Is the Platelet Serotonin Transporter Different in Venous <Emphasis Type="Italic">vs</Emphasis>. Arterial Blood?

The binding of labelled paroxetine to the serotonin transporter (SERT) of platelet membranes has been studied in both venous and mixed venous/arterial blood of the rat. In addition, we studied the inhibition of paroxetine binding to SERT by quipazine and N-methyl-quipazine (NMQ). The results indicate differences in affinity for the two test drugs, quipazine and NMQ, in venous vs. mixed venous/arterial blood. This suggests different post-translational modifications of SERT in platelets of arterial vs. venous blood.     

Decrease of Serotonin Transporters in Blood Platelets after Epileptic Seizures

Serotonin transporter (SERT) was studied by [³H]-paroxetine binding in blood platelets from controls and epileptic patients with generalized convulsive seizures. The average K_D and B_Max were not different in the two cases. However, a significant decrease was found in the serotonin transporter density in the platelet membranes from patients having undergone an epileptic seizure less than 4 days before. This circumstance may indicate a homeostatic reaction to the epileptic attack.     

Subclasses of Adenosine Receptors in Brain Membranes from Adult Tissue and from Primary Cultures of Chick Embryo

Abstract: Membranes from adult chicken brain have high-affinity binding sites for N⁶-cyclohexyl[³H]adenosine (CHA) (K_D= 4 nM, Bmax = 0.6 pmol/mg protein). This CHA binding could be attributed to adenosine receptors of the A₁ type, since substituted adenosine analogs, e.g. N⁶-(l -2-phenylisopropyl)adeno sine (IC50 = 60 nM), were very potent displacers. Binding sites for 1,3-diethyl- 8-[³H]phenylxanthine (DPX) in adult brain membranes have a moderate affinity (K_D= 50 nM, Bmax = 1.5 pmol/mg). The association of DPX with these sites could be completely displaced by 8-phenyltheophylline (IC₅₀= 300 nM) and other xanthines, but only 45% of specific DPX binding could be displaced by phenylisopropyladenosine. This suggests that about half of DPX sites are putative A₁ receptors and the other half are of the A₂ type. Primary cultures of pure glial and neuronal cells from chick embryo brain were also examined for adenosine receptors. Specific binding of CHA could not be detected in these preparations, but both glial and neuronal membranes have specific sites for DPX. At a [³H]DPX concentration of 20 nM, specific binding was 50% higher (per mg protein) in glial than in neuronal membranes. The maximum binding of DPX to glial membranes (B_max= 1.6 pmol/mg) was comparable to values for adult brain, but the glial affinity (K_D= 90 nM) was somewhat less. Phenylisopropyladenosine was able to displace less than 20% of the total glial sites for DPX. This finding was in accord with the lack of CHA sites and demonstrates that A₁ receptors make little contribution to DPX binding in glial membranes. In decreasing order of potency, 8-phenyltheophylline, CHA, theophylline, caffeine, and 3-isobutyl-I-methylxanthine completely displace DPX association with glia. DPX binding to glial membranes thus appears due to a single class of receptors, which may prove to be of the A₂ type.     

Characterization, Regional Distribution, and Subcellular Distribution of 125I-Tyr1-Somatostatin Binding Sites in Rat Brain

Abstract: ¹²⁵I-Tyr₁-somatostatin binds reversibly, in a saturable manner, and with high affinity to membranes from rat brain. Kinetic and saturation data measured at equilibrium lead to K_Dvalues of 0.4 nM for cortical membranes. The binding is not affected significantly by seven neuropeptides and drugs unrelated structurally to somatostatin (SRIF) while native SRIF, Tyr₁-SRIF, and D-Trp⁸-D-Cys¹⁴-SRIF displace ¹²⁵I-Tyr₁-SRIF in a dose-dependent manner, with K_i of 0.23 nM, 0.90 nM, and 0.11 nM, respectively. Binding sites for ¹²⁵I-Tyr₁-SRIF were found in 9 out of 11 central structures; there was a significant correlation between binding capacity and endogenous SRIF levels measured by radioimmunoassay. In each of the two structures containing the most binding sites, the cortex and the preoptic area, Scatchard analysis suggests a single population of sites with apparent affinities of 0.8 nM and 1.4 nM, respectively. Subcellular fractionation of these two regions reveals that more than 60% of ¹²⁵I-Tyr₁-SRIF specific binding of the homogenate is found in the crude mitochondrial pellet (P₂), which contains synaptosomes. When P₂ is further fractionated on a discontinuous sucrose gradient, most of the initial P₂ binding is recovered from membrane fractions. Each of nine SRIF analogs, with a single alanine substitution, displaces ¹²⁵I-Tyr₁-SRIF binding on cortical membranes in the same order of potency as on adenohypophyseal membranes (r= 0.84). The data demonstrate the presence of SRIF binding sites in the rat brain, with kinetic characteristics comparable to those found in the adenohypophysis, and they provide a biochemical basis for the multiple functions of SRIF in brain.     

