Allosteric Modulation of Flunitrazepam Binding to Rat Brain Benzodiazepine Receptors by Methyl β-Carboline-3-Carboxylate

The inhibition of flunitrazepam (FNP) binding to rat brain benzodiazepine (BZ) receptors by methyl beta-carboline-3-carboxylate (MCC) was studied. Biphasic dissociation was observed for [3H]FNP and [3H]MCC in cerebral cortex, cerebellum, and hippocampus, although the dissociation of [3H]MCC was much faster. The dissociation rate of [3H]FNP was increased by MCC in the cerebellum, but was not altered in cerebral cortex or hippocampus. [3H]FNP binding stimulated by gamma-aminobutyric acid was enhanced in the presence of MCC in all three regions examined. These results indicate that MCC exerts these effects by interacting with allosteric sites that are different from the FNP recognition sites on the BZ receptors.     

Solubilization of adrenal medullary opioid receptors

The opioid-binding activity of digitonin extract of bovine adrenal medullary membranes was studied. [3H]Diprenorphine binding to the solubilized material was rapid and saturable. The dissociation constant of [3H]diprenorphine binding was 0.76 nM. Several opioids displaced the [3H]diprenorphine binding. The complex of [3H]diprenorphine and the solubilized binding sites was eluted as a single peak on a Sepharose 6B column and showed an apparent molecular weight of 200,000. These results indicate that active opioid receptors are solubilized with digitonin from bovine adrenal medullary membranes.     

Kinetics and Physical Parameters of Rat Brain Opioid Receptors Solubilized by Digitonin and CHAPS

Rat brain opioid receptors were solubilized with digitonin and a zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS). The yield of solubilization was 70-75% with digitonin and 30-35% with CHAPS. Kinetic and equilibrium studies performed from digitonin extracts resulted in KD values comparable with those of the membrane fractions. Two [3H]naloxone binding sites were obtained in the extracts similarly to membrane fractions. The rank order potency of drugs used in the competition experiments did not change during solubilization. The distributions of mu, delta, and kappa opioid receptor binding sites were similar in membrane and digitonin-solubilized fractions (48-50% mu, 35-37% kappa, and 13-17% delta subtypes). The hydrodynamic properties of digitonin- and CHAPS-solubilized preparations were studied by sucrose density gradient centrifugation and Sepharose-6B chromatography. In all cases, two receptor populations were identified with the following parameters: sedimentation coefficients for the digitonin extracts were 9.2S and 13.2S and for CHAPS extract 8S and 15.6S; the Stokes radii were 45 A and 65A for the digitonin extract and 31A and 76A for the CHAPS-solubilized preparation.     

The peripheral-type benzodiazepine receptor. Localization to the mitochondrial outer membrane

We have investigated the subcellular localization of the peripheral-type benzodiazepine receptor in rat adrenal gland using the high affinity ligand 3H-labeled 1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide ([3H]PK11195). The autoradiographic pattern of [3H]PK11195 binding sites in tissue sections of adrenal gland is similar to the histochemical distribution of the mitochondrial marker enzymes, cytochrome oxidase and monoamine oxidase, which are present in high concentrations only in the cortex. Subcellular fractionation studies of homogenates of adrenal gland indicate that the recovery and enrichment of [3H]PK11195 binding sites in the nuclear, mitochondrial, microsomal, and soluble fractions correlate closely with cytochrome oxidase activity, but not with markers for the nuclei, lysosomes, peroxysomes, endoplasmic reticulum, plasma membrane, or cytoplasm, indicating an association of the peripheral-type benzodiazepine receptor with the mitochondrial compartment. Titration of isolated mitochondria with digitonin results in the simultaneous release of the peripheral-type benzodiazepine receptor and of monoamine oxidase, but not cytochrome oxidase, indicating association of the peripheral-type benzodiazepine receptor with the mitochondrial outer membrane. Scatchard analysis and drug displacement studies of the binding of [3H] PK11195 to intact mitochondria and to the outer membrane-enriched digitonin extract further confirm the localization of the peripheral-type benzodiazepine receptor to the mitochondrial outer membrane.     

