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PubMed1988

Modulation of red cell band 4.1 function by cAMP-dependent kinase and protein kinase C phosphorylation.

Human erythrocyte protein 4.1 is phosphorylated in vivo by several protein kinases including protein kinase C and cAMP-dependent kinase. We have used cAMP-dependent kinase purified from red cells and protein kinase C purified from brain to test the effects of phosphorylation on band 4.1 function. In solution, each kinase catalyzed the incorporation of 1-4 mol of PO4/mol of band 4.1. Phosphorylation of band 4.1 by each kinase resulted in a significant (50-80%) reduction in the ability of band 4.1 to promote spectrin binding to F-actin. Direct measurement of spectrin-band 4.1 binding showed that phosphorylation by each kinase also caused dramatic reduction in this association. Phosphorylation of band 4.1 by each kinase for increasing time periods enabled us to demonstrate an approximately linear inverse relationship between PO4 incorporation into band 4.1 and spectrin binding. These results show that phosphorylation of band 4.1 by cAMP-dependent kinase and protein kinase C may be central to the regulation of red cell cytoskeletal organization and membrane mechanical properties.

Animals↗
PubMed1986

Disintegration of red cell membrane cytoskeleton by hemin.

Spectrin and actin were isolated and their oligomeric state after association with hemin at various conditions was studied. Intact cytoskeletons were prepared by Triton X-100 extraction of red blood cells and incubated with hemin and their stability analyzed by the appearance of dissociated proteins in the supernatant. The cytoskeletons dissociated in a time, temperature and hemin concentration-dependent manner. Following 18 hours incubation in the presence of 0.3 mM hemin there was no dissociation at 4 degrees C, while at the same hemin concentration after 2 hours complete dissociation of the cytoskeletons occurred at 37 degrees C. Microscopy indicated that the cytoskeletons incubated with hemin lost their "cell like" shapes in a time dependent manner. Hemin applied to intact cells also caused dissociation of their cytoskeletons as judged by the failure to separate integer cytoskeletons from red cells treated with hemin. From hemin-induced dissociation profiles of separated actin, spectrin and whole cytoskeletons under various conditions, a mechanism of cytoskeleton breakdown was analyzed, as a release of band 4.1 in the first step which is followed by spectrin dimerization and eventually dissociation of the entire cytoskeletons.

Actins↗
PubMed1985

The structural organization of skeletal proteins influences lipid translocation across erythrocyte membrane.

In order to define the influence of skeletal protein organization on transmembrane phospholipid movement in erythrocyte membranes, we measured the translocation rate of lysophosphatidylcholine in pathologic red cells. A simple method based on the differential extraction of lysophosphatidylcholine from the red cell membrane by saline and albumin solutions was used to quantitate the translocation rate. Two groups of pathologic red cells were chosen for these studies: red cells with quantitative deficiencies of the skeletal proteins, spectrin and protein 4.1, and sickle erythrocytes in which controlled reorganization of the membrane was induced by hemoglobin polymerization. Marked increase in lipid translocation rate was seen in red cells having quantitative deficiencies of spectrin and protein 4.1. The magnitude of the increase in translocation rate in spectrin-deficient red cells was related to the magnitude of protein deficiency. Translocation rate in sickle erythrocyte membranes increased by 50% upon deoxygenation as a result of sickle hemoglobin polymerization. No increase in translocation rate was seen in normal cells upon deoxygenation. By manipulating the extent of membrane reorganization that occurred following deoxygenation of sickle cells, we have been able to show that skeletal reorganization induced by hemoglobin polymerization and not hemoglobin polymerization per se is responsible for the increase in translocation rate. Together, these findings imply that the structural organization of membrane skeletal proteins plays an important role in regulating the rate of transbilayer movement of lipids across the erythrocyte membrane.

Anemia, Sickle Cell↗
PubMed1979

ATP-induced endocytosis in human erythrocyte ghosts. Characterization of the process and isolation of the endocytosed vesicles.

