Rat Brain Microsomal Fraction Research Articles

Prospects for Intranasal Delivery of Neuropeptides to the Brain

Intranasal administration (INA) of medicines has the advantages of being noninvasive, painless, simple, and convenient. The basic approaches to solving problems with effective delivery of peptides to the brain through nasal membranes are discussed with respect to nasal cavity anatomy and physiology. INA can bypass the blood—brain barrier although proteolysis of the peptides by olfactory epithelium components, i.e., the enzyme barrier, is an obstacle. Pro-Gly-Pro, Pro-Gly-Pro-Leu, Semax, and Selank were used as reference peptides for in vivo experiments. The maximum contents in rat blood plasma of Pro-Gly-Pro-Leu, Semax, Pro-Gly-Pro, and Selank were 0.54, 1.69, 1.30, and 1.04%, respectively, of the administered amount of labeled peptide. The corresponding values in rat brain for Pro-Gly-Pro-Leu, Semax, Pro-Gly-Pro, and Selank reached 0.0013, 0.13, 0.04, and 0.16%. The methionine in Semax was replaced by alanine, glycine, threonine, and tryptophan to minimize its proteolysis during transversal of the enzyme barrier. Liposomes and acetylation of the N-terminal amino acids were also used to improve the stability of Semax. Use of N-acetyl-Semax was shown to be most promising. In vitro experiments used leucine aminopeptidase (incubation medium contained 12.6% Semax after 60 min), dipeptidyl peptidase (95.4%), carboxypeptidases B (96.8%) and Y(51.0%), nasal mucus enzymes (4.0%), and microsomal fractions of rat brain (9.0%) and blood plasma (0.7%). Proteolysis of Semax formed mainly His-Phe-Pro-Gly-Pro, Phe-Pro-Gly-Pro, and Pro-Gly-Pro.

Перспективы использования интраназального введения для доставки нейропептидов в головной мозг

Преимуществами интраназального метода введения (ИМВ) лекарственных препаратов является неинвазивность, безболезненность, простота и удобство применения. Показаны основные подходы, связанные с решением задач по эффективной доставке в мозг пептидов через слизистую носа с учетом особенности анатомии и физиологии носовой полости. ИМВ позволяет обойти гематоэнцефалический барьер, но сталкивается с проблемой протеолиза пептидов под действием компонентов слизи обонятельного эпителия — с ферментативным барьером. В качестве реперных пептидов для экспериментов in vivo использовали Pro-Gly-Pro, Pro-Gly-Pro-Leu, семакс, селанк. В плазме крови крыс максимальное содержание Pro-Gly-Pro-Leu, семакса, Pro-Gly-Pro и селанка составило 0,54, 1,69, 1,30 и 1,04 % от введенного количества меченого пептида. В мозге крыс соответствующие значения для Pro-Gly-Pro-Leu, семакса, Pro-Gly-Pro и селанка достигали 0,0013, 0,13, 0,04 и 0,16 %. Для уменьшения протеолиза семакса при преодолении им ферментативного барьера в нем заменяли метионин на аланин, глицин, треонин и триптофан. Для повышения устойчивости семакса использовали также липосомы и ацетилирование N-концевой аминокислоты. Показано, что использование N-ацетил-семакса наиболее перспективно. Для экспериментов in vitro использовали лейцинаминопептидазу (в инкубационной среде через 60 мин содержалось 12,6 % семакса), дипептидную пептидазу (95,4 %), карбоксипептидазу B (96,8 %) и Y (51,0 %), ферменты назальной слизи (4,0 %), микросомальной фракции мозга (9,0 %) и плазму крови (0,7 %) крыс. При протеолизе семакса в основном образуются His-Phe-Pro-Gly-Pro, Phe-Pro-Gly-Pro, Pro-Gly-Pro.

