bluuebunny/crossref_metadata_2025 huggingface dataset:
Calpain Inhibitor I Prevents Rapid Postmortem Degradation of Benzodiazepine Binding Proteins: Fluorographic and Immunological Evidence
Authors: Reichelt Ralf , Möhler Hanns , Hebebrand Johannes
Published: October 2006

Abstract: Endogenous proteolysis of the major central benzodiazepine (BZ) binding protein of 53K occurs rapidly postmortem and leads to a fragment of 47K. To determine indirectly the protease responsible for this proteolysis, membranes of porcine cortex were prepared from homogenates. which were either frozen immediately or left at room temperature for 12 h in the presence or absence of various representative protease inhibitors. Membranes were subsequently photolabeled with [3H]flunitrazepam, and subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and fluorography or immunoblotted using an α‐subunit‐specific monoclonal antibody bd‐24. Both fluorographs and imraunoblots revealed that calpain inhibitor I, Ep‐459 (E‐64 analogue), and EDTA (±1 mM) prevent endogenous proteolysis. In future studies one of these inhibitors should be added to receptor preparations. The results indicate that calpain is the responsible protease.
Serotonin Stimulates Both Cytosolic and Membrane‐Bound Guanylate Cyclase in NG108–15 Cells
Authors: Tohda Michihisa , Nomura Yasuyuki
Published: October 2006

Abstract: The cyclic GMP (cGMP) content was rapidly (>30 s) increased by serotonin [5‐hydroxytryptamine (5‐HT)] (EC50= 10 μM), and the increase lasted for > 10 min in NG108–15 cells. The 5‐HT‐induced elevation of cGMP level (EC50= 10 μM) at 20 s (“fast” elevation) was inhibited by ICS 205–930 or MDL 72,222 and by Ca2+ deficiency in the reaction medium but not by organic Ca2+ antagonists. The 5‐HT effect at 10 min (“slow” elevation) was not inhibited by several antagonists for 5‐HT receptors of the IA, IB, IC., ID, 2, and 3 subtypes and was independent from external Ca2+ concentration. The fast and slow effects of 5‐HT were similar to the effects of bradykinin and atrial natriuretic peptide (ANP), respectively, in aspects of both Ca2+ dependency and time course of the effects. Bradykinin transiently stimulated formation of inositol phosphates as well as accumulation of cGMP, a finding suggesting that intracellular Ca2+ is involved in bradykinin‐induced cGMP accumulation as shown in the fast response to 5‐HT. ANP. an activator of membrane‐associated guanylate cyclase (mGC), slowly (∼60 s) increased the cGMP content (EC50= 10 nAf), a result lasting for >10 min, and the effects were independent from external Ca2+, as shown in the slow response to 5‐HT. 5‐HT and ANP did not induce formation of inositol phosphates. These results suggest that (a) the fast effects of 5‐HT on cGMP level elevation are mediated by 5‐HT3 receptors, which activate cytosolic guanylate cyclase through Ca2+ entry via ion channels other than voltage‐sensitive Ca24 channels, and (b) the slow effects seem to be due to an unidentified subtype of 5‐HT receptor that activates ANP‐sensitive mGC.
Ca2+ Mobilized by Caffeine from the Inositol 1,4,5‐Trisphosphate‐Insensitive Pool of Ca2+ in Somatic Regions of Sympathetic Neurons Does Not Evoke [3H]Norepinephrine Release
Authors: Wakade Taruna D. , Bhave Sanjiv V. , Bhave Anjali , Przywara Dennis A. , Wakade Arun R.
Published: October 2006

Abstract: The effects of electrical stimulation, muscarinic and serotonergic agonists, and caffeine on [3H]inositol 1,4,5‐trisphosphate ([3H]Ins(1,4,5)P3) content, intracellular free Ca2+ concentration ([Ca2+]i), and release of [3H]norepinephrine ([3H]NE) were studied in cultured sympathetic neurons. Neuronal cell body [Ca2+]i was unaffected by muscarinic or serotonergic receptor stimulation, which significantly increased [3H]Ins(1,4,5)P3 content. Stimulation at 2 Hz and caffeine had no effect on [3H]Ins(1,4,5)P3, but caused greater than two‐fold increase in [Ca2+]i. Only 2‐Hz stimulation released [3H]NE. Caffeine had no effect on the release. When [Ca2+]i was measured in growth cones, only electrical stimulation produced an increase in [Ca2+]i. The other agents had no effect on Ca2+ at the terminal regions of the neurons. We conclude that Ins(1,4,5)P3‐insensitive, but caffeine‐sensitive Ca2+ stores in sympathetic neurons are located only in the cell body and are not coupled to [3H]NE release.
Early Changes in Nuclear Proteins Following Axotomy
Authors: Buriani Alessandro , Savage Mary J. , Burmeister Donald W. , Goldberg Daniel J.
Published: October 2006

Abstract: Changes in protein synthesis are thought to be important in the response of the neuron to axotomy. Certain axonally transported proteins whose synthesis increases probably play important roles in regeneration of the axon. Although little is known about the regulation of these changes, the cell often controls its production of proteins at the nuclear level, where transactivating proteins modulate the transcription of specific genes. Thus, changes in nuclear proteins might be expected to be among the early events following axotomy, but such changes have not yet been described. We have addressed this issue by dissecting out single nuclei from [35S]methionine‐labeled giant R2 neurons of Aplysia and analyzing the proteins by two‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. This procedure was used to avoid contamination with nonneuronal and nonnuclear proteins. Our results demonstrate large increases in two nuclear proteins (56 kDa and 41 kDa) and decreases in two others (77 kDa and 46 kDa) 5 h after axotomy. These are the earliest postaxotomy changes in [35S]methionine‐labeled proteins that have been reported.
2‐Phenylethylamine: A Modulator of Catecholamine Transmission in the Mammalian Central Nervous System?
Authors: Paterson I. A. , Juorio A. V. , Boulton A. A.
Published: October 2006

