol/kg i.v.) is a powerful vasoconstrictor agent in the pithed rat. However, at a dose of 3 nmol/kg i.v. all the pithed animals "died" following a gradual decrease in mean arterial blood pressure and pulse pressure and changes in the form of the electrocardiogram (ECG). The predominant feature of the change in the ECG was a progressive decrease in the amplitude of the T wave resulting in a depression of the curve representing repolarization. Investigations in isolated perfused SH rat hearts showed that ET powerfully reduces coronary flow concentration-dependently (IC50 2.1 +/- 0.3 nM) an effect associated with sinus bradycardia and a decrease in coronary pressure amplitude. No overt ECG changes were seen. Control experiments with mechanical flow restriction suggest that bradycardia is a consequence of reduced coronary flow and that the ECG changes observed in vivo can be explained on the basis of coronary insufficiency and resulting myocardial hypoxia. Vasoconstrictor responses to angiotensin II (0.4 microgram/kg i.v.), phenylephrine (8 micrograms/kg i.v.) and ET (0.5 nmol/kg i.v.) were antagonised by around 70% by isradipine (0.03 mg/kg i.a.). The results suggest that endothelin-induced vasoconstriction may involve receptor operated channel activation and opening of voltage sensitive Ca2+ channels.

1 Palmitoyl carnitine, a lipid metabolite which accumulates in cytoplasmic membranes during ischaemia, has been shown to resemble the Ca2+ channel activator, Bay K 8644, in K+-depolarized smooth muscle. Palmitoyl carnitine caused concentration-dependent (1-1000 mumol l-1) augmentations in the sensitivity to Ca2+ of K+-depolarized taenia preparations from the guinea-pig caecum. The (+/-)-isomer was equieffective with the (-)-isomer, whereas carnitine was ineffective and palmitic acid relaxed the tissues. The shift to the left of Ca2+ concentration-response curves induced by palmitoyl carnitine (100 mumol l-1) was additive with that of Bay K 8644 (1 mumol l-1). 2 The interactions of palmitoyl carnitine with the different classes of calcium-antagonist were similar to those seen with Bay K 8644. Schild plots of the calcium-antagonist effects of nifedipine were shifted to the right following preincubation of the taenia with palmitoyl carnitine (30-300 mumol l-1). The inhibitory effects of verapamil were especially sensitive to palmitoyl carnitine (100 mumol l-1). Whereas the potency of diltiazem as a calcium-antagonist was reduced by palmitoyl carnitine (100 mumol l-1), the inhibitory effects of the lipophilic class III calcium-antagonists, cinnarizine and flunarizine, were entirely resistant to palmitoyl carnitine (100 mumol l-1). 3 Although palmitoyl carnitine has detergent properties in high concentrations and lyses red blood cells, these effects were not Ca2+-dependent, nor were they modified by calcium-antagonists. Other detergents did not have selective interactions with Ca2+ channels. 4 Palmitoyl carnitine inhibited [3H]-nitrendipine, [3H]-verapamil and [3H]-diltiazem binding to rat cortical membranes with IC50 values (mumol l-1) of 120 +/- 1, 95 +/- 17 and 120 +/- 15 mumol l-1 respectively. The inhibition showed little temperature-dependence, in contrast to that of Bay K 8644, except for a small reduction in the IC50 value for [3H]-verapamil binding at 37 degrees C (42 +/- 5 mumol l-1). Palmitoyl carnitine interacted selectively with the Ca2+ channel, in that effects on ligand binding to alpha-adrenoceptors, beta-adrenoceptors and 5-HT1A receptors occurred only at 5-10 fold higher concentrations. 5 It is concluded that palmitoyl carnitine, at concentrations which have previously been shown to occur in the cytoplasm during myocardial ischaemia, may interact directly with Ca2+ channels and may therefore be considered as an endogenous modulator of channel function. The site of action differs from that of other agents.

