Previous Binding Studies Research Articles

The cholecystokininB receptor is coupled to two effector pathways through pertussis toxin-sensitive and -insensitive G proteins.

Previous binding studies have suggested the existence of two affinity states for type B cholecystokinin receptors (CCK(B)R), which could correspond to different coupling states of the receptor to G proteins. To test this hypothesis, we have further investigated signal transduction pathways coupled to rat CCK(B)R stably transfected in Chinese hamster ovary cells. We show that CCK(B)R are coupled to two distinct transduction pathways involving two different G proteins, a pertussis toxin-insensitive/phospholipase C pathway leading to the production of inositol phosphate and arachidonic acid, and a pertussis toxin-sensitive/phospholipase A2 pathway leading to the release of arachidonic acid. We further demonstrate that the relative degree of activation of each effector pathway by different specific CCK(B)R agonists is the same, and that a specific CCK(B)R antagonist, RB213, can differentially antagonize the two signal transduction pathways elicited by these agonists. Taken all together, these data could be explained by the recently proposed theory assuming that the receptor can exist in a three-state model in which two active conformations corresponding to the complex formed by the receptor with two different G proteins coexist. According to this model, agonists or antagonists could recognize preferentially either conformation of the activated receptor, leading to variable behavior in a system containing a single receptor type.

Interfacial membrane docking of cytosolic phospholipase A2 C2 domain using electrostatic potential-modulated spin relaxation magnetic resonance.

The C2 domain of cytosolic phospholipase A2 (C2cPLA2) plays an important role in calcium-dependent transfer of the protein from the cytosol to internal cellular membranes as a prelude for arachidonate release from membrane phospholipids. By using a recently developed electron paramagnetic resonance approach together with 13 site-specifically nitroxide spin labeled C2cPLA2s and membrane-permeant and -impermeant spin relaxants, we have determined the orientation of C2cPLA2 with respect to the surface of vesicles of the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphomethanol. The structure reveals that the two calcium-binding regions on C2cPLA2 that display hydrophobic residues, CBR1 and CBR3, are partially inserted into the core of the membrane. CBR2 that contains predominantly hydrophilic residues is close to the membrane but not inserted. The long axis of the cylindrical C2cPLA2 molecule is tilted with respect to the bilayer normal, which brings a cluster of basic protein residues close to the phospholipid headgroups. Such an orientation places the two bound calcium ions close to the membrane surface. All together, the results provide structural support for previous proposals that binding of C2cPLA2 to the membrane interface is driven in part by insertion of hydrophobic surface loops into the membrane core. The results are contrasted with previous studies of the interfacial binding of the first C2 domain of synaptotagmin I, which has shorter surface loops that display basic residues for electrostatic interaction with the bilayer surface.

A novel mutation in the CAG triplet region of exon 1 of androgen receptor gene causes complete androgen insensitivity syndrome in a large kindred.

Complete androgen insensitivity syndrome (CAIS) is an X-linked inherited disease caused by mutations in the androgen receptor (AR) gene. We have previously reported the largest kindred of CAIS, with 17 46,XY psychosexual and phenotypic females who lack secondary sexual hair. Analysis of AR binding indicated a receptor-negative form of complete androgen insensitivity, and DNA linkage analysis indicated that the absent binding was not caused by a large AR gene deletion. Using PCR-single-strand DNA conformational polymorphism, PCR-denaturing gradient gel electrophoresis, and DNA sequencing, we have identified a novel mutation in the polymorphic CAG trinucleotide region of exon 1 of the AR gene, where a single adenine is inserted, or equivalently, a GC-dinucleotide is deleted at this region of the gene. The mutation results in a frameshift at amino acid 60 and a premature termination of the receptor downstream of the mutation. This predicts a mutant AR with only 79 amino acids in the amino-terminal of AR protein, prohibiting binding to the ligand, as well as the cognate DNA. The rest of the encoding regions of the AR gene in the affected subjects are normal. These results are consistent with previous ligand binding and DNA linkage analysis studies. This new mutation in the CAG trinucleotide area of exon 1 of the AR gene represents the first example of a defect in a CAG repeat causing CAIS in this large kindred. All previous reported variants in this region are changes in the number of triplet repeats.

