Kinetics of [3H]prazosin and [3H]dihydroalprenolol binding to α1- and β-adrenoreceptors in rat brain cortex membranes

The binding of nonselective α₁- and β-adrenoreceptor antagonists [³H]prazosin and [³H]dihydroalprenolol ([³H]DHA) to rat cerebral cortex synaptosomal membranes has been studied. It is found that ligand-receptor interactions of α₁-adrenoreceptors fit into a single receptor pool model, which assumes the binding of two ligand molecules to one receptor molecule. The parameters of [³H]prazosin binding to α₁-adrenoreceptors are as follows: K _d = 2.58 ± 0.20 nM; B _m = 2.95 ± 1.12 fmol/mg protein; Hill coefficient, n = 2. For β-adrenoreceptors, ligand-receptor interactions fit into a model assuming the presence of two receptor pools in the same effector system and binding of two ligand molecules to one receptor molecule. The corresponding parameters of the [³H]DHA binding to β-adrenoreceptors are as follows: K _d1 = 0.74 ± 0.09 nM; K _d2 = 7.63 ± 0.70 nM; B _m1 = 25 ± 2 fmol/mg, B _m2 = 48 ± 2 fmol/mg, n ₁ = 2; n ₂ = 2. We suggest that in rat cerebral cortex membranes α-and β-adrenoreceptors exist as dimers.     

d,l-α-fluoropalmitic acid inhibits sphingosine base formation and accumulates in membrane lipids of cultured mammalian cells

d,l-α-Fluoropalmitic acid was synthesized by tosylation of methyI-d,l-α-hydroxypalmitate, and displacement of the tosylated function by tetrabutylammonium fluoride in acetonitrile. Uptake and utilization of the compound by cultured Balb/c 3T3 cells were studied after presentation of the fluoro fatty acid analogue complexed with bovine serum albumin. A concentration of 0.28 mM had very little effect on cell growth over several days of incubation, and cell morphology was unchanged. Chromatographie and mass spectrometric analyses at 6 and 12 h of incubation showed that d,l-α-fluoropalmitic acid was taken up by the cells and incorporated without modification as a fatty acyl moiety into select lipids. Significant levels of the compound were found at 12 h in phosphatidylcholine (1.6%) and sphingomyelin (0.6%) fatty acids, but not in those of other phospholipids or neutral lipids. d,l-α-Fluoropalmitic acid represented a significant percentage of the fatty acids of neutral glycosphingolipids (1.4%) and ceramides (0.8%) by 12 h. The fluoro fatty acid was not incorporated into long-chain sphingolipid bases, and mass spectrometry failed to reveal additional carbon-2 fluorine-substituted compounds in cellular lipids. Cellular levels of triacylglycerols and phosphatidylcholine remained essentially unchanged, or were slightly increased, while amounts of ceramide and gangliosides were decreased. Comparison of labeled palmitate incorporation into sphingolipid bases and fatty acids of sphingomyelin suggested inhibition of sphingosine synthesis by the fluoro fatty acid. d,l-α-Fluoropalmitic acid inhibited the formation of palmitoyl-CoA by Balb/c 3T3 long-chain acyl-CoA synthetase in vitro. The results support involvement of CoA thiol ester-independent steps in modification of membrane lipids.     

Effect of morphine and abstinence syndrome on [3H]bromoxidine binding to α2-adrenoreceptors in rat brain

At 4 days after the implantation of two subcutaneous 75 mg morphine pellets in the back skin, rats were morphine-dependent. In the three layers studied in the occipital cortex we found that the values of the ₂-adrenergic agonist [³H]bromoxidine binding increased with respect to animals implanted with placebo pellets. Typical behavioral and physiological symptoms of the abstinence syndrome appeared 30 minutes after administration of naloxone, [³H]bromoxidine binding values being similar to those obtained in animals implanted with placebo pellets. The pattern of response of the [³H]bromoxidine binding was similar in the hippocampus and the superficial gray layer of the superior colliculus of the mesencephalon, but the differences were not statistically significant in these areas. This paper concludes that exist brain regional differences in the ₂-adrenoreceptors response under morphine-treatment and possibly under naloxone-induced morphine abstinence syndrome.     

