Different specific binding sites of [3H]glycine and [3H]strychnine in synaptosomal membranes isolated from frog retina

Synaptosomal fractions were isolated from frog retina: a fraction enriched in photoreceptor terminals (P1) and a second one (P2) containing interneurons terminals. We compared the binding of [³H]glycine and [³H]strychine to membranes of these synaptosomes. The binding of both radioactive ligands was saturable and Na⁺-independent. [³H]Glycine bound to a single site in P1 and P2 synaptosomal fractions, with K_D=12 and 82 nM and B_Max=3.1 and 3.06 pmol/mg protein respectively. [³H]Strychnine bound to two sites in each one of the synaptosomal fractions. For P1 K_D values were 3.9 and 18.7 nM, and B_Max values were 1.1 and 7.1 pmol/mg protein, respecitively. Membranes from the P2 synaptosomal fraction showed K_D's of 0.6 and 48 nM and B_Max's of 0.4 and 4.5 pmol/mg. Specific [³H]glycine binding was displaced by -alanine, l-serine, d-serine and HA966, but not by strychnine 7-chlorokynurenic or 5,7-dichloro-kynurenic acids. Specific [³H]strychnine, binding was partially displaced by glycine and related aminoacids and totally displaced only by 2-NH₂-strychnine. Our results indicate the presence of high affinity binding sites for glycine and strychnine in frog retinal synaptosomal membranes. The pharmacological binding pattern indicates the presence of the strychnine sensitive glycine receptor as well as other sites. These might not include the NMDA receptor-associated glycine site.     

Endogenous inhibitor of GABA binding in mammalian brain

Binding of radioactive gamma-aminobutyric acid to homogenates of mammalian brain was detected by a centrifugation assay. The binding capacity of the tissue was maximal and stable with time only if the tissue was thoroughly washed to remove an endogenous inhibitor of binding. With such washed tissue, binding to total rat or cow brain appeared to involve two populations of sites in the absence of sodium ions, the major site having a dissociation constant of 150 nM and saturating at 80 pmol/g brain, and a minor site with a K_D of 20 nM and saturating at 20 pmol/g wet tissue. This sodium-independent GABA binding as a whole was localized in the crude mitochondrial, microsomal, and synaptosomal membrane fractions.     

Binding of l-[3H]glutamate to repeatedly frozen-thawed rat brain membranes

l-[³H]Glutamate binding to synaptic plasma membranes from rat cerebral cortices was carried out at 2–4°C in 50 mM Tris-acetate buffer (pH 7.4) using a microfuge centrifugation method. Binding was increased by repeated freezing-thawing and washing in either crude or partially purified synaptic membranes. Scatchard analysis showed a single binding site (dissociation constant, K_D = 697 nM; maximal binding capacity, B_max = 7.5 pmol/mg protein) in four times distilled water washed crude synaptic membrane. After six times freezing-thawing and washing, a new high affinity site (K_D1 = 26 nM, B_max1 = 1.8 pmol/mg protein) appeared and the number of low affinity site was increased with no apparent change in affinity (K_D2 = 662 nM, B_max2 = 10.5 pmol/mg protein). l-[³H]Glutamate binding was inhibited by acidic amino acid analogues that interact with N-methyl-d-aspartate- and quisqualate-sensitive sites of glutamate receptors. Binding was marginally inhibited by kainate and l-2-amino-4-phosphonobutyrate. These results indicate that repeatedly frozen-thawed and washed synaptic plasma membrane is suitable for studying the subtypes and regulation of glutamate receptors.     

SUBCELLULAR DISTRIBUTION OF [3H]QUINUCLIDINYL BENZYLATE BINDING ACTIVITY IN VERTEBRATE RETINA AND ITS RELATIONSHIP TO OTHER CHOLINERGIC MARKERS

