Excitatory amino acids activate calpain I and induce structural protein breakdown in vivo.

Neuronal activity regulates the catabolism of specific structural proteins in adult mammalian brain. Pharmacological stimulation of rat hippocampal neurons by systemic or intraventricular administration of the excitatory amino acids kainate or N-methyl-D-aspartate induces selective loss of brain spectrin and the microtubule-associated protein MAP2, as determined by quantitative immunoblotting, but not of actin, the high molecular weight neurofilament polypeptide, or glial fibrillary acidic protein. The spectrin decrease occurs primarily by enhanced proteolysis, as levels of the major breakdown products of the alpha-subunit increase more than 7-fold. This proteolysis may occur from activation of the calcium-dependent neutral protease calpain I. The immunopeptide maps produced by alpha-spectrin degradation, selective loss of spectrin and MAP2, and decrease in calpain I levels are all consistent with calpain I activation accompanied by autoproteolysis. We propose that calcium influx and calpain I activation provide a mechanism by which neuronal activity regulates the degradation of specific neuronal structural proteins and may thereby modify neuronal morphology.

Expression of beta-amyloid precursor protein in reactive astrocytes following neuronal damage.

Although the beta-amyloid peptide is an established core component of neuritic plaques that accumulate in Alzheimer's disease, the mechanisms responsible for its deposition are not well understood. We now report that lesions of rat hippocampal neurons cause a time-dependent, long-lasting elevation of immunoreactivity for the beta-amyloid precursor protein (APP) in neighboring astrocytes, a cell type not normally containing the protein. The increase represents astroglial expression of the protein rather than a scavenging of APP released by damaged neurons. Immunoelectron microscopy confirmed that APP-containing cells are reactive astroglia, both surrounding capillaries and within the neuropil. These results demonstrate that neuronal damage stimulates APP expression in adult brain and suggest that reactive astrocytes may be a source of the beta-amyloid that forms neuropathological plaques in Alzheimer's disease.

Behaviour of a full-scale expanded bed reactor with overlaid anaerobic and aerobic zones for domestic wastewater treatment.

This paper presents the behaviour of a full-scale expanded bed reactor (160 m3) with overlaid anaerobic and aerobic zones used for municipal wastewater treatment. The research was carried out in two experimental steps: anaerobic and anaerobic-aerobic conditions, and the experimental results presented in this paper refer to four months of reactor operation. In the anaerobic condition, after inoculation and 60 days of operation, the reactor treating 3.40 kg CODm(-3)d(-1) for thetaH of 2.69 h, reached mean removal efficiencies of 76% for BOD, 72% for COD, and 80% for TSS, when the effluent presented mean values of 225 mg.L(-1) of COD, 98 mg.L(-1) of BOD and 35 mg.L(-1) of TSS. Under these conditions, for nitrogen loading of 0.27 kgN.m(-3)d(-1), the reactor generated an effluent with mean N-org. of 8 mg.L(-1) and N-ammon. of 37 mg.L(-1), demonstrating high potential of ammonification. For the anaerobic-aerobic condition (118th day) the system was operated with thetaH of 5.38 h presented mean removal efficiencies of 84% for BOD, 79% for COD, 76% for TSS, and 30% for TKN. The reactor's operation time was less than two months, which was not long enough to reach nitrification. Regarding the obtained results, this research confirmed that this reactor is configured as a flexible and adequate alternative for the treatment of sewage, requiring relatively small area and only thetaH of 10 h that can be adjusted to the local circumstances.

Presenilin-1 P264L knock-in mutation: differential effects on abeta production, amyloid deposition, and neuronal vulnerability.