A pharmacological comparison of [3H]GBR12935 binding to rodent striatal and kidney homogenates: binding to dopamine transporters?

Binding of [3H]GBR12935 to homogenates of mouse and rat striatum and kidney was studied. [3H]GBR12935 bound to both tissue preparations with high affinity (mouse striatum Kd = 2.4 +/- 0.4 nM, n = 4; mouse kidney Kd = 3.8 +/- 0.9 nM, n = 4), in a saturable (striatal Bmax = 1.5 +/- 0.4 pmol/mg protein; kidney Bmax = 4.9 +/- 0.5 pmol/mg protein) and reversible manner. Saturation experiments revealed the presence of a single class of high affinity binding sites in both tissues of both species. Mouse kidney appeared to possess a greater density of [3H]GBR12935 binding sites than the striatum while the reverse situation prevailed for the rat. Although two dopamine uptake inhibitors, namely GBR12909 and benztropine, displaced [3H]GBR12935 binding from striatal and kidney homogenates with a similar affinity in both tissues of these species, unlabelled mazindol, (+/-)cocaine, nomifensine and amfonelic acid were significantly (P < 0.001-0.02) more potent inhibitors of [3H]GBR12935 binding in the striatum than in the kidney. While the pharmacological profile of [3H]GBR12935 binding in the rodent striatum compared well with that of the dopamine transporter reported previously, the pharmacology in the kidney was considerably different to that in the striatum. GBR12909 (1-30 mg/kg, i.p.), a close analog of GBR12935, induced significant antidiuretic and antinatriuretic effects in spontaneously hypertensive rats. These data suggest that while [3H]GBR12935 labels the dopamine uptake sites in the brain, it does not appear to label similar sites in the kidney. The mechanism of action of GBR12909 on sodium and water excretion remains to be determined.     

A re-examination of the Na+-independent binding of [3H]β-alanine to rat brain stem-spinal cord

The Na⁺-independent binding of [³H]-alanine to rat brain stem plus spinal cord was reinvestigated, in order to study in more detail the characteristics of previously described -alanine binding processes. Binding was absent when amino acid-free postnuclear supernatants or crude synaptic membranes were used. Experiments performed with several other Na⁺-free preparations showed a sole binding component, irrespective of the preparation used. Biochemical characterization of this Na⁺-independent binding, using frozen/thawed/washed synaptosomal-mitochodrial fractions, showed that binding reached a plateau between 7 min and 13 min, increasing thereafter. Binding was linear with fraction protein over a range of 200–415 g/ml incubation medium. Binding was completely inhibited by glycine, alanine, -aminobutyric acid, -aminoisobutyric acid, hypotaurine and strychnine, and to a lesser extent by 2,2-dimethyl--alanine, brucine and gelsemine. It was insensitive to taurine, -aminobutyric acid (GABA), 2-guanidinoethanesulfonic acid (GES), carnosine, and bicuculline methiodide. Binding was reversible, saturable (K _D 20 M), and heat sensitive.     