Stability In Vitro of 5-oxo-Pro-Arg-Pro, 5-oxo-Pro-His-Pro-Gly-ProNH2, and Phe-Pro-Leu-Pro-Ala

Proteolysis of 5-oxo-Pro-Arg-Pro, 5-oxo-Pro-His-Pro-Gly-ProNH2, and Phe-Pro-Leu-Pro-Ala in the presence of leucine aminopeptidase and carboxypeptidases Y and B was compared. It was found that 5-oxo-Pro-Arg-Pro was stable in the presence of the peptidases. This peptide was hydrolyzed only by carboxypeptidase Y and by ~50% over 26 h. 5-Oxo-Pro-His-Pro-Gly-ProNH2 was stable in the presence of leucine aminopeptidase and carboxypeptidase Y whereas Phe-Pro-Leu-Pro-Ala was stable only in the presence of carboxypeptidase B. 5-Oxo-Pro-Arg-Pro was the most stable peptide in the presence of nasal mucus, blood, and the rat-brain microsomal fraction. The blood enzyme system was most effective for hydrolysis of all three peptides. Several metabolites of the aforementioned peptides were detected and identified. Conclusions about the main hydrolysis pathways of 5-oxo-Pro-His-Pro-Gly-ProNH2 and Phe-Pro-Leu-Pro-Ala were made and suggested a set of peptides that could be formed in experiments in vivo.

Direct or indirect influence of triphenyl-lead on activity of Na+/K+-ATPase

We have studied the effect of triphenyl-lead chloride on the lipid phase of erythrocyte membranes, on lipid monomolecular layers and Na + /K + -ATPase of the microsomal fraction of rat brain. It was found that the haemolytic effect induced by this compound occurs when its concentration exceeds 30 μM. The minimal lead concentration inducing measurable effects in monomolecular lecithin layers is about 1 μM. Inhibition of Na + /K + -ATPase activity begins at a concentration exceeding 0.5 μM. Maximum inhibition is observed at around 40 μM-a concentration at which haemolysis also occurs. It can thus be thought that at very low lead concentrations the main (or exclusive) role in modifying membrane function is played by direct interaction between lead and the sulphydryl groups of ATPase, whereas at higher concentrations two effects seem to overlap: direct interaction between lead and enzymic proteins via their sulphydryl groups and as indirect influence on the proteins via changes in the organization of the lipid phase of the membrane.

Analysis of two constitutive forms of microsomal heme oxygenase in different rat tissues.

To isolate and purify the heme oxygenase (HO) isoform in microsomal fractions of Sprague-Dawley rat liver and brain in order to understand the characteristics of the two constitutive forms and the mechanism of the occurrence of hyperbilirubinemia. After induction by hematin and phenylhydrazine, the rat liver and brain microsomal fractions were isolated and purified by DEAE-Sephacel and hydroxyapatite. Activity and the apparent molecular weight of the two isoforms [heme oxygenase 1 (HO-1) and heme oxygenase-2 (HO-2)] were measured. Kunming mice were used to prepare antiserum against purified liver HO-2. Rat liver HO-1 and brain HO-2 preparations were analyzed by the western immunoblotting technique. Two isoforms were purified and identified in the treated rat liver, and HO-1 was the predominant form with a ratio of 2:1. In the native state, HO-2 activity was detectable but HO-1 activity was increased in response to hematin and phenylhydrazine, while HO-2 activity was fully refractory to these agents. The apparent molecular weights of HO-1 and HO-2 were about Mr 30000 and Mr 36000 under reducing conditions, respectively. In the untreated liver and treated brain, only one peak of HO activity exhibiting an elution profile similar to that of HO-2 of the treated liver was detected. The presence of an activity peak was not found in the elution profile at the region where the inducible isoform of HO (HO-1) was expected. The apparent molecular weight in treated brain preparation was identical to that of the purified liver HO-2. Cross-reactivity of HO-2 in the brain microsomal preparation was established, but a reactivity of HO-1 in the liver was not observed by western immunoblotting analysis when antiserum to liver HO-2 was applied. Two constitutive forms of HO, designated as HO-1 and HO-2, exist in the treated rat liver. HO-1 is an inducible enzyme. In the treated rat brain only HO-2 exists and is a molecular entity similar to that found in liver. The two constitutive forms were different in molecular weight and in inducibility and immunochemical properties.

Preferential in vivo incorporation of [3H]arachidonic acid from blood in rat brain synaptosomal fractions before and after cholinergic stimulation.

