Is Alzheimer's senile dementia associated with the phenomena of reactive synaptogenesis in the hippocampus?

The densities of specific binding sites for glutamate or its analogue kainate were determined in the hippocampi of patients having suffered of senile dementia of the Alzheimer type (SDAT) and compared to age matched controls. The densities of sites were reduced in SDAT cases. These results indicate a lack of hippocampal plasticity in senile dementia of Alzheimer type.     

Galanin-Like Immunoreactivity Is Increased in the Postmortem Cerebral Cortex from Patients with Alzheimer's Disease

Abstract: Galanin is a peptide that is associated with cholinergic neurons of the basal forebrain and, thus, of interest for the neuropathology of Alzheimer's disease. In the present study, human galanin-like immunoreactivity was measured in postmortem human cerebral cortical tissues by using a homologous radioimmunoassay. In an initial study, six cerebral cortical regions were evaluated from nine elderly controls, 13 neuropathologically verified Alzheimer's disease patients, and 19 elderly schizophrenics. A significant 65% increase in galanin was found in frontal cortex Brodmann area 8 of Alzheimer's disease patients compared with controls. In contrast, cerebral cortical tissues from elderly schizophrenics were not different from those from elderly controls in any region. In a second study, 10 cerebral cortical regions were evaluated from 50 neuropathologically verified Alzheimer's disease patients and nine elderly controls. Concentrations of galanin were increased significantly 26–61% in six of 10 cerebral cortical regions examined (Brodmann areas F8, F44, T20, T21, T36, and P22). Purification of brain extracts by size-exclusion Sephadex G-50 chromatography revealed that human galanin-like immunoreactivity eluted in two peaks of different molecular weights. These studies reveal increased concentrations of galanin in the cerebral cortex of Alzheimer's disease, similar to previous findings in basal forebrain tissue. Because galanin inhibits cholinergic neurotransmission, these findings may have important implications in the understanding of Alzheimer's disease neuropathology and associated cognitive deficits.     

Intracerebroventricular administration of galanin or galanin receptor subtype 1 agonist M617 induces c-Fos activation in central amygdala and dorsomedial hypothalamus

The neuropeptide galanin and galanin receptors are widespread throughout cortical, limbic and midbrain areas implicated in reward, learning/memory, pain, drinking and feeding. While many studies have shown that galanin produces a variety of presynaptic and post-synaptic responses, work studying the effects of galanin on neural activation is limited. The present study examined patterns of c-Fos immunoreactivity resulting from intracerebroventricular administration of galanin versus saline injection in awake rats. An initial comprehensive qualitative survey was conducted to identify regions of high c-Fos expression followed up with quantitative analysis. Galanin induced a significant increase in c-Fos levels relative to saline-treated controls in dorsomedial hypothalamus and in the central nucleus of the amygdala. This pattern of activation was also produced by galanin receptor type 1 agonist M617. The present findings confirm that galanin upregulates c-Fos activation in hypothalamic nuclei, and supports roles for galanin in central amygdala-mediated regulation of stress-responses, food intake, and Pavlovian conditioning.     

Normal cortical concentration of cholecystokinin-like immunoreactivity with reduced choline acetyltransferase activity in senile dementia of Alzheimer type

Choline acetyltransferase (ChAT) activity and cholecystokinin immunoreactivity (CCK-I) were determined in ten brains from patients dying with a diagnosis of senile dementia of Alzheimer type (SDAT) and in ten brains from control cases. The post-mortem stability of CCK-I was high, as determined using a mouse brain model. Although ChAT activity was significantly reduced in the cerebral cortex, hippocampus and caudate nucleus in the SDAT cases, there was no difference in CCK-I content between the two groups in any brain area. Thus the population of intrinsic cortical cells which contains CCK-I does not appear to be significantly affected in SDAT.     

Galanin binding sites in rat gastric and jejunal smooth muscle membrane preparations.

