TCP Binding Research Articles

Guanine nucleotide modulation of [ 3H]TCP binding to the NMDA receptor complex

Guanine nucleotides have been examined for their effect on [ 3H]-[1-(2-thienyl)-cyclohexyl]-piperidine ([ 3H]TCP) binding to rat forebrain synaptic plasma membranes (SPM). We report that of the series of guanine nucleotides tested, GTP, GDP, 5′-guanylylimidodiphosphate (Gpp(NH)p) and 5′-guanylylmethylenediphosphate (Gpp(CH 2)p) are significantly more potent at decreasing [ 3H]TCA binding than GMP, cyclic GMP, and guanosine. GTP, the most potent compound tested, inhibited basal [ 3H]TCP binding with an IC 50 of 38.7 μM. Stimulation of [ 3H]TCP binding with either the N-methyl-D-aspartate (NMDA) agonist, L-glutamate, or Mg 2+ was also inhibited by GTP. Addition of GTP resulted in a rightward shift in the glutamate dose-response curve and a decrease in the maximum level of stimulation. The Mg 2+ stimulation of [ 3H]TCP binding was completely blocked by the addition of GTP. These results, coupled with the previous findings that guanine nucleotides inhibit the binding of L-[ 3H]glutamate to the NMDA recognition site (Monahan et al., 1988), indicate that guanine nucleotides antagonize NMDA receptor-mediated neurotransmission, at least in part, through their action (direct or indirect) on the NMDA recognition site and thus may be endogenous negative modulators of the NMDA receptor.

Identification of a domain in cytochrome C that displaces [ 3H]TCP binding from rat brain membrane receptors: Synthesis of β-neuroprotectin

1. 1. A stereospecific radioreceptor binding assay for the phencyclidine analogue, [ 3H]TCP, was utilized to screen for inhibition of binding in extracts of rat brain. 2. 2. Extracts were prepared from rat cerebral cortex and hippocampus by methods employing aqueous acid. The extracts were fractionated by reversed phase-HPLC (RP-HPLC) and tested for activity in the radioreceptor assay. Three zones of activity were detected. The middle zone was further purified by high performance-size exclusion chromatography (HP-SEC). 3. 3. Size exclusion chromatography revealed a single zone of activity corresponding to mol. wts of ca 12,000–31,000 daltons. A fraction from this zone was digested with trypsin, and the resulting enzyme fragments, isolated by a combination of HP-SEC and RP-HPLC, were identified as fragments of rat cytochrome C. 4. 4. Horse cytochrome C was digested with trypsin and the fragments were similarly purified on the basis of the [ 3H]TCP binding displacement assay. The fragments were sequenced and found to be trypsin cleavage products of a single largely invariant domain of the cytochrome C molecule: Lys-Lys-Lys-Asp-Glu-Arg-Ala-Asp-Leu-Ile-Ala-Tyr-Leu-Lys-Lys. 5. 5. β-neuroprotectin ( d)-Ala-Asp-Leu-Ile-Ala-Tyr-Leu-NH 2, inhibits [ 3H]TCP binding and provides protection against NMDA mediated neuronal cell death at low concentrations.

Isolation and identification of a peptide from rat brain which inhibits [ 3H]TCP binding

1. 1. A stereospecific radioreceptor binding assay for the phencyclidine analogue [ 3H]TCP was utilized to screen for inhibitors of binding in extracts of rat brain. 2. 2. Extracts were prepared from rat cortex and hippocampus by methods employing aqueous acid or acidified methanol. Samples were fractionated by reversed phase-HPLC (RP-HPLC) and tested for activity in the radioreceptor assay. Three zones of activity were detected. The most active fraction was further purified by high performance-size exclusion chromatography. 3. 3. Size exclusion chromatography revealed two zones of activity, corresponding to mol. wts of 4000–8000 Da and 1000–2000 Da. Final purification of the lower molecular weight material was achieved by RP-HPLC. 4. 4. Two well-separated peaks were shown to be homogeneous. Their amino acid sequences were determined by automated Edman degradation and data base searching identified these two peaks as the undecapeptide Substance P and its oxidized counterpart (Substance P sulfoxide). 5. 5. Comparative HPLC of synthetic Substance P, or its sulfoxide, as well as spectral analysis confirmed the identity of the isolated peptides. 6. 6. Synthetic Substance P inhibits specific [ 3H]TCP binding in the radioreceptor assay.

