Identification of alpha adrenoceptor subtypes in dog arteries by (3H) yohimbine and (3H) prazosin

Binding of the alpha adrenergic antagonists (³H) prazosin and (³H) yohimbine to membranes of dog arteries exhibit the characteristics expected of alpha adrenoceptors. Binding of both ligands is saturable with dissociation constants of 0.19nM and 1.15nM for (³H) prazosin and (³H) yohimbine respectively. A series of catecholamines inhibit binding of both ligands with a potency in the order $\text{epinephrine}\text{>}\text{norepinephrine}\text{a}\text{?}\text{isoproterenol}$, corresponding with the activity of these agents at alpha adrenoceptors in blood vessels. Competition for binding in both instances is stereoselective. ?-Phenylephrine has similar potencies in inhibiting (³H) prazosin and (³H) yohimbine specific binding whilst the imidazoline related partial alpha adrenergic agonists clonidine and guanfacine are more potent in inhibiting (³H) yohimbine specific binding. The affinity of prazosin for the (³H) yohimbine binding site is approximately 2500 times less than for the (³H) prazosin site whilst yohimbine is approximately 150 times more potent in inhibiting (³H) yohimbine than (³H) prazosin specific binding. Non-selective alpha adrenergic antagonists have similar affinities for both binding sites. The concentrations of (³H) yohimbine binding sites in different arteries vary about two fold whilst for (³H) prazosin the variation was about three fold. These results indicate that there are two discrete noradrenergic binding sites in the major arteries of dog which have binding properties expected of alpha₁ and alpha₂ adrenoceptors.     

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.     

Differential binding of 3H-imipramine and 3H-mianserin in rat cerebral cortex

Drug competition profiles, effect of raphé lesion, and sodium dependency of the binding of two antidepressant drugs ³H-imipramine and ³H-mianserin to rat cerebral cortex homogenate were compared to examine whether the drugs bound to a common “antidepressant receptor.” Of the neurotransmitters tested, only serotonin displaced binding of both ³H-imipramine and ³H-mianserin. ³H-mianserin binding was potently displaced by serotonin S₂ antagonists and exhibited a profile similar to that of ³H-spiperone binding. In the presence of the serotonin S₂ antagonist spiperone, antihistamines (H₁) potently displaced ³H-mianserin binding. ³H-Imipramine binding was displaced potently by serotonin uptake inhibitors. The order of potency of serotonergic drugs in displacing ³H-imipramine binding was not similar to their order in displacing ³H-spiperone or ³H-serotonin binding. Prior midbrain raphé lesions greatly decreased the binding of ³H-imipramine but did not alter binding of ³H-mianserin. Binding of ³H-imipramine but not ³H-mianserin was sodium dependent. These results show that ³H-imipramine and ³H-mianserin bind to different receptors. ³H-Imipramine binds to a presynaptic serotonin receptor which is probably related to a serotonin uptake recognition site, the binding of which is sodium dependent. ³H-Mianserin binds to postsynaptic receptors, possibly both serotonin S₂ and histamine H₁ receptors, the binding of which is sodium independent.     

Comparative labelling of α1-acid glycoprotein by (3H)-borohydride or (125I)-iodide for use in a radioimmunoassay

The labelling of α₁-acid glycoprotein (AGP) with (³H)-sodium borohydride was compared to the labelling with (¹²⁵I)-sodium iodide by the chloramine T method in view to its use in a radioimmunoassay. The tritium labelling allowed to reach a high specific radioactivity similar to that obtained with iodide ((³H)-AGP: 29.8 mCi/mg; (¹²⁵I)-AGP: 30.5 mCi/mg). Each mole of sialic acid residue of AGP contains one atom of tritium. The stability of (³H)-AGP was better than that of (¹²⁵I)-AGP as indicated by its immunoreactivity as a function of time. Immunoreactivities and standard curves were similar for the two tracers but affinity of antiserum was higher for (¹²⁵I)-AGP than for (³H)-AGP. Tritium labelling by (³H)-borohydride will be very useful for glycoprotein antigens which cannot be labelled with (¹²⁵I)-iodide.     

Modulation of [3H]-dopamine binding by cholecystokinin octapeptide (CCK-8)

Cholecystokinin-octapeptide (CCK-8) is a putative neurotransmitter which has been demonstrated previously to occur in midbrain dopamine neurones. We observe that CCK-8 causes changes in both the affinity and density of binding sites for [³H]-dopamine in rat striatal homogenates, in vitro, upon incubation with the peptide at a concentration of 1 micromolar. A dose-response study of the competetion of CCK-8 with [³H]-dopamine binding indicates an IC₅₀ for the peptide of 450 nM; desulfated CCK-8 and the related peptide caerulin are at least 4-fold less active than CCK-8. CCK-8 was also administered to rats in a separate study; the binding of [³H]-dopamine was evaluated to homogenates of striata and olfactory tubercles obtained from these animals, which had been treated with systemic injection at a dose of 20 micrograms/kg, daily, for four days. A decrease in the number of striatal binding sites for the radioligand was observed, with a concomitant increase in the number of binding sites in the olfactory tubercle. These data collectively suggest a possible regulatory role for CCK-8 in the ascending dopamine systems.     