The pathogenic mechanism linking presenilin-1 (PS-1) gene mutations to familial Alzheimer's disease (FAD) is uncertain, but has been proposed to include increased neuronal sensitivity to degeneration and enhanced amyloidogenic processing of the beta-amyloid precursor protein (APP). We investigated this issue by using gene targeting with the Cre-lox system to introduce an FAD-linked P264L mutation into the endogenous mouse PS-1 gene, an approach that maintains normal regulatory controls over expression. Primary cortical neurons derived from PS-1 homozygous mutant knock-in mice exhibit basal neurodegeneration similar to their PS-1 wild-type counterparts. Staurosporine and Abeta1-42 induce apoptosis, and neither the dose dependence nor maximal extent of cell death is altered by the PS-1 knock-in mutation. Similarly, glutamate-induced neuronal necrosis is unaffected by the PS-1P264L mutation. The lack of effect of the PS-1P264L mutation is confirmed by measures of basal- and toxin-induced caspase and calpain activation, biochemical indices of apoptotic and necrotic signaling, respectively. To analyze the influence of the PS-1P264L knock-in mutation on APP processing and the development of AD-type neuropathology, we created mouse lines carrying mutations in both PS-1 and APP. In contrast to the lack of effect on neuronal vulnerability, cortical neurons cultured from PS-1P264L homozygous mutant mice secrete Abeta42 at an increased rate, whereas secretion of Abeta40 is reduced. Moreover, the PS-1 knock-in mutation selectively increases Abeta42 levels in the mouse brain and accelerates the onset of amyloid deposition and its attendant reactive gliosis, even as a single mutant allele. We conclude that expression of an FAD-linked mutant PS-1 at normal levels does not generally increase cortical neuronal sensitivity to degeneration. Instead, enhanced amyloidogenic processing of APP likely is critical to the pathogenesis of PS-1-linked FAD.

Novel dipeptidyl proteasome inhibitors overcome Bcl-2 protective function and selectively accumulate the cyclin-dependent kinase inhibitor p27 and induce apoptosis in transformed, but not normal, human fibroblasts.

It has been suggested that overexpression of the Bcl-2 oncoprotein in human cancer cells contributes to their resistance to apoptosis induced by chemotherapy. We report here that a novel dipeptidyl proteasome inhibitor, CEP1612, at low concentrations rapidly induces apoptosis in human Jurkat T cells overexpressing Bcl-2 and also in all human prostate, breast, tongue and brain tumor cell lines we have tested to date, without exception. In contrast, etoposide, a standard anticancer drug, fails to kill these cells when employed under the same conditions. The apoptosis-inducing abilities of CEP1612 and its analogous compounds match precisely their order for inhibition of the proteasome chymotrypsin-like activity. CEP1612-induced apoptosis is p53-independent, inhibitable by a tetrapeptide caspase inhibitor, and associated with accumulation of the cyclin-dependent kinase inhibitors p21 and p27. Furthermore, CEP1612 selectively accumulates p27 and induces apoptosis in simian virus 40-transformed, but not the parental normal, human fibroblasts. Proteasome inhibitors such as those investigated herein might therefore have potential use as novel anticancer drugs.

Strategies to alter the progression of Alzheimer's disease.

Recent advances in the genetics of familial Alzheimer's disease provide direction for therapeutic strategies to alter the progressive neurodegeneration. The rationale is particularly strong for targeting the deposition of amyloid into neuritic plaques, but attention has also turned to abnormalities in apoptosis and signal-transduction processes.

The role of calpain-mediated spectrin proteolysis in traumatically induced axonal injury.