Alterations in rat brain [ 3H]-TCP binding following chronic phencyclidine administration

Rats were chronically infused with phencyclidine (PCP, 13.3 mg PCP·HCl/kg/day) or saline, s.c., for 10 days using osmotic minipumps (n = 5 for each group). Twenty-four hours after the cessation of dosing, the rats were sacrificed, and brains were removed for analysis of PCP receptor binding. Saturation studies of the binding of [ 3H]-TCP to brain homogenates revealed statistically significant increases in the maximum binding capacity ( B max) and decreases in the affinity for [ 3H]-TCP in the PCP-treated group.

Ligand-binding characteristics of [ 3H]QNB, [ 3H]prazosin, [ 3H]rauwolscine, [ 3H]TCP and [ 3H]nitrendipine to cerebral cortical and hippocampal membranes of senescence accelerated mouse

The senescence accelerated mouse (SAM) is known as a murine model of aging and memory dysfunction. In the cerebral cortical membranes of male 9-month-old SAM mice, the B max values of [ 3H]rauwolscine and [ 3H]nitrendipine binding, and the values of both K d and B max of [ 3H]TCP binding in the accelerated aging strain SAM-P 8 were significantly increased compared with the values in the control strain SAM-R 1 . In hippocampal membranes, however, the B max values of [ 3H]quinuclidinyl benzilate and [ 3H]nitrendipine binding were significantly decreased in SAM-P 8 compared with those in SAM-R 1 . These results suggest that muscarinic acetylcholine receptors, α 2-adrenoceptors, N- methyl- d-aspartate receptor channels and L-type Ca 2+ channels are changed in cerebral cortex and hippocampus in SAM-P 8 at 9 months.

Embryonic and postnatal development of [formula omitted] binding sites in rat forebrain homogenates and slices

The development of N-(1-[2- thienyl]-cyclohexyl)[ 3 H]piperidine ([ 3H]TCP) binding to phencyclidine (PCP) receptors in both brain homogenates and slices has been investigated in the rat. The specific binding sites for [ 3H]TCP in the homogenate were already detected at prenatal stages and steadily increased after birth. A similar developmental pattern was seen in the autoradiography of the [ 3H]TCP binding to the brain slice in which the distribution of the binding in the young is more homogeneous than that in adult. There was an increase in the B max without changes in the K d of the [ 3H]TCP binding and there was no change in inhibition of the binding by TCP, PCP and d-(−)-2-amino-5-phosphonovalerate during postnatal maturation. These findings suggest an increase in the density with no change in the affinity of PCP receptors and the absence of a change in the interaction between the PCP and N- methyl- d-aspartate receptors in the developing rat forebrain.

Effects of amygdaloid kindling on NMDA receptor function and regulation

These studies were conducted to determine whether amygdaloid kindling results in the long-term alteration of NMDA receptors which could explain the persistent reduction in seizure threshold seen in this phenomenon. NMDA-induced [ 3H]norepinephrine (NE) release, NMDA-sensitive l-[ 3H]glutamate binding, and NMDA and glycine-enhanced [ 3H]TCP binding were measured in brain tissue from kindled rats and nonstimulated control rats 3 to 6 weeks after the last seizure. There was no difference in the ability of NMDA to induce [ 3H]NE release from kindled or control slices of amygdala or hippocampus. There was also no difference in the ability of phencyclidine (PCP) or Mg 2+ to inhibit [ 3H]NE release induced by 100 μ M NMDA. Equilibrium saturation experiments of NMDA-sensitive l-[ 3H]glutamate binding revealed no differences in K D or B max values between control and kindled cortex, amygdala, and hippocampus. The K i values for NMDA displacement of l-[ 3H]glutamate binding also did not differ in kindled tissue. NMDA-enhanced [ 3H]TCP binding was similar in cortex, amygdala, and hippocampus of kindled and control tissues. Finally, glycine-enhanced [ 3H]TCP binding was not different in control or kindled tissues. These studies suggest that the NMDA recognition site and the modulation of the NMDA receptor/ion channel complex by magnesium, PCP, and glycine are not altered several weeks after the last seizure. Even though NMDA-mediated electrophysiological responses are reportedly enhanced in kindled tissue at that time, the mechanism(s) underlying the enhancement remains to be determined.