Since the identification of 2‐phenylethylamine (β‐phenylethylamine; PE) as a biogenic amine, there has been much discussion about what role, if any, it may have in the CNS. Indeed, the low endogenous concentration of PE in the brain and its relatively low potency in behavioral and pharmacological experiments have led some researchers to conclude that perhaps PE possessed no physiological role at all but that it was merely a metabolic by‐product. Our findings have caused us to conclude otherwise, and in this article we review the neurochemical, neuropharmacological, and neurophysiological findings that lead us to propose that PE is a neuromodulator of catecholamine neurotransmission in the CNS.
Labeling of Retina and Optic Tectum Phospholipids in Chickens Exposed to Light or Dark
Authors: Guido M. E. , Caputto Beatriz L.
Published: October 2006

Abstract: The labeling of retina ganglion cell and optic tectum phospholipids was determined in chickens given an intraocular injection of 32P and then either exposed to light or maintained in the dark. Significantly higher labeling was found in the optic tectum phospholipids of light‐exposed compared with dark‐maintained animals after 3–24 h of labeling. In the ganglion cells, the labeling of phospholipids increased in dark with respect to light at 15 and 30 min of labeling; from 60 min to 24 h, the labeling of phospholipids was significantly higher in light with respect to dark, even if the precursor pool showed a higher labeling in dark at all times studied. When labeling was allowed to proceed in the dark for 30 min and then half of the animals were exposed to light for 15 min, the labeling of ganglion cell phospholipids of light‐exposed animals was significantly higher than those of animals kept in the dark. No individual phospholipid accounted for the differences observed in the labeling of the total phospholipid pool. These results are interpreted as an increase in the biosynthesis of phospholipids in the ganglion cell somas in light with respect to dark.
N‐Methyl‐d‐Aspartate‐Sensitive Glutamate Receptors Induce Calcium‐Mediated Arachidonic Acid Release in Primary Cultures of Cerebellar Granule Cells
Authors: Lazarewicz J. W. , Wroblewski J. T. , Costa E.
Published: October 2006

Abstract: In primary cultures of cerebellar granule cells, glutamate, aspartate, and N‐methyl‐d‐aspartate (NMDA) induced a dose‐dependent release of [3H]arachidonic acid ([3H]AA) which was selective for these agonists and was inhibited by NMDA receptor antagonists. The agonist‐induced [3H]AA release was reduced by quinacrine at concentrations that inhibited phospholipase A2 (PLA2) but affected neither the activity of phospholipase C (PLC) nor the hydrolysis of phosphoinositides induced by glutamate or quisqualate. Thus, the increased formation of AA was due to the receptor‐mediated activation of PLA2 rather than to the action of PLC followed by diacylglycerol lipase. The receptor‐mediated [3H]AA release was dependent on the presence of extracellular Ca2+ and was mimicked by the Ca2+ ionophore ionomycin. Pretreatment of granule cells with either pertussis or cholera toxin failed to inhibit the receptor‐mediated [3H]AA release. Hence, in cerebellar granule cells, the stimulation of NMDA‐sensitive glutamate receptors leads to the activation of PLA2 that is mediated by Ca2+ ions entering through the cationic channels functioning as effectors of NMDA receptors. A coupling through a toxin‐sensitive GTP‐binding protein can be excluded.
The Glycine Antagonist 7‐Chlorokynurenic Acid Blocks the Effects of N‐Methyl‐d‐Aspartate on Agonist‐Stimulated Phosphoinositide Hydrolysis in Guinea‐Pig Brain Slices
Authors: Kendall David A. , Robinson Jean P.
Published: October 2006

Abstract: Despite having no effect on basal phosphoinositide hydrolysis, N‐methyl‐d‐aspartate (NMDA) inhibited carbachol‐stimulated accumulation of 3H‐inositol phosphates and enhanced that due to noradrenaline in guinea‐pig cerebral cortex slices. The glycine antagonist 7‐chlorokynurenic acid inhibited the effects of NMDA and this inhibition was reversed by glycine. The action of 7‐chlorokynurenic acid was not mimicked by strychnine or HA 966 (l‐hydroxy‐3‐aminopyrrolid‐2‐one). l‐Glutamate also inhibited carbacholstimulated accumulation of 3H‐inositol phosphates, but this inhibition was not blocked by 7‐chlorokynurenic acid. The data are consistent with glycine maintaining tonic control over NMDA receptor activity in guinea‐pig brain.
Decreased Protein Kinase C Activity During Cerebral Ischemia and After Reperfusion in the Adult Rat
Authors: Crumrine R. Christian , Dubyak George , LaManna Joseph C.
Published: October 2006

Abstract: The possible activation of protein kinase C (PKC) during total cerebral ischemia was investigated in the rat. Translocation of PKC activity from the soluble to the particulate fraction was used as an index of PKC activation. There was a drop in the proportion of particulate PKC activity from 30% for controls to 20% by 30 min of ischemia (p < 0.01). By 20 min of cardiac arrest, there was a 40% decline of the total cellular PKC activity (p < 0.01). This was not accompanied by an increase in activator‐independent activity, a finding indicating PKC was not being converted to protein kinase M. These data suggest that PKC was not activated during ischemia, but rather that ischemia causes a reduction in cellular PKC activity. Translocation of PKC activity to the particulate fraction was not observed in the cerebral cortex or hippocampus of reperfused brain for up to 6 h of recovery following 11–13 min of total cerebral ischemia. The level of total, soluble, and particulate PKC activity in the cerebral cortex was reduced (p <0.05), corresponding to the decrease observed by 15 min of ischemia without reflow. A similar decline in activity was also observed in the hippocampus. No increase in activator‐independent activity was observed. These data suggest that PKC was inhibited during cerebral ischemia and that this reduced level of PKC activity was maintained throughout 6 h of recovery. We conclude that pathological activation of PKC was not responsible for the evolution of ischemic brain damage.
Developmental Expression of HNK‐1‐Reactive Antigens in the Rat Cerebellum and Localization of Sulfoglucuronyl Glycolipids in Molecular Layer and Deep Cerebellar Nuclei
Authors: Prasadarao Nemani , Koul Omanand , Tobet Stuart A. , Chou Denise K. H. , Jungalwala Firoze B.
Published: October 2006