ATP-induced endocytosis in human erythrocyte ghosts has been studied, and a procedure for the isolation of the endocytotic vesicles is described. Under isotonic conditions and 37 degrees C, optimal endocytosis occurs with concentrations of 4 to 10 mM MgATP. Within 30 min, up to 45% of the membrane is removed from the surface and converted into sealed inside-out vesicles. Local anesthetics, such as chlorpromazine, potentiate ATP-induced endocytosis in ghosts. Forcing cells containing endocytotic vesicles through a hypodermic needle leads to the exclusive fragmentation of the outermost plasma membrane. The endocytosed vesicles can then be separated from these fragments by centrifugation on a gradient of dextran T70. Biochemical analyses indicate that endocytotic vesicles contain full complements of the major membrane proteins (i.e. also spectrin and actin), common phospholipids, fatty acids, and cholesterol. Furthermore, they exhibit a fully intact spectrin component 2 phosphorylation machinery. In contrast, MgATPase activity is largely excluded from these vesicles. The novel inside-out vesicles described have properties different from those of previously analyzed fragments of the erythrocyte membrane. They will permit a detailed study of a native spectrin-actin network now exposed to the outside.

Acetylcholinesterase↗
PubMed1982

Further characterization of the erythrocyte membrane protein abnormality in megaloblastic anemia.

Erythrocyte membranes obtained from patients with severe megaloblastic anemia contain spectrin as indicated by double immunodiffusion against anti-spectrin raised in rabbits. These membranes have normal protein patterns determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after solubilization at 100 degrees C for 3 min in SDS. When incubation is carried out at 37 degrees C for 3 hours, however, the membrane protein patterns become grossly abnormal, lacking spectrin, band 3 and having several diffuse smaller bands. Cross-linking of these membranes with SH oxidizing agents, such as diamide prevented this dissociation phenomenon. The SH group content of megaloblastic membranes, both from severe and mild megaloblastic anemia, was significantly higher than that of control membranes. This was also confirmed by the greater incorporation of 3H-N-ethylmalemide into red cell membranes from mild and severe megaloblastic anemia than controls. Incorporation of this probe was greatest into the band 3 region. These findings indicate that the erythrocyte membrane proteins in megaloblastic anemia have an intrinsic abnormality which renders them more susceptible to degradation by their endogenous proteases.

Anemia, Macrocytic↗
PubMed1982

Polyamines and membrane proteins in sickle cell disease.

The polyamines, putrescine, spermidine, and spermine have been implicated in cellular growth and membrane stabilization. We have shown that RBC stroma and lysate polyamines are significantly elevated in sickle cell disease (SS and SC) relative to controls (AA). The crude spectrin fraction of the red cell stroma has associated with it a large fraction of the total stroma polyamines, suggesting that the polyamines in the stroma are preferentially bound to spectrin. The high levels of polyamines associated with the spectrin of young and ISC's relative to normal may contribute to an alteration of the physical-chemical properties of the sickle RBC membrane. Spermine levels were high in the SS lysates but not detectable in SC or AA lysates. Levels in SC patients were intermediate. In preliminary experiments using washed red cells obtained from SS and AA subjects, there was extensive incorporation of 14C putrescine into red cell protein. Labelled N-(gamma-glutamyl) putrescine was isolated, consistent with covalent binding of the polyamines to protein.

Anemia, Sickle Cell↗
PubMed1981

The construction of the red cell cytoskeleton.

The state of actin in the erythrocyte membrane cytoskeleton has been examined. The presence of ADP, rather than ATP, as the predominant nucleotide species reinforces the view that actin occurs in the polymerized form. Redeterminations of the amounts of the three cytoskeletal proteins, spectrin, actin and 4.1, present in the cell allow evaluation of some of the stoichiometric constraints on the construction of the cytoskeleton. The available evidence is compatible with a network consisting of spectrin tetramers as the structural members, attached at both ends to junctions consisting of 4.1 and short filaments (or 'protofilaments') of actin. Electron micrographs of isolated cytoskeletons support such a picture. The dynamic state of the actin has been studied, using the DNAase assay method. The results indicate that whereas native monomeric actin is present in the cell, only a small proportion of the subunits of the protofilament can enter into an equilibrium with this pool. A considerable proportion of the cytoskeletal actin is not liberated under conditions that dissociate F-actin and is evidently tightly associated with spectrin and 4.1. Attention is drawn to the possible consequences of ATP depletion on the state of the actin and thus of the cytoskeleton.

Actins↗
PubMed1994

Stress exacerbates neuron loss and cytoskeletal pathology in the hippocampus.

