Decreased [3H]serotonin and [3H]spiperone binding consequent to lipid peroxidation in rat cortical membranes

The effect of lipid peroxidation on two types of serotonin binding sites was investigated. Incubation of rat cortical membranes with an ascobate-dependent peroxidizing system resulted in the formation of malonyldialdehyde and significant decreases in the specific binding of [³H]serotonin and [³H]spiperone to the treated membranes. When ascorbate concentrations were varied from 0.025 to 6.0 mM, malonyldialdehyde production increased to a maximum at 0.5 mM ascorbate and then declined. Conversely, the specific binding of [³H]serotonin and [³H]spiperone decreased to a minimum at 0.5 mM ascorbate and then increased. Regression analysis of the data revealed that the decrease in the two binding sites was linearly correlated to lipid peroxidation.     

High affinity binding sites for [3H]-nitrendipine in rabbit uterine smooth muscle

The binding properties of the high affinity binding site for [³H]-nitrendipine on rabbit uteri membranes were investigated. The specific component had a dissociation constant of 0.46±.07nM and a maximal binding of 175±11 pmol/mg. A variety of other calcium channel blockers inhibited the binding of [³H]nitrendipine with varying potencies. Flunarizine demonstrated a high potency (IC₅₀ = 0.30 uM) in inhibiting binding while verapamil and bepridil were less potent with IC₅₀'s of approximately 0.6–1.5 uM. Diltiazem did not displace nitrendipine even at very high concentrations. Verapamil displayed negative cooperative inhibition suggesting that a second site exists on uterine membranes for the binding of other calcium channel blockers.     

Decrease in lymphocyte [3H]spiroperidol binding sites in parkinsonism

[³H]spiroperidol binding has been measured in lymphocytes from patients with Parkinson's disease and age matched healthy volunteers. A dramatic decrease (73%) in the number of binding sites (^Bmax) without any variation of the affinity (^KD) has been observed in Parkinsonian patients. This decrease in ^Bmax is linearly correlated with the degree of disability of the Parkinsonian patients (r = 0.891, p <0.001). This decrease appeared to be relatively selective since no variation was observed with patients suffering of other neurological disorders (vascular hemiplegia, Alzeihmer's disease, olivopontocerebellar degeneration, Huntington's chorea).     

Intracellular disposition of [3H]-cocaine, [3H]-norcocaine, [3H]-benzoylecgonine and [3H]-benzoylnorecgonine in the brain of rats

[³H]-cocaine, [³H]-norcocaine, [³H]-benzoylecgonine and [³H]-benzoylnorecgonine were administered i.c. in equi-potent pharmacologic doses and the intracellular disposition and metabolism of each drug determined. Norcocaine and cocaine rapidly entered and egressed from the brain so that 4.8–6.1% of the radioactivity present in brain at one minute was observed at 30 minutes. The highest levels of subcellular radioactivity were generally found in the microsomal plus supernatant, followed by the nuclear and shocked mitochondrial fractions. No apparent localization of the radioactivity occured in synaptic membranes. The brain/plasma (B/P) ratio curves for cocaine and norcocaine were similar; however, the norcocaine values were considerably higher at each time interval. Benzoylecgonine and benzoylnorecgonine had higher comparative B/P ratios than cocaine or norcocaine and persisted in brain for a longer period of time so that 0.6–2.1% of the radioactivity present in brain at 1 hour was detected at 24 hours. Cocaine and norcocaine were extensively metabolized to the benzoylmetabolites. Benzoylecgonine was metabolized to benzoylnorecgonine and benzoylnorecgonine was unmetabolized. The brain disposition data and B/P ratios agreed quite well with the overall pharmacologic action of cocaine and its metabolites.     

Identification of stereospecific [3H]spiroperidol binding sites in mammalian lymphocytes

Direct binding to intact rat lymphocytes has been shown for the potent dopaminergic antagonist [³H]spiroperidol. The specific binding is saturable with two components (K_D1 = 1.9 nM, K_D2 = 36.2 nM). Determination of the K_D by kinetic studies measuring rate constants for association and dissociation provided K_D values similar to those obtained in equilibrium experiments. The specific binding is proportional to cell concentration and temperature dependent with a maximum at 37°C. [³H]spiroperidol binding is stereospecific since (+)butaclamol was more effective than (?)butaclamol. The relative potencies of different antidopaminergic agents in competing for [³H]spiroperidol binding sites parallel their activity in the striatum. Dopaminergic receptors have also been demonstrated in other mammalian lymphocytes (rabbit, dog, human). Lymphocyte dopaminergic receptors could be implicated in lymphocytes mediated immune response.     

