In order to investigate possible differences between NMDA receptor-coupled ion channels in the spinal cord and in the cerebral cortex, we have characterized [3H]MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine] binding and its regulation by glutamate and glycine in membrane preparations of the rat spinal cord and cerebral cortex. The K(D) value of [3H]MK-801 binding was higher in the spinal cord than in the cerebral cortex, mainly due to a lower association rate constant. When corrected for the concentrations of residual endogenous amino acids, the EC50 values for glycine were lower at spinal NMDA receptors compared to those in the cerebral cortex, whereas the EC50 values for glutamate were similar in both regions. The IC50 values of D-((3)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (D-CPP) were significantly lower in the spinal cord in the presence of saturating concentrations of glutamate. The IC50 values of 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(H)-quinoline (L-701,324) were significantly lower in the spinal cord under all conditions. These results suggest that NMDA receptors in the spinal cord display low affinity for MK-801, which may correspond to a lower affinity of the voltage-dependent Mg2+ block. Furthermore, NMDA receptors in the spinal cord appear to display high sensitivity to glycine and to glutamate and glycine antagonists.

Using a receptor binding assay for [H-3](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-10-imine (MK-801) the pharmacology of spinal cord NMDA receptors was compared to that of NMDA receptors in the cerebral cortex. The affinities of glutamate site agonists L-glutamate, L-aspartate, ibotenic acid, NMDA and quinolinic acid for stimulation of [H-3]MK-801 binding were 6-10 times lower in the spinal cord and the efficacy of quinolinic acid was 50% of that of the other agonists in this region. Also the affinities of glycine site agonists glycine, D-serine, D-alanine and L-serine were lower in the spinal cord as were the affinities of the non-competitive antagonists phencyclidine, (+/-)-cyclazocine and dextromethorphan. The divalent cations Zn2+, Mg2+ and Ca2+ had 4-8 times lower affinity for spinal NMDA receptors while the affinity of Co2+ was 50 times lower. The affinity of [H-3]MK-801 was 2.5-fold lower in the spinal cord. These data show that spinal cord NMDA receptors show qualitative and quantitative differences compared to those in the cerebral cortex.

We have studied the effects of various cations on [3H]MK-801 binding to N-methyl-d-aspartate (NMDA) receptors in membrane preparations of the rat cerebral cortex. Low concentrations of Tris, K+, Na+, Mg2+, and Ca2+ enhanced submaximally stimulated [3H]MK-801 binding. At high concentrations, all compounds inhibited [3H]MK-801 binding, possibly by a direct competitive effect. H+ decreased the observed association rate of [3H]MK-801 binding observed as a decreased [3H]MK-801 binding under nonequilibrium conditions, apparently by decreasing the sensitivity of the glutamate and glycine effects on the association rate. In addition, Tris, Na+, Mg2+, and possibly K+ at very high concentrations, permitted glutamate and glycine to decrease [3H]MK-801 binding, probably reflecting a decreased affinity of [3H]MK-801 binding. In contrast, Ca2+ and H+ antagonized these glutamate- and glycine-induced decreases of [3H]MK-801 binding observed in the presence of Mg2+, possibly by a direct competitive action on the permissive Mg2+ effect. These Ca2+ and H+ -induced increases in [3H]MK-801 binding in the presence of Mg2+ may correspond to an increase in the potency of the Mg2+ block. Copyright © 1996 Elsevier Science Ltd.

In sagittal brain sections of newborn male rats (1-day-old) there were no regional differences in the IC50 values of dopamine at [125I]iodosulpride binding sites. In contrast, in 20- and 60-day-old rats, there was a selective increase in the IC50 values of dopamine in the nucleus accumbens, caudate-putamen, and olfactory tubercule. The IC50 values of these regions decreased in 262-day-old rats. Some of the other brain areas appeared to behave in a similar, but much less pronounced, fashion. Thus, there were significant regional differences in the IC50 values of young, adult, and old rats. In addition, there was a rapid increase in [125I]iodosulpride binding between the newborn and the 20-day-old rats, which leveled off thereafter, and selectively decreased in the substantia nigra of the 262-day-old rats. In conclusion, these results indicate that a biphasic decrease-increase in the affinity of D2 agonist binding sites occurs selectively in the basal ganglia. These findings may be of relevance for developmental diseases in which dopaminergic mechanisms have been implicated, such as schizophrenia and Parkinson's disease.