Glucocorticoids (GCs), the adrenal steroids secreted during stress, endanger the hippocampus, compromising its ability to survive neurological insults. GCs probably do so by disrupting energetics in the hippocampus, thus impairing its ability to contain damaging fluxes of excitatory amino acids and calcium. Superficially, these observations suggest that stress itself should also exacerbate the toxicity of neurological insults. However, most studies have involved unphysiologic GC manipulations, limiting speculations about the endangering effects of stress. In this study, rats were infused with the excitotoxin kainic acid (KA) after either having been adrenalectomized and replaced with a range of physiologic concentrations of GCs, or having been stressed intermittently. We observed that within the CA3 region, increasing CORT concentrations exacerbated the KA-induced neuron loss, the extent of tau immunoreactivity, and of spectrin proteolysis. The transitions from low to high basal GC concentrations and from high basal to stress GC values were both associated with significant exacerbation of neuron loss and tau immunoreactivity; the extent of spectrin proteolysis was less sensitive to increments in GCs. As would be expected from these data, exposure to intermittent stress prior to KA infusion also exacerbated neuron loss, tau immunoreactivity, and spectrin proteolysis in CA3. Thus, physiological elevations of GCs, and stress itself, can exacerbate hippocampal neuron loss and the attendant degenerative markers following an excitotoxic insult. Of significance, seizure and hypoxia-ischemia provoke considerable GC stress responses, which may thus worsen the resultant damage. Furthermore, a number of neuropsychiatric disorders, as well as aging, are associated with elevated basal GC concentrations, which may endanger the hippocampus in the event of neurological insult.

Animals↗
PubMed1996

Abnormal assembly of membrane proteins in erythroid progenitors of patients with beta-thalassemia major.

The life threatening anemia in beta-thalassemia major (Cooley's anemia) is characterized by profound intramedullary lysis, the cause of which is incompletely understood. Using marrow obtained from beta thalassemia major patients undergoing allogeneic bone marrow transplantation in Pesaro Italy, it became possible to directly study the mechanism of the intramedullary hemolysis. Based on our previous studies, we hypothesized that the unmatched alpha globin chains would interfere with normal assembly of erythroid precursor membrane proteins. Patient and control erythroid precursors were reacted with monospecific polyclonal rabbit antibodies directed against spectrin, band 3, and band 4.1 and with a monoclonal anti-alpha globin chain antibody. Using laser confocal fluorescence microscopy, normal erythroid precursors show no alpha globin chain accumulation and exhibited uniformly smooth rim fluorescence of the three membrane proteins. In some thalassemic precursors, spectrin appeared to interact with large alpha globin accumulations, and in many of these cells the spectin appeared clumped and discontinuous. Band 4.1 interacted strongly with accumulations of alpha globin in thalassemic precursors to produce bizarrely clumped zones of abnormal band 4.1 distribution. Band 3 was incorporated smoothly into thalassemic erythroblast membranes. However, the proerythroblasts and basophilic erythroblasts were significantly deficient in band 3. Thus, accumulations of alpha globin in beta-thalassemia major colocalized with and disrupt band 4.1 and spectrin assembly into the membrane. The cause of deficient band 3 incorporation into thalassemic proerythroblast membranes remains unknown. These profound membrane alterations would likely contribute to the intramedullary lysis seen in Cooley's anemia.

Animals↗
PubMed1995

Chronology of the formation of vesicles and membrane protein aggregates during erythrocyte aging.

The aim of the present work was to study the changes undergone by proteins in ghost cells, vesicles and membrane protein aggregates during erythrocyte aging. As a model of cell, whole blood collected into CPD was stored for eight weeks at +4 degrees C. SDS-PAGE and immunoblotting with antibodies against spectrin and band 3 showed that vesicles were mainly composed of band 3 and were devoid of spectrin, whereas membrane protein aggregates contained several membrane proteins but in a more advanced state of degradation. A study of spectrin and band 3 in the ghosts, vesicles and protein aggregates revealed increasing fragmentation of both proteins with storage time. Since this degradation was most important in membrane protein aggregates, it was possible to establish the chronological order of appearance of vesicles and aggregates. In view of our observations, we propose that membrane protein aggregates may be regarded as replacement structures resulting from membrane rearrangements occurring after the emission of vesicles.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1997

Thrombosis and secondary hemochromatosis play major roles in the pathogenesis of jaundiced and spherocytic mice, murine models for hereditary spherocytosis.

