Localization of [H]nitrobenzylthioinosine binding sites in rat spinal cord and primary afferent neurons

The distribution of [3H]nitrobenzylthioinosine ([3H]NBI) binding to nucleoside transport sites in rat spinal cord and spinal roots was examined using membrane binding and autoradiographic techniques. A single class of high affinity binding sites having dissociation constants (KD) between 0.42 +/- 0.05 and 0.088 +/- 0.012 nM was observed in dorsal and ventral spinal cord and their associated roots. The maximal number of binding sites (Bmax) in dorsal and ventral spinal cord was 110.1 +/- 7.1 and 73.6 +/- 7.5 fmol/mg protein, respectively. The highest levels of [3H]NBI binding were found in the dorsal grey matter of the cervical and lumbar enlargements. Autoradiographic studies showed that [3H]NBI sites were especially concentrated in the substantia gelatinosa of the dorsal spinal cord and the nucleus caudalis of the spinal trigeminal nucleus. The level of these binding sites in dorsal roots was nearly 4 times that observed in ventral roots; 98.5 and 23.0 fmol/mg protein, respectively. Adult animals depleted of unmyelinated sensory fibers by neonatal capsaicin treatment showed significantly reduced numbers of [3H]NBI sites (35%) in dorsal roots but not ventral roots, while KD values were unaffected. These results indicate that [3H]NBI sites are enriched in areas of the spinal cord and brainstem which subserve sensory functions and that these sites are located, in part, on unmyelinated primary afferent fibers.     

Binding of [3H]prazosin and [3H]p-aminoclonidine to alpha-adrenoceptors in rat spinal cord

alpha-Adrenoceptors in spinal cord appear to play a role in a number of physiologic processes including the control of blood pressure, pain and motor function. In order to evaluate more clearly these potential roles, the characteristics of binding of [3H]prazosin ([3H]PRZ) to spinal alpha 1 adrenoceptors and [3H]p-aminoclonidine ([3H]PAC) to spinal alpha 2 adrenoceptors were determined. Binding of each ligand to their respective adrenoceptors was saturable and Scatchard analysis revealed binding of each to a single class of adrenoceptors with characteristics of [3H]PRZ binding of Bmax = 78 fmol/mg protein and Kd = 0.75 nM and [3H]PAC binding Bmax = 70 fmol/mg protein and Kd = 1.39 nM. Whereas [3H]PRZ specific binding (Bmax) was unaltered by guanine nucleotides. [3H]PAC binding was increased with addition of 10 microM guanosine triphosphate (GTP) (P less than 0.05) and decreased with either 50 microM GTP or guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] (P less than 0.01). Competition for specific [3H]PRZ and [3H]PAC binding by various alpha 1 and alpha 2 adrenoceptor agonists and antagonists of known pharmacologic activity revealed that [3H]PRZ defines alpha 1 adrenoceptors (Ki = 2.1 nM for prazosin vs 4300 nM for yohimbine) and [3H]PAC defines alpha 2 adrenoceptors (Ki = 1.06 nM for yohimbine vs 15480 nM for prazosin). Regional spinal cord studies demonstrated that dorsal spinal cord in the lumbar region contains the highest density of both [3H]PRZ (Bmax = 93 +/- 14 fmol/mg protein) and [3H]PAC (Bmax = 101 +/- 6 fmol/mg protein) binding. In contrast, lowest binding was evident in thoracic cord with equal levels in both dorsal and ventral regions (Bmax = 44-48 fmol/mg protein). The regional distribution of both alpha 1 and alpha 2 adrenoceptors in spinal cord compares to the localization previously classified functionally utilizing various pharmacological agonists and antagonists at norepinephrine receptors.     