Frontal decortication decreases the affinity ofN-(1-[2-thienyl]cyclohexyl)[ 3H]piperidine binding to rat striatum Frontal decortication; Striatum

Bilateral ablation or transection of the corticostriatal pathways of the rat causede a reduction inN-(1-[2-thienyl]cyclohexyl)-[ 3H]piperidine ([ 3H]TCP) binding to the striatum at 5–7, but not 3 and 14–28, days postlesion with a persistent decrease in striatal glutamate content 3–28 days after the decortication. This reduction was found to be due to an increase inK d without changes inB max of the [ 3H]TCP binding. The present binding suggest that interruption of striatal glutamatergic transmission following frontal decortication may produce a temporal reduction in the affinity of phencyclidine receptor in th striatum.

Glutamine mimics glycine to enhance [ 3H]TCP binding at the NMDA receptor complex

Investigation of the molecular site of L- and D-glutamine effects at the N-methyl-D-aspartate complex in neuronal membranes of rats by use of [ 3 H]TCP, N-(1-[2-thenyl]-cyclohexyl)-3,4-piperidine, binding

N-methyl-D-aspartate receptor plasticity in kindling: quantitative and qualitative alterations in the N-methyl-D-aspartate receptor-channel complex.

Kindling is an animal model of epilepsy and neuronal plasticity produced by periodic electrical stimulation of the brain. Electrophysiologic studies indicate that this phenomenon is associated with increased participation of N-methyl-D-aspartate (NMDA) receptors in excitatory synaptic transmission. Biochemical studies suggest that a change intrinsic to the NMDA receptor-channel complex may contribute to the increase in NMDA receptor-mediated synaptic transmission. We tested this idea by measuring the binding of 3-[(+)-2-(carboxypiperazin-4-yl)][1,2-3H]propyl-1-phosphonic acid ([3H]CPP), [3H]glycine, and tritiated N-[(1-thienyl)cyclohexyl]piperidine [( 3H]TCP) to rat hippocampal membranes. In this preparation these ligands are selective for the NMDA receptor, the strychnine-insensitive glycine receptor, and the NMDA receptor-gated ion channel, respectively. Kindling increased the density of CPP, glycine, and TCP binding sites in hippocampal membranes by 47%, 42%, and 25%, respectively. No significant changes were detected in the affinity of these binding sites. Surprisingly, alterations in the glycine binding site were detected in animals sacrificed 1 month but not 1 day after the final kindling stimulation. Thus, delayed upregulation of the NMDA receptor-channel complex may be one molecular mechanism that maintains the long-lasting hyperexcitability of hippocampal neurons in kindled animals.

Characterization df a [3H]Glycine Recognition Site as a Modulatory Site of the N‐Methyl‐D‐Aspartate Receptor Complex

A [3H]glycine recognition site in rat brain synaptic plasma membranes (SPM) has been identified, having characteristics expected of a modulatory component of the N-methyl-D-aspartate receptor complex. Incubation of SPM with [3H]glycine for 10 min at 2 degrees C results in saturable, reversible binding with a KD of 0.234 microM and a Bmax of 9.18 pmol/mg. A pharmacological analysis of this binding site indicates that D-serine (Ki = 0.27 microM), D-alanine (Ki = 1.02 microM), and D-cycloserine (Ki = 2.33 microM) are potent inhibitors of binding, whereas the corresponding L isomers have significantly less activity (Ki = 25.4 microM, 15.9 microM, and greater than 100 microM, respectively). Inactive at concentrations of up to 100 microM were strychnine, L-valine, N,N-dimethylglycine, aminomethylphosphonate, and aminomethylsulfonate. The active compounds were analyzed further for their ability to stimulate [3H]1-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) binding to Triton X-100-washed SPM. Results indicate that the affinity of the compounds for the [3H]glycine recognition site correlates with the ability of these analogues to stimulate [3H]TCP binding.