Jaundiced mice, ja/ja, suffer from a severe hemolytic anemia caused by a complete deficiency of erythroid beta-spectrin. We used these mice as a model to investigate the pathophysiological consequences of the deficiency, including the effects in the nonerythroid tissues where this protein is expressed. Because the ja/ja mice rarely survive beyond the fourth postnatal day, methods were assessed for extending lifespan into adulthood. Neonatal transfusion increased lifespan to a mean of 3.7 months, allowing a more complete characterization of the pathophysiology. Blood parameters and histopathology of the jaundiced mouse were compared with that from spherocytic mice, which have a hemolytic anemia caused by deficiency of erythroid alpha-spectrin, yet can survive the postnatal period transfusion free. The adult jaundiced and spherocytic mice present with greatly decreased hematocrit and red blood cell counts, reticulocytosis, and bilirubinemia, leading secondarily to hepatosplenomegaly and cardiomegaly. Jaundiced and spherocytic mice were analyzed histopathologically between 1.0 and 9.5 months of age. Interestingly, the complete absence of erythroid beta-spectrin in jaundiced mice leads to no detectable structural defects in brain, cardiac, or skeletal muscles. However, fibrotic lesions and lymphocytic infiltration were observed in cardiac tissue from 4 of 13 jaundiced mice and 15 of 15 spherocytic mice, and thrombi were detected at either the atrioventricular valves or within the atria of 2 of 13 jaundiced mice and 15 of 15 spherocytic mice. In addition, all affected mice had a progressive renal hemosiderosis concurrent with hydronephrosis and glomerulonephritis. The severity of the renal disease and its presence in all moribund mice suggests kidney failure rather than the fibrotic heart lesions as the major cause of death in these mice.

Alternative Splicing↗
PubMed1989

Immunoelectron-microscopic localization of the 180 kD component of the neural cell adhesion molecule N-CAM in postsynaptic membranes.

In order to investigate the expression of cell adhesion molecules in synapses, we have studied the localization of the neural cell adhesion molecule N-CAM in the cerebellum and hippocampus of adult mice by immunocytological and immunochemical methods. Of the three molecular components of N-CAM with relative molecular masses (Mr) of 120, 140, and 180 kD, N-CAM 120 is not detectable in synaptosomal membranes, whereas N-CAM 140 is expressed on both pre- and postsynaptic membranes and N-CAM 180 is restricted to postsynaptic sites, with localization of the N-CAM 180-specific epitope in postsynaptic densities. Specificity of immunoreactivity is indicated by the observation that antibodies to the neural cell adhesion molecule L1 do not label synaptic membranes, whereas antibodies to two major components of postsynaptic densities, actin and erythrocyte spectrin, react with synaptic structures. Interestingly, N-CAM 180 is only detectable in subpopulations of synapses in the intact tissue. Isolated synaptosomes, opened for unimpeded accessibility of antibody by hypoosmotic treatment, also reveal a partial expression of N-CAM 180 in that 67% are labeled by antibodies to N-CAM 180, while antibodies to actin and erythrocyte spectrin react with 95% and 88% of all synaptosomes, respectively. N-CAM 180 does not appear to be differentially expressed in synapses of a particular morphological type, but is detectable in all types of synapses in the cerebellum and hippocampus, except for mossy fiber synapses and synapses between basket and Purkinje cells, which are generally N-CAM 180-negative. Since N-CAM 180 has been shown to be characteristic of stabilized or stabilizing cell contacts, possibly by its association with the cytoskeleton-membrane linker protein spectrin (Pollerberg et al.: J. Cell Biol. 101:1921-1929, '85; Nature 324:462-465, '86; Cell Tissue Res. 250:227-236, '87), we would like to suggest N-CAM 180 plays an important role in determining the stability of contacts between pre- and postsynaptic membranes and state of synaptic activity.

Animals↗
PubMed1988

Differential immunoreactivity and Ca2+-dependent degradation of vimentin in human fibroblasts and fibrosarcoma cells.

Immunostaining of normal human fibroblasts with a monoclonal antibody (MAb) (V22AC12) revealed typical cytoplasmic arrays of vimentin filaments in both mitotic and interphase cells. In human A8387 fibrosarcoma cells and SV40-virus-transformed human fibroblasts, the same antibody showed positivity only in mitotic cells and in interphase cells only after treatment of the fixed cells with alkaline phosphatase. Upon immunoblotting with the MAb, an Mr 57,000 vimentin polypeptide was seen in normal fibroblasts. In fibrosarcoma cells the same polypeptide was revealed by this antibody only after treatment with alkaline phosphatase. The Mr 57,000 vimentin polypeptide was a major cytoskeletal protein in both fibroblasts and fibrosarcoma cells. Inclusion of Ca2+ into the cytoskeleton extraction medium brought about a somewhat increased degradation of vimentin in fibroblasts. In fibrosarcoma cells, such treatment caused a quantitative disappearance of the Mr 57,000 protein with a concomitant appearance of 3 distinct, low-molecular-weight degradation products in the detergent-soluble fraction. Another Ca2+-induced change in the polypeptide profile of fibrosarcoma cells was the disappearance of the Mr 240,000 non-erythroid alpha-spectrin and the concomitant appearance of a prominent Mr 140,000 degradation product. Inclusion of proteolysis inhibitors in the Ca2+-supplemented extraction medium inhibited degradation of both vimentin and alpha-spectrin polypeptides. The results suggest differences in the composition of the cytoskeletons of normal fibroblasts and fibrosarcoma cells, manifested in the differential Ca2+-susceptibility of vimentin and non-erythroid alpha-spectrin. Results with MAb V22AC12 suggest that differential phosphorylation of vimentin could account for at least part of this difference.

