Specific opioid binding sites for dermorphin in rat brain. A radioreceptor assay using the tritiated hormone as primary ligand

Dermorphin, a heptapeptide amide isolated from amphibian skin, is the most potent of the naturally occurring opioid peptides. (3H)-dermorphin (52 Ci/mmol, 1294 GBq/mmol) was prepared by catalytic tritiation of the synthetic (2,5-iodotyrosyl 1,5)-dermorphin precursor. High affinity specific binding sites for dermorphin were labeled in rat brain membranes using tritiated dermorphin as primary ligand. The binding was saturable and time-dependent. Scatchard analysis revealed a single population of non-interacting high affinity sites (Kd = 0.86 nM). Dermorphin and the specific opiate antagonist naloxone inhibited specific (3H)-dermorphin binding in a concentration dependent manner. The displacement curves could be fit to a simple competitive model assuming only one population of binding sites, with IC 50 of 1.6 nM and 3.4 nM for dermorphin and naloxone, respectively. The use of tritiated dermorphin will be helpful to ascertain unequivocally the selectivity of dermorphin for the different opioid receptor subtypes in the central nervous system.     

Effects of oxymorphazone in frogs: long lasting antinociception in vivo, and apparently irreversible binding in vitro

Oxymorphazone (at doses of 50-200 mg/kg) was found to be a relatively weak antinociceptive drug in intact frog (Rana esculenta) when acetic acid was used as pain stimulus. Frogs remained analgesic for at least 48 hrs following oxymorphazone (200 mg/kg) administration. The ligand increased the latency of wiping reflex in spinal frogs too. These effects were blocked by naloxone. In equilibrium binding studies (3H)oxymorphazone had high affinity to the opioid receptors of frog brain and spinal cord as well (apparent Kd values were 8.9 and 10.6 nM, respectively). Kinetic experiments show that only 25% of the bound (3H)oxymorphazone is readily dissociable. Preincubation of the membranes with labeled oxymorphazone results in a washing resistant inhibition of the opioid binding sites. At least 70% of the (3H)oxymorphazone specific binding is apparently irreversible after reaction at 5 nM ligand concentration, and this can be enhanced by a higher concentration of tritiated ligand.     

Identification and properties of a novel type of Na+-permeable amiloride-sensitive channel in thyroid cells

Amiloride-sensitive cationic channels are present in the apical membrane of porcine thyroid cells in primary culture. An amiloride-sensitive (K0.5 = 150 +/- 28 nM where K0.5 is the concentration of unlabelled ligand which reduces the specific binding of the same labelled ligand by 50%) 22Na+-flux component (Km for Na+ at 18 mM) has been identified which was also blocked by the potent amiloride derivative phenamil (K0.5 = 47 +/- 21 nM). The most potent inhibitor of Na+/H+ exchange, ethylisopropyl-amiloride, hardly inhibited this 22Na+-influx component at a concentration of 21 microM. Amiloride binding sites were characterized using [3H]phenamil. The tritiated ligand binds to a single family of binding sites in thyroid membranes with a Kd value of 50 +/- 10 nM and a maximal binding capacity of 5 +/- 1 pmol/mg protein. Patch-clamp experiments have directly demonstrated the existence of a phenamil- and amiloride-sensitive cationic channel, with a conductance of 2.6 pS, which is permeable to sodium, but not very selective (PNa+/PK+ = 1.2). This channel is an important element in the regulation of the resting membrane potential of thyroid cells.     

Radioligand binding properties of VV-hemorphin 7, an atypical opioid peptide

Receptor binding properties of the hemoglobin-derived nonapeptide VV-hemorphin 7 (Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe-OH) were studied using both the unlabelled form and tritium-labelled derivative of the peptide. In binding studies using selective opioid radioligands, VV-hemorphin 7 exhibited a rank order of potency of mu > kappa > delta. VV-hemorphin 7 was tritiated resulting in a compound with 1.03 TBq/mmol (27.8 Ci/mmol) specific radioactivity. The maximal number of binding sites was found to be 66.5 pmol/mg protein with an affinity of 82.1 nM in rat brain membranes. In competition studies, marked similarity was observed to the binding profile of the naturally occurring opioid heptapeptide Met-enkephalin-Arg-Phe (MERF) and its analogues to their naloxone-insensitive binding site. The common -Arg-Phe sequence at the carboxyl terminal end, which is similar to those of other endogenous peptides (-Arg-Phe-NH(2) in neuropeptide FF and FMRF-NH(2)) brings attention to the C-terminal end of the molecule and points to the possible existence of a common nonopioid binding site in mammals.     