Receptors for galanin in membranes from the rat gastric and jejunal smooth muscle were studied using [125I] radioiodinated synthetic porcine galanin. Specific binding was time and temperature dependent. At 32 degrees C radioligand was degraded in the presence of smooth muscle membranes in a time-dependent manner. At optimal experimental conditions, the equilibrium binding analyses showed the presence of a single population of high affinity binding sites in both the rat stomach and jejunum (Kd value of 2.77 +/- 0.78 nM and 4.93 +/- 1.74 nM for stomach and jejunal smooth muscle membranes, respectively). The concentration of the high affinity binding sites was 58.19 +/- 11.04 and 32.36 +/- 5.68 fmol/mg protein, for gastric and jejunal preparations, respectively. Specific binding was completely inhibited by 10(-6) M of nonradioactive galanin; was 75% blocked by 1 microM of galanin(9-29); it was 10% blocked by 1 microM of galanin(15-29). Galanin(1-15) at a concentration of 1 microM was ineffective for inhibiting [125I]galanin binding. Deletion of four C-terminal amino acid residues from galanin(9-29) to give galanin(9-25) also resulted in almost complete loss of affinity. Radioiodinated galanin and N-terminally deleted fragments had receptor binding potency in the following order: galanin(1-29) greater than galanin(9-29) greater than galanin(15-29). We conclude that the C-terminal part of the galanin chain is important for the rat gastric and jejunal smooth muscle membrane receptor recognition and binding and that N-terminal amino acid sequences are probably not so important, since galanin(1-15) was not active but galanin(9-29) retained most of the receptor binding activity.     

Characterization of l-[3H]Nicotine Binding in Human Cerebral Cortex: Comparison Between Alzheimer''s Disease and the Normal

Putative nicotine receptors in the human cerebral cortex were characterized with L-[3H]nicotine, L-[3H]Nicotine binding was enhanced by the addition of Ca2+ and abolished in the presence of Na3EDTA. Association and dissociation of the ligand were rapid at 25 degrees C with t1/2 values of 2 and 3 min, respectively. Saturation binding analysis revealed an apparent single class of sites with a dissociation constant of 5.6 nM and a Hill coefficient of 1.05. There was no effect of postmortem interval on the density of binding sites assayed up to 24 h in rat frontoparietal cortex. Nicotine binding in human cortical samples was also unaltered by increasing sampling delay. In human cortical membranes, binding site density decreased with normal aging. Receptor affinity and concentration in samples of frontal cortex (Brodmann area 10) from patients with Alzheimer's disease were comparable to age-matched control values. Samples of infratemporal cortex (Brodmann area 38) from patients with Alzheimer's disease had a 50% reduction in the number of L-[3H]nicotine sites. Choline acetyltransferase activity was significantly decreased in both cortical areas. Enzyme activities in the temporal pole were reduced to 20% of control values. These data indicate that postsynaptic nicotine receptors are spared in the frontal cortex in Alzheimer's disease. In the infratemporal cortex, significant numbers of receptors remain despite the severe reduction in choline acetyltransferase activity. Replacement therapy directed at these sites may be warranted in Alzheimer's disease.     

125I]-galanin binding sites in the human nodose ganglion

The cell bodies of centrally-projecting vagal afferent neurons are contained in the inferior vagal (nodose) ganglion. Although binding sites for a number of different neuropeptides/modulators have been detected in the human nodose ganglion, the presence of galanin binding sites has not been reported. In vitro receptor autoradiography using [125I]-galanin enabled visualisation of binding sites for galanin in the human nodose ganglion. The presence of such binding sites suggests a potential role for galanin in the neuromodulation of vagal transmission in humans.     

Cholinergic deficit in Alzheimer's disease: A study based on CSF and autopsy data

Cholinesterase (ChE) activity was measured as a possible marker of cholinergic neurotransmission of the brain in CSF of 93 patients with probable Alzheimer's disease/senile dementia of the Alzheimer type (AD/SDAT) and of 29 control patients. ChE activity in CSF was decreased significantly in the AD/SDAT patients as compared to the controls. This reduction correlated significantly with the various measures of the severity of dementia. However, the reduction of ChE activity was only moderate (25–30%) even in patients with the most severe dementia and nonsignificant in patients with early symptoms of AD/SDAT. The significance of various confounding factors, which may interfere with CSF ChE measurements is discussed. Our findings seem to indicate that the deficiency of cholinergic neurons is not directly reflected in CSF and that the measurements of ChE activities in CSF are not helpful in diagnosing AD/SDAT. In the autopsy study the activities of cholineacetyltransferase (ChAT) and ChE were determined for ten brain areas of 20 AD/SDAT patients and of 14 controls. In AD/SDAT patients ChAT activity was profoundly decreased (50–85% decrease) in the cortical areas and hippocampus, but was unchanged or only mildly reduced in other subcortical brain areas. This study further confirms that the affection of cholinergic neurons is limited to projections from nucleus basalis to cortex and hippocampus, whereas other cholinergic neurons, like in striatum, seem to be relatively spared. In general, the activities of ChAT and ChE were lower in Alzheimer patients dying at younger age suggesting more severe disease process with these patients.     