Ontogenesis of adenosine receptors in the central nervous system of the rat

The ontogeny of adenosine receptors was studied in rat brain and spinal cord using the specific ligand [3H]cyclohexyladenosine [( 3H]CHA). The [3H]CHA affinity constant (Kd) and the maximum receptor binding capacity (Bmax) were analyzed at all ages and in all CNS regions studied. Throughout development the Kd of [3H]CHA binding remained relatively stable and for cortex, cerebellum, subcortex, midbrain, brainstem and spinal cord ranged from 2.2 +/- 0.2 to 5.5 +/- 0.6 nM (mean +/- S.E.M.). In contrast, the Bmax values from 1- and 90-day animals increased by as little as 2-fold in subcortical regions and by as much as 9- and 16-fold in cortex and cerebellum, respectively. The highest density of binding sites was observed in subcortical structures and the lowest in brainstem and midbrain. In cortex, a steady increase in receptor number began at day 1 and stopped at the adult level by 21 days. In cerebellum, maximum receptor proliferation began at about 14 days and continued to adulthood. Other CNS regions showed intermediate rates of receptor development. These differences may reflect both the pattern of postnatal neurogenesis in the rat CNS and the maturation of those neural elements containing adenosine receptors.     

Localization of [3H]cyclohexyladenosine and [3H]nitrobenzylthioinosine binding sites in rat striatum and superior colliculus

The localization of adenosine receptors labelled with [3H]cyclohexyladenosine ([3H]CHA) and adenosine transport sites labelled with [3H]nitrobenzylthioinosine ([3H]NBI) was examined in striatum and superior colliculus (SC) using radioligand binding and lesioning methods. Striatal kainic acid lesions significantly reduced the number (Bmax) of a single class of high affinity binding sites for [3H]CHA by 50% and that for [3H]NBI by 15% without altering Kd values for either ligand. In SC, enucleations significantly reduced both high and low affinity [3H]CHA binding sites by about 60% while levels of [3H]NBI binding were unaffected. Thus, adenosine receptors are present on striatal interneurons and retinal projections to the SC and some [3H]NBI binding sites are located on striatal interneurons.     

Cyclohexyladenosine binding in rat striatum

N6-Cyclohexyl-[3H]adenosine [( 3H]CHA) binds specifically to rat brain membranes prepared from the caudate-putamen complex with a Kd value of 2.50 +/- 0.39 nM and Bmax of 458 +/- 51 fmol/mg protein. Lesioning the nigrostriatal dopaminergic pathway using 6-hydroxydopamine failed to alter [3H]CHA binding characteristics. Intrastriatal kainate lesions reduced the binding capacity of [3H]CHA by 28% though this was not statistically significant (0.1 less than P greater than 0.05). In kainate-lesioned striata, however, 2-deoxyglucose uptake was reduced by only 39%.     

Localization of 5-HT3 receptors in the rat brain using [3H]LY278584

5-HT3 receptors have been localized in the rat brain using the selective antagonist ligand [3H]LY278584. The binding of this ligand to slide mounted tissue sections was characterized by a Kd value of 1.5 nM and a Bmax value of 110 fmol/mg tissue dry weight. The specific binding was displaced by 5-HT or a number of 5-HT3 antagonist compounds. High densities of 5-HT3 receptors were detected in the nucleus of the solitary tract, dorsal motor nucleus of the vagus and area postrema. Moderate levels of binding were found in the glomerular layer of the olfactory bulb, substantia gelatinosa of the trigeminal nucleus and spinal cord and various nuclei of the amygdala. Low levels of binding were found in the superficial laminae of the cerebral cortex and relatively evenly distributed in the hippocampus. These results indicate that [3H]LY278584 is a useful ligand to study 5-HT3 receptors by quantitative autoradiography.     