Abstract: Monoclonal antibody HNK‐1‐reactive carbohydrate epitope is expressed on proteins, proteoglycans, and sulfoglucuronyl glycolipids (SGGLs). The developmental expression of these HNK‐1‐reactive antigens was studied in rat cerebellum. The expression of sulfoglucuronyl lacto‐N‐neotetraosylceramide (SGGL‐1) was biphasic with an initial maximum at postnatal day one (PD 1), followed by a second rise in the level at PD 20. The level of sulfoglucuronyl lacto‐N‐norhexaosyl ceramide (SGGL‐2) in cerebellum was low until PD 15 and then increased to a plateau at PD 20. The levels of SGGLs increased during postnatal development of the cerebellum, contrary to their diminishing expression in the cerebral cortex. The expression of HNK‐1‐reactive glycoproteins decreased with development of the rat cerebellum from PD 1. Several HNK‐1‐reactive glycoproteins with apparent molecular masses between 150 and 325 kDa were visualized between PD 1 and PD 10. However, beyond PD 10, only two HNK‐1‐reactive bands at 160 and 180 kDa remained. The latter appeared to be neural cell adhesion molecule, N‐CAM‐180. A diffuse HNK‐1‐reactive band seen at the top of polyacrylamide electrophoretic gels was due mostly to proteoglycans. This band increased in its reactivity to HNK‐1 between PD 15 and PD 25 and then decreased in the adult cerebellum. The lipid antigens were shown by two complementary methodologies to be localized primarily in the molecular layer and deep cerebellar nuclei as opposed to the granular layer and white matter. A fixation procedure which eliminates HNK‐1‐reactive epitope on glycoproteins and proteoglycans, but does not affect glycolipids, allowed selective immunoreactivity in the molecular layer and deep cerebellar nuclei. In order to confirm this localization, SGGLs were analyzed by HPTLC‐immunoverlay method in micro‐dissected cerebellar layers from freeze‐dried cryocut sections: they were found primarily in the molecular layer and deep cerebellar nuclei and were undetectable in the granule cell layer and white matter. These results, along with the lack of SGGLs and disialosyl lacto‐N‐neotetraosylceramide (LD1) in several Purkinje cell‐deficient murine mutants reported previously, indicate that these glycolipids are associated specifically with Purkinje cell dendrites in the molecular layer and Purkinje cell axon synapses in deep cerebellar nuclei.
α‐Latrotoxin Releases Both Vesicular and Cytoplasmic Glutamate from Isolated Nerve Terminals
Authors: McMahon Harvey T. , Rosenthal Luann , Meldolesi Jacopo , Nicholls David G.
Published: October 2006

Abstract: α‐Latrotoxin causes a massive release of endogenous glutamate from guinea‐pig cerebrocortical synaptosomes. There appear to be two components to the release. In the first 2 min following addition of 1.3 nMα‐latrotoxin, glutamate release is largely energy dependent. Superimposed upon this release is a more slowly developing but ultimately much more extensive release of cytoplasmic glutamate together with γ‐aminobutyric acid and nonvesicular amino acids such as aspartate and α‐aminoisobutyrate. In parallel with this cytoplasmic release there is an extensive depletion of ATP, a massive rise in cytoplasmic free Ca2+ concentration, and a severe restriction of synaptosomal respiratory capacity. The cytoplasmic release is only partially Na+ dependent. eliminating a simple reversal of the plasma membrane acidic amino acid carrier. It is concluded that α‐latrotoxin releases both transmitter and cytoplasmic pools of amino acids in synaptosomes and causes a major disruption of terminal integrity.
Volume Regulation of Nerve Terminals
Authors: Babila T. , Atlan H. , Fromer I. , Schwalb H. , Uretzky G. , Lichtstein D.
Published: October 2006

Abstract: Pinched‐off presynaptic nerve terminals (synaptosomes) possess significant regulatory volume increase (RVI) and regulatory volume decrease (RVD) capabilities. Following a swelling induced by a hypotonic challenge, the synaptosomes regulate their volume and adjust it, in 2 min, to within 5% of its initial value (RVD) at an initial rate of ‐0.77 ± 0.10%/s (mean ± SEM). Following a shrinking induced by a hypertonic challenge, the synaptosomes also regulate their volume at an initial rate of 0.18 ± 0.02%/s (RVI), resulting in a new steady state, reached within 5–10 min, with a synaptosomal volume below the original volume. The omission of Na+ or K+ ions from the extrasynaptosomal medium reduces the initial rate of RVI by 72.5 and 66.5%, respectively. The “loop diureties” bumetanide and furosemide significantly inhibited the RVI of the synaptosomes. In contrast, ouabain, amiloride, or 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid did not have any significant effect on RVI parameters. Furthermore, bumetanide‐sensitive 86Rb uptake by rat brain synaptosomes was stimulated threefold by a hypertonic perturbation of 30%. Thus we conclude that the RVI of synaptosomes is mainly due to a stimulation of the Na+, K+, Cl− co‐transport system induced by the synaptosomal shrinking following the hypertonic challenge.
Embryonic Neural Cell Adhesion Molecule in Cerebrospinal Fluid of Younger Children: Age‐Dependent Decrease During the First Year
Authors: Weisgerber Ch. , Husmann M. , Frosch M. , Rheinheimer C. , Peuckert W. , Görgen I. , Bitter‐Suermann D.
Published: October 2006