The use of protamine to study [6,7-3H]-oestradiol-17β binding in rat uterus

1. The binding of [6,7-(3)H]oestradiol-17beta to uteri has been studied by using sucrose-gradient analysis and also the property of oestradiol receptors to form insoluble complexes with protamine. 2. Protamine precipitates the 8S and part of the 4S oestradiol-binding proteins in uterine cytoplasm from mature rats. It does not precipitate the oestradiol-17beta-binding proteins present in cytoplasm from non-target tissues or serum. No tritium-labelled material was precipitated by protamine after equilibration of [6,7-(3)H]oestradiol-17beta with either serum albumin or phosvitin. 3. Protamine precipitated a small amount of progesterone but not testosterone or cortisol that had been equilibrated with uterine cytoplasm. It did not precipitate any tritium radioactivity from muscle cytoplasm that had been equilibrated with either [1,2-(3)H]testosterone sulphate or [1,2-(3)H]dehydroepiandrosterone. 4. A simple method has been devised for measuring binding constants of tissue extracts for [6,7-(3)H]oestradiol-17beta, based on precipitation with protamine. Reasonable agreement was obtained between the values obtained by this method and those obtained by sucrose-gradient analysis. 5. This method has been used to study the effect of maturity, ovariectomy, adrenalectomy and hypophysectomy on the cytoplasmic binding of [6,7-(3)H]oestradiol-17beta. None of these procedures affected the dissociation constant K(d) or the number of binding sites/mg of cytoplasmic protein. When measured per uterus or per mg of DNA, ovariectomy and hypophysectomy decreased the number of binding sites. Adrenalectomy had no effect. 6. The properties of the 4S oestradiol-binding protein present in cytoplasm from mature uteri have been studied. It is not present in uteri from immature, ovariectomized, or hypophysectomized rats and it does not bind testosterone or cortisol. Unlabelled oestradiol-17beta, U-11,100A, N-ethylmaleimide and N-bromosuccinimide all decrease the binding of [6,7-(3)H]oestradiol-17beta to both 8S and 4S receptors. Binding to both 8S and 4S receptors decreases when oestradiol is transported to the nucleus. The 4S receptor is not the same as the 4S binding component formed by salt dissociation of the 8S receptor.     

[3H]norharman ([3H]β-carboline) binds reversibly and with high affinity to a specific binding site in rat liver

In addition to the known binding of norharman (NH) to monoamine oxidase (MAO) and benzodiazepine (BZ) binding sites (at M concentrations), a distinct class of high-affinity NH binding sites was discovered in rat brain (1,2). Investigations of several organs of the rat led to the discovery of high affinity binding sites in the liver, which successfully could be solubilized from P₂ membrane homogenate (0.25% w/v Triton X-100). Scatchard analysis revealed an apparent K_D value of 26±8 nM and a maximum number of binding sites of 11±3 pmol/mg protein (n=14). Association kinetics showed that equilibrium was nearly reached after two hours. Dissociaton was totally complete only after more than 16 hours. The MAO-inhibitors examined did not influence the binding characteristics. No displacement of specific binding could be found by haloperidol.     

Rabit distal colon epithelium: II. Characterization of (Na+, K+, Cl−)-cotransport and [3H]-bumetanide binding

Summary Loop diuretic-sensitive (Na⁺,K⁺,Cl^–)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of grandients of all three ions was essential for optimal transport activity (Na⁺,K⁺) gradien-driven³⁶Cl fluxes weree half-maximally inhibited by 0.14 m bumetanide and 44 m furosimide. While⁸⁶Rb uptake rates showed hyperbolic dependencies on Na⁺ and K⁺ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl^– concentration, Hill coefficient 1.8, indicating a 1 Na⁺: 1 K⁺:2 Cl^– stoichiometry of ion transport.The interaction of putative (Na⁺, K⁺, Cl^–)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [³H]-bumetanide. The requirement for the simulataneous presence of Na⁺,K⁺, and Cl^–, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na⁺, K⁺, Cl^–)-cotransporter. [³H]-bumetanide recognizes a minimum of two classes of diuretic receptors sites. high-affinity (K _D1=0.13 m;B _max1 =6.4 pmol/mg of protein) and low-affinity (K _D2=34 m;B _max2=153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl^– (K ₁=60mm), whereas low-affinity sites seem to be unaffected by Cl^–. We have shown that only high-affinity [³H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium.     