Pharmacological characterization of two specific binding sites for neurohypophyseal hormones in hippocampal synaptic plasma membranes of the rat

Synaptic plasma membranes containing binding sites for tritiated oxytocin and arginine vasopressin were isolated from rat hippocampus. The binding parameters for oxytocin and vasopressin sites were determined and statistically analysed. The fitted curve for oxytocin binding was compatible with a model where the ligand interacts with two classes of receptors with different capacities and affinities. The sites with low binding capacity had an apparent dissociation constant at equilibrium of 1.8 nM and a maximal binding capacity of 17 fmol/mg protein. By contrast, the Scatchard plot failed to reveal a marked heterogeneity in the population of sites labelled with [3H]vasopressin with an affinity of 1.5 nM and a maximal binding capacity of 39 fmol/mg protein. The specificity of these binding sites, tested in competition experiments, revealed that these neurohypophyseal hormones labelled two distinct populations of sites. One population with a high affinity for vasopressin, oxytocin and vasotocin, the other population with a high affinity for vasopressin and vasotocin and a low affinity for oxytocin. Adenylate cyclase activity was not affected by arginine-vasopressin or oxytocin. These receptors are compared with previously characterized peripheral receptors.     

Neurotensin receptors on the rat liver plasma membranes

Neurotensin (NT) is now classified as a brain-gut peptide in the central nervous system and gastrointestinal tract. In the present study, we characterized the NT receptors on the rat liver plasma membranes. The specific binding of [3H]NT was time dependent, reversible, and saturable. Scatchard analysis of the specific binding data yielded two classes of binding sites, a high affinity site and a low affinity site. The average maximum number of binding sites (Bmax) amounted to 13.3 +/- 1.1 fmol/mg protein at high affinity site and 122.3 +/- 21.5 fmol/mg protein at low affinity site, respectively. The dissociation constant (Kd) had values of 0.39 +/- 0.01 nM at high affinity site and 8.1 +/- 1.1 nM at low affinity site, respectively. The amount of specifically bound [3H]NT was significantly reduced in the presence of mono and divalent cations, EDTA, EGTA and a peptidase inhibitor bacitracin, NT1-13 competed with [3H]NT for its binding site with an IC50 of 0.19 nM at high affinity site (0.2 nM concentration of [3H]NT) and 0.7 nM at low affinity site (4.0 nM concentration of [3H]NT). Xenopsin, a NT analogue separated from the skin of Xenopus laevis, was equipotent (IC50 0.75 nM) with NT1-13 at 4.0 nM concentration of [3H]NT. C-terminal sequence of NT contains the structure necessary for interaction with NT binding sites whereas N-terminal sequence had no binding activity. Since NT has a hyperglysemic and a hypercholesterolemic effects in rats, these NT receptors on the rat liver plasma membranes may be involved in the hyperglycemia and/or hypercholesteroremia induced by NT.     

Characteristics of 3H-substance P binding sites in rat brain membranes

Binding characteristics of 3H-Substance P (SP) were studied with rat brain membranes using a method applied to peripheral tissues by Lee and Snyder [15]. This method was well applicable to central nervous system (CNS) tissues. The results in the present study indicate that specific 3H-SP binding reaches a plateau only after 20 minutes of incubation, and the binding sites are saturable at a relatively low concentration of 3H-SP. Scatchard analysis of specific binding data reveals a single class of binding sites with a high affinity (Kd = 0.30 nM) and a low density (Bmax = 27.7 fmol/mg protein) in rat brain membranes. A Hill plot of the displacement curve of 3H-SP with unlabelled SP showed no indication for cooperativity (nH = 0.83). The relative potencies of binding of various SP fragments at 3H-SP binding sites were fairly parallel to the length of the C-terminal fragments. Neurotransmitters not structurally related to SP produced no effect on 3H-SP binding even when used at micromolar concentrations.     