Antibodies, Monoclonal↗
PubMed2002

Novel UNC-44 AO13 ankyrin is required for axonal guidance in C. elegans, contains six highly repetitive STEP blocks separated by seven potential transmembrane domains, and is localized to neuronal processes and the periphery of neural cell bodies.

Conventional ankyrins are cortical cytoskeletal proteins that form an ankyrin-spectrin meshwork underlying the plasma membrane. We report here the unusual structure of a novel ankyrin (AO13 ankyrin, 775,369 Da, 6994 aa, pI = 4.45) that is required for proper axonal guidance in Caenorhabditis elegans. AO13 ankyrin contains the ANK repeat and spectrin-binding domains found in other ankyrins, but differs from all others in that the acidic carboxyl region contains six blocks of serine/threonine/glutamic acid/proline rich (STEP) repeats separated by seven hydrophobic domains. The STEP repeat blocks are composed primarily of sequences related to ETTTTTTVTREHFEPED(E/D)X(n)VVESEEYSASGSPVPSE (E/K)DVE(H/R)VI, and the hydrophobic domains contain sequences related to PESGEESDGEGFGSKVLGFAKK[AGMVAGGVVAAPVALAAVGA]KAAYDALKKDDDEE, which includes a potential transmembrane domain (in brackets). Recombinant protein fragments of AO13 ankyrin were used to prepare polyclonal antisera against the spectrin-binding domain (AO271 Ab), the conventional ankyrin regulatory domain (AO280 Ab), the AO13 ankyrin STEP domain (AO346 Ab), the AO13 ankyrin STEP + hydrophobic domain (AO289 Ab), and against two carboxyl terminal domain fragments (AO263 Ab and AO327 Ab). Western blot analysis with these Ab probes demonstrated multiple protein isoforms. By immunofluorescence microscopy, the antispectrin-binding and regulatory domain (AO271 and AO280) antibodies recognized many cell types, including neurons, and stained the junctions between cells. The AO13 ankyrin-specific (AO289 and AO346) antibodies showed a neurally restricted pattern, staining nerve processes and the periphery of neural cell bodies. These results are consistent with a role for AO13 ankyrin in neural development.

Amino Acid Sequence↗
PubMed1993

Interactions of IgG with specific erythrocyte membrane proteins in affinity electrophoresis are highly dependent on low ionic strength conditions.

Agarose gel affinity electrophoresis has been used to demonstrate interactions between autologous IgG and specific erythrocyte membrane proteins. These binding phenomena are here further examined by combining affinity electrophoresis with affinity chromatography, absorption experiments, and immunoblotting. It is demonstrated that the interactions are highly dependent on polyreactive IgG binding favored by the low ionic strength conditions of the electrophoretic assay. Thus, about 25% of normal IgG under low ionic strength conditions bound to the purified cytoskeletal protein, spectrin, immobilized on Sepharose. This IgG reacted in affinity electrophoresis in a polyspecific fashion with the same array of membrane proteins as before the low ionic strength-affinity chromatography. Further, the binding seen in affinity electrophoresis, including the interaction with spectrin, was completely abolished by preabsorption of the IgG with spectrin-devoid membranes. The charge characteristics of an IgG subclass might be responsible for the observed binding. However, the observed precipitate formation suggested an interaction involving at least two binding sites on each molecule and the binding appears to require structurally intact IgG because reductive treatment with dithiothreitol diminished the reactivity considerably. Conclusively, under the conditions of affinity electrophoresis with ligand present in the gel, electrostatic interactions are amplified. The degree of binding of IgG to erythrocyte membrane proteins that take place under these conditions does not reflect binding which would occur to the same extent under physiological ionic strength conditions.

Blood Platelets↗
PubMed1998

Altered membrane skeleton of hydroxyethylstarch-cryopreserved human erythrocytes.

