Effect of angiotensin-(1-7) on ATPase activities in several tissues

The present investigation was undertaken to determine whether Ang-(1-7) is able to modify ATPase activities in membrane fractions prepared from several tissues. In the presence of 10(-6) M Ang-(1-7), total (Na , K+, Mg2+)-ATPase activity decreased 31% in rat atrium and 13% in sheep atrium but was unmodified in sheep liver, rat ventricle or crude brain membranes. In rat brain synaptosomal membranes, Ang-(1-7) at 10(-8) and 10(-7) M concentrations activated Na+, K+-ATPase 20 and 24%, respectively. Rat kidney Na+, K+-ATPase activity decreased roughly 40-70% with 10(-10)-10(-6) M Ang-(1-7)), but increased 22% with 10(-12) M peptide concentration, thus indicating a biphasic effect. Our findings showing that ATPase from several tissues responds differently to Ang-(1-7) are attributable to enzyme tissue specificity.     

Membrane mass spectrometer inlet for quantitation of nitric oxide

Cholesterol domains and transport have been well-studied in non-neuronal membranes in contrast to neuronal membranes. The purpose of the experiments reported in this paper was to determine: (1) exchangeable and non-exchangeable cholesterol domains or pools were present in brain synaptosomal membranes; (2) effects of hydrolysis of sphingomyelin on cholesterol pools, that has previously been shown to alter membrane cholesterol in non-neuronal membranes and; (3) sphingomyelin hydrolysis and enzyme activity. Cholesterol pools were determined using cholesterol exchange between radiolabeled small unilamellar vesicles and mouse synaptosomes. Activity of Ca(2+)+Mg(2+)-ATPase and Na(+)+K(+)-ATPase were measured in synaptosomal membranes following treatment with sphingomyelinase. The size of the exchangeable pool of synaptosomal membrane cholesterol was approx 50% of total membrane cholesterol when measured at 37 degrees C. The t1/2 of cholesterol exchange at 37 degrees C in synaptosomes was approx 10 h. Lowering the incubation temperature to 25 degrees C, significantly reduced the size of the exchangeable pool and significantly increased the t1/2 of cholesterol exchange. Sphingomyelinase treatment of synaptosomes significantly slowed cholesterol exchange but did not modify the size of the exchangeable pool of cholesterol. Ca(2+)+Mg(2+)-ATPase activity was significantly inhibited by sphingomyelinase treatment as compared to Na(+)+K(+)-ATPase activity. Cholesterol domains were described in neuronal tissue and the size and kinetics of those pools were altered by temperature-induced changes in fluidity and hydrolysis of sphingomyelin. Sphingomyelinase-induced changes in Ca(2+)+Mg(2+)-ATPase activity were not affected by hydrolysis of sphingomyelin but appeared to be associated with a reduction in cytofacial phosphatidylinositol.     

Evidence for the existence of imidazoline-specific binding sites in synaptosomal plasma membranes of the bovine brainstem

Nonadrenergic imidazoline-specific binding sites were characterized pharmacologically in crude cerebral membrane preparations, but little is known about their subcellular localization in neurons. As in the brainstem these sites are involved in cardiovascular regulation and peripherally imidazolines modulate neurotransmitter release, we tried to determine a possible (pre)synaptic localization in brainstem. We found a specific enrichment in (entire) synaptosome, purified synaptosomal plasma membrane (37 fmol/mg), and mitochondrial (83 fmol/mg) fractions as compared with other membrane fractions (3-8 fmol/mg). Synaptosomes appeared to be free of postsynaptic structures, and purified synaptosomal plasma membranes were devoid of mitochondrial material, as determined by electron microscopy and by comparison with the distribution of marker enzymes such as monoamine oxidase. These results show for the first time that these extramitochondrial imidazoline-specific sites are neuronal and are located on presynaptic terminals. We found high affinities for unlabeled p-iodoclonidine (subnanomolar), clonidine (0.2 nM), and efaroxan (11 nM), but idazoxan did not compete significantly for the p-[125I]iodoclonidine binding in these membranes. Therefore, these sites can be classified as I1 imidazoline receptors. In summary, we describe for the first time that high-affinity I1 receptors of the bovine brainstem are located on (pre)synaptic membranes.     