Differential effect of cholesterol on two types of 5-hydroxytryptamine binding sites

The specific binding of [3H]5-hydroxytryptamine ([3H]5-HT, [3H]serotonin) to rat cerebral cortex is increased approximately 1.5 to 2.0 fold by cholesterol hydrogen succinate (CHS) and is solubilized into the supernatant fraction by 12 mM CHS. [3H]5-HT binding sites can be constituted by incubating the supernatant fraction obtained from CHS-treated cerebral cortex with cerebellum in which no significant [3H]5-HT binding is detectable. The constituted [3H]5-HT binding could be displaced by unlabeled 5-HT, d-lysergic acid diethylamide (d-LSD) and spiperone as could the binding to cortex membranes. Unlabeled 5-HT, d-LSD and spiperone each inhibited specific [3H]5-HT binding to constituted binding sites by 50% at about 1 X 10(-9) M. Specific [3H]spiperone binding was not detectable in the constituted membranes. Stearic acid which is reported to have similar effects on membrane fluidity as cholesterol also increased specific [3H]5-HT binding in cortical membranes. Stearic acid does not affect specific [3H]spiperone binding. These results suggest that [3H]5-HT and [3H]spiperone binding sites are affected differently by membrane fluidity.     

Pre- Versus Postsynaptic Localization of Benzodiazepine and β-Carboline Binding Sites

[3H]Flunitrazepam (FNP) and [3H]methyl beta-carboline-3-carboxylate (MCC) binding was examined in soluble and particulate fractions from membranes solubilized with Triton X-100 or in subfractions of synaptosomal membranes obtained by a physical separation technique. Results using both methods demonstrate that benzodiazepine and beta-carboline sites reside on both pre- and postsynaptic membranes. Further, subfractionation experiments indicate that the binding sites for both ligands are unequally distributed within the synapse and among brain regions. For example, in cerebral cortical presynaptic membranes there are twice as many FNP as MCC sites whereas in postsynaptic membranes this ratio is reversed. The number of FNP and MCC sites are equal in the presynaptic fraction from cerebellum. The postsynaptic membranes derived from cerebellum have three times the number of FNP compared to MCC sites. In hippocampus this ratio varies between 1.5 and 2.8 in each subfraction. These results support the idea that benzodiazepine and beta-carboline binding sites represent different recognition sites.     

5'-Guanylylimidodiphosphate decreases affinity for agonists and apparent molecular size of a frog brain opioid receptor in digitonin solution

When assayed for specific opiate binding in the presence of 120 mM NaCl, digitonin extracts from frog (Rana ridibunda) brain membranes were found to contain about the same quantity (0.5 pmol/mg of protein) of high (Kdh = 0.4 nM) and of lower (Kdl = 15-20 nM) affinity sites for the opiate agonist [3H]etorphine. The two classes of [3H]etorphine binding sites displayed equally high (Kd = 0.3 nM) affinity for the opiate antagonist [3H]diprenorphine. 5'-Guanylylimidodiphosphate (GppNHp) selectively and potently (IC50 = 0.1 microM) inhibited high affinity binding of the tritiated agonist, and this inhibition resulted from the GppNHp-induced conversion of the high into the lower affinity sites for [3H]etorphine. Following centrifugation of the digitonin extract in sucrose gradients, opioid binding activity was found to be associated with two clearly separated macromolecular components of apparent sedimentation coefficients 11.5 and 9.7 S, respectively. The two components bound [3H]diprenorphine equally well, whereas the fast sedimented component bound [3H]etorphine better than did the slower sedimented one. In addition, labeling of the component of bigger apparent size with [3H]etorphine was considerably reduced in the presence of 50 microM GppNHp. Finally, in soluble extracts which had been (i) preincubated with and (ii) centrifuged in the presence of GppNHp, the fast sedimented component was no longer observed while there was about twice as much of the component of smaller apparent size as in control (no GppNHp) extracts. Together, these results demonstrated the existence of an opioid receptor-G protein complex which, in digitonin solution, was still amenable to regulation (dissociation) by guanine nucleotides.     