Visualizing Dopamine and Serotonin Transporters in the Human Brain with the Potent Cocaine Analogue [125I]RTI-55: In Vitro Binding and Autoradiographic Characterization

Abstract: The cocaine analogue RTI-55 was evaluated as a probe for in vitro labeling and localization of dopamine and serotonin transporters after death in the human brain. Kinetic, saturation, and competition binding experiments indicated complex interactions of the radioligand with the identification of multiple recognition sites. In membrane binding assays, the association of [¹²⁵I]RTI-55 at 25°C to putamen membranes was monophasic. In contrast, dissociation of [¹²⁵I]RTI-55 occurred in two phases with t_1/2 values of 9.4 and 36.5 min, respectively. Saturation analysis of [¹²⁵I]RTI-55 binding demonstrated two binding sites in the human putamen with K_D values of 0.10 ± 0.02 and 1.81 ± 0.46 nM. The binding of [¹²⁵I]RTI-55 was displaced by a wide range of cocaine analogues and monoamine uptake inhibitors. The rank order of potency demonstrated in competition assays with human putamen membranes indicates that the radioligand labels cocaine recognition sites on the dopamine transporter (mazindol > GBR 12909 > GBR 12935 > paroxetine > nisoxetine > desipramine ≥ fluoxetine > citalopram). In the human occipital cortex, [¹²⁵I]RTI-55 recognized multiple binding sites with K_D values of 0.02 ± 0.01 and 4.18 ± 0.46 nM. The rank order of potency for inhibition of [¹²⁵I]RTI-55 binding to cerebral cortex membranes (paroxetine > citalopram > GBR 12909 ≥ mazindol ≥ nisoxetine > benztropine) suggests that [¹²⁵I]RTI-55 labels the serotonin transporter in the human occipital cortex. Autoradiographic mapping of [¹²⁵I]RTI-55 revealed very high densities of cocaine recognition sites over areas known to be rich in dopaminergic innervation, including the caudate, putamen, and nucleus accumbens. Moderately elevated densities of [¹²⁵I]RTI-55 binding sites were also seen throughout the thalamus, hypothalamus, and substantia nigra. [¹²⁵I]RTI-55 binding sites were prevalent throughout the cerebral cortex and amygdala. In autoradiographic studies, the addition of the selective serotonin transport blocker citalopram completely prevented [¹²⁵I]RTI-55 labeling in the thalamus, hypothalamus, and throughout most of the cerebral cortex. In the presence of citalopram, [¹²⁵I]RTI-55 binding site densities remained elevated over the striatum and substantia nigra, with selective residual labeling also seen in the external segment of the globus pallidus and the lateral nucleus of the amygdala. These results demonstrate that in the human brain, [¹²⁵I]RTI-55 labels multiple recognition sites on dopamine and serotonin transporters.     

Pre- and Postsynaptic Localization of 3H-Imipramine Binding Sites in Rat Cerebral Cortex

Abstract

The subcellular localization of ³H-imipramine binding sites in brain was investigated with the aim of learning about the possible mechanism of action of this antidepressant. The rat cerebral cortex was submitted to a systematic fractionation and both the nuclear and the synaptosomal fractions were purified by gradient centrifugation. Using a centrifugation assay for the binding, we found that the synaptosomal membranes had the highest specific activity and showed two binding sites, one of high affinity with a K_D of 14 nM and a Bmax of 3.1 pmol per mg protein, and another of lower affinity with a K_D of 99 nM and a Bmax of 14.2 pmol per mg protein. Purified nuclei have a lower specific activity than the synaptosomal membrane, specially when expressed per g tissue. On the other hand, myelin and capillaries have few binding sites. Synaptosomal membranes were treated with 0.1, 0.2 and 0.5% Triton X-100 to dissolve the pre- and post-synaptic membrane and submitted to ³H-imipramine binding in the presence of the detergent or after washing of the residue. The results obtained suggest that although most ³H-imipramine binding sites are localized pre-synaptically, a certain proportion are post-synaptic. These findings are discussed in relation to previous studies from this laboratory on the localization of central receptors with reference to the synaptic region and to the antidepressant action of imipramine.     

