bluuebunny/crossref_metadata_2025 huggingface dataset:
The unc‐18 Gene Encodes a Novel Protein Affecting the Kinetics of Acetylcholine Metabolism in the Nematode Caenorhabditis elegans
Authors: Hosono R. , Hekimi S. , Kamiya Y. , Sassa T. , Murakami S. , Nishiwaki K. , Miwa J. , Taketo A. , Kodaira K.‐I.
Published: October 2006

Abstract: Genes affecting acetylcholine (ACh) levels without influencing choline acetyltransferase activity have been identified in Caenorhabditis elegans. We have examined one such gene, unc‐18. We isolated a transposon‐insertion allele for unc‐18 and used it to clone a genomic region containing the unc‐18 locus. The unc‐18 location within this region was determined by rescuing the unc‐18 mutant phenotype in a germline transformation experiment and identifying transcripts affected by four independent unc‐18 mutations. A single‐sized poly(A)+ RNA was synthesized from the gene. Expression of the transcript appears to be stage specific: The transcript is found in abundance at the early larval stage but in decreased amounts at the fourth larval and the adult stages. These results show that the unc‐18 gene plays a role in development as well as in the kinetics of ACh metabolism.
Regulation of Tyrosine Hydroxylase Gene Expression in the Rat Carotid Body by Hypoxia
Authors: Czyzyk‐Krzeska Maria F. , Bayliss Douglas A. , Lawson Edward E. , Millhorn David E.
Published: October 2006

Abstract: The activity (Vmax) of tyrosine hydroxylase (TH; EC 1.14.16.2), the rate limiting enzyme in the synthesis of catecholamines, is increased in carotid body, superior cervical ganglion, and the adrenal medulla during hypoxia (i.e., reduced Pao2). The present study was undertaken to determine if the increase in TH activity in these tissues during hypoxia is regulated at the level of TH mRNA. Adult rats were exposed to hypoxia (10% O2) or room air for periods lasting from 1 to 48 h. The carotid bodies, superior cervical ganglia, and adrenals were removed and processed for in situ hybridization using 35S‐labeled oligonucleotide probes. The concentration of TH mRNA was increased by hypoxia at all time points in carotid body type I cells, but not in cells of either superior cervical ganglion or adrenal medulla. The increase in TH mRNA in carotid body during hypoxia did not require innervation of the carotid body or intact adrenal glands. In addition, hypercapnia, another physiological stimulus of carotid body activity, failed to induce an increase in TH mRNA in type I cells. Our findings suggest that hypoxia stimulates TH gene expression in the carotid body by a mechanism that is intrinsic to type I cells.
Potentiation of N‐Methyl‐d‐Aspartate‐Stimulated Dopamine Release from Rat Brain Slices by Aluminum Fluoride and Carbachol
Authors: Woodward John J. , Harms John
Published: October 2006

Abstract: N‐Methyl‐d‐aspartate (NMDA) stimulated the release of endogenous dopamine from striatal slices prepared from adult Sprague‐Dawley rats. A mixture of sodium fluoride and aluminum chloride (AlF4‐) added to the slices significantly potentiated the NMDA‐stimulated release of dopamine in a concentration‐ and timedependent manner. The NF4‐ mixture had no effect on the nonstimulated basal efflux of dopamine, and no increases in NMDA‐stimulated release were observed when NaF was replaced with NaCl. Similarly, AlCl3 or a mixture of NaCl and AlCl3 had no effect on NMDA‐stimulated release. The A1F4‐‐induced increase in NMDA‐stimulated dopamine release was totally blocked by magnesium or the selective NMDA glycine antagonist 7‐chlorokynurenic acid. Striatal slices depolarized with KCl (15 mM) also released dopamine and this release was similarly potentiated by AlF4‐. However, KCI‐stimulated dopamine release from striatal synaptosomes was not potentiated by concentrations of AlF4‐ that greatly increased release from striatal slices. NMDA did not stimulate the release of dopamine from striatal synaptosomes in the absence or presence of aluminum fluoride. Modulators of adenylate cyclase (for‐skolin) and protein kinase C (phorbol esters) did not enhance NMDA‐stimulated dopamine release. The protein kinase C inhibitor H‐7 also did not reduce the potentiating effects of AlF4‐. The mixed cholinergic agonist carbachol and the calcium ionophore A23187 mimicked the AlF4‐ effect although the increase in NMDA‐stimulated dopamine release produced by these agents was less than that seen with AlF4‐. Atropine blocked the carbachol response, but did not alter the effect of AlF4‐ on NMDA‐stimulated release. Neither pertussis toxin (500 ng/ml; 12 h) or tiazofurin (100 μM; 3 h) significantly attenuated the ability of AlF4‐ or carbachol to stimulate release. However, the calmodulin antagonist W‐7 (15–50 μM) significantly reduced the AlF4‐‐ or carbachol‐induced increase in NMDA‐stimulated release. These results suggest that aluminum fluoride may enhance depolarization‐induced dopamine release from striatal slices through activation of a pertussis toxin‐insensitive GTP‐binding protein, which may be linked to the mobilization of intracellular calcium.
Acute and Chronic Ethanol Exposure Alters the Function of Hippocampal Kainate Receptors Expressed in Xenopus Oocytes
Authors: Dildy‐Mayfield J. E. , Harris R. A.
Published: October 2006

