Polyamine analogue regulation of NMDA MK-801 binding: a structure-activity study

A series of analogues and homologues of spermine were synthesized, and their impact on MK-801 binding to the N-methyl-D-aspartate (NMDA) receptor was evaluated. These tetraamines encompass both linear and cyclic compounds. The linear molecules include norspermine, N1, N11-diethylnorspermine, N1,N12-bis(2,2,2-trifluoroethyl)spermine, homospermine, and N1,N14-diethylhomospermine. The cyclic tetraamines consist of the piperidine analogues N1,N3-bis(4-piperidinyl)-1,3-diaminopropane, N1,N4-bis(4-piperidinyl)-1,4-diaminobutane, N1,N4-bis(4-piperidinylmethyl)-1,4-diaminobutane, and N1,N4-bis[2-(4-piperidinyl)ethyl]-1,4-diaminobutane and the pyridine analogues N1,N3-bis(4-pyridyl)-1,3-diaminopropane, N1,N4-bis(4-pyridyl)-1,4-diaminobutane, N1,N4-bis(4-pyridylmethyl)-1,4-diaminobutane, and N1,N4-bis[2-(4-pyridyl)-ethyl]-1,4-diaminobutane. This structure-activity set makes it possible to establish the importance of charge, intercharge distance, and terminal nitrogen substitution on polyamine-regulated MK-801 binding in the NMDA channel. Four families of tetraamines are included in this set: norspermines, spermines, homospermines, and tetraazaoctadecanes. Calculations employing a SYBYL modeling program revealed that the distance between terminal nitrogens ranges between 12.62 and 19.61 A. The tetraamines are constructed such that within families cyclics and acyclics have similar lengths but different nitrogen pKa's and thus different protonation, or charge, states at physiological pH. The pKa values for all nitrogens of each molecule and its protonation state at physiological pH are described. The modifications at the terminal nitrogens include introduction of ethyl and beta,beta,beta-trifluoroethyl groups and incorporation into piperidinyl or pyridyl systems. The studies clearly indicate that polyamine length, charge, and terminal nitrogen substitution have a significant effect on how the tetraamine regulates MK-801 binding to the NMDA receptor. Thus a structure-activity basis set on which future design of MK-801 agonists and antagonists can be based is now available.     

Modulation of [3H]quinpirole binding to dopaminergic receptors by adenosine A2A receptors

Alteration of ligand binding to dopamine D2 receptors through activation of adenosine A2A receptors in rat striatal membranes has been studied by means of kinetic analysis. The binding of dopaminergic agonist [3H]quinpirole to rat striatal membranes was characterized by the constants Kd = 1.50+/-0.09 nM and Bmax = 115+/-2 fmol/mg of protein. The kinetic analyses revealed that the binding had at least two consecutive and kinetically distinguishable steps, the fast equilibrium of complex formation between receptor and agonist (KA = 5.9+/-1.7 nM), followed by a slow isomerization equilibrium (Ki = 0.06). Activation of adenosine A2A receptors by CGS 21680 caused enhancement of the rate [3H]quinpirole binding, altering mainly the formation of the receptor-ligand complexes (KA) as well as the isomerization rate of this complexes (ki), while the deisomerization rate (k[-i]) and the apparent dissociation rate remained unchanged.     

Neurosteroid modulation of [3H]flunitrazepam binding in the medulla: an autoradiographic study

Neurosteroids bind to unique sites on the GABA(A) receptor complex and modulate receptor function. The effects of neurosteroids on GABA(A) receptors have been well characterized in forebrain regions. However, little is known about their effects on GABA(A) receptors in the medulla, especially those areas involved in autonomic reflex pathways. Stimulation of [3H]flunitrazepam binding to the GABA(A) receptor by two progesterone metabolites, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha-OH-DHP) and 3beta-hydroxy-5alpha-pregnan-20-one (3beta-OH-DHP), was studied using autoradiographic methods in the medulla and cerebellum of female rats at estrus. [3H]Flunitrazepam binding was enhanced by 3alpha-OH-DHP in every nucleus examined in the medulla and cerebellum. This effect was stereoselective since 3beta-OH-DHP had no effect on binding in any region. No differences were observed in the degree of stimulation of [3H]flunitrazepam binding by 3alpha-OH-DHP among medullary brain regions. However, in the cerebellum, the stimulation of binding was significantly greater in the granular layer than in the molecular layer. Stimulation of [3H]flunitrazepam binding by 3alpha-OH-DHP in nuclei involved in the baroreflex pathways supports previous studies which report that neurosteroids modulate autonomic regulation of blood pressure. These actions may also underlie alterations in autonomic function during pregnancy.     