Relationship Between Opioid-Receptor Occupancy and Stimulation of Low-Km GTPase in Brain Membranes

Treatment of rat brain membranes with the irreversible opioid ligand cis-3-methylfentanylisothiocyanate (Superfit) was used to reduce gradually the number of available binding sites for the delta-selective agonist [3H][D-Ser2,Leu5]enkephalin-Thr6 ([ 3H]DSLET). Subsequently, the correlation between ligand binding and low-Km GTPase was investigated. Alkylation with 10 microM and 25 microM Superfit inactivated 66% and 71% of high-affinity (KD, 1 nM) binding sites without decreasing the affinity of the remaining sites and the stimulation of low-Km GTPase by DSLET. Following exposure of the membranes to 50 microM and 75 microM Superfit, ligand binding was confined to the low-affinity (KD, 20 nM) sites. In these membranes, the delta-agonists DSLET and [D-Pen2,D-Pen5]enkephalin still stimulated low-Km GTPase, and these effects were blocked by ICI 174864 (N,N-diallyl-Tyr-AIB-AIB-Phe-Leu-OH; AIB, alpha-aminoisobutyric acid), a delta-selective antagonist. A similar relationship between low-affinity ligand binding and GTPase stimulation was observed following alkylation of the delta-opioid receptor with the non-selective irreversible antagonist beta-chlornaltrexamine in the presence of protective concentrations of DSLET. The results reveal spare receptor sites in the coupling of the delta-opioid receptor to low-Km GTPase in brain and identify low-affinity ligand binding as a functional component in the process.     

Characterization of the alpha adrenergic receptor population in hippocampus up regulated by serotonergic raphe deafferentiation

Serotonergic raphe deafferentiation elicits an up regulation of a nM (3H)WB-4101 binding site in rat hippocampus for which norepinephrine displays high affinity and prazosin displays low affinity. Guanine nucleotide affects the nM binding to hippocampal alpha-1 adrenergic receptors. Firstly, Gpp(NH)p, a nonhydrolyzable analog of GTP, inhibits (3H)WB-4101 binding at 3 nM concentration of the radioligand, the ligand concentration labelling the lower affinity, nM, binding site. Secondly, the addition of Gpp(NH)p causes recovery of the heterogeneity of binding sites lost upon preincubation of the membranes with 100 microM epinephrine, apparently by decreasing the affinity of the nM (3H)WB-4101 binding site for the adrenergic receptors. The phenomenon was still observed in the presence of saturating concentrations of the alpha-2 antagonist, yohimbine, and the beta antagonist, propranolol. The results imply that Gpp(NH)p regulates ligand binding to hippocampal alpha-1 agonist sites. It is likely that agonist and antagonist binding sites for the alpha-1 receptor exist in hippocampus with the agonist site being modulated by serotonin.     

Photoaffinity-labelling of the glycine receptor of rat spinal cord

The irreversible incorporation upon ultraviolet illumination of the glycine receptor antagonist, [3H]strychnine, into synaptic membrane fractions of rat spinal cord has been investigated. The specificity of this photoaffinity-labelling reaction for the glycine receptor was demonstrated by the following results: (a) the Kd value (9.7 nM) of the glycine-displaceable irreversible incorporation of [3H]strychnine was similar to the previously reported Kd of [3H]strychnine binding to the glycine receptor; (b) pre-illumination of the membranes with unlabelled strychnine led to a corresponding reduction in the number, but not the affinity, of reversible glycine-displaceable [3H]strychnine binding sites; (c) the ultraviolet light-induced incorporation into the membranes of [3H]strychnine was inhibited by different glycine receptor agonists; other neurotransmitter substances had little or no effect. Also, [3H]strychnine alone was shown to be stable upon illumination with ultraviolet light; this suggests that photocrosslinking of [3H]strychnine may require energy transfer from specific groups of its high-affinity receptor binding site. Upon sodium dodecyl sulphate/polyacrylamide gel electrophoresis a single labelled polypeptide with a relative molecular mass of 48000 was revealed from spinal cord membranes photoaffinity-labelled with [3H]strychnine. Spinal cord membranes photoaffinity-labelled with the gamma-aminobutyric acid receptor ligand [3H]flunitrazepam, however, gave a single polypeptide with a relative molecular mass of 5- 0000. Treatment of membranes, labelled with [3H]strychnine, by endoglycosidase H did not alter the relative molecular mass of the 48000-Mr labelled polypeptide. Trypsin treatment, on the other hand, successively produced major fragments of relative molecular masses of 42000 and 37000. Also, even after extensive treatment with trypsin or chymotrypsin, greater than or equal to 90% of the radioactivity incorporated into the labelled membranes remained membrane-associated. It is concluded that the strychnine binding site of the glycine receptor is located on a protease-inaccessible, i.e. probably hydrophobic domain of the 48000-Mr subunit.     