Abstract: Poly‐α‐2,8‐N‐acetylneuraminic acid (poly‐α‐2,8‐NeuAc) is developmentally expressed in neural tissue of higher animals, where it is covalently attached to the neural cell adhesion molecule (NCAM), a large integral membrane glycoprotein mediating cell‐cell adhesion during neuronal development. NCAM exists in several molecular forms, of which only embryonic NCAM carries lengthy chains (n > 5) of poly‐α‐2,8‐NeuAc. Chemically identical poly‐α‐2,8‐NeuAc of bacterial origin is an important virulence factor in infections caused by Neisseria meningitidis group B and Escherichia coli K1, the predominant pathogens of bacterial meningitis. A quantitative enzyme‐linked immunoassay was developed using monoclonal antibody (MAb) 735, an MAb specifically recognizing poly‐α‐2,8‐NeuAc, and applied to CSF specimens from younger children. Poly‐α‐2,8‐NeuAc contents were within the range of 20‐0.2 μg/ml, decreasing from day 1 to day 300. Immunoprecipitation, immunoblot with a rabbit anti‐mouse NCAM serum recognizing the protein part of human NCAM by cross‐reactivity, affinity enrichment using immobilized MAb 735, and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed that poly‐α‐2,8‐NeuAc in CSF is bound to human NCAM, probably NCAM‐120.
Effect of Tetanus Toxin on Oxytocin and Vasopressin Release from Nerve Endings of the Neurohypophysis
Authors: Halpern Jane L. , Habig William H. , Trenchard Holly , Russell James T.
Published: October 2006

Abstract: The effect of tetanus toxin on neuropeptide hormone release from isolated nerve endings of the neural lobe of rat pituitaries (neurosecretosomes) was measured in a perfusion system. Tetanus toxin inhibited depolarization‐evoked release of oxytocin and vasopressin in a time‐ and dose‐dependent manner. At 1 μg/ml, tetanus toxin blocked stimulated release by 85%. Tetanus toxin that was preincubated with a neutralizing monoclonal antibody or heated to 100°C had no effect on hormone release. The ionophores A23187 and ionomycin were potent stimulators of hormone release in control nerve endings, but were not able to overcome the effect of tetanus toxin in intoxicated nerve endings. 8‐Bromo cyclic GMP. which has been reported to reverse the action of tetanus toxin in PC12 cells, had no effect on the action of tetanus toxin in neurosecretosomes. Neurosecretosomes are the first system in which tetanus toxin has been shown to block release from peptidergic nerve terminals. They appear to be a valuable in vitro system for studying the biochemical mechanism of tetanus toxin action.
Purinergic Modulation of Hippocampal Acetylcholine Release Involves α‐Dendrotoxin‐Sensitive Potassium Channels
Authors: Benishin Christina G.
Published: October 2006

Abstract: Modulation of acetylcholine (ACh) release from superfused hippocampal slices was examined when the release of ACh was stimulated by exposure of slices to elevated K+ concentration. Evoked release was not sensitive to inhibition by 0.1 μM tetrodotoxin, but it could be inhibited in a dose‐dependent manner by a muscarinic agonist (10–100 nM oxotremorine) and a purinergic agonist (10–100 nM 2‐chloroadenosine). The α‐dendrotoxin (100 nM), which selectively blocks voltage‐gated inactivating K+ channels in nerve endings, did not affect the release of ACh under resting or depolarized conditions. However, α‐dendrotoxin reduced the 2‐chloroadenosine‐induced inhibition of release, but did not alter the oxotremorine‐induced inhibition. These results suggest that an α‐dendrotoxin‐sensitive K+ channel may be activated as an obligatory step in the modulation of ACh release by presynaptic purinergic receptor activation, but not in the modulation by presynaptic muscarinic receptors.
Gangliosides Prevent Ischemia‐Induced Down‐Regulation of Protein Kinase C in Fetal Rat Brain
Authors: Magal Ella , Louis Jean‐Claude , And Jose Aguilera. , Yavin Ephraim
Published: October 2006

Abstract: Complete obstruction of the maternal blood flow to fetal rats at 20 days of gestation for a period of 10 min causes a significant shift of ∼22% in protein kinase C (PKC) activity from a cytosolic to a membrane‐bound form in the fetal brain. This translocation can be entirely reversed without losses in activity by a single intraperitoneal injection into the gravid rat of either a mixture of disialo‐ and trisialoganglioside [polysialoganglioside (PSG)] or by GM1 (50 mg/kg of body weight) given 3 h before onset of the ischemic episode. Cessation of blood flow for 15 min followed by a reperfusion period of 24 h results in a 47% loss in total PKC activity. This down‐regulation can be almost entirely prevented upon intraperitoneal administration of GM1 3 h before, but also during and even 90 min after the onset of ischemia. The PSG mixture is also effective, particularly when given 3 h before the insult. Down‐regulation of PKC is accompanied by an increase in a Ca2+‐phosphatidylserinc‐indepcndent kinase [protein kinase M (PKM)] activity, which rises from 30 pmol/min/mg of protein in control animals to a maximal value of 83.1 pmol/min/mg of protein after 15 min of ischemia and 6 h of reperfusion. By 24 h, PKM activity is 46.8 pmol/min/mg of protein. Administration of GM1 blocks completely the appearance of PKM, a result suggesting that PKC down‐regulation and PKM activity elevation are intimately associated events and that both are regulated by GM1 ganglioside.
The General Anesthetic Propofol Enhances the Function of γ‐Aminobutyric Acid‐Coupled Chloride Channel in the Rat Cerebral Cortex
Authors: Concas A. , Santoro G. , Mascia M. P. , Serra M. , Sanna E. , Biggio G.
Published: October 2006