Abstract— The subcellular distribution of binding sites for tritiated quinuclidinyl benzylate ([³H]QNB) was studied in cow retina. Primary fractions showing higher specific activity than homogenate were P₂ (synaptosomal-mitochondrial) and P₃ (microsomal). P₂ was subfractionated on a Ficoll gradient with the P₂B subfraction exhibiting the greatest enrichment in [³H]QNB binding. Similar subfractionation of P₂ on a discontinuous sucrose gradient showed that fractions of particles banding in 0.8m and in the 0.8-1.0 m -sucrose interface also exhibit the greatest enrichment of [³H]QNB binding. When subjected to Scatchard analysis, this reaction shows a density of sites equal to 0.212-0.294 pmol per mg of protein. By plotting the apparent dissociation constant (K_D) values vs protein concentration a‘true’K_D value of 0.73 nM was obtained. Only one set of binding sites was found using three different concentrations of protein. The reaction was specificially antagonized by atropine (DI₅₀= 7 nM) and scopolamine (DI₅₀= 9 nM) whereas carbamylcholine and d-tubocurarine exhibited DI⁵⁰'s of 0.4 and 0.15 mM, respectively. For P₃ the binding of [³H]QNB is characterized by one set of binding sites with n_i= 0.250 pmol per mg of protein and an apparent K_D of 8.2 nM, and a DI₅₀ for atropine of 15 nM. The [³H]QNB binding sites showed a subcellular distribution similar to that of acetylcholinesterase and choline acetyltransferase. P₁ fractions accounted for 40–60% of the total activity of the three cholinergic markers. Purification of the crude P₁ yielded an additional fraction in which the cholinergic markers showed an enrichment with respect to homogenate and P₁. Synaptosomes isolated from this fraction exhibited the unusual ultrastructure expected from nerve endings in the outer synaptic layer of retina. The possible location of the muscarinic cholinergic transmitter system in the vertebrate retina is discussed.     

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 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.     

Demonstration of alpha 1-adrenoceptors in guinea pig lung using 3H-prazosin.

³H-prazosin, a new radioligand of high specific radioactivity (33 Ci/mmol) was used to characterise postsynaptic (α₁) adrenoceptors in guinea pig lung membranes. Binding was saturable and of high affinity (K_D 0.24 nM) with a Bmax of 54 fmol/mg protein. Adrenergic agonists competed for binding in the order (?)-epinephrine > (?)-norepinephrine ? (?)-phenyl-ephrine > (?)-isoproterenol. (+)-norepinephrine was 100x less potent than (?)-norepinephrine. α-Adrenergic antagonists competed in the order prazosin > WB 4101 > indoramin > phentolamine > haloperidol > chlorpromazine ? piperoxan > yohimbine, indicating that ³H-prazosin binding is probably to α₁-adrenoceptors. Propranolol, methysergide and sulpiride inhibited binding only at high concentrations. Binding of (?)-³H-dihydroalprenolol under identical experimental conditions gave a K_D of 0.93 nM and a Bmax of 870 fmol/mg protein, giving a ratio of beta : α-adrenoceptor binding sites of 16 : 1 in this lung membrane preparation. ³H-prazosin appears to be a useful ligand in studying α₁-adrenoceptors.     

[3H] verapamil binding sites in skeletal muscle tranverse tubule membranes

[³H]verapamil binding to muscle tubule membrane has the following properties. K_D = 27 ± 5 nM and maximum binding capacity B_max = 50 ± 5 pmol/mg of protein. A 1 = 1 stoichiometry of binding was found for the ratio of [³H]verapamil versus [³H] nitrendipine binding sites. The dissociation constant found at equilibrium is near that determined from the ratio of the rate constants for association (k₁) and dissociation (k_?1). Antiarrhythmic drugs like D600, diltiazem and bepridil are competitive inhibitors of [³H]verapamil binding with K_D values between 40 and 200 nM. Dihydropyridine analogs are apparent non competitive inhibitors of [³H]verapamil binding with half-maximum inhibition values (K_0.5) between 1 and 5 nM.     

