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The cleavage of amyloid precursor protein by [gamma]-secretase is an important aspect of the pathogenesis of Alzheimer's disease. [gamma]-Secretase also cleaves other membrane proteins (eg, Notch), which control cell development and homeostasis. Presenilin 1 and 2 are considered important determinants of the [gamma]-secretase catalytic site. Our aim was to investigate whether [gamma]-secretase can be important for microglial phagocytosis of Alzheimer's disease [beta]-amyloid. [ABSTRACT FROM AUTHOR]

Activation of the m1 muscarinic receptor subtype in rat pheochromocytoma (PC12) cells stably expressing cloned m1 muscarinic acetylcholine receptors was previously shown to induce morphological changes and growth arrest. However, the signaling pathways which lead to these effects were not identified. In an attempt to characterize the intracellular signaling that might be involved in the muscarinic-induced effects, we investigated the role of reactive oxygen species in the regulation of these processes. Stimulation of the muscarinic receptor in these cells increased the intracellular concentrations of reactive oxygen species. Muscarinic activation induced intracellular signaling pathways that involve activation of Ras, extracellular signal-regulated kinase (ERK), and p38. These pathways were partially blocked when reactive oxygen species (ROS) production was prevented by the antioxidant N-acetylcysteine. Other muscarinic-induced signals, such as activation of c-Jun NH[sub 2]-terminal kinase (JNK) or an increase in the binding activity of the transcription factors nuclear factor-κB and activator protein-1, were inhibited by the antioxidant dicoumarol. N-Acetylcysteine also blocked the growth arrest and changes in cell shape induced by stimulation of the muscarinic receptor in PC12M1 cells. These findings suggest that ROS act as second messengers in muscarinic-induced cellular signaling. Moreover, generation of ROS appears to be an early and critical intermediary event, which occurs immediately after stimulation of the muscarinic receptor and affects in a variety of mechanisms the muscarinic-mediated cellular signaling. [ABSTRACT FROM AUTHOR]

Recent research in the field of bioinformatics and molecular biology has revealed the immense complexity and uniqueness of microbiomes, while also showcasing the impact of the symbiosis between a microbiome and its host or environment. A core property influencing this process is horizontal gene transfer between members of the bacterial community used to maintain genetic variation. The essential effect of this mechanism is the exposure of genetic information to a wide array of members of the community, creating an additional "layer" of information in the microbiome named the "plasmidome". From an engineering perspective, introduction of genetic information to an environment must be facilitated into chosen species which will be able to carry out the desired effect instead of competing and inhibiting it. Moreover, this process of information transfer imposes concerns for the biosafety of genetic engineering of microbiomes as exposure of genetic information into unwanted hosts can have unprecedented ecological impacts. Current technologies are usually experimentally developed for a specific host/environment, and only deal with the transformation process itself at best, ignoring the impact of horizontal gene transfer and gene-microbiome interactions that occur over larger periods of time in uncontrolled environments. The goal of this research was to design new microbiome-specific versions of engineered genetic information, providing an additional layer of compatibility to existing engineering techniques. The engineering framework is entirely computational and is agnostic to the selected microbiome or gene by reducing the problem into the following set up: microbiome species can be defined as wanted or unwanted hosts of the modification. Then, every element related to gene expression (e.g., promoters, coding regions, etc.) and regulation is individually examined and engineered by novel algorithms to provide the defined expression preferences. Additionally, the synergistic effect of the combination of engineered gene blocks facilitates robustness to random mutations that might occur over time. This method has been validated using both computational and experimental tools, stemming from the research done in the iGEM 2021 competition, by the TAU group.

Ataxia-telangiectasia (A-T) is a multisystem autosomal recessive disease caused by mutations in the ATM gene and characterized by cerebellar atrophy, progressive ataxia, immunodeficiency, male and female sterility, radiosensitivity, cancer predisposition, growth retardation, insulin-resistant diabetes, and premature aging. ATM phosphorylates more than 1500 target proteins, which are involved in cell cycle control, DNA repair, apoptosis, modulation of chromatin structure, and other cytoplasmic as well as mitochondrial processes. In our quest to better understand the mechanisms by which ATM deficiency causes cerebellar degeneration, we hypothesized that specific vulnerabilities of cerebellar microglia underlie the etiology of A-T. Our hypothesis is based on the recent finding that dysfunction of glial cells affect a variety of process leading to impaired neuronal functionality (Song et al., 2019). Whereas astrocytes and neurons descend from the neural tube, microglia originate from the hematopoietic system, invade the brain at early embryonic stage, and become the innate immune cells of the central nervous system and important participants in development of synaptic plasticity. Here we demonstrate that microglia derived from Atm -/- mouse cerebellum display accelerated cell migration and are severely impaired in phagocytosis, secretion of neurotrophic factors, and mitochondrial activity, suggestive of apoptotic processes. Interestingly, no microglial impairment was detected in Atm-deficient cerebral cortex, and Atm deficiency had less impact on astroglia than microglia. Collectively, our findings validate the roles of glial cells in cerebellar attrition in A-T., (© 2021 Wiley Periodicals LLC.)