Calcium changes induced by presynaptic 5-hydroxytryptamine-3 serotonin receptors on isolated terminals from various regions of the rat brain

The serotonin 5-hydroxytryptamine-3 receptor is a ligand-gated ion channel that is distributed widely in the nervous system. Within the CNS, a significant portion of the 5-hydroxytryptamine-3 receptors appears to be present on presynaptic nerve terminals and, using an imaging approach, it was shown previously that presynaptic 5-hydroxytryptamine-3 receptors on individual isolated nerve terminals (synaptosomes) from rat corpus striatum display a distinctive set of properties—slow onset, little desensitization and high apparent permeability for Ca 2+—when compared to those observed for 5-hydroxytryptamine-3 receptors localized at postsynaptic sites on neuronal cell bodies. To consider whether their characteristic nature is a common feature of presynaptic 5-hydroxytryptamine-3 receptors across the brain, we used confocal microscopy to measure changes in intracellular Ca 2+ concentration resulting from 5-hydroxytryptamine-3 agonist-induced responses in synaptosomes from representative rat brain regions, ranging in expression of overall levels of 5-hydroxytryptamine-3 receptors from relatively low (cerebellum) to intermediate (corpus striatum and hippocampus) to high (amygdala). Application of 100 nM m-chlorophenyl biguanide, a specific 5-hydroxytryptamine-3 receptor agonist, induced changes in relative intracellular Ca 2+ concentration in subsets of synaptosomes from the corpus striatum (∼6% of total), hippocampus (∼3% of total), amygdala (∼30% of total) and cerebellum (∼32% of total). In order to assure the viability of the synaptosomes that did not respond to 5-hydroxytryptamine-3 agonist stimulation, KCl (45 mM) was subsequently added to depolarize the same population of synaptosomes, and increases in intracellular Ca 2+ concentration were then seen in 80–90% of the synaptosomes from all four regions. The kinetics of the intra synaptosomal Ca 2+ changes produced by K +-evoked depolarization were similar in all regions, showing a rapid rise to a peak followed by an apparent plateau phase. In contrast, the changes in intracellular Ca 2+ concentration evoked by m-chlorophenyl biguanide displayed substantially slower kinetics, similar to previous findings, but which varied among responding synaptosomes from one region to another. In particular, m-chlorophenyl biguanide-induced changes were notably slower in synaptosomes from the amygdala (rise time constant, τ=25 s), when compared to responses in synaptosomes from other regions (striatum, τ=12 s; hippocampus, τ=9.6 s; cerebellum, τ=7 s). To independently demonstrate the presence of 5-hydroxytryptamine-3 receptors on nerve terminals in the various regions using a molecular approach, we double-immunostained the synaptosomes for the 5-hydroxytryptamine-3 receptor and the synaptic vesicle protein synaptophysin, using, respectively, a polyclonal antibody raised against an N-terminal peptide of the 5-hydroxytryptamine-3 receptor and a monoclonal anti-synaptophysin antibody, and observed 5-hydroxytryptamine-3 receptors in varying subsets of the synaptosomes from each region, providing direct support for the results obtained in our functional experiments. These results suggest that the distinctive properties of presynaptic 5-hydroxytryptamine-3 receptors are found throughout the brain, with evident differences in the kinetics of the responses to agonist stimulation observed across the brain regions studied. As expected, the proportion of the synaptosomal population that responded on application of 5-hydroxytryptamine-3 agonist varied in preparations from one region to another; however, the presence of a relatively high proportion of presynaptic 5-hydroxytryptamine-3 receptors in the cerebellum contrasts with previous binding studies demonstrating a relatively low overall density of 5-hydroxytryptamine-3 receptors in this region. We hypothesize that presynaptic 5-hydroxytryptamine-3 receptors present on nerve terminals regulate the functioning of select synapses throughout the rat brain.

