bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphatidic Acid Accumulation and Catecholamine Release in Adrenal Chromaffin Cells: Stimulation by High Potassium and by Nicotine, and Effect of a Diacylglycerol Kinase Inhibitor R 59 022
Authors: Owen P. Jane , Jones J. Alison , Boarder Michael R.
Published: October 2006

Using primary cultures of bovine adrenal chromaffin cells labelled with 32Pi, we show that stimulation with bradykinin, nicotine, or a depolarising concentration of potassium stimulates the accumulation of [32P]phosphatidic acid. The effects of nicotine and potassium are smaller than the effect of bradykinin, and are dependent entirely on extracellular calcium. The diacylglycerol kinase inhibitor R 59 022 attenuates the formation of phosphatidic acid by nicotine and depolarising concentrations of potassium. This inhibitor also blocks the nicotine and potassium stimulation of nor‐ adrenaline release from Chromaffin cells. Using 45Ca2+ influx studies, we show that the nicotine‐evoked calcium influx is also attenuated by R 59 022. These observations contrast with those in another report in which we showed that bradykinin stimulation of either [32P]phosphatidic acid accumulation or noradrenaline release is not affected by R 59 022. It is likely that the calcium influx produced by nicotine and depolarising potassium is blocked by R 59 022 by a mechanism that is independent of its ability to block diacylglycerol kinase. The nicotine‐ and potassium‐stimulated [32P]phosphatidic acid accumulation is a consequence of this calcium influx and presumably reflects calcium activation of either phospholipase C or phospholipase D.
The Mouse Dopamine D2A Receptor Gene: Sequence Homology with the Rat and Human Genes and Expression of Alternative Transcripts
Authors: Mack Kenneth J. , Todd Richard D. , O'Malley Karen L.
Published: October 2006

To understand the possible involvement of dopamine receptors in the pathogenesis of various neurological disorders, we have cloned and sequenced a dopamine D2A receptor gene from the mouse. A mouse genomic library was screened with probes derived from the published sequence of a rat D2A receptor cDNA. Using restriction endonuclease mapping, Southern blotting, and DNA sequencing, we have determined the cDNA sequence and genomic organization of the mouse D2A receptor gene. Unlike other guanine nucleotide‐binding protein‐coupled receptors, but similar to its rat and human counterparts, the mouse D2A receptor gene has seven introns and spans at least 30 kb of genomic DNA. The mouse D2A sequence shows 99% amino acid homology with the rat and 95% amino acid homology with the human sequence. As would be predicted, sequence differences are significantly more frequent outside of the hypothesized transmembrane spanning domain regions of the protein. Using the polymerase chain reaction with primers made from neighboring exons, we have identified two alternatively spliced D2A transcripts in the mouse. However, in contrast to the other species studied, the mouse expresses primarily the mRNA representing the larger, 444‐amino‐acid form of the receptor. Mouse pituitary expresses only the mRNA of the 444‐amino‐acid form of the D2A receptor. Hence, the mouse may offer the best model to study the in vivo physiology of the long form of the D2A receptor.
Mechanism of Neurotransmitter Release Elicited by the Preferential α1‐Adrenergic Receptor Agonist Phenylephrine in Superfused Superior Cervical Ganglion Cells in Culture
Authors: Schwartz Dean D. , Malik K. U.
Published: October 2006

Phenylephrine increased [3H]norepinephrine efflux and accumulation of cyclic AMP in cultured rat superior cervical ganglion cells supervised with Tyrode's solution. The purpose of this study was to determine the mechanism and relationship between these two events. Electrical stimulation (1–2 Hz), potassium chloride (50 mM), and the preferential α1‐adreneargic receptor agonist phenylephrine (1–100 μM) increased fractional tritium efflux, whereas methoxamine, cirazoline, and amidephrine were relatively ineffective. Phenylephrine, but not methoxamine and cirazoline, also iacreased cyclk AMP accumulation. Phenylephrine‐induced tritium efflux was not altered by α‐and β‐adrenergic receptor antagonists or by removal of extracellular calcium. Phenylephrine‐induced cyclic AMP accumulation was blocked by the β‐adrenergic receptor antagonists propranolol and atenokd. Focskolin (10 μM) and the nonhydrolyzable cyclic AMP analogue 8‐(4‐chlorophenylthio)cyclic AMP (100 μM) had minimal effect on tritium efflux. However, phenylephrine‐evoked increase in tritium efflux was dose dependency attenuated by the neuronal uptake blocker cocaine, and phenylephrine dose‐dependently inhibited the incorporation of [3H]norepinephrine into neuronal stores. We conclude that the increase in tritium efflux induced by Phenylephrine is independent of cyclic AMP accumulation and appears to be mediated by uptake of phenylephrine via the neuronal carrier‐mediated amine transport process, which in tarn promotes efflux of the adrenergic transmitter from its storage sites.
Comparative Incorporation of Proenkephalin‐Derived Peptides, Chromogranin A, and Dopamine β‐Hydroxylase into Chromaffin Vesicles
Authors: Wilson Steven P. , Corcoran James J. , Kirshner Norman
Published: October 2006

The incorporation of enkephalin‐containing peptides (ECPs) derived from proenkephalin into chromaffin vesicles was examined in primary cultures of adrenal medullary chromaffin cells. Cells were pulse‐labeled with [35S]methionine and chased for periods up to 24 h. Chromaffin vesicles in cell homogenates were then fractionated by density gradient centrifugation and the presence of [35S]Met‐enkephalin sequences in gradient fractions determined. 35S‐ECPs were incorporated into particles suggestive of immature vesicles within 1–2 h after radiolabeling. Vesicle maturation, measured by co‐equilibration of 35S‐ECPs and total ECPs in the gradients, was complete within 9–12 h and was unaffected by treatments that increase proenkephalin synthesis. Incorporation of [35S]chromogranin A into chromaffin vesicles followed a similar time course, but 35S‐labeled dopamine β‐hydroxylase was much more slowly incorporated, possibly reflecting differences in incorporation of membrane and soluble components. In summary, the data demonstrate that ECPs are rapidly sequestered in immature chromaffin vesicles, a process unaltered by changing rates of proenkephalin synthesis.
Brain Micro vessel 12‐Hydroxyeicosatetraenoic Acid Is the (S) Enantiomer and Is Lipoxygenase Derived
Authors: Moore Steven A. , Giordano Michael J. , Kim Hee‐Yong , Salem Norman , Spector Arthur A.
Published: October 2006