Differential Regulation of Transferrin Receptor Gene Expression in Human Hemopoietic Cells: Molecular and Cellular Aspects

Abstract

As we have shown earlier (-)¹²⁵lodocyanopindolol (¹²⁵ICYP) binding to β-adrenoceptors (β-AR) in human mononuclear leucocytes (MNL) yields evidence for the existence of high affinity (B_hiaff) and low affinity (B_loaff) binding sites. We studied the regulation of these 2 classes of binding sites during 240 min of (-)-epinephrine (EPI) infusion (0.1 μg/kg/min) (n=8) in male healthy volunteers. Saturation experiments were performed on MNL membranes with ¹²⁵ICYP over a large concentration range (1–550 pmol/l). Binding parameters were calculated by computer analysis assuming 2 classes of binding sites. We found a preinfusion value of 830±50 [sites/cell] (K_D=1.5±0.2 pmol/l) of B_hiaff binding sites and 5210±510 [sites/cell] (K_D=420±80 pmol/l) of B_loaff. During EPI infusion we observed biphasic modulation of the B_hiaff and an inverse modulation of the B_loaff. After 40 min of EPI B_hiaff increased to 1970±280 [sites/cell] (K_D=4.2±0.8 pmol/l), whereas B_loaff decreased to 2720±280 [sites/cell] (K_D=140±70 pmol/l); despite constant plasma epinephrine concentration (PEC) after 240 min of EPI B_hiaff changed to 1310±240 [sites/cell] (K_D=2.8±1.0 pmol/l) vs. 4370±760 [sites/cell] (K_D=190±100 pmol/l) B_loaff. These results suggest an interdependent inverse modulation of the 2 classes of binding sites for ¹²⁵ICYP on MNL during EPI infusion.     

Benzodiazepine receptor: Heterogeneity in rabbit brain

Binding characteristics of benzodiazepine receptors were studied with synaptosomal and microsomal membranes from rabbit brain $\text{in}$$\text{vitro}$ utilizing [methyl-³H]diazepam. In synaptosomal membranes, both high and low affinity binding sites were identified with the dissociation constants (K_d) of 4.92 nM and 83.8 nM, respectively. However, only the high affinity site was identified with K_d of 3.96 nM with microsomal membranes. Benzodiazepine binding sites appear to include at least two subpopulations of receptors, one with high affinity and another with low affinity binding site.     

alpha 1-Adrenergic receptors in guinea pig myocardium: identification by binding of a new radioligand, (3H)-prazosin.

Binding of (³H)-prazosin to adrenoceptors in guinea pig myocardial membranes was rapid, readily reversible, stereospecific and saturable. By Scatchard analysis (n = 6) Bmax was 58 fmol of (³H)-prazosin bound/mg protein and the K_D was 0.58 nm. The Hill number was 1.05. Adrenergic agonists competed with (³H)-prazosin as follows: (?)adrenaline > (?)noradrenaline > (?)phenylephrine ? ($\text{+}$)isoprenaline > (+)noradrenaline; antagonists competed in the order: non-radioactive prazosin > phentolamine ? piperoxan > yohimbine > sulpiride > propranolol. The K_D for beta-adrenoceptors assessed by (?³H)-dihydroalprenolol was 0.86 nM and the Bmax (96 fmol/mg protein) was almost twice that of alpha-adrenoceptors. (³H)-prazosin appears to be a useful radioligand for the study of post-synaptic (alpha₁) adrenoceptors in myocardial tissue.     

High-affinity binding of 3H-imipramine in brain and platelets and its relevance to the biochemistry of affective disorders

Specific high-affinity binding of ³H-imipramine has been demonstrated in the brain of various species including man. These binding sites have many of the characteristics to be expected for a pharmacological receptor and appear to be associated with the neuronal uptake mechanism for serotonin. Different antidepressant treatments like chronic administration of tricyclic antidepressants, chronic electroshock or sleep-deprivation result in decreases in the density of ³H-imipramine binding sites in normal animals. ³H-imipramine binding sites have also been found in blood platelets from different species including man. These sites are identical to those described in the brain. Clinical studies have shown that untreated severely depressed patients have a lower density of ³H-imipramine binding sites in their platelets when compared with control volunteers of the same age and sex. Longitudinal studies indicate that the low density of ³H-imipramine binding sites does not change during treatment with tricyclic antidepressant drugs and the subsequent clinical recovery from depression. ³H-imipramine binding in brain and platelets is proposed as a useful research tool in biochemical and clinical studies in affective disorders.     

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.     