Regulation of CAK kinase activity by p53

The growth suppressor p53 is an important key element which controls cell cycle progression in response to cellular stress like DNA damage. Its ability to act as transcriptional activator or repressor links transcription and cell cycle control. Several target genes selectively transactivated by p53 are implicated in growth control, apoptosis and DNA repair. Here we report the interaction of p53 with another important dual player of cell cycle control and transcription, the protein kinase complex CDK7/cyclin H/Mat1 (CDK activating kinase, CAK kinase). This is implicated in the activating phosphorylation of CDK2/cyclin A kinase required to allow cells to proceed through the G1/S transition, and on the other hand, as a component of the basal transcription factor TFIIH found to be necessary for CTD phosphorylation of RNA polymerase II in order to allow elongation of transcription. Based on previous binding studies of p53 with other C-terminal interaction partners of p53 we demonstrate a direct physical interaction of p53 with cyclin H in vitro and in vivo. As a consequence of this interaction we tested the influence of p53 on the kinase activity of CAK kinase for CTD and CDK2 phosphorylation. The addition of wild type p53 to the kinase reactions resulted in a significant downregulation of CDK2 phosphorylation and CTD phosphorylation by the CDK activating kinase. On the other hand addition of a mutant p53His175 failed to downregulate CDK2 and CTD phosphorylation by the CDK activating kinase. In an attempt to support our findings in vivo we measured CAK kinase activity in p21-/- and p53-/- mice embryonal fibroblasts under conditions when p53 gets activated by irradiation. In the case of p21-/- cells this led to a significant reduction of CTD phosphorylation activity of the CDK activating kinase by irradiation of the cells. On the other hand in p53 cells no downregulation of CTD phosphorylation activity of CAK kinase was observed indicating that this kind of negative regulation of CAK kinase activity is exclusively due to a functional p53. These findings imply a direct involvement of p53 in triggering growth arrest by its interaction with the CDK activating kinase complex without the need of cyclin-dependent kinase inhibitors (CKIs) and potentially suggest a new mechanism for p53-dependent apoptosis.

Distinct modifications by neurokinin 1 (SR140333) and neurokinin 2 (SR48968) tachykinin receptor antagonists of the N-methyl- d-aspartate-evoked release of acetylcholine in striosomes and matrix of the rat striatum

The effects of SR140333 and SR48968 (neurokinin 1 and neurokinin, tachykinin receptor antagonists, respectively) on the N-methyl- d-aspartate-evoked release of [ 3H]acetylcholine (previously formed from [ 3H]choline) were investigated in striosome-enriched areas and in the matrix of the rat striatum using an in vitro microsuperfusion method. In both striatal compartments, SR140333 and SR48968 did not modify the 50 μM N-methyl- d-aspartate-evoked release of [ 3H]acetylcholine. However, in low concentrations, both SR140333 (0.1 μM to 1 pM) and SR48968 (0.1 μM to 0.1 nM) markedly enhanced the 1 mM N-methyl- d-aspartate (+10 μM d-serine)-evoked release of [ 3H]acetylcholine in striosome-enriched areas. These responses were dopamine-dependent since they were not observed any more following the local blockade of D 2 receptors by sulpiride or of dopamine synthesis by α-methyl-p-tyrosine. A dopamine-dependent disinhibitory effect (of lower amplitude) on the 1 mM N-methyl- d-aspartate (+10 μM d-serine)-evoked release of [ 3H]acetylcholine was also induced by SR48968 (0.1 μM to 0.1 nM) (but not by SR140333) in the matrix. In addition, in the matrix, as shown only in the presence of α-methyl-p-tyrosine, both SR140333 and SR48968 reduced the 1 mM N-methyl- d-aspartate (+10 μM d-serine)-evoked response and these non-dopamine-mediated inhibitory effects only occurred at the highest tested concentration (0.1 μM) of the antagonists. Indicating the specificity of these responses, the effects of SR140333 were reproduced by RP67580, another neurokinin, receptor antagonist and, as expected from previous binding studies, corresponding SR140333 and SR48968 enantiomers were without effect. These results suggest that under potent stimulation of N-methyl- d-aspartate receptors, endogenously released substance P and neurokinin A (or related tachykinins) regulate differently the N-methyl- d-aspartate-evoked release of [ 3H]acetylcholine in striosomes and in the matrix. The inhibitory effects of these tachykinins on the evoked release of [ 3H]acetylcholine are mediated by dopamine. On the contrary, their facilitatory responses are only observed in the matrix under blockade of dopamine transmission.