Purpose: Semaphorin 3A (Sema-3A) is a secreted protein that deflects axons from inappropriate regions and induces neuronal cell death. Intravitreal application of polyclonal antibodies against Sema-3A prevents loss of retinal ganglion cells ensuing from axotomy of optic nerves. This suggested a therapeutic approach for neuroprotection via inhibition of the Sema-3A pathway., Methods: To develop potent and specific Sema-3A antagonists, we isolated monoclonal anti-Sema-3A antibodies from a human antibody phage display library and optimized low-molecular weight Sema-3A signaling inhibitors. The best inhibitors were identified using in vitro scratch assays and semiquantitative repulsion assays., Results: A therapeutic approach for neuroprotection must have a long duration of action. Therefore, antibodies and low-molecular weight inhibitors were formulated in extruded implants to allow controlled and prolonged release. Following release from the implants, Sema-3A inhibitors antagonized Sema-3A effects in scratch and repulsion assays and protected retinal ganglion cells in animal models of optic nerve injury, retinal ischemia, and glaucoma., Conclusions and Translational Relevance: Collectively, our findings indicate that the identified Sema-3A inhibitors should be further evaluated as therapeutic candidates for the treatment of Sema-3A-driven central nervous system degenerative processes.

Evidence suggests that astrocytes play key roles in structural and functional organization of neuronal circuits. To understand how astrocytes influence the physiopathology of cerebellar circuits, we cultured cells from cerebella of mice that lack the ATM gene. Mutations in ATM are causative of the human cerebellar degenerative disease ataxia-telangiectasia. Cerebellar cultures grown from Atm -/- mice had disrupted network synchronization, atrophied astrocytic arborizations, reduced autophagy levels, and higher numbers of synapses per neuron than wild-type cultures. Chimeric circuitries composed of wild-type astrocytes and Atm -/- neurons were indistinguishable from wild-type cultures. Adult cerebellar characterizations confirmed disrupted astrocyte morphology, increased GABAergic synaptic markers, and reduced autophagy in Atm -/- compared with wild-type mice. These results indicate that astrocytes can impact neuronal circuits at levels ranging from synaptic expression to global dynamics., Competing Interests: The authors declare no conflict of interest.

In Alzheimer's disease (AD), astrocytes undergo morphological changes ranging from atrophy to hypertrophy, but the effect of such changes at the functional level is still largely unknown. Here, we aimed to investigate whether alterations in astrocyte activity in AD are transient and depend on their microenvironment, or whether they are irreversible. We established and characterized a new protocol for the isolation of adult astrocytes and discovered that astrocytes isolated from old 5xFAD mice have higher GFAP expression than astrocytes derived from WT mice, as observed in vivo. We found high C1q levels in brain sections from old 5xFAD mice in close vicinity to amyloid plaques and astrocyte processes. Interestingly, while old 5xFAD astrocytes are impaired in uptake of soluble Aβ42, this effect was reversed upon an addition of exogenous C1q, suggesting a potential role for C1q in astrocyte-mediated Aβ clearance. Our results suggest that scavenger receptor B1 plays a role in C1q-facilitated Aβ uptake by astrocytes and that expression of scavenger receptor B1 is reduced in adult old 5xFAD astrocytes. Furthermore, old 5xFAD astrocytes show impairment in support of neuronal growth in co-culture and neurotoxicity concomitant with an elevation in IL-6 expression. Further understanding of the impact of astrocyte impairment on AD pathology may provide insights into the etiology of AD., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Brain-machine interfaces (BMI) were born to control "actions from thoughts" in order to recover motor capability of patients with impaired functional connectivity between the central and peripheral nervous system. The final goal of our studies is the development of a new proof-of-concept BMI-a neuromorphic chip for brain repair-to reproduce the functional organization of a damaged part of the central nervous system. To reach this ambitious goal, we implemented a multidisciplinary "bottom-up" approach in which in vitro networks are the paradigm for the development of an in silico model to be incorporated into a neuromorphic device. In this paper we present the overall strategy and focus on the different building blocks of our studies: (i) the experimental characterization and modeling of "finite size networks" which represent the smallest and most general self-organized circuits capable of generating spontaneous collective dynamics; (ii) the induction of lesions in neuronal networks and the whole brain preparation with special attention on the impact on the functional organization of the circuits; (iii) the first production of a neuromorphic chip able to implement a real-time model of neuronal networks. A dynamical characterization of the finite size circuits with single cell resolution is provided. A neural network model based on Izhikevich neurons was able to replicate the experimental observations. Changes in the dynamics of the neuronal circuits induced by optical and ischemic lesions are presented respectively for in vitro neuronal networks and for a whole brain preparation. Finally the implementation of a neuromorphic chip reproducing the network dynamics in quasi-real time (10 ns precision) is presented.