12‐Hydroxyeicosatetraenoic acid (12‐HETE) production from arachidonic acid by cerebral microvessels isolated from perfused adult murine brain was reduced by the lipoxygenase inhibitors baicalein, esculetin, gossypol, nordihydroguaiaretic acid, and quercetin. Except for quercetin and gossypol, the IC50 did not exceed 10 μM. Each inhibitor, except baicalein, also decreased microvessel prostaglandin production when present in concentrations above their IC50 value for 12‐HETE. In contrast, inhibitors of the cytochrome P450 monooxygenase system, clotrimazole. metyrapone, and proadifen (SKF‐525A), had little effect on microvessel 12‐HETE production. Chiral phase HPLC analysis revealed that only the (5) enantiomer of 12‐HETE was formed. The major microvessel metabolite of eicosapentaenoic acid co‐eluted with 12‐hydroxyeicosapentaenoic acid (12‐HEPE) on reverse‐phase HPLC and the (S) enantiomer of 12‐HEPE on chiral phase HPLC. Furthermore, like 12‐HETE. 12‐HEPE production was blocked by lipoxygenase inhibitors. These studies demonstrate that brain microvessels produce only the (5) enantiomeric 12‐hydroxy derivatives of both arachidonic acid and eicosapentaenoic acid by the action of a lipoxygenase that can be selectively inhibited by baicalein. Since arachidonic acid and eicosapentaenoic acid are available to cerebral blood vessels in certain pathological settings, these 12‐hydroxy acid lipoxygenase products may mediate some of the cere‐brovascular dysfunction that occurs following stroke, brain trauma, or seizures.
Saturable Transport of Manganese(II) Across the Rat Blood‐Brain Barrier
Authors: Murphy Vincent A. , Wadhwani Kishena C. , Smith Quentin R. , Rapoport Stanley I.
Published: October 2006

Unanesthetized adult male rats were infused intravenously with solutions containing 54Mn (II) and one of six concentrations of stable Mn(II). The infusion was timed to produce a near constant [Mn] in plasma for up to 20 min. Plasma was collected serially and on termination of the experiment, samples of CSF, eight brain regions, and choroid plexus (CP) were obtained. Influx of Mn (JMn) was calculated from uptake of 54Mn into tissues and CSF at two different times. Plasma [Mn] was varied 1,000‐fold (0.076–78 nmol/ml). Over this plasma concentration range, JMn increased 123 times into CP, 18–120 times into brain, and 706 times into CSF. CP and brain JMn values fit saturation kinetics with Km (nmol/ml) equal to 15 for CP and 0.7–2.1 for brain, and Vmix (10–2 nmol · g−1· s−1) of 27 for CP and 0.025–0.054 for brain. Brain JMn except at cerebral cortex had a nonsaturable component. CSF JMn varied linearly with plasma [Mn]. These findings suggest that Mn transport into brain and CP is saturable, but transport into CSF is nonsaturable.
Adenosine Inhibits Histamine‐Induced Phosphoinositide Hydrolysis Mediated via Pertussis Toxin‐Sensitive G Protein in Human Astrocytoma Cells
Authors: Nakahata Norimichi , Abe Marilene T. , Matsuoka Isao , Ono Tomoyuki , Nakanishi Hironori
Published: October 2006

The effect of adenosine on phosphoinositide hydrolysis was examined in 1321N1 human astrocytoma cells. Adenosine, L‐N6‐phenylisopropyladenosine (l‐PIA), and 5‐ (N‐ethylcarboxamido)adenosine (NECA) inhibited histaminc‐stimulated accumulation of inositol phosphates in a concentration‐dependent manner. The potency order of adenosine analogues for inhibition of inositol phosphate accumulation was l‐PIA > adenosine > NECA, a finding indicating that A1‐class adenosine receptors are involved in the inhibition. The reduction in inositol phosphate accumulation by l‐PIA was blocked by an adenosine receptor antagonist, 8‐phenyltheophylline. Stimulation of A1‐class adenosine receptors inhibited isoproterenol‐stimulated cyclic AMP accumulation as well as histamine‐induced inositol phosphate accumulation. Both inhibitory effects were blocked by pretreatment of the cells with pertussis toxin [islet‐activating protein (IAP)]. l‐PIA also inhibited guanosine 5′‐(γ‐thio)triphosphate (GTPγ)‐stimulated accumulation of inositol phosphates in membrane preparations, and 8‐phenyltheophylline antagonized the inhibition. l‐PIA could not inhibit GTPγS‐induced accumulation of inositol phosphates in IAP‐treated membranes. Gi/G0, purified from rabbit brain, inhibited GTPγS‐stimulated accumulation of inositol phosphates in a concentration‐dependent manner in membrane preparations. These results suggest that stimulation of Ar class adenosine receptors interacts with the IAP‐sensitive G protein(s), resulting in the inhibitions of phospholipase C as well as adenylate cyclase in human astrocytoma cells.
Characterization of Delta‐Sleep‐Inducing Peptide‐Evoked Release of Met‐Enkephalin from Brain Synaptosomes in Rats
Authors: Nakamura Akihiro , Sakai Kenji , Takahashi Yukie , Shiomi Hirohito
Published: October 2006