[3H]Vasopressin binding to rat hippocampal synaptic plasma membrane: Kinetic and pharmacological characterization

Characterization of specific vasopressin binding sites to rat hippocampal membranes has been assayed using tritiated lysine-vasopressin labelled on the tyrosyl residue. At 30°C specific [³H]vasopressin binding was saturable. The estimated equilibrium dissociation constant was 7.1 nM, the mean maximal binding capacity was 78 fmol/mg protein. Arginine-vasopressin has a high affinity (K_d = 2.8 nM) and dDAVP has a low affinity (K_d = 249 nM) for hippocampal synaptic membranes. (OH)AVP and Phe²Orn⁸VT are at least as active as AVP in inhibiting [³H]vasopressin binding. Adenylate cyclase was activated by VIP and inhibited by PIA, but not affected by lysine-vasopressin.     

Affinity labelling of alpha-adrenoceptors in intact liver cells by [3H]phenoxybenzamine.

[³H]phenoxybenzamine of high specific activity (5.3 Ci/mmol) was synthesized and its binding to isolated, viable rat liver cells was studied. Phentolamine suppressible binding of [³H]phenoxybenzamine was irreversible and saturable (EC50: 10 nM, b_max: 200 fmol/mg wet cell weight). Competition-inhibition studies showed structural and stereoselectivity compatible with α-receptors. The IC50 of unlabelled phenoxybenzamine to reduce specific binding (9 nM) or to block adrenaline-induced phosphorylase activation in the same cells (2 nM) was similar, whereas the IC50 of agonists to suppress binding was higher than their EC50's for phosphorylase activation. The results represent the first example of labelling α-adrenoceptors in intact liver cells. The sites labelled by [³H]phenoxybenzamine mediate the block of phosphorylase activation by α-adrenoceptor antagonists. However, the relationship of these sites to receptors that mediate responses to physiological, low concentrations of catecholamines remains to be clarified.     

Synthesis and binding characteristics of [3H] H-Tyr-Ticpsi[CH2-NH] Cha-Phe-OH, a highly specific and stable delta-opioid antagonist.

Substitution of the Phe3 aromatic ring in H-Tyr-Ticpsi[CH2-NH]Phe-Phe-OH with cyclohexylalanine (Cha) has been reported to result in a compound, H-Tyr-Ticpsi[CH2-NH]Cha-Phe-OH (TICP[psi]), showing substantially increased delta-opioid antagonist potency and high delta selectivity. TICP[psi] was radiolabeled by catalytic tritiation of its precursor Tyr(3',5'-I2)1TICP[psi]. Binding characteristics of the new tritiated pseudopeptide were determined using the radioligand binding assay in rat brain membranes. On the basis of the results of saturation binding studies performed at 25 degrees C, an equilibrium dissociation constant (Kd) of 0.35 nM and a receptor density (Bmax) of 112 fmol/mg protein were calculated. This new tritiated ligand exhibits high affinity for delta-opioid receptors, whereas its binding to mu and kappa receptors is weak. A study of [H3]TICP[psi] binding displacement by various receptor-selective opioids showed the following rank order of potency: delta > kappa = mu. These receptor binding characteristics of the ligand, together with its high specific radioactivity (41.3 Ci/mmol) and stability, makes it a useful tool for labeling delta-opioid receptors, both in vitro and in vivo.     

Stimulation by polydeoxyribonucleotide of histone phosphorylation by guanosine 3':5'-monophosphate-dependent protein kinase.

[³H]Dihydroalprenolol bound to a single population of high affinity sites in rat myocardial membranes when the concentration of the radioligand was below 5 nM. These sites displayed characteristics which would be expected of binding to the β-receptor. Kinetic- and Scatchard-derived dissociation constants were 0.6 and 2.0 nM, respectively. Binding was to a limited number of sites, 60 fmols/mg protein. Scatchard analysis using radioligand concentrations in excess of 5 nM resulted in concave upward plots suggestive of more than one population of binding sites. The lower affinity sites (labeled at high radioligand concentration) were non-stereospecific in nature and became a progressively larger fraction of “specific binding” as the concentration of dihydroalprenolol was increased above 5 nM.     