Distribution and elimination of RGH-5002 in rats

In this study we analysed the changes in the properties of rat cerebral cortex Na+K(+)-ATPase in streptozotocin induced diabetes (STZ-diabetes). Special attempt was made to determine whether insulin treatment of diabetic animals could restore the altered parameters of this enzyme. Na+/K(+)-ATPase activity was found to be decreased by 15% after 2 weeks, and by 37% after 4 weeks in diabetic rat brains with a parallel decrease in maximal capacity of low affinity ouabain binding sites. There was no significant change in the high affinity ouabain binding sites. The Kd values did not change significantly. Western blot analysis of brain Na+/K(+)-ATPase isoforms indicated a 61 +/- 5.8% and 20 +/- 2.8% decrease of the alpha 1 and alpha 3 isoforms, respectively in 4 weeks diabetic animals. Change in the amount of the alpha 2 isoform proved to be less characteristic. Both types of beta subunit isoform showed a significant decrease in four weeks diabetic rats. Our data indicate a good correlation in diabetic rats between changes in Na-/K(+)-ATPase activity, low affinity ouabain binding capacity and the level of alpha 1 isoform. While insulin treatment of diabetic animals restored the blood glucose level to normal, a complete reversal of diabetes induced changes in Na+/K(+)-ATPase activity, ouabain binding capacity and Na+/K(+)-ATPase isoform composition could not be achieved.     

Neural dysfunction and metabolic imbalances in diabetic rats. Prevention by acetyl-L-carnitine

The rationale for these experiments is that administration of L-carnitine and/or short-chain acylcarnitines attenuates myocardial dysfunction 1) in hearts from diabetic animals (in which L-carnitine levels are decreased); 2) induced by ischemia-reperfusion in hearts from nondiabetic animals; and 3) in nondiabetic humans with ischemic heart disease. The objective of these studies was to investigate whether imbalances in carnitine metabolism play a role in the pathogenesis of diabetic peripheral neuropathy. The major findings in rats with streptozotocin-induced diabetes of 4-6 weeks duration were that 24-h urinary carnitine excretion was increased approximately twofold and L-carnitine levels were decreased in plasma (46%) and sciatic nerve endoneurium (31%). These changes in carnitine levels/excretion were associated with decreased caudal nerve conduction velocity (10-15%) and sciatic nerve changes in Na(+)-K(+)-ATPase activity (decreased 50%), Mg(2+)-ATPase (decreased 65%), 1,2-diacyl-sn-glycerol (DAG) (decreased 40%), vascular albumin permeation (increased 60%), and blood flow (increased 65%). Treatment with acetyl-L-carnitine normalized plasma and endoneurial L-carnitine levels and prevented all of these metabolic and functional changes except the increased blood flow, which was unaffected, and the reduction in DAG, which decreased another 40%. In conclusion, these observations 1) demonstrate a link between imbalances in carnitine metabolism and several metabolic and functional abnormalities associated with diabetic polyneuropathy and 2) indicate that decreased sciatic nerve endoneurial ATPase activity (ouabain-sensitive and insensitive) in this model of diabetes is associated with decreased DAG.     

Nicotine in the crude synaptosomal fraction of the rat brain

The in vitro uptake of nicotine into the crude synaptosomal fraction of rat brain and spinal cord was studied. The tissue/midium ratio was low and the changing of incubation time or [14C]nicotine concentration did not affect the ratio, nor did a metabolic inhibitor, sodium fluoride. A lowered ratio was obtained at 0degrees C, but this decrease may be attributable to an altered pKa of the drug at low temperature. Nicotine antagonists, mecamylamine and hexamethonium, did not affect the ratio when incubating the crude synaptosomal fraction of either adult or infant rat brain. These results suggest that the uptake of nicotine into the synaptomal fraction is not an active process. When mecamylamine and nicotine were injected in vivo, the mecamylamine antagonism was also demonstrated as lowered nicotine concentrations in infant ray synaptosomes. Since the newborn rat cortex lacks glial tissue, the nicotine concentrations in the crude synaptosomal fraction of infant rats may reflect the receptor level effects better than in adult brains. The pretreatment of infant rats with mecamylamine also lowered blood nicotine levels, suggesting that mecamylamine affected nicotine brain levels also in an unspecific way.     