Interaction of [3H]AMP with liver cells and their plasma membranes

Specific binding of [3H]AMP to rat hepatocytes and their plasma membranes was studied. It was shown that the time course of this binding reached a maximum within the first 15 seconds. An equilibrium binding study revealed the presence of a single class of binding sites with Kd of 20 microM both in hepatocytes and in plasma membranes. The [3H]AMP binding sites were inactivated by treatment with trypsin as well as by heating. 5'-Phosphorylated derivatives of adenosine (ATP, ADP) effectively competed with [3H]AMP for the binding sites, while adenosine, beta-glycerophosphate and 3'-AMP were inactive. The binding of [3H]AMP increased by 400% in the presence of concanavalin A, a specific inhibitor of plasma membrane 5'-nucleotidase. It was concluded that the catalytic center of 5'-nucleotidase is a receptor for adenine nucleotides.     

Specific binding sites on human phagocytic blood cells for Gly-Leu-Phe and Val-Glu-Pro-Ile-Pro-Tyr, immunostimulating peptides from human milk proteins.

Two immunostimulating peptides were isolated from human milk proteins by enzymatic digestion, the tripeptide GLF and the hexapeptide VEPIPY. These peptides increased the phagocytosis of human and murine macrophages and protected mice against Klebsiella pneumoniae infection. The present study showed that this activity may be correlated to the presence of specific binding sites on human blood phagocytic cells. The receptor molecules implicated were different for the two peptides. [3H]GLF specifically bound to PMNL and monocytes, whereas [3H]VEPIPY only bound to monocytes. The leukemic promyelocytic cell line HL-60 differentiated into granulocytes or into macrophages (depending on inducer used) coroborated these results. Specific binding of [3H]GLF on plasma membrane preparations of human PMNL (20 degrees C) was saturable and Scatchard analysis indicated two classes of binding sites: high-affinity sites of Kd 2.3 +/- 1.0 nM and Bm 60 +/- 9 fmol/mg protein and low-affinity sites of Kd 26.0 +/- 3.5 nM and Bm 208 +/- 45 fmol/mg protein. [3H]GLF binding was inhibited in a concentration-dependent manner by various analogous peptides, such as LLF, GLY, LLY and RGDGLF, but not by RGD, RGDS, VEPIPY and the chemotactic peptide f-Met-Leu-Phe (f-MLF). Only at high concentrations the direct analog MLF competed with labeled GLF. An important inhibitory effect was also observed with C1q component of the complement whereas C3 and BSA were uneffective. Specific binding of [3H]VEPIPY on monocyte membranes (20 degrees C) was saturable and Scatchard analysis was consistent with one class of binding sites of Kd 3.7 +/- 0.3 nM and Bm 150 +/- 6 fmol/mg protein.     

Two distinct (-)nicotine binding sites in goldfish brain. Identification and characterization of putative neuronal nicotinic acetylcholine receptor subtypes

The binding of (-)-[3H]nicotine to membrane fragments and a detergent solubilized fraction of goldfish brain was characterized. (-)-[3H]nicotine binding was not displaced by alpha-bungarotoxin, but was displaced by (-)nicotine and carbamoylcholine with Ki of approximately 8.6 and 86 nM, respectively. Preincubation of solubilized membrane extract with alpha-bungarotoxin-coupled Sepharose resulted in the loss of approximately 50% of the (-)-[3H]nicotine binding protein from the eluent and an increase in (-)-[3H]nicotine binding to the gel compared to control, non-alpha-bungarotoxin Sepharose. 125I-alpha-bungarotoxin binding protein in the eluent from the same preincubation experiments was totally removed. In addition, incubation of the solubilized tissue extracts with alpha-bungarotoxin-coupled Sepharose resulted in an increase in the affinity for (-)-[3H]nicotine in the eluent (mean KD = 3.1) compared to control solubilized tissue extracts (KD = 6.4 nM). Specific (-)-[3H]nicotine binding sites could be eluted from the alpha-bungarotoxin-coupled Sepharose with carbamoylcholine and D-tubocurarine. Similar to previously reported 125I-alpha-bungarotoxin binding data, eye removal resulted in an approximately 40% decrease in (-)-[3H]nicotine binding in the contralateral tectum compared to that in the ipsilateral tectum. These data indicate that at least two distinct subtypes of (-)nicotine binding sites may be present in goldfish brain, one which binds alpha-bungarotoxin and (-)nicotine and another which binds only (-)nicotine.     