The growing recognition that brain pathologies do not affect neurons only but rather are, to a large extent, pathologies of glial cells as well as of the vasculature opens to new perspectives in our understanding of genetic disorders of the CNS. To validate the role of the neuron-glial-vascular unit in the etiology of genome instability disorders, we report about cell death and morphological aspects of neuroglia networks and the associated vasculature in a mouse model of Ataxia Telangiectasia (A-T), a human genetic disorder that induces severe motor impairment. We found that A-T-mutated protein deficiency was consistent with aberrant astrocytic morphology and alterations of the vasculature, often accompanied by reactive gliosis. Interestingly similar findings could also be reported in the case of other genetic disorders. These observations bolster the notion that astrocyte-specific pathologies, hampered vascularization and astrocyte-endothelium interactions in the CNS could play a crucial role in the etiology of genome instability brain disorders and could underlie neurodegeneration.

Pronounced neuropathology is a feature of ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS), which are both genomic instability syndromes. The Nbs1 protein, which is defective in NBS, is a component of the Mre11/RAD50/NBS1 (MRN) complex. This complex plays a major role in the early phase of the cellular response to double strand breaks (DSBs) in the DNA. Among others, MRN is required for timely activation of the protein kinase ATM (A-T mutated), which is disrupted in patients with A-T. Earlier reports show that Atm-deficient mice exhibit severe degeneration of tyrosine hydroxylase (TH)-positive dopaminergic nigro-striatal neurons and their terminals in the striatum. This cell loss is accompanied by a large reduction in immunoreactivity for the dopamine transporter protein (DAT) in the striatum. To test whether Nbs1 inactivation also affects the integrity of the nigro-striatal pathway, we examined this pathway in a murine model with conditional inactivation of the Nbs1 gene in central nervous system (Nbs1-CNS-Δ). We report that this model has a reduction in TH-positive cells in the substantia nigra. This phenomenon was seen at very early age, while Atm-/- mice showed a progressive age-dependent reduction. Furthermore, we observed an age-dependent increase in the level of TH in the striatum of Atm-/- and Nbs1-CNS-Δ mice. In addition to the altered expression of TH, we also found a reduction of DAT in the striatum of both Atm-/- and Nbs1-CNS-Δ mice at 60 days of age. Finally, microglial recruitment and alterations in the levels of various neurotrophic factors were also observed. These results indicate that malfunctioning DNA damage response severely affects the integrity of the nigro-striatal pathway and suggest a new neurodegenerative pathway in Parkinsonian syndromes.

Nijmegen breakage syndrome (NBS) is a genomic instability disorder caused by hypomorphic mutations in the Nbs1 gene. When Nbs1 is conditionally inactivated in the central nervous system of mice (Nbs1-CNS-Δ), they suffer from severe cerebellar atrophy, ataxia, and white matter damage. Here, we show that conditional inactivation of the murine Nbs1 gene has a profound effect on the integrity and the functionality of the glial cells, which suggests their crucial role in the pathogenesis of NBS. Interestingly, in Nbs1-CNS-Δ mice, the dramatic reduction in the numbers of Purkinje and granule cells was also linked to a reduction of microglial cells but not to astrocytes (GFAP+), suggesting an impairment in astrocytic functionality. Nbs1 levels were dramatically reduced in adult astrocyte isolated from Nbs1-CNS-Δ mice, suggesting a major role in cerebellar pathology. In order to investigate the effect of Nbs1 deletion on astrocyte activity, we investigated glutamine synthetase levels in astrocyte and discovered 40% reduction as compared to WT. Furthermore, we found a significant reduction in the secretion of neurotrophic factors, such as brain-derived neurotrophic factor and neurotrophin 3. Understanding the contribution of malfunctioning astrocytes to the etiology of NBS can elucidate a hitherto unknown aspect of this disorder.