Awake adult male rats were infused intravenously with [3H]arachidonic acid for 5 min, with or without prior administration of an M1 cholinergic agonist, arecoline (15 mg/kg i.p.). Methylatropine was also administered (4 mg/kg s.c.) to control and arecoline-treated animals. At 15 min postinfusion, the animals were killed, brains were removed and frozen, and subcellular fractions were obtained from homogenates of whole brain. Total radioactivity and radioactivity in various lipid classes were determined for each fraction following normalization for exposure by use of a unidirectional incorporation coefficient, k*brain. In control animals, incorporation was greatest in synaptosomal and microsomal fractions, accounting for 50 and 30% of total label incorporated into membrane lipids, respectively. Arecoline increased incorporation in these two fractions by up to 400% but did not increase incorporation into the myelin, mitochondrial, or cytosolic fractions. Of the incorporated radioactivity, 50-80% was in phospholipid in microsomal and synaptosomal fractions, indicating that phospholipid is the major lipid affected by cholinergic stimulation. These results demonstrate that plasma [3H]arachidonic acid is preferentially incorporated into phospholipids of synaptosomal and microsomal fractions of rat brain. Cholinergic stimulation increases incorporation into these fractions, likely by activation of phospholipase A2 and/or C in association with acyltransferase activity, Thus, intravenously infused radiolabeled arachidonic acid can be used to examine synapse-mediated changes in brain phospholipid metabolism in vivo.

Binding of cerebrosides and sulfatides to saposins A-D.

Saposins are a family of four small glycoproteins, all of which are derived from prosaposin, and are involved in the lysosomal hydrolysis of various sphingolipids. Results from this investigation demonstrate that saposins A-D bind to galactosyl- and glucosylceramide. The binding was highly dependent on the solution pH; maximum binding of glucosylceramide to all saposins occurred at pH 7. Maximum binding of galactosylceramide to saposins B and D occurred at a more basic pH (8.5). The binding of glucosylceramide to saposins was significantly inhibited by Mg2+, Ca2+, or Zn2+. Although maximum binding of sulfatide to saposins A, C, and D occurred at acidic pH, the binding to saposin B was maximum at pH 8.5. Saposin A also bound sphingomyelin or phosphatidylcholine at neutral pH. No significant binding was evident between these lipids and saposins B-D at any pH value. The existence of saposin-lipid complexes was further confirmed in selected samples by gel filtration, isoelectric focusing, and a TLC binding assay. We have also shown that galactosylceramide bound to saposins A-D was efficiently transported to a rat brain microsomal fraction. This result suggests that saposins and possibly their precursor, prosaposin, may be involved in membrane biogenesis such as the assembly of myelin and plasma membranes.

Brain cytochrome P450 and testosterone metabolism by rat brain subcellular fractions: Presence of cytochrome P450 3A immunoreactive protein in rat brain mitochondria

The hydroxylation of testosterone by rat brain subcellular fractions has been studied using an HPLC method with an enhanced resolution for the separation of testosterone and its monohydroxy derivatives. Although the analysis time is longer than that reported for earlier methods, a baseline separation was obtained between all hydroxytestosterones, excepting 6α-hydroxytestosterone and 15β-hydroxytestosterone, which were separated using a second chromatography system. This separation was important as rat brain microsomes metabolized testosterone to 15α-, 6β-, 15β-, 16β-, 2β-, 1β-hydroxytestosterone and androstenedione. Testosterone metabolism was found to be linear with time and protein concentration. The rat brain mitochondrial fraction metabolized testosterone to androstenedione. Small amounts of immunoreactive bands comigrating with purified cytochromes P450j, P450b, and P450p were detected by Western blot analysis in rat brain microsomes, while only an immunoreactive protein related to cytochrome P450p was found in the mitochondrial fractions. Immunoinhibition studies showed that BEA33, a monoclonal antibody to cytochrome P450b and simultaneously recognizing cytochromes P450e and P450a, was able to inhibit the metabolism of testosterone to the 1β-, 15α-, 2β-, and 6α-hydroxylated metabolites, whereas polyclonal anticytochrome P450p did not inhibit the formation of the 6β-hydroxytestosterone by rat brain microsomes. The metabolism of testosterone by rat brain microsomal or mitochondrial fractions was refractory to induction by 3-methylcholanthrene or pregnenolone-16α-carbonitrile. Thus, in the brain multiple isozymes of cytochrome P450 are constitutively expressed in different subcellular fractions, which suggests that brain cytochrome P450 may play an important role in the metabolism of endogenous compounds. The significance and role of cytochrome P450p-related protein in the rat brain mitochondrial fraction are yet to be determined.