Binding of [3H]cytochalasin B and [3H]colchicine to isolated liver plasma membranes

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([³H]cytochalasin B and [³H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [³H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB³H₄. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insentive to changes of pH or ionic strength. At 10^?6 M [³H]cytochalasin B, glucose or p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 Å) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10^?5 M [³H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [³H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes.[³H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [³H]cytochalasin B binding and cytochalasin B stimulated ³H colchicine binding. [³H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

[3H]muscimol binding in the rat retina

Crude membrane fractions were prepared from rat retinae and used to study the specific binding of [³H]muscimol, a potent GABA agonist. Specific [³H]muscimol binding was enhanced 2–3 fold by pretreatment of the membranes with 0.025% Triton X-100. Two muscimol binding sites were demonstrated with K_D values of 4.4 and 12.3 nM. GABA, muscimol, and 3-aminopropanesulfonic acid were the most potent inhibitors of specific [³H]muscimol binding with K_I values of 15, 10, and 50 nM, respectively. These data are consistent with binding to the synaptic GABA receptor.     

Effect of phencyclidine on [3H]QNB binding

Intraperitoneal injection of phencyclidine before intravenous injection of [³H] Quinuclidinyl benzilate (QNE, 1.6 μg/kg) significantly increased the amount of radioactivity found in the brains of female C57BL/6J mice one hour after the ³H-QNB administration. This effect was found in hypothalamus, cortex, hippocampus and striatum and was decreased by pretreatment of the animal with atropine. The magnitude of the enhancement varied as a function of dose but did not change across the time span studied. These data are in contrast to our findings and those of others of inhibition of the specific binding of ³H-QNB to muscarinic cholinergic receptors by PCP $\text{in vitro}$. When atropine or PCP was administered $\text{in vivo}$ and the tissue later analyzed $\text{in vitro}$, no effects of the drugs were observed on ³H-QNB binding. The reasons for the differences remain a matter of speculation.     

2-Nitroimipramine: A selective irreversible inhibitor of [3H] serotonin uptake and [3H] imipramine binding in platelets

The effect(s) of a new imipramine analogue, 2-nitroimipramine, on high affinity [³H] imipramine binding and [³H] serotonin uptake in human platelets were studied. 2-Nitroimipramine was found not only to be a very potent inhibitor of [³H] imipramine binding and [³H] serotonin uptake but was found to irreversibly inhibit binding and uptake simultaneously. This finding supports previous observations from our laboratory and others that high affinity imipramine binding labels serotonin uptake or transport sites. 2-Nitroimipramine should prove an important tool for subsequent studies of the molecular mechanism(s) involved in the transport of serotonin and the binding of imipramine to platelet and brain membranes.     

Localization of [3H]-imipramine binding sites in rat brain by light microscopic autoradiography

The binding characteristics of [³H]-imipramine in slide mounted tissue sections of rat forebrain have been studied to ascertain the optimal binding conditions for labeling the sites prior to autoradiographic localization. The conditions for the experiments and the kinetics of the imipramine binding correspond reasonably well with those used in membrane preparations to initially define the imipramine binding site. Subsequent labeling of sections, using these parameters, allowed the autoradiographic localization of high concentrations of imipramine binding sites in such areas as the cerebral cortex, striatum, and several limbic and visual system structures. In addition, there was a marked overlap between regions demonstrating imipramine binding and areas known to be innervated by serotonergic neurons. This study outlines the potential sites of action of imipramine in the brain and defines areas for future investigations which attempt to localize brain regions involved in the etiology of depression and areas involved in the side effects of antidepressant drug therapy.     

Light microscopic autoradiographic visualization of [3H]-arginine vasopressin binding sites in rat brain

Specific [³H]-arginine vasopressin ([³H]-AVP) binding sites were identified in the rat brain by light microscopic autoradiography. Discrete intrahypothalamic nuclei were densely labelled by [³H]-AVP. High specific binding was observed in the paraventricular and supraoptic nuclei. These binding sites may represent specific receptors for AVP, postulated to exist in the mammalian central nervous system.     