Formyltetrahydrofolates associated with mitochondria have longer polyglutamate chains than the methyltetrahydrofolates associated with cytoplasm in rat brain

The subcellular distribution of folate coenzymes in the brain is unknown. Brain folate concentrations are low and hence require a sensitive assay to determine the subcellular distribution. Rat brain was fractionated by differential centrifugation into cytoplasmic, mitochondrial and crude synaptosomal fractions. The compositions of the folate pools in these subcellular fractions were determined by differential conversion of one-carbon forms enzymatically to 5,10-methylenetetrahydrofolate (5,10CH2H4PteGlu(n)) followed by reaction of the 5,10CH2H4PteGlu(n) with thymidylate synthetase and [3H]fluorodeoxyuridylate to form ternary complexes, which were then separated as a function of polyglutamate chain length by isoelectric focusing, visualized by fluorography and quantified by densitometry. The distribution of the pteridine derivatives in brain was very similar to the distribution of these derivatives in liver. Cytoplasm contained primarily 5-methyltetrahydropteroylpolyglutamates with smaller amounts of unsubstituted tetrahydropteroylpolyglutamates, whereas mitochondria contained approximately equal concentrations of unsubstituted and formyl-substituted tetrahydropteroylpolyglutamates. The subcellular distribution of polyglutamate derivatives in brain, however, was different from that in liver. In the brain, the mitochondrial folates exhibited longer polyglutamate chains than did the cytoplasmic folates, a pattern opposite to that in the liver. Whereas the brain cytoplasmic pteroylpolyglutamates were primarily penta and hexa glutamates, the brain mitochondrial pteroylpolyglutamates were primarily hexa and hepta glutamates. The brain also contained small but measurable levels of oxidized folates, which were seen in crude synaptosomal fractions but not in cytoplasmic or mitochondrial fractions.     

Characterization of neurotoxic effects of NMDA and the novel neuroprotection by phytopolyphenols in mice.

Excitotoxicity plays a major role in various neurological disorders. In this study, we explored the behavioral and neurotoxic effects of intraventricular NMDA administration in mice. After NMDA injection, acute seizures were followed by impairments in locomotor activity, motor performance on a rotarod, and climbing ability. Mice killed 1 day after NMDA administration showed increased synaptosomal reactive oxygen species ROS production and calcium concentration and decreased mitochondrial membrane potential, mitochondrial reductase activities, and neuronal membrane Na+, K+ -ATPase and mg2+ -ATPase activities. One and 3 days after excitotoxic injury, Golgi stains showed that dendritic length and spine density were significantly decreased in neurons of the hippocampal dentate gyrus. Some mice received honokiol, tea polyphenol plus memantine, and honokiol plus memantine prior to NMDA treatment; the occurrence of generalized seizures was attenuated, seizure scores were reduced, latency to generalized seizures was prolonged, and motor impairments were lessened. Moreover, all of the neurochemical changes of the synaptosomes were also ameliorated. In conclusion, the behavioral and neurotoxic effects of intracerebroventricular injection of NMDA were ameliorated by treatment with honokiol alone or combined treatment with either tea polyphenol plus memantine or honokiol plus memantine, but only partly by either tea polyphenol or memantine alone. The therapeutic potential of these neuroprotective regimens in treating excitotoxicity-related diseases merits for further investigation. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Opioid tolerance in neonates: mechanisms, diagnosis, assessment, and management

Na+,K+-ATPase activity of rat brain synaptosomal membranes was evaluated in the presence of an inhibitory fraction II-E (termed endobain E), isolated by gel filtration and anionic exchange HPLC of a rat brain soluble fraction. We studied endobain E aging, analyzed its inhibitory potency in the absence or presence of ouabain as well as its ability to block high affinity [3H]ouabain binding to cerebral cortex membranes. Similar loss of endobain E activity was observed when samples were stored either dried or in solution. Endobain E fraction inhibited synaptosomal membrane Na+,K+-ATPase activity in a concentration-dependent manner and the slope of the corresponding curve strongly resembled that of ouabain. Assays performed in the presence of endobain E and ouabain indicated that the inhibitory effect was additive or less than additive, depending on their respective concentrations during preincubation and/or incubation. High affinity [3H]ouabain binding to cerebral cortex membranes proved concentration-dependent from 0.10 to 0.50 mg protein per ml; binding inhibition by endobain E was independent of protein concentration within the above range. [3H]ouabain binding inhibition by endobain E was concentration-dependent over a 10-fold range, an effect similar to that found for Na+,K+-ATPase inhibition. The extent of endobain E effect on Na+,K+-ATPase inhibition was much higher (90-100%) than that on [3H]ouabain binding blockade (50%). Findings suggest some type of interaction between endobain E and ouabain inhibitory mechanisms and favour the view that the former behaves as an endogenous ouabain.     