Delta‐sleep‐inducing peptide (DSIP) stimulates the release of Met‐enkephalin (Met‐ENK) from superfused slices of the rodent lower brainstem in vitro. In our present study, DSIP (10–10–10–9M) induced a significant release of Met‐ENK from medullary synaptosomes of rats. This DSIP‐evoked release of Met‐ENK was Ca2+ dependent and tetro‐dotoxin (TTX) insensitive. Furthermore, DSIP (10–11–10–9M) significantly increased 45Ca2+ uptake in medullary synaptosomes. These results demonstrate that DSIP acts directly on the nerve endings of Met‐ENK‐containing neurons to release this pentapeptide by generating a Ca2+ influx into these neurons. Effects of DSIP on Met‐ENK release in other discrete brain regions were also studied. Significant DSIP‐evoked Met‐ENK release from synaptosomes was observed in the cortex, hypothalamus, and midbrain (at concentrations of 10–10 and 10–9M) and in the hippocampus and thalamus (only at 10–9M), but not in the striatum. In the hypothalamus, the release of Leu‐enkephalin from its synaptosomes was slightly, but not significantly, enhanced by DSIP (10–10–10–8M). Our findings demonstrate that DSIP triggered a Ca2+ influx in nerve endings to induce a subsequent release of Met‐ENK from neurons in only certain brain regions.
Splicing Pattern of Gsα mRNA in Human and Rat Brain
Authors: Granneman James G. , Bannon Michael J.
Published: October 2006

Gsα, the GTP‐binding protein that mediates receptor stimulation of adenylyl cyclase, exists in small (Gsα‐s) and large (Gsα‐L) molecular forms. Previous work has established that these Gsα isoforms are products of alternatively spliced Gsα pre‐mRNA. The present work examined the splicing pattern of Gsα mRNA in rat and human brain using a method based upon the polymerase chain reaction. In rat brain, transcripts encoding Gsα‐L were most abundant and constituted 92% of total Gsα mRNA in hippocampus and cerebral cortex and 84% in striatum. Compared to rat brain, the relative abundance of Gsα‐L mRNA was less in human brain and constituted 84% of total Gsα mRNA in hippocampus and cortex and 66% in striatum. In addition, it was found that approximately 40% of the Gsα‐S mRNA utilizes an alternative splice site that encodes serine at amino acid 72. No regional variations in this splicing pattern were found.
High Expression of the HNK‐1/L2 Carbohydrate Epitope in the Major Glycoproteins of Shark Myelin
Authors: Zand Dina , Hammer Jeffrey , Gould Robert , Quarles Richard
Published: October 2006

The major 24‐ and 28‐kDa glycoproteins in shark PNS and CNS myelin express high levels of the adhesion‐associated HNK‐1/L2 carbohydrate epitope. The 28‐kDa protein, but not the 24‐kDa protein, cross‐reacts strongly with one of two anti‐bovine PO antisera not previously tested against fish myelin proteins. Shark PNS and CNS myelin also contains smaller amounts of high‐molecular‐weight HNK‐1‐positive proteins, including a prominent broad band in the 65–85‐kDa range. Although myelin‐associated glycoprotein (MAG) is well known to react with HNK‐1 in some mammals, monoclonal and polyclonal anti‐MAG antibodies did not react with the high‐molecular‐weight HNK‐1‐positive material in shark myelin, a result suggesting that it is not a MAG‐like protein. The high expression of the HNK‐1/L2 epitope in glycoproteins of shark myelin, including the major Po‐related ones, suggests that this adhesion‐related carbohydrate structure may have had an important role in the molecular evolution of the myelinating process.
Role of Dendritic Dopamine of the Substantia Nigra in the Modulation of Nigrocollicular γ‐Aminobutyric Acid Release: In Vivo Studies in the Rat
Authors: Le Boulch N. Lantin , Truong‐Ngoc N. A. , Gauchy C.
Published: October 2006

The release of γ‐[3H]aminobutyric acid ([3H]GABA) newly synthesized from [3H]glutamine was estimated in the superior colliculus of ketamine‐anesthetized rats superfused via a push‐pull cannula. A significant amount of [3H]GABA was spontaneously released in the superior colliculus (582 ± 49 pCi/10 min). A major part of the large K+‐evoked increase of the [3H]GABA release was Ca2+ dependent. When neuronal activity of the substantia nigra was enhanced by nigral application of K+ (30 mM) or bicuculline (10–4M), a persistent increase of the collicular [3H]GABA release was observed (60 and 80%, respectively). Conversely, when nigral activity was reduced by nigral application of GABA (10–4M) or superfusion with a Ca2+ free medium, a sustained decrease of the collicular [3H]GABA release was observed (‐30 and ‐40%. respectively). Following the nigral application of a selective D2‐receptor agonist, RU 24926 (10–5M), for 30 min in 6‐hydroxydopamine‐lesioned rats, a phasic increase (60%) of the collicular [3H]GABA release was detected. This effect could result from an activation of nigrocollicular GABAergic neurons by D2‐receptor stimulation, because nigral activity and collicular release of [3H]GABA changed in a parallel direction.
Acetylation of Serotonin in the Rabbit Pineal Gland: An N‐Acetyltransferase with Properties Distinct from NAT1 and NAT2 Is Responsible
Authors: Heim M. H. , Blum M. , Beer M. , Meyer U. A.
Published: October 2006

Two rabbit arylamine N‐acetyltransferases (NAT1 and NAT2, EC 2.3.1.5) have been cloned and characterized recently in this laboratory. They catalyze the acetylation of primary arylamine and hydrazine drugs and other substrates in the liver, including sulfamethazine, ρ‐aminosalicylic acid, and ρ‐aminobenzoic acid. In the pineal gland, serotonin is metabolized to N‐acetylserotonin by an unknown N‐acetyltransferase. Similarity of the liver enzymes and the pineal gland arylalkylamine N‐acetyltransferase (AA‐NAT) has been suggested, because pineal gland homogenates were shown to metabolize arylamine substrates as ρ‐phenetidine, aniline, or phenylethylamine, and liver homogenates or partially purified liver enzyme preparations catalyzed the N‐acetylation of serotonin. The present study was undertaken to elucidate the possible role of NAT1 or NAT2 in serotonin acetylation in the pineal gland. We transiently expressed rNAT1 and rNAT2 genes in COS cells, studied the kinetics of the enzymes produced with various substrates, and compared these data with activities of rabbit pineal glands and livers. These enzymatic studies were complemented with western blot analysis with antibodies against NAT1 and NAT2. Cross‐hybridization of rNAT1 or rNAT2 to the gene for the pineal gland AA‐NAT was tested by Southern blot studies of genomic rabbit DNA. Our results indicate that although NAT1 is expressed in the pineal gland, it is not involved in the physiologically important step of N‐acetylation of serotonin.
Amphiphilic and Nonamphiphilic Forms of Bovine and Human Dopamine β‐Hydroxylase
Authors: Bon Suzanne , Lamouroux Annie , Vigny Annette , Massoulié Jean , Mallet Jacques , Henry Jean‐Pierre
Published: October 2006