Sodium-dependent [3H]imipramine binding in rat hippocampus and its relationship to serotonin uptake

The relationship of [3H]imipramine recognition sites and serotonergic function was investigated by simultaneously determining the desipramine-defined and sodium-dependent components of [3H]imipramine binding and the serotonin levels and uptake in hippocampus of rats without and with selective lesion of serotonergic neurons with 5,7-dihydroxytryptamine. In control rats, the desipramine-defined [3H]imipramine binding to hippocampal membranes showed a high affinity (Kd = 2 nM) and low affinity (Kd = 31 nM) component. In contrast, the Scatchard analysis of sodium-dependent binding revealed a single class of sites of high affinity (Kd = 1.5 nM). Displacement of sodium-dependent [3H]imipramine binding by cold imipramine resulted in a steep curve best fitted to a one-site model. Sodium-dependent binding of [3H]imipramine at 4 nM concentration represented only about 38% of desipramine-defined binding. 5,7-Dihydroxytryptamine treatment resulted in marked reduction of hippocampal serotonin concentration and uptake without any changes in norepinephrine levels. Virtually only the low affinity component of desipramine-defined [3H]imipramine binding was detected by Scatchard analysis in 5,7-dihydroxytryptamine lesioned rats. The desipramine-defined "specific" [3H]imipramine binding in hippocampi of lesioned rats was decreased by 46%, whereas the sodium-dependent binding was only 18% of that seen in controls. Desipramine-defined specific binding in absence of sodium was not altered by lesion to serotonergic neurons. The results suggest that desipramine-defined specific [3H]imipramine binding may not be appropriate for studying the role of imipramine sites in relation to serotonin neuronal uptake and that determination of sodium-dependent binding components of both [3H]imipramine binding and serotonin uptake should be used in future studies.     

Interaction of ACTH synthetic fragments with rat adrenal cortex membranes.

Synthetic peptide, corresponding to the amino acid sequence 11-24 of human adrenocorticotropic hormone (ACTH), was labeled with tritium (specific activity of 22 Ci/mmol). [(3)H]ACTH (11-24) was found to bind to rat adrenal cortex membranes with high affinity and specificity (K(d) = 1.8 +/- 0.1 nM). Twenty nine fragments of ACTH (11-24) have been synthesized and their ability to inhibit the specific binding of [(3)H]ACTH (11-24) to adrenocortical membranes has been investigated. Unlabeled fragment ACTH 15-18 (KKRR) was found to replace in a concentration-dependent manner [(3)H]ACTH (11-24) in the receptor-ligand complex (K(i) = 2.3 +/- 0.2 nM). ACTH (15-18) was labeled with tritium (specific activity of 20 Ci/mmol). [(3)H]ACTH (15-18) was found to bind to rat adrenal cortex membranes with high affinity (K(d) = 2.1 +/- 0.1 nM). The specific binding of [(3)H]ACTH (15-18) was inhibited by unlabeled ACTH (11-24) (K(i) = 2.2 +/- 0.1 nM). ACTH (15-18) at the concentration range of 1-1000 nM did not affect the adenylate cyclase activity in adrenocortical membranes.     

The Na+ channel in mammalian cardiac cells. Two kinds of tetrodotoxin receptors in rat heart membranes

The properties of interaction of both tetrodotoxin (TTX) and tritiated ethylenediamine tetrodotoxin [3H] en-TTX) were studied in rat heart membranes at different stages of development and in cultured cells. Studies by electrophysiology and by 22Na+ flux measurements on cardiac cultured cells indicate that the functional form of the Na+ channel is of low affinity for TTX (250-700 nM). Binding experiments (bioassay and [3H]en-TTX binding) on cultured cardiac cells from newborn rats indicate the presence of both high and low affinity binding sites for TTX with dissociation constants (Kd) of 1.6 and 135 nM, respectively. On homogenates of hearts taken just after birth, [3H]en-TTX binding reveals no high affinity binding site for TTX but the presence of a low affinity binding site with a Kd of 125 nM. This result was confirmed by kinetic studies and competition experiments. Conversely, binding studies on homogenates and extensively purified membranes from adult ventricles reveal the presence of both high and low affinity binding sites for TTX with Kd values of 1.5 and 170 nM, respectively. The maximum binding capacity for the low affinity binding sites is 45 times higher than that of the high affinity binding sites. High affinity sites do not exist at the fetal stage or at birth, but after 5 days their number gradually increases to reach a maximum level around 45 days after birth. Conversely, the number of low affinity binding sites is essentially invariant between birth and adulthood. Monolayers of cardiac cells from hearts at 2 days after birth which have no high affinity TTX-binding sites in vivo develop both high and low affinity binding sites for TTX in vitro. The results presented here are the first direct demonstration of the coexistence in rat heart plasma membrane of two families of binding sites for TTX.     