Pre- and Postsynaptic Localization of 3H-Imipramine Binding Sites: Action of 5–7 Dihydroxytryptamine and Triton X-100 on Subcellular Fractions of Rat Brain

Abstract

In rats injected intraventricularly with 5–7 dihydroxytryptamine there was a considerable reduction of 5-hydroxytryptamine and 5-hydroxyindol acetic acid content in cerebral cortex and hippocampus. After cell fractionation of these structures, a 37% reduction of ³H-imipramine binding was observed in the crude mitochondrial fraction of the treated rats, that contains the synaptosomes. In purified synaptosomal membranes the reduction was about 20%. Dissolution of the presynaptic membrane with 0.1 and 0.2% Triton X-100 on the treated membranes further reduced ³H-imipramine binding respectively by 25% and 40%, values similar to those obtained on control synaptosomal membranes. These findings were further substantiated using saturation experiments for the high affinity site of ³H-imipramine. The results obtained are discussed in relation to the possible mechanism of action of antidepressant drugs at the synaptic region, and the possible postsynaptic effect is emphasized.     

Imipramine treatment differentially affects platelet 3H-imipramine binding and serotonin uptake in depressed patients

Uptake of serotonin and 3H-imipramine binding in platelets of depressed patients were investigated simultaneously with changes in clinical state. Both Vmax for serotonin uptake and Bmax for 3H-imipramine binding were significantly lower in unmedicated depressed patients with respect to normal subjects. Successful treatment with imipramine led to a significant increase in Bmax for 3H-imipramine binding, without significant change in Vmax for serotonin uptake. Bmax values increased to the normal range following complete, rather than partial clinical improvement. These data indicate that successful antidepressant treatment may increase the density of 3H-imipramine binding sites on platelets by a process which is independent of the uptake of serotonin.     

Characterization of a dopamine-sensitive [3H]LSD binding site in honeybee (Apis mellifera) brain

[³H]Lysergic acid diethylamide ([³H]LSD) binds on membrane homogenate of honeybee brain to both a dopamine-sensitive site (D-site) and a serotonin-sensitive site (S-site). Under suitable conditions the properties of the two sites can be studied separately. Specific binding of [³H]LSD to both the D-site and the S-site has high affinity and is saturable. The mean equilibrium dissociation constants (K_D) were 3.8 nM for the D- and 0.89 nM for the S-site. The densities (B_max values) of both binding sites were 1.7 pmol/mg protein for the D-site and 0.79 pmol/mg protein for the S-site. [³H]LSD binding to the D-site was reversible and reached equilibrium in about 30 min. Pharmacological displacement studies display a high binding affinity of the putative natural agonist dopamine to the D-site (K_i = 22 nM). The most potent displacers of D-site binding were lisuride, (+)-bromocriptine, chlorpromazine, S(+)-butaclamol, and 6,7-ADTN. The [³H]LSD labelled D-site seems to be G-protein coupled, since addition of the stable GTP analogue GTPγS or NaCl to the incubation medium evoked a decrease of specific [³H]LSD binding to the D-site.     

Characterization of quisqualate recognition sites in rat brain tissue usingDl-[3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and a filtration assay

The binding of [³H]AMPA (Dl--amino-3-hydroxy-5-methylisoxazole-4-propionic acid), a ligand for the putative quisqualate excitatory amino acid receptor subtype, was evaluated using centrifugation and filtration receptor binding techniques in rat brain crude synaptosomal membrane preparations. Maximal specific binding of [³H]AMPA occurred in Triton X-100 treated membranes in the presence of the chaotropic agent potassium thiocyanate (KSCN). The effects of KSCN on binding were reversible and optimal at 100 mM. Supernatant obtained from detergent-treated membranes inhibited specific [³H]AMPA and [³H]kainic acid binding, suggesting the presence of an inhibitory agent which was tentatively identified as glutamate. Using centrifugation, saturation analysis revealed two distinct binding sites in both the absence and presence of KSCN. The chaotrope was most effective in increasing binding at the low affinity binding site, enhancing the affinity (K _d) without a concommitant change in the total number of binding sites. Using filtration, a single binding site was detected in Triton-treated membranes. Like the data obtained by centrifugation, KSCN enhanced the affinity of the receptor (K _d value=10 nM) without altering the number of binding sites (B _max=1.2 pmol/mg protein). The rank order of potency of various glutamate analogs in the [³H]AMPA binding assay was quisqualate > AMPA > l-glutamate > kainate > d-glutamate, consistent with the labeling of a quisqualate-type excitatory amino acid receptor subtype.l-glutamic acid diethylester, and 2-amino-7-phosphonoheptanoic acid (AP₇) were inactive. The present technique provides a rapid, reliable assay for the evaluation of quisqualate-type excitatory amino acid agonists and/or antagonists that may be used to discover more potent and selective agents.     