Abstract: The effects of acute and extended ethanol exposure on N‐methyl‐d‐aspartate‐ and kainate‐induced currents were examined electrophysiologically in Xenopus oocytes expressing rat hippocampal mRNA. Ethanol inhibited responses stimulated by low and high concentrations of N‐methyl‐d‐aspartate to a similar degree. However, responses produced by low or high concentrations of kainate were differentially inhibited by ethanol. Low kainate concentration responses were much more sensitive to ethanol than high kainate concentrations (e.g., 50 mM ethanol inhibited 12.5 μM kainate responses by 45% compared to 15% inhibition of 400 μM kainate responses). In oocytes cultured in 100 mM ethanol for 1–5 days, the ethanol inhibition of maximum N‐methyl‐d‐aspartate and kainate responses was not different from that in non–ethanol‐exposed oocytes. Ethanol treatment, however, selectively decreased the ethanol sensitivity of low kainate concentration responses. Currents stimulated by N‐methyl‐d‐aspartate or kainate were not different between control and ethanol‐treated oocytes, indicating that ethanol exposure did not interfere with channel expression. The selective actions of acute and extended ethanol exposure on low kainate responses may indicate selective actions of ethanol on subtypes of kainate receptors expressed in oocytes.
Chromatographic Evidence for the Presence of Multiple Tachykinins in the Bovine Adrenal Medulla
Authors: Basile Silvio , Cheung Nam Sang , Livett Bruce G.
Published: October 2006

Abstract: Among the mammalian tachykinins, substance P (SP) has been shown to be the most potent at modulating the response due to nicotinic acetylcholine receptor stimulation of bovine adrenal chromaffin cells. SP‐like immunoreactivity has been detected in nerve terminals innervating the adrenal medulla; however, little is known of the presence of other tachykinins in this tissue. In this study, reverse‐phase HPLC was used to fractionate peptides in bovine adrenal medullary extracts, and the fractions were analyzed by radioimmunoassay using antisera to SP or neurokinin A (NKA). The results show that both NKA‐ and SP‐like immunoreactivities are present in the adrenal medulla. The presence of neurokinin B is also indicated. The presence of multiple tachykinins in this tissue raises questions as to their functions in the adrenal medulla.
Polyamine Metabolism in Experimental Brain Tumors of Rat
Authors: Ernestus Ralf‐Ingo , Röhn Gabriele , Hossmann Konstantin‐Alexander , Paschen Wulf
Published: October 2006

Abstract: Biosynthesis and accumulation of the polyamines putrescine, spermidine, and spermine are closely associated with cellular growth processes. We examined polyamine levels and the activity of their first rate‐limiting enzyme, ornithine decarboxylase (ODC), in stereotactically induced experimental gliomas of the rat brain 1 and 2 weeks after implantation. Regional ODC activity and polyamine levels were determined in the tumor and in the ipsi‐ and contralateral striatum, white matter, and cerebral cortex. In the tumor, both ODC activity and polyamine levels markedly increased with progressive tumor growth, as compared to those in the white matter of the opposite hemisphere. In the peritumoral brain tissue, ODC activity did not change, but there was a marked increase of putrescine and, to a lesser degree, of spermidine and spermine almost throughout the whole ipsilateral hemisphere. ODC activity, therefore, seems to be a reliable marker of neoplastic growth in the brain, which may be of use for new clinical concepts of the diagnosis and therapy of brain tumors. The more diffuse distribution of polyamines, however, may be associated with the formation and spreading of edema, which would explain some of the biological effects of tumors on distant brain tissue.
Amphetamine and Other Weak Bases Act to Promote Reverse Transport of Dopamine in Ventral Midbrain Neurons
Authors: Sulzer David , Maidment Nigel T. , Rayport Stephen
Published: October 2006

Abstract: Amphetamine‐like psychostimulants are thought to produce rewarding effects by increasing dopamine levels at mesolimbic synapses. Paradoxically, dopamine uptake blockers, which generally increase extracellular dopamine, inhibit amphetamine‐induced dopamine overflow. This effect could be due to either inhibition of amphetamine uptake or inhibition of dopamine efflux through the transporter (reverse transport). We used weak bases and dopamine uptake blockers in ventral midbrain neuron cultures to separate the effects on blockade of amphetamine uptake from reverse transport of dopamine. Amphetamine, ammonium chloride, tributylamine, and monensin, at concentrations that produce similar reductions in acidic pH gradients, increased dopamine release. This effect was inhibited by uptake blockers. Although in the case of amphetamine the inhibition of release could have been due to blockade of amphetamine uptake, inhibition also occurred with weak bases that are not transporter substrates. This suggests that reduction of vesicular pH gradients increases cytoplasmic dopamine which in turn promotes reverse transport. Consistent with this model, extracellular 3,4‐dihydroxyphenylacetic acid was increased by ammonium chloride and monensin, as would be expected with elevated cytoplasmic dopamine levels. These findings extend the weak base mechanism of amphetamine action, in which amphetamine reduces vesicular pH gradients resulting in increased cytoplasmic dopamine that promotes reverse transport.
Involvement of a Disulfide Bond in the Binding of Flunitrazepam to the Benzodiazepine Receptor from Bovine Cerebral Cortex
Authors: Otero de Bengtsson Marcela S. , Lacorazza H. Daniel , Biscoglio de Jiménez Bonino Mirtha J. , Medina Jorge H.
Published: October 2006