We show that human and bovine dopamine β‐hydroxylases (DBH) exist under three main molecular forms: a soluble nonamphiphilic form and two amphiphilic forms. Sedimentation in sucrose gradients and electrophoresis under nondenaturing conditions, by comparison with acetylcholin‐esterase (AChE), suggest that the three forms are tetramers of the DBH catalytic subunit and bind either no detergent, one detergent micelle, or two detergent micelles. By analogy with the Gna4 and Ga4 AChE forms, we propose to call the nonamphiphilic tetramer Dna4 and the amphiphilic tetramers Da4I and Da4II. In addition to the major tetrameric forms, DBH dimers occur as very minor species, both amphiphilic and nonamphiphilic. Reduction under nondenaturing conditions leads to a partial dissociation of tetramers into dimers, retaining their amphiphilic character. This suggests that the hydrophobic domain is not linked to the subunits through disulfide bonds. The two amphiphilic tetramers are insensitive to phosphatidylinositol phospholipase C, but may be converted into soluble DBH by proteolysis in a stepwise manner; Da4II ‐Da4I‐S. Incubation of soluble DBH with various phospholipids did not produce any amphiphilic form. Several bands corresponding to the catalytic subunits of bovine DBH were observed in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, but this multiplicity was not simply correlated with the amphiphilic character of the enzyme. In the case of human DBH, we observed two bands of 78 and 84 kDa. As previously reported by others, the presence of the heavy subunit characterizes the amphiphilic forms of the enzyme. We discuss the nature of the hydrophobic domain, which could be an uncleaved signal peptide. and the organization of the different amphiphilic and nonamphiphilic DBH forms. We present two models in which dimers may possess either one hydrophobic domain or two domains belonging to each subunit; in both cases, a single detergent micelle would be bound per dimer.
The Synthesis and Release of Acetylcholine by Depolarized Hippocampal Slices Is Increased by Increased Choline Available In Vitro Prior to Stimulation
Authors: Wecker Lynn
Published: October 2006

The objective of these experiments was to determine whether preincubating hippocampal slices with choline provides precursor that can be used during a subsequent incubation to support or enhance the synthesis of acetylcholine (ACh). Slices were preincubated for 60 min with 0, 10, 25, or 50 μM choline, washed, resuspended, and then incubated for 10 min in choline‐free buffer containing 4.74 (Krebs‐Ringer bicarbonate, KRB) or 25 mM KC1. The tissue contents of ACh and choline were determined prior to and after the preincubation, as well as after the incubation; the amounts of ACh and choline released were measured, and ACh synthesis was calculated. Preincubation in the absence of choline increased the tissue content of ACh to 242% of original levels; preincubation with 10 μM choline did not lead to a further increase, but preincubation with 25 or 50 μM choline increased the ACh content to 272% of original levels, significantly greater than that of slices preincubated with either 0 or 10 μM choline. When tissues were subsequently incubated for 10 min with either KRB or 25 μM KC1, ACh release from slices preincubated with 50 μM choline was greater than from slices preincubated with 0, 10, or 25 μM choline. Incubation of slices with KRB did not alter the tissue content of ACh, but when tissues were incubated with 25 mM KC1, the ACh content of slices preincubated with 0 or 10 μM choline decreased significantly, whereas that of slices preincubated with 25 or 50 μM choline did not. Although ACh synthesis by slices incubated with KRB did not differ significantly among groups, synthesis by slices incubated with 25 mM KC1 was significantly greater for tissues preincubated with 50 μM choline than for those preincubated with 0, 10, or 25 μM choline. Preincubation in the absence of choline decreased choline levels by 43%, whereas preincubation with 50 μM choline increased levels by 55%; choline levels were unaltered by preincubation with 10 μM choline. Following incubation with 25 mM KCl, the choline content of slices preincubated with 50 μM choline decreased by 0.59 nmol, whereas the content of slices preincubated with 10 μM choline decreased by 0.33 nmol, suggesting that the former used more of its tissue choline to support ACh synthesis; choline levels did not change in slices preincubated in the absence of choline. These results suggest that prior exposure of hippocampal slices to choline provides precursor that is used during a subsequent incubation with 25 mM KCl to support the synthesis of ACh.
Ontogeny of the Benzodiazepine Receptor in Human Brain: Fluorographic, Immunochemical, and Reversible Binding Studies
Authors: Reichelt Ralf , Hofmann Dietmar , Födisch Hans‐Jörg , Möhler Hanns , Knapp Michael , Hebebrand Johannes
Published: October 2006