Ionotropic glutamate receptor expression in human spinal cord during first trimester development

Quantitative receptor autoradiography and immunoblotting were used to study the expression and distribution of AMPA, kainate and NMDA receptors in first trimester human spinal cord obtained from elective abortions ranging from 4 to 11.5 weeks of gestational age. Spinal cord tissue sections were processed for receptor autoradiography with the ligands [3H]AMPA, [3H]kainate and [3H]MK-801 and the optical density was measured separately in a dorsal region (alar plate) and ventral region (basal plate) of the autoradiographs. Binding sites for all three ligands were demonstrated already at 4-5.5 weeks of gestation and increased continuously during the first trimester both in the dorsal and ventral regions. [3H]AMPA binding to both high- and low-affinity sites increased from undetectable levels to about 35 and 400 fmol/mg tissue, respectively, during this period. A temporal difference in the distribution of [3H]AMPA binding sites was observed. The early homogeneous pattern of [3H]AMPA binding in both alar and basal plates had changed to a heterogeneous pattern at 11 weeks of gestation with the highest density of [3H]AMPA binding sites in the superficial layers of the immature dorsal horn. [3H]kainate and [3H]MK-801 binding sites were densely and homogeneously distributed already at 4 weeks, and steadily increased six- and two-fold, respectively, to about 100 fmol/mg tissue at 11.5 weeks of gestation. Immunoreactive bands corresponding to the NMDA receptor subunits NR1, NR2A, NR2B, NR2C and NR2D were demonstrated by immunoblotting at the earliest between 4.5 and 7 weeks and increasing concentrations were seen up to 11 weeks of gestation. These results suggest that AMPA, kainate and NMDA receptors are expressed in the human spinal cord early in embryogenesis.     

Characterization of [3H]rauwolscine binding to alpha 2-adrenoceptor sites in the lumbar spinal cord of the cat: comparison to such binding sites in the cat frontal cerebral cortex

The binding of the selective alpha 2-adrenoceptor antagonist radioligand [3H]rauwolscine ([3H]RAUW) to homogenates of cat frontal cerebral cortex and cat lumbar spinal cord was investigated. Experiments were performed at 20 degrees C in 50 mM Tris HCl/l mM Na2EDTA buffer (pH 6.9 at 20 degrees C). At this temperature, specific [3H]RAUW binding, defined as the difference between the amount of [3H]RAUW bound in the absence and presence of 1 microM rauwolscine or 1 microM rauwolscine or 1 microM yohimbine, reaches equilibrium values by approximately 60 min and is reversible with a mean t1/2 of dissociation of 15 min in cortex and 20 min in spinal cord. The kinetically determined Kd of [3H]RAUW (mean K-1/mean K1) was 0.59 nM and 1.68 nM in cortex and spinal cord, respectively. The results of equilibrium saturation experiments, routinely performed at [3H]RAUW concentrations between 0.1 nM and 6.0 nM, indicate that [3H]RAUW binds to saturable sites in both CNS regions of the cat. Scatchard plots of saturation isotherm data were consistently linear and the mean Kd value determined from 10 such experiments was 0.72 nM in frontal cortex and 0.82 nM in lumbar spinal cord. A mean Bmax value of 230 fmol/mg protein was determined for saturable [3H]RAUW binding sites in the cat frontal cortex. In teh cat lumbar spinal gray, a mean Bmax value for saturable [3H]RAUW binding sites of 75 fmol/mg protein was obtained. Saturable [3H]RAUW binding sites in the cat lumbar spinal gray are present at apparently equal density in dorsal and ventral horns. Inhibition experiments, performed at 0.2 nM or 0.4 nM [3H]RAUW, indicate that the binding sites labeled by [3H] RAUW possess a pharmacology characteristic of alpha-adrenoceptors. Thus, rauwolscine, yohimbine, and phentolamine compete for specific [3H]RAUW binding with high affinity and are much more potent inhibitors than corynanthine, prazosin, and propranolol. Mean Hill coefficients, calculated from logit-log plots of competition data, were close to one for all antagonists examined. L-Epinephrine and L-norepinephrine were 15-20 times more potent inhibitors of specific [3H]RAUW binding than were their corresponding D-isomers. The agonist inhibitor potency series: p-aminoclonidine = clonidine = L-epinephrine greater than L-norepinephrine much greater than isoproterenol, is that expected of alpha 2-adrenoceptor sites. Mean Hill coefficients efficients for all agonists were considerably less than one.(ABSTRACT TRUNCATED AT 400 WORDS)     

