Differential distribution of liposome-entrapped [H]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.     

Regional distribution of [3H] kainic acid after intraventricular injection

[³H] Kainic acid was administered intraventricularly to rats at a dose that selectively destroys the pyramidal cells of hippocampal area CA3. Only about one-third of the injected radioactivity was recovered in the brain 15 min later, but the residual radioactivity disappeared at a much slower rate. [³H]-Kainic acid distributed rather evenly throughout the brain; there was no correlation between accumulation of radioactivity and neurotoxicity. Almost 90% of the radioactivity in sucrose homogenates was recovered in the high-speed supernatant. No cerebral metabolism of [³H] kainic acid was detected by thin-layer chromatography. These data rule out the possibility that a lethal accumulation of the toxin by hippocampus accounts for the preferential vulnerability of hippocampal pyramidal cells.     

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.     

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.     

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.     

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.     

The distribution of radioactivity in rats given [3H]bishydroxymethyl-1-methylpyrrole and its relationship to that from [3H]synthanecine a bis-N-ethylcarbamate

The distribution of radioactivity was measured in rats various times after single intravenous doses of tritium-labelled 2,3-bis-hydroxymethyl-1-methylpyrrole (BHMP). Over half the dose was excreted in urine during the first day; less than a seventh this amount was found in the faeces. The level in glandular stomach was much higher than in any other organ and there was evidence that this was related to the acidity of this tissue. With this exception, the radioactivity in other tissues was lower than after an equivalent dose of tritiated synthanecine A bis-N-ethylcarbamate, and that in liver tissue was more easily solubilised than after the latter compound. The results indicate it is unlikely that more than a small amount of free BHMP is released into the bloodstreams of rats given synthanecine A bis-N-ethylcarbamate.When [³H]BHMP is given by stomach tube much radioactivity remains within the gut and there is no exceptional binding to glandular stomach tissue. Whereas the tissue binding as well as chemical and toxicological properties of BHMP are similar to those of dehydroretronecine, the binding properties of synthanecine A bis-N-ethylcarbamate are likely to resemble those of monocrotaline and similar pyrrolizidine alkaloids.     

The effect of elimination of macrophages on the tissue distribution of liposomes containing [3H]methotrexate

In the present study the tissue distribution of [³H]methotrexate was studied after intravenous injection of [³H]methotrexate-containing liposomes in normal and macrophage-depleted mice. Elimination of macrophages was performed by treatment with dichloromethylene diphosphonate- (DMDP)-containing liposomes. After thorough elimination of the macrophages from spleen and liver, by two intravenous injections of DMDP liposomes 6 and 4 days before tissue distribution studies, we found dramatic changes in the localization pattern of [³H]methotrexate liposomes in the blood, due to a decreased uptake of [³H]methotrexate liposomes by the DMDP liposome-treated liver. Because of the absence of these macrophages that are able to clear the blood of liposomes, and because of the resulting higher blood level of liposomes, we found an enhanced uptake of [³H]methotrexate liposomes by the spleen. It may be concluded that, in the spleen, apart from uptake of liposomes by macrophages, at least one other mechanism is responsible for the clearance of liposomes from the circulation. When comparing cholesterol-rich with cholesterol-poor liposomes, we found basically the same results, although uptake of cholesterol-rich liposomes by macrophages was smaller than that of cholesterol-poor liposomes, as found in several other studies. We suggest that pretreatment with DMDP liposomes can help to maintain a high level of intravenous-injected liposome-entrapped material in the blood, which otherwise would be removed by macrophages.     

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.     

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.     

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.     

[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.     

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.     

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.     

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.     

Differential modulation by muscimol of [3H] flunitrazepam binding to benzodiazepine binding site subtypes

The effects of the GABA agonist, muscimol on [³H]flunitrazepam binding were examined in cerebellum and hippocampus regions proposed to contain different populations of benzodiazepine binding site subtypes. Quantitative analysis was made of the contribution of different components of [³H]flunitrazepam binding by utilising the selective affinities of propyl β-carboline-3-carboxylate for these sites. The influence of muscimol on each of these components was determined and the results provide clear evidence that GABA receptors interact with only some subtypes of benzodiazepine binding sites; for example, whilst the cerebellar site and the low affinity hippocampal site are influenced, the high affinity site in hippocampus appears to be quite unaffected.     

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).     

Photoincorporation of [3H]-chloropromazine into a solubilized bovine striatal preparation

Photolysis at 300 nm of [³H]-chlorpromazine (CPZ) in the presence of a digitonin-solubilized bovine striatal homogenate results in the formation of a high molecular mass component (>200,000 daltons) which does not arise upon photolysis of either [³H]-CPZ or the solubilized preparation separately. Additional components of low molecular mass are observed in both photolyzed and unphotolyzed preparations. The high molecular mass component to which [³H]-CPZ binds irreversibly contains a significant fraction of the initial [³H] activity. The formation of the high molecular mass component is inhibited by 35 ± 9% when the original photolysis is performed in the presence of 500 nM (+)-butaclamol, an active antagonist of specific binding to postsynaptic dopamine receptors, compared to photolysis in the presence of 500 nM (?)-butaclamol, which has negligible specific affinity for these dopamine receptors. EGTA at a concentration of 5mM has no effect on the photolabeling by [³H]-CPZ. SDS-polyacrylamide gel electrophoretic separation of the high molecular mass component shows a major band at ～61,000 daltons which is not evidenced in the low molecular mass fraction. These observations suggest that upon photoexcitation CPZ may photocrosslink a complex of membrane proteins which includes a neuronal DA receptor.     

Synthesis and stability of [3H] 5—demethoxyubiquinone-9

Radioactive [³H]5-demethoxyubiquinone-9 (3-methyl-2-nonaprenyl-6-methoxy-1,4-benzo-quione), an intermediate in the biosynthesis of ubiquione-9 by selected microorganisms and by the rat, has been synthesized. 4-Methyl-3-nitrophenol was converted to the corresponding anisole with [³H]methyl iodide and the anisole was then reduced to the corresponding aniline. Oxidation of 6-methyl-3-methoxy [³H]aniline with chromic acid gave the corresponding 1,4-benzo-quinone which was reduced and alkylated with solanesol in the presence of boron trifluorideetherate. Oxidation with ferric chloride gave two isomers, 5-demethoxyubiquinone-9 and 6-methyl-2-nonaprenyl-3-methoxy-1,4-benzoquinone which were separated by thin layer chromatography. The [³H]methoxyl-5-demethoxyubiquinone-9 prepared had a specific radioactivity of 100 mCi/mmole.