Ataxia-telangiectasia is a multifaceted syndrome caused by null mutations in the ATM gene, which encodes the protein kinase ATM, a key participant in the DNA damage response. Retinal neurons are highly susceptible to DNA damage because they are terminally differentiated and have the highest metabolic activity in the central nervous system. In this study, we characterized the retina in young and aged Atm-deficient mice (Atm(-/-)). At 2 months of age, angiography revealed faint retinal vasculature in Atm(-/-) animals relative to wild-type controls. This finding was accompanied by increased expression of vascular endothelial growth factor protein and mRNA. Fibrinogen, generally absent from wild-type retinal tissue, was evident in Atm(-/-) retinas, whereas mRNA of the tight junction protein occludin was significantly decreased. Immunohistochemistry labeling for occludin in 6-month-old mice showed that this decrease persists in advanced stages of the disease. Concurrently, we noticed vascular leakage in Atm(-/-) retinas. Labeling for glial fibrillary acidic protein demonstrated morphological alterations in glial cells in Atm(-/-) retinas. Electroretinographic examination revealed amplitude aberrations in 2-month-old Atm(-/-) mice, which progressed to significant functional deficits in the older mice. These results suggest that impaired vascularization and astrocyte-endothelial cell interactions in the central nervous system play an important role in the etiology of ataxia-telangiectasia and that vascular abnormalities may underlie or aggravate neurodegeneration., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are related genomic instability syndromes characterized by neurological deficits. The NBS1 protein that is defective in NBS is a component of the Mre11/RAD50/NBS1 (MRN) complex, which plays a major role in the early phase of the complex cellular response to double strand breaks (DSBs) in the DNA. Among others, Mre11/RAD50/NBS1 is required for timely activation of the protein kinase ATM (A-T, mutated), which is missing or inactivated in patients with A-T. Understanding the molecular pathology of A-T, primarily its cardinal symptom, cerebellar degeneration, requires investigation of the DSB response in cerebellar neurons, particularly Purkinje cells, which are the first to be lost in A-T patients. Cerebellar cultures derived from mice with different mutations in DNA damage response genes is a useful experimental system to study malfunctioning of the damage response in the nervous system. To clarify the interrelations between murine Nbs1 and Atm, we generated a mouse strain with specific disruption of the Nbs1 gene in the central nervous system on the background of general Atm deficiency (Nbs1-CNS-Δ//Atm(-/-)). This genotype exacerbated several features of both conditions and led to a markedly reduced life span, dramatic decline in the number of cerebellar granule neurons with considerable cerebellar disorganization, abolishment of the white matter, severe reduction in glial cell proliferation, and delayed DSB repair in cerebellar tissue. Combined loss of Nbs1 and Atm in the CNS significantly abrogated the DSB response compared with the single mutation genotypes. Importantly, the data indicate that Atm has cellular roles not regulated by Nbs1 in the murine cerebellum.

Objective: The cleavage of amyloid precursor protein by γ-secretase is an important aspect of the pathogenesis of Alzheimer's disease. γ-Secretase also cleaves other membrane proteins (eg, Notch), which control cell development and homeostasis. Presenilin 1 and 2 are considered important determinants of the γ-secretase catalytic site. Our aim was to investigate whether γ-secretase can be important for microglial phagocytosis of Alzheimer's disease β-amyloid., Methods: We investigated the role of γ-secretase in microglia activity toward β-amyloid phagocytosis in cell culture using γ-secretase inhibitors and small hairpin RNA and presenilin-deficient mice., Results: We found that γ-secretase inhibitors impair microglial activity as measured in gene expression, protein levels, and migration ability, which resulted in a reduction of soluble β-amyloid phagocytosis. Moreover, microglia deficient in presenilin 1 and 2 showed impairment in phagocytosis of soluble β-amyloid. Dysfunction in the γ-secretase catalytic site led to an impairment in clearing insoluble β-amyloid from brain sections taken from an Alzheimer's disease mouse model when compared to microglia from wild-type mice., Interpretation: We suggest for the first time, a dual role for γ-secretase in Alzheimer's disease. One role is the cleavage of the amyloid precursor protein for pathologic β-amyloid production and the other is to regulate microglia activity that is important for clearing neurotoxic β-amyloid deposits. Further studies of γ-secretase-mediated cellular pathways in microglia may provide useful insights into the development of Alzheimer's disease and other neurodegenerative diseases, providing future avenues for therapeutic intervention., (Copyright © 2010 American Neurological Association.)

Background: Neurons of adult mammalian CNS are prevented from regenerating injured axons due to formation of a non-permissive environment. The retinal ganglion cells (RGC), which are part of the CNS, share this characteristic. In sharp contrast, the RGC of lower vertebrates, such as fish, are capable of re-growing injured optic nerve axons, and achieve, through a complex multi-factorial process, functional vision after injury. Semaphorin-3A (sema-3A), a member of the class 3 semaphorins known for its repellent and apoptotic activities, has previously been shown to play a key role in the formation of a non-permissive environment after CNS injury in mammalians., Methods: The expression of sema-3A and its effect on regenerative processes in injured gold fish retina and optic nerve were investigated in this study. Unilateral optic nerve axotomy or crush was induced in goldfish. 2 microl sema-3A was injected intraviterally 48 hours post injury. Neuronal viability was measured using the lipophilic neurotracer dye 4-Di-10-Asp. Axonal regeneration was initiated using the anterograde dye dextran. Retinas and optic nerves were collected at intervals of 2, 3, 7, 14 and 28 days after the procedure. Using Western blot and immunohistochemical analysis, the expression levels of semaphorin-3A, axonal regeneration, the removal of myelin debris and macrophage invasion were studied., Results: We found a decrease in sema-3A levels in the retina at an early stage after optic nerve injury, but no change in sema-3A levels in the injured optic nerve. Intravitreal injection of sema-3A to goldfish eye, shortly after optic nerve injury, led to destructive effects on several pathways of the regenerative processes, including the survival of retinal ganglion cells, axonal growth, and clearance of myelin debris from the lesion site by macrophages., Conclusions: Exogenous administration of sema-3A in fish indirectly interferes with the regeneration process of the optic nerve. The findings corroborate our previous findings in mammals, and further validate sema-3A as a key factor in the generation of a non-permissive environment after transection of the optic nerve.