Glucocorticoid Receptors Are Differentially Expressed in the Cells and Tissues of the Immune System

Cytosolic glucocorticoid receptor (GR) binding studies on immune tissues demonstrate that the thymus exhibits three to four times higher levels of GR protein than the spleen. High levels of GR are consistent with the exquisite sensitivity of the thymus to glucocorticoid exposure. Nevertheless, whole cell binding studies reveal similar levels of GR in immature thymic T lymphocytes and more mature, splenic T lymphocytes. Moreover, whole cell binding techniques indicate that neutrophils (which represent roughly 30% of splenic leukocytes) exhibit higher GR than both T and B lymphocytes, further contradicting results from cytosolic binding studies. To address these inconsistencies, GR protein was assessed in immune cells and tissues using cytosolic radioligand binding, Western blot analysis, and immunocytochemistry. Consistent with previous cytosolic receptor binding studies on immune tissue homogenates, thymic T cells were found to have higher levels of GR than T cells isolated from the spleen. In addition, neutrophils were found to have fewer GR than lymphocytes and monocytes. These results indicate a meaningful relationship between receptor expression and known sensitivity to glucocorticoids.

The MotA transcriptional activator of bacteriophage T4 binds to its specific DNA site as a monomer.

During bacteriophage T4 middle mode gene expression, the MotA transcription factor binds to T4 middle promoters at a -30 mot box consensus sequence to allow activation. Previous binding studies showed that MotA forms multiple gel-shifted complexes with DNA, and structural evidence suggested that MotA dimerizes upon DNA binding. We have shown that a short (13 bp) mot box DNA substrate binds MotA protein but fails to form slower migrating complexes. Therefore, the slower migrating complexes in gel shift assays are caused by DNA-mediated binding events. Competition experiments indicate that the slower migrating complexes are formed by nonspecific binding events, while the first-shifted complex is caused by specific binding to the mot box. Saturation binding experiments revealed that the stoichiometry of MotA binding to DNA is 1:1 in the first-shifted complex, while the slower complexes apparently contain MotA multimers. Gel shift assays using mixtures of MotA and a GST-MotA fusion protein supported the conclusion that the first-shifted complex contains one protein molecule bound to DNA. Furthermore, MotA monomers were cross-linked by glutaraldehyde under conditions where slower complexes exist, but not under conditions that lead to only the first-shifted complex. We conclude that MotA binds specifically to the mot box as a monomer and that additional nonspecific binding events require flanking DNA.

Cocaine toxicity: concurrent influence of dopaminergic, muscarinic and sigma receptors in mediating cocaine-induced lethality.

The concurrent influence of dopaminergic, sigma and muscarinic neurotransmitter systems in mediating cocaine-induced lethality is predicted by previous receptor binding results from our laboratories. The present results demonstrate that pharmacological manipulations of these predicted neurotransmitter systems alter the occurrence of cocaine-induced lethality in C57BL/6J mice. The dopamine reuptake inhibitor bupropion increased the number of occurrences of lethality produced by (-) cocaine, while the dopaminergic D1 antagonist SCH 23390, the muscarinic M1 antagonist pirenzepine, and the sigma ligand (+) SKF 10047 all significantly, but only partially, antagonized (-) cocaine-induced lethality. In addition, the protective effects against lethality of the drug combinations SCH 23390 + pirenzepine or SCH 23390 + SKF 10047 were greater than any of these drugs used alone. These results are consistent with those from the previous receptor binding studies, and provide converging supportive evidence that the lethal effects of cocaine depend upon concurrent interactions with dopaminergic, muscarinic M1 and sigma receptor sites.

Cocaine-induced convulsions: pharmacological antagonism at serotonergic, muscarinic and sigma receptors.

The concurrent influence of multiple neurotransmitter systems in mediating cocaine-induced convulsions is predicted by the results of previous receptor binding studies. The present results demonstrate that pharmacological manipulations of these predicted neurotransmitter systems alters the occurrence of cocaine-induced convulsions. The 5-HT reuptake inhibitor fluoxetine enhanced the occurrence and severity of convulsions produced by 100 mg/kg (-) cocaine, while the 5-HT2 receptor antagonists cinanserin, ketanserin and pirenperone antagonized cocaine-induced convulsions in a dose-dependent manner. Further, the M1 receptor antagonist pirenzepine antagonized cocaine-induced convulsions, but atropine did not. In addition, both the (+) and (-) stereoisomers of the sigma ligand SKF 10047 significantly attenuated cocaine-induced convulsions. (+)SKF 10047 was more potent than (-)SKF 10047 in this effect, suggesting a stereoselective effect at sigma receptor sites. In constrast, DA and NE neurotransmission do not appear to modulate the proconvulsant effects of cocaine in a specific, dose-dependent manner. Thus, of the CNS binding sites with which cocaine is known to interact, the results are consistent with the conclusion that 5-HT transporters and 5-HT2 receptor sites appear to be direct and primary sites related to cocaine-induced convulsions, while M1 and sigma binding sites appear to play important but secondary and modulatory roles in this response.