To investigate whether adenosine A2a agonists modulate dopamine D2 receptor binding in vivo, we have analyzed the effects of intraperitoneally administered 2-[p-(carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS 21680) on the ability of dopamine to compete at [125I]iodosulpride (0.25 nM) binding sites in filter-wiped cryostat sections of the rat forebrain and on [3H]L-(−)-N-propylnorapomorphine ([3H]NPA) binding (1 nM) using quantitative receptor autoradiography. CGS 21680 (1–3 mg/kg) decreased the IC50 value of dopamine on [125I]iodosulpride binding, and the decrease at 1 mg/kg was blocked by the A2 antagonist 3,7-dimethyl-1-propargylxanthine (DMPX; 5 mg/kg). The decrease in the IC50 value of dopamine was due to a decrease in the KL value whereas the KH value and the proportion of high-affinity binding sites were unaffected. The binding of [3H]NPA was significantly increased in the rostral and caudal parts of the caudate-putamen and in the rostral part of the olfactory tubercle, whereas no change could be demonstrated in the nucleus accumbens and in the caudal part of the olfactory tubercle. These results indicate that stimulation of A2a receptors in vivo causes alterations in the binding characteristics of D2 receptors in certain regions of the basal ganglia.

GTP or G protein inactivation by N-ethylmaleimide reduced the Bmax value but not the KD value of 7-[3H]hydroxy-N,N-di-n-propyl-2-aminotetralin ([3H]7-OH-DPAT) binding in the rat subcortical limbic area. Neurotensin (10 nM) increased the KD and the Bmax values of [3H]7-OH-DPAT binding, and these effects persisted also following N-ethylmaleimide pretreatment. N-Propylnorapomorphine, quinpirole, raclopride, and remoxipride inhibited [3H]7-OH-DPAT binding with Ki values of 0.093, 1.97, 10.6, and 710 nM, respectively. These findings indicate that the D3 receptor is coupled to G proteins in the brain, and that neurotensin can modulate D3 agonist binding by a G protein-independent mechanism.

The pharmacological properties of [3H]7-hydroxy-N, N-di--n-propyl-2-aminotetralin ([3H]7-OH-DPAT) binding sites in the islands of Calleja of the rat were studied using quantitative receptor autoradiography. The KD and the Bmax values of [3H]7-OH-DPAT binding were about 1.6 nM and 100 fmol/mg protein, respectively. The rank order of potency was R(−)-propylnorapomorphine > 7-OH-DPAT ≈ haloperidol > raclopride > dopamine > remoxipride. Remoxipride injected in vivo (0.03–100 μmol/kg i.p., 1 h before decapitation) did not inhibit subsequent [3H]7-OH-DPAT binding. These results indicate that the pharmacological profile of dopamine D3 receptors in the islands of Calleja of the rat resembles that obtained from cell lines or membrane preparations.

Neurotoxicology 15(3): 621-624, 1994. In the present study we have investigated the effects of toluene exposure (80 ppm, 4 weeks, 5 day/week, 6 h/day) on the serum levels of prolactin, and elaborated our earlier findings about persistent effects of toluene exposure on apomorphine-induced (1 mg/kg, s.c.) locomotor activity. We found that the serum levels of prolactin were increased by 67% in the toluene-exposed rats, as analyzed 17 days after the last exposure. The locomotor activity counts of the control rats were not normally distributed before log-transformation, since most rats showed a low level of activity and only a few showed a very high activity level. The toluene-exposed rats showed a higher level of apomorphine-induced locomotion and motility but not rearing, as analyzed 17 days after the last exposure, whereas spontaneous locomotor activity was unaffected. These results indicate that subacute exposure to 80 ppm of toluene causes persistent impairments in dopamine-mediated neurotransmission.

To further elucidate the previously demonstrated protective actions of nicotine on lesioned nigrostriatal dopamine (DA) systems (Janson and Moller, Neuroscience, 57 (1993) 931-941), the present receptor binding experiments were carried out. Rats were partially hemitransected at the meso-diencephalic junction and the effects of chronic continuous (-)nicotine treatment (osmotic pumps s.c., 0.125 mg/kg/h, 14 days) on [H-3]N-propylnorapomorphine ([H-3]NPA) and [H-3]methylcarbamylcholine ([H-3]MCC) binding were investigated in striatal coronal sections to study the agonist binding sites of DA D-2 receptors and nicotinic cholinoceptors, respectively. In saline-treated but not in nicotine-treated rats, the lesion led to an increased B-max value of [H-3]NPA binding. The B-max value of [H-3]MCC binding was increased by nicotine treatment and decreased by the partial hemitransection. These results indicate that chronic nicotine treatment counteracts the lesion-induced upregulation of the high-affinity agonist binding site of the DA D-2 receptor, which may be explained by an increased presence of DA via a protective effect of nicotine on neostriatal DA terminals. This action of nicotine may be of interest in the treatment of neurodegenerative diseases such as Parkinson's disease.