Axonal Transport and Metabolism of Glycoproteins in Rat Sciatic Nerve

The distribution of 5'-nucleotidase activity, dopaminergic [3H]spiperone binding sites, and [3H]quinuclidinyl benzilate (QNB) binding sites in different subcellular fractions of bovine caudate nucleus has been studied. Each activity was enriched in a microsomal (P3) preparation from that tissue. The microsomal preparation was further fractionated by different techniques. First, the P3 fraction, or a sonicated P3 fraction, was fractionated on a discontinuous sucrose density gradient. Second, the P3 fraction, or a digitonin pretreated P3 fraction, was fractionated on a continuous sucrose density gradient. The results obtained demonstrate that 5'-nucleotidase activity does not cofractionate with radioligand binding activity, although no difference between the distributions of [3H]spiperone binding and [3H]QNB binding were seen. It is concluded that the two radioligand binding activities are located on nonglial membranes.     

Differential effect of detergents on [3H]Ro 5-4864 and [3H]PK 11195 binding to peripheral-type benzodiazepine-binding sites

The present study demonstrates a differential effect of various detergent treatments on [3H]Ro 5-4864 and [3H]PK 11195 binding to peripheral benzodiazepine binding sites (PBS). Triton X-100 (0.0125%) caused a decrease of about 70% in [3H]Ro 5-4864 binding to membranes from various peripheral tissues of rat, but had only a negligible effect on [3H]PK 11195 binding. A similar effect of Triton X-100 was observed on guinea pig and rabbit kidney membranes. The decrease in [3H]Ro 5-4864 binding after treatment with Triton X-100 was apparently due to a decrease in the density of PBS, since the affinity remained unaltered. The detergents 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), Tween 20, deoxycholic acid, or digitonin (0.0125%) caused only a minor change in [3H]Ro 5-4864 and [3H]PK 11195 binding to rat kidney membranes; but when concentrations were substantially increased (0.1%), all detergents caused a decrease of at least 50% in [3H]Ro 5-4864 binding, while [3H]PK 11195 binding to rat kidney membranes remained unaffected by the first three detergents, with only a minor decrease (15%) after treatment with digitonin. These results may further support the assumption that Ro 5-4864 and PK 11195 are agonist and antagonist, respectively, of PBS and interact with two different conformations or domains in the peripheral-type benzodiazepine binding site molecule.     

Specific [3H]Piflutixol Binding to CHAPS-Solubilised Rat Striatal Preparations Involves Dopamine D-2 but Not D-1 Binding Sites

[3H]Piflutixol binding to rat striatal membrane preparations identifies both D-1 and D-2 sites. We used [3H]piflutixol to characterise those binding sites present in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)-solubilised rat striatal preparations. The specific binding of [3H]piflutixol, as defined using cis-flupenthixol, to CHAPS-solubilised rat striatal tissue was saturable and of high affinity. Specific [3H]piflutixol binding to the solubilised preparations was displaced stereoselectively by the isomers of butaclamol and to an equal extent by both cis-flupenthixol and (+/-)-sulpiride. A positive correlation was found between the capacity of a range of drugs to displace [3H]piflutixol binding and the displacement of [3H]spiperone to the same preparations. The Bmax of [3H]piflutixol binding was not different from that of [3H]spiperone binding to the same preparation. These studies suggest that, in contrast to specific binding of membrane preparations, the specific binding of [3H]piflutixol to CHAPS-solubilised preparations involves mainly D-2 sites. Specific [3H]piflutixol binding, in contrast to [3H]spiperone binding, showed only slow dissociation from soluble preparations. The binding of [3H]piflutixol to CHAPS-solubilised preparations was retained during passage through a gel filtration column. This prelabelling of solubilised striatal preparations using [3H]piflutixol may aid in the purification of CHAPS-solubilised rat striatal D-2 sites.     

Partial purification of the 5-hydroxytryptamine-reuptake system from human blood platelets using a citalopram-derived affinity resin [corrected

This paper describes a procedure for the synthesis and application of a citalopram-derived affinity resin in purifying the 5HT-reuptake system from human blood platelets. A two-step scheme has been developed for partial purification, based on wheat germ agglutinin-lectin (WGA) affinity and citalopram affinity chromatographies. Upon solubilization of the carrier with 1% digitonin, a 50-70-fold increase in specific [3H]imipramine binding activity with a 70% recovery could be accomplished through WGA-lectin chromatography. The WGA pool was then subjected to affinity chromatography on citalopram-agarose. At least 90% of the binding capacity adsorbed to the column. Specific elution using 10 microM citalopram resulted in a 22% recovery of binding activity. A 10,000-fold overall purification was obtained by using this two-step procedure. Analysis of the fractions on SDS-PAGE after 125I labeling revealed specific elution of 78- and 55-kDa proteins concomitant with the appearance of [3H]imipramine binding activity. The pharmacological profile of the partially purified reuptake system correlated well with that derived from the crude membrane-bound reuptake system, suggesting a copurification of the 5HT binding activity and [3H]imipramine binding activity.     