Dual modulation of dopamine release from anterior nucleus accumbens through cholecystokinin-B receptor subsites.

Previous binding studies have suggested the existence of two affinity states for cholecystokinin-B (CCK-B) receptor. One study, using BC 197 and BC 264, two highly selective CCK-B agonists, has shown that BC 197 is selective for one subsite, B1, and that BC 264 has the same affinity for the two subsites, B1 and B2. Therefore, the possible involvement of CCK-B subsites in the modulation of endogenous dopamine (DA) release from slices of the anterior part of the nucleus accumbens was investigated with these two agonists in order to associate a functional response with activation of each subsite. The selective B1 agonist BC 197 produced a dose-dependent increase of 35 mM K(+)-stimulated DA release. In contrast, at a low concentration (20 nM), BC 264 inhibited the K(+)-evoked DA release, whereas at a higher concentration (1 microM), it stimulated the DA release. These two opposing effects were suppressed by the CCK-B antagonist PD-134,308, but not by the CCK-A antagonist L-364,718 and were not prevented by tetrodotoxin, a Na(+)-channel blocker. Moreover, BC 264 at 20 nM, in the presence of PD-134,308 at a concentration that would block the B2 subsites (0.1 nM), increased the evoked DA release. All together, these results support further the existence of distinct CCK-B subsites and suggest that, in the anterior nucleus accumbens, their stimulation mediates opposite effects on K(+)-stimulated DA release via a presynaptic mechanism.

Viewing AIDS from a glycobiological perspective: potential linkages to the human fetoembryonic defence system hypothesis.

The primary molecular changes that lead to development of acquired immunodeficiency syndrome (AIDS) are very poorly understood, as are the mechanisms underlying the protection of the developing human from the maternal immune response. Recent data that the human immunodeficiency virus (HIV) may be using the glycosylation system of the T lymphocytes to acquire glycans for its glycoproteins that enable it to disrupt carbohydrate dependent immune cell interactions or induce aberrant immune reactions. Consistent with this hypothesis, gp120 from HIV infected human H9 lymphoblastoid cells expresses biantennary N-linked glycans with a bisecting GlcNAc sequence on 11% of their total oligosaccharides. This specific carbohydrate sequence has recently been shown to protect K562 erythroleukemic cells from natural killer (NK) cell responses when presented on the cell surface. We have recently demonstrated that bisecting biantennary type N-linked glycans are also expressed on the human zona pellucida (ZP); previous lectin binding studies indicate that is also expressed on human spermatozoa. Thus both the human gametes and HIV produced by H9 cells carry this same protective carbohydrate epitope on their outer surfaces. Human alpha-fetoprotein expressed in the developing human also carries the bisecting GlcNAc sequence, indicating that it may be suppressing the emerging fetal immune response by using its carbohydrate sequence as a functional group. We have suggested that the developing human and the gametes are also protected by soluble immunosuppressive glycoproteins found in the amniotic fluid and seminal plasma known as glycodelin-A (GdA) and glycodelin-S (GdS) respectively. Structural analysis of their N-linked oligosaccharides combined with other functional studies suggest that GdA and GdS employ their very unusual carbohydrate sequences as functional groups that enable them to manifest their immunosuppressive activities. GdA and GdS are significant components of our recently proposed model for the protection of the developing human and gametes designated the human fetoembryonic defence system hypothesis. A striking relationship now emerging is that the same unusual carbohydrate sequences associated with these immunosuppressive glycodelins are also specifically expressed on intravascular helminthic parasites, Helicobacter pylori, human tumour cells, and HIV infected T lymphocytes. The information presented in this review suggests that two new corollaries should be added to our recently proposed defence system hypothesis: (i) mimicry or acquisition of glycans that are used in this protective system by pathogens or tumour cells may enable them to either subvert or misdirect the human immune response, thereby greatly increasing their pathogenicity; and (ii) expression of glycoproteins used in this system by normal cells and tissues outside the reproductive system may protect them from immune responses, especially in those cases where major histocompatibility recognition is either absent or minimal. A better understanding of this hypothesis and its corollaries may enable us to address the molecular mechanisms underlying not only AIDS but also a host of other very serious pathological conditions in the human.

