Rat Brain Synaptosomal Preparations Research Articles

Sarcosine-Based Glycine Transporter Type-1 (GlyT-1) Inhibitors Containing Pyridazine Moiety: A Further Search for Drugs with Potential to Influence Schizophrenia Negative Symptoms.

We have synthesized a novel series of N-substituted sarcosines, analogues of NFPS (N-[3-(biphenyl-4- yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine), as type-1 glycine transporter (GlyT-1) inhibitors. Several compounds incorporated a diazine ring inhibited recombinant hGlyT-1b expressed permanently in CHO cells and GlyT-1 in rat brain synaptosomal preparations. A structure-activity relationship for the newly synthesized compounds was obtained and discussed on the ground of their GlyT-1 inhibitory potencies. Replacement of the biphenyl-4-yloxy moiety in NFPS with a 5-pyridazinylphenoxy moiety (compounds 3, 4, 5, and 6) or a 2-phenyl-5- pyridazinyloxy moiety (compounds 10, 11, and 12) afforded compounds exhibiting potent inhibition on GlyT-1 activity. The GlyT-1 inhibitory properties of NFPS analogues, in which sarcosine was closed into a ring forming (methylamino)pyridazine-3-(2H)-one, were markedly reduced (compounds 13 and 14). The pyridazine-containing GlyT-1 inhibitors with in vitro GlyT-1 inhibitory potency also enhanced extracellular glycine concentrations in conscious rat striatum as was measured by microdialysis technique. In contrast to NFPS, sarcosine-based pyridazine containing GlyT-1 inhibitors failed to evoke compulsive running behavior whereas they inhibited phencyclidine- induced hypermotility in mice. It is believed that increase of extracellular concentrations of glycine by inhibition of its reuptake may probably influence positively glutamate N-methyl-D-aspartate (NMDA)-type ionotropic receptors in the central nervous system. This may have importance in the treatment of neuropsychiatric disorders associated with hypofunctional NMDA receptor-mediated glutamatergic neurochemical transmission. Thus, impaired NMDA receptor functions have been shown to be involved in the development of the negative symptoms and the cognitive deficit of schizophrenia and the treatment of these symptoms is the possible clinical indication of GlyT-1 inhibitors including those containing pyridazine moiety.

Characterizing synaptic vesicle proteins using synaptosomal fractions and cultured hippocampal neurons.

Cloning and characterization of synaptic vesicle proteins and their binding counterparts on the presynaptic plasma membrane have greatly advanced our understanding of the molecular mechanisms involved in the synaptic vesicle cycle and neurotransmitter release. This unit discusses multidisciplinary approaches to characterize proteins from synaptosome-enriched subcellular fractions and localize them within cultured neurons. The first approach regroups methods used to isolate synaptic vesicles from rat brain synaptosomal preparations, allowing for specific biochemical investigation of synaptic vesicle proteins. The second is a detailed procedure for pre-embedding immunogold staining and electron microscopic observation, which permits the morphological identification of proteins in individual vesicles at intact synapses. Additionally, this chapter proposes methods for light microscopic examination of hippocampal neurons. It includes procedures for embryonic and postnatal hippocampal neuron culture and describes an immunocytochemical staining protocol used to investigate synaptic vesicle protein localization with respect to other proteins or subcellular structures.

The effect of nefopam and its enantiomers on the uptake of 5-hydroxytryptamine, noradrenaline and dopamine in crude rat brain synaptosomal preparations

The effect of (+/-), (+) and (-)-nefopam on the uptake of 5-hydroxytryptamine (5-HT), noradrenaline and dopamine in synaptosomal preparations from rat forebrain, hippocampus and striatum has been investigated. All three forms of nefopam inhibited the amine uptake in the investigated structures, the order of potency being (+) greater than (+/-) greater than (-). (+)-Nefopam was 7-30 times more potent than (-)-nefopam. The same order of potency has also been found for the antinociceptive effect of these three forms, however, the differences were smaller. Inhibition of 5-HT and noradrenaline uptake may not be the sole mechanism underlying the analgesic effect of nefopam.