Abstract: The effects of chemical modification of a disulfide bond(s) (‐SS‐) or sulfhydryl group(s) (‐SH) on the [3H]‐flunitrazepam ([3H]FNZ) binding to membrane‐bound or immunoprecipitated benzodiazepine (BZD) receptors (BZD‐R) from bovine cerebral cortex were examined. Reduction of ‐SS‐ with dithiothreitol (DTT) brought about a reversible, time‐ and dose‐dependent inhibition of [3H]FNZ binding to the membrane‐bound BZD‐R. Alkylation of the membranes with the ‐SH‐modifying reagent iodoacetamide (IAA) or 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (DTNB) produced a slight inhibition of [3H]FNZ binding in a dose‐dependent manner. Scatchard analysis of saturation curves of [3H]FNZ binding in the presence and absence of 5 mM DTT revealed changes in affinity without modification in the maximal binding capacity, thus indicating a competitive mode of interaction. DTT pretreatment of both the membrane‐bound and the immunoprecipitated BZD‐R led to [3H]FNZ binding inhibition. Consistent with the modification of a binding site is the observation that reduction of ‐SS‐ does not bear on the binding affinity, but rather reduces the number of sites. Complete protection from DTT inhibition of [3H]FNZ binding by FNZ (an agonist) or by Ro 15–1788 (an antagonist) suggests the presence of ‐SS‐ at, or very close to, the BZD recognition binding site. No protection against IAA or DTNB inhibition was provided by FNZ. Photoaffinity labeling experiments with [3H]FNZ revealed a clear‐cut band of 50 kDa in native and alkylated membranes but an extremely weak label in 5 mM DTT/IAA‐treated membranes. The present results provide evidence for the participation of a disulfide bond in the recognition binding site of the bovine cerebral cortex BZD‐R.
Repeated Reserpine Increases Striatal Dopamine Receptor and Guanine Nucleotide Binding Protein RNA
Authors: Butkerait Paul , Friedman Eitan
Published: October 2006

Abstract: In the present study the effects of repeated administration of reserpine on striatal dopamine receptor and guanine nucleotide binding protein mRNAs were determined. Twenty‐four hours after seven consecutive daily injections of reserpine—a treatment that is known to produce functional sensitization of D1 and D2 dopamine receptors—the level of striatal D1 dopamine receptor mRNA was unchanged. However, the level of mRNA for the G protein Gsα was increased by 127%. After extended reserpine treatment for 14 days, levels of both striatal D1 DA receptor and Gsα mRNAs were elevated by 99 and 78%, respectively. Seven days of reserpine treatment also increased levels of mRNA of the striatal D2 dopamine receptor and of G proteins Gi2α and Goα by 200, 79, and 32%, respectively. After 14 days of reserpine treatment the level of striatal D2 dopamine receptor mRNA was increased by twofold. In contrast, levels of the mRNAs coding for the G proteins Gi2α and Goα were unchanged. These data suggest that dopamine receptors and their respective G proteins play important roles in the development of sensitization of striatal dopamine receptors during repeated reserpine treatment. Furthermore, the persistent increase in level of striatal Gsα mRNA suggests that this G protein is necessary to maintain supersensitivity of the striatal D1 dopamine receptor system following long‐term dopamine depletion.
Glucose Deprivation Results in a Lactate Preventable Increase in Adenosine and Depression of Synaptic Transmission in Rat Hippocampal Slices
Authors: Fowler John C.
Published: October 2006

Abstract: The effect of glucose deprivation on adenosine levels and on synaptic transmission was investigated in rat hippocampal slices. Incubation of hippocampal slices either in glucose‐free medium or in the presence of the glucose transport inhibitor cytochalasin B (50 μM) increased bath adenosine levels and depressed the extracellularly recorded synaptic potential or population spike. The addition of lactate (10 mM), a precursor for mitochondrial ATP generation, prevented the elevation in adenosine and the depression of the population spike. These results indicate that the neuroinhibitory modulator adenosine is elevated during glucose deprivation and contributes to the hypoglycemic depression of synaptic transmission. The increase in adenosine during glucose deprivation can be prevented by providing substrate for mitochondrial ATP generation. The present results indicate an interaction between lactate and adenosine such that an increase in lactate may contribute to a decline in adenosine production.
Brain Hydroxyl Radical Generation in Acute Experimental Head Injury
Authors: Hall Edward D. , Andrus Paula K. , Yonkers Patricia A.
Published: October 2006