Synthetic peptide KKRR corresponding to the human ACTH fragment 15-18 is an antagonist of the ACTH receptor

Tritium-labeled synthetic fragments of human adrenocorticotropic hormone (ACTH) [3H]ACTH (11-24) and [3H]ACTH (15-18) with a specific activity of 22 and 26 Ci/mmol, respectively, were obtained. It was found that [3H]ACTH (11-24) binds to membranes of the rat adrenal cortex with high affinity and high specificity (Kd 1.8 +/- 0.1 nM). Twenty nine fragments of ACTH (11-24) were synthesized, and their ability to inhibit the specific binding of [3H]ACTH (11-24) to adrenocortical membranes was investigated. The shortest active peptide was found to be an ACTH fragment (15-18) (KKRR) (Ki 2.3 +/- 0.2 nM), whose [3H] labeled derivative binds to rat adrenocortical membranes (Kd 2.1 +/- 0.1 nM) with a high affinity. The specific binding of [3H]ACTH-(15-18) was inhibited by 100% by unlabeled ACTH (11-24) (Ki 2.0 +/- 0.1 nM). ACTH (15-18) in the concentration range of 1-1000 nM did not affect the adenylate cyclase activity of adrenocortical membranes and, therefore, is an antagonist of the ACTH receptor.     

14 beta-(Bromoacetamido)morphine irreversibly labels mu opioid receptors in rat brain membranes

The binding properties of 14 beta-(bromoacetamido)morphine (BAM) and the ability of BAM to irreversibly inhibit opioid binding to rat brain membranes were examined to characterize the affinity and selectivity of BAM as an irreversible affinity ligand for opioid receptors. BAM had the same receptor selectivity as morphine, with a 3-5-fold decrease in affinity for the different types of opioid receptors. When brain membranes were incubated with BAM, followed by extensive washing, opioid binding was restored to control levels. However, when membranes were incubated with dithiothreitol (DTT), followed by BAM, and subsequently washed, 90% of the 0.25 nM [3H] [D-Ala2,(Me)Phe4,Gly(ol)5]enkephalin (DAGO) binding was irreversibly inhibited as a result of the specific alkylation of a sulfhydryl group at the mu binding site. This inhibition was dependent on the concentrations of both DTT and BAM. The mu receptor specificity of BAM alkylation was demonstrated by the ability of BAM alkylated membranes to still bind the delta-selective peptide [3H] [D-penicillamine2,D-penicillamine5]enkephalin (DPDPE) and (-)-[3H]bremazocine in the presence of mu and delta blockers, selective for kappa binding sites. Under conditions where 90% of the 0.25 nM [3H]DAGO binding sites were blocked, 80% of the 0.8 nM [3H]naloxone binding and 50% of the 0.25 nM 125I-labeled beta h-endorphin binding were inhibited by BAM alkylation. Morphine and naloxone partially protected the binding site from alkylation with BAM, while ligands that did not bind to the mu site did not afford protection.2+hese studies have demonstrated that when a disulfide bond     

Photoaffinity labeling of D-2 dopamine binding subunits from rat striatum, anterior pituitary and olfactory bulb with a new probe, [3H]azidosulpride

A novel photoaffinity probe [3H] azidosulpride has been developed for biochemical studies of D-2 dopamine receptors. This ligand binds to the receptors with high affinity (Kd = 3.1 +/- 0.2 nM) and, upon photoactivation, about 20% of the radioactivity bound to membranes becomes covalently incorporated. More than 90% of this irreversible binding is protectable by dopaminergic agents including D-2 selective compounds, whereas D-1 selective and non-dopaminergic compounds are ineffective. Analysis by sodium dodecylsulfate polyacrylamide gel electrophoresis reveals a single band at Mr = 85 kDa for labeled receptors in striatum, anterior pituitary or olfactory bulb, where pharmacologically distinct binding sites have been previously detected.