Adenosine 3′:5′-cyclic monophosphate production and steroidogenesis by isolated rat adrenal glomerulosa cells. Effects of angiotensin II and [Sar1,Ala8]angiotensin II

Angiotensin II effects on cyclic AMP production and steroid output were studied in a sensitive preparation of isolated rat adrenal glomerulosa cells. With increasing concentrations of angiotensin II logarithmic dose-response curves for aldosterone and cyclic AMP production were similar. The minimum effective dose (0.2nm) for stimulation of aldosterone production also significantly (P<0.001) increased cyclic AMP output. For both aldosterone and cyclic AMP production, the peptide hormone concentration eliciting maximal response (0.2mum) and the ED(50) (median effective dose) values (1nm) were the same; this is consistent with cyclic AMP acting as an intracellular mediator for angiotensin II-stimulated aldosterone production by glomerulosa cells. The angiotensin II antagonist [Sar(1),Ala(8)]angiotensin II inhibited angiotensin II-stimulated corticosterone and aldosterone production in these cells. An equimolar concentration of antagonist halved the response to 20nm-angiotensin II, and complete inhibition was observed with 0.2mum-antagonist. In contrast, [Sar(1),Ala(8)]angiotensin II had no effect on maximally stimulated steroidogenesis induced by serotonin and a raised extracellular K(+) concentration. Increasing concentrations of [Sar(1),Ala(8)]angiotensin II alone decreased corticosterone and aldosterone outputs significantly (P<0.05) at concentrations of 20nm and 2nm of antagonist respectively. A significant (P<0.001) decrease in cyclic AMP production occurred with 2mum antagonist and this was comparable with the decrease in aldosterone production. It is concluded that [Sar(1),Ala(8)]angiotensin II can independently affect glomerulosa-cell steroidogenesis, possibly by modulating adenylate cyclase activity.     

Simultaneous determination of leu-enkephalin localization and [3H]γ-aminobutyric acid uptake in rat striatal cell cultures

The purpose of this study was to investigate the coexistence of gamma-aminobutyric acid (GABA) and leu-enkephalin in single neurons from the corpus striatum. Monolayer cell cultures, started from newborn rat corpus striatum, were grown in serum-free medium and examined using GABA autoradiography and leu-enkephalin immunocytochemistry in a double-label protocol. Examples of cells were found which were positive for one or the other neurotransmitter or for neither transmitter, but not for both. Furthermore, cells which appear similar by morphological criteria alone differed in transmitter specificity. We conclude that the two transmitters tested are not localized within single cells and that morphology alone is inadequate to identify functional cell classes in this area.     

NPRA promotes fatty acid metabolism and proliferation of gastric cancer cells by binding to PPARα

Among cancers, gastric cancer (GC) ranks third globally in morbidity and mortality, particularly in East Asia. Natriuretic peptide receptor A (NPRA), a receptor for guanylate cyclase, plays important roles in regulating water and sodium balance. Recent studies have suggested that NPRA is involved in tumorigenesis, but its role in GC development remains unclear. Herein, we showed that the expression level of NPRA was positively correlated with gastric tumor size and clinical stage. Patients with high NPRA expression had a lower five-year survival rate than those with low expression, and NPRA was identified as an independent predictor of GC prognosis. NPRA knockdown suppressed GC cell proliferation, migration and invasion. NPRA overexpression enhanced cell malignant behavior. Immunohistochemistry of collected tumor samples showed that tumors with high NPRA expression had higher peroxisome proliferator-activated receptor α (PPARα) levels. In vivo and in vitro studies showed that NPRA promotes fatty acid oxidation and tumor cell metastasis. Co-IP showed that NPRA binds to PPARα and prevents PPARα degradation. PPARα upregulation under NPRA protection activates arnitine palmitoyl transferase 1B (CPT1B) to promote fatty acid oxidation. In this study, new mechanisms by which NPRA promotes the development of GC and new regulatory mechanisms of PPARα were identified.     

