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In addition to its role in hydrolyzing the neurotransmitter acetylcholine, the synaptically enriched enzyme acetylcholinesterase (AChE) has been reported to play an important role in the development and remodelling of neural processes and synapses. We have shown previously that AChE causes an increase in binding of the specific AMPA receptor ligand (S)-[(3)H]-5-fluorowillardiine ([(3)H]-FW) to rat brain membranes. In this study we have used quantitative autoradiography to investigate the regional distribution and age-dependence of AChE-evoked increases in the binding of [(3)H]-FW in rat brain. Pretreatment of rat brain sections with AChE caused a marked enhancement of [(3)H]-FW binding to many, but not all, brain areas. The increased [(3)H]-FW binding was blocked by the specific AChE inhibitor BW 284c51. The maximal potentiation of [(3)H]-FW binding occurred at different developmental age-points in different regions with a profile consistent with the peak periods for synaptogenesis in any given region. In addition to its effects on brain sections, AChE also strongly potentiated [(3)H]-FW binding to detergent solubilized AMPA receptors suggesting a direct action on the receptors themselves rather than an indirect effect on the plasma membrane. These findings suggest that modulation of AMPA receptors could provide one molecular mechanism for the previously reported effects of AChE on synapse formation, synaptic plasticity and neurodegeneration.

The dynamins comprise an expanding family of ubiquitously expressed 100-kD GTPases that have been implicated in severing clathrin-coated pits during receptor-mediated endocytosis. Currently, it is unclear whether the different dynamin isoforms perform redundant functions or participate in distinct endocytic processes. To define the function of dynamin II in mammalian epithelial cells, we have generated and characterized peptide-specific antibodies to domains that either are unique to this isoform or conserved within the dynamin family. When microinjected into cultured hepatocytes these affinity-purified antibodies inhibited clathrin-mediated endocytosis and induced the formation of long plasmalemmal invaginations with attached clathrin-coated pits. In addition, clusters of distinct, nonclathrin-coated, flask-shaped invaginations resembling caveolae accumulated at the plasma membrane of antibody-injected cells. In support of this, caveola-mediated endocytosis of labeled cholera toxin B was inhibited in antibody-injected hepatocytes. Using immunoisolation techniques an anti-dynamin antibody isolated caveolar membranes directly from a hepatocyte postnuclear membrane fraction. Finally, double label immunofluorescence microscopy revealed a striking colocalization between dynamin and the caveolar coat protein caveolin. Thus, functional in vivo studies as well as ultrastructural and biochemical analyses indicate that dynamin mediates both clathrin-dependent endocytosis and the internalization of caveolae in mammalian cells.

The paired helical filaments (PHFs) in Alzheimer's disease neurofibrillary tangles are composed of PHF-tau which is thought to be hyperphosphorylated because several residues in postmortem samples of PHF-tau and human fetal tau are phosphorylated while the corresponding sites are not phosphorylated in autopsy-derived normal adult human brain tau. To determine how the phosphorylation of these sites is regulated, we isolated tau from rat brains at different embryonic and postnatal ages in the presence of okadaic acid to obtain tau in its most native in situ phosphorylation state. Fetal tau was highly phosphorylated from embryonic day 18 (E18) until postnatal day 11 (P11). Thereafter, the levels of fetal tau diminished as did its phosphorylation state concomitant with the appearance of the five adult tau isoforms. Several phosphorylation-dependent antibodies (i.e. AT270, AT8, AT180, T3P, and PHF1) that recognize PHF-tau also recognized these tau isoforms, albeit at reduced levels in the mature rat brain. This suggests that Thr172, Ser193, Thr222, Ser387, and Ser395 are normal sites of phosphorylation in rat brain tau. The inclusion of OK in the microtubule assembly buffers did not alter the ability of tau to bind microtubules at any age. However, phosphatases were activated and kinases were down-regulated in the rat brain after P12 since adult tau proteins were partially dephosphorylated at and beyond this time in the absence of OK. Protein phosphatase 2A (PP2A) and 2B (PP2B) activities in the adult rat brain extracts dephosphorylated tau efficiently, but protein phosphatases in extracts of the P6 rat brain did not have a similar effect. This suggests that the sensitivity of tau to OK after P12 may be regulated by the de novo induction of adult brain phosphatases. Finally, PP2A and/or PP2B in adult rat brain extracts dephosphorylated tau in a site-specific manner. Thus, PP2A and PP2B (or closely related phosphatases) may regulate the phosphorylation state of adult tau isoforms in vivo, and the generation of PHF-tau in the AD brain may result from the abnormal inactivation of similar phosphatases.