The effects of subchronic inhalation exposure to toluene (80 ppm, for 4 weeks, 5 days/week, 6 h/day) was studied on spatial learning (postexposure days 3–6) and memory (postexposure day 14) using a water maze, on spontaneous and apomorphine-induced (1 mg/kg, subcutaneously (s.c.)) locomotor activity (postexposyre day 17) and on the binding parameters of the dopamine D2 agonist S(−)[N-propyl-3H(N)]propylnorapomorphine ([H]NPA) in membrane preparations of the neostriatum of the rat. Toluene treatment was found to cause a statistically significant impairment in acquisition and retention of the spatial learning task. Furthermore, toluene significantly increased (2-fold) apomorphine-induced locomotion and caused a trend for a 50–60% increase in motility without any significant effect on rearing. Spontaneous locomotion, motility and rearing were not affected by toluene. Toluene treatment produced a significant 30–40% increase in the Bmax values of [3H]NPA and a trend for a 20–30% increase in the KD values. These results indicate that subchronic exposure to toluene in low concentrations causes a slight but persistent deficit in spatial learning and memory, a persistent increase in dopamine-mediated locomotor activity and an increase in the number of dopamine D2 receptors in the rat.

Combined perfusion of the neostriatum with 1 nM of cholecystokinin octapeptide (CCK-8) and 0.01, 0.1 or 1 nM of neurotensin was done in the halothane-anesthetized rat after systemic apomorphine treatment (0.05 mg/kg, s.c.). Neurotensin (1 nM) plus CCK-8 (1 nM) effectively counteracted the apomorphine-induced inhibition of neostriatal perfusate levels of dopamine (DA). With a constant concentration of CCK-8 (1 nM), the apomorphine-induced inhibition of DA release was counteracted dose relatedly by neurotensin in concentrations of 0.01, 0.1 and 1 nM. The results of binding experiments demonstrated that threshold concentrations of CCK-8 and neurotensin significantly increased the KD values of the high-affinity D2 receptors without significant alterations in the low-affinity D2 receptors or in the proportion of D2 receptors in the high-affinity state. Thus, neurotensin and CCK receptors may regulate synergistically, via intramembrane interactions with the D2 receptors, the binding characteristics and the signal transduction of D2 autoreceptors in the neostriatum. The combined presence of very low concentrations of CCK-8 and neurotensin in the extracellular fluid may be sufficient to regulate D2 receptor transduction, underlining the important role of these peptide receptor interactions with the D2 receptors.

In the concentration range of 1–10 nM, neuromedin N produced a significant concentration-related increase in the Kd values of [3H]l-(−)-N-propylnorapomorphine binding sites in rat neostriatal membranes with a peak action at 10 nM (36% increase versus the control group mean value). The Bmax values were not affected by neuromedin N. Neurotensin at 10 nM induced an increase in the Kd values, which was not affected by a threshold concentration of neuromedin N (0.1 nM). In view of the higher potency of neuromedin N versus neurotensin to modulate neostriatal D2 receptors in contrast to the higher potency of neurotensin versus neuromedin N to bind to the cloned neurotensin receptors, it seems possible that the neuromedin N activated neostriatal neurotensin receptors controlling the D2 receptors represent a distinct subtype of neurotensin receptors.

From behavioural and biochemical experiments, we have found evidence for the existence of a specific adenosine A2a receptor/dopamine D2 receptor interaction in the brain. Behavioural data show that stimulation A2a receptors inhibits and their blockade potentiates a D2‐mediated locomotor activation in mice and that stimulation of D2 receptors counteracts on A2a mediated cataleptic effect in rats. Biochemical data show that, in rat striatal membrane preparations, A2a receptor stimulation decreases the affinity of D2 receptors and the transduction of the signal from the D2 receptor to the G‐protein. Based on these findings it is postulated that this A2a/D2 interaction might be the main mechanism responsible for the central effects of adenosine agonists and antagonists, like methylxanthines. The increased behavioural effect of methylxanthines after dopamine denervation could be explained by an increased interaction between adenosine A2a and dopamine D2 receptors. Membrane preparations‐denervated striatum are more sensitive to the effect of adenosine A2a receptor stimulation. Our results underline the potential antiparkinsonian action of adenosine A2a antagonists and the potential antipsychotic of adenosine A2a agonists. © 1993 Wiley‐Liss, Inc.