Constrained glycopeptide ligands for MPRs. Limitations of unprotected phosphorylated building blocks

A new methodology for the synthesis of cyclic and phosphorylated glycopeptide templates was developed. First, fully protected building blocks containing mannose and mannose disaccharides with bis-trichloroethyl phosphate on Fmoc-Thr-OPfp were synthesized. These were used in solid-phase assembly through side chain anchoring of glycosylated hexa- and octa-peptides protected at the C-terminal car☐ylate as the allyl ester. Selective allyl ester cleavage and head-to-tail cyclization under pseudodilution conditions gave a high yield of pure cyclic peptide templates. Unprotected phosphate in the building block was evaluated as an alternative to the problematic trichloroethyl group. It was found that one unprotected phosphate is readily incorporated, whereas the second unprotected phosphorylated building block react very slowly due to electrostatic repulsion in the solid-phase synthesis. For comparison with previous binding studies modified glycopeptide templates containing only phosphorylated mannose monosaccharides or templates modified in the peptide part were synthesized. All the structures were tested for their binding to the mannose 6-phosphate receptor, and it was found that although mannose disaccharides are required for optimal interaction, the detailed structure of the peptide template has a strong influence on binding to the receptor. The restricted conformations of the cyclic peptides decreased the binding considerably.

Actions of picrodendrin antagonists on dieldrin-sensitive and -resistant Drosophila GABA receptors.

1. A series of terpenoid compounds, recently isolated from Picrodendron baccatum, share a picrotoxane skeleton with picrotoxinin, an antagonist of ionotropic GABA receptors. Referred to as picrodendrins, they inhibit the binding of [35S]-tert-butylbicyclophosphorothionate (TBPS) to rat GABAA receptors. Hitherto, their effects on GABA receptors have not been investigated electrophysiologically. Under two-electrode voltage-clamp, the actions of picrodendrins and related terpenoids have been assayed on homooligomeric GABA receptors formed by the expression of a Drosophila GABA receptor subunit (RDLac) in Xenopus oocytes. 2. All the terpenoids tested, dose-dependently antagonized currents induced by 30 microM (EC50) GABA. 3. Tutin and its analogues (dihydrotutin and isohyenanchin) differ in the structure of their axial C4 substituents. Of these compounds, tutin, which bears an isopropenyl group at this carbon atom, was the most potent antagonist of RDLac homo-oligomers, whereas isohyenanchin, which bears a hydroxyisopropyl group, was the least potent antagonist tested. 4. Picrodendrins differ mainly in the structure of their C9 substituents. The IC50s of picrodendrins ranged from 17 +/- 1.3 nM (picrodendrin-Q) to 1006 +/- 1.3 nM (picrodendrin-O). As such, the most potent picrodendrins (Q, A and B) were approximately equipotent with picrotoxinin as antagonists of RDLac homo-oligomers. 5. Certain picrodendrin compounds effected a use-dependent blockade of RDLac homo-oligomers. Such a biphasic block was not observed with tutin analogues. 6. Picrotoxin-resistant RDLacA3025 homo-oligomers, which have a single amino acid substitution (A302S) in the 2nd transmembrane region, were markedly less sensitive to picrodendrin-O than the wild-type, dieldrin-sensitive, homo-oligomers. 7. The relative potency of tutin analogues demonstrates that the structure-activity relationship of the C4 substituent of picrotoxane-based compounds is conserved in vertebrates and insects. However, the relative order of potency of picrodendrins on RDLac homo-oligomers is distinctly different from that observed in previous radioligand binding studies performed on vertebrate GABAA receptors. As picrodendrin compounds differ in the structure of their C9 substituents, these data suggest that the optimal convulsant pharmacophores of vertebrate GABAA receptors and RDLac homo-oligomers differ with respect to this substituent.

Re-examination of crotoxin-membrane interactions

The interaction of crotoxin with synaptic membranes from Torpedo marmorata has been re-examined, using radioiodinated toxin. In competition experiments, the ‘saturable binding’ is usually calculated by subtracting the non-saturable binding, determined in the presence of an excess of unlabelled crotoxin, from total binding. Paradoxically, we observed a notable increase of the ‘saturable binding’ of 125I-crotoxin, defined in this manner in the presence of a high concentration of certain competitors (e.g. crotoxin subunit B, agkistrodotoxin, ammodytoxin and ammodytin I 2). This potentiation effect was analysed by competition and cross-linking experiments. The dissociation of the basic crotoxin subunit CB, which differs markedly from crotoxin in its membrane- binding characteristics, was found to be the main reason for the observed phenomenon. 125I-CB could be released from 125I-crotoxin by exchange with a molecule having sufficient affinity towards CA (e.g. CB or agkistrodotoxin) or it could be dissociated from the specific crotoxin membrane-binding site(s) by a competitor molecule. Our results, therefore, suggest a reinterpretation of previous 125I-crotoxin binding studies.