Direct photoaffinity labeling of the primary region of the ouabain binding site of (Na+ + K+)-ATPase with [3H]ouabain, [3H]digitoxin and [3H]digitoxigenin

The tritiated cardiotonic steroids, ouabain, digitoxin, and digitoxigenin are shown to photolabel the large polypeptide but not the glycoprotein or proteolipid component of the (Na⁺ + K⁺)-ATPase when they are bound to the inhibitory site and exposed to light of 220 or 254 nm. The extent of photolabeling is low, less than 1%, and is limited by photocross-linking of the enzyme. The mechanism of photoincorporation does not appear to be either photolysis of the lactone ring in ouabain or photolysis of tryptophan or tyrosine residues in the polypeptide.     

The synthesis of [3H]gibberellin A3 and [3H]gibberellin A1 by the palladium-catalyzed actions of carrier-free tritium on gibberellin A3

Reaction of gibberellin A₃ (GA₃) with carrier-free tritium gas and 5% palladium on calcium carbonate as catalyst gave a complex mixture of products, several of which were isolated and identified. Three of the purified products are the radioactive forms of naturally occurring gibberellins: [³H]GA₃ (1), [³H]GA₁ (2) and [³H]tetrahydro GA₃ (4). Another substance was isolated and tentatively identified as [³H]16,17-dihydro GA₃ (3). GLC was used to determine the specific activities of 1 and 2. [³H]GA₃ likely arises from palladium catalysed nonspecific exchange of GA₃ alkane hydrogen atoms with tritium. [³H]GA₁ is also exchange labeled but most of its radioactivity is due to tritium addition to the C-1,2 olefinic bond of GA₃.     

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.     

Double-label autoradiography on polyacrylamide gels with 3H and 14C

A method of autoradiography is deseribed which allows discrimination between ³H- and ¹⁴C-labeled materials on polyacrylamide gels. The method relies on a very simple procedure for switching from fluorography to autoradiography which results in a decrease in ¹⁴C detection sensitivity of about 10-fold but a concomitant decrease in ³H sensitivity of over 800-fold. With the proper ratio of ³H to ¹⁴C radioactivities, there is little or no “spillover” of one isotope during the detection of the other. No special equipment is required.     

Hormone (3-indoleacetic acid)-phospholipid interaction: 1H, 13C and 31P nuclear magnetic resonance studies

The interaction between the plant hormone, 3-indoleacetic acid (IAA), and some phospholipids in CDCL₃ has been studied by ¹H, ¹³C and ³¹P nuclear magnetic resonance (NMR) spectroscopy. Upon interaction with IAA, significant changes occurred in resonance positions of the phospholipid head group nuclei. Alteration of the fatty acid composition influenced the effects of IAA on these nuclei. These effects were observed in the ethanolamine and phosphate groups of the phosphatidylethanolamines, and in the choline, phosphate and glycerol groups of the phosphatidylcholines. Changes in resonance positions of the phospholipid head group nuclei were used for the determination of dissociation constants (K_d). In all cases, K_d values were approx. 10^?2 molal for 1 : 1 interaction. The NMR results suggest an interaction orientation in which the aromatic ring system of IAA interacts with the quaternary nitrogen function of the head group, and the phosphate group becomes hydrogen-bonded to the NH or carboxyl proton of 1AA.     

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.     

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.     

Differential distribution of liposome-entrapped [3H]methotrexate and labelled lipids after intravenous injection in a primate