Comparative Alterations of Nicotinic and Muscarinic Binding Sites in Alzheimer''s and Parkinson''s Diseases

We have recently reported on the differential alterations of various cholinergic markers in cortical and subcortical regions in Alzheimer's disease (AD). The main purpose of the present study was to determine if cholinergic deficits observed in patients with AD are unique to this disorder or can be generalized to others such as idiopathic Parkinson's disease (PD) and PD with Alzheimer-type dementia (PD/AD). Muscarinic M1, M2, and nicotinic receptor binding parameters (KD and Bmax) were determined in various cortical and subcortical areas using selective radioligands ([3H]pirenzepine, [3H]AF-DX 116, and N[3H]methylcarbamylcholine). Choline acetyltransferase activity was also determined as a marker of the integrity of cholinergic innervation. Alterations of cholinergic markers are comparable in cortical areas in AD, PD, and PD/AD brains. In frontal and temporal cortices, as well as in the hippocampus, choline acetyltransferase activity and binding capacities of M2 and nicotinic binding sites are similarly decreased in these three disorders compared with age-matched control values. M1 receptor binding parameters are not significantly modified in cortical areas in patients with these disorders. In contrast, important differences between AD and PD brain tissues are found in subcortical areas such as the striatum and the thalamus. The density of M1 sites is significantly increased in striatal areas only in patients with AD, whereas densities of nicotinic sites are decreased in thalamus and striatum in PD and PD/AD, but not AD, brain tissues. The binding capacity of M2 sites is apparently unchanged in subcortical areas in all three disorders, although tendencies toward reductions are observed in the striatum of PD and PD/AD patients. Thus, although comparable alterations of various cholinergic markers are observed in cortical areas in the three neurological disorders investigated in the present study, important differences are seen in subcortical areas. This may be relevant to the respective etiological and clinical profiles of AD and PD.     

Solubilization of Serotonin1a and Serotonin1b Binding Sites from Bovine Brain

Serotonin1 (5-hydroxytryptamine1, 5-HT1) binding sites have been solubilized from bovine brain cortex using a mixture of 0.1% Nonidet P-40 and 0.3% digitonin in a low-salt buffer containing 0.1% ascorbic acid. The affinity of [3H]5-HT for the soluble cortical binding sites (2.1 nM) is identical to its affinity at membrane-bound binding sites (2.1 nM). [3H]8-Hydroxy-2-(di-n-propylamino)tetralin ([3H]DPAT), a selective 5-HT1a radioligand, also binds to soluble cortical binding sites with high affinity (1.8 nM) comparable with its affinity in the crude membranes (1.7 nM). A significant correlation exists in the rank order potency of serotonergic agents for [3H]5-HT binding and for [3H]DPAT binding to crude and soluble membranes. The density of [3H]DPAT binding sites relative to the [3H]5-HT sites in the solubilized cortical membranes (35%) corresponds well with the proportion of 5-HT1a sites in the crude membranes determined by spiperone displacement (33%), suggesting that both the 5-HT1a and 5-HT1b binding sites have been cosolubilized. [3H]5-HT binding in the soluble preparations was inhibited by GTP, suggesting that a receptor complex may have been solubilized. [3H]Spiperone-specific binding was not detectable in this preparation, suggesting that 5-HT2 sites were not cosolubilized.     

Influence of Region-Specific Alterations of Neuropeptidase Content on the Catabolic Fates of Neuropeptides in Alzheimer's Disease

Abstract: We established the cartography of 11 exo- and endopeptidases in the frontal and parietal cortices and in the cerebellum of brains of patients diagnosed with a senile dementia of the Alzheimer's type (SDAT). Comparison with those of four subjects who had died without known neurologic or psychiatric illness indicated that there existed a region-specific alteration of the peptidase contents in the disease. In the frontal area of SDAT brains, postproline dipeptidyl aminopeptidase and aminopeptidase M activities were significantly reduced. In the parietal cortex of SDAT brain, activities of three additional endopeptidases—angiotensin-converting enzyme, proline endopeptidase, and endopeptidase 24.15—were also drastically reduced. In contrast, the cerebellum displayed a set of proteolytic activities that remained unaffected in SDAT brain. The putative influence of the disease on the catabolic fates of neurotensin, neuropeptide Y, and somatostatin(1–14) was investigated. Neurotensin was catabolized at identical rates in the frontal and parietal cortices in nondemented and SDAT brains. In contrast, neuropeptide Y metabolism was slowed down in SDAT brains in the frontal but not in the parietal cortex. Finally, the degradation velocities of somatostatin(1–14) were lowered in both cortical areas of SDAT brains. It is interesting that, by means of specific peptidase inhibitors, we demonstrated that endopeptidase 24.15 participated in somatostatin(1–14) inactivation in the parietal but not in the frontal cortex. It is suggested that the lowering of the rate of somatostatin(1–14) inactivation in the parietal cortex of SDAT brains likely results from the depletion of endopeptidase 24.15 in this brain region.     