Goldfish brain is a widely used model system for the study of the mechanisms involved in neuronal regeneration and synaptic plasticity. Because of the proposed role of glutamate receptors in these processes we have investigated the anatomical localisations of [3H]AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate), [3H]kainate, [3H]CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and [3H]L-glutamate binding sites in horizontal and sagittal sections. Binding sites for [3H]L-glutamate were the most widespread and both NMDA (N-methyl-D-aspartate) and non-NMDA sensitive components were detected. The density of [3H]kainate binding was very high in the cerebellum compared to other regions and in comparison with the other radioligands used. Conversely, relatively low amounts of [3H]AMPA binding were present with the telencephalon being the most densely labelled structure. [3H]CNQX binding was most densely localised in the tectum with the cerebellum also possessing high binding. In addition, there was a small population of [3H]CNQX binding sites located in the telencephalon and lobus vagi that appeared insensitive to AMPA and kainate.

The rate of production of cAMP by the adenylyl cyclase enzyme from goldfish brain was linear with time and with protein concentration. In agreement with mammalian adenylyl cyclase systems the enzyme is divalent cation dependent, being activated in the presence of either Mg2+ or Mn2+. Forskolin also stimulated the rate of reaction in a dose-dependent manner with a half-maximal effect of 1 microM. The activated enzyme was inhibited by high concentrations of Ca2+ but was independent of Na+ concentration. The presence of guanine nucleotide binding proteins (G-proteins) was demonstrated by the fact that both NaF and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) stimulated the basal rate. In addition, the p[NH]ppG dose-response curve of the forskolin-stimulated enzyme was biphasic, similar to that observed for other systems. At low concentrations of p[NH]ppG a small inhibition was observed while higher concentrations produced a stimulation. These data suggest that the goldfish brain adenylyl cyclase enzyme complex includes both stimulatory and inhibitory G-proteins in addition to the catalytic unit. A series of known and putative goldfish neurotransmitter substances failed to either stimulate or inhibit the adenylyl cyclase activity. The endogenous neurotransmitters which interact with this second messenger system remain to be determined.

Recent reports have suggested that a major proportion of [3H]kainate binding in goldfish brain is to a novel form of G-protein-linked glutamate receptor. Here we confirm that guanine nucleotides decrease [3H]kainate binding in goldfish brain membranes, but that binding is also reduced to a similar extent under conditions where G-protein modulation should be minimised. Inclusion of GTP gamma S resulted in an approximately twofold decrease in the affinity of [3H]kainate binding and a 50% reduction in the apparent Bmax values in both Mg2+/Na+ and Mg2+/Na(+)-free buffer when assayed at 0 degrees C. The pharmacology of [3H]kainate binding is similar to that of well-characterised ionotropic kainate receptors but unlike that of known metabotropic glutamate receptors, with neither 1S,3R-amino-1,3-cyclopentanedicarboxylic acid (1S,3R-ACPD) nor ibotenic acid being effective competitors. The molecular mass of the [3H]kainate binding protein, as determined by radiation inactivation, was 40 kDa, similar to the subunit sizes of other lower vertebrate kainate binding proteins that are believed to comprise ligand-gated ion channels. Furthermore, GTP gamma S also inhibited the binding of the non-NMDA receptor-selective antagonist 6-[3H]cyano-7-nitroquinoxaline-2,3-dione. These data strongly suggest that the regulatory interaction between guanine nucleotides and [3H]kainate and 6-[3H]cyano-7-nitroquinoxaline-2,3-dione binding is complex and involves competition at the agonist/antagonist binding site in addition to any G-protein-mediated modulation.