Characterization of [3H]clozapine binding sites in rat brain

We examined the characteristics of [3H]clozapine binding sites in four rat brain regions (frontal cortex, limbic area, hippocampus and striatum) in order to elucidate the pharmacological profile of this unique atypical antipsychotic drug. The specific [3H]clozapine binding was found to be saturable and reversible in all these brain regions. Scatchard analysis of the saturation data indicated that the specific binding consisted of high- and low-affinity components. Displacement experiments showed that the muscarinic cholinergic receptor represented about 50% of [3H]clozapine binding in each brain area. Serotonin 5-HT2 and dopamine D4 receptor binding sites could also be detected by displacement experiments using ketanserin and nemonapride, respectively, in frontal cortex and limbic area, but not in hippocampus or striatum. Alpha-1, alpha-2, histamine H1, dopamine D1, D2, or D3 receptor components could not be determined within the high-affinity [3H]clozapine binding sites in any brain region. It is possible that the atypical property of clozapine may depend on the modulatory effect on dopaminergic function via 5-HT2 receptor blockade and/or may be mediated via D4 receptor blockade in the mesocortical and mesolimbic area.     

Autoradiographic study on [3H]-[D-Ala2]-deltorphin-I binding sites in the rat brain

Previous biochemical and pharmacological studies have shown that [D-Ala2]-deltorphin-I (DADTI) has a high affinity and selectivity for delta-opioid receptors. In this study, designed to provide morphological details, the distribution of DADTI binding sites was examined by autoradiography on coronal, sagittal and horizontal frozen sections of adult rat brain. The sections were incubated with tritiated DADTI solution and exposed for 12 weeks to a 3H-sensitive film. DADTI labelling clearly demonstrated selective and high affinity binding sites of delta-opioid type in several brain regions, including olfactory system, neostriatum, nucleus accumbens, and cortical layers I-II and V-VI.     

Selective in vivo binding of [3H]naltriben to delta-opioid receptors in mouse brain

Naltriben (NTB) is a selective antagonist for the putative delta2-opioid receptor. We have determined the regional kinetics and pharmacological profile of [3H]naltriben in vivo in mouse brain. After i.v. administration to CD1 mice, [3H]naltriben uptake and retention were high in striatum, cortical regions and olfactory tubercles, and low in superior colliculi and cerebellum. Robust rank order correlation was found between [3H]naltriben uptake in discrete brain regions and prior delta-opioid receptor binding determinations in vitro and in vivo. [3H]Naltriben binding in vivo was saturable, and was blocked by the delta-opioid receptor antagonist naltrindole, but not by the mu-opioid receptor antagonist cyprodime or the K-opioid receptor agonist (trans)-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]ben zeneacetamide mesylate (U50,488H). (E)-7-Benzylidenenaltrexone (BNTX), a selective antagonist for the putative delta1-opioid receptor, was 9.6- to 12.9-fold less potent than naltriben as an inhibitor of [3H]naltriben binding. Thus, the sites labeled by [3H]naltriben in vivo may correspond to the delta2-opioid receptor subtype. Such assignment is not definitive, particularly considering the 4-fold higher brain uptake of naltriben as compared to (E)-7-benzylidenenaltrexone. Moreover, the regional distribution of [3H]naltriben in brains from CXB-7/BY (CXBK) mice, a strain that shows supraspinal delta1- but not delta2-opioid receptor agonist effects, was quite similar to that found for CD1 mice.     