In animals and man, traumatic brain injury (TBI) results in axonal injury (AI) that contributes to morbidity and mortality. Such injured axons show progressive change leading to axonal disconnection. Although several theories implicate calcium in the pathogenesis of AI, experimental studies have failed to confirm its pivotal role. To explore the contribution of Ca2+-induced proteolysis to axonal injury, this study was undertaken in an animal model of TBI employing antibodies targeting both calpain-mediated spectrin proteolysis (CMSP) and focal neurofilament compaction (NFC), a marker of intra-axonal cytoskeletal perturbation, at 15-120 minutes (min) postinjury. Light microscopy (LM) revealed that TBI consistently evoked focal, intra-axonal CMSP that was spatially and temporally correlated with NFC. These changes were seen at 15 min postinjury with significantly increasing number of axons demonstrating CMSP immunoreactivity over time postinjury. Electron microscopy (EM) demonstrated that at 15 min postinjury CMSP was confined primarily to the subaxolemmal network. With increasing survival (30-120 min) CMSP filled the axoplasm proper. These findings provide the first direct evidence for focal CMSP in the pathogenesis of generalized/diffuse AI. Importantly, they also reveal an initial subaxolemmal involvement prior to induction of a more widespread axoplasmic change indicating a spatial-temporal compartmentalization of the calcium-induced proteolytic process that may be amenable to rapid therapeutic intervention.

Prolonged calpain-mediated spectrin breakdown occurs regionally following experimental brain injury in the rat.

Calpain, a calcium-activated neutral protease family, has been implicated in the neuropathologic sequelae accompanying various neurological disorders. We have characterized the distribution and time course of calpain activation following brain injury in the rat, using a monoclonal antibody that recognizes calpain-generated breakdown products (BDPs) of spectrin. Adult male Sprague-Dawley rats received lateral fluid percussion brain injury of moderate severity (2.2-2.4 atm, n = 35) or served as controls (uninjured, n = 12). One group of animals (n = 21) were sacrificed at either 30 minutes (min), 1 day, or 3 days post-injury, and selected brain regions were prepared for Western blot analysis. The remaining animals (n = 26) were sacrificed at 90 min, 4 hours (h), 1 day, or 7 days post-injury, and immunohistochemistry was performed. Spectrin BDPs were found predominantly in the hemisphere ipsilateral to the injury site, located primarily in cortical and hippocampal regions which exhibit neuronal death. Calpain-mediated spectrin breakdown was detected at 90 min in dendrites and axons, and by 4 h in neuronal perikarya. By 1 day post-injury, cortical and hippocampal regions of calpain activation had increased in size. Delayed spectrin breakdown was observed in the thalamus, both at 3 days and 7 days after injury. These results suggest that calpain may play an important role in the neurodegenerative process following brain injury.

Perikaryal accumulation and proteolysis of neurofilament proteins in the post-mortem rat brain.

Investigations of neurofilament alterations in neurodegenerative disorders utilize postmortem human tissues obtained at autopsy. To determine if alterations in the levels or distribution of neurofilament proteins might occur during the interval between death and autopsy, the postmortem cooling curve of the human brain was modeled in Sprague-Dawley rats and neurofilament proteins were examined by immunocytochemistry and immunoblots. One hour after death, enhanced perikaryal immunostaining of NF-M and both phosphorylated and nonphosphorylated NF-H epitopes was observed throughout the hippocampal formation. A greater number of neurons exhibited increased somatic immunostaining 4-h postmortem. In addition, loss of neurofilament protein immunostaining was observed in the neuropil, particularly in the molecular layer of the dentate gyrus. This corresponded with, but lagged behind, the pattern of calpain activation determined using an antibody against calpain-cleaved alpha-spectrin. Immunoblots confirmed the postmortem loss of neurofilament proteins in both triton-soluble and insoluble fractions. These results demonstrate that the levels and localization of neurofilament proteins observed in tissues obtained at autopsy even with short postmortem intervals may not accurately reflect the premortem condition.

Diffuse plaques contain C-terminal A beta 42 and not A beta 40: evidence from cats and dogs.