Abstract: The time course and intensity of brain hydroxyl radical (•OH) generation were examined in male CF‐1 mice during the first hour after moderate or severe concussive head injury. Hydroxyl radical production was measured using the salicylate trapping method in which the production of 2,3‐ and/or 2,5‐dihydroxybenzoic acid (DHBA) in brain 15 min after salicylate administration was used as an index of •OH formation. In mice injured with a concussion of moderate severity as defined by the 1‐h posttraumatic neurologic recovery (grip score), a 60% increase in 2,5‐DHBA formation was observed by 1 min after injury compared with that observed in uninjured mice. The peak in DHBA formation occurred at 15 min after injury (+67.5%; p < 0.02, compared with uninjured). At 30 min, the increase in DHBA lost significance, indicating that the posttraumatic increase in brain •OH formation is a transient phenomenon. In severely injured mice, the peak increase in DHBA (both 2,3‐ and 2,5‐) was observed at 30 min after injury, but also fell off thereafter as with the moderate injury severity. Preinjury dosing of the mice with SKF‐525A (50 mg/kg i.p.), an inhibitor of microsomal drug oxidations, did not blunt the posttraumatic increase in salicylate‐derived 2,5‐DHBA, thus showing that it is not due to increased metabolic hydroxylation. Neither injury nor SKF‐525A administration affected the DHBA plasma levels. However, saline perfusion of the injured mice to remove the intravascular blood before brain removal eliminated the injury‐induced increase in 2,5‐DHBA, but did not affect the baseline levels seen in uninjured mice. This implies that the source of the increased DHBA in the injured mice is the microvasculature, probably the endothelium. The administration of the 21‐aminosteroid lipid antioxidant, tirilazad mesylate, which possesses •OH scavenging properties, also attenuated the posttraumatic increase in DHBA, further supporting that it reflects an increase in •OH radical formation. These results are the first direct demonstration of the occurrence and time course of increased •OH production in injured brain.
Characterization of Membrane‐Bound and Solubilized High‐Affinity Binding Sites for 5′‐N‐Ethylcarboxamido[3H]adenosine from Bovine Cerebral Cortex
Authors: Lorenzen Anna , Nitsch‐Kirsch Monika , Vogt Heidrun , Schwabe Ulrich
Published: October 2006

Abstract: A high‐affinity binding site for 5′‐N‐ethylcarboxamido[3H]adenosine ([3H]NECA) from bovine cerebral cortex has been characterized in its membrane‐bound and solubilized state after gel filtration on Sepharose CL‐6B. For detection of this site in membranes, it was necessary to remove metabolites with high affinities for this site enzymatically, e.g., adenosine by addition of adenosine deaminase and inosine by addition of nucleoside phosphorylase. The pore‐forming peptide antibiotic alamethicin further enhanced binding of [3H]NECA to this site in membranes. In contrast to adenosine receptors and the adenotin‐like low‐affinity binding protein, this novel site was extremely sensitive against treatment with the sulfhydryl alkylating agent N‐ethylmaleimide. In competition experiments, this site could be differentiated from adenosine receptors by its high affinity for adenine nucleotides and its lack of affinity for adenosine receptor antagonists. Inosine and its derivative S‐(4‐nitrobenzyl)‐6‐thioinosine were relatively potent ligands with Ki values in the high nano‐ and low micromolar range, respectively. We conclude that the high‐affinity NECA binding site described previously in bovine striatum is not exclusively located in the striatum, but can also be detected in membrane preparations and soluble extracts of bovine brain cortex.
Effects of Nucleotides on [3H]Bradykinin Binding in Guinea Pig: Further Evidence for Multiple B2 Receptor Subtypes
Authors: Seguin L. , Widdowson P. S.
Published: October 2006

Abstract: We have suggested recently the existence of three subtypes of B2 bradykinin receptors in tissues of guinea pigs. We have classified these B2 bradykinin receptors into B2a, B2b, and B2c subtypes depending on their affinity for various bradykinin antagonists. Because the actions of bradykinin in different cell systems appear to be both dependent on and independent of G proteins, we sought to determine whether the binding of [3H]bradykinin to the B2 subtypes is sensitive to guanine nucleotides and, therefore, possibly coupled to G proteins. In the ileum, where we have demonstrated B2a and B2b subtypes, specific [3H]bradykinin binding was reduced with GDP (100 μM) and the nonmetabolized analogue of GTP, guanyl‐5′‐yl‐imidodiphosphate (GppNHp; 100 μM). Competition studies with bradykinin and with [Hyp3]‐bradykinin, which shows approximately 20‐fold greater selectivity for the B2a subtype than bradykinin, were performed in the presence or absence of GppNHp (100 μM). The competition experiments demonstrated that binding to the B2a subtype, which has higher affinity for [Hyp3]‐bradykinin and bradykinin than the B2b subtype, was lost in the presence of GppNHp, whereas binding to the B2b subtype was unaffected. In contrast, GppNHp (100 μM) and GDP (100 μM) failed to alter specific [3H]bradykinin binding to B2b and B2c subtypes in lung. [3H]Bradykinin binding was unaffected by AMP, ADP, ATP, and GMP (100 μM each). Based on this evidence, we suggest that the B2a bradykinin subtype is coupled to G proteins. The B2b and B2c subtypes are either not coupled to G proteins, or may be coupled to the Go‐type GTP binding proteins, which have been suggested to be less sensitive to guanine nucleotides. These data provide further evidence for three subtypes of B2‐type bradykinin receptors in guinea pig.
Protection Against Acute MPTP‐Induced Dopamine Depletion in Mice by Adenosine A1 Agonist
Authors: Lau Yuen‐Sum , Mouradian M. Maral
Published: October 2006