Myosin5a Tail Associates Directly with Rab3A-containing Compartments in Neurons

Myosin-Va (Myo5a) is a motor protein associated with synaptic vesicles (SVs) but the mechanism by which it interacts has not yet been identified. A potential class of binding partners are Rab GTPases and Rab3A is known to associate with SVs and is involved in SV trafficking. We performed experiments to determine whether Rab3A interacts with Myo5a and whether it is required for transport of neuronal vesicles. In vitro motility assays performed with axoplasm from the squid giant axon showed a requirement for a Rab GTPase in Myo5a-dependent vesicle transport. Furthermore, mouse recombinant Myo5a tail revealed that it associated with Rab3A in rat brain synaptosomal preparations in vitro and the association was confirmed by immunofluorescence imaging of primary neurons isolated from the frontal cortex of mouse brains. Synaptosomal Rab3A was retained on recombinant GST-tagged Myo5a tail affinity columns in a GTP-dependent manner. Finally, the direct interaction of Myo5a and Rab3A was determined by sedimentation velocity analytical ultracentrifugation using recombinant mouse Myo5a tail and human Rab3A. When both proteins were incubated in the presence of 1 mm GTPγS, Myo5a tail and Rab3A formed a complex and a direct interaction was observed. Further analysis revealed that GTP-bound Rab3A interacts with both the monomeric and dimeric species of the Myo5a tail. However, the interaction between Myo5a tail and nucleotide-free Rab3A did not occur. Thus, our results show that Myo5a and Rab3A are direct binding partners and interact on SVs and that the Myo5a/Rab3A complex is involved in transport of neuronal vesicles.

Antidepressant-like effects of Trichilia catigua (Catuaba) extract: evidence for dopaminergic-mediated mechanisms

Currently available therapy for depression treatment is often associated with several undesirable side effects, and it is effective only in a certain portion of the population. Therefore, the identification of alternative therapeutic tools for the treatment of depression is still needed. The present study analyzed the possible antidepressant-like effects of the Brazilian medicinal plant, Trichilia catigua, in rodents. Attempts were also made to investigate some of the possible mechanisms implicated in its actions. The antidepressant-like effects of T. catigua extract were assessed in two species of rodents (mice and rats) by means of in vivo (forced swimming test) and in vitro (monoamine reuptake and release in synaptosomal preparations) approaches. Acute oral treatment with the extract of T. catigua produced antidepressant-like effects in the forced swimming model in both mice and rats. Anti-immobility actions of T. catigua extract in mice were significantly reversed by haloperidol or by chlorpromazine, but not by pimozide, ketanserin, spiroxatrine or p-chlorophenylalanine. In vitro, T. catigua extract concentration-dependently inhibited the uptake and increased the release of serotonin, and especially of dopamine, from rat brain synaptosomal preparations. The present study provides convincing evidence for a dopamine-mediated antidepressant-like effect of the active principle(s) present in the hydroalcoholic extract of T. catigua in mice and rats when in vivo and in vitro strategies were employed. Therefore, a standardized T. catigua extract or its purified constituents could be of potential interest for the treatment of depressive disorders.

Selective inhibitors of GABA uptake: synthesis and molecular pharmacology of 4- N-methylamino-4,5,6,7-tetrahydrobenzo[ d]isoxazol-3-ol analogues