Distribution of indoleamines and [3H]paroxetine binding in rat brain regions following acute or perinatal Δ9-tetrahydrocannabinol treatments

The effects of ⁹-tetrahydrocannabinol (⁹-tetrahydrocannabinol-THC) administration on the central serotoninergic system were evaluated by biochemical assays of tissue levels of indoleamines; a measure of the serotonin (5-HT) innervation was obtained by using [³H]paroxetine as a maker of 5-HT uptake sites. Two different ⁹-THC treatments were chosen, i.e: acute and chronic perinatal maternal exposure. Following acute treatment (5mg/kg), the 5-HT content increased in dorsal hippocampus (+35%), Substantia nigra (+61%) and neostriatum (+62%) but remained unchanged in cingulate cortex, Raphe nuclei, Locus coeruleus and anterior hypothalamus. Endogenous 5-hydroxyindole-3-acetic acid (5-HIAA) decreased in anterior hypothalamus (–23%) and Raphe nuclei (–21%). Following maternal exposure to ⁹-THC (5 mg/kg per day; from gestational day 13 to postnatal day 7), levels of 5-HT were increased in the neostriatum (+22%) but decreased in anterior hypothalamus (–25%), Raphe nuclei (–29%) and Locus coeruleus (–20%) of the litters. Tissue 5-HIAA was increased in anterior hypothalamus (+23%) and Substantia nigra (+48%). There were no changes in 5-HT uptake site density, determined by [³H]paroxetine binding, except for an increase (+50%) in the cingulate cortex of perinatal-treated rats when compared to acutely-treated animals. The present results show that acute and maternal exposure to ⁹-THC produced different effects on the central 5-HT system of the offspring, with a clear regional especifity, but with no changes in the densities of 5-HT uptake sites.Abbreviations Hypo anterior hypothalamus - Cin cingulate cortex - dHipp dorsal hippocampus - 5-HIAA 5-hydroxyindole-3-acetic acid - HPLC high-performance liquid chromatography - 5-HT serotonin - 5-HTP 5 hydroxy-1-tryptophan - LC Locus coeruleus - rNS rostral neostriatum - MRN midbrain Raphe nuclei region - SN Substantia nigra - ⁹-THC ⁹-Tetrahydrocannabinol     

Depletion of 9L rat brain tumor cell polyamine content by treatment with d,l-α-difluoromethylornithine inhibits proliferation and the G1 to S transition

d,l-α-Difluoromethylornithine (DFMO), an irreversible inactivator of ornithine decarboxylase, inhibited 9L monolayer culture rat brain tumor cell proliferation at concentrations as low as 1 mM DFMO to about 25% of control growth when cells were seeded at an initial density of 5 × 10⁵/flask. DFMO reduced intracellular putrescine content to <5% of control by 8 h and spermidine content to <5 % of control by 48 h post-treatment. Cytostasis caused by 10 or 25 mM DFMO could both be reversed and blocked by addition of exogenous putrescine. Cells pretreated for 48 h with DFMO and then replated in its absence could not enter exponential growth until polyamine production resumed. Addition of exogenous putrescine at the time of replating allowed pretreated cells to resume exponential growth at the same time as controls. Flow cytometry revealed that the fraction of cells in G1 increased until polyamine accumulation resumed, implying the presence of a G1-S block. Within 6 h of replating, there was a decrease in the fraction of control cells in G1. These observations support the hypothesis that entry of 9L cells into S phase depends on an adequate intracellular pool of polyamines.     

An in vitro assay of the effect of lysine oxidation end-product, α-aminoadipic acid,on the redox status and gene expression in probiotic Lactobacillus reuteri PL503

Amino Acids - This study was designed to gain information about the underlying mechanisms of the effects of a food-occurring free oxidized amino acid, α-aminoadipic acid (AAA), on the...     

Analysis of [2H7]methionine, [2H4]methionine,methionine, [2H4]homocysteine and homocysteine in plasma by gas chromatography–mass spectrometry to follow the fate of administered [2H7]methionine