Abstract: The effects of the adenosine A1 agonist N6‐cyclohexyladenosine (CHA) on MPTP‐induced dopamine (DA) depletion in the striatum of C57BL/6 mice were studied. Twenty hours after a single injection of MPTP (30 mg/kg, s.c.), the toxin caused 62% depletion of striatal DA. CHA (0.2–3 mg/kg, s.c.), when given together with MPTP, prevented the toxin‐induced DA depletion in a dose‐dependent manner. This protective action was apparently mediated by the A1 receptors, because this effect was selectively antagonized by pretreating the animals with the A1 antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (25 mg/kg, i.p.) but not with the A2 antagonist 1,3‐dipropyl‐7‐methylxanthine (25 mg/kg, i.p.). When CHA (3 mg/kg) was injected 5 h after MPTP administration, at which point striatal DA levels were already reduced significantly, a rapid and complete recovery of the striatal DA levels occurred. These neurochemical data suggest that the A1 agonist CHA is potentially useful as a neuroprotective agent against MPTP‐induced toxicity.
Regulation of Neuropeptide Expression in Cultured Cerebral Cortical Neurons by Brain‐Derived Neurotrophic Factor
Authors: Nawa Hiroyuki , Bessho Yasumasa , Carnahan Josette , Nakanishi Shigetada , Mizuno Keiko
Published: October 2006

Abstract: The neuropeptide‐inducing activity of neurotrophic factors was tested in cultured cerebral cortical neurons. Brain‐derived neurotrophic factor (BDNF) specifically increased contents of the neuropeptides somatostatin (SOM) and neuropeptide Y (NPY), but its effect on contents of cholecystokinin octapeptide and GABA was much less significant. The maximal induction of NPY content (15‐fold increase) was achieved by 20 ng/ml of BDNF. These changes were also reproduced at the mRNA level. In contrast, neurotrophin‐3 was much less potent at increasing NPY and SOM contents, and nerve growth factor had no effect on them. The expression of mRNA for NPY and SOM was fully dependent on the presence of BDNF in culture but irrelevant to the survival‐promoting activity of BDNF, which has been reported previously. Most of the NPY immunoreactivity induced by BDNF was colocalized with glutamate decarboxylase immunoreactivity in cultured cortical neurons. These results suggest that BDNF regulates the peptidergic expression of GABAergic neurons in the cerebral cortex.
Prolactin‐Stimulated Mitogenesis of Cultured Astrocytes Is Mediated by a Protein Kinase C‐Dependent Mechanism
Authors: DeVito William J. , Avakian Crystal , Stone Scott , Okulicz William C.
Published: October 2006

Abstract: Prolactin (PRL) has been reported to activate cellular proliferation in nonreproductive tissue, such as liver, spleen, and thymus. Recently, we have extended the possible role of PRL as a mammalian mitogen by demonstrating a mitogenic effect of PRL in cultured astrocytes. Although the cellular mechanisms by which PRL regulates cell growth are not fully understood, protein kinase C (PKC) has been implicated as one of the transmembrane signaling systems involved in the regulation of PRL‐induced cell proliferation in Nb2 lymphoma cells and liver. In the present studies, we examined the possible role of PKC in PRL‐induced proliferation of cultured astrocytes. Incubation of cultured astrocytes with 1 nM PRL resulted in a rapid translocation of PKC from the cytosol to the membrane, with maximal PKC activity in the membrane occurring 30 min after exposure to PRL. Translocation of PKC activity occurred over a physiological range of PRL, with maximal PKC activation occurring at 1 nM. At concentrations greater than 10 nM PRL, there was a decrease in the amount of PKC activity associated with the membrane fraction compared with that of cells stimulated with 1 nM PRL. Incubation of astrocytes with PRL in the presence of the PKC inhibitors staurosporine, 1‐(‐5‐isoquinolinesulfonyl)‐2‐methylpiperazine, or polymyxin B blocked the PRL‐induced increase in cell number with IC50 values of approximately 2 nM, 10 μM, and 6 μM, respectively. PKC is the only known cellular receptor for 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), which stimulates the translocation of PKC from the cytosol to the membrane. Incubation of astrocytes with 20 nM TPA resulted in an increase in the expression of proliferating cell nuclear antigen and cell number, whereas 4α‐phorbol 12,13‐didecanoate, an inactive phorbol ester, was ineffective. To examine further the effect of TPA and PRL on cellular proliferation, cultured astrocytes were incubated with increasing concentrations of TPA in the presence or absence of a minimal effective dose of PRL (100 pM). In the absence of PRL, incubation with TPA resulted in an inverted U‐shaped dose‐response curve, with 100 nM TPA resulting in a maximal increase in cell number. In the presence of 100 pM PRL, the TPA dose‐response curve was shifted to the left, with maximal activity occurring with 10 nM TPA. Chronic stimulation of astrocytes with 500 nM TPA depleted the cells of PKC and blocked the PRL‐induced increase in cell number. Finally, TPA treatment decreased cell‐surface binding of 125I‐PRL. These data indicate that the PKC is involved in the mitogenic effect of PRL in cultured astrocytes.
N‐Methyl‐d‐Aspartate‐ and Non‐N‐Methyl‐d‐Aspartate‐Evoked Adenosine Release from Rat Cortical Slices: Distinct Purinergic Sources and Mechanisms of Release
Authors: Craig Constance G. , White Thomas D.
Published: October 2006