Adenosine uptake and [3H]nitrobenzylthioinosine binding in developing rat brain

The ontogenesis of adenosine transport sites as labelled with [3H]nitrobenzylthioinosine ([3H]NBI) was examined using radioligand binding and membrane preparations from whole brain and 4 brain regions of rats between the postnatal ages of one day through to adulthood. In whole brain, cerebral cortex and cerebellum, [3H]NBI binding was two-fold higher in 6-day-old than in 50-day-old rats. In contrast, [3H]NBI binding was higher in adults than in one-day-old rats by 4-fold in hypothalamus and 8-fold in superior colliculus. In cortex and hypothalamus, the levels of [3H]NBI binding in newborn and adult rats were reflected by changes in Bmax and not Kd values. As a measure of the utility of [3H]NBI as a probe for identifying functional adenosine transport sites, we examined [3H]NBI binding to and [3H]adenosine accumulation by intact brain cells prepared from adult and newborn rats. For [3H]NBI binding to brain cells from adult rats, the values of Kd were 0.092 nM and of Bmax were 274 fmol/mg protein. For newborns, slightly higher Kd and Bmax values were observed; 0.2 nM and 395 fmol/mg protein, respectively. [3H]Adenosine accumulation was higher in brain cells from one-day-old than from adult rat brains. Kinetically this uptake was best described by a two-component model: the Vmax values for the high- and low-affinity uptake, and the Km value for the high-affinity component in one-day-old rats were greater than in adults.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of nitrobenzylthioinosine binding to nucleoside transport sites selective for adenosine in rat brain

Nucleoside transport sites in rat brain membrane preparations were labeled with [3H]nitrobenzylthioinosine ([3H] NBI), a potent inhibitor of nucleoside transport systems. The membranes contained a single class of very high affinity binding sites with KD and Bmax values of 0.06 nM and 147 fmol/mg of protein, respectively. The displacement of [3H]NBI binding by various nucleosides, adenosine receptor agonists and antagonists, and known nucleoside transport inhibitors was examined. The Ki values (micromolar concentration) of [3H]NBI displacement by the nucleosides tested were: adenosine, 3.0; inosine, 160; thymidine, 240; uridine, 390; guanosine, 460; and cytidine, 1000. These nucleosides displayed parallel displacement curves indicating their interaction with a common site labeled by [3H]NBI. The nucleobases, hypoxanthine and adenine, exhibited Ki values of 220 and 3640 microM, respectively. Adenosine receptor agonists exhibited moderate affinities for the [3H]NBI site, whereas the adenosine receptor antagonists, caffeine, theophylline, and enprofylline, were ineffective displacers. The Ki values for cyclohexyladenosine, (+)- and (-)-phenylisopropyladenosine, 2-chloroadenosine, and adenosine 5'-ethylcarboxamide were 0.8, 0.9, 2.6, 12, and 54 microM, respectively. These affinities and the rank order of potencies indicate that [3H]NBI does not label any known class of adenosine receptors (i.e., A1, A2, and P). The Ki values of other nucleoside transport inhibitors were: nitrobenzylthioguanosine, 0.05 nM; dipyridamole, 16 nM; papaverine, 3 microM; and 2'-deoxyadenosine, 22 microM. These results indicate that [3H]NBI binds to a nucleoside transporter in brain which specifically recognizes adenosine as its preferred endogenous substrate. This ligand may aid in the identification of CNS neural systems that selectively accumulate adenosine and thereby control "adenosinergic" function.     