α-Bungarotoxin binding properties of a central nervous system nicotinic acetylcholine receptor

High-affinity, specific binding of radiolabeled α-bungarotoxin to particulate fractions derived from rat brain shows saturability (B_max ≈ 37fmol/mg, K_D^app = 1.7 nM) and insensitivity to ionic strength, and is essentially irreversible (K_on = 5 · 10⁶ min^?1 · mol^?1; K_displacement = 1.9 · 10^?4 min^?1, τ_1/2 = 62 h). Subcellular distribution of specific sites is consistent with their location on synaptic junctional complex and post-synaptic membranes. These membrane-bound binding sites exhibit unique sensitivity to cholinergic ligands; pretreatment of membranes with cholinergic agonists (but not antagonists) induces transformation of α-bungarotoxin binding sites to a high affinity form toward agonist. The effect is most marked for the natural agonist, acetylcholine. These results strongly support the notion that the entity under study is an authentic nicotinic acetylcholine receptor.     

Binding of DL-[3H]-alpha-Amino-3-Hydroxy-5-Methyl-Isoxazole-4-Propionic Acid (AMPA) to Rat Cortex Membranes Reveals Two Sites or Affinity States

Abstract

A method for measuring [³H]-AMPA binding in rat cortex membranes is described. Specific binding was saturable and accounted for 95% of total binding at 5 nM of [³H]-AMPA. Non linear curve fitting of [³H]-AMPA saturation isotherms suggested the presence of two binding sites: the high affinity site showed a pKd of 8.26 ± 0.07 (Kd = 5.49 nM) and a Bmax of 0.19 ± 0.03 pmol/mg protein, whereas the low affinity site indicated a pKd of 7.28 ± 0.05 (Kd = 52 nM) and a Bmax of 1.30 ± 0.23 pmol/mg protein. The pharmacological profile of [³H]-AMPA binding has been determined by studying a series of compounds in binding displacement experiments: Quisqualate was the most potent inhibitor of [³H]-AMPA binding (IC₅₀ = 9.7 nM), followed by AMPA (19 nM), CNQX, DNQX and L-Glutamate (272–373 nM). Kainate was a moderate displacer (6.2 μM); Ibotenic acid and glycine were very weak inhibitors (74 and 92 μM, respectively). CPP, GAMS and L-Aspartic acid showed IC₅₀-values of over 400 μM and MK-801, DL-AP5 and NMDA were almost inactive at the maximal concentration used in our experiments.     

Specific calcium antagonist binding sites in brain

The binding of the calcium antagonist [³H] nitrendipine ([³H] NDP) to brain and heart is described and the brain site is characterized. The binding is saturable, specific and of very high affinity with K_D values of 0.16 nM in brain and 0.21 nM in heart. Our kinetic results are similar to those recently reported by two other groups (1,2), indicating a saturable, high affinity binding site in brain. In brain the binding sites are enriched in crude nuclear and synaptosomal fractions. The highest levels of binding are seen in the hippocampus, caudate and cerebral cortex with much lower levels in the cerebellum and pons. Calcium has a marked stimulatory effect on [³H] NDP binding at 10^?4 M. Addition of 0.5 mM CaCl₂ to EDTA treated membranes nearly doubles the number of binding sites. Of the many drugs and neurotransmitters tested only other calcium antagonists, i.e., verapamil, inhibit binding (IC₅₀ = 250 nM). The inhibition of [³H] NDP binding by verapamil is apparently non-competitive and not complete, suggesting that [³H] NDP binds to several sites, only some of which are inhibited by verapamil. The [³H] NDP binding site is probably a protein since it is very sensitive to trypsin, heat and sulfhydryl reagents.