In vitro transformation of dehydroepiandrosterone or its sulphate into androstenediol or its sulphate by rat brain and blood preparations

Abstract— –Enzymic transformation of [4-¹⁴C]dehydroepiandrosterone or [4-¹⁴C]dehydro-epiandrosterone sulphate to androstenediol or its sulphate occurred when incubated with a microsomal preparation of rat brain or a whole rat blood homogenate. The brain enzyme which appeared to cause this transformation had a pH optimum at 60, was NADPH₂-dependent, and had an apparent K_m of 4·6 × 10^?6m . When the subcellular fractions of rat brain were compared for transformation, microsomes had the highest specific activity, followed by the cytosol. The crude nuclear and mitochondrial fractions had no significant activity. The level of enzymic activity in the brain microsomes increased from that for rats sacrificed at 7 days of postnatal age to a maximum for rats sacrificed at 1 month of age; then the activity appeared to level off in rats older than 1 month. Microsomes obtained from the cerebellum had the highest specific activity in comparison to that obtained from the cerebral cortex, the diencephalon, and the brain stem. The incubated preparations of rat brain also converted dehydroepiandrosterone sulphate to androstenediol sulphate without hydrolysis. The enzyme in rat blood which was similar to that in the brain was also partially characterized. The blood enzyme had a pH optimum at 6–5, was nearly exclusively present in erythrocytes, was also NADPH₂-dependent, and had an apparent K_m of 2·7 × 10^?4m . The developmental pattern of the blood enzyme specific activity was similar to that of the rat brain enzyme. Upon haemolysis, most activity was recovered in the haemolysate.     

Binding of synthetic peptide TPLVTLFK to nonopioid beta‐endorphin receptor on rat brain membranes

The synthetic peptide TPLVTLFK corresponding to the sequence 12–19 of β‐endorphin (referred to as octarphin) was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from the rat brain cortex (K_d = 2.6 ± 0.2 nM ). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to α‐endorphin, γ‐endorphin, [Met⁵]enkephalin, and [Leu⁵]enkephalin, as well. The [³H]octarphin specific binding with brain membranes was inhibited by unlabeled β‐endorphin (K_i = 2.4 ± 0.2 nM ) and a selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin SLTCLVKGFY (K_i = 2.9 ± 0.2 nM ). At the same time, unlabeled octarphin completely (by 100%) inhibited the specific binding of [³H]immunorphin with membranes (K_i = 2.8 ± 0.2 nM ). Thus, octarphin binds with a high affinity and specificity to nonopioid receptor of β‐endorphin on rat brain cortex membranes. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Specific binding sites for corticosterone in isolated cells and plasma membrane from rat liver

Summary The specific binding of [³H]corticosterone to hepatocytes is a nonsaturable, reversible and temperature-dependent process. The binding to liver purified plasma membrane fraction is also specific, reversible and temperature dependent but it is saturable. Two types of independent and equivalent binding sites have been determined from hepatocytes. One of them has high affinity and low binding capacity (K _D=8.8nm andB _max=1477 fmol/mg protein) and the other one has low affinity and high binding capacity (K _D=91nm andB _max=9015 fmol/mg). In plasma membrane only one type of binding site has been characterized (K _D=11.2nm andB _max=1982 fmol/mg). As it can be deduced from displacement data obtained in hepatocytes and plasma membrane the high affinity binding sites are different from the glucocorticoid, progesterone nuclear receptors and the Na⁺,K⁺-ATPase digitalis receptor. Probably it is of the same nature that the one determinate for [³H]cortisol and [³H]corticosterone in mouse liver plasma membrane. Beta-and alpha-adrenergic antagonists as propranolol and phentolamine did not affect [³H]corticosterone binding to hepatocytes and plasma membranes; therefore, these binding sites are independent of adrenergic receptors. The binding sites in hepatocytes and plasma membranes are not exclusive for corticosterone but other steroids are also bound with very different affinities.     