The prenatal and postnatal human ontogeny of the central benzodiazepine receptor was investigated in six different brain regions between week 24 postconception and age 14 years. Binding studies, which were performed with [3H]flunitrazepam ([3H]FNZ), revealed a steep increase in receptor density postnatally in frontal cortex and cerebellum. Bmax values were higher in medulla oblongata, pons, and thalamus than in cortex and cerebellum up to week 26. After that, receptor densities declined significantly in medulla and olive. The same tendency was apparent in pons, whereas receptor density remained unchanged in thalamus. The early ontogeny of the benzodiazepine receptor was also evaluated in fluorographs ([3H]FNZ) and immunoblots using the αrsubunit‐specific monoclonal antibody (mAb) bd‐24. Specific radiolabeled proteins with molecular weights of 53K and 59K were visible in cortical membranes from gestational week 8, the earliest time investigated. During further development, the intensity of the 53K band increased without changes in the 59K band. As in other species, postmortem proteolysis in human brain led to a specifically labeled peptide of 47K. The mAb bd‐24 immunolabeled only the 53K protein and the 47K peptide.
Nondestructive Detection of Glutamate by 1H Nuclear Magnetic Resonance Spectroscopy in Cortical Brain Slices from the Guinea Pig: Evidence for Changes in Detectability During Severe Anoxic Insults
Authors: Kauppinen Risto A. , Williams Stephen R.
Published: October 2006

31P and 1H nuclear magnetic resonance spectroscopy (NMR) was used to study the metabolism of intact superfused cortical brain slices during normoxia and anoxia. Attention was focused on quantification of 1H NMR‐detected glutamate by a water‐suppressed spin‐echo method, using N‐acetyl aspartate as an internal concentration reference. To quantify the 1H NMR signals, the spin‐spin relaxation times and saturation effects were estimated for given metabolites. In addition, absolute concentrations of metabolites were determined by biochemical methods from acid extracts of the preparations after NMR experiments. Under aerobic conditions, 1H NMR detected 79% of the glutamate determined biochemically from the brain slice extracts. During anoxia in the absence of glucose when a severe energetic failure was evident, both 1H NMR and biochemical assays gave closely matching levels for glutamate. We conclude that in the brain cortex 21% of glutamate is located in an intracellular compartment in which this amino acid does not contribute to the 1H NMR signal. However, during severe anoxia an intracellular reorganization occurs increasing the detectability of this amino acid neurotransmitter by NMR.
Complex Interactions Between Polyamines and Calpain‐Mediated Proteolysis in Rat Brain
Authors: Najm Imad , Vanderklish Peter , Etebari Amir , Lynch Gary , Baudry Michel
Published: October 2006

Polyamine synthesis is induced by various extracellular signals, and it is widely held that this biochemical response participates in cell growth and differentiation. Certain of the triggers for synthesis in brain tissues also increase the breakdown of high‐molecular‐weight structural proteins, apparently by activating calcium‐dependent proteases (cal‐pains). The present experiments tested the possibility that calpain activity is modulated by polyamines. Spermine, spermidine, and putrescine all increased calcium‐dependent proteolysis of [14C]casein by soluble fractions of rat brain. The order of potency was spermine > spermidine > putrescine, with apparent affinities of 30, 300, and 6.000 μM, respectively. Each of the three polyamines at physiological concentrations also potentiated the calcium‐dependent breakdown of two endogenous high‐molecular‐weight structural proteins known to be substrates of calpain, in both supernatant and membrane fractions. The thiol protease inhibitor leupeptin, a known calpain inhibitor, also inhibited calcium‐dependent proteolysis in the presence and absence of polyamines. The polyamines did not increase the activity of purified calpain I or calpain II determined with either [14C]casein or purified spectrin as the substrate, nor did they interfere with the inhibitory effects of calpastatin, an endogenous inhibitor of calpain. However, polyamines potentiated the stimulation of endogenous but not purified calpain activity produced by an endogenous calpain activator. These results suggest a rote for polyamines in protein degradation as well as protein synthesis.
Postnatal Age and Protein Tyrosine Phosphorylation at Synapses in the Developing Rat Brain
Authors: Cudmore Stephen B. , Gurd James W.
Published: October 2006

The relationship between postnatal age and protein tyrosine kinase activity in synaptosomes prepared from the rat forebrain was studied. Synaptosomal particulate and soluble fractions, as well as total homogenates, the cell soluble fraction, and P3, were prepared from rats ranging in postnatal age from 5 to 60 days and analyzed for (a) tyrosine kinase activity using polyglutamyltyrosine (4:1) as the substrate, (b) the presence of endogenous substrates for tyrosine phosphorylation using polyclonal antibodies specific for phosphotyrosine, and (c) levels of pp60src. Enzyme activity, expressed per milligram of protein, in the total homogenate, P3, and both the cell and synaptosomal soluble fractions was highest in the brains of young animals (postnatal days 5–10) and decreased thereafter to adult levels. In contrast, tyrosine kinase activity in the synaptosomal particulate fraction exhibited a unique biphasic developmental profile, increasing to maxima at postnatal days 10 and 20 before decreasing to adult values. Endogenous substrates for tyrosine phosphorylation were identified by incubating subcellular fractions with 2 mM ATP in the presence of sodium orthovanadate and probing nitrocellulose blots of proteins separated by gel electrophoresis with antiphosphotyrosine antibodies. Several phosphotyrosine‐containing proteins were detected in the synaptosomal particulate and P3 fractions, including proteins of Mr 180K, 145K, 120K, 100K, 77K, 68K, 62K, 54K, 52K, and 42K. In the cell soluble fraction a protein doublet of Mr 54/52K and a 120K protein were the major phosphotyrosine‐containing proteins. The 54/52K doublet was the major protein tyrosine kinase substrate in the synaptosomal soluble fraction. There was a general decrease in the immunostaining of phosphotyrosine‐containing proteins in the P3 fraction after postnatal day 10. In contrast, the tyrosine phosphorylation of several proteins in the synaptosomal particulate fraction increased to maximal levels during the third and fourth week after birth before declining to adult levels. Western blotting using monoclonal antibody 327, specific for pp60src, showed that pp60src levels decreased throughout development in both homogenate and P3 fractions. The concentration of pp60src in the synaptosomal particulate fraction and postsynaptic densities, in contrast, increased 80–100% during the third and fourth weeks before decreasing slowly to adult levels. The results indicate that the expression of tyrosine kinase and the phosphorylation of synaptic proteins on tyrosine residues at the developing synapse are transiently increased during the latter stages of synaptogenesis and suggest that the phosphorylation of proteins on tyrosine residues may be involved in the regulation of synapse formation.
Characterization and Ca2+ Requirement of Histamine‐Induced Catecholamine Secretion in Cultured Bovine Chromaffin Cells
Authors: Goh Yasumasa , Kurosawa Atsushi
Published: October 2006