Physicochemical Properties of Serotonin 5-HT3 Binding Sites Solubilized from Membranes of NG 108-15 Neuroblastoma-Glioma Cells

Specific binding sites with pharmacological properties typical of serotonin 5-HT3 receptors were identified in membranes of the murine hybridoma cell line NG 108-15, using [3H]zacopride as a ligand. Optimal solubilization of these sites (yield, 50%) could be achieved using the detergent 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) at 24 mM plus 0.5 M NaCl in 25 mM Tris-HCl, pH 7.4. Specific [3H]zacopride binding to soluble sites in the 100,000-g CHAPS extract was saturable and showed characteristics (Bmax = 425 +/- 81 fmol/mg of protein; KD = 0.19 +/- 0.02 nM) closely related to those of membrane-bound sites (Bmax = 932 +/- 183 fmol/mg of protein; KD = 0.60 +/- 0.03 nM). Determination of association (k+1 = 0.17 nM min-1) and dissociation (k-1 = 0.02 min-1) rate constants for the soluble sites gave a KD value of 0.12 nM, a result consistent with that calculated from saturation studies. As assessed from the displacement potencies (IC50) of 10 different drugs, the pharmacological profile of [3H]zacopride specific binding sites was essentially the same (r = 0.99) in the CHAPS-soluble extract and in cell membranes, although some increase in the affinity for 5-HT3 antagonists (zacopride, ICS 205-930, and MDL 72222) and decrease in the affinity for 5-HT3 agonists (2-methyl-5-hydroxytryptamine and phenylbiguanide) were noted for the soluble sites. Sucrose density gradient sedimentation of the CHAPS-soluble extract gave a Svedberg coefficient of 12S for the material with [3H]zacopride specific binding capacity. Chromatographic analyses using Sephacryl S-400 and wheat germ agglutinin-agarose columns indicated marked enrichment (by 2.5- and 10-fold, respectively) in [3H]zacopride specific binding activity in the corresponding eluates compared with the starting soluble extract, a finding suggesting that both steps are of potential interest for the partial purification of solubilized 5-HT3 receptors. Two soluble materials with apparent molecular masses of approximately 600 and approximately 36 kDa were found to bind [3H]zacopride specifically in the Sephacryl S-400 eluate. Interestingly, molecular mass determination by radiation inactivation of [3H]zacopride binding sites in frozen NG 108-15 cells gave a value of approximately 35 kDa.     

Effects of an adenosine analogue administration on the striatal NMDA receptors in an experimental model of epilepsy

Specific [3H]-MK801 binding to rat NMDA receptors following the administration of the convulsant drug 3-mercaptopropionic acid (MP) and the adenosine analogue cyclopentyladenosine (CPA) was studied in striatal membrane fractions. MP administration (150 mg/kg, i.p.) caused an increase of 53% and 82% in [3H]-MK801 binding during seizure and the postseizure period respectively. Administration of CPA (2 mg/kg, i.p.) raised [3H]-MK801 binding by 72%. When CPA was administered 30 min before MP and rats sacrificed at seizure (CPA + MPc), an increase of 64%, was observed. Saturation results indicate that receptor sites increased their maximal binding capacity (Bmax) in all treatments while the apparent dissociation constant (Kd) remained unchanged. MP administration brought about an increase of 52% and 42% in [3H]-MK801 binding sites during seizure and postseizure respectively. Administration of CPA raised receptor density by 75%. When CPA was administered 30 min before MP and rats sacrificed at seizure (CPA + MPc), an increase of 62%, was observed. These results show that striatal NMDA receptors have a selective role in seizure activity in the basal ganglia and that the adenosine analogue administration may modify [3H]-MK801 binding in a way similar to that of the convulsant drug.     