Molecular target size analyses of the NMDA-receptor complex in rat cortex

The molecular weights of different subunits of the NMDA-receptor complex were determined by high-energy radiation inactivation analyses of the binding of [ 3H]L-glutamate, [ 3 H](3-(±)-2-( carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), [ 3H]N-(1-[2-thienyl]cyclohexyl)3,4-piperidine (TCP) and [ 3H]glycine to rat cortical membranes. The molecular target sizes of [ 3H]L-glutamate binding (the recognition site), [ 3H]TCP binding (the ionophore) and [ 3H]glycine (a modulatory unit) were similar: 121 000, 118 000 and 115 000 Da, respectively. These results suggest that the three subunits are on the same protein. The molecular weigth of [ 3H]CPP binding was 209 000 Da. This suggests that in order to bind [ 3H]CPP (a competitive antagonist) with high affinity an additional macrmolecule may be associated to the agonist site.

Ifenprodil and SL 82.0715 are antagonists at the polyamine site of the N-methyl-D-aspartate (NMDA) receptor

In the present study, we confirm that spermidine increases [ 3 H]TCP binding (in the presence of glutamate) to well washed rat brain membranes, and show that this effect is potently antagonised by ifenprodil and SL 82.0715 at concentrations that themselves have no effect on [ 3 H]TCP binding in the absence of spermidine

Expression of (+)-3-PPP binding sites in the PC12 pheochromocytoma cell line

Phencyclidine binds with high affinity to both PCP and σ receptors. We investigated whether the clonal cell line PC12 expressed either of these receptors, and found that these cells contain a haloperidol-sensitive (+)-[ 3H]3-PPP binding site with a K D of 56 nM, but no PCP binding sites. The (+)3-PPP binding sites in PC12 cells displayed a reversed stereoselectivity for the benzomorphan opiates compared to CNS σ receptors. Neither nerve growth factor nor sodium butyrate treatment affected the expression of either (+)-3-PPP or TCP binding sites in PC12 cells.

A Glycine Site Associated with N‐Methyl‐d‐Aspartic Acid Receptors: Characterization and Identification of a New Class of Antagonists

Membranes from rat telencephalon contain a single class of strychnine-insensitive glycine sites. That these sites are associated with N-methyl-D-aspartic acid (NMDA) receptors is indicated by the observations that [3H]glycine binding is selectively modulated by NMDA receptor ligands and, conversely, that several amino acids interacting with the glycine sites increase [3H]N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding to the phencyclidine site of the NMDA receptor. The endogenous compound kynurenate and several related quinoline and quinoxaline derivatives inhibit glycine binding with affinities that are much higher than their affinities for glutamate binding sites. In contrast to glycine, kynurenate-type compounds inhibit [3H]TCP binding and thus are suggested to form a novel class of antagonists of the NMDA receptor acting through the glycine site. These results suggest the existence of a dual and opposite modulation of NMDA receptors by endogenous ligands.

Anaesthetic properties of phencyclidine (PCP) and analogues may be related to their interaction with Na + channels

Among other properties, phencyclidine (PCP) and analogues display anaesthetic and anticonvulsant properties. Interaction of PCP and some analogues with the voltage-sensitive Na + channels have been investigated and compared with their interaction with the PCP receptor. PCP and TCP inhibit apparently in a competitive manner the veratridine stimulated 22Na + synaptosomal uptake with K i values of 8.6 and 12.7 μM, respectively, close to those obtained in the inhibition of [ 3H]BTX-B binding (IC 50 = 4.1 and 3.8 μM, respectively). The specific [ 3H]TCP binding to synaptosomes in ionic near physiological conditions is inhibited by PCP and TCP with IC 50 values of 1.25 and 0.29 μM, respectively. Other PCP derivatives (GK3 and GK4) and PCP-like drugs (ketamine and MK801) inhibit 22Na + uptake in an order of potency (GK3 > GK4 > PCP > TCP > MK801 > ketamine) which is different from that obtained in the inhibition of [ 3H]TCP binding (MK801 > TCP > PCP > ketamine > GK4 > GK3). Ketamine inhibits the veratridine-stimulated Na + uptake at a concentration where its anesthetic effect occurs. It was concluded that the interaction of these drugs with the Na + channel may reflect their anaesthetic properties while the interaction with the PCP receptors may be mainly related to their anticonvulsant and ataxic properties.