Abstract: The effect of the general anesthetic propofol on t‐[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to unwashed membrane preparations from rat cerebral cortex was studied and compared to that of other general anesthetics (pentobarbital, alphaxalone) which are known to enhance GABAergic transmission. Propofol produced a concentration‐dependent complete inhibition of [35S]TBPS binding, an effect similar to that induced by pentobarbital and alphaxalone, although these agents differ markedly in potency (alphaxalone > propofol > pentobarbital). The concomitant addition of propofol either with alphaxalone or pentobarbital produced an additive inhibition of [35S]TBPS binding, suggesting separate sites of action or different mechanisms of these drugs. Moreover, although bicuculline (0.1 μM) completely antagonized the propofol‐induced inhibition of [35S]TBPS binding, the effect of this anesthetic was not due to a direct interaction with the γ‐aminobutyric acidA (GABAA) recognition site. In fact, propofol, like alphaxalone and pentobarbital, markedly enhanced [3H]GABA binding in the rat cerebral cortex. Finally, propofol was able to enhance [3H]GABA binding in membranes previously incubated with the specific chloride channel blocker picrotoxin. Taken together these data strongly suggest that propofol, like other anesthetics and positive modulators of GABAergic transmission, might exert its pharmacological effects by enhancing the function of the GABA‐activated chloride channel.
A Novel Potential Metallopeptidase Derived from the Enkephalinase Gene by Alternative Splicing
Authors: Llorens‐Cortes Catherine , Giros Bruno , Schwartz Jean‐Charles
Published: October 2006

Abstract: Amplification of rat intestine mRNAs was performed by the reverse transcriptase‐polymerase chain reaction (RT‐PCR) using various oligonucleotide primers mainly corresponding to the translated region of the enkephalinase (EC 3.4.24.11, membrane metalloendopeptidase, MME 1) gene. In addition to the expected transcript, a shorter one was identified and its sequence indicated that it corresponds to an alternatively spliced mRNA from which exons 5–18 of MME I are deleted. It encodes a deduced 255 amino acid protein, MME II, instead of the 742 amino acid sequence of enkephalinase. The deduced structure of MME II is consistent with its being a membrane‐bound, zinc‐containing glycoprotein with a modified peptidase activity. MME II mRNA is also expressed, together with MME I mRNA, in brain and thyroid in a tissue‐specific manner.
Both A1 and A2a Purine Receptors Regulate Striatal Acetylcholine Release
Authors: Brown Susan J. , James Stephen , Reddington Martin , Richardson Peter J.
Published: October 2006

Abstract: The receptors responsible for the adenosine‐mediated control of acetylcholine release from immunoaffinitypurified rat striatal cholinergic nerve terminals have been characterized. The relative affinities of three analogues for the inhibitory receptor were (R)‐phenylisopropyladenosine > cyclohexyladenosine > N‐ethylcarboxamidoadenosine (NECA), with binding being dependent of the presence of Mg2+ and inhibited by 5′‐guanylylimidodiphosphate [Gpp(NH)p] and adenosine receptor antagonists. Adenosine A1 receptor agonists inhibited forskolin‐stimulated cholinergic adenylate cyclase activity, with an IC50 of 0.5 nM for (R)‐phenylisopropyladenosine and 500 nM for (S)‐phenylisopropyladenosine. A1 agonists inhibited acetylcholine release at concentrations approximately 10% of those required to inhibit the cholinergic adenylate cyclase. High concentrations (1 μM) of adenosine A1 agonists were less effective in inhibiting both adenylate cyclase and acetylcholine release, due to the presence of a lower affinity stimulatory A2 receptor. Blockade of the A1 receptor with 8‐cyclopentyl‐1,3‐dipropylxanthine revealed a half‐maximal stimulation by NECA of the adenylate cyclase at 10 nM, and of acetylcholine release at approximately 100 nM. NECA‐stimulated adenylate cyclase activity copurified with choline acetyltransferase in the preparation of the cholinergic nerve terminals, suggesting that the striatal A2 receptor is localized to cholinergic neurones. The possible role of feedback inhibitory and stimulatory receptors on cholinergic nerve terminals is discussed.
Membrane Viscosity Correlates with α1‐Adrenergic Signal Transduction of the Aged Rat Cerebral Cortex
Authors: Miyamoto Atsushi , Araiso Tsunehisa , Koyama Tomiyasu , Ohshika Hideyo
Published: October 2006

Abstract: We investigated, using adult (2‐month‐old) and senescent (12‐ and 24‐month‐old) rats, the effects of aging on the relationship between the α1‐adrenergic coupling system and the membrane viscosity of the cerebral cortex. There was no age‐related difference in the KD values of [3H]prazosin binding on the membranes. The Bmax values of [3H]prazosin binding were reduced with advanced age. Norepinephrine induced formation of 3H‐labeled inositol phosphates (3H‐IPs) in the slices increased with advanced age. The EC50 values for norepinephrine to stimulate the formation of 3H‐IPs at advanced age were lower than that at adult age. The cholesterol content in membranes increased with advanced age. No changes in the phospholipid content in membranes were observed with advanced age. Concomitantly, an increase of the molar ratio of cholesterol to phospholipids was observed with advanced age. The membrane viscosity as measured by 1,6 diphenyl‐1,3,5‐hexatriene increased with advanced age. These results indicate that the altered cholesterol content and/or viscosity in cortical membranes of the aged rat may account for the loss of α1‐adrenergic receptor density and/or compensatory changes in the receptor‐phospholipase C coupling system.
Molecular Cloning, Structure, and Expression of Dopamine‐β–Hydroxylase from Bovine Adrenal Medulla
Authors: Wu Hong‐Jiang , Parmer Robert J. , Koop Allen H. , Rozansky David J. , O'Connor Daniel T.
Published: October 2006

Abstract: Dopamine‐β‐hydroxylase (DBH), the enzyme that catalyzes the conversion of dopamine to norepinephrine, remains the topic of many unanswered questions. We isolated DBH cDNA clones from a bovine adrenal medulla cDNA library in the vector λgt10. The longest cDNA had an open reading frame encoding an entire mature DBH 578 amino acid (64,808 dalton) polypeptide chain, though lacking a portion of the signal peptide. Additional 5'clones, obtained by the polymerase chain reaction, established the sequence of a 19 amino acid signal peptide. The mature protein sequence was 84% homologous to that of human pheochromocytoma DBH, including preservation of four potential copper ligand sites (HH or HXH) and substrate binding domains. There were no hydrophobic (putative membrane spanning) domains, other than the signal peptide. All available DBH peptide and protein sequence data can be accounted for by the cDNA‐deduced 578 amino acid mature protein primary structure. Prokaryotic DBH expression yielded a 65‐kilodalton DBH‐immunoreactive peptide that differed from eukaryotic adrenal DBH only in N‐linked, endoglycosidase F‐sensitive glycosylation in the latter. Southern analysis suggested one DBH gene, whereas Northern analysis suggested a single 2.6 kbp tissue‐specific DBH transcript. Comparison of the DBH primary structure with other reported sequences [Protein Identification Resource (PIR), New Atlas (NEWAT)] did not indicate that DBH is a member of any known gene family. The results suggest that a single DBH gene encodes a message specifying a single DBH polypeptide chain.
Gallium‐67 as a Potential Marker for Aluminium Transport in Rat Brain: Implications for Alzheimer's Disease
Authors: Pullen R. G. L. , Candy J. M. , Morris C. M. , Taylor G. , Keith A. B. , Edwardson J. A.
Published: October 2006