Attempts have been made to use hydroxyethylstarch (HES) as an alternative to glycerol for cryopreservation of erythrocytes. However, HES cryopreservation causes significant transient rheological alterations in erythrocytes. Membrane proteins play a critical role for erythrocyte rheology. This study was undertaken to analyze erythrocyte membrane proteins during HES cryopreservation. Erythrocyte membranes with submembrane skeleton (ghosts) and the submembrane skeleton alone (unstripped skeletons) were prepared before freezing (native), after thawing and following 3 h reconditioning in glucose-enriched Ringer's solution (Ringer plus glucose), or in autologous fresh frozen plasma (AFFP). After electrophoresis protein concentrations (percentage of total protein) were determined by densitometry. In ghosts, no significant changes were found, whereas in unstripped skeletons the following results could be seen: beta-Spectrin: 31.8 +/- 2.2% (native), 22.1 +/- 0.8% (postthawing, P < 0.05 vs native), 22.4 +/- 1.6% (Ringer plus glucose, P < 0.05 vs native), 31.0 +/- 2.8% (AFFP). Other proteins remained unchanged. Since a significant decrease in beta-spectrin concentration after HES cryopreservation and after subsequent reconditioning in Ringer's solution with glucose was only detected in unstripped skeletons, this cannot be interpreted as in vivo protein loss. More likely, HES cryopreservation may have created changes in protein-protein associations. The course of beta-spectrin concentration parallels certain rheological and biochemical changes and might explain the transient rheological changes seen after HES cryopreservation.

Cryopreservation↗
PubMed2000

Structure of the utrophin actin-binding domain bound to F-actin reveals binding by an induced fit mechanism.

Utrophin is a large ubiquitously expressed cytoskeletal protein, homologous to dystrophin, the protein disrupted in Duchenne muscular dystrophy. The association of both proteins with the actin cytoskeleton is functionally important and is mediated by a domain at their N termini, conserved in members of the spectrin superfamily, including alpha-actinin, beta-spectrin and fimbrin. We present the structure of the actin-binding domain of utrophin in complex with F-actin, determined by cryo-electron microscopy and helical reconstruction, and a pseudo-atomic model of the complex, generated by docking the crystal structures of the utrophin domain and F-actin into the reconstruction. In contrast to the model of actin binding proposed for fimbrin, the utrophin actin-binding domain appears to associate with actin in an extended conformation. This conformation places residues that are highly conserved in utrophin and other members of the spectrin superfamily at the utrophin interface with actin, confirming the likelihood of this binding orientation. This model emphasises the importance of protein flexibility in modeling interactions and presents the fascinating possibility of a diversity of actin-binding mechanisms among related proteins.

Actins↗
PubMed1992

Alterations of choline acetyltransferase, phosphoinositide hydrolysis, and cytoskeletal proteins in rat brain in response to colchicine administration.

Colchicine, a microtubule disrupting toxin, was administered intracerebroventricularly to rats, followed by measurements of (i) the activity of choline acetyltransferase, a biochemical marker of cholinergic neurons, (ii) cytoskeletal protein concentrations, including tau, MAP-2, spectrin, and tubulin, and (iii) the activity of the second messenger-generating system, receptor-coupled phosphoinositide hydrolysis. One week after colchicine treatment there was a 60% decrease in choline acetyltransferase activity in the hippocampus, which was followed by a gradual increase in only a 29% deficit after 12 weeks. In the striatum and cerebral cortex, choline acetyltransferase activity was slightly reduced (by 13% and 19%, respectively) 1 week after colchicine treatment followed by increases to control values. The concentrations of tau and tubulin in the hippocampus were unaltered by colchicine treatment, and MAP-2 and spectrin were only slightly reduced 4 weeks after colchicine. Hippocampal phosphoinositide hydrolysis induced by norepinephrine was elevated approximately 28% 1 and 2 weeks after colchicine treatment and that induced by ibotenate was increased by 53% 2 weeks after colchicine. These results demonstrate that colchicine causes a severe depletion of choline acetyltransferase 1 week after administration. There was not a significant reduction of the concentration of any of the cytoskeletal proteins after 1 week, possibly due to the cell-selectivity of the toxic effect of colchicine, but there was a delayed, and temporary, decline of MAP-2 and spectrin concentrations. Associated with the decreased choline acetyltransferase activity after 1 week was an enhanced phosphoinositide hydrolysis in response to norepinephrine, and after 2 weeks there were enhanced responses to norepinephrine and to ibotenate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