GYKI 52466 and related 2,3-benzodiazepines are highly selective antagonists of AMPA-evoked responses in rat CNS. However, these compounds do not compete with [3H]AMPA binding and their site of action remains unclear. Here we show that [3H]GYKI 53405 binds specifically to Xenopus brain membranes with KD and Bmax values of 4.5 microM and 35 pmol mg-1 protein respectively. Binding is increased in the presence of Mg2+ and is unaffected by AMPA or kainate. This is the first report of the binding of a GYKI 52466-related radioligand to any tissue and these results provide an initial step towards the characterization of novel recognition sites which are of considerable therapeutic potential.

The anatomical distribution of binding sites for the non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist [3H]6-cyano-7-nitroquinoxaline-2,3-dione ([3H]CNQX) in 1-day-old chick brain was investigated. Specific [3H]CNQX binding sites were widely distributed but were particularly densely localised in the molecular layer of the cerebellum. In the midbrain, the binding was comparatively low, except for relatively high levels in the nucleus isthmi of the optic lobe. The distribution of [3H]CNQX binding was markedly different to that of the NMDA receptor ligand [3H]MK-801. Overall, the localisation of [3H]CNQX binding was similar to the combined distributions of [3H]AMPA and [3H]kainate binding sites. Kainate inhibited [3H]CNQX binding throughout the brain. AMPA also inhibited [3H]CNQX binding in the fore- and midbrain, but the dense binding in the molecular layer of the cerebellum was notable in being AMPA-insensitive.

The binding of (-)-[3H]nicotine to membrane fragments and a detergent solubilized fraction of goldfish brain was characterized. (-)-[3H]nicotine binding was not displaced by alpha-bungarotoxin, but was displaced by (-)nicotine and carbamoylcholine with Ki of approximately 8.6 and 86 nM, respectively. Preincubation of solubilized membrane extract with alpha-bungarotoxin-coupled Sepharose resulted in the loss of approximately 50% of the (-)-[3H]nicotine binding protein from the eluent and an increase in (-)-[3H]nicotine binding to the gel compared to control, non-alpha-bungarotoxin Sepharose. 125I-alpha-bungarotoxin binding protein in the eluent from the same preincubation experiments was totally removed. In addition, incubation of the solubilized tissue extracts with alpha-bungarotoxin-coupled Sepharose resulted in an increase in the affinity for (-)-[3H]nicotine in the eluent (mean KD = 3.1) compared to control solubilized tissue extracts (KD = 6.4 nM). Specific (-)-[3H]nicotine binding sites could be eluted from the alpha-bungarotoxin-coupled Sepharose with carbamoylcholine and D-tubocurarine. Similar to previously reported 125I-alpha-bungarotoxin binding data, eye removal resulted in an approximately 40% decrease in (-)-[3H]nicotine binding in the contralateral tectum compared to that in the ipsilateral tectum. These data indicate that at least two distinct subtypes of (-)nicotine binding sites may be present in goldfish brain, one which binds alpha-bungarotoxin and (-)nicotine and another which binds only (-)nicotine.

The brains of 1-day-old chicks were shown to be a rich source of binding sites with the pharmacological characteristics expected of a quisqualate-type glutamate receptor. alpha-[3H]Amino-3-hydroxy-5-methylisoxazolepropionate ([3H]AMPA) bound with KD and Bmax values, measured at 0 degree C in the presence of the chaotrope potassium thiocyanate, of 55 nM and 2.6 pmol/mg protein. The regional localisations of [3H]AMPA and [3H]kainate binding sites were manifestly different. The membrane-bound [3H]AMPA binding sites were efficiently solubilised by N-octyl-beta-D-glucopyranoside (1%) in the presence of 0.2 M thiocyanate. In the detergent extract the affinity was 69 nM and there was an apparent increase in the number of sites (Bmax, 4.6 pmol/mg protein). The rank order of potency for competitive ligands in displacing [3H]AMPA binding was quisqualate approximately AMPA greater than 6-cyano-7-nitroquinoxaline-2,3-dione greater than L-glutamate greater than kainate and was identical for the membrane-bound and solubilised sites. Dissociation was biphasic with rate constants of 0.117 min-1 and 0.015 min-1. The association rate constants for [3H]AMPA at the solubilised sites were 1.45 x 10(6) M-1 min-1 and 6.55 x 10(6) M-1 min-1. The kinetically derived KD values were 80.7 nM and 2.3 nM. The detection of higher affinity binding sites by kinetic analysis but not by equilibrium binding may be explained by the greater sensitivity of dissociation data to small populations of high-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)