Recent reports have suggested that beta-amyloid (A beta) species of variable length C-termini are differentially deposited within early and late-stage plaques and the cerebrovasculature. Specifically, longer C-terminal length A beta 42/3 fragments (i.e., A beta forms extending to residues 42 and/or 43) are thought to be predominant within diffuse plaques while both A beta 42/3 and A beta 40 (A beta forms terminating at residue 40) are present within a subset of neuritic plaques and cerebrovascular deposits. We sought to clarify the issue of differential A beta deposition using aged canines, a partial animal model of Alzheimer's disease that exhibits extensive diffuse plaques and frequent vascular amyloid, but does not contain neuritic plaques or neurofibrillary tangles. We examined the brains of 20 aged canines, 3 aged felines, and 17 humans for the presence of A beta immunoreactive plaques, using antibodies to A beta 1(-17), A beta 17(-24), A beta 1(-28), A beta 40, and A beta 42. We report that plaques within the canine and feline brain are immunopositive for A beta 42 but not A beta 40. This is the first observation of nascent AD pathology in the aged feline brain. Canine plaques also contained epitopes within A beta 1(-17), A beta 17(-24), and A beta 1(-28). In all species examined, vascular deposits were immunopositive for both A beta 40 and A beta 42. In the human brain, diffuse plaques were preferentially A beta 42 immunopositive, while neuritic plaques and vascular deposits were both A beta 40 and A beta 42 immunopositive. However, not all neuritic plaques contain A beta 40 epitopes.

Mutant and native human beta-amyloid precursor proteins in transgenic mouse brain.

Human beta-amyloid precursor protein (beta APP) has been targeted to transgenic neurons using synapsin I promoter-based chimeric transgenes. Native human beta APP was introduced as well as beta APP containing mutations genetically linked to familial Alzheimer's disease (AD) and to hereditary cerebral hemorrhage with amyloidosis-Dutch type. In mouse brain, human beta APP RNA was up to 60% as abundant as total endogenous beta APP RNA. Human beta APP gene expression was most abundant in the CA subfields of the hippocampus and in the piriform cortex. Correct processing of human beta APP at the beta-secretase cleavage site was demonstrated in transgenic mouse brains. Despite a 40% increase in total beta APP immunoreactivity in lines expressing mutant human beta APP, no evidence of amyloid deposition was found in brains of mice up to 14 months in age. Higher levels of mutant human beta APP, increased age, or other factors may be necessary to elicit beta-amyloid-related neuropathologies in the rodent brain.

Synthesis and biological activity of a series of potent fluoromethyl ketone inhibitors of recombinant human calpain I.

Calpain I, an intracellular cysteine protease, has been implicated in the neurodegeneration following an episode of stroke. In this paper, we report on a series of potent dipeptide fluoromethyl ketone inhibitors of recombinant human calpain I (rh calpain I). SAR studies revealed that while calpain I tolerates a variety of hydrophobic groups at the P1 site, Leu at P2 is preferred. However, the nature of the N-terminal capping group has a significant effect on the inhibitory activity of this series of compounds. Compound 4e [(1,2,3,4-tetrahydroisoquinolin-2-yl)carbonyl-Leu-D,L-Phe-CH2F+ ++], having a tetrahydroisoquinoline containing urea as the N-terminal capping group, is the most potent dipeptide fluoromethyl ketone inhibitor of calpain I (with a second-order rate constant for inactivation of 276,000 M-1 s-1) yet reported; tripeptide 4k (Cbz-Leu-Leu-D,L-Phe-CH2F) is equipotent. A number of compounds presented in this study displayed excellent selectivity for calpain I over cathepsins B and L, two related cysteine proteases. Compounds which exhibited good inhibitory activity in the assay against isolated rh calpain I also inhibited intracellular calpain I in a human cell line. Thus, in an intact cell assay, compounds 4e and 4k inhibited calpain I with IC50 values of 0.2 and 0.1 microM, respectively. Finally, we also disclose the first example of fluorination of a dipeptide enol silyl ether to generate the corresponding dipeptide fluoromethyl ketone.

Gamma-secretase subunit composition and distribution in the presenilin wild-type and mutant mouse brain.