Positive liposomes consisting of phosphatidylcholine, cholesterol and stearylamine and negatively charged liposomes consisting of phosphatidylcholine, cholesterol and phosphatidylserine, were double labelled with either ³H-labelled dipalmitoyl phosphatidylcholine and [¹⁴C]cholesterol or with [¹⁴C]cholesterol and [³H]methotrexate entrapped in the aqueous phase. The plasma levels and urinary excretion of radioactivity from sonicated and non-sonicated liposomes were then compared with the levels of radioactivity from free [³H]methotrexate during a 4 h experimental period after an initial intravenous injection in cynomolgous monkeys. Tissue uptake at the completion of the 4 h experimental period was also measured.It was found that plasma radioactivity from [³H]methotrexate and [¹⁴C]cholesterol in sonicated positive liposomes was cleared more slowly than from comparable non-sonicated liposomes, and considerably slower than from free [³H]methotrexate. Radioactivity from sonicated negative liposomes was cleared more rapidly than from positive sonicated liposomes. Positive liposomes captured considerably more [³H]methotrexate than negative liposomes and showed very low permeability to [³H]methotrexate in in vitro studies, even in the presence of high concentrations of serum.[¹⁴C]Cholesterol radioactivity was cleared more rapidly from plasma than ³H-radioactivity from liposome-entrapped [³H]methotrexate for double-labelled sonicated liposomes and generally showed greater uptake into tissues and red blood cells. ³H-labelled dipalmitoyl phosphatidylcholine in sonicated positive liposomes was cleared faster than [¹⁴C]cholesterol during the first 3 h. The more rapid disappearance of [¹⁴C]cholesterol from the plasma was complemented by greater uptake into a number of tissues, and positive non-sonicated liposomes were taken up to a greater extent by the spleen than equivalent sonicated liposomes.Renal excretion of ³H from liposome-entrapped [³H]methotrexate was considerably less than that of ³H from free [³H]methotrexate. There was insignificant excretion, however, of ¹⁴C from cholesterol in the urine.Entrapment in liposomes completely prevented the otherwise considerable breakdown of free methotrexate to ³H-containing products in plasma and partially prevented its breakdown in tissues.These studies indicate marked differences in the distribution of liposomes in vivo due to surface charge and size, and some degree of exchange of the lipid components of the liposome bilayer independent of the distribution of the entrapped species. They also show that entrapment in liposomes can reduce metabolic degradation of a drug, maintain high plasma levels and reduce its renal excretion.     

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.     

Modification of morphine-induced change in striatal (3H)-spiroperidol binding and stereotype behavior by cyclo(Leu-Gly)

Recent reports from our laboratories have indicated that the peptide cyclo(Leu-Gly), an analog of MIF (Pro-Leu-Gly-NH₂), administered prior to chronic exposure to morphine, prevents the development of both analgesic tolerance and some signs of physical dependence. The same peptide treatment also prevented the development of morphine-induced increases in certain behavioral responses to the dopamine agonist apomorphine. The present study investigated behavioral (stereotypy) and neurochemical receptor changes (specific (³H)-spiroperidol binding) occuring in the rat striatal dopamine (DA) system following chronic morphine treatment with and without prior cyclo(Leu-Gly)administration. While chronic morphine treatment (s.c. 5 pellet implant for 3 days, each pellet contained 65 mg morphine free base) did not alter the total number of high-affinity striatal (³H)-spiroperidol binding sites (28 fmol/mg tissue), it did increase the affinity of the receptor for the ligand (K_D decreased from 40 to 24 pM). Cyclo(Leu-Gly) (8 mg/kg) prevented the morphine induced increase in dopamine receptor affinity. In parallel, cyclo(Leu-Gly) prevented the increase in apomorphine-induced stereotypy which was observed in chronic morphine treated rats. The peptide alone did not alter any of the binding characteristics. These data suggest that the ability of the peptide to block the development of physical dependence induced by morphine may involve the ability of the peptide to interfere with morphine-induced changes in dopaminergic systems.     

The incorporation of [3H]glucosamine, [3H]fucose and [14C]mannose into protein in the rat anterior pituitary incubated in vitro

Rat anterior hemipituitaries incubated in vitro rapidly take up and incorporate into protein D-[6-³H]-glucosamine · HCl, D-[1-¹⁴C]mannose and L-[G-³H]fucose. The newly labeled protein was only slowly released into a Krebs-Ringer bicarbonate incubation medium. Glucosamine- or mannose-labeled protein was barely detectable in the medium after a 30–60 min incubation whereas about 4% of all fucose-labeled protein had already been released into the incubation medium by 30 min. Puromycin · 2HCl (1 mM) inhibited incorporation of glucosamine or mannose into protein to 40% or less of control values within 30 min; fucose incorporation was not significantly inhibited before 45 min. Acid hydrolysis followed by amino acid analysis of glucosamine-labeled protein yielded significant amounts of label in glucosamine, galactosamine and apparent glucosamine-degradation products but no significant amount of label in any amino acid.     

Beta-adrenergic receptors in early human placenta characterization of [3H]-dihydroalprenolol binding

The binding characteristics of beta-adrenergic ligand [³H]-dihydroalprenolol (DHA) were determined in particulate membranes of early human placenta (8 – 12 weeks of gestation). [³H]-DHA binding to crude membrane fractions was rapid, reversible, saturable and linearly correlated with the membrane protein concentration. Scatchard analysis of saturation experiments showed a K_D of 2.80 ± 0.9 nM and a density of binding sites of 330.30 ± 93.5 fmol/mg protein. Agonist potency isoproterenol epinephrine norepinephrine indicated that early human placenta contains an adrenergic receptor of beta-2 subtype.     

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.