The stimulation of cultured bovine chromaffin cells with histamine induced a continuous catecholamine secretion (EC50= 3 × 10−7M) via the H1 receptor, in addition to an initial catecholamine burst due to a nonspecific stimulatory effect at higher doses (≥10−4M). The continuous secretion showed little desensitization and lasted for more than 1 h. In fura‐2‐loaded cells, the stimulation with histamine evoked a transient rise of intracellular free Ca2+ concentration ([Ca2+]i) which lasted only for a few minutes and was followed by a sustained [Ca2+]i rise which continued for more than 20 min. The addition of an activator for the L‐type voltage‐sensitive Ca2+ channel, i.e., Bay K 8644 (1 μM), facilitated the sustained [Ca2+]i rise, as well as the secretion, whereas the addition of relatively high concentrations of Ca2+‐channel blockers (10 μM) suppressed the sustained [Ca2+]i rise and part of the secretion. Removal of extracellular Ca2+ completely abolished continuous secretion and sustained [Ca2+]irise. When the external Ca2+ level was elevated, both sustained (Ca2]i rise and continuous secretion were enhanced in a similar Ca2+‐dependent manner, showing saturation with around 1–3 mM Ca2+. This Ca2+ dependence was clearly different from that observed with high K+ and nicotine, which is mediated by the L‐type Ca2+ channel, in which the responses showed little or no saturation when the Ca2+ level was increased. The results indicate that stimulation with histamine induces a continuous secretion via the H1 receptor, in addition to a transient and nonspecific secretion at higher doses. At least part of the continuous secretion is induced by a sustained [Ca2+]i rise due to the continuous Ca2+ influx through Ca2+ channels. These channels are sensitive to dihydropyridines, but do not appear to be identical to the L‐type voltage‐sensitive Ca2+ channel.
Somatostatin Release from Rat Cerebral Cortex Synaptosomes
Authors: Bonanno G. , Parodi B. , Cafaggi S. , Raiteri M.
Published: October 2006

Rat cerebral cortex synaptosomes were exposed in superfusion to various depolarizing stimuli and the release of somatostatin‐like immunoreactivity (SRIF‐LI) was measured by means of a radioimmunoassay procedure. High KC1 (9‐50 mM) concentration dependently evoked SRIF‐LI release; the evoked overflow reached a plateau at 25 mM KC1 and was completely abolished when Ca2+ ions were omitted from the superfusion medium, independently of the concentration of KC1 used. The 15 mM K+‐evoked release of SRIF‐ LI increased sharply as the Ca2+ concentration was raised to 0.8 mM, then leveled off and reached a plateau at 1.2 mM. The 15 mM K+‐evoked overflow, but not the spontaneous outflow, was partially decreased (50%) by 1 μM tetrodotoxin. The presence in the superfusion fluid of a mixture of peptidase inhibitors did not improve the recovery of SRIF‐LI both in the absence and in the presence of high K+. Exposure of synaptosomes to veratrine (1‐50 μM) induced release of SRIF‐LI in a concentration‐dependent way. The effect of the alkaloid was strictly Ca2+ and tetrodotoxin sensitive. Replacement of extracellular Na+ by sucrose caused an acceleration of the spontaneous SRIF‐LI outflow that was inversely correlated to the Na+ content in the superfusion medium. The release evoked by the sodium‐deprived media did not exhibit any calcium dependence. HPLC analysis of the samples collected during superfusion showed that >90% of the SRIF‐LI released either during the spontaneous outflow or by 15 mM KC1 was represented by SRIF‐14 (SRIF‐2814‐28). These values reflected the ratio SRIF‐14/SRIF‐28 found in synaptosomes at the end of the experiments.
Effects of Monensin and Veratridine on Acetylcholine Release and Cytosolic Free Ca2+ Levels in Cerebrocortical Synaptosomes of Rats
Authors: Satoh Eiki , Nakazato Yoshikazu
Published: October 2006

Monensin (10−8‐10−4M) caused a dose‐dependent increase in the release of [3H] acetylcholine ([3H] ACh) from purified rat cerebrocortical synaptosomes, with an EC50 of ∼1.6 × 10−6M. Extracellular Na+, but not Ca2+, was required for a monensin‐induced increase in the release of [3H] ACh. Monensin also increased the cytosolic free Ca2+ concentration ([Ca2+]i) and uptake of 22Na+ in a dose‐dependent manner. Monensin continued to cause a dose‐dependent increase in [Ca2+]i in the absence of extracellular Ca2+, although an ∼50% reduction was noted at concentrations of >10−5M. The EC50 for the monensin‐induced increase in [Ca2+]i was similar to that noted in the release of [3H]ACh. Veratridine exhibited effects similar to those of monensin, but a large portion of the increase in [Ca2+]i and [3H] ACh release was dependent on extracellular Ca2+. Measurements of rhodamine 6G fluorescence indicated that monensin and veratridine caused synaptosomal hyperpolarization and depolarization, respectively. Tetrodotoxin (10−6M) completely blocked all the effects of veratridine but had no effect on the activity of monensin. These results suggest that monensin increases the release of ACh at least in part by increasing [Ca2+]i, resulting from the increase in the Na+ influx through tetrodotoxin‐insensitive mechanisms in rat cerebrocortical synaptosomes.
In Vivo Studies Identify 5a‐Pregnan‐3a‐o1‐20‐one as an Active Anesthetic Agent
Authors: Mok Wai Man , Herschkowitz Sylvia , Krieger Neil R.
Published: October 2006