The NMDA receptor is coupled to a cation-selective ion channel, which has been implicated in important brain functions such as long-term potentiation and burst firing, and in neuronal death associated with stroke and epilepsy. We have investigated the binding properties of [3H]MK-801, which binds selectively to the open state of the NMDA channel, at physiological concentrations of Mg2+ in membrane preparations of the rat cerebral cortex. Glutamate and glycine were found to enhance [3H]MK-801 binding at low concentrations and inhibit [3H]MK-801 binding at high concentrations. The inhibition of [3H]MK-801 binding was due to an enhancement of the dissociation rate constant and was reversed by competitive glutamate and glycine antagonists. These findings could be explained by a glutamate- and glycine-induced decrease in the affinity of [3H]MK-801 binding sites within activated NMDA channels, in the presence of Mg2+. This decrease in [3H]MK-801 affinity may correspond to a decreased affinity of the site where Mg2+ causes a voltage-dependent block of the NMDA channel.

The effects of neurotensin fragments and of neurotensin itself on the characteristics of neostriatal dopamine D2 agonist binding were studied in competition experiments with dopamine using the D2 antagonist, [3H]raclopride. The biologically active neurotensin-(8–13) fragment, but not the inactive neurotensin-(1–7) fragment, caused a concentration-related increase in the KH and KL values of dopamine with a maximal increase by 110 and 97%, respectively, at 1 nM, while neurotensin-(1–13) only induced such changes at 10 nM. In view of the higher potency and the increased ability of neurotensin-(8–13) versus neurotensin (1–13) to reduce the affinities of the high- and low-affinity states of the neostriatal D2 receptors, the C-terminal neurotensin fragments may be among the endogenous ligands of the neostriatal neurotensin receptors.

At normal pH, glutamate and glycine are known to inhibit [3H]MK-801 binding in the presence of Mg2+. We found, however, that at pH 5.4 glutamate and glycine enhance [3H]MK-801 binding in rat cerebrocortical membranes. Furthermore, H+, similarly to Mg2+, inhibited basal [3H]MK-801 binding. These results indicate that H+ competes with binding sites for Mg2+ and [3H]MK-801, and may be correlated to the depression in neuronal activity observed during acidosis.

To elucidate possible actions of nicotine on dopamine D2 receptor binding, the effect of chronic continuous (-)nicotine treatment (osmotic pumps s.c., 0.125 mg kg h-2, 14 days) was studied in the binding of [3H]N-propylnorapomorphine ([3H]NPA) and [125I]sulpride in coronal cryostat sections in the rat. Quantitative autoradiography showed that nicotine decreased the binding of [3H]NPA in the basal ganglia, preferentially in the nucleus accumbens and olfactory tubercle. In contrast, [125I]sulpride binding was not affected. Nicotine decreased the KD value of [3H]NPA by 27% and decreased the Bmax value by 17%, using filter-wiped sections. These results indicate that chronic continuous nicotine treatment affects the D2 receptor and that this effect may be involved in the development of nicotine dependence.

A trypsin inhibitor-like peptide PEC-60 reduces the affinity of dopamine D2 agonist binding sites in rat neostriatal membranes

The effects of dopamine on [32P]ATP-labelled phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidic acid were analyzed by TLC in synaptosomal membranes of the rat neostriatum. The incorporation of 32P into these compounds was found to be stable within 1 min and was maintained during the 30 min of incubation. Dopamine (0.1-10 microM) was found to attenuate the levels of phosphatidylinositol 4,5-bisphosphate without affecting the levels of phosphatidylinositol 4-phosphate or phosphatidic acid. The maximal decrease (-35 +/- 4%) was reached at 10 microM of dopamine after 30 min of incubation. The dopamine (0.1 microM)-induced decrease was blocked by the D2 selective antagonist raclopride (1 microM), but not by the D1 selective antagonist SCH 23390 (1 microM). These findings indicate the existence of an intramembrane coupling of dopamine D2 receptors to phosphoinositide turnover and may underlie some of the physiological effects of D2 receptor stimulation.