Abstract: Excitatory amino acids, acting at both Nmethyl‐d‐aspartate (NMDA) and non‐NMDA receptors, release the inhibitory neuromodulator adenosine from superfused rat cortical slices. This study was initiated to investigate the possible purinergic sources and mechanisms of release for the adenosine release evoked by NMDA and non‐NMDA receptor activation. Inhibition of the bidirectional nucleo‐side transporter with dipyridamole greatly enhanced adenosine release evoked by glutamate, NMDA, kainate, and (RS‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA). Inhibition of ecto‐5′‐nucleotidase with α,β‐methylene ADP and GMP had no effect on either kainateor AMPA‐evoked adenosine release, but it decreased glutamate‐ and NMDA‐evoked adenosine release by 23 and 68%, respectively. A similar inhibition of NMDA‐evoked adenosine release was observed with α,β‐methylene ADP alone, indicating that the inhibitory effect was not due to the reported competitive inhibition of NMDA receptors by GMP. Finally, NMDA‐evoked adenosine release, but not kainate‐ or AMPA‐evoked release, was Ca2+ dependent. These results indicate that activation of non‐NMDA receptors releases adenosine per se in a Ca2+‐independent manner. In contrast, NMDA receptor activation releases primarily a nucleotide that is subsequently converted extracellularly to adenosine; in this case, release is Ca2+ dependent. Although neither NMDA‐ nor non‐NMDA‐evoked adenosine release occurs via the nucleoside transporter, this transporter does appear to be a major route for removal of adenosine from the extracellular space.
Measurement of Receptor‐Activated Phosphoinositide Turnover in Rat Brain: Nonequivalence of Inositol Phosphate and CDP‐Diacylglycerol Formation
Authors: Heacock Anne M. , Seguin Edward B. , Agranoff Bernard W.
Published: October 2006

Abstract: Two methods for the measurement of receptor‐activated phosphoinositide turnover were evaluated for their degree of correspondence in slices of rat brain; they involved the Li+‐dependent accumulations of either [3H]‐inositol‐labeled inositol phosphates or [3H]cytidine‐labeled CDP‐diacylglycerol. In contrast to the expectation that the ratio of these two responses would remain approximately constant, varying degrees of correspondence were obtained. The two extremes are exemplified by carbachol, which elicited large increases in both inositol phosphate and CDP‐diacylglycerol labeling, and endothelin, which gave a robust inositol phosphate response with little or no accumulation of 3H‐CDP‐diacylglycerol. No instance of the presence of the latter response in the absence of 3H‐inositol phosphate accumulation was observed. Measurement of 3H‐CDP‐diacylglycerol accumulation thus may add additional insight into the regulation of phosphoinositide turnover and the complex actions of Li+.
Cocaine Regulation of Brain Preprothyrotropin‐Releasing Hormone mRNA
Authors: Sevarino Kevin A. , Primus Renée J.
Published: October 2006

Abstract: Clinical and preclinical evidence supports a possible role for thyrotropin‐releasing hormone (TRH) in cocaine action. However, the interaction between cocaine and TRH has not been directly examined. In the following report we describe a solution hybridization RNase protection assay that can sensitively detect mRNA for the TRH precursor, prepro‐TRH (ppTRH). Using this assay, we examined ppTRH mRNA levels in rat brain regions implicated in cocaine reinforcement, including the nucleus accumbens, hypothalamus, amygdala, hippocampus, and thalamus. Acute cocaine treatment (15 mg/kg) resulted in significant decreases in ppTRH mRNA levels in the amygdala and hippocampus, but not in the hypothalamus, nucleus accumbens, or thalamus, 45 min postinjection. Chronic cocaine treatment (15 mg/kg twice daily for 14 days) resulted in marked regulation in all regions but the thalamus. Regulation was strongly dependent on the length of cocaine withdrawal and persisted up to 72 h postinjection in the amygdala. These studies support the hypothesis that TRH or other ppTRH‐derived peptides are involved in cocaine action, especially in the extrahypothalamic regions of the amygdala and hippocampus.
Nerve Regeneration Occurs in the Absence of Apolipoprotein E in Mice
Authors: Popko Brian , Goodrum Jeffry F. , Bouldin Thomas W. , Zhang Sunny H. , Maeda Nobuyo
Published: October 2006