Nijmegen breakage syndrome (NBS) is a genomic instability disease caused by hypomorphic mutations in the NBS1 gene encoding the Nbs1 (nibrin) protein. Nbs1 is a component of the Mre11/Rad50/Nbs1 (MRN) complex that acts as a sensor of double strand breaks (DSBs) in the DNA and is critical for proper activation of the broad cellular response to DSBs. Conditional disruption of the murine ortholog of the human NBS1, Nbs1, in the CNS of mice was previously reported to cause microcephaly, severe cerebellar atrophy and ataxia. Here we report that conditional targeted disruption of the murine NBS1 gene in the CNS results in mal-development, degeneration, disorganization and dysfunction of the murine visual system, especially in the optic nerve. Nbs1 deletion resulted in reduced diameters of Nbs1-CNS-Delta eye and optic nerve. MRI analysis revealed defective white matter development and organization. Nbs1 inactivation altered the morphology and organization of the glial cells. Interestingly, at the age of two-month-old the levels of the axonal guidance molecule semaphorin-3A and its receptor neuropilin-1 were up-regulated in the retina of the mutant mice, a typical injury response. Electroretinogram analysis revealed marked reduction in a- and b-waves, indicative of decreased retinal function. Our study points to a novel role for Nbs1 in the development, organization and function of the visual system.

Nijmegen breakage syndrome (NBS) is a genomic instability disease caused by hypomorphic mutations in the NBS1 gene encoding the Nbs1 (nibrin) protein. Nbs1 is a component of the Mre11/Rad50/Nbs1 (MRN) complex that acts as a sensor of double strand breaks (DSBs) in the DNA and is critical for proper activation of the broad cellular response to DSBs. Conditional disruption of the murine ortholog of NBS1, Nbn, in the CNS of mice was previously reported to cause microcephaly, severe cerebellar atrophy and ataxia. In this study we used MRI to study the brain morphology and organization of Nbn deleted mice. Using conventional T(2)-weighted magnetic resonance, we found that the brains of the mutant mice (Nbs1-CNS-del) were significantly smaller than those of the wild-type animals, with marked mal-development of the cerebellum. Region of interest analysis of the T(2) maps revealed significant T(2) increase in the areas of white matter (corpus callosum, internal capsule and midbrain), with minor changes, if any, in gray matter. Diffusion tensor imaging (DTI) data confirmed that fractional anisotropy values were significantly reduced in these areas, mainly due to increased radial diffusivity (water diffusion perpendicular to neuronal fibers). Biochemical analysis showed low and dispersed staining for MBP and GalC in Nbs1-CNS-del brains, indicating defects in myelin formation and oligodendrocyte development. Myelin index and protein levels were significantly reduced in these brains. Our results point to a novel function of Nbs1 in the development and organization of the white matter.

Ataxia-telangiectasia (A-T) is a multisystem, cancer-predisposing genetic disorder caused by deficiency of the ATM protein. To dissect the A-T phenotype, we augmented specific features of the human disease by generating mouse strains that combine Atm deficiency with dysfunction of other proteins. Increasing oxidative stress by combining deficiencies in Atm and superoxide dismutase 1 (Sod1) exacerbated growth retardation and markedly reduced the mean survival time following ionizing radiation. In contrast, increasing genomic instability by combining deficiencies of Atm and the mismatch repair protein Mlh1 caused a moderate increase in radiation sensitivity and dramatic increase in aggressive lymphomas, compared with thes Atm-/- single knockout. Remarkably, Atm, Mlh1 or Mlh1/Atm single or double heterozygosity did not significantly affect the life span of the various genotypes. Mlh1/Atm double null tumors were polyclonal, whereas the tumors in other genotypes were mono- or oligoclonal, demonstrating the high predisposition of thymocytes with this genotype to become malignant. Chromosomal aberrations in the tumors were localized mainly in chromosomes 12 and 15. The genomic region on chromosome 15, which contains the gene for the c-Myc oncoprotein, was commonly amplified, and elevated levels of the c-Myc protein were subsequently observed in the tumors. Our data suggest that impaired genomic instability is an important contributing factor to cancer predisposition in A-T, whereas oxidative stress is more important in the radiation sensitivity and growth retardation facets of this disease.