Characterization of the Binding of the GABA Agonist [3H]Piperidine-4-Sulphonic Acid to Bovine Brain Synaptic Membranes

Abstract: The binding of radioactive piperidine-4-sulphonic acid ([³H]P4S) to thoroughly washed, frozen, and thawed membranes isolated from cow and rat brains has been studied. Quantitative computer analysis of the binding curves for four regions of bovine brain revealed the general presence of two binding sites. In these brain regions less satisfactory computer fits were obtained for receptor models showing one or three binding sites or negative cooperativity. With the use of Tris-citrate buffer at 0°C the two affinity classes for P4S in bovine cortex membranes revealed the following binding parameters: K_D= 17 ± 7 nM (B_max= 0.15 ± 0.07 pmol/mg protein) and K_D= 237 ± 100 nM (B_max= 0.80 ± 0.20 pmol/mg protein). Heterogeneity was also observed for association and dissociation rates of [³H]P4S. The slow binding component (k_on= 5.6 × 10⁷ or 8.8 × 10⁷ M^-1 min^-1, k_Off= 0.83 min^-1, and K_D= 14.7 or 9.4 nM, determined by two different methods in phosphate buffer containing potassium chloride) corresponds to the high-affinity component of the equilibrium binding curve (K_D= 11 nM, B_max= 0.12 pmol/mg protein in the same buffer system). The association and dissociation rates for the subpopulation of rapidly dissociating sites, apparently corresponding to the low-affinity sites, were too rapid to be measured accurately. The binding of [³H]P4S appears to involve the same two populations of sites with B_max values similar to those for [³H]GABA binding to the same tissue, although the kinetic parameters for the two ligands are somewhat different. Furthermore, comparative studies on the inhibition of [³H]P4S and [³H]GABA binding by various GABA analogues, strongly suggest that P4S binds to the GABA receptors. The different effects of P4S and GABA on benzodiazepine binding are discussed.     

High affinity specific antiestrogen binding sites are concentrated in rough microsomal membranes of rat liver

Saturation and competitive binding analyses demonstrated the presence of a high affinity (K_D = 0.92 nM), specific antiestrogen binding site (AEBS) in rat liver microsomes and at least 75% of total liver AEBS was recovered in this fraction. When microsomes were further separated into smooth and rough fractions, AEBS was concentrated in the latter. Subsequent dissociation of ribosomes from the rough membranes revealed that AEBS was associated with the membrane and not the ribosomal fraction. Antiestrogen binding activity could not be extracted from membranes with 1 M KCl or 0.5 M acetic acid but could be solubilized with sodium cholate. These data indicate that AEBS is an integral membrane component of the rough microsomal fraction of rat liver.     

Optimal Conditions for [3H]Apomorphine Binding and Anomalous Equilibrium Binding of [3H]Apomorphine and [3H]Spiperone to Rat Striatal Membranes: Involvement of Surface Phenomena Versus Multiple Binding Sites

I. Binding of [³H]apomorphine to dopaminergic receptors in rat striatum was most reproducible and clearly detectable when incubations were run at 25°C in Tris-HCl buffer, pH 7.5, containing 1 mM-EDTA and 0.01% ascorbic acid, using a washed total-membrane fraction. The receptor binding was stereospecifically inhibited by (+)-butaclamol, and dopamine agonists and antagonists showed high binding affinity for these sites. Unlabelled apomorphine inhibited an additional nonstereospecific binding site, which was unrelated to dopamine receptors. EDTA in the incubation mixture considerably lowered nonstereospecific [³H]apomorphine binding, apparently by preventing the complexation of the catechol moiety with metal ions which were demonstrated in membrane preparations. Stereospecific [³H]apomorphine binding was not detectable in the frontal cortex, whereas in the absence of EDTA much saturable nonstereospecific binding occurred. II. Kinetic patterns of stereospecific [³H]spiperone and [³H] apomorphine binding to rat striatal membranes and the inhibition patterns of a dopamine antagonist and an agonist were evaluated at different temperatures in high-ionic-strength Tris buffer with salts added and low-ionic-strength Tris buffer with EDTA. Apparent K_D, values of spiperone decreased with decreasing tissue concentrations. K_D, values of both spiperone and apomorphine were little influenced by temperature changes. Scatchard plots of the stereospecific binding changed from linear to curved; the amount of nonstereospecific binding of the ³H ligands varied considerably, but in opposite directions for spiperone and apomorphine in the different buffers. In various assay conditions, interactions between agonists, and between antagonists, appeared fully competitive, but agonist-antagonist interactions were of mixed type. The anomalous binding patterns are interpreted in terms of surface phenomena occurring upon reactions of a ligand with complex physicochemical properties and nonsolubilized sites on membranes suspended in a buffered aqueous solution. It is concluded that anomalous binding patterns are not necessarily an indication of binding to multiple sites or involvement of distinct receptors for high-affinity agonist and antagonist binding.     