A series of lipophilic diaromatic derivatives of the glia-selective GABA uptake inhibitor ( R)-4-amino-4,5,6,7-tetrahydrobenzo[ d]isoxazol-3-ol [( R)- exo-THPO, 4] were synthesized via reductive amination of 3-ethoxy-4,5,6,7-tetrahydrobenzo[ d]isoxazol-4-one ( 9) or via N-alkylation of O-alkylatedracemic 4. The effects of the target compounds on GABA uptake mechanisms in vitro were measured using a rat brain synaptosomal preparation or primary cultures of mouse cortical neurons and glia cells (astrocytes), as well as HEK cells transfected with cloned mouse GABA transporter subtypes (GAT1-4). The activity against isoniazid-induced convulsions in mice after subcutaneous administration of the compounds was determined. All of the compounds were potent inhibitors of synaptosomal uptake the most potent compound being ( RS)-4-[ N-(1,1-diphenylbut-1-en-4-yl)amino]-4,5,6,7-tetrahydrobenzo[ d]isoxazol-3-ol ( 17a, IC 50 = 0.14 μM). The majority of the compounds showed a weak preference for glial, as compared to neuronal, GABA uptake. The highest degree of selectivity was 10-fold corresponding to the glia selectivity of ( R)- N-methyl- exo-THPO ( 5). All derivatives showed a preference for the GAT1 transporter, as compared with GAT2-4, with the exception of ( RS)-4-[ N-[1,1-bis(3-methyl-2-thienyl)but-1-en-4-yl]- N-methylamino]-4,5,6,7-tetrahydrobenzo[ d]isoxazol-3-ol ( 28d), which quite surprisingly turned out to be more potent than GABA at both GAT1 and GAT2 subtypes. The GAT1 activity was shown to reside in ( R)- 28d whereas ( R)- 28d and ( S)- 28d contributed equally to GAT2 activity. This makes ( S)- 28d a GAT2 selective compound, and ( R)- 28d equally effective in inhibition of GAT1 and GAT2 mediated GABA transport. All compounds tested were effective as anticonvulsant reflecting that these compounds have blood–brain barrier permeating ability.

Multidisciplinary Approaches for Characterizing Synaptic Vesicle Proteins

Investigation of synaptic vesicle membrane proteins using multidisciplinary approaches, particularly to characterize synaptic vesicle proteins in synapses, can greatly advance our knowledge of the molecular mechanisms involved in synaptic vesicle exocytosis and neurotransmission. Three approaches are presented in this unit to identify and characterize synaptic vesicle proteins. The first is a subcellular fractionation method used to isolate synaptic vesicles from rat brain synaptosomal preparations, which can then be used in a variety of biochemical studies on synaptic vesicle proteins. The second is a detailed procedure for pre-embedding immunogold staining and electron microscopic observation, techniques that permit the morphological identification of synaptic vesicle proteins in individual vesicles at the intact synapse. Finally, a protocol for immunocytochemical staining of cultured hippocampal neurons for light microscopic examination is provided, which allows one to stain multisynaptic vesicle proteins and determine their localization in relation to other proteins or subcellular structures in synapses.

Pharmacologic and radioligand binding analysis of the actions of 1,4-dihydropyridine activators related to Bay K 8644 in smooth muscle, cardiac muscle and neuronal preparations

The structure-activity relationships of a series of 1,4-dihydropyridine Ca2+ channel activators, including Bay K 8644, have been determined by pharmacologic and radioligand binding techniques. Pharmacologic techniques included tension responses and the measurement of pA2 values for nifedipine antagonism of Bay K 8644 responses in guinea pig ileal, rat femoral and rat atrial and papillary muscle preparations. Radioligand binding experiments employed competition against [3H]nitrendipine binding in ileal smooth muscle and rat ventricular membranes and rat brain synaptosomal preparations. The series of compounds was employed as the racemates. Binding affinities were not significantly different between smooth muscle, cardiac muscle and brain preparations and the same rank order of pharmacologic activities is observed in smooth and cardiac muscle, where the effects of the 4-phenyl substituents, o greater than or equal to m greater than p, parallel those observed for 1,4-dihydropyridine antagonists. In the ileal and femoral artery smooth muscle preparations a 1:1 correlation is observed between pharmacologic and radioligand binding affinities. However, in the cardiac muscle preparations, left atrium and papillary muscle, there is an approximately 10-fold difference between the binding affinities and the lower pharmacologic affinities. A similar difference between smooth and cardiac muscle is observed with the pA2 values of 6.97 and 7.06 in atrial and papillary muscle respectively, which are significantly lower than the values of 8.54 and 8.72 measured in ileal and femoral artery respectively. The structure-activity expressions measured for this small series of 1,4-dihydropyridine activators parallel those observed in the larger series of 1,4-dihydropyridine antagonists. This is consistent with proposals that activators and antagonists interact at common binding sites that are components of a voltage-dependent Ca2+ channel.