Endothelins have been implicated in the regulation of cell proliferation, differentiation, and apoptosis, but the mechanisms of these complex events are not yet fully understood. Although the nuclear factor-kappaB (NF-kappaB) was shown to play a prominent role in the above processes, its participation in endothelin receptor A (ET(A)R) signaling has not been previously demonstrated. This study provides evidence that NF-kappaB is involved in ET(A)R-induced proliferation and inhibition of apoptosis. Endothelin (ET)-1, ET-3, and sarafotoxin b induce cell proliferation and prevent apoptosis induced by serum deprivation in a Chinese hamster lung (CCL39) cell line that stably expresses ET(A)R (CCL39ET(A)). Activation of ET(A)R resulted in enhanced DNA-binding activity of NF-kappaB and degradation of IkappaB-alpha. Expression of the dominant negative form of IkappaB-alpha (IkappaB deltaN) inhibited the proliferative activities mediated by ET(A)R as well as its anti-apoptotic activities. Treatment of the cells with prostaglandin A1, an inhibitor of IkappaB kinase-beta, reduced ET-1-induced proliferation and its anti-apoptotic effect. These findings indicate that the regulation of cell proliferation and apoptosis by ET(A)R is mediated by the ET(A)R-activated NF-kappaB.

Background and Purpose: Endothelin 1 (ET-1), a highly potent endogenous vasoactive peptide, exerts a sustained vasoconstrictive effect on cerebral vessels. Elevation of ET-1 in plasma has been reported 1 to 3 days after ischemic stroke. Since we assumed that a much faster and more intense response may be observed in the cerebrospinal fluid (CSF) and since an increase in concentration of ET-1 in the CSF may cause constriction of cerebral vessels and eventually influence the neurological outcome, we measured ET-1 values in the CSF within 18 hours of stroke onset and compared the values with those in the plasma., Methods: Twenty-six consecutive patients with acute stroke were clinically evaluated according to the modified Matthew Scale and underwent two repeat CT scans. Within 5 to 18 hours of stroke onset, lumbar puncture and blood samples were concomitantly obtained and tested; ET-1 levels in CSF and plasma of these patients were analyzed by radioimmunoassay and compared with the levels of a control group of patients with no neurological disease., Results: The mean CSF concentration of ET-1 in the CSF of stroke patients was 16.06 +/- 4.9 pg/mL, compared with 5.51 +/- 1.47 pg/mL in the control group (P < .001). It was significantly higher in cortical infarcts (mean, 17.7 +/- 4.1 pg/mL) than in subcortical lesions (mean, 10.77 +/- 4.1 pg/mL) (P < .001) and significantly correlated with the volume of the lesion (P = .003). The correlation between ET-1 levels in the CSF and the Matthew Scale score was less significant (P = .05). Plasma ET-1 level was not elevated in any group., Conclusions: ET-1 is found to be significantly elevated in the CSF of stroke patients during the 18 hours after stroke. No elevation was demonstrated in plasma at this time period. ET-1 may be used as an additional indicator of ischemic vascular events in the early diagnosis of stroke. The dissimilarity between the CSF and plasma ET-1 concentrations may lead also to an hypothesis that there is a vasoconstrictive effect on the cerebral vessels or a neuronal effect caused by ET-1 in the mechanism of the progression of brain ischemia.

Involvement of a cyclic GMP pathway in signal transduction stimulated by endothelins (ETs) and sarafotoxins (SRTXs) was explored using rat cerebellar slices. These peptides activated the same receptor binding sites (ET-1 and SRTX-b at the picomolar sites; ET-3 and SRTX-c at the nanomolar sites) to produce cyclic GMP, but their signaling pathways differed. The endothelins (ET-1 and ET-3) were found to signal via nitric oxide formation and to involve pertussis toxin-sensitive G-protein(s). The SRTXs (b and c), while also stimulating cyclic GMP production, did so via a pathway which is not L-arginine-dependent, i.e., carbon monoxide formation, and did not involve pertussis-toxin-sensitive G-protein(s). This is the first demonstration that the signaling pathways of endothelins and sarafotoxins may differ, even though they share the same binding sites.

The possible involvement of a cAMP pathway in endothelin (ET) signal transduction was explored using rat atrial slices. We show that ET-1 induces both stimulation and inhibition of cAMP formation, depending on its concentration. Unexpectedly, the effects of ET-3 and of sarafotoxins b and c (SRTX-b and SRTX-c) on this pathway differ from that of ET-1. Moreover, we show that the ET-1-induced formation of cAMP results from catecholamine release in a process mediated by a Ca2+ channel coupled to a pertussis toxin sensitive G-protein. It is concluded that this pathway is mediated by a new ETA receptor subtype (probably presynaptic), for which ET-1 is an agonist and ET-3, SRTX-b, and SRTX-c are antagonists.