Toluene-induced oxidative stress in several brain regions and other organs

The in vivo dose-response relationship between toluene and reactive oxygen species (ROS) formation in rat brain, liver, kidney, and lung, and the time-course of these effects has been characterized. The rate of oxygen radical formation was measured using the probe 2',7'-dichlorofluorescin diacetate. In vivo exposure to various doses of toluene (0.5, 1.0, and 1.5 g/kg ip) elicited a dose-dependent elevation of ROS generation within crude mitochondrial fractions obtained from rat lung and kidney, and within crude synaptosomal fractions from cerebellum. ROS formation in crude mitochondrial fractions from liver, and crude synaptosomal fractions from striatum and hippocampus, reached a maximum value at relatively low doses of toluene. Of the brain regions, the hippocampus had the highest induced levels of ROS. In vivo exposure to a single dose of toluene (1.5 g/kg ip), revealed that toluene-induced ROS reached a peak within 2 h, which correlated directly with measured toluene blood levels. This elevated oxidative activity was maintained throughout the next 24 h, even though blood values of toluene decreased to negligible amounts. These results demonstrate that exposure to toluene results in broad systemic elevation in the normal rate of oxygen radical generation, with such effects persisting in the tissues despite a rapid decline in toluene blood levels. Acute exposure to toluene may lead to extended ROS-related changes, and this may account for some of the clinical observations made in chronic toluene abusers.     

Differential serologic response to Mycoplasma ovipneumoniae and Mycoplasma arginini in lambs affected with chronic respiratory disease

BM 17.0744 (2,2-dichloro-12-(p-chlorophenyl)-dodecanoic acid) is a substance from a group of omega-substituted alkyl carboxylic acids with the general formula, ring-spacer-carboxylic acid. With BM 17.0744-a compound structurally unrelated to thiazolidinediones--antihyperglycemic and antihyperinsulinemic potency has been demonstrated in various animal models of type II diabetes. The antidiabetic effect is independent of the genetic background of the disease, gender, and animal species. The 24-hour blood glucose profile was dose- and time-dependently improved in ob/ob mice after a single and fourth oral administration of 0.3, 1, and 3 mg/kg/d. A dose-dependent reduction of hyperglycemia (10%, 15%, 28%, and 66%) was found in db/db mice after the fifth oral administration of 3, 10, 30, and 100 mg/kg/d. Hyperinsulinemia was reduced dose-dependently in yellow KK mice by 1%, 24%, 34%, and 66% after the fifth oral administration of 0.3, 1, 3, and 10 mg/kg/d. Overall glucose metabolism was predominantly higher in euglycemic-hyperinsulinemic clamp studies in obese fa/fa rats pretreated for 14 days with 10 mg/kg/d BM 17.0744. The data in diabetic and insulin-resistant animals suggest an improvement of insulin action that is supported by enhancement of insulin effects in vitro. There is no evidence of a risk for hypoglycemia in diabetic and metabolically healthy animals. Triglyceride (TG) and cholesterol were reduced in the serum of metabolically healthy rats, as well as serum lipids in db/db mice, which suggests this effect is independent of amelioration of the diabetic status. Lipid-lowering effects in diabetic and healthy animals show an additional property of BM 17.0744. Because of its antidiabetic and lipid-lowering potency, the substance is of great interest in treating the metabolic syndrome. Lipid decreases in rats are associated with a dose-dependent increase in carnitine acetyltransferase activity in the liver to about 100-fold (12.5 mg/kg/d). This together with hepatomegaly in small rodents may indicate peroxisomal proliferation, a phenomenon considered species-specific. Its relevance for humans is well documented for other classes of compounds including fibrates. Specific side effects of insulin sensitizers of the thiazolidinedione type, such as an increase in body weight and heart weight, could not be observed after 4-week oral application of BM 17.0744 in rats. In general, BM 17.0744 was well tolerated in the pharmacological dose range in all species tested.     