Autoradiographic localization of binding sites for galanin and VIP in small intestine

The distribution of the binding sites for [125I]galanin and [125I]iodotyrosyl VIP in sections of small intestine from rat, rabbit, guinea pig and man was investigated using in vitro receptor autoradiography. The specific binding sites for [125I]galanin were most concentrated in the myenteric plexus and longitudinal muscle layers. The distribution of [125I]VIP binding was similar, with the highest level of binding in the myenteric plexus. Some VIP binding was also seen in the mucosa. The abundance of binding sites for both peptides in the myenteric plexus suggests that this is the anatomical site of the functional antagonism between these endogenous peptides in the mammalian intestine.     

Binding of [3H]SCH23390 in Rat Brain: Regional Distribution and Effects of Assay Conditions and GTP Suggest Interactions at a D1-Like Dopamine Receptor

The compound [9-3H]SCH23390 [R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7- ol] was synthesized, and the binding of this purportedly selective antagonist of D1 3,4-dihydroxyphenylethylamine (dopamine) receptors was characterized. The regional distribution of high-affinity, specific [3H]SCH23390 binding sites in the rat brain correlated well with levels of endogenous dopamine. Receptor densities were greatest in corpus striatum, nucleus accumbens, and olfactory tubercle; intermediate levels were found in several limbic and cortical areas, whereas few sites were detectable in cerebellum, brainstem, and ol-factory bulb. Specific binding in caudate-putamen was found to be both temperature- and pH-dependent, with optima at 25-30 degrees C and pH 7.8-8.0. Scatchard or Woolf analyses of binding in caudate-putamen suggest that most of the sites are either of a single class or of classes with similar characteristics (KD = 0.7 +/- 0.1 nM; Bmax = 347 +/- 35 fmol/mg of protein). Both dopamine and cis-flupenthixol altered the slope but not the intercept of lines generated by Scatchard analysis, suggesting a competitive mode of inhibition of [3H]SCH23390 binding. Competition for binding by dopamine or the D1 agonist SKF38393 was inhibited by guanine nucleotides, whereas GTP had little effect on the competition for binding by the antagonist cis-flupenthixol. The competition for [3H]SCH23390 binding sites by dopamine was much more sensitive to GTP than was competition for [3H]spiperone binding. These data support the hypotheses that [3H]SCH23390 binds to recognition sites that differ from those previously described using other radiolabeled dopamine antagonists and that these sites have the characteristics expected of dopamine receptors.     

Galanin receptor and its ligands in the rat hippocampus

Receptors for the 29-amino-acid peptide, galanin, in membranes from the rat ventral hippocampus were examined using chloramine-T-iodinated porcine galanin as ligand. The equilibrium binding of 125I-galanin showed the presence of a high-affinity binding site (Kd = 1.91 +/- 0.40 nM). The concentration of the high-affinity-binding sites was 107 +/- 15 fmol/mg membrane protein. The on rate constant was estimated to be 2.6 +/- 0.1 M-1 min-1 at 37 degrees C. The affinity of rat galanin (differing in three amino acid residues from the porcine protein) was equal to that of porcine galanin. The 125I--galanin-binding site is a trypsin-sensitive membrane protein, which is heat-denaturated at 60 degrees C within 5 min. The effect of GTP and its analogs and of pertussis-toxin-catalyzed ADP-ribosylation on the binding of 125I-galanin suggest that the galanin receptor is coupled to an inhibitory G protein (Gi protein). 127I-galanin was shown to be a ligand with affinity equal to that of galanin in displacing 125I-galanin. The 125I-galanin-binding site in the ventral hippocampus recognizes as a ligand the tryptic fragments 1-20 and 21-29 of rat galanin and the synthetic fragments 12-29, 18-29 and 21-29 of porcine galanin. None of these afforded full inhibition of the binding of fragment 1-29 of 125I-galanin at a concentration of 1 microM.     

Solubilization and molecular characterization of active galanin receptors from rat brain.