Studies conducted in cell culture indicate that the gamma-secretase involved in amyloid beta-formation and Notch signaling is a multisubunit aspartic protease. Little is known, however, of the structure, function, or localization of gamma-secretase in the adult brain, or possible effects of familial Alzheimer's disease (FAD)-causing mutations on the brain protease. We report here that mouse brain contains a complex composed of gamma-secretase subunits presenilin-1 N-terminal fragment, presenilin-1 C-terminal fragment, Nicastrin, Aph-1a and Pen-2. A homozygous FAD-linked Presenilin-1 knock-in mutation does not alter relative subunit levels. Immunocytochemical localization of gamma-secretase subunits revealed overlapping but distinct regional and subcellular distributions. All subunits are expressed throughout the neuraxis predominantly in neurons, and are present in axons. Their distributions and levels of expression are unaffected by mutant presenilin-1. In a presenilin-1/amyloid precursor protein double knock-in mouse, subunits are associated with plaques, but are expressed at similar levels in amyloid-rich and -poor regions. gamma-Secretase subunits are distributed much more extensively than circumscribed amyloid deposits, suggesting the importance of other factors for localized amyloid deposition. These results indicate a widespread neuronal function for gamma-secretase in the adult brain, and suggest the pathogenic mechanism of FAD-linked mutations does not involve alterations in the composition, expression or brain distribution of the protease. The subcellular localization of gamma-secretase subunits is consistent with a nerve terminal source for amyloid aggregates.

Excitatory amino acid neurotoxicity in the hippocampal slice preparation.

The effect of sustained activation of excitatory amino acid receptors on neuronal survival was studied using slices of adult rat hippocampus and light and electron microscopy. Kainate, N-methyl-D-aspartate, quisqualate, and ibotenate all produce signs of severe neurotoxicity within 90 min. Neuronal damage occurs in the form of perikaryal and dendritic swelling, cytoplasmic and nucleoplasmic disintegration, and plasma and nuclear membrane ruffling and collapse. The toxicity is restricted to intrinsic neuronal somata, dendrites and spines, while afferent axons, boutons and glia are spared. Although damage is generally distributed throughout all areas of hippocampus, kainate has little effect on pyramidal neurons in the CA2 region. Quantitative analysis of neuronal survival indicates that agonists induce dose-dependent damage over concentration ranges known to be excitatory. Based on selective antagonism by DL-aminophosphonoheptanoate and the patterns of damage produced by each, N-methyl-D-aspartate, kainate, and quisqualate trigger neurotoxicity by acting on distinct receptor classes. It is concluded that, in hippocampal slices, excitatory amino acids induce neurotoxicity in a similar manner to their actions in vivo. The results support the hypothesis that hippocampal neurotoxicity is initiated by excessive excitation, and provide another example of the capacity of adult hippocampal neurons for rapid structural modification.

Immunolocalization of caspase proteolysis in situ: evidence for widespread caspase-mediated apoptosis of neurons and glia in the postnatal rat brain.

Activation of a member of the caspase family of cysteine proteases is thought to be required for the execution of apoptosis in neurons and other cell types. We describe here an antibody (Ab127) reactive with a neoantigenic site on caspase substrate proteins degraded during apoptosis, and its characterization as a biochemical and histochemical probe for apoptosis-associated proteolysis in growth factor-deprived neural cells in vitro and the developing postnatal rat brain. Neuronally differentiated PC12 cells became strongly Ab127 immunoreactive only during apoptosis following nerve growth factor withdrawal. Apoptosis-associated caspase proteolysis was detectable on western blots as markedly increased immunoreactivity of a approximately 46,000 mol. wt polypeptide, a product also generated by caspase-3 treatment of cell-free extracts. In the postnatal rat brain, intense immunoreactivity indicative of caspase activation was exhibited by small proportions of neurons and glia in distinct regional and temporal patterns. The degenerating nature of these cells was confirmed by their argyrophilia, cytoplasmic immunoreactivity for c-jun and fragmented processes. Combined immunofluorescence and Hoechst 33342 staining demonstrated that cells immunopositive for caspase activation have apoptotic nuclear morphologies. Caspase proteolysis was observed throughout the neuraxis in a minority of progenitor cells in germinal zones, postmitotic neurons in the parenchyma, and glia in the corpus callosum and other white matter tracts, but was observed rarely in the adult brain. These data characterize a new approach for evaluating apoptosis in physiological and pathological neurodegeneration, and demonstrate that caspase-associated apoptosis is a widespread mechanism for the programmed death of neurons and glia in the postnatal rat brain.