In situ hybridization analysis of the distribution of neurokinin-3 mRNA in the rat central nervous system.

The tachykinin family of neuropeptides, which includes substance P, neurokinin A, and neurokinin B, have three distinct receptors: NK-1, NK-2, and NK-3. With the cloning of the rat NK-3 cDNA, it is now possible to evaluate the distribution of NK-3 mRNA in the rat brain. Female rat brains were sectioned and hybridized with a riboprobe complimentary to NK-3 mRNA. The results of these studies revealed an extensive distribution of NK-3 mRNA throughout the rostral-caudal extent of the brain, spinal cord, and retina. In agreement with previous binding studies, we observed NK-3 mRNA in the cortex, the amygdala, the hippocampus, the medial habenula, the zona incerta, the paraventricular and supraoptic nuclei of the hypothalamus, the substantia nigra, the ventral tegmental area, the interpeduncular nucleus, the raphe nuclei, the dorsal tegmental nucleus, and the nucleus of the solitary tract. In contrast with binding data, only a few NK-3 mRNA cells were detected in the striatum. In addition, the present study detected NK-3 mRNA in the olfactory bulb, the dentate gyrus and subiculum, the medial septum, the diagonal band of Broca, the ventral pallidum, the globus pallidus, the bed nucleus of the stria terminalis, the arcuate, the premammillary and mammillary nuclei, the dorsal and lateral regions of the posterior hypothalamus, the central gray, the cerebellum, the parabrachial nuclei, the nucleus of the spinal trigeminal tract, the dorsal horn of the spinal cord, and the retina. The results of these in situ hybridization histochemical studies have provided detailed and novel information about the distribution of NK-3 mRNA and have elucidated the putative sites of neurokinin B action in the rat central nervous system.

In situ hybridization analysis of the distribution of neurokinin‐3 mRNA in the rat central nervous system

The tachykinin family of neuropeptides, which includes substance P, neurokinin A, and neurokinin B, have three distinct receptors; NK-1, NK-2, and NK-3. With the cloning of the rat NK-3 cDNA, it is now possible to evaluate the distribution of NK-3 mRNA in the rat brain. Female rat brains were sectioned and hybridized with a riboprobe complimentary to NK-3 mRNA. The results of these studies revealed an extensive distribution of NK-3 mRNA throughout the rostral-caudal extent of the brain, spinal cord, and retina. In agreement with previous binding studies, we observed NK-3 mRNA in the cortex, the amygdala, the hippocampus, the medial habenula, the zona incerta, the paraventricular and supraoptic nuclei of the hypothalamus, the substantia nigra, the ventral tegmental area, the interpeduncular nucleus, the raphe nuclei, the dorsal tegmental nucleus, and the nucleus of the solitary tract. In contrast with binding data, only a few NK-3 mRNA cells were detected in the striatum. In addition, the present study detected NK-3 mRNA in the olfactory bulb, the dentate gyrus and subiculum, the medial septum, the diagonal band of Broca, the ventral pallidum, the globus pallidus, the bed nucleus of the stria terminalis, the arcuate, the premammillary and mammillary nuclei, the dorsal and lateral regions of the posterior hypothalamus, the central gray, the cerebellum, the parabrachial nuclei, the nucleus of the spinal trigeminal tract, the dorsal horn of the spinal cord, and the retina. The results of these in situ hybridization histochemical studies have provided detailed and novel information about the distribution of NK-3 mRNA and have elucidated the putative sites of neurokinin B action in the rat central nervous system. © 1996 Wiley-Liss, Inc.