A fatty acid-induced decrease in pyruvate dehydrogenase activity is an important determinant of beta-cell dysfunction in the obese diabetic db/db mouse

We studied the effects of fatty acid oxidation on insulin secretion of db/db mice and underlying molecular mechanisms of these effects. At 2-3 months of age, db/db mice were markedly obese, hyperglycemic, and hyperinsulinemic. Serum free fatty acid (FFA) levels were increased in 2-month-old (1.5 +/- 0.1 vs. 1.1 +/- 0.1 mmol/l, P < 0.05) and 3-month-old (1.9 +/- 0.1 vs. 1.2 +/- 0.1 mmol/l, P < 0.01) mice compared with the age and sex-matched db/+ mice serving as controls. Glucose-induced insulin release from db/db islets was markedly decreased compared with that from db/+ islets and was specifically ameliorated (by 54% in 2-month-old and 38% in 3-month-old mice) by exposure to a carnitine palmitoyltransferase I inhibitor, etomoxir (1 micromol/l). Etomoxir failed to affect the insulin response to alpha-ketoisocaproate. The effect of etomoxir on glucose-induced insulin release was lost after culturing db/db islets in RPMI medium containing 22 mmol/l glucose but no fatty acid. Culture of db/+ islets with 0.125 mmol/l palmitate led to a decrease in glucose-induced insulin secretion, which was partially reversible by etomoxir. Both islet glucose oxidation and the ratio of glucose oxidation to utilization were decreased in db/db islets. Etomoxir significantly enhanced glucose oxidation by 60% and also the ratio of oxidation to glucose utilization (from 27 +/- 2.5 to 37 +/-3.0%, P < 0.05). Pyruvate dehydrogenase (PDH) activity was decreased in islets of db/db mice (75 +/-4.2 vs. 91 +/- 2.9 nU/ng DNA, P < 0.01), whereas PDH kinase activity was increased (rate of PDH inactivation -0.25 +/- 0.02 vs. - 0.11 +/- 0.02/min, P < 0.0 1). These abnormalities were partly but not wholly reversed by a 2-h preexposure to etomoxir. In conclusion, elevated FFA levels in the db/db mouse diminish glucose-induced insulin secretion by a glucose-fatty acid cycle in which fatty acid oxidation inhibits glucose oxidation by decreasing PDH activity and increasing PDH kinase activities.     

Depletion of cytosolic GSH decreases the ATP levels and viability of synaptosomes from aged mice but not from young mice

The effect of glutathione depletion on the viability of freshly isolated synaptosomes from whole brain was investigated in young and aged mice. Aging did not influence the GSH and ATP levels and the viability of these synaptosomes. However depletion of glutathione caused by the cytosolic glutathione inhibitor diethyl maleate (1 mM) resulted in a significant decline, after 60 min of incubation, in ATP levels and viability in the synaptosomes from aged mice but not in those from young mice. When synaptosomes were incubated in the presence of the mitochondrial glutathione inhibitor ethacrynic acid (0.2 mM) there was a similar decline in glutathione, ATP levels and synaptosomal viability, both in young and aged mice. These results emphasize the relative importance of the cytosolic glutathione pool for the maintenance of the plasma membrane integrity in synaptosomes from aged mice.     

Identification of specific changes in the pattern of brain protein synthesis after training

Double labeling studies with [3H]valine and [14C]valine were used to investigate the pattern of protein synthesis in the brains of goldfish. The protein fractions in three bands (alpha, beta, and gamma) on sodium dodecyl sulfate-polyacrylamide gels indicate that more valine was incorporated in the brains of goldfish that had been trained in a vestibular conditioning task than in the brains of untrained fish or fish trained in a variety of control behavioral situation. Changes in the pattern of labeling were localized in the cytoplasmic fraction of the brain; no increases in labeling occurred in either the nuclear or synaptosomal components. The results suggest that a specific change occurs in the pattern of protein synthesis in the brain after the acquistion of a new behavior.     

Ethanol disordering of GM1-enriched short-sleep synaptosomal plasma membranes

The Long-Sleep (LS) and Short-Sleep (SS) mouse synaptosomal plasma membranes differ in ethanol sensitivity at superficial membrane regions, which corresponds with the behavioral response of the mice to ethanol hypnosis. The only significant difference between these synaptosomal plasma membranes is the synaptosomal monosialoganglioside (GM1) content, LS > SS. Here, GM1 was examined as a parameter for increasing membrane sensitivity to ethanol effects in the ethanol-resistant SS membranes. Synaptosomal plasma membranes from SS mice were allowed to incorporate exogenous GM1. Membrane order was then studied at the surface, intermediate, and interior regions of the membranes by delayed Fourier transform proton NMR in the presence and absence of perdeuterated ethanol. Differences in membrane order were observed in all three membrane regions with increasing perdeuterated ethanol concentrations depending on the synaptosomal GM1 content.     