Inhibition of N6-[3H]cyclohexyladenosine binding by carbamazepine

The mechanism of action of carbamazepine (CBZ) (Tegretol), despite widespread use in the management of partial and tonic-clonic seizures in adults, is not completely understood. In animals, adenosine and adenosine analogues have anticonvulsant effects that may be due to interactions with central A1 adenosine receptors. CBZ (at therapeutically relevant concentrations) inhibits the binding of agonists and antagonists to brain A1 adenosine receptors, but whether as an agonist/antagonist is not clear. The adenosine agonist, N6-[3H]cyclohexyladenosine ([3H]CHA), binds to membranes from rat cortex and hippocampus at two nanomolar binding sites or states. To clarify the actions of carbamazepine at the A1 adenosine receptor, its inhibitory actions were compared with those of known adenosine agonists and xanthine antagonists using 0.1 nM[3H]CHA, in which almost all binding is to the higher affinity state, or 10 nM [3H]CHA, in which there is a substantial contribution of binding from both states. The ratios of the IC50 values (concentration that inhibits specific binding by 50%) at 10 nM [3H]CHA to the IC50 values at 0.1 nM [3H]CHA were 18-31 for the agonists and 4-10 for the xanthine antagonists. CBZ had a ratio of 3. The inhibitory effects of GTP on [3H]CHA binding were less in the presence of the adenosine agonist, 2-chloroadenosine than were inhibitory effects in the presence of the xanthine antagonist theophylline or CBZ in both cortex and hippocampus. These in vitro studies indicate that CBZ is an antagonist at A1 adenosine receptors in cerebral cortical and hippocampal membranes from rat brain. Agonist activity at A1 adenosine receptors would have been compatible with the sedative anticonvulsant effects of CBZ, but these data do not support a role of the anticonvulsant action of carbamazepine on A1 adenosine receptors in cerebral cortex or hippocampus.     

Characterization of glucocorticoid receptors in whole and cellular subfractions of embryonic chick spinal cord

Steroid-specific binding sites for tritiated corticosterone have been localized, via autoradiography, to motoneurons in the lateral motor columns of the adult rat spinal cord. Binding sites in adult rat spinal cord have been characterized biochemically and shown to possess the properties of a putative glucocorticoid receptor. The presence of receptors for glucocorticoids in embryonic chick spinal cord was determined and their characterization undertaken as a prelude to the study of the functions under regulation by glucocorticoids during development. Assay conditions were defined and binding of [3H]dexamethasone [( 3H]Dex) to cytosols of 6- and 10-day embryonic spinal cord and cellular subfractions of 6-day spinal cord determined. Saturable, high-affinity binding of [3H]Dex to cytosols prepared from both whole 6- and 10-day spinal cords and cells of all 3 cellular subfractions of 6-day spinal cords was observed. The binding component in 10-day cytosols was (1) proteinaceous, as binding was eliminated by heating cytosols, and (2) a macromolecular species, as it displayed a sedimentation coefficient of 8S upon centrifugation in sucrose gradients. The putative receptor displayed binding specific for glucocorticoids in a competition assay, with the exception that some inhibition of binding by the androgen ligand, methyltrienolone (R1881) was observed. The binding affinity decreased as the values for KD increased from 3.4 +/- 0.9 nM on day 6 to 15.7 +/- 1.8 nM on day 10, while the values for Bmax increased from 270 to 855 fmol/mg protein over the same period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synchronized overproduction of AMPA,kainate, and NMDA glutamate receptors during human spinal cord development

Quantitative receptor autoradiography was used to map the distribution in the developing human spinal cord of the three types of ionotropic glutamate receptors. N-methyl-D-Aspartate (NMDA) receptors were labeled with [³H]glutamate, kainic acid (KA) receptors were labeled with [³H]KA, and α-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA) receptors were labeled with [³H]AMPA. In the adult, labeling of all three receptor subtypes is largely restricted to the substantia gelatinosa (SG) in the dorsal horn, with very low level labeling elsewhere in the spinal gray matter. In marked distinction, in late fetal life, high level ligand binding is seen throughout the spinal gray matter. In early postnatal life, binding sites diminish in all regions, but least so in the SG, until the adult pattern emerges. Thus a coordinated transient high level of ionotropic glutamate receptor expression occurs within the developing spinal cord. Saturation analysis of ligand binding shows that the affinity of [³H]KA and [³H]AMPA binding is not developmentally regulated. In contrast, the affinity of [³H]glutamate binding to the NMDA receptor in the fetal ventral horn is three-fold greater than in the adult ventral horn. Thus, in addition to quantitative changes in glutamate receptor expression, qualitative changes occur in the expression of NMDA receptors during development. The distinct glutamate receptor phenotype of fetal and early postnatal spinal cord cells suggests that alterations in the excitable properties of these cells plays an important role in activity-dependent development and in susceptibility to excitotoxic injury. J. Comp. Neurol. 384:200-210, 1997. © 1997 Wiley-Liss, Inc.     