Properties of PAF‐synthesizing phosphocholinetransferase and evidence for lysoPAF acetyltransferase activity in rat brain

Several reports have indicated that platelet-activating factor (PAF) may play a role in the physiopathology of nervous tissue. We previously have demonstrated the presence, in the microsomal fractions of rat brain, of a phosphocholinetransferase which is able to synthesize PAF by the de novo pathway. The presence of dithiothreitol in the medium increases the rate of PAF biosynthesis, whereas it inhibits the synthesis of long-chain alkylacyl- and diacyl-glycerophosphocholines (GPC), including dioctanoyl-GPC. This and other properties, such as pH dependence and thermal stability, indicate that rat brain may have two distinct enzymes for the synthesis of PAF and other choline phospholipids. The affinity of these enzymes for CDPcholine is similar to that reported for other tissues, the Km being 42 microns and 55 microns with alkylacetylglycerol and dioctanoylglycerol as lipid substrates, respectively. The Vmax values were 3.0 and 2.2 nmol/mg prot/min for PAF and dioctanoyl-GPC, respectively. In addition, it was shown that the microsomal fraction of rat brain contains an acetyltransferase which can convert lysoPAF to PAF. Since it has been reported previously that brain tissue possesses phospholipase A2 activity that can hydrolyze alkylacyl-GPC to lysoPAF, we conclude that brain tissue has all enzymic activities for the synthesis of PAF by the "remodeling pathway". The role of the two routes of PAF biosynthesis in nervous tissue remains to be established.

Pharmacological characterization of the specific binding of [ 3H]ryanodine to rat brain microsomal membranes

High-affinity binding of [ 3H]ryanodine has been characterized in rat brain microsomal fractions. Membrane fractions from 4 brain regions (cerebral cortex, cerebellum, hippocampus and brainstem) have been isolated using sucrose density gradient purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed the presence of a high-molecular weight protein ( M r ∼320kDa ), similar to that of ryanodine receptor from muscle sarcoplasmic reticulum (SR). In the presence of high salt (1 M KCl), [ 3H]ryanodine binds to low density (0.8 M sucrose) cortical microsomal fraction with high affinity ( K d 1.5nM ), and with the highest capacity ( B max 330fmol/mg protein). Kinetic analysis of the binding suggests multiple available binding sites for ryanodine. Binding of ryanodine is Ca 2+ dependent (ED 50 1 μM) and inhibited by Mg 2+ and Ruthenium red. Adenine nucleotides have a biphasic effect on the binding of [ 3H]ryanodine. At low Ca 2+ concentration caffeine and daunorubicin enhance the binding of [ 3H]ryanodine. The inositol 1,4,5-trisphosphate (IP 3) binding inhibitor, heparin, has no effect on ryanodine binding, and ryanodine and caffeine do not influence the binding of [ 3H]IP 3, which is enriched in the cerebellar fractions. These data demonstrate significant quantitative differences in the pharmacology of brain and muscle receptors and raise the question as to the physiological role of ryanodine binding proteins in the central nervous system and whether it is coupled to an endoplasmatic reticulum (ER) Ca 2+ release channel.

Brain acetylcholinesterase as an in vitro detector of organophosphorus and carbamate insecticides in water

An inexpensive but accurate enzymatic method is proposed for the detection of carbamate and organophosphorus pesticides contaminating water supplies. The method uses an acetylcholinesterase preparation obtained after extraction of rat brain microsomal fraction with Triton X-100. The method is based on inhibition of acetylcholinesterase in the presence of the pesticides. Some phosphorothionate insecticides (e.g. parathion, malathion), which are not direct acetylcholinesterase inhibitors, can also be activated by preincubation with the enzyme preparation. Enzyme assay is performed by a potentiometric method based on the formation of acetic acid in the incubation mixture. Interference of any eventual buffering capacity of the sample can be easily corrected. Malathion, parathion, diazinon and deoxicarbamate inhibited the enzyme at least 20% when they were added to the medium in the limit concentration recommended for public water supplies (0.1 mg/l). The method was evaluated in samples collected from selected locations of Paraiba do Sul river, Rio de Janeiro, Brazil, and it proved to be sufficiently practical and accurate as an alarm routine test for such pesticide classes.

Properties of Ca 2+ + Mg 2+-ATPase from rat brain and its inhibition by pyrethroids

Ca 2+ + Mg 2+-ATPase from microsomal fractions of rat brain was studied. The enzyme was activated by either Ca 2+ or Mg 2+ reaching the peak at the Ca 2+ concentration of 0.3 m M. Maximal activation occurred at an ATP concentration of 5 m M with an apparent K m of 0.66 m M, a V max of 62.5 μmol inorganic phosphate/mg protein/hr, and a pH between 8.1 and 8.5. The enzyme was found to be ouabain insensitive but was inhibited by ruthenium red and lanthanum with I 50 values of 10 −5 and 10 −6 M, respectively. The enzyme was highly sensitive to the actions of certain pyrethroid insecticides under in vitro conditions. The cyano-containing pyrethroids, karate and baythroid, exerted a greater inhibitory effect on the enzyme ( K i = 1.7 and 2 μM) than the noncyano-containing pyrethroids, permethrin and bioallethrin ( K i = 7 and 8.5 μM).