Elevation of [3H]cimetidine binding by CuCl2 in brain membranes of rats

Influences of dithiothreitol (DTT), p-chloromercuriphenyl sulfonate (PCMPS) and ascorbate on CuCl₂-induced elevation of [³H]cimetidine binding were investigated in brain membranes of rats. CuCl₂ (10–500 μM) elevated specific [³H]cimetidine binding in a concentration-dependent manner. There were two types of [³H]cimetidine binding in the presence of 50 μM CuCl₂: high affinity binding with K_d = 1.97 nM and low affinity with K_d = 21.6 nM. PCMPS (10 and 100 μM) reduced the binding in both media with and without CuCl₂. DTT (1–30 μM) or ascorbate (0.1 and 1.0 mM) markedly elevated the binding in the presence of CuCl₂ but showed no effect and ascorbate rather inhibited the binding in the absence of CuCl₂. DTT (0.1 mM) diminished the binding in the presence and absence of CuCl₂. CuCl₂ (50 μM) significantly (P < 0.01) increased the IC₅₀ of histamine for [³H]cimetidine binding and the effect was greater than that from 100 μM GTP. It is suggested that sulfhydryl groups sensitive to PCMPS could interact with Cu²⁺ and thus be involved in an elevation of cimetidine binding. Cu²⁺ seems to regulate affinity of agonist binding for cimetidine binding sites presumably by acting on cimetidine binding sites and/or GTP binding regulatory proteins.     

Demonstration of specific high affinity binding sites for [3H] imipramine in human brain

High affinity binding sites (Kd = 1.7 nM) for [³H] imipramine have been characterized in membranes prepared from human brain. The binding of [³H] imipramine was found to be saturable, reversible, and inhibited by pharmacologically active tricyclic antidepressants. Other psychoactive compounds as well as most neurotransmitter substances were ineffective in inhibiting [³H] imipramine binding at concentrations up to 10 μM. The hypothalamus was found to contain a relatively high density of these binding sites and is enriched approximately 4-fold when compared to cerebral and cerebellar cortex. A very good correlation (r = 0.97) p < 0.001 was found between the abilities of a series of clinically active tricyclic antidepressants in displacing specifically bound [³H] imipramine from human brain and platelet membranes, suggesting that the binding sites from these two tissues are very similar.     

Subcellular distribution of [3H]-prostaglandin E2 binding sites in porcine gastric mucosa

The distribution of PGE₂ binding sites in four subcellular fractions (F1–F4) from porcine fundic mucosa obtained by gradient centrifugation was examined. Binding of HPGE₂ to fractions F2–F4 was specific, dissociable, saturable and pH dependent. A significant degree of specific binding was not evident in F1. The Scatchard analysis of binding to F2 and F3 revealed heterogenous populations of binding sites with similar dissociation constants but greater concentrations of binding sites than was evident in the initial 30,000 xg homogenate protein. A single class of low affinity binding sites was evident in F4. The ratio of total: nonspecific binding was approximately equal in F2 and F3. The ratio was considerably smaller in F4. The activity of 5' nucleotidase the marker enzyme for plasma membranes followed this ratio. There was no correlation between the binding ratio and marker enzyme activities for mitochondrial membranes and endoplasmic reticulum. These data suggest that high affinity PGE₂ binding sites occur predominantly on the plasma membrane from gastric mucosal tissue.     

Pituitary binding sites for [3H]-labelled luteinizing hormone releasing factor (LRF)

Normal rat anterior pituitary cells in culture possess two binding sites for [³H]LRF. One is a high affinity (2 × 10^?9M), low capacity site which corresponds to the half-maximal biological potency of LRF. The second site has low affinity (2 × 10^?8M) and an enormously higher capacity to bind LRF. This second site shows only partial specificity in that inactive LRF-analogues, although not TRF, compete for binding of [³H]LRF. The high affinity site is competible by LRF, LRF-agonists and LRF-antagonists even in the presence of an excess of an inactive LRF-analogue which saturates the low affinity binding site.     

Preferential release of newly synthesized [3H]GABA from striatal slices to preloaded [3H]GABA

The release of [³H]GABA which is newly synthesized from [³H]l-glutamic acid (GLU) has been examined using striatal slices obtained from the rat brain. It was found that 8–10% of [³H]GLU transported was converted to [³H]GABA during the incubation of striatal slices in the presence of nipecotic acid (5 × 10^?5 M). Nipecotic acid was added to the medium in order to prevent possible reuptake of [³H]GABA released during its synthesis, and it was found to have no significant effect on the formation of [³H]GABA from [³H]GLU as well as on the uptake of [³H]GLU. The application of high potassium (60 mM) stimulation exhibited a significant enhancement of the release of this newly synthesized [³H]GABA in a Ca²⁺ dependent manner. Kinetic analysis revealed that the evoked release of newly synthesized [³H]GABA was approximately two times greater than that of previously-loaded [³H]GABA, whereas no significant difference was observed in the spontaneous release. An immobilization stress in water failed to affect the release of newly synthesized [³H]GABA from striatal slices despite the occurrence of a significant enhancement of GABA formation in this structure.These results suggest that newly synthesized GABA may be preferentially released from its nerve terminals in response to the excitation of neurons at least in the striatum as compared with previously accumulated GABA.     