Occurrence of Sialyltransferase Activity in the Synaptosomal Membranes Prepared from Calf Brain Cortex

The possible occurrence of sialyltransferase activity in the plasma membranes surrounding nerve endings (synaptosomal membranes) was studied, using calf brain cortex. The synaptosomal membranes were prepared by an improved procedure which provided: (a) a ?nerve ending fraction” consisting of at least 85% well-preserved nerve endings and containing only small quantities of membranes of intracellular origin; (b) a ?synaptosomal membrane fraction” carrying high amounts of authentic plasma membrane markers (Na⁺-K⁺ ATPase, 5′-nucleotidase, sialidase, gangliosides) with values of specific activity four to fivefold higher than those in the ?nerve ending fraction” and very small amounts of cerebroside sulphotransferase, marker of the Golgi apparatus, and of other markers of intracellular membranes (rotenone-insensitive NADH and NADPH: cytochrome c reductases), the specific activities of which were, respectively, 0.5- and 0.7-fold that in the ?nerve ending fraction”. Thus the preparation of synaptosomal membranes used had the characteristics of plasma membranes and carried a negligible contamination of membranes of intracellular origin. The distribution of sialyltransferase activity in the main brain subcellular fractions (microsomes; P₂ fraction; nerve ending fraction; mitochondria) resembled most closely that of thiamine pyrophosphatase, the enzyme known to be linked to the Golgi apparatus and the plasma membranes and of acetylcholine esterase, the enzyme known to be linked to either intracellular or plasma membranes. The enrichment of sialyltransferase activity in the ?synaptosomal membrane fraction”, referred to the ?nerve ending fraction”, was practically the same as that exhibited by authentic plasma membrane markers. All this is consistent with the hypothesis that in calf brain cortex sialyltransferase has two different subcellular locations: one at the level of intracellular structures, most likely the Golgi apparatus (as described by other authors), the other in the synaptosomal plasma membranes. The basic properties (pH optimum, V/S, V/t and V/protein relationships) and detergent requirements of the synaptosomal membrane-bound sialyltransferase were established. The highest enzyme activities were recorded on exogenous acceptors, lactosylceramide and ds -fetuin. The K_m values for CMP-NeuNAc were different using lactosylceramide and ds -fetuin as acceptor substrates (0.57 and 0.135 mm , respectively); the thermal stability of the enzyme acting on glycolipid acceptor was higher than that on the glycoprotein acceptor; the effect of detergents was different when using glycoprotein from glycolipid acceptors; no competition was observed between lactosylceramide and ds -fetuin. Thus the synaptosomal membranes carry at least two different sialyltransferase activities: one acting on lactosylceramide (and glycolipid acceptors), the other working on ds -fetuin (and glycoprotein acceptors). Ganglioside GM₃ was recognized as the product of synaptosomal membrane-bound sialyltransferase activity working on lactosylceramide as acceptor substrate.     

Hypodensity of platelet serotonin uptake sites in posttraumatic stress disorder: Associated clinical features

We have previously reported that binding to blood platelets of paroxetine, a selective serotonin (5-HT) reuptake inhibitor which binds to 5-HT uptake sites, is decreased in patients with posttraumatic stress disorder (PTSD). Specifically, we found a lower number of platelet ³H-paroxetine binding sites (B_max) and a lower dissociation constant (K_d) for ³H-paroxetine binding in combat veterans with PTSD compared to normal control subjects. In the current study we assessed the relationship of platelet ³H-paroxetine binding to clinical features in 41 Vietnam combat veterans with SCID-diagnosed PTSD. The results indicated that B_max of platelet ³H-paroxetine binding was negatively correlated with both state and trait anxiety, as well as with depressive and overall PTSD symptoms. However, there was no evidence that platelet ³H-paroxetine binding differed as a function of comorbid psychiatric diagnoses including major depression, other anxiety disorders and substance abuse in these patients.     