Homocysteine plays a key role in several pathophysiological conditions. To assess the methionine–homocysteine kinetics by stable isotope methodology, we developed a simultaneous quantification method of [²H₇]methionine, [²H₄]methionine, methionine, [²H₄]homocysteine and homocysteine in rat plasma by gas chromatography–mass spectrometry (GC–MS). [¹³C]Methionine and [¹³C]homocysteine were used as analytical internal standards to account for losses associated with the extraction, derivatization and chromatography. For labeled and non-labeled homocysteine measurements, disulfide bonds between homocysteine and other thiols or proteins were reduced by dithiothreitol. The reduced homocysteine and methionine species were purified by cation-exchange chromatography and derivatized with isobutyl chlorocarbonate in water–ethanol–pyridine. Quantification was carried out by selected ion monitoring of the molecular-related ions of N(O,S)-isobutyloxycarbonyl ethyl ester derivatives on the chemical ionization mode. The intra- and inter-day precision of the assay was less than 6% for all labeled and non-labeled methionine and homocysteine species. The method is sensitive enough to determine pharmacokinetics of labeled methionine and homocysteine.     

High-affinity uptake of γ-[3H]aminobutyric acid by isolated mouse oligodendrocytes in culture

Oligodendrocytes were isolated from mixed glial cultures of neonatal mouse forebrain and further grown in serum-free hormone supplemented culture medium. Cell populations were identified by indirect immunofluorescence using a range of specific antibodies, revealing a predominantly immature population of oligodendrocytes, the majority expressing the myelin glycolipids galactocerebroside and sulfatide on their plasma membrane. Astroglial contamination was found to be minimal. Simultaneous autoradiography and immunofluorescence demonstrated the presence of a transport system for the major inhibitory neurotransmitter GABA in the oligodendrocytes. The transport system was found to be energy, sodium and temperature dependent. Kinetic analysis revealed a high affinity system, with aK _m of 6.27 M and aV _max of 0.714 nmol/min/mg protein, which is comparable to that found previously for CNS neurons and astrocytes.Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Quantification of in vivo binding of [3H]RX 821002 in rat brain: Evaluation as a radioligand for central α2-adrenoceptors

On the basis of its established in vitro characteristics, [³H]RX 821002 was evaluated in rats as an in vivo radioligand for central α₂-adrenoceptors. Estimates for in vivo binding potential, obtained by compartmental analyses of time-radioactivity data, ranged between 1.9 for hypothalamus and 0.2 for cerebellum, with a regional distribution in brain which was similar to that observed in vitro. Selectivity and specificity of the signal were checked by predosing with either the α₂-antagonists, idazoxan or yohimbine, the α₂-agonist, clonidine, or the α₁-antagonist, prazosin. Pretreatment of the rats with the selective neurotoxin, DSP-4, had no significant effect on [³H]RX 821002 binding, suggesting that the majority of labelled sites were situated post-junctionally. The studies indicate that [³H]RX 821002 can be used experimentally as an in vivo marker for central α₂-adrenoceptors. The size and rate of expression of the specific signal encourage the development and assessment of [¹¹C]RX 821002 for clinical PET studies.     

Incorporation of [3H] uridine into the ribonucleic acid of rat uterus during pseudopregnancy and in the presence of I.C.I. 46474 [trans-1-(p-β-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1 -ene]

1. The incorporation of [(3)H]uridine into RNA of rat uterine tissue has been measured during pseudopregnancy and in rats receiving different doses of an anti-implantation compound [trans-1-(p-beta-dimethylaminoethoxyphenyl)-1,2-diphenylbut- 1-ene, I.C.I. 46474]. 2. In the uterus of the pseudopregnant rat uridine incorporation into RNA increased markedly on day 3 of pseudopregnancy, remained high until day 5 and decreased sharply by day 6, rising again by day 9. 3. This pattern of change was similar to, but not identical with, that previously found in the pregnant rat. 4. Rats receiving I.C.I. 46474 at a dose concentration below that preventing implantation showed a pattern of RNA synthesis similar to that found in untreated control rats. 5. Rats treated with doses of I.C.I. 46474 sufficient to inhibit implantation revealed a totally different pattern of incorporation of [(3)H]uridine into uterine RNA. 6. The results are discussed in terms of previous findings with the normally pregnant rat. It is concluded that the increasing uterine RNA synthesis found on day 3 in the pregnant rat is an important requirement for the occurrence of the subsequent implantation.