[3H]norepinephrine binding and lipolysis by isolated fat cells

[³H]norepinephrine was shown to bind to specific sites on isolated fat cells. A Scatchard plot of norepinephrine binding showed two apparent K_a of 1.9 · 10⁶ and 1.2 · 10⁵ LM^?. 1.4 · 10^?4 M Norepinephrine covalently-linked to agarose beads reduced [³H]norepinephrine binding by over 50%. Several structurally related drugs were compared as inhibitors of [³H]norepinephrine binding and as stimulators of lipolysis in preparations of similarly prepared cells. Dose-response curves for norepinephrine, epinephrine and isoproterenol showed the affinities for binding inhibition and for stimulation of lipolysis to be in the same range of 6 · 10^?7-2 · 10^?6 M. Dopamine and dopa were potent inhibitors of [³H]norepinephrine binding at 8.5 · 10^?7 M and 2.0 · 10^?6 M respectively, but did not stimulate lipolysis even at 10^?4 M. Propranolol, a β-adrenergic antagonist, had no effect on [³H]norepinephrine binding at 10^?4 M but completely inhibited catecholamine-stimulated lipolysis at 10^?5 M. Phentolamine, an α-adrenergic antagonist, did not inhibit binding or catecholamine-stimulated lipolysis at 10^?4 M. Ephedrine, metaraminol, phenylephrine and normetanephrine were also ineffective both as [³H]norepinephrine binding inhibitors and as stimulators of lipolysis. The results suggested the catechol ring of catecholamines is more important than the ethanolamine side chain as a requirement for binding, while both an intact catechol moiety and ethanolamine function appear necessary for physiological effect.     

Binding of [3H]estradiol- and [3H]H1285-receptor complexes to rabbit uterine chromatin

In the present study we investigated the binding characteristics of estrogen and antiestrogen-receptor complexes to rabbit uterine chromatin. Activated or nonactivated estrogen receptors were partially purified by DEAE-cellulose chromatography using low (1 mM) or high (10 mM) concentrations of sodium molybdate. Activated [³H]estradiol-receptor complexes showed enhanced binding to chromatin acceptor sites unmasked by 1 M, 4 M and 6 M guanidine hydrochloride. We also examined the chromatin-binding characteristics of the estrogen receptors when bound by the high-affinity triphenylethylene antiestrogen, H1285. The acceptor site activity for the [³H]H1285-receptor complexes was markedly decreased at sites unmasked by 4 M and 6 M guanidine hydrochloride. Further, the nonactivated receptor complexes showed very low binding to deproteinized chromatin. The estrogen-receptor chromatin-acceptor sites were tissue specific and saturable. These chromatin acceptor sites differ in their affinity and capacity (number of binding sites per cell) for the estrogen- and antiestrogen-receptor complexes. Thus, we suggest that the differences in the physiological and physicochemical properties of estrogens and antiestrogens may be related to their differential interaction with uterine chromatin subfractions.     

Characteristics of high-affinity [3H]adenosine binding to rat brain synaptosomes and turkey erythrocyte membranes

High affinity binding sites for [³H]adenosine in rat brain and in turkey erythrocytes can be identified by binding experiments. Displacement experiments using a number of adenosine analogs indicate that these high affinity sites do not represent the R-type adenosine receptors which mediate activation of adenylate cyclase, although the binding is theophylline sensitive. Similarly, the binding of [³H]adenosine is not to the P-site, which mediates inhibition of adenylate cyclase, since the high affinity binding persists in the presence of 2′,5′-dideoxyadenosine. Furthermore, these results remain qualitatively similar also in the presence of dipyridamole which blocks adenosine transport sites. We conclude that theophylline sensitivity does not indicate that [³H]adenosine binding sites correspond to adenosine receptors coupled to adenylate cyclase.     

Characterization of [3H] corticosterone binding activity in adrenal incubation media

Glucocorticoid-binding activity in adrenal incubation media was investigated with regard to characterization of a protein-like ligand. Scatchard analysis of corticosterone binding activity indicated the presence of a single non-interacting protein with a dissociation constant (Kd) of 8.81 × 10?¹⁰ M (0°C), a value which is different from that of plasma and cytoplasmic glucocorticoid binding proteins. In addition, an observed lack of affinity of the protein for dexamethasone distinguishes the protein from Type II cytoplasmic receptor proteins. Thus our data suggest a glucocorticoid-binding protein which is distinct from the two known groups of glucocorticoid-binding proteins, corticosteroid-binding globulin (CBG) and cytoplasmic receptors.