A calpain inhibitor attenuates cortical cytoskeletal protein loss after experimental traumatic brain injury in the rat.

The capacity of a calpain inhibitor to reduce losses of neurofilament 200-, neurofilament 68- and calpain 1-mediated spectrin breakdown products was examined following traumatic brain injury in the rat. Twenty-four hours after unilateral cortical impact injury, western blot analyses detected neurofilament 200 losses of 65% (ipsilateral) and 36% (contralateral) of levels observed in naive, uninjured rat cortices. Neurofilament 68 protein levels decreased only in the ipsilateral cortex by 35% relative to naive protein levels. Calpain inhibitor 2, administered 10 min after injury via continuous arterial infusion into the right external carotid artery for 24 h, significantly reduced neurofilament 200 losses to 17% and 3% relative to naive neurofilament 200 protein levels in the ipsilateral and contralateral cortices, respectively. Calpain inhibitor administration abolished neurofilament 68 loss in the ipsilateral cortex and was accompanied by a reduction of putative calpain-mediated neurofilament 68 breakdown products. Spectrin breakdown products mediated by calpain 1 activation were detectable in both hemispheres 24 h after traumatic brain injury and were substantially reduced in animals treated with calpain inhibitor 2 both ipsilaterally and contralaterally to the site of injury. Qualitative immunofluorescence studies of neurofilament 200 and neurofilament 68 confirmed western blot data, demonstrating morphological protection of neuronal structure throughout cortical regions of the traumatically injured brain. Morphological protection included preservation of dendritic structure and reduction of axonal retraction balls. In addition, histopathological studies employing hematoxylin and eosin staining indicated reduced extent of contusion at the injury site. These data indicate that calpain inhibitors could represent a viable strategy for preserving the cytoskeletal structure of injured neurons after experimental traumatic brain injury in vivo.

Cathepsin G: localization in human cerebral cortex and generation of amyloidogenic fragments from the beta-amyloid precursor protein.

Amyloid deposits in Alzheimer's disease, Down's syndrome and aged brain are composed largely of A beta protein, which is generated by proteolytic processing of beta-amyloid precursor protein. Proteases responsible for liberating the A beta protein from the precursor have not yet been identified. Here, we examined the ability of cathepsin G, a chymotrypsin-like protease, to cleave two protease substrates: (i) a fluorogenic hexapeptide, whose sequence spans the cleavage site in the precursor for generating the A beta NH2-terminus, and (ii) recombinant human beta-amyloid precursor protein purified from a baculovirus expression system. Unlike two other members of the chymotrypsin family, cathepsin G readily degraded the hexapeptide. Furthermore, cathepsin G cleaved the beta-amyloid precursor protein to generate several breakdown products, including a prominent 11,500 mol. wt fragment immunoreactive with antibodies directed against the COOH-terminus of the protein. This COOH-terminal fragment co-migrated using two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis with C-100, a recombinant COOH-terminal segment of the beta-amyloid precursor, whose NH2-terminus is one residue upstream of the NH2-terminus of the A beta domain. We also examined the localization of cathepsin G in human brain. The distribution of cathepsin G-containing cells was examined by immunohistochemistry in the temporal cortex of both Alzheimer's and aged control samples. Cathepsin G-like immunoreactivity was contained specifically within neutrophils. As visualized by double-labeling with antibodies to cathepsin G and Factor VIII, neutrophils were most frequently found within meningeal or cortical blood vessels. In addition, occasional neutrophils could be identified without an apparent vascular surround, in the brain parenchyma. By simultaneous labeling with antibodies to cathepsin G and A beta protein, neutrophils were also sometimes found associated with both parenchymal and vessel amyloid deposits; however, these associations were rare. These findings indicate that cathepsin G is capable of cleaving the beta-amyloid precursor protein to liberate the free NH2-terminus of the A beta protein and may have access to areas where this material is deposited in Alzheimer's disease. However, since there is no physical association between neutrophils and deposited amyloid and no increase in the number of neutrophils in an Alzheimer's brain, cathepsin G seems to be an unlikely mediator of amyloid deposition in this disease.