Galanin receptors were solubilized from rat brain using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). Binding of 125I-galanin to the soluble fraction was time- and temperature-dependent, saturable, and reversible. Scatchard analysis of binding data indicated that the soluble extract contained a single class of galanin binding sites with a Kd of 0.8 nM and a Bmax of 26 fmol/mg of protein. Unlabeled galanin and its fragments galanin(2-29) and galanin(1-15) antagonized the binding of 125I-galanin to CHAPS-solubilized extracts with relative potencies similar to those observed with membrane receptors. Galanin(3-29) was found inactive. Binding of 125I-galanin to CHAPS extracts was inhibited by guanine nucleotides with the following rank order of potency: GMP-P-(NH)P greater than GTP greater than GDP. Molecular analysis of the soluble galanin receptor by covalent cross-linking of 125I-galanin to CHAPS extracts using disuccinimidyl tartrate and further identification on SDS-PAGE indicated that the soluble galanin binding site behaves as a protein of Mr 54,000. After incubation of CHAPS extracts with 125I-galanin, gel filtration on Sephacryl S-300 followed by ultracentrifugation on sucrose density gradient revealed a binding component with the following hydrodynamic parameters: Stokes radius, 5 nm; s20,w, 4.5 S; Mr, 98,000; frictional ratio, 1.6. GMP-P(NH)P treatment of CHAPS extracts gave rise to a molecular form with the following characteristics: Stokes radius, 4 nm; s20,w, 3.3 S; Mr, 57,000; frictional ratio, 1.4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of the basal nucleus of Meynert in senile dementia of Alzheimer's type. A preliminary report

The basal nuclei of Meynert are the principal sources of cholinergic innervation of the cerebral cortex. It has been hypothesized that the depressed cortical glucose metabolic activity in senile dementia of Alzheimer's type (SDAT) may result primarily from diminished activity and loss of these cells. The present study was designed to test the hypothesis that electrical stimulation of the basal nuclei would bring about clinical improvement and increase cortical glucose metabolic activity in SDAT. An electrode was implanted in September 1984 into the left basal nucleus of a 74-year-old man with SDAT. Repetitive cycles of stimulation for 9 months since have had no definite effect clinically but a follow-up positron emission tomography scan shows that cortical glucose metabolic activity was preserved in the ipsilateral temporal and parietal lobes while it declined elsewhere in the cortex.     

[3H]Nitrobenzylthioinosine Binding to the Guinea Pig CNS Nucleoside Transport System: A Pharmacological Characterization

The binding of [3H]nitrobenzylthioinosine (NBMPR) to specific membrane sites in guinea pig brain was rapid, reversible, and saturable, and was dependent upon protein concentration, pH, and temperature. Mass law analysis of the binding data for cortical membranes indicated that NBMPR bound with high affinity to a single class of sites at which the equilibrium dissociation constant (KD) for NBMPR was 0.10-0.25 nM and which possessed a maximum binding capacity (Bmax) per mg of protein of 300 fmol of NBMPR. Kinetic analysis of the site-specific binding of NBMPR yielded an independent estimate of the KD of 0.16 nM. A relatively homogeneous subcellular distribution of the sites for NBMPR was found in cortical tissue. Recognized inhibitors of nucleoside transport were potent, competitive inhibitors of the binding of NBMPR in guinea pig CNS membranes whereas benzodiazepines and phenothiazines have low affinity for the sites. NBMPR sites in guinea pig cortical membranes have characteristics similar to those for NBMPR in human erythrocytes, the occupation of which is associated with inhibition of nucleoside transport. The comparable affinities for a range of agents for sites in human erythrocytes and guinea pig CNS membranes suggest that NBMPR also binds to transport inhibitory elements of the guinea pig CNS nucleoside transport system. It is proposed that the study of the binding of NBMPR provides an effective method by which to examine drug interactions with the membrane-located nucleoside transport system in CNS membranes.     