Presynaptic and postsynaptic alpha 2-adrenergic receptors in human cerebral arteries and their alteration after subarachnoid hemorrhage

The nature of alpha-adrenergic receptors in human cerebral arteries was characterized, and alteration of these receptors after subarachnoid hemorrhage was examined using a radioligand binding assay. Norepinephrine content of control arteries was also analyzed and compared with that of arteries after subarachnoid hemorrhage. Norepinephrine content in human cerebral arteries in cases of subarachnoid hemorrhage was about 5% of the control group. Specific binding of [3H]yohimbine, a selective alpha 2-antagonist, to cerebral arteries of the control group indicated two classes of binding sites: high-affinity sites with KD of 0.5 nM and Bmax of 18 fmol/mg protein and low-affinity sites with KD of 29 nM and Bmax of 248 fmol/mg protein. In cerebral arteries obtained from the subarachnoid hemorrhage group, [3H]yohimbine binding sites were of a single class with KD of 53 nM and Bmax of 456 fmol/mg protein. These results suggest that sympathetic denervation and subsequent alterations in alpha 2-adrenergic receptors occurred after subarachnoid hemorrhage in human cerebral arteries. These changes in sympathetic innervation to cerebral arteries were considered to be one of the antecedents of delayed vasospasm after subarachnoid hemorrhage.     

A novel GABAA antagonist [3H]SR 95531: microscopic analysis of binding in the rat brain and allosteric modulation by several benzodiazepine and barbiturate receptor ligands

Recent reports have demonstrated that the synthetic gamma-aminobutyric acid (GABA)-derivative, SR 95531 [2-(3'-carbethoxy-2'-propyl)-3-amino-6-paramethoxy-phenyl-pyrid azinium bromide], possesses selective GABAA antagonistic properties. Because of its potency for recognition of GABAA sites, this agent has been used to identify GABAA receptors. In the present investigation, we studied the binding of [3H]SR 95531 to tissue sections of rat brain using microscopic analysis of receptor localization. The appropriate binding conditions for defining GABAA receptors with this radioligand were obtained by determining the dissociation and association kinetics, and performing saturation and displacement studies. Using membrane preparations from whole rat brain (or brain regions representing cortex, striatum, hippocampus, midbrain-thalamus, medulla-pons and cerebellum), saturation and displacement studies were analyzed, and allosteric modulation of [3H]SR 95531 binding was examined by including several benzodiazepine and barbiturate receptor ligands in the incubation media. To assess the stereoselective properties of [3H]SR 95531 binding in rat membranes, numerous barbiturates were added during the incubation. The binding of [3H]SR 95531 was demonstrated to be saturable, specific and to bind with relatively high affinity to low-affinity GABAA sites. Scatchard analysis performed on saturation data of binding to tissue sections showed a dissociation constant (KD) of 42.4 nM and a maximum number of binding sites (Bmax) of 105.8 fmol/mg tissue. Microscopic analysis showed that intermediate to high densities of [3H]SR 95531 binding occurred in brain regions containing intermediate to high densities of low-affinity GABAA receptor sites. The binding of [3H]SR 95531 to membranes also appeared to occur at low-affinity GABAA sites. Results from competition studies demonstrated that [3H]SR 95531 is displaceable by GABAA agents and displaced preferentially by GABAA antagonists. Scatchard analysis of saturation experiments from membrane preparations indicated that the KD and Bmax from the centrifugation assay was 53.0 nM and 4.26 pmol/mg protein, respectively. Using the filtration assay, binding to membranes yielded a KD value of 45.6 nM and a Bmax of 0.77 pmol/mg protein. The allosteric modulation data demonstrated that numerous benzodiazepine and barbiturate agents inhibited [3H]SR 95531 binding and this varied according to brain region. Several barbiturates included in the incubation media exhibited a stereoselective inhibition of [3H]SR 95531 binding to whole rat brain membranes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prostaglandin E2 Receptors in the Chicken Spinal Cord: 1. Biochemical Characterization