Heparin promotes beta-secretase cleavage of the Alzheimer's amyloid precursor protein.

In Alzheimer's disease, abnormal processing of the amyloid precursor protein (APP) is thought to play an important role in amyloid deposition. We investigated the effect of heparin, a highly sulfated glycosaminoglycan related to heparan sulfate, on the secretion of the beta-secretase cleavage product of APP (sAPP beta) in a human neuroblastoma cell line. Heparin induced an increase in the secretion of total APP, and an even greater relative increase in the secretion of sAPP beta. The effect on sAPP beta was specific to heparin. These data support the hypothesis that highly sulfated heparan sulfate proteoglycans may promote amyloidogenic pathways of APP metabolism.

Cyclosporin A limits calcium-induced axonal damage following traumatic brain injury.

In traumatic axonal injury, Ca2+ influx across a focally damaged axolemma precipitates local mitochondrial failure, degradation of the subaxolemmal spectrin network and compaction of neurofilaments, which collectively contribute to axonal failure. In previous studies, cyclosporin A pretreatment preserved mitochondrial integrity and attenuated axonal failure following trauma. Here we investigate whether this CsA-linked protection was related to the concomitant blunting of intra-axonal, Ca2+-induced cytoskeletal changes in traumatic axonal injury, assessed with antibodies targeting spectrin proteolysis and neurofilament compaction. CsA pretreatment dramatically reduced Ca2+-induced cytoskeletal damage following injury; CsA-treated rats, compared with vehicle-treated rats, displayed a 70% decrease in immunoreactive/damaged profiles. We suggest that CsA-mediated preservation of mitochondrial integrity enables the restoration of ionic and metabolic homeostasis thereby short-circuiting Ca2+-induced proteolysis in injured axons.

Differential regulation of muscarinic and nicotinic receptors by cholinergic stimulation in cultured avian retina cells.

Sustained cholinergic stimulation of retina cells grown in primary aggregate and monolayer cultures regulated the concentration of muscarinic but not nicotinic receptors. Muscarinic receptor sites, quantified by the binding of [3H]quinuclidinyl benzilate to membranes and the binding of [3H]N-methyl-scopolamine to intact cells, decreased up to 84% following long-term incubation of cultures in muscarinic agonists. This decrease was blocked by atropine and was not induced by chronic nicotine treatment. The rate of the muscarinic response was biphasic. A rapid binding decrease of 30% occurred within 15 min. The slower phase was half-maximal by 6 h and was complete by 24 h. Neither the fast nor the slow receptor loss was reversed by the guanine nucleotide GppNp. Three different depolarizing agents (gramicidin D, protoveratrine, and ouabain) blocked the cholinergic-induced receptor loss, but the hyperpolarizing ionophore valinomycin had no effect. In contrast to the muscarinic response, nicotinic receptor binding was not altered by chronic receptor stimulation. Exposure to receptor-saturating doses of carbamylcholine or nicotine for 48 h did not change [125I]alpha-bungarotoxin or [3H]bromoacetylcholine binding. Differential regulation of acetylcholine receptors is discussed in relation to the possible physiological role of receptor regulation by receptor activity.