Mice were anesthetized with 5a‐[3H]pregnan‐3a‐o1‐20‐one. Brain levels for 5α‐pregnan‐3α‐o1–20‐one and its five major metabolites (5α‐pregnanedione, ko, k1, k2, k3) were compared at behavioral endpoints that are characteristic of the anesthetized state. The results support the hypothesis that 5α‐pregnan‐3α‐o1–20‐one mediates the anesthetic response, and they weigh against the hypothesis that any of its metabolites is solely responsible for the onset or the maintenance of the anesthetized state. For an administered dose of 3 mg/kg, brain levels (means ± SEM) for 5α‐pregnan‐3α‐o1–20‐one at the time of the loss of the righting response (n = 10) and at the time of the return of the righting response (n = 6) were 7.24 ± 0.61 pmol/mg of brain tissue and 3.63 ± 0.26 pmol/mg of brain tissue, respectively. No metabolite level was lower at the return of the righting response than at the loss of the righting response. 5α‐Pregnan‐3′‐o1–20‐one brain levels increased consistently with the percentage of anesthetized mice. This was not the case for any of the metabolites. Fifty percent of the mice were anesthetized when the 5α‐pregnan‐3α‐o1–20‐one level was 4.5 pmol/mg of brain tissue.
Okadaic Acid‐Induced Inhibition of B‐50 Dephosphorylation by Presynaptic Membrane‐Associated Protein Phosphatases
Authors: Han Yi‐fan , Dokas Linda A.
Published: October 2006

The neuronal tissue‐specific protein kinase C (PKC) substrate B‐50 can be dephosphorylated by endogenous protein phosphatases (PPs) in synaptic plasma membranes (SPMs). The present study characterizes membrane‐associated B‐50 phosphatase activity by using okadaic acid (OA) and purified 32P‐labeled substrates. At a low concentration of [γ‐32P] ATP, PKC‐mediated [32P]phosphate incorporation into B‐50 in SPMs reached a maximal value at 30 s, followed by dephosphorylation. OA, added 30 s after the initiation of phosphorylation, partially prevented the dephosphorylation of B‐50 at 2 nM, a dose that inhibits PP‐2A. At the higher concentration of 1 μM, a dose of OA that inhibits PP‐1 as well as PP‐2A, a nearly complete blockade of B‐50 dephosphorylation was seen. Heat‐stable PP inhibitor‐2 (1–2) also inhibited dephosphorylation of B‐50. The effects of OA and 1–2 on B‐50 phosphatase activity were additive. Endogenous PP‐1‐ and PP‐2A‐like activities in SPMs were also demonstrated by their capabilities of dephosphorylating [32P] phosphorylase a and [32P] casein. With these exogenous substrates, sensitivities of the membrane‐bound phosphatases to OA and 1–2 were found to be similar to those of purified forms of these enzymes. These results indicate that PP‐1‐ and PP‐2A‐like enzymes are the major B‐50 phosphatases in SPMs.
Characterization of a Novel Receptor in Toad Retina with Dual Specificity for Insulin and Insulin‐Like Growth Factor I
Authors: Zetterstrom Charlotta , Fang Carrie , Benjamin Ann , Rosenzweig Steven A.
Published: October 2006

The biochemical properties of insulin receptors from toad retinal membranes were examined in an effort to gain insight into the role this receptor plays in the retina. Competition binding assays revealed that toad retinal membranes contained binding sites that displayed an equal affinity for insulin and insulin‐like growth factor I (IGF‐I). Affinity labeling of toad retinal membrane proteins with I25I‐insulin resulted in the specific labeling of insulin receptor α‐subunits of ∼ 105 kDa. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of partially reduced (αβ‐heterodimer) receptors affinity‐labeled with I25I‐insulin indicated the presence of a disulfide‐linked β‐subunit of ∼95 kDa. Endoglycosidase F digestion of the affinity‐labeled a‐subunits increased their mobility by reducing their apparent mass to ∼83 kDa. This receptor was not detected by immunoblot analysis with a site‐specific antipeptide antibody directed against residues 657–670 of the carboxy terminal of the human insulin receptor α‐subunit, whereas this antibody did label insulin receptor a‐subunits from pig, cow, rabbit, and chick retinas. In in vitro autophosphorylation assays insulin stimulated the tyrosine phosphorylation of toad retina insulin receptor β‐ subunits. These data indicate that toad retinal insulin receptors have a heterotetrameric structure whose a‐subunits are smaller than other previously reported neuronal insulin receptors. They further suggest that a single receptor may account for both the insulin and IGF‐I binding activities associated with toad retinal membranes.
Insulin‐Like Growth Factor‐I Enhances Tyrosine Hydroxylase Activation in Bovine Chromaffin Cells
Authors: Dahmer Mary K. , Hart P. Michelle , Perlman Robert L.
Published: October 2006

Previous studies have shown that insulin‐like growth factor‐I (IGF‐I) enhances secretagogue‐stimulated Ca2+ uptake and catecholamine release in bovine chromaffin cells. This report describes the effect of IGF‐I on the activity of tyrosine hydroxylase (tyrosine 3‐monooxygenase, EC 1.14.16.2), the major regulatory enzyme in the pathway of catecholamine biosynthesis. Tyrosine hydroxylase activity was assayed by measuring 3, 4‐dihydroxyphenylalanine (Dopa) accumulation in the presence of brocresine, an inhibitor of Dopa decarboxylase. Chromaffin cells cultured in serum‐free medium produced ∼40% less Dopa when stimulated by 55 mM K+ than did cells that had been cultured in the presence of serum. Incubation of cells for 3 days in serum‐free medium containing 10 nM IGF‐I restored high K+‐stimulated Dopa accumulation to a level comparable to that seen in cells cultured continuously in serum‐containing medium. In eight experiments, IGF‐I increased high K+‐stimulated Dopa accumulation (expressed as picomoles per minute per milligram of protein) by 96 ± 13%. IGF‐I increased the protein content of chromaffin cells by ∼30%; consequently, its effect on tyrosine hydroxylase activity was even greater when Dopa synthesis was expressed as picomoles per minute per 107 cells. IGF‐I also enhanced the rate of Dopa accumulation in cells stimulated by dimethylphenyl‐piperazinium, 8‐bromo‐cyclic AMP, phorbol 12, 13‐dibutyrate, or Ba2+. The effect of IGF‐I on high K+‐stimulated tyrosine hydroxylase activity was measurable when enzyme activity was assayed in vitro, suggesting that this effect was due to a stable modification of the enzyme. Immunoblotting studies indicated that cells cultured in the presence of IGF‐I had a small selective increase (36 ±12%) in the amount of tyrosine hydroxylase per unit of protein. The effect of IGF‐I on tyrosine hydroxylase activity and catecholamine synthesis appears to be due to an integrated response of the cells to this agonist: IGF‐I increases the amount of tyrosine hydroxylase in the cells and promotes the activation of this enzyme by a variety of agents.
An Ion Channel Locus for the Protein Kinase C Potentiation of Transmitter Glutamate Release from Guinea Pig Cerebrocortical Synaptosomes
Authors: Barrie Anne P. , Nicholls David G. , Sanchez‐Prieto Jose , Sihra Talvinder S.
Published: October 2006