Age-related changes of sodium-dependent D-[3H]aspartate and [3H]FK506 binding in rat brain

We investigated age-related changes in excitatory amino acid transport sites and FK506 binding protein (FKBP) in 3-week-, and 6-, 12-, 18- and 24-month-old Fischer 344 rat brains using receptor autoradiography. Sodium-dependent D-[3H]aspartate and [3H]FK506 were used to label excitatory amino acid transport sites and immunophilin (FKBP), respectively. In immature rats (3-week-old), sodium-dependent D-[3H]aspartate binding was lower in the frontal cortex, parietal cortex, striatum, nucleus accumbens, whole hippocampus, thalamus and cerebellum as compared to adult animals (6-month-old), whereas [3H]FK506 binding was significantly lower in only the hippocampus, thalamus and cerebellum. 3[H]FK506 binding exhibited no significant change in the brain regions examined during aging. However, sodium-dependent D-[3H]aspartate binding showed a conspicuous reduction in the substantia nigra in 18-month-old rats. Thereafter, a significant reduction in sodium-dependent D-[3H]aspartate binding was found in the thalamus, substantia nigra and cerebellum in 24-month-old rats. Other regions also showed about 10-25% reduction in sodium-dependent D-[3H]aspartate binding. The results indicate that excitatory amino acid transport sites are more susceptible to aging process than immunophilin. Further, our findings demonstrate the conspicuous differences in the developmental pattern between excitatory amino acid transport sites and immunophilin in immature rat brain.     

Dopamine transporter development in postnatal rat striatum: an autoradiographic study with [3H]WIN 35,428

The dopamine transporter mediates the reinforcing effects of cocaine, thus playing a central role in human cocaine addiction, and perhaps providing the mechanism for inducing the effects of prenatal cocaine exposure. This possibility has stimulated growing interest in the normal and abnormal development of this transporter. [3H]WIN 35,428 is a cocaine analog that is useful for studying the distribution and density of the dopamine transporter in striatum and other brain regions. The postnatal development of the dopamine transporter in the rat striatum was measured by quantitative autoradiography with [3H]WIN 35,428. Dopamine transporter levels were low at birth, increased through day 15, followed by much more rapid growth in late postnatal development. The majority of the transporter sites appeared after day 15. Lateral to medial and anterior to posterior gradients in transporter density were established early during development, and there was also an early concentration of transporter in striosomes that became difficult to identify by day 15. Differences between the developmental patterns described here and studies using other ligands for the dopamine transporter suggest there are significant differences in the transporter binding sites for these drugs. These differences in transporter ligand binding characteristics may reflect developmental changes in post-translational modification of the transporter and/or changes in the functional activity rather than simply the presence of the transporter.     

Spermine inhibits [3H]glycine binding at the NMDA receptors from plexiform layers of chick retina

Saturable specific binding of glycine to synaptosomal membranes from plexiform layers of the retina has been described, which seems to correspond to the modulatory site on NMDA-receptors (26). Spermine inhibited specific [3H]glycine binding to membranes from synaptosomal fractions from the outer (P1) and the inner (P2) plexiform layers of 1-3 day-old chick retinas in a dose-dependent manner with an IC50 = 35 microM for the P1 fraction and 32 microM for the P2 fraction. Kinetic experiments and non-linear regression analysis of [3H]glycine-specific binding showed a Kd approximately 100-150 nM in both fractions, and a higher Bmax (4.11 +/- 0.47 pmol/mg protein) for the inner plexiform layer compared to the outer plexiform layer (Bmax = 2.76 +/- 0.25 pmol/mg protein). Strychnine-insensitive [3H]glycine binding was inhibited by 100 microM spermine, due to a reduction in Bmax (P1 = 0.84 +/- 0.16 pmol/mg protein; P2 = 0.81 +/- 0.16 pmol/mg protein) without affecting the Kd. Association and dissociation constants in the absence and presence of 50 microM spermine remained unchanged. Results demonstrate the presence of a single modulatory site for spermine on NMDA receptors, in both synaptic layers of the chick retina.     

Differential binding of [3H]MK-801 to brain regions and spinal cord of mice treated chronically with morphine

Motor and sensory nerve conduction velocity, H-reflex and F-response have been studied in the age group showing maximum changes i.e. neonates and infants. The nerve conduction velocity in upper and lower limbs was 25 M/S and 23.75 M/S respectively in neonate age group; 34.4 M/S and 32.4 M/S respectively in infant group. A significant relationship of age with nerve conduction parameters (velocity, terminal latency) has been observed in infants group but not so in neonate group. H-reflex (late response) was elicited in both Abductor Pollicis Brevis and Soleus. It was present in small muscles of hand (i.e. APB) in all the neonates and 55% of the infants only. This could be attributed to immaturity of nervous system. However, in the lower limb, H-reflex could be elicited in 100% of infants and neonates. In the present study, the relationship of age and height with different nerve conduction parameters as well as H-reflex (latency) has been highlighted.     