Abstract: The concentration of apolipoprotein E (apoE), a high‐affinity ligand for the low‐density lipoprotein receptor, increases dramatically in peripheral nerve following injury. This endoneurial apoE is thought to play an important role in the redistribution of lipids from the degenerating axonal and myelin membranes to the regenerating axons and myelin sheaths. The importance of apoE in nerve repair was examined using mutant mice that lack apoE. We show that at 2 and 4 weeks following sciatic nerve crush, regenerating nerves in apoE‐deficient mice were morphologically similar to regenerating nerves in control animals, indicating that apoE is not essential for peripheral nerve repair. Moreover, cholesterol synthesis was reduced in regenerating nerves of apoE‐deficient mice as much as in regenerating nerves of control animals. These results suggest that the intraneural conservation and reutilization of cholesterol following nerve injury do not require apoE.
Cyclothiazide Treatment Unmasks AMPA Excitotoxicity in Rat Primary Hippocampal Cultures
Authors: May Patrick C. , Robison Paula M.
Published: October 2006

Abstract: Mechanisms of non‐NMDA receptor‐mediated excitotoxicity were studied in embryonic rat hippocampal cultures using kainic acid (KA) and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) as agonists. Under basal culture conditions, overnight treatment with AMPA resulted in negligible excitotoxicity as assessed by phase‐contrast microscopy and measurement of lactate dehydrogenase (LDH) release. In contrast, similar treatment with KA resulted in marked excitotoxic morphologic changes and release of LDH. Cotreatment of cultures with AMPA but not NMDA effectively blocked KA toxicity, suggesting that AMPA‐induced rapid desensitization of the AMPA/KA receptor could account for the lack of prominent direct toxicity as well as AMPA's ability to block KA toxicity. To test this hypothesis, cultures were briefly pretreated with 10 μM cyclothiazide, a drug reported to block desensitization of the AMPA/KA receptor, and then exposed overnight to cyclothiazide plus AMPA and/or KA. Cyclothiazide‐treated cultures were now vulnerable to AMPA as well as KA; moreover, AMPA was unable to block KA toxicity completely, suggesting that cyclothiazide impaired AMPA/KA receptor desensitization. These and related studies suggest that a regulatory site may exist on the AMPA/KA receptor that modulates non‐NMDA receptor‐mediated excitotoxicity.
High‐Affinity Choline Transport Sites: Use of [3H]Hemicholinium‐3 as a Quantitative Marker
Authors: Happe H. Kevin , Murrin L. Charles
Published: October 2006

Abstract: High‐affinity choline transport (HAChT), the rate‐limiting and regulatory step in acetylcholine (ACh) synthesis, is selectively localized to cholinergic neurons. Hemicholinium‐3 (HC3), a potent and selective inhibitor of HAChT, has been used as a specific radioligand to quantify HAChT sites in membrane binding and autoradiographic studies. Because both HAChT velocity and [3H]HC3 binding change as in vivo activity of cholinergic neurons is altered, these markers are also useful measures of cholinergic neuronal activity. Evidence that [3H]HC3 is a specific ligand for HAChT sites on cholinergic terminals is reviewed. The ion requirements of HAChT and [3H]HC3 binding indicate that sodium and chloride are required for recognition of both choline and [3H]HC3. A common recognition site is also indicated by the close correspondence of the potency of HC3 and choline analogues for inhibiting both HAChT and [3H]HC3 binding. The parallel regional distributions of both markers in adult brain, during development and after specific lesions, all indicate specific cholinergic localization. The close association of HAChT and [3H]HC3 binding sites is also supported by parallel regulatory changes occurring after in vivo drug treatments and in vitro depolarization. Overall, the data indicate a close association between HAChT and [3H]HC3 binding and are consistent with the sites being identical. Methodologic considerations in using [3H]HC3 as a ligand and considerations in interpretation of results are also discussed.
Excitatory Amino Acid‐Mediated Cytotoxicity and Calcium Homeostasis in Cultured Neurons
Authors: Frandsen Aase , Schousboe Arne
Published: October 2006