The Na,K-ATPase beta 2 subunit is expressed in rat brain and copurifies with Na,K-ATPase activity.

We have used a cloned fusion protein as antigen to generate an antiserum specific for the rat Na,K-ATPase beta 2 subunit. Utilizing this antiserum, we analyzed some of the structural features and tissue distribution of the beta 2 subunit. Treatment of a rat brain microsomal membrane fraction with N-glycanase F revealed that the beta 2 subunit is composed of an approximately 32 kDa core protein and at least two N-linked carbohydrate chains. The beta 2 subunit also was found to copurify with ouabain-inhibitable Na,K-ATPase activity from rat brain. Western blot analysis of rat tissue microsomes showed that beta 2 subunits were expressed in brain, pineal gland, and thymus. However, no beta 2 subunits were detected in kidney, heart, spleen, liver, mammary gland, or lung. These results suggest that the beta 2 subunit is a functional component of the rat brain Na,K-ATPase. The restricted tissue distribution of beta 2 subunits may reflect important differences in the functions of individual beta subunit isoforms.

Decreased peroxidative potential in rat brain microsomal fractions during ageing

Rough and smooth microsomes of brain in senescent rats showed less sensitivity to ascorbate-NADPH- and cumene hydroperoxide-induced peroxidative damage compared with those of young adults. The observed decrease in peroxidative potential in senescent rats seemed to be due to decrease in the substrate for peroxidation in the form of phospholipids and increase in the level of antioxidants such as reduced glutathione and superoxide dismutase.

Resolution of the rat brain heme oxygenase activity: Absence of a detectable amount of the inducible form (HO-1)

In the present study we report on the detection of a distinct pattern of heme oxygenase isoform composition in the rat brain. In this organ only the noninducible form of heme oxygenase, HO-2, could be clearly detected. This pattern of composition distinguishes the brain from other organs tested to date, namely the liver, testis, and spleen. The rat brain microsomal fraction displayed a rather impressive rate of heme oxygenase activity. This fraction also exhibited a rate of NADPH-cytochrome P-450 reductase activity that was sufficient to fully support the oxygenase activity. The brain microsomal fraction was solubilized and subjected to ion-exchange chromatography on DEAE-Sephacel. The Chromatographie elution pattern of heme oxygenase activity was compared with those of the liver and testis. In the brain only one peak of heme oxygenase activity was detected. The peak exhibited an elution profile similar to that of HO-2 of the liver and the testis. The presence of an activity peak was not detected in the elution profile at the region where the inducible isoform of heme oxygenase, HO-1, was expected. Cross-reactivity was observed between the solubilized brain microsomal fraction and antiserum to the testis HO-2 when subjected to Ouchterlony double diffusion immunoanalysis. A reaction was not observed when antiserum to liver HO-1 was employed. The presence of HO-2 in the brain microsomal preparation was also established by Western immunoblotting analysis. A protein having a mobility that was identical to the purified testicular HO-2 ( M r 36,000) was present in the brain microsomal preparation when probed with antiserum to HO-2. However, our attempts to demonstrate the presence of HO-1 in the brain microsomal preparation by a similar technique, but using antiserum to HO-1, were not successful. It is proposed that HO-2 is responsible for the bulk, if not all, of the brain microsomal heme oxygenase activity. It is further proposed that tissue-specific regulatory mechanisms are responsible for both the refractory response of the brain heme oxygenase to known metallic inducers and the absence of a detectable amount of the HO-1 isoform.

Inhibition of Lipid Peroxidation by Dihydroquinoline-Type Antioxidant (CH 402)

The in vitro effect of a non-toxic, water soluble, low molecular weight, stable dihydroquinoline-type antioxidant, CH 402 (Na (2,2-dimethyl-1,2-dihydroquinoline-4-yl)-methane sulphonic acid) was studied on free radical reactions in brain subcellular fractions. Experiments were performed using rat and mouse brain homogenate and microsomal fractions. Non-enzymatically induced lipid peroxidation by ascorbic acid was studied in correlation with ascorbic acid and CH 402 concentrations and incubation time. Malondialdehyde production during lipid peroxidation was measured by the thiobarbituric acid test. In a concentration range of 10(-2)-10(-5) M CH 402 dose-dependently inhibited the ascorbic acid induced in vitro lipid peroxidation in mouse and rat brain subcellular fractions.