Abstract: Evidence of a link between aluminium and Alzheimer's disease, parkinsonism‐dementia of Guam, and dialysis encephalopathy raises questions regarding the role of this element in the pathogenesis of these conditions. Therefore, we have investigated the use of gallium‐67 (67Ga) as a marker for brain uptake of aluminium. The binding of 67Ga to plasma proteins has been studied, and the blood‐brain barrier permeability and autoradiographic distribution of this isotope in rat brain determined in vivo. The autoradiographic distribution of 125I‐Fe‐transferrin receptors in rat brain has also been determined in vitro. Results show that 67Ga was bound to plasma transferrin, entered the brain with a blood‐brain barrier permeability of 2.48 X 10‐6 ml/min/g, and showed a marked regional distribution that was very similar to that of 125I‐Fe‐transferrin receptors. Our data suggest that the vulnerability of the hippocampus, amygdala, and cerebral cortex in conditions such as those mentioned above may be partly due to an increased uptake and deposition of aluminium in these regions by the iron transport system.
Stimulation of Dopamine Release from Cultured Rat Mesencephalic Cells by Naturally Occurring Excitatory Amino Acids: Involvement of Both N‐Methyl‐D‐Aspartate (NMDA) and Non‐NMDA Receptor Subtypes
Authors: Mount Howard , Quirion Rémi , Chaudieu Isabelle , Boksa Patricia
Published: October 2006

Abstract: In rat mesencephalic cell cultures, L‐glutamate at concentrations ranging from 100 μM to 1 mM stimulated release of [3H]dopamine that was attenuated by the non‐N‐methyl‐D‐aspartate (non‐NMDA) receptor antagonist 6,7‐dinitroquinoxalinedione, but not by the selective NMDA receptor antagonists (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine hydrogen maleate (MK‐801; 10 μM) and 3‐(2‐carboxypiperazine‐4‐yl)propyl‐1‐phosphonate (300 μM). Even at 1 mM glutamate, this release was Ca2+ dependent. These observations suggest that the release was mediated by a non‐NMDA receptor. Only release stimulated by a lower concentration (10 μM) of glutamate was inhibited by MK‐801 (10 μM), indicating that glutamate at this concentration activates the NMDA receptor. By contrast, L‐aspartate at concentrations of 10 μM to 1 mM evoked [3H]dopamine release that was completely inhibited by MK‐801 (10 μM) and was also Ca2+ dependent (tested at 1 and 10 mM aspartate). Thus, effects of aspartate involved activation of the NMDA receptor. Sulfur‐containing amino acids (L‐homocysteate, L‐homocysteine sulfinate, L‐cysteate, L‐cysteine sulfinate) also evoked [3H]dopamine release. Release evoked by submillimolar concentrations of these amino acids was attenuated by MK‐801 (10 μM), indicating involvement of the NMDA receptor. Higher concentrations of the sulfur‐containing amino acids (≥1 μM L‐homocysteate, ≥ 1 mM L‐homocysteine sulfinate, ≥ 10 mM L‐cysteate, ≥ 10 mM L‐cysteine sulfinate) evoked [3H]dopamine release that was Ca2+ dependent (largely Ca2+ dependent for 10 mM L‐cysteine sulfinate) and inhibited by 6,7‐dinitroquinoxalinedione (100 μM), but unaffected by MK‐801 (10 or 100 μM). Thus, like glutamate, higher concentrations of the sulfur‐containing amino acids interact with non‐NMDA receptors, while non‐NMDA receptor involvement was not observed with aspartate.
Basic Fibroblast Growth Factor (bFGF) Immunoreactivity Is Present in Chromaffin Granules
Authors: Westermann Reiner , Johannsen Marianne , Unsicker Klaus , Grothe Claudia
Published: October 2006

Abstract: Basic fibroblast growth factor (bFGF) has recently been isolated from bovine adrenal glands. Immunohistological data revealed its presence in both adrenal cortex and adrenal medulla. Using immuno‐electronmicroscopy, we found that in medullary chromaffin cells bFGF‐immunoreactivity is localized in the secretory granules. Immuno‐reactivity also was observed by electronmicroscopy in isolated granules. Western blot analysis revealed the presence of the typical 18‐kDa bFGF and additional immunoreactive materials with molecular masses of ∼ 24, 30, and 46 kDa in whole bovine adrenal, and in cortex and medulla. Similar results were obtained with proteins from bovine chromaffin granules, with the following two exceptions: the 46‐kDa immunoreactivity was found to be highly enriched when compared with medulla or cortex, and the 18‐kDa band could be detected with only an antiserum against a synthetic peptide comprising the 24 NH2‐terminal amino acids of bFGF, and not with an antiserum against purified bovine pituitary bFGF. All fractions enriched for bFGF‐immunoreactivity showed neurotrophic activity for chick ciliary ganglion neurons, which could be blocked by antibodies. These results demonstrate for the first time the localization and occurrence of bFGF in a cellular secretory organelle, and present further evidence for the existence of higher molecular weight immunoreactive forms of bFGF.
Regulation of Calcium Concentrations in Synaptosomes: α2‐Adrenoceptors Reduce Free Ca2+ by Closure of N‐Type Ca2+ Channels
Authors: Xiang Jian‐Zhong , Morton John , Brammer Michael J. , Campbell Iain C.
Published: October 2006