Homologous desensitization of human corticotropin-releasing factor, receptor in stable transfected mouse fibroblast cells

Previous radioligand binding and second messenger studies have shown that corticotropin-releasing factor (CRF) modulates its receptor following both in vivo and in vitro treatment. In the present study, we determined the sequence of events leading to CRF-induced downregulation and desensitization of cloned CRF receptors in murine fibroblast cells (Ltk −) stably transfected with CRF, DNA (from human pituitary). Treatment of cells with rat/human CRF produced a dose- and time-dependent decrease in [ 125I]Tyr o-ovine CRF ([ 125I]oCRF) binding and a concomitant decrease in CRF-stimulated adenylate cyclase activity. Significant decreases in [ 125I]oCRF binding and agonist-stimulated cAMP production were evident minutes after CRF treatment with maximal (60–80%) reductions seen following 1 h of CRF treatment. Scatchard analysis revealed that the decrease in [ 125I]oCRF binding was due to the downregulation of the receptor with no significant alteration seen in the affinity of the ligand. Since the transfected cell line is engineered using an artificial promoter, we did not detect any significant changes in CRF 1 receptor mRNA levels following CRF treatment for up to 24 h.

Binding and Hydrolysis of TNP-ATP by Escherichia coli F1-ATPase

It had previously been suggested that Vmax hydrolysis rate of 2', 3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) by F1-ATPase required filling of only two catalytic sites on the enzyme (Grubmeyer, C., and Penefsky, H. S. (1981) J. Biol. Chem. 256, 3718-3727), whereas recently it was shown that Vmax rate of ATP hydrolysis requires that all three catalytic sites are filled (Weber, J., Wilke-Mounts, S., Lee, R. S. F., Grell, E., and Senior, A. E. (1993) J. Biol. Chem. 268, 20126-20133). To resolve this apparent discrepancy, we measured equilibrium binding and hydrolysis of MgTNP-ATP under identical conditions, using betaY331W mutant Escherichia coli F1-ATPase, in which the genetically engineered tryptophan provides a direct fluorescent probe of catalytic site occupancy. We found that MgTNP-ATP hydrolysis at Vmax rate did require filling of all three catalytic sites, but in contrast to the situation with MgATP, "bisite hydrolysis" of MgTNP-ATP amounted to a substantial fraction (approximately 40%) of Vmax. Binding of MgTNP-ATP to the three catalytic sites showed strong binding cooperativity (Kd1 < 1 nm, Kd2 = 23 nm, Kd3 = 1.4 microM). Free TNP-ATP (i.e. in presence of EDTA) bound to all three catalytic sites with lower affinity but was not hydrolyzed. These data emphasize that the presence of Mg2+ is critical for cooperativity of substrate binding, formation of the very high affinity first catalytic site, and hydrolytic activity in F1-ATPases and that these three properties are strongly correlated.

Expression of NK-1 receptor mRNA in murine T lymphocytes.

Substance P (SP), one of the best characterized neurogenic mediators of immune hyperactivity, stimulates the production of a variety of cytokines, and acts as a general proinflammatory agent inducing proliferation and activation of immune cells Neurokinin 1 receptor (NK-1R), a G-protein-coupled receptor with high affinity for SP, has been cloned from nonlymphoid tissues. Although previous binding studies indicated the presence of high affinity SP receptors on immune cells, recent studies questioned the existence of specific NK-1R on lymphocytes and monocytes. In this study we investigate the expression of the NK-1R gene in murine T lymphocytes and T cell lines. The expression of NK-1R mRNA was determined by reverse transcription polymerase chain reaction (RT-PCR), with two separate sets of specific primers, and by nuclease protection assays. The NK-1R nature of the lymphocytic amplified fragments was confirmed by Southern blots with specific murine brain NK-1R probes. Both T cells lines (EL-4.IL-2 and LBRM-T6G) which were previously shown to respond to SP by increased IL-2 production express NK-1R mRNA constitutively. Normal spleen cells and purified CD4+ T lymphocytes which also responded to SP by increased IL-2 production express NK-1R mRNA. In addition to CD4+ T cells, NK-1R message is also expressed in murine splenic B lymphocytes. Sequencing of the RT-PCR amplified fragments from EL-4.IL-2 cells, purified CD4+ T lymphocytes, and murine brain showed complete identity to the published murine NK-1R sequence. This study indicates that murine CD4+ T lymphocytes possess the molecular template for the production of NK-1R. Together with previous binding studies, and with pharmacological studies regarding the effect of SP and of selective NK-1R antagonists on cytokine production, the present study supports the hypothesis that the response of CD4+ T cells to SP and related tachykinins is mediated through specific NK-1 receptors.