Objective: We investigated the binding properties of the endothelin receptors in the human myometrium in clinical situations associated with different ovarian steroid levels., Subjects and Methods: Binding properties of the endothelin receptors were studied in myometrial membranes from post-menopausal women (n = 12), myomatous premenopausal women (n = 14) and pregnant women (n = 14), using 125I-labelled endothelin-1., Results: The mean (+/- SD) maximal receptor density (Bmax) was significantly higher in samples from premenopausal and pregnant women than from post-menopausal women (983 +/- 196, 1116 +/- 201 and 490 +/- 145 pmol/g protein, respectively). Receptor affinity (Kd) did not differ significantly between these groups. Among the pregnant women, mean Bmax and Kd values were similar in those who electively underwent Caesarean section prior to the onset of labour and those operated on during the second stage of spontaneous labour. Binding properties of myometrial membranes of either pre or post-menopausal women were unaffected by the presence of high levels of beta-oestradiol or progesterone in the medium. Among samples of premenopausal women, no significant difference was found in binding properties between those operated on either during mid-follicular phase or during mid-luteal phase., Conclusions: In clinical situations associated with relatively high levels of ovarian steroids, the density of endothelin receptors in the myometrium is higher than in situations associated with low ovarian steroid level. Ovarian steroids may exert their influence via the production of other mediators. Changes in density of the endothelin receptor, induced by change in ovarian steroids activity, might play a role in the regulation of myometrial contractility.

Densities and affinities of tissue protein receptor sites for endothelin-1 in placental and fetal membranes of six preeclamptic and 16 normotensive women in the 36-41st week of gestation were determined by the use of a binding assay with [125I]endothelin-1. The mean maximal density of receptor sites (Bmax) was significantly higher in the placentas of the pre-eclamptic than of the normotensive women (905 +/- 107 vs. 539 +/- 140 fmol/mg protein, p < 0.0001). No differences were found between the two groups with respect to the mean affinity (Kd) of placental receptors, or the mean Bmax and the mean Kd of fetal chorionic samples. In the normotensive group, there were no differences in mean placental Bmax values or in mean Kd values between women who went into spontaneous labor (whether delivered vaginally or abdominally) and those who were electively operated on prior to labor onset. No binding sites were detected in the fetal amniotic membranes of any of the women. Our results suggest that an increase in the maximal density of receptor sites to endothelin-1 in the placenta may play a role in the pathophysiology of pre-eclampsia by contributing to the placental insufficiency that characterizes this disorder.

Seven of the eight known isopeptides of the endothelin/sarafotoxin (ET/SRTX) family were tested on the isolated guinea pig ileum and found to cause a concentration-dependent increase in basal tone. The rate or the amplitude of the spontaneous rhythmic contractions of the ileal smooth muscle were essentially not affected by any of the peptides. The maximum contraction elicited by vasoactive intestinal contractor (VIC) was slightly stronger than that induced by endothelin-1 (ET-1) or sarafotoxin-b (SRTX-b), and significantly stronger than the maximal contractions elicited by sarafotoxin-a (SRTX-a), sarafotoxin-c (SRTX-c), or endothelin-3 (ET-3). Sarafotoxin-d (SRTX-d) caused, essentially, no contraction but a rather marked relaxation. The potencies of the various peptides to induce the increase in tension, in terms of EC50 values (cumulative effective concentrations that induce half-maximum response), ranged between 6 and 95 nM depending on the peptide. VIC, ET-1, SRTX-b and SRTX-a had similar potencies and were significantly more potent than SRTX-c and ET-3. A high concentration of SRTX-b elicited no additional response when applied to the organ bath after one of the other peptides had shown a maximal effect. Binding experiments with ileal membranes revealed similar binding properties for the various peptides. Competition with iodinated SRTX-b showed no meaningful differences between the various peptides. It is concluded that all the ET/SRTX peptides compete for the same receptor subtype in the ileum. In terms of efficacy, VIC can be considered as a full agonist of this receptor, SRTX-d is probably an antagonist, while all the other peptides behave as partial agonists.

Kinetics of ligand/receptor interactions using ET-1, ET-3 and SRTX-b were studied and cross-linking experiments carried out in guinea pig ileum and rat cerebellar preparations. Dissociation studies indicate that the two regions are characterized by different receptor subtypes and different modes of ligand binding. Autoradiographic patterns obtained following cross-linking of ET-1 and ET-3 to the different tissues support these conclusions.

The induction of phosphoinositide hydrolysis (PI) by endothelin/sarafotoxin (ET/SRTX) receptors in rat heart myocytes was investigated by the use of bacterial toxins as well as a phorbol ester. Both pertussis- and choleratoxin enhanced the stimulation of PI hydrolysis. Phorbol ester treatment of the myocytes for short periods distinguished between two types of PI-hydrolysis, the one induced by endothelins and the other by sarafotoxins. The possible mediation of G-protein (s) in the induction by ET/SRTX receptors of PI-hydrolysis is discussed.