Covalent structure and some pharmacological features of native and cleaved alpha-KTx12-1, a four disulfide-bridged toxin from Tityus serrulatus venom.

A toxin with four disulfide bridges from Tityus serrulatus venom was able to compete with 125I-kaliotoxin on rat brain synaptosomal preparations, with an IC50 of 46 nM. The obtained amino acid sequence and molecular mass are identical to the previously described butantoxin. Enzymatic cleavages in the native peptide followed by mass spectrometry peptide mapping analysis were used to determine the disulfide bridge pattern of alpha-KTx12-1. Also, after the cleavage of the first six N-terminal residues, including the unusual disulfide bridge which forms an N-terminus ring, the potency of the cleaved peptide was found to decrease about 100 fold compared with the native protein.

Selective Association of TRPC Channel Subunits in Rat Brain Synaptosomes

TRPC genes encode a ubiquitous family of ion channel proteins responsible for Ca(2+) influx following stimulation of G-protein-coupled membrane receptors linked to phospholipase C. These channels may be localized to large multimeric signaling complexes via association with PDZ-containing scaffolding proteins. Based on sequence homology, the TRPC channel family can be divided into two major subgroups: TRPC1, -C4, and -C5 and TRPC3, -C6, and -C7. Although TRPC channels are thought to be tetramers, the actual subunit composition remains unknown. To determine subunit arrangement, individual TRPC channel pairs were heterologously expressed in Sf9 insect cells and immunoprecipitated using affinity-purified rabbit polyclonal antibodies specific for each channel subtype. Reciprocal co-immunoprecipitations showed that TRPC1, -C4, and -C5 co-associate and that TRPC3, -C6, and -C7 co-associate but that cross-association between the two major subgroups does not occur. Additionally, the interaction between each TRPC channel and the PDZ-containing protein, INAD (protein responsible for the inactivation-no-after-potential Drosophila mutant), was examined. TRPC1, -C4, and -C5 co-immunoprecipitated with INAD, whereas TRPC3, -C6, and -C7 did not. To define channel subunit interactions in vivo, immunoprecipitations were performed from isolated rat brain synaptosomal preparations. The results revealed that TRPC1, -C4, and -C5 co-associate and that TRPC3, -C6, and -C7 co-associate in both cortex and cerebellum but that cross-association between the two major subgroups does not occur. These results demonstrate that TRPC channels are present in nerve terminals and provide the first direct evidence for selective assembly of channel subunits in vivo.

Selective inhibitors of glial GABA uptake: synthesis, absolute stereochemistry, and pharmacology of the enantiomers of 3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazole (exo-THPO) and analogues.