Prostaglandins (PGs) are neuroactive substances which act in the vicinity of their site of synthesis through receptors coupled to G-proteins. Since large amounts of PGE2 can be synthesized by chicken spinal cord, binding sites for PGE2 were looked for in various cell fractions of spinal cord. In the 17 000 g pellet incubated with 0.3 nM [3H]PGE2, 70% of ligand was specifically bound. Two types of PGE2 binding site were characterized (i) high affinity, low capacity binding sites (KD1 1.34 nM, Bmax1 34.5 fmol/mg prot); (ii) low affinity, high capacity binding sites (KD2 2.23 microM, Bmax2 13.2 pmol/mg prot). The high affinity binding sites fulfil several requirements for being receptors to PGE2: (i) since the KD1 is increased in the presence of the GTP analogue, Gpp(NH)p, these binding sites would be regulated by a G-protein; (ii) a desensitization was obtained by an excess of unlabelled PGE2 and reversed by Gpp(NH)p; (iii) the competition experiments between PGE2 and various prostanoids pointed to PGE2 receptors such as EP2 or EP3. The receptor characteristics of the low-affinity binding sites were not investigated. Hence, our results support the presence of two types of PGE2 binding site in the chicken spinal cord; a high affinity site, which corresponds to a PGE2 receptor responding to nanomolar concentrations and a low affinity site sensitive to micromolar concentrations of PGE2.     

Heterogeneous binding of [3H]4-DAMP to muscarinic cholinergic sites in the rat brain: evidence from membrane binding and autoradiographic studies.

The present study shows that [3H]4-DAMP binds specifically, saturably, and with high affinity to muscarinic receptor sites in the rat brain. In homogenates of hippocampus, cerebral cortex, striatum, and thalamus, [3H]4-DAMP appears to bind two sub-populations of muscarinic sites: one class of high-affinity, low capacity sites (Kd less than 1 nM; Bmax = 45-152 fmol/mg protein) and a second class of lower-affinity, high capacity sites (Kd greater than 50 nM; Bmax = 263-929 fmol/mg protein). In cerebellar homogenates, the Bmax of [3H]4-DAMP binding sites was 20 +/- 2 and 141 +/- 21 fmol/mg protein for the high- and the lower-affinity site, respectively. The ligand selectivity profile for [3H]4-DAMP binding to its sites was similar for both the high- and lower-affinity sites; atropine = (-)QNB = 4-DAMP much greater than pirenzepine greater than AF-DX 116, although pirenzepine was more potent (16-fold) at the lower- than at the high-affinity sites. The autoradiographic distribution of [3H]4-DAMP sites revealed a discrete pattern of labeling in the rat brain, with the highest densities of [3H]4-DAMP sites present in the CA1 sub-field of Ammon's horn of the hippocampus, the dentate gyrus, the olfactory tubercle, the external plexiform layer of the olfactory bulb and layers I-II of the frontoparietal cortex. Although the distribution of [3H]pirenzepine sites was similar to that of [3H]4-DAMP sites in many brain regions, significant distinctions were apparent. Thus, both the ligand selectivity pattern of [3H]4-DAMP binding and the autoradiographic distribution of sites suggest that although the high-affinity [3H]4-DAMP sites may consist primarily of muscarinic-M3 receptors, the lower-affinity [3H]4-DAMP sites may be composed of a large proportion of muscarinic-M1 receptors.     

Differential radioligand binding properties of [3H]5-hydroxytryptamine and [3H]mesulergine in a clonal 5-hydroxytryptamine1C cell line.