Lipolytic response of "diabetic" mice (db/db) to isoproterenol and propranolol in vivo

The genetically diabetic and obese db/db mice responded lipolytically to isoproterenol and propranolol similarly to normal mice in vivo. However, considering the large amount of triglyceride in a db/db mouse, we conclude that the in vivo response of db/db adipose tissue is deficient in magnitude.     

Principles of organization of a neonatal intensive care unit from scratch

The relative amounts of brain-specific antigens, S-100, glial fibrillary acidic protein (GFA), 14.3.2, synaptin C1, D1, D2 and D3 were determined by crossed immunoelectrophoresis in bulk-prepared neuronal, glial and synaptosomal fractions from 35-day-old rat brains. Little enrichment was ontained for any antigen in the neuronal perikaryal fraction. The glial fraction showed a 3.5-fold enrichment in GFA but had levels similar to whole brain with respect to S-100. Synaptosomes were somewhat enriched in the synaptin C1 protein, but did not differ markedly from whole brain with respect to the most antigens. The extent of cross-contamination in the various fractions could be judged only tentatively, partly due to differences in extractability of the proteins.     

Protein kinase C and calmodulin effects on the plasma membrane Ca2+-ATPase from excitable and nonexcitable cells

We have purified Ca2+-ATPase from synaptosomal membranes (SM)1 from rat cerebellum by calmodulin affinity chromatography. The enzyme was identified as plasma membrane Ca2+-ATPase by its interaction with calmodulin and monoclonal antibodies produced against red blood cell (RBC) Ca2+-ATPase, and by thapsigargin insensitivity. The purpose of the study was to establish whether two regulators of the RBC Ca2+-ATPase, calmodulin and protein kinase C (PKC), affect the Ca2+-ATPase isolated from excitable cells and whether their effects are comparable to those on the RBC Ca2+-ATPase. We found that calmodulin and PKC activated both enzymes. There were significant quantitative differences in the phosphorylation and activation of the SM versus RBC Ca2+-ATPase. The steady-state Ca2+-ATPase activity of SM Ca2+-ATPase was approximately 3 fold lower and significantly less stimulated by calmodulin. The initial rate of PKC catalyzed phosphorylation (in the presence of 12-myristate 13-acetate phorbol) was approximately two times slower for SM enzyme. While phosphorylation of RBC Ca2+-ATPase approached maximum level at around 5 min, comparable level of phosphorylation of SM Ca2+-ATPase was observed only after 30 min. The PKC-catalyzed phosphorylation resulted in a statistically significant increase in Ca2+-ATPase activity of up to 20-40%, higher in the SM Ca2+-ATPase. The differences may be associated with diversities in Ca2+-ATPase function in erythrocytes and neuronal cells and different isoforms composition.     

3H]naltrindole: a potent and selective ligand for labeling delta-opioid receptors

Naltrindole (NTI) is a selective and potent delta-opioid antagonist which preferentially antagonizes a subset of selective delta-opioid agonists. The purpose of this study was to evaluate whether [3H]NTI, the first radiolabeled delta-opioid antagonist, could selectively label delta-opioid receptors in a synaptosomal preparation. Increasing temperature and protein concentration (0.1-1.6 mg protein) increased the specific binding of [3H]NTI. Monovalent and divalent cations (0.01-100 mM) had minimal effects on the binding properties of [3H]NTI, in contrast to their effects on binding of the delta agonists [3H]DPDPE and [3H]DSLET. Subfractionation of rat brain homogenates revealed that [3H]NTI and [3H]DSLET primarily labeled binding sites in synaptosomal and microsomal fractions, whereas [3H]DPDPE labelled half as many sites in synaptosomal fraction. The Bmax determined for [3H]NTI in crude synaptosomal fraction was 95 +/- 12 fmol/mg. The dissociation constant (Kd) was determined from three different methods to be 0.08 +/- 0.02 nM (Scatchard analysis), 0.07 +/- 0.02 nM (competition study) and 0.03 +/- 0.005 nM (kinetic analysis). [3H]NTI binding was not significantly inhibited by mu- or kappa-opioid ligands or by nonopioid compounds. These results demonstrate that [3H]NTI is a potent and selective radioligand for delta-opioid receptors in rat brain preparations.