3-Methoxy-4,5,6,7-tetrahydro-1,2-benzisoxazol-4-one (20a), or the corresponding 3-ethoxy analogue (20b), and 3-chloro-4,5,6, 7-tetrahydro-1,2-benzisothiazol-4-one (51) were synthesized by regioselective chromic acid oxidation of the respective bicyclic tetrahydrobenzenes 19a,b and 50, and they were used as key intermediates for the syntheses of the target zwitterionic 3-isoxazolols 8-15 and 3-isothiazolols 16 and 17, respectively. These reaction sequences involved different reductive processes. Whereas (RS)-4-amino-3-hydroxy-4,5,6,7-tetrahydro-1,2-benzisoxazole (8, exo-THPO) was synthesized via aluminum amalgam reduction of oxime 22a or 22b, compounds 9, 11-13, and 15-17 were obtained via reductive aminations. Compound 10 was synthesized via N-ethylation of the N-Boc-protected primary amine 25. The enantiomers of 8 were obtained in high enantiomeric purities (ee >/= 99.1%) via the diastereomeric amides 32 and 33, synthesized from the primary amine 23b and (R)-alpha-methoxyphenylacetyl chloride and subsequent separation by preparative HPLC. The enantiomers of 9 were prepared analogously from the secondary amine 27. On the basis of X-ray crystallographic analyses, the configuration of oxime 22a was shown to be E and the absolute configurations of (-)-8 x HCl and (+)-9 x HBr were established to be R. The effects of the target compounds on GABA uptake mechanisms in vitro were measured using a rat brain synaptosomal preparation and primary cultures of mouse cortical neurons and glia cells (astrocytes). Whereas the classical GABA uptake inhibitor, (R)-nipecotic acid (2), nonselectively inhibits neuronal (IC(50) = 12 microM) and glial (IC(50) = 16 microM) GABA uptake and 4,5,6,7-tetrahydroisoxazolo¿4,5-cpyridin-3-ol (1, THPO) shows some selectivity for glial (IC(50) = 268 microM) versus neuronal (IC(50) = 530 microM) GABA uptake, exo-THPO (8) was shown to be more potent as an inhibitor of glial (IC(50) = 200 microM) rather than neuronal (IC(50) = 900 microM) GABA uptake. This selectivity was more pronounced for 9, which showed IC(50) values of 40 and 500 microM as an inhibitor of glial and neuronal GABA uptake, respectively. These effects of 8 and 9 proved to be enantioselective, (R)-(-)-8 and (R)-(+)-9 being the active inhibitors of both uptake systems. The selectivity of 9 as a glial GABA uptake inhibitor was largely lost by replacing the N-methyl group of 9 by an ethyl group, compound 10 being an almost equipotent inhibitor of glial (IC(50) = 280 microM) and neuronal (IC(50) = 400 microM) GABA uptake. The remaining target compounds, 11-17, were very weak or inactive as inhibitors of both uptake systems. Compounds 9-13 and 15 were shown to be essentially inactive against isoniazide-induced convulsions in mice after subcutaneous administration. The isomeric pivaloyloxymethyl derivatives of 9, compounds 43 and 44, were synthesized and tested as potential prodrugs in the isoniazide animal model. Both 43 (ED(50) = 150 micromol/kg) and 44 (ED(50) = 220 micromol/kg) showed anticonvulsant effects, and this effect of 43 was shown to reside in the (R)-(+)-enantiomer, 45 (ED(50) = 44 micromol/kg). Compound 9 also showed anticonvulsant activity when administered intracerebroventricularly (ED(50) = 59 nmol).

Role of lysine and tryptophan residues in the biological activity of toxin VII (Ts gamma) from the scorpion Tityus serrulatus.

Toxin VII (TsVII), also known as Ts gamma, is the most potent neurotoxin in the venom of the Brazilian scorpion Tityus serrulatus. It has been purified to homogeneity using a new fast and efficient method. Chemical modification of TsVII with the tryptophan-specific reagent o-nitrophenylsulfenyl chloride yielded three modified derivatives (residues Trp39, Trp50 and Trp54). Acetylation of TsVII mostly generated the monoacetylated Lys12 derivative. No side reactions were detected, as indicated by endoproteinase Lys-C peptide mapping, Edman degradation and electrospray mass spectrometry. Circular dichroism and fluorimetric measurements showed that none of the chemical modifications altered the overall structure of the derivatives. The acetylation of Lys12 or the sulfenylation of Trp39 or Trp54 led to a loss of both toxicity in mice and apparent binding affinity for rat brain and cockroach synaptosomal preparations. Sulfenylation of Trp50, however, moderately affected the toxicity of TsVII in mice and had almost no effect on its binding properties. A 3-dimensional model of TsVII was constructed by homology modeling. It suggests that the most reactive residues (Lys12 and Trp39 and Trp54) are all important in the functional disruption of neuronal sodium channels by TsVII, and are close to each other in the hydrophobic conserved region.