[3H]5-Hydroxytryptamine ([3H]5-HT) and [3H]mesulergine were used to label 5-HT1C receptors expressed in NIH 3T3 mouse fibroblast cells. Using a rapid filtration assay, saturation analysis of the [3H]5-HT radioligand data indicate that the binding is biphasic. Based on computerized analysis of the data, a 2-site model of radioligand binding is significantly more consistent with the data than a one-site model (P less than 0.01). The KD values of [3H]5-HT for the 2 populations are 0.5 +/- 0.1 nM and 31 +/- 15 nM, while the Bmax values are 400 +/- 90 pmol/g protein and 3,000 +/- 600 pmol/g protein, respectively. A biphasic binding pattern is also observed with [3H]5-HT using a centrifugation assay (KD1 = 0.6 +/- 0.06 nM, KD2 = 60 +/- 10 nM; Bmax1 = 740 +/- 90 pmol/g, Bmax2 = 4,000 +/- 700 pmol/g). By contrast, saturation analysis of [3H]mesulergine binding is monophasic (KD = 4.7 +/- 0.7 nM) with a Bmax value (6,800 +/- 1,000 pmol/g protein) that is significantly greater than that obtained using [3H]5-HT (P less than 0.01). Drug competition studies confirm that both [3H]5-HT and [3H]mesulergine label at least 2 subpopulations of expressed 5-HT1C receptors in NIH 3T3 cells. 10(-4) M GTP eliminates the high affinity [3H]5-HT-labeled binding sites with minimal effect on the low affinity [3H]5-HT-labeled sites and no effect on [3H]mesulergine-labeled sites. These data demonstrate that at least 2 distinct subpopulations of 5-HT1C receptors in NIH 3T3 cells can be differentiated using radioligand binding techniques.     

Quantitative autoradiographic distribution of multiple neurokinin binding sites in rat spinal cord

As a means of evaluating the role of neurokinins (NKs) in spinal function, the present study examines the quantitative autoradiographic distribution in the rat spinal cord of [125I]Bolton-Hunter-substance P, (2-[125I]iodohistidyl1)-neurokinin A and [125I]Bolton-Hunter-eledoisin as respective radioligands for NK-1, NK-2 and NK-3 receptors. These putative NK receptor sub-types are clearly differentially distributed at the various levels of the spinal cord. NK-1 sites represent the most abundant population of spinal NK receptors. They are most concentrated in the dorsal and ventromedial borders of the dorsal horn, the intermediolateral nucleus of the thoracic cord and the phrenic motor nucleus in the cervical ventral horn. NK-2 and NK-3 sites are also present in the spinal cord, although in much lower quantities than NK-1 sites. NK-2 sites are mostly found along the dorsal and ventromedial borders of the dorsal horn, in a narrow band connecting the two lateral horns of the thoracic cord, around the central canal of the lumbar and sacral segments and lamina IX of the cervical ventral horn. NK-3 sites are most dense in the dorsal border of the dorsal horn, with moderate amounts in the lateral horn of the thoracic cord and around the central canal of lumbar and sacral segments. The differential distribution of these 3 classes of NK sites in the spinal cord suggests that each NK receptor sub-type could mediate specific sensory, autonomic and/or motor functions at the spinal level.     

Prominent binding of the dopamine D3 agonist [3H]PD 128907 in the caudate-putamen of the adult rat

We have analyzed the binding properties of the selective D3 receptor agonist [3H]PD 128907 in 120 days old rats. In tissue sections, we found high numbers of binding sites for [3H]PD 128907 both in the islands of Calleja and the caudate-putamen (Bmax values being 500 and 1000 fmol/mg protein, respectively). The KD values were higher in the caudate-putamen than in the islands of Calleja. Similar regional differences in Bmax and KD values were observed in membranes from the caudate-putamen and the subcortical limbic region. The distribution of [3H]PD 128907 in adult rats is markedly different from that observed in young rats. Taken together, the present results suggest a prominent presence of D3 receptors in the caudate-putamen of adult, but not young, rats. Hence, these findings may have important physiological, pathophysiological, and clinical implications.