Differences in NMDA receptor antagonist-induced locomotor activity and [3H]MK-801 binding sites in short-sleep and long-sleep mice

Short-Sleep (SS) and Long-Sleep (LS) mice differ in initial sensitivity to ethanol. Ethanol acts as an antagonist at N-methyl D-aspartate receptors (NMDARs). Therefore, we tested whether SS and LS mice also differ in initial sensitivity to NMDAR antagonists. Systemic injection (intraperitoneal) of either the noncompetitive NMDAR antagonist MK-801 (dizocilpine) or the competitive NMDAR antagonist 2-carboxypiperazin-4-yl-propyl-1-phosphonic acid (CPP) produced similar results. At lower drug doses, SS mice showed greater locomotor activation than LS mice; and at higher doses, SS mice continued to be activated whereas LS mice became sedated. Brain levels of [3H]MK-801 were 40% higher in SS, compared with LS, mice. However, blood levels of [3H]MK-801 and [3H]CPP and brain levels of [3H]CPP were similar in the two lines. NMDARs were measured using quantitative autoradiographic analysis of in vitro [3H]MK-801 binding to SS and LS mouse brains. Significantly higher (20 to 30%) receptor densities were observed in the hippocampus and cerebral cortex of SS mice. Our results support the hypothesis that SS and LS mice differ in initial sensitivity to NMDAR antagonists and suggest that the line differences in the dose-response relationships for MK-801- and CPP-induced locomotor activity are qualitatively similar to those reported for ethanol. Differences in pharmacokinetics and number of NMDARs may contribute to, but are unlikely to entirely account for, the differential behavioral responsiveness of SS and LS mice to MK-801 and CPP.     

Pharmacology and subcellular distribution of [3H]rilmenidine binding sites in rat brain

We have previously reported that in rat brain membranes, [3H]rilmenidine, in addition to labelling alpha2-adrenoceptors and the I2B-subtype of imidazoline receptor binding site (I2B-RBS), may label an additional I-RBS population, distinct from previously classified I1-RBS and I2-RBS. In this study, using crude or fractionated rat brain membranes we examined the possible association of [3H]rilmenidine-labelled I-RBS with the A- and B-isoforms of monoamine oxidase (MAO) by studying the inhibition of [3H]rilmenidine binding by a number of MAO inhibitors; and comparing the maximal binding density (Bmax) and subcellular distribution of [3H]rilmenidine binding sites with that of MAO-A and MAO-B catalytic sites labelled by [3H]RO41-1049 and [3H]RO19-6327 and 12-RBS labelled by [3H]2-BFI. Inhibition of [3H]rilmenidine binding by all MAO inhibitors tested produced very shallow curves (slope 0.29-0.56). Clorgyline and moclobemide (selective MAO-A inhibitors) displayed moderate affinities (60-140 nM), while pargyline (non-selective MAO-inhibitor), RO41-1049 (selective MAO-A inhibitor) and RO19-6327 (selective MAO-B inhibitor) exhibited very low affinities (> 2 microM) for 50-75% of [3H]rilmenidine-labelled I-RBS in crude brain membranes and even lower affinity for the remaining binding. Under identical buffer conditions, the Bmax of [3H]rilmenidine-labelled I-RBS (1.45+/-0.14 pmol/mg protein) was considerably lower than those of MAO-A (13.10+/-0.15 pmol/mg) and MAO-B (10.35+/-0.50 pmol/mg) sites. These results suggest that [3H]rilmenidine does not interact directly with the active catalytic site of either MAO enzyme and could at best only associate with a subpopulation of MAO molecules. Binding studies on five fractions of rat cortex homogenates-nuclear (N), heavy (M) and light (L) mitochondrial, microsomal non-mitochondrial (P), and soluble cytosolic (S) fractions-revealed that 45% of total [3H]rilmenidine binding was present in the P fraction cf. 20 and 23% in the M and L fractions, in contrast to [3H]RO19-6327 and [3H]2-BFI which bound 11-13% in the P fraction and 36-38% and 35-44% in the M and L fractions, respectively. Binding of all ligands in the N fraction was 6-15% of total. These studies reveal that [3H]rilmenidine-labelled I-RBS, unlike the I2-RBS, are not predominantly associated with mitochondrial fractions containing the MAO enzymes (and cytochrome oxidase activity), but appear to be distributed in both the mitochondrial and plasma membrane fractions in rat cerebral cortex.     