Abstract: The relationship between intrasynaptosomal total (CaT) and free ([Ca2+]i) calcium and 45Ca accumulation was studied under physiological and K+‐depolarised conditions in rat cortical synaptosomes. Under physiological conditions, CaT (10.7 mM) was ∼ 10,000 times higher than [(Ca2+]i (118 nM), showing that there is a large reservoir of sequestered calcium in synaptosomes. 45Ca accumulation was rapid (initial rate, 3.4 nmol/mg protein/min), substantial (7 nmol/mg protein in 2 min), and depolarisation dependent, and reached equilibrium after 5 min. At equilibrium, only 10% of CaT was freely exchangeable. This pool was much larger than the free Ca2+ pool. CaT, [Ca2+]i, and 45Ca accumulations were directly related to the Ca2+ concentration in the buffer, suggesting that [Ca2+]i is not highly conserved but is maintained by simple equilibria between the various pools. Clonidine reduced 45Ca accumulation in a time‐ and dose‐dependent manner. Maximum inhibition (40% at 100 μM) occurred at 2 min and the IC50 was 80 nM The reduction caused by clonidine (1 μM) reached equilibrium after 5 min, but this equilibrium value was lower than in controls, suggesting that clonidine changes the exchangeable Ca2+ pool size. The effects of clonidine (1 μM) on [Ca2+]i (26% reduction) and on 45Ca accumulation (24% reduction) were most apparent under physiological conditions. However, while it was not dependent on depolarisation, it did not occur in physiological buffer containing low K+ concentration (0.1–1 mM). The inhibitory effect of clonidine on 45Ca accumulation is receptor mediated as it was antagonised by idazoxan (1 μM). Under nondepolarised conditions (5 mM K+), ω‐conotoxin fraction GVIA (ω‐CgTx; 0.5 μM) caused a 35% reduction in 45Ca accumulation in a 2‐min incubation, whereas nifedipine (1 μM) had no significant effect. In contrast, both ω‐CgTx and nifedipine effectively blocked 45Ca entry into depolarised synaptosomes. However, the effect of ω‐CgTx was not additive with that of clonidine, whereas that of nifedipine was. Finally, idazoxan (1 μM) did not alter the effect caused by nifedipine. These data show (a) that clonidine decreases 45Ca accumulation by inhibiting N‐ but not L‐type voltage‐sensitive Ca2+ channels, (b) that specific Ca2+ channels on synaptosomes display a range of voltage sensitivities, and (c) that L‐type Ca2+ channels are operational under depolarising conditions.
Involvement of Lactic Acidosis in Anoxia‐Induced Perturbations of Synaptosomal Function
Authors: White Elizabeth J. , Clark John B.
Published: October 2006

Abstract: L‐Lactate (4–32 mM) added exogenously to resting or depolarised rat forebrain synaptosomes led to a significant decrease in intrasynaptosomal pH. Similarly depolarisation‐induced increases in intrasynaptosomal calcium, calcium uptake, and acetylcholine release were all inhibited. These effects mimicked those previously observed in synaptosomes under anoxic conditions and suggest that lactate may be involved in limiting the damage due to calcium accumulation occurring during ischaemia. D‐Lactate (added exogenously up to 32 mM) did not produce similar effects on these parameters even though the concentrations of intrasynaptosomal D‐lactate reached levels comparable to those obtained with L‐lactate (at 8–16 mM exogenous concentration). The results suggest that the mechanism of action of lactate on these parameters is stereospecific for the L‐enantiomer. The effect of glucose availability on lactate production was assessed to explore the role of substrate availability on ischaemia/anoxic events. When exogenous glucose was increased (10–60 mM), there was no further increase in lactate production in normoxic synaptosomes, which suggests that glucose is not limiting under these conditions. When glucose was removed, as may occur in complete ischaemia, there was a significant decrease in lactate production after 60 min under anoxic or normoxic conditions. It would seem likely therefore that the mechanism underlying the changes observed in synaptosomes incubated under conditions reflecting complete ischaemia does not involve lactate.
Changes in Microtubule‐Associated Protein MAP1B Phosphorylation During Rat Brain Development
Authors: Fischer Itzhak , Romano‐Clarke Giuseppina
Published: October 2006

Abstract: Microtubule‐associated protein MAP1B from neonatal rat brain was separated on sodium dodecyl sulfate‐containing polyacrylamide gels into two isoforms (high and low MAP1B), both of which were recognized by a panel of monoclonal and polyclonal antibodies against MAP1B. In addition, SMI31, a monoclonal antibody directed against phosphorylated epitopes of the neurofilament proteins, showed phosphatase‐sensitive reactivity against the high isoform of MAP1B. The antigenic relationship between the phosphorylated isoform of MAP1B and neurofilaments was confirmed by the reactivity of SMI31 with the immunoprecipitated MAP1B protein. After dephosphorylation of MAP1B with alkaline phosphatase, the higher‐molecular‐weight isoform of MAP1B was no longer detectable with phosphate‐insensitive anti‐MAP1B antibodies, whereas there was a significant increase in the immunoreactivity of the lower‐molecular‐weight MAP1B isoform. These data suggest that the structural microheterogeneity of MAP1B is due to differences in phosphorylation. The two isoforms were present in all brain regions of the young rat. During brain development, the general decrease in MAP1B levels was accompanied by changes in the relative amount of the two isoforms. In particular, the phosphorylated isoform of MAP1B decreased dramatically to almost undetectable levels in adult brain. This conclusion was further supported by immunoblotting analysis that showed the disappearance of phosphorylated epitopes of MAP1B early during brain development. In addition, dephosphorylation experiments demonstrated the phosphatase sensitivity of the phosphorylated isoform throughout development.
Increased Cerebrospinal Fluid Quinolinic Acid, Kynurenic Acid, and L‐Kynurenine in Acute Septicemia
Authors: Heyes Melvyn P. , Lackner Andrew
Published: October 2006