Isolation, identification and synthesis of an endogenous arachidonic amide that inhibits calcium channel antagonist 1,4-dihydropyridine binding

This study was part of a broad search for endogenous regulators of L-type calcium channels. The screening for active fractions was done by measuring inhibition [³H]1,4-dihydropyridine (DHP) binding to rat cardiac and cortex membranes. An inhibitory fraction, termed lyophilized brain hexane-extractable inhibitor (LBHI), was isolated from hexane extracts of lyophilized calf brain. The active substance was purified by a series of chromatographic steps. ¹³C nuclear magnetic resonance (NMR), ¹H coherence spectroscopy (COSY) NMR and fast atom bombardment (FAB) mass spectroscopy suggested that LBHI was N-arachidonic acid-2-hydroxyethylamide. Synthesis of this substance and subsequent high performance liquid chromatography (HPLC) and NMR analysis confirmed this structure. Synthetic LBHI (SLBHI) inhibited [³H]DHP binding to rat cortex membranes with an IC₅₀ value of 15 μM and a Hill coefficient of 2. Saturation analysis in the presence of SLBHI showed a change in K_D (equilibrium dissociation constant), but not maximal binding capacity (B_max). SLBHI produced an increased dissociation rate, which, along with the Hill slope of > 1, suggested a non-competitive interacton with the DHP binding site. The results suggest that arachidonic acid derivatives may be endogenous modifiers of the DHP calcium antagonist binding site.     

An [3H]oxotremorine binding method reveals regulatory changes by guanine nucleotides in cholinergic muscarinic receptors of cerebral cortex

A rapid, reliable filtration method for [³H]oxotremorine binding to membranes of the cerebral cortex that allows the direct study of regulation by guanine nucleotides of muscarinic receptors was developed. [³H]Oxotremorine binds to cerebral cortex membranes with high affinity (K _D, 1.9 nM) and low capacity (B _max, 187 pmol/g protein). These sites, which represent only about 18% of those labeled with [³H]quinuclidinyl benzilate, constitute a population of GTP-sensitive binding sites. Association and dissociation binding experiments revealed a similar value ofK _D (2.3 nM). Displacement studies with 1–4000 nM oxotremorine showed the existence of a second binding site of low affinity (K _D, 1.2 M) and large capacity (B _max, 1904 pmol/g protein). Gpp(NH)p, added in vitro, produced a striking inhibition of [³H]oxotremorine binding with an IC 50 of 0.3 M. Saturation assays, in the presence of 0.5 M Gpp(NH)p, revealed a non-competitive inhibition of the binding with little change in affinity. These results are discussed from the viewpoint of conflicting reports in the literature about guanine nucleotide regulation of muscarinic receptors in reconstituted systems and membranes from different tissues.     

Substance P: Regional distribution and specific binding to synaptic membranes in rabbit central nervous system

Regional distribution of endogenous substance P and the specific [³H]substance P binding to synaptic membranes in rabbit central nervous system were investigated. The highest level of substance P was found in mesencephalon, followed by diencephalon, corpus striatum, hippocampus, pons-medulla and cortex. In spinal cord, much higher amount of substance P existed in dorsal half than in ventral half. Most of the substance P present in the areas enriched in substance P was located in crude mitochondrial P₂ fractions containing nerve endings. A saturable, high affinity, specific binding of [³H]substance P in synaptic membranes was found. The apparent maximal number of binding sites was 95.7 fmole/mg protein, while the dissociation constant (K_D) was 2.74 nM. The binding was displaced by substance P sequence fragments and the related peptides with relative potencies generally parallelizing their pharmacological activities. The distribution of such specific binding generally correlated with endogenous substance P. The presence of such binding sites for substance P in synaptic membranes suggests a possible role for substance P as a transmitter or modulator of neural function.     