3H]Nicotine binding in rat brain: alteration after chronic acetylcholinesterase inhibition

(+/-)-[3H]Nicotine binds specifically to rat brain membranes. The binding is stereospecific, (+)-nicotine being 57 times less potent than (-)-nicotine in displacing labeled (+/-)-nicotine. Saturation binding experiments revealed the presence of two binding sites with dissociation constant (Kd) values of 23.7 and 590 nM, and binding site density (Bmax) values of 76 and 646 fmol/mg of protein, respectively. The substrate specificity of the binding site suggests that it represents the nicotinic cholinergic receptor. [3H] Nicotine binding was found to be highest in the hypothalamus and hippocampus and lowest in the cerebellum. Chronic treatment with the acetylcholinesterase inhibitor disulfoton (2 mg/kg/day for 10 days) decreased the number of cholinergic muscarinic and nicotinic binding sites in rat brain. Moreover, the antinociceptive effect of nicotine was found to be markedly reduced in rats chronically treated with disulfoton.     

Characterization of A2A adenosine receptors in human lymphocyte membranes by [3H]-SCH 58261 binding

1. The present study describes for the first time the characterization of the adenosine A2A receptor in human lymphocyte membranes with the new potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4 triazolo [1,5-c] pyrimidine, ([3H]-SCH 58261). In addition, both receptor affinity and potency of reference adenosine receptor agonists and antagonists were determined in binding and adenylyl cyclase studies. 2. Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 0.85 nM and 35 fmol mg-1 protein, respectively. A series of adenosine receptor ligands were found to compete for the binding of 0.8 nM [3H]-SCH 58261 to human lymphocyte membranes with a rank order of potency consistent with that typically found for interactions with the A2A-adenosine receptor. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency similar to that observed in binding experiments. 3. Thermodynamic data indicate that [3H]-SCH 58261 binding to human lymphocytes is entropy and enthalpy-driven, a finding in agreement with the thermodynamic behaviour of antagonists for rat striatal A2A-adenosine receptors. 4. It is concluded that in human lymphocyte membranes [3H]-SCH 58261 directly labels binding sites showing the characteristic properties of the adenosine A2A-receptor. The presence of A2A-receptors in peripheral tissue such as human lymphocytes strongly suggests an important role for adenosine in modulating immune and inflammatory responses.     

Characterization of [3H]staurosporine binding in protein kinase C-II purified from rat brain

The type II protein kinase C (PKC-II) densely present in mammalian brain plays functional roles in CNS. We examined the characteristics of [3H]staurosporine binding to PKC-II purified from rat brain, compared to [3H]phorbol 12, 13-dibutyrate (PDBu) binding. In brief, [3H]staurosporine binding increased by phosphatidylserine (PtdSer) in a concentration-dependent manner and the binding was enhanced by Ca2+ and phorbol 12-myristate 13-acetate (PMA). In the presence of Ca2+, PMA and PtdSer, Bmax of these bindings markedly increased, but KD did not change. These characteristics of binding were similar to [3H]PDBu binding to PKC-II. Although [3H]PDBu binding was not affected by protein kinase inhibitors such as staurosporine, H-7, K-252a and K-252b, [3H]staurosporine binding was inhibited by these inhibitors. [3H]staurosporine binding was inhibited by several ATP analogues, but was not by guanine nucleotides. PtdSer-induced increase in [3H]PDBu binding was inhibited by Zn2+, but Zn2+ induced increase in [3H]staurosporine binding as well as PtdSer and/or Ca2+. Staurosporine would thus appear to bind to a domain different from phorbol ester-binding one in PKC, interactions between both domains may regulate kinase activity, and 1 mol staurosporine and 4 mol phorbol ester may bind to 1 mol PKC-II.