Abstract: Increases in brain quinolinic acid have been implicated in neurodegeneration and convulsions that may accompany infectious diseases. In three rhesus macaques (Macaca mulatta) with septicemia, both CSF and serum quinolinic acid concentrations were markedly elevated and were accompanied by increases in CSF kynurenic acid levels that were of a smaller magnitude. Elevated serum and CSF L‐kynurenine concentrations also occurred and are consistent with activation of indoleamine‐2,3‐dioxygenase and increased substrate flux through the kynurenine pathway. Although it is probable that the marked increases in CSF quinolinic acid and kynurenic acid concentrations are reflected in the extracellular fluid space of brain, it remains to be determined whether the magnitude of such increases influences the activity of excitatory amino acid receptors in brain to produce excitotoxic pathology or noncytolytic disruption of functions mediated by excitatory amino acid receptors.
Differential Effects of Angiotensin II and Eledoisin on Monoamine Oxidase A and B Activities in Rat Brain
Authors: Tomaszewicz Maria , Micossi Luigi G. , Bielarczyk Hanna , Luszawska Danuta , Santarelli Ivano , Szutowicz Andrzej
Published: October 2006

Abstract: Intracerebroventricular injections of angiotensin II caused 108, 62, and 54% increases in monoamine oxidase A activities in rat hippocampus, hypothalamus, and striatum respectively. These activatory effects were abolished by simultaneous injections of eledoisin. No significant changes of monoamine oxidase B activities were found under the same experimental conditions. Neither angiotensin II nor eledoisin changed substrate/inhibitor affinities of both isoenzymes. These data indicate that angiotensin II and tachykinin transmitter systems may exert opposite, long‐term regulatory effects on monoaminergic neurons in rat brain.
Galanin Reduces Carbachol Stimulation of Phosphoinositide Turnover in Rat Ventral Hippocampus by Lowering Ca2+ Influx Through Voltage‐Sensitive Ca2+ Channels
Authors: Palazzi E. , Felinska S. , Zambelli M. , Fisone G. , Bartfai T. , Consolo S.
Published: October 2006

Abstract: The 29‐amino‐acid peptide galanin (GAL) caused concentration‐dependent inhibition of the accumulation of 3H‐inositol phosphates (3H‐InsPs) induced by the muscarinic agonist carbachol (CARB; 10‐3‐10‐5M) in the presence of 5 mM lithium, specifically in tissue miniprisms from rat ventral hippocampus. The inhibitory effect of GAL involved the mono‐, bis‐, tris‐, and tetrakisphosphates formed during activation for 2 min of phospholipase C by CARB (1 mM) in the absence of lithium. GAL (1 μM) did not affect α‐adrenergic or serotonergic type 2 receptor‐mediated phosphoinositide (PI) breakdown in the same tissue. GAL by itself neither acted on basal levels of 3H‐InsPs nor affected muscarinic receptors in binding studies. Blockade of the T‐, N‐, and L‐types of voltage‐sensitive calcium channel (VSCC) with 200 μM Cd2+ reduced muscarinic receptor‐mediated PI breakdown by 50% and abolished the inhibitory effect of GAL (1 μM). Reduction of the extracellular Ca2+ concentration from 1.3 mM to 0.49 μM abolished the GAL inhibition of CARB‐stimulated PI hydrolysis. Ca2+ influx promoted by 18 mM K+ depolarization or by 1 μM Bay K 8644, a selective agonist of the L‐type VSCC, prevented the inhibitory effect of GAL. Blockade of the L‐type VSCC with nifedipine (1 μM) potentiated the inhibitory effects of GAL without affecting muscarinic stimulation of PI breakdown. The neurotoxin ω‐conotoxin (2 μM), a blocker of both L‐ and N‐types of VSCC, by itself reduced CARB‐mediated breakdown of PIs by ∼25%, and when it was added before GAL (1 μM) there was no summation of the two individual inhibitory effects, a result suggesting a common site of action for GAL and ω‐conotoxin. The data presented thus indicate that GAL modulation of muscarinic stimulation of the phospholipase C activity is mediated by a reduction of Ca2+ entry through VSCCs, presumably of the N type.
Characterization of Histamine Release from the Rat Hypothalamus as Measured by In Vivo Microdialysis
Authors: Itoh Yoshinori , Oishi Ryozo , Nishibori Masahiro , Saeki Kiyomi
Published: October 2006

Abstract: The release of endogenous histamine (HA) from the hypothalamus of anesthetized rats was measured by in vivo microdialysis coupled with HPLC with fluorescence detection. Freshly prepared Ringer's solution was perfused at a rate of 1 μl/min immediately after insertion of a dialysis probe into the medial hypothalamus, and brain perfusates were collected every 30 min into microtubes containing 0.2 M perchloric acid. The basal HA output was almost constant between 30 min and 7 h after the start of perfusion, with the mean value being 7.1 pg/30 min. Thus, the extracellular HA concentration was assumed to be 7.8 nM, by a calculation from in vitro recovery through the dialysis membrane. Perfusion with a high K+ (100 mM)‐containing medium increased the HA output by 170% in the presence of Ca2+. Systemic administration of either thioperamide (5 mg/kg, i.p.), a selective H3 receptor antagonist, or metoprine (10 mg/kg, i.p.), an inhibitor of HA‐N‐methyltransferase, caused an approximately twofold increase in the HA output 30–60 min after treatment. The combined treatment with thioperamide and metoprine produced a marked increase (650%) in the HA output. The HA output decreased by ∼70% 4–5 h after treatment with α‐fluoromethylhistidine (α‐FMH; 100 mg/kg, i.p.), an inhibitor of histidine decarboxylase. Furthermore, the effect of combined treatment with thioperamide and metoprine was no longer observed in α‐FMH‐treated rats. These results suggest that both HA‐N‐methyltransferase and H3 autoreceptors are involved in maintaining a constant level of extracellular HA and that their blockade effectively results in a higher activity level of the endogenous histaminergic system in the CNS.