Formation of phosphatidylethanol in rat brain by phospholipase D

The mechanism of phosphatidyl [ 14C]ethanol formation was studied in rat brain microsomal fraction. Phospholipase D and base-exchange enzymes were assayed with [ 14C]ethanol as substrate. Phospholipase D was found to catalyse the formation of phosphatidylethanol. The reaction was dependent on sodium-oleate as activating factor. Phosphatidylethanol formation by phospholipase D has previously only been reported to occur in plant tissues. Stimulation of base-exchange enzymes with calcium in the presence of [ 14C]ethanol did not induce any formation of phosphatidylethanol. These findings indicate that phosphatidylethanol formation in ethanol intoxicated rats is catalysed by phospholipase D.

The monomethylethanolamine- and dimethylethanolamine-base exchange reactions of a rat-brain microsomal fraction

The ability of crude rat-brain microsome preparations to convert DME and MME to their corresponding phospholipid was explored. In common with the other base-exchange reactions, the incorporations of DME and MME were stimulated by about 1–4 mM Ca 2+, possessed slightly alkaline pH optima, were energy independent and were unaffected by exogenous phospholipids. The K m values were 0.97 mM and 0.5 mM and the V max values were 9.6 nmol/mg protein per h and 6.25 nmol/mg protein per h for DME and MME, respectively. The P3 fraction of the brain and heart had the highest specific activities of particles prepared from several tissues.

Axonal transport of antibodies to subcellular and protein fractions of rat brain

Experiments examined the feasibility of using the axonal transport of antibodies as a possible means to characterize nerve membrane composition and the fate of internalized macromolecules. Polyspecific antibodies were generated in rabbits against rat brain synaptosomal and microsomal subcellular fractions and against wheat germ agglutinin-binding proteins isolated by lectin affinity chromatography. Antisera were injected into the vitreal chamber of the eye and into the facial musculature of anesthetized rats to test, respectively, for anterograde transport in retinotectal neurons and for retrograde transport in facial motoneurons. Control injections of preimmune serum were made into the opposite side. After survival for 4-168 h, animals were perfused and the axonally transported rabbit immunoglobulins detected in frozen sections of the brainstem using a modified peroxidase-antiperoxidase immunocytochemical procedure. Antisera against all 3 classes of neuronal antigens contained antibodies that underwent retrograde axonal transport. No evidence of anterograde transport was seen. Neurons containing retrogradely transported immunoglobulins exhibited punctate as well as diffuse staining of the cytoplasm and proximal dendrites, exclusive of the nucleus. Following retrograde transport of antibodies to the synaptosomal fraction, staining of the neuropil around motoneurons was also observed, suggesting transcellular transport of these antibodies. Concentrations of injected antibodies as low as 1% of whole antiserum led to detectable retrograde transport. Increasing concentrations of antibodies above the amount in whole antiserum did not increase the intensity of staining in retrogradely labeled neurons, suggesting saturation. The findings support the view that antibodies to neural membranes are taken up and transported by binding to specific sites on nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the stimulation of neuronal Na +, K +-ATPase activity by low concentrations of ouabain

Low concentrations (< 10 −7 M) of ouabain stimulate the activity of Na +, K +-ATPase in whole homogenates of rat brain. The magnitude of this stimulation varies from 5 to 70%. The concentrations of ouabain which induces maximal stimulation is also highly variable and ranges between 10 −9 to 10 −7 M. The ouabain stimulation disappears following 1:50 dilution and 2 h preincubation or freezing and thawing of the membranes or their treatment with deoxycholate. “Aging” of a preparation of ATPase also results in loss of its ability to be stimulated by ouabain but ouabain inhibition is preserved. No stimulation of enzyme activity by ouabain is observed in rat brain microsomal fraction. The β-adrenergic blocker propranolol does not inhibit the ouabain induced stimulation of ATPase activity. It is suggested that the stimulation of Na +, K +-ATPase activity by low concentrations of cardiac glycosides if a result of either the displacement of an endogenous ouabain-like compound from the enzyme or an indirect effect by changing membrane surrounding environment of the Na +, K +-ATPase.