Nitric Oxide Modification of Rat Brain Neurogranin Affects Its Phosphorylation by Protein Kinase C and Affinity for Calmodulin

Neurogranin (Ng) is a prominent protein kinase C (PKC) substrate which binds calmodulin (CaM) in the absence of Ca2+. Rat brain Ng contains four cysteine residues that were readily oxidized by nitric oxide (NO) donors, 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEANO) and sodium nitroprusside, and by oxidants, H2O2 and o-iodosobenzoic acid. NO oxidation of Ng resulted in a conformational change detectable by increased electrophoretic mobility upon SDS-polyacrylamide gel electrophoresis. The NO-mediated mobility shift was reversed by treatment with dithiothreitol and was blocked by modification of Ng sulfhydryl groups with 4-vinylpyridine. Both the nonphosphorylated and PKC-phosphorylated Ng were susceptible to NO oxidation. Modification of Ng by DEANO was blocked by CaM in the absence of Ca2+; while in the presence of Ca2+, CaM did not protect Ng from oxidation by DEANO. CaM also failed to protect DEANO-mediated oxidation of PKC-phosphorylated Ng with or without Ca2+. Oxidation of Ng by the various oxidants apparently resulted in the formation of intramolecular disulfide bond(s) as judged by a reduction of apparent Mr on SDS-polyacrylamide gel electrophoresis; this oxidized form, unlike the reduced form, did not bind to CaM-affinity column. The oxidized Ng was also a poorer substrate for PKC; both the reduced and oxidized forms had similar Km values, but the Vmax of the oxidized form was about one-fourth of the reduced one. When comparing the rate of DEANO-mediated nitrosation of Ng with other sulfhydryl-containing compounds, it became evident that Ng ranked as one of the best NO acceptors among those tested, including serum albumin, glutathione, and dithiothreitol. Ng present in the rat brain synaptosomal preparations was also oxidized by DEANO in a dose-dependent manner when analyzed by immunoblot with a polyclonal antibody against this protein. These results suggest that Ng is a likely target of NO and other oxidants and that oxidation/reduction may serve as a mechanism for controlling both the PKC phosphorylation and the CaM-binding affinity of this protein.

Synthesis and characterization of N-(acenaphth-5-yl)-N'-(4-methoxynaphth-1-yl)guanidine as a glutamate release inhibitor and potential anti-ischemic agent

Structure-activity relationship (SAR) studies of N,N'-diarylguanidines as glutamate release inhibitors led to the synthesis of N-(acenaphth-5-yl)-N'-(4-methoxynaphth-1-yl)guanidine ( 6). Compound 6, with IC 50 values of 1.3 and 2.2 μM in 14C-guanidine flux and 45Ca 2+- flux assays respectively, effectively inhibited both veratridine and K +-evoked glutamate release in rat brain synaptosomal preparations.

Involvement of Bidirectional Adenosine Transporters in the Release of l‐[3H]Adenosine from Rat Brain Synaptosomal Preparations