(E)-beta-Fluoromethylene-m-tyrosine (MDL 72394) is not per se an inhibitor of monoamine oxidase (MAO) but is a substrate of aromatic L-amino acid decarboxylase (AADC) which liberates the potent MAO inhibitor (E)-beta-fluoromethylene-m-tyramine (MDL 72392). When co-administered to animals with the peripherally selective AADC inhibitor, carbidopa, MDL 72394 inhibited MAO selectively in the brain. Chronic (14 days plus 3 days withdrawal) administration of 0.5 mg/kg per day p.o. MDL 72394, 0.1 mg/kg per day p.o. MDL 72394 combined with 10 mg/kg per day p.o. carbidopa or 50 mg/kg per day p.o. pargyline produced equivalent inhibition of rat brain MAO and decreased the binding of [3H]clonidine and [3H]RX 781094 to the alpha 2-adrenoceptor and of [3H]dihydroalprenolol to the beta-adrenoceptor without changing binding of [3H]prazosin to the alpha 1-adrenoceptor. The locomotor depressant effect of clonidine was attenuated without attenuation of the hypotensive effect in rats treated chronically with the MAO inhibitors. Neither the sensitivity of the alpha 2-autoreceptor nor of the alpha 2-heteroreceptor was decreased in brain slices. However, the sensitivity of adenylate cyclase to activation by both noradrenaline and isoprenaline was significantly reduced. The number of 5-HT2 and 5-HT1A binding sites was decreased: the 5-HT1B binding sites remained unchanged. The effect of chronic MAO inhibitor treatment on 5-HT1A receptors was associated with a decrease in the behavioural response to 8-hydroxy-2-(di-n-propylamino)tetralin and the decrease in 5-HT2 binding was related to a small reduction in the sensitivity of the inositol phosphate system to stimulation by 5-HT. The lack of effect of chronic MAO treatment on the 5-HT autoreceptor measured in cortical slices corresponded to a lack of effect on the 5-HT1B binding site except that chronic administration of pargyline produced a small but significant decrease in 5-HT autoreceptor sensitivity. Overall, the data show that chronic administration of MDL 72394 has a profile of effects on central monoamine receptor binding and function similar to that seen following chronic administration of a number of clinically effective antidepressants.

A conformational study of four 5-HT1A (serotonin) receptor ligands ((R-(-)-methiothepin, spiperone, (S)-(-)-propranolol, and buspirone) led to the definition of a pharmacophore and a three-dimensional map of the 5-HT1A antagonist recognition site. These models were used to design new compounds and successfully predict their potency, stereospecificity, and selectivity. For example, 8-[4-[(1,4-benzodioxan-2-ylmethyl)amino] butyl]-8-azaspiro[4.5]decane-7,9-dione (1, MDL 72832) has nanomolar affinity (pIC50 = 9.14) for the 5-HT1A binding site in rat frontal cortex. As predicted, the S-(-) enantiomer of 1 was more active than its R-(+) enantiomer (pIC50 = 9.21 and 7.66, respectively) and a naphthalene analogue of 1 displayed the expected improved selectivity.

The subcutaneous injection of isoprenaline, salbutamol, histamine, and adrenaline to rats, which were subsequently killed by microwave irradiation, resulted in a rapid increase in the cyclic AMP content of the carotid body. On the other hand, noradrenaline, dopamine, adenosine, and 5-hydroxytryptamine, at doses at least 100 times greater than that of isoprenaline, did not significantly alter the cyclic nucleotide content in vivo. The response to isoprenaline was dose related, with an ED50 of 15 micrograms X kg-1, and reached a peak level 1-1.5 min after injection. Incubation of intact carotid bodies with isoprenaline (10(-5) M) in vitro also resulted in a 10-fold increase in cyclic AMP content. The in vivo response to isoprenaline could be blocked stereo-selectively by propranolol, and ICI 118.551, a beta 2-selective antagonist, blocks the isoprenaline-elicited increase in cyclic AMP completely at a dose of 30 micrograms X kg-1; whereas betaxolol, a beta 1-selective antagonist, was ineffective, even at a dose of 300 micrograms X kg-1. Hypoxia (5% oxygen in 95% N2) did not result in a significant increase in the cyclic AMP content, nor did it significantly alter the isoprenaline-stimulated increase in the cyclic AMP content of the rat carotid body. These results suggest that some catecholamines may stimulate cyclic AMP formation by interacting with a beta 2-adrenoceptor in the rat carotid body.