D-Cycloserine: A ligand for the coupled glycine receptor has partial agonist characteristics

We have previously shown that D-cycloserine displaces [3H]glycine binding to a recognition site with properties consistent with an N-methyl-D-aspartate (NMDA) receptor modulatory site. Additionally, D-cycloserine positively modulates the NMDA receptor as evidenced by its dose-dependent enhancement of [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding to the NMDA receptor-coupled ionophore. Further evaluation of this compound indicates that the maximal stimulation of [3H]TCP binding induced by D-cycloserine is lower than that produced by other compounds acting at the NMDA receptor associated glycine modulatory site (glycine and D-serine). Moreover, the stimulation of [3H]TCP binding induced by D-cycloserine in the presence of various fixed concentrations of glycine results in a family of dose-response curves which asymptotically converge to 40-50% of the maximal stimulation induced by glycine alone. These results are consistent with D-cycloserine acting as a partial agonist of the NMDA receptor via its interaction with the coupled glycine modulatory site.

Solubilization of phencyclidine receptors from rat cerebral cortex in an active ligand binding state

A phencyclidine (PCP) receptor binding site has been solubilized in an active ligand-binding state from rat cerebral cortical membranes with sodium deoxycholate. Optimal receptor solubilization occurs at a detergent/protein ratio of 0.5 (w/w); for 5 mg protein/ml solubilized with 0.25% sodium deoxycholate, about 60% of the protein and 25% of the receptor is solubilized. Specific binding of either [ 3H]-N-[1-(2-thienyl) cyclohexyl]piperidine ([ 3H]TCP) or [ 3H]MK-801 is measurable by filtration through Sephadex G-50 columns or glass fiber filters; more than 60% of the binding activity is stable after 48 h at 4°C. In the presence of detergent, [ 3H]TCP binding exhibits a K d of 250 nM, a B max of 0.56 pmol/mg protein, and a pharmacological profile consistent with that of the membrane-bound PCP receptor, although most drugs bind with affinities 2 to 8 fold lower than in membranes. Upon reduction of detergent concentration, binding parameters approximate those for the membrane-bound receptor ([ 3H]TCP binding: K d = 48 nM, B max = 1.13 pmol/mg protein).

Comparison of [ 3H] Phencyclidine ([ 3H] PCP) and [ 3H] N- [1 - (2-thienyl) cyclohexyl] Piperidine ([ 3H] TCP) binding properties to rat and human brain membranes.

The investigation of [ 3H] PCP and [ 3H] TCP binding properties to rat cerebrum and cerebellum resulted in the demonstration of multiple binding sites for the two drugs. In the two tissue preparations PCP had a lower affinity than TCP. In membranes from the cerebrum an equal number of high affinity binding sites were present for [ 3H] PCP and [ 3H] TCP. However, low affinity binding sites were two times more numerous for [ 3H] PCP than for [ 3H] TCP. In the cerebellum, the number of high and low affinity sites labeled by the two radioligands was identical, but the number of high affinity sites was about 7 fold lower than in the cerebrum. Taken together these results may indicate that in the cerebrum [ 3H] PCP labels other sites than NMDA/PCP receptor(s), maybe sigma receptors and/or the dopamine uptake complex. In human cerebral cortex samples [ 3H] TCP also bound to two different sites. The number of high and low affinity sites were 12 and 3 times, respectively, less abundant than in the rat cerebrum. Low affinity sites were of higher affinity (5 times) than corresponding sites in the rat brain. In the human cerebellum [ 3H] TCP binding parameters were identical to those measured in the same region in the rat.

P-408 - Enhancement by glutamate and glycine of [3H]TCP binding activity

P-408 - Enhancement by glutamate and glycine of [3H]TCP binding activity