Adenosine transport inhibitors as enhancers of extracellular levels of endogenous adenosine would, presumably, only be effective if, for example, (1) the inhibitors block influx to a greater degree than efflux (release) of intracellular adenosine or (2) the inhibitors block equally well the influx and efflux of adenosine, but significant amounts of adenosine are formed as a result of dephosphorylation of released adenine nucleotides. Limited information is available regarding the directional symmetry of adenosine transporters in neural cells. Using rat brain crude P2 synaptosomal preparations preloaded with L-[3H]adenosine, our objectives here were to determine (1) if L-[3H]adenosine, a substrate for adenosine transporters that is more metabolically stable than physiological D-adenosine, was being released from synaptosomal preparations, (2) the optimal conditions necessary to observe the release, and (3) the degree to which this release was mediated by efflux through bidirectional nucleoside transporters. L-[3H]Adenosine release was found to be concentration and time dependent, temperature sensitive, and linear with synaptosomal protein. L-[3H]Adenosine release was inhibited dose-dependently by dipyridamole, nitrobenzylthioinosine, and dilazep; at concentrations of 100 microM inhibition was at least 40% for dipyridamole, 52% for nitrobenzylthioinosine, and 49% for dilazep. After loading with L-[3H]adenosine alone or I-[3H]adenosine plus unlabeled L-adenosine, D-adenosine, or uridine, L-[3H]adenosine release was inhibited 42% by L-adenosine, 69% by uridine, and 81% by D-adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the Binding of Omega-Conopeptides to Different Classes of Non-L-Type Neuronal Calcium Channels

The interaction of two synthetic ω-conopeptides SNX111 (MVIIA) and SNX-230 (MVIIC) both derived from the marine snail Conus magus, with non-L-type neuronal voltage-sensitive calcium channels (VSCC) in rat brain synaptosomal preparations has been investigated with the aid of well-characterized 125I derivatives of the two peptides. To assess the effects of iodination on the binding characteristics of SNX-111 and SNX-230, the corresponding peptides containing monoiodotyrosine in place of tyrosine, namely, SNX-259 ([ 127I]SNX-111) and SNX260 ([ 127I]SNX-230), respectively, were prepared by solid-phase synthesis. Saturation analysis showed that [ 127I]SNX-111 and [ 127I]SNX-230 bound to two distinct classes of high-affinity sites with apparent K d's of 9 and 11 p M and B max's of 0.54 and 2.2 pmol/mg protein, respectively. Kinetic analysis revealed that both peptides exhibited high association rates as well as rapid dissociation rates in contrast to the 125I derivative of the synthetic ω-conopeptide from Conus geographus, GVIA (SNX- 124), which binds irreversibly to N-type channels in rat brain synaptosomes. Competition binding experiments with [ 125I]SNX-111 and [ 125I]SNX-124 established that both of them bind to the same site, namely, N-type VSCC. The site detected by the binding of [ 125I]SNX-230 is distinct from N-type VSCC since SNX-111 has very low affinity ( K i = 135 n M) in competition studies. Recent findings that a novel high-voltage-activated calcium channel in rat cerebellar granule neurons is resistant to blockers of L-, N-, and P-type VSCC but is highly sensitive to SNX-230 suggest that the [ 125I]SNX-230 binding site may represent this novel type of calcium channel or another, as yet undescribed, VSCC.

Systematic study of substance P analogs

The multipin peptide synthesis technique, a method for simultaneous multiple peptide synthesis, was developed for large-scale screening of oligopeptides [Geysen et al. (1984) Proc. Natl. Acad. Sci. USA, 81, 3998-4002]. A modification of the technique allows the peptides assembled on polyethylene pins to be cleaved in their native amide form and reconstituted into physiologically compatible solutions. In this study, the suitability of these peptides for quantitative receptor binding assay was evaluated. Substance P and 18 analogs, including a set of N-terminal truncated substance P and a set of naturally occurring substance P analogs, were synthesized by the multipin methods. An average yield of 20 +/- 3 nmol of peptide per pin was obtained. The purity of the peptides was estimated to be ca. 90%. The binding activities of these peptides were determined in a competition assay against 125I-BHSP binding to a rat brain synaptosome preparation. The rank order of the affinities of these peptides depicted a typical pharmacological profile of central NK1 receptor. The IC50 values obtained were also in good agreement with data reported by other groups using similar experimental conditions, except that bulk synthesized peptides were used. This study demonstrates that the peptides synthesized with the multipin technique are suitable for quantitative receptor studies, particularly for a high-volume screening of bioactive peptides.