Preferential activation by galanin 1–15 fragment of the GalR1 protomer of a GalR1–GalR2 heteroreceptor complex

The three cloned galanin receptors show a higher affinity for galanin than for galanin N-terminal fragments. Galanin fragment (1–15) binding sites were discovered in the rat Central Nervous System, especially in dorsal hippocampus, indicating a relevant role of galanin fragments in central galanin communication. The hypothesis was introduced that these N-terminal galanin fragment preferring sites are formed through the formation of GalR1–GalR2 heteromers which may play a significant role in mediating galanin fragment (1–15) signaling. In HEK293T cells evidence for the existence of GalR1–GalR2 heteroreceptor complexes were obtained with proximity ligation and BRET² assays. PLA positive blobs representing GalR1–GalR2 heteroreceptor complexes were also observed in the raphe-hippocampal system. In CRE luciferase reporter gene assays, galanin (1–15) was more potent than galanin (1–29) in inhibiting the forskolin-induced increase of luciferase activity in GalR1–GalR2 transfected cells. The inhibition of CREB by 50 nM of galanin (1–15) and of galanin (1–29) was fully counteracted by the non-selective galanin antagonist M35 and the selective GalR2 antagonist M871. These results suggested that the orthosteric agonist binding site of GalR1 protomer may have an increased affinity for the galanin (1–15) vs galanin (1–29) which can lead to its demonstrated increase in potency to inhibit CREB vs galanin (1–29). In contrast, in NFAT reporter gene assays galanin (1–29) shows a higher efficacy than galanin (1–15) in increasing Gq/11 mediated signaling over the GalR2 of these heteroreceptor complexes. This disbalance in the signaling of the GalR1–GalR2 heteroreceptor complexes induced by galanin (1–15) may contribute to depression-like actions since GalR1 agonists produce such effects.     

Organization,development, and effects of infraorbital nerve transection on galanin binding sites in the trigeminal brainstem complex

Previous experiments from this laboratory have indicated that transection of the infraorbital nerve (ION, the trigeminal \[V] branch that supplies the mystacial vibrissae follicles) at birth and in adulthood has markedly different effects on galanin immunoreactivity in the V brainstem complex. Adult nerve transection increases galanin immunoreactivity in the superficial layers of V subnucleus caudalis (SpC) only, while neonatal nerve transection results in increased galanin expression in vibrissae-related primary afferents throughout the V brainstem complex. The present study describes the distribution of binding sites for this peptide in the mature and developing V ganglion and brainstem complex and determines the effects of neonatal and adult ION damage and the associated changes in galanin levels upon their distribution and density. Galanin binding sites are densely distributed in all V brainstem subnuclei and are particularly dense in V subnucleus interpolaris and the superficial layers of SpC. They are present at birth (P-0) and their distribution is similar to that in adult animals. Transection of the ION in adulthood and examination of brainstem 7 days later indicated marked reductions in the density of galanin binding sites in the V brainstem complex. With the exception of the superficial laminae of SpC, the same reduction in density remained apparent in rats that survived 45 days after nerve cuts. Transection of the ION on P-0 resulted in no change in the density of galanin binding sites in the brainstem after either 7 or 60 days survival. These results indicate that densely distributed galanin binding sites are present in the V brainstem complex of both neonatal and adult rats, that they are located in regions not innervated by galanin-positive axons, and that their density is not significantly influenced by large lesion-induced changes in the primary afferent content of their natural ligand.     

Species Differences in the Binding of [3H]Nitrobenzylthioinosine to the Nucleoside Transport System in Mammalian Central Nervous System Membranes: Evidence for Interconvertible Conformations of the Binding Site/Transporter Complex

The binding of [3H]nitrobenzylthioinosine (NBMPR) to specific sites in CNS membranes was investigated using cortical tissue from a variety of mammalian species. Mass law analysis of the site-specific binding of NBMPR data revealed that rat, mouse, guinea pig, and dog cortical membranes each contained an apparent single class of high-affinity (KD 0.11-4.9 nM) binding sites for NBMPR; rabbit cortical membranes, however, exhibited two distinct classes of NBMPR binding sites with KD values of 0.4 nM and 13.8 nM. Dipyridamole, a potent inhibitor of nucleoside transport, produced a biphasic profile of inhibition of the binding of NBMPR to guinea pig, rabbit, and dog membranes (IC50 less than 20 nM and IC50 greater than 6 microM for NBMPR binding sites displaying high and low affinity for dipyridamole, respectively). These results are indicative of heterogeneity of NBMPR binding sites in mammalian cortical membranes. Rat and mouse cortical membranes appear to possess only one type of NBMPR binding site, which has low affinity for dipyridamole. Detailed analysis of inhibitor-induced dissociation of NBMPR from its sites in each species led to the conclusion that these multiple forms of NBMPR binding sites are different conformations of a single site associated with the CNS nucleoside transport system, rather than two distinct sites. It is also suggested that the affinity of dipyridamole for each conformation of NBMPR site indicates the susceptibility of that conformation of the nucleoside transport system to inhibition by dipyridamole.