Aging of rat heart myocytes in culture is accompanied by approximately 50% reduction in endothelin (ET)/sarafotoxin (SRTX) receptor-binding capacity as well as in the induction of phosphoinositide (PI) hydrolysis. Treatment of aged cultures under conditions yielding myocytes with a lipid composition similar to that in young cultures restored all the ET/SRTX receptors; at the same time it re-established only the endothelin-induced but not the sarafotoxin-induced PI-hydrolysis response. Thus more than one mechanism may stimulate PI metabolism.

A study of the effects of bisquaternary pyridinium oximes on calcium-dependent potassium-evoked [3H]acetylcholine release from rat brain slices revealed that at presynaptic autoreceptors these drugs function like muscarinic agonists, as they mimic the effects of acetylcholine in their inhibition of the evoked [3H]-acetylcholine release in an atropine-sensitive and dose-dependent manner. Since the bisquaternary pyridinium oximes are mild muscarinic antagonists at postsynaptic muscarinic receptors, they constitute a category of muscarinic ligands that are characterized by inverse dual activity at pre- and postsynaptic muscarinic receptors. These drugs may have dual function on cholinergic transmission by acting as presynaptic agonists and as postsynaptic antagonists. The most potent inhibitor of the evoked [3H]acetylcholine release was 1,1'-(4-hydroxyiminopyridinium)trimethylene (TMB-4) (I50 = 8 microM) and the weakest were 1-(2-hydroxyiminoethylpyridinium) 1-(3-cyclohexylcarboxypyridinium) dimethylether (HGG-42) and 1-(2-hydroxyiminoethylpyridinium) 1-(3-phenylcarboxypyridinium) dimethylether (HGG-12) (I50 = 150 microM). As postsynaptic antagonists, the latter drugs are more potent (K1 = 1.3-3.3 microM) than TMB-4 (K1 = 50 microM). Combined therapy with two drugs such as TMB-4 and HGG-12 might be effective in blocking severe hyperactivity of the cholinergic system.

Chemical modification of muscarinic receptors of rat cerebral cortex, brain stem and atria by a carboxyl-group-specific reagent, namely trimethyloxonium ion (TMO+) reduces the number of tritium-labeled antagonist- and agonist-binding sites in a dose-dependent way. No such effect is observed when modification is carried out in the presence of atropine, oxotremorine or carbamylcholine. These findings suggest that TMO+ specifically methylates the carboxyl residue(s) positioned at the binding site in members of the M1 and M2 receptor family.

Functional receptors for the peptides of the endothelin (ET) and sarafotoxin (SRTX) family were characterized in newborn rat heart myocytes using human and rat endothelins (ET-1 and ET-3, respectively), SRTX-b and SRTX-c. Binding studies in intact cells and homogenates revealed significantly higher affinities of ET-1 and SRTX-b than of ET-3 and SRTX-c towards these receptors. This binding profile of ET/SRTX peptides points to their interaction with the receptor subtype designated E-S alpha. All four peptides induced time- and dose-dependent phosphoinositide hydrolysis with the following rank order of potency: ET-1 greater than SRTX-b greater than SRTX-c greater than ET-3. Thus, ET-3 which possesses an intermediate affinity toward the receptor was the least effective with regard to this response. These results confirm and extend our earlier report that the ET/SRTX peptides interact with a newly characterized receptor(s) associated with phosphoinositide metabolism and Ca2+ mobilization. The initiation of inositol phosphate formation is largely independent of extracellular Ca2+, verapamil and nifedipine, indicating that the ET/SRTX peptides are not agonists for the voltage-dependent Ca2+-channels.

Binding properties of submaxillary gland muscarinic receptors and agonist-induced saliva secretion were studied in rats subjected to heat acclimation. The maximal binding capacity for the muscarinic antagonist N-[3H]methyl-4-piperidyl benzilate was increased from control value of 0.21 to 0.40 pmol/mg protein within 1-2 days of heat acclimation. The increase in the number of muscarinic receptors per gland (100%) was by far higher than the increase in tissue weight (20%), indicating higher density of receptors in the acinar cells of the treated rats. High levels of receptors coincided with the appearance of high-affinity binding sites for muscarinic agonists (oxotremorine, pilocarpine and carbamylcholine), and with reduced tissue sensitivity to pilocarpine. After 4-8 weeks of heat acclimation, the number of receptors as well as tissue response to pilocarpine returned to control levels. These results suggest a functional correlation between the transient upregulation muscarinic receptors in the submaxillary gland and the physiological activity in salivary secretion, and indicate that the high-affinity muscarinic receptors may attenuate saliva secretion during the initial phase of heat acclimation.