Activation of phosphoinositide/protein kinase C pathway in rat brain tissue by pyrethroids

We have investigated the effects of a type II pyrethroid insecticide, deltamethrin, on changes in the protein phosphorylation pattern associated with neurotransmitter release in rat brain synaptosomal preparations. Deltamethrin was found to stimulate directly the activity of the protein kinase C/ phosphoinositide pathway at very low concentrations. This action resulted in an increase in the intracellular concentration of inositol 1,4,5-triphosphate (IP 3) and free calcium, as well as an increase in overall and specific protein phosphorylation within the synapse. Particularly noticeable was the deltamethrin-induced increase in phosphorylation on two very acidic proteins (87 and 48 kDa proteins) and one basic 38 kDa protein. These results are consistent with those of a previously reported study in which deltamethrin caused an increase in neurotransmitter release which was accompanied by increased intrasynaptosomal free Ca 2+ levels and protein phosphorylation activities. Together all these observations support the view that calcium-sensitive proteins involving synaptic transmission are the major action targets of type II pyrethroids.

Characterization of a cholecystokinin 8-generating endoprotease purified from rat brain synaptosomes.

An endoproteolytic activity that specifically cleaves CCK 33, producing CCK 8, has been purified from a rat brain synaptosome preparation. The purification, which included anion exchange, chromatofocusing, hydroxyapatite, and gel filtration chromatography, resulted in a greater than 3000-fold increase in specific activity. This neutral endoprotease (pH optimum 8) exists as a 90-kDa species, which can be dissociated into active 40-kDa species. The enzyme is a non-trypsin serine protease, which is inhibited by diisopropyl-fluorophosphate and p-aminobenzamidine but not by soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, aprotinin, or a number of thiol or metalloprotease inhibitors. It is highly substrate-specific and cleaves neither trypsin, enteropeptidase, kallikrein substrates, nor analogues of mono- or dibasic cleavage sites of prohormones other than pro-CCK. The endoprotease will not cleave CCK 12 desulfate or CCK (20-29), although these peptides contain common sequences with CCK-33. The protease does cleave [Glu27]CCK (20-29), a peptide in which the glutamate mimics the negative charge normally present on tyrosine sulfate. This suggests that the negative charge at position 27 is important in substrate recognition. The enzyme will also cleave CCK 33 and CCK (1-21) on the carboxyl-terminal side of a single lysine residue in position 11. The subcellular location and specificity of this endoprotease make it a good candidate for a CCK-processing protease.

Effects of GTPγS and other nucleotides on phosphoinositide metabolism in crude rat brain synaptosomal preparations

Crude nerve-ending preparations from rat brain were labeled with either [(32)P]phosphate or myo[2-(3)H]inositol in order to observe effects of guanosine 5?-[?-thio]triphosphate (GTP?S) and other nucleotides on phosphoinositides, phosphatidate and inositol phosphates. This system exhibited typical responses to muscarinic agonists, including acetylcholine-a decrease in net labeling of [(32)P]polyphosphoinositides, an increase in labeling of [(32)P]phosphatidate, and a stimulation of [(3)H]inositol phosphate formation. GTP?S and other nucleotides may not readily penetrate intact synaptosomal membranes to cause activation of phospholipase C via an interaction with G proteins, and, as might be expected, there was no indication that G-protein interaction occurred in these preparations. However, other effects were observed. GTP?S decreased net [(32)P] incorporation in phosphatidylinositol (PI) and polyphosphoinositides in a dose-dependent manner. GTP?S also caused an initial marked stimulation of [(32)P] labeling of phosphatidate, suggesting a possible inhibition in the conversion of phosphatidic acid to PI. Other nucleotides [GTP, ATP, Gpp(NH)p, GMP] produced qualitatively similar effects on phosphoinositides. Thus GTP?S and other nucleotides, at physiologically relevant concentrations, may influence phosphoinositide turnover via extracellular or other mechanisms, in addition to the proposed interaction of GTP with G-proteins within membranes.