The mechanism by which protein kinase C (PKC) activates transmitter release from guinea pig cerebrocortical synaptosomes was investigated by employing parallel fluorescent assays of glutamate release, cytoplasmic free Ca2+, and plasma membrane potential. 4β‐Phorbol dibutyrate (4β‐ PDBu) enhances the Ca2+‐dependent, 4‐aminopyridine (4AP)‐evoked release of glutamate from synaptosomes, the 4AP‐evoked elevation of cytoplasmic free Ca2+, and the 4AP‐ evoked depolarization of the plasma membrane. 4β‐PDBu itself causes a slow depolarization, which may underlie the small effect of 4β‐PDBu on spontaneous, KCI‐evoked, and Ca2+‐independent/4AP‐evoked glutamate release. Because 4AP (but not KCI) generates spontaneous, tetrodotoxin‐sensitive action potentials in synaptosomes, a major locus of presynaptic PKC action is to enhance these action potentials, perhaps by inhibiting delayed rectifier K+ channels.
Identification and Immunolocalization of Phospholipase C in Bovine Rod Outer Segments
Authors: Ghalayini Abdallah J. , Tarver Alan P. , Mackin William M. , Koutz Cynthia A. , Anderson Robert E.
Published: October 2006

Bovine rod outer segments (ROS) contain a phospholipase C (PLC) that hydrolyzes phosphatidylinositol 4, 5‐bisphosphate. Approximately 60–70% of PLC activity is recovered in soluble extracts of ROS. Moreover, the specific activity of this soluble PLC is approximately 10‐fold higher than that of resealed ROS enzyme activity. Peptide‐specific antiserum (Ab 1109) directed against a highly conserved sequence of the Y‐region found in several PLC isozymes was used to detect any PLC belonging to this family. This antibody specifically recognized a protein of apparent molecular mass of ∼ 140 kDa present in immunoblots of soluble extracts of both ROS and whole retina. The elution profile of this 140‐kDa antigen from a Sephadex G‐150 column coincided with the peak of PLC activity, suggesting PLC activity is associated with the 140‐kDa protein. Immunocytochemical studies of bovine retina using Ab 1109 showed pronounced immunoreactive labeling in the photoreceptor layer. In resealed ROS and washed ROS membranes, Ab 1109 recognized an additional protein of apparent molecular mass of 70 kDa not usually detectable in soluble extracts of ROS, suggesting the presence of at least two isozymes of PLC in ROS.
Membrane‐Associated Phospholipase D Activity in Rat Sciatic Nerve
Authors: Chattopadhyay Jyotiprakas , Natarajan V. , Schmid Harald H. O.
Published: October 2006

Rat sciatic nerve contains a membrane‐bound phospholipase D that catalyzes the hydrolysis of exogenous phosphatidylcholine (PC) to phosphatidic acid (PA) and choline. The enzyme is associated with a particulate fraction consisting primarily of microsomes and myelin. This fraction also contains phosphatidate phosphohydrolase activity leading to the production of diacylglycerols (DAG). The phosphohydrolase activity can be completely inhibited by NaF. Hydrolysis of exogenous PC requires detergent and is linear up to about 40 μg of protein at a pH optimum of 6.5. In the absence of NaF, the sum of PA and DAG increases linearly for 40 min, whereas in its presence, PA production is linear for only 15 min. At optimum conditions, PC hydrolysis proceeds at 15 nmol/h/mg ot protein. Addition of increasing amounts of ethanol to the incubation system leads to the generation of increasing amounts of phosphatidylethanol, indicating transphosphatidylation activity. At an ethanol concentration of 0.4 M, phosphatidylethanol represents about one‐half of the reaction products generated at approximately the same rate of enzymic activity observed in the absence of ethanol. Higher ethanol concentrations are inhibitory.
In Vivo Study of the Elimination from Rat Brain of an Intracerebrally Formed Xenobiotic Metabolite, 1‐Naphthyl‐β‐D‐Glucuronide
Authors: Leininger Brigitte , Ghersi‐Egea Jean‐François , Siest Gérard , Minn Alain
Published: October 2006

Abstract: Among the drug‐metabolizing enzymes present in the rat brain, one form of UDP‐glucuronyltransferase catalyzes the formation of the polar metabolite 1‐naphthyl‐β‐D‐glucuronide from 1‐naphthol. We measured the activity of this isoform in different brain regions and showed its heterogeneous distribution. Conjugation activities were found to be the highest in the olfactory bulbs (25.4 nmol/h/mg protein) and lowest in the cerebellum (4.5 nmol/h/mg protein). As the blood–brain barrier prevents the passage of hydrosoluble molecules, we studied in vivo the characteristics of the efflux of labeled 1‐naphthyl‐β‐D‐glucuronide injected into the lateral ventricle and the cortex tissue, using tritiated water and labeled inulin as reference compounds. The results reported here indicate that intracerebrally formed glucuronide is cleared from brain tissue by both diffusion and a saturable efflux process.