Properties of [3H]diazepam binding sites on rat blood platelets

Intact rat blood platelets are shown to possess benzodiazepine binding sites of the peripheral type, binding of [³H]diazepam being strongly inhibited by Ro5-4864 (K_i = 3.6 ± 0.5 nM) but only weakly inhibited by clonazepam (K_i = 35.1 ± 18.2 μM). Binding of [³H]diazepam is specific and saturable. Scatchard analysis reveals a single class of binding sites with K_D = 14.7 ± 1.0 nM and B_max = 564 ± 75 fmoles/10⁸ platelets. The Hill coefficient is 0.94, indicating a lack of binding site heterogeneity or negative cooperativity. Binding reaches equiliibrium at 6 min, with k₊₁ = 2.9 × 10⁷ M^?1 min^?1, and is rapidly reversible ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\; =\; 2.2\; </mtext><mtext>min</mtext>}}$ with K_?1 = 0.315 min^?1. K_D derived from the rate constants agrees with that estimated by Scatchard analysis. K_D of the crude membrane fraction of platelets is also close to that of intact platelets. Binding of [³H]diazepam is linear with platelet number (between 0.25–2 × 10⁸ platelets), is temperature sensitive with maximum binding at 0°C, and has a broad optimal pH range between pH 5–9.     

Dopamine Receptors in Subcellular Fractions from Bovine Caudate: Enrichment of [3H]Spiperone Binding in a Postsynaptic Membrane Fraction

Abstract: Specific binding of tritiated dopamine, spiperone, and N-propylnorapomorphine was examined in subcellular fractions from bovine caudate nucleus. All fractions contained at least two sets of specific binding sites for [³H]spiperone (K_{D 1}^aPP= 0.2 nM, K_{D 2}^aPP= 2.2 nM), the higher affinity sites accounting for one-third to one-eighth of the total. [³H]Spiperone binding was slightly enriched over the total particulate fraction in P₂, P₃, SPM, and a crude fraction of synaptic mitochondria. A microsomal subfraction (P₃B₂) exhibited the highest specific binding capacity obtained, representing a fourfold enrichment over the total particulate fraction. [³H]Dopamine exhibited apparent binding to a single class of high-affinity sites in all fractions examined (K_D^aPP= 4.0 nM). A greater than twofold enrichment was observed in all fractions except myelin and P₃, with a fivefold enrichment in SPM and P₃B₂. At least two classes of receptors were labeled by [³H]-N-propylnorapomorphine (K_{D 1}^aPP= 0.55 nM, K_{D 2}^aPP= 20 nM), using 50 nM-spiperone together with 100 nM-dopamine to define nonspecific binding. Although binding to the higher affinity site was displaced by spiperone, and lower affinity binding by dopamine, comparison of receptor densities with values obtained by using [³H]spiperone and [³H]dopamine directly suggested that [³H]-N-propylnorapomorphine labeled additional sites. We have also examined a postsynaptic membrane (PSM) fraction obtained from SPM by successive extraction with salt and EGTA followed by sonication and separation on a density gradient. [³H]Spiperone binding in PSM was enriched two- to threefold over unfractionated SPM with a concomitant decrease in [³H]dopamine binding. The enrichment in spiperone receptors was almost entirely due to an increase in the number of lower affinity binding sites, suggesting that these sites may be associated with the postsynaptic membrane.     

Endogenous and Exogenous Regulation of Human α- and β-Adrenergic Receptors

Abstract

Regulation of human β2-adrenergic receptors in lymphocytes (determined by (±)-125 iodocyanopindolol (ICYP) binding) and α₂-adrenergic receptors in platelets (determined by ³H-yohimbine binding) was studied. While α₂-adrenergic receptor number did not change with age, a significant negative correlation between the number of α₂-adrenergic receptors and age was found; plasma catecholamines, on the contrary, were elevated in the elderly.In healthy women during normal menstrual cycle the number of α₂-adrenergic receptors decreased with increasing plasma estradiol levels.Incubation of lymphocyte membranes with isoprenaline (100 μM) and of platelet membranes with clonidine (1-100 μM) led to a reduction of the number of β₂- and α₂-receptors, respectively, without changes in the K_D-values. Treatment of hypertensive patients with clonidine (3x150 μg/die) for 7 days reduced the number of α₂-adrenergic receptors in platelets. In platelet membranes from such treated patients inhibition of ³H-yohimbine binding by clonidine and adrenaline was not affected by 10-⁴MGTP. It is concluded, that human α- and β-adrenergic receptors undergo regulatory mechanisms similar to those recently described for adrenergic receptors in a variety of animal models.