The binding of several ligands that selectively interact with glutamate receptor subtypes was characterized in extensively washed synaptosomal membrane preparations from goldfish brain. The binding affinity (Kd), an estimate of the number of sites (Bmax), the rank-order potency of glutamatergic ligands at inhibiting binding, and the regional localization of binding sites were determined. In whole brain preparations, 3H-kainate had a Kd of 136 nM and a Bmax of 63 pmol/mg protein, 3H-AMPA had a Kd of 26 nM and a Bmax of 0.4 pmol/mg protein, and 3H-L-glutamate bound with an apparent affinity of 323 nM and a Bmax of 5 pmol/mg protein. Most of the binding sites for each of the glutamate analogs were present in the cortex and the fewest were in the cerebellum, except for 3H-kainate binding sites, which were most prevalent in the cerebellum and least abundant in the cortex. The proposed neuronal nicotinic acetylcholine receptor (nAChR) ligands 3H-(-)nicotine and 125I-alpha-Bgt were also investigated. 125I-alpha-Bgt had a Kd of 0.08 nM and a Bmax of 132 fmol/mg protein. 3H-(-)nicotine did not bind to the extensively washed membrane preparations, so a less stringently washed P2 tissue fraction was used. In this tissue preparation, 3H-(-)nicotine had a Kd of 9 nM and a Bmax of 84 fmol/mg protein. Eye removal resulted in a time-dependent decrease in the number of 3H-(-)nicotine and 125I-alpha-Bgt binding sites in the contralateral optic tectum, but no reduction in the number of binding sites for any of the glutamatergic ligands. The results suggest that both 3H-(-)nicotine and 125I-alpha-Bgt binding sites are similarly regulated in the optic tectum, which supports previously reported data indicating that the binding sites are located on closely related proteins or may, at least partially, be colocalized on the same protein (Henley and Oswald, 1987).(ABSTRACT TRUNCATED AT 250 WORDS)

The binding of [3H]kainate to goldfish brain membrane fragments was investigated. Scatchard analysis revealed a single class of binding sites in Tris-HCl buffer with a Kd of 352 nM and a Bmax of 3.1 pmol/mg wet weight. In Ringer's saline, [3H]kainate bound with a Bmax of 1.8 pmol/mg wet weight and a Kd of 214 nM. Binding in Ringer's saline, but not Tris-HCl buffer, displayed positive cooperativity with a Hill coefficient of 1.15. The [3H]kainate binding sites were solubilized in Ringer's saline using the nonionic detergent n-octyl-beta-D-glucopyranoside. Approximately 30-50% of the total number of membrane-bound binding sites were recovered on solubilization. The Kd of [3H]kainate for solubilized binding sites was approximately 200 nM. The rank order of potency for glutamatergic ligands at inhibiting [3H]kainate binding was identical and the competitive ligands had similar Ki values in both membranes and solubilized extracts. In membrane preparations, [3H]kainate displayed a two component off-rate with koff values of 0.97 min-1 and 0.07 min-1; in solubilized extracts, however, only a single off-rate (koff = 0.52 min-1) was observed. The hydrodynamic properties of n-octyl-beta-D-glucopyranoside solubilized [3H]kainate binding sites was investigated by sucrose density centrifugation. A single well defined peak was detected which yielded a sedimentation coefficient of 8.3 S. The results presented in this report suggest that goldfish brain may provide an ideal system in which to study kainate receptor biochemistry.

A series-type enzyme deactivation/activation model involving active enzyme states is utilized to theoretically quantify the influence of regional brain development and drugs on enzyme activity levels in rats. Continuous hexachlorobenzene administration with, or without, phenobarbitone pretreatment has different effects on the deactivation/activation kinetics of porphyrinogen carboxylase, delta-aminolaevulinate synthase and delta-aminolaevulinate dehydratase. The deactivation/activation kinetics exhibited by pyruvate dehydrogenase, citrate synthase, and D-3-hydroxybutyrate dehydrogenase during the development of the medulla oblongata, mid-brain, striatum, and hypothalamus sections exhibit similarities as well as discrepancies. These are identified and made more quantitative.