Solubilization of Peripheral-Type Benzodiazepine Binding Sites from Cat Cerebral Cortex

The present study demonstrates for the first time the solubilization of peripheral-type benzodiazepine binding sites (PBS) from cat cerebral cortex. Of all detergents tested [digitonin, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), Tween 20, deoxycholate, and Triton X-100] in the presence of NaCl, the best solubilization (15% of initial activity) was obtained using 0.5% of the zwitterionic detergent CHAPS plus 2 M NaCl. Specific binding of [3H]PK 11195 to membrane-bound and solubilized PBS was saturable, yielding equilibrium dissociation constants (KD) of 1.3 +/- 0.2 and 1.9 +/- 0.3 nM, respectively, and maximal numbers of binding sites of 1,435 +/- 150 and 980 +/- 126 fmol/mg protein, respectively. The KD value of PK 11195 binding to solubilized PBS obtained from experimental kinetic analysis was 0.95 +/- 0.09 nM. The relative potencies of various compounds (PK 11195, Ro 5-4864, diazepam, flunitrazepam, clonazepam, methyl-beta-carboline-3-carboxylate, and Ro 15-1788) in displacing [3H]PK 11195 specific binding from membrane-bound and solubilized PBS were similar. Most of the solubilized binding activity was destroyed by heating at 60 degrees C for 30 min or by treatment with 2 M guanidinium chloride, which indicates the presence of a protein-binding site in the solubilized preparation. Over 85% of the solubilized binding activity was retained after 1 week at 4 degrees C, which will enable future application of purification procedures without major concern for stability of the material.     

Effects of pentylenetetrazol on GABA-A/benzodiazepine/picrotoxinin receptor complexes in rat brain regions

The effects of acute convulsive doses of pentylentetrazol (PTZ) on [³⁵S]t-butyl-bicyclophosphorothionate (TBPS), [³H]flunitrazepam (FNP), [³H]muscimol, and [³H]-aminobutyric acid (GABA) binding sites were examined in well-washed homogenates of various brain regions of rat. Except for a significant increase in the number of striatal [³⁵S]TBPS binding sites, no significant change in [³⁵S]TBPS, [³H]FNP, [³H]muscimol, and [³H]GABA binding was found in various brain regions 30 min after subcutaneous injection of PTZ at 90 or 100 mg/kg. Similarly there were no significant changes in [³⁵S]TBPS and [³H]FNP binding to unwashed P₂ membranes of cerebral cortices 30 min following administration of convulsive doses of PTZ. These experiments failed to demonstrate acute modulation of GABA-A/benzodiazepine/picrotoxinin receptor complex by PTZ in the various brain regions examined except striatum. The significance of the increased [³⁵S]TBPS binding in striatum caused by PTZ remains unclear.     

Characteristics of adenosine binding sites in atrial sarcolemmal membranes

The studies reported here involve an exploration of the sites on atrial myocyte membranes with which adenosine interacts to produce its potent physiological effects in atrial muscle. Specific, high affinity binding of the stable adenosine analogs 2-chloro[3H]adenosine (2-ClAdo) and [3H]adenosine 5'-N-ethylcarboxamide (NECA) to atrial sarcolemmal membranes was measured in kinetic and equilibrium studies at 4 degrees C and 35 degrees C. Analysis of the [3H]2-ClAdo binding isotherm indicated the presence of two classes of binding site with equilibrium Kassoc values estimated to be 5.7 X 10(7) M-1 and 2.7 X 10(6) M-1. Displacement of bound [3H]2-ClAdo by adenosine 5'-N-cyclopropylcarboxamide (NCPCA) and by several N6-substituted adenosine analogs confirmed the presence of two classes of binding site. Analysis of the [3H]NECA binding also revealed the presence of two types of binding site for this ligand. The methylxanthines isobutylmethylxanthine and theophylline displaced bound [3H]2-ClAdo whereas adenosine uptake inhibitors and several other purines showed little activity. These atrial membrane binding sites exhibit many of the characteristics of the physiological adenosine receptors studied in intact atria. Furthermore, the [3H]2-ClAdo binding sites were sensitive to treatment with proteolytic enzymes, suggesting that these sites exist on sarcolemmal membrane proteins.     

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.