Abstract: A large body of evidence suggests that disturbances of Ca2+ homeostasis may be a causative factor in the neurotoxicity induced by excitatory amino acids (EAAs). The route or routes by which an increase in intracellular calcium concentration ([Ca2+]i) is mediated in vivo are presently not clarified. This may partly reflect the complexity of intact nervous tissue in combination with the relative unspecific action of the available “calcium antagonists,” e.g., blockers of voltage‐sensitive calcium channels. By using primary cultures of cortical neurons as a model system, it has been found that all EAAs stimulate increases in [Ca2+]i but via different mechanisms. By using the drug dantrolene, it has been shown that 2‐amino‐3‐(3‐hydroxy‐5‐methylisoxazol‐4‐yl)propionate (AMPA) apparently exclusively stimulates Ca2+ influx through agonist‐operated calcium channels and voltage‐operated calcium channels. Increased [Ca2+]i due to exposure to kainate (KA) is for the major part caused by influx, as in the case of AMPA, but a small part of the increase in [Ca2+]i may be attributed to a release of Ca2+ from intracellular stores. Quisqualate (QA) stimulates Ca2+ release from an intracellular store that is independent of Ca2+ influx; presumably this store is activated by inositol phosphates. The increase in [Ca2+]i due to exposure to glutamate or N‐methyl‐d‐aspartate (NMDA) may be compartmentalized into three components, one of which is related to influx and the other two to Ca2+ release from internal stores. Only one of the latter stores is dependent on Ca2+ influx with regard to release of Ca2+, whereas the other is activated by some other second messengers or, alternatively, directly coupled to the receptor. In muscles dantrolene is known to inhibit Ca2+ release from the sarcoplasmic reticulum, and also in neurons dantrolene inhibits an equivalent release from one or more hitherto unidentified internal Ca2+ pool(s). By using this drug it has been possible to show to what extent these Ca2+ stores are involved in the toxicity observed subsequent to exposure to the EAAs. It turned out that dantrolene, even under conditions allowing Ca2+ influx, inhibited toxicity induced by QA, NMDA, and glutamate, whereas that induced by AMPA or KA was unaffected. In combination with the findings that dantrolene inhibited release from the intracellular stores activated by QA, NMDA, and glutamate, it may be concluded that Ca2+ influx per se is not the primary event causing toxicity following exposure to these EAAs in these neurons. However, it may certainly be involved in the cases of toxicity induced by AMPA and KA. Finally, it should be pointed out that this model only serves as a much simplified working hypothesis and that the situation in vivo is much more complex.
Fast and Local Electrochemical Monitoring of Noradrenaline Release from Sympathetic Terminals in Isolated Rat Tail Artery
Authors: Gonon F. , Bao J. X. , Msghina M. , Suaud‐Chagny M. F. , Stjärne L.
Published: October 2006

Abstract: Noradrenaline release from sympathetic nerve terminals was evoked by electrical nerve stimulation of an isolated segment of rat tail artery. This release was recorded by a carbon fiber electrode combined with differential pulse amperometry. The active part of the electrode (one carbon fiber 8 μm in diameter and 50 μm in length) was placed in close contact with the arterial surface. The oxidation current appearing at +120 mV and corresponding to the local noradrenaline concentration at the electrode surface was recorded every 0.5 s. No oxidation current was detected under resting conditions, but electrical stimulation evoked an immediate increase in this current. This response was suppressed when tetrodotoxin was added to the perfusion medium and was enhanced when noradrenaline reuptake was inhibited by cocaine. The amplitude of the response was increased with increasing stimulation frequencies (2–25 Hz) and train lengths (1–16 pulses). Finally, the time resolution of the method (0.5 s) was good enough to show that noradrenaline release precedes the postsynaptic response, i.e., the electrically evoked contraction of the artery.
Limited Proteolysis of Myelin Basic Protein in a System Mimetic of the Myelin Interlamellar Aqueous Space
Authors: Nicot C. , Vacher M. , Denoroy L. , Kahn P. C. , Waks M.
Published: October 2006

Abstract: We have investigated the early steps of myelin basic protein (MBP) degradation in a membrane mimetic system (reverse micelles), resembling the interlamellar aqueous spaces where the protein is located in the myelin sheath. MBP, unfolded in buffer, refolds on incorporation into the micelles, resulting in reduced accessibility to three proteolytic enzymes, trypsin, cathespin D, and Staphylococcus aureus V8 protease, in comparison with aqueous solution. Eleven cleavage sites seen in buffer are removed from proteolytic attack in micellar solution. These sites delineate a protected protein domain displaying a potential β‐sheet structure capable of interacting with the myelin membrane. An additional site not seen in buffer is attacked in the micelles. Experiments with a structure inducer, 15% 1‐propanol in buffer, reveal that the refolding pattern of MBP in reverse micelles is specific to the membrane biomimetic system and is not produced by organic solvent per se. Micellar digestions of MBP generate long peptides, two of which, isolated after tryptic digestion, have been found to be immunodominant in multiple sclerosis patients. The findings suggest the structure induced in MBP by the micelles resembles that leading to production of the self‐peptides recognized by T cells during proteolytic breakdown of MBP in autoimmune demyelinating diseases.
Sodium‐Dependent Antiporters in Choroid Plexus Epithelial Cultures from Rabbit
Authors: Mayer Steven E. , Sanders‐Bush Elaine
Published: October 2006

Abstract: The mechanism of recovery from an acid load in primary cultures of rabbit choroid plexus epithelium (CPE) was examined, with emphasis on Na+‐dependent antiports. Cells were incubated in saline solutions buffered to pH 7.38 with either HEPES or HCO3− plus 95% O2/5% CO2. Intracellular pH (pHi) was determined from the steady‐state distribution of [14C]benzoate. Recovery after acidification with NH4Cl was rapid (t1/2= 5 min) and was dependent on external Na+ (EC50= 12 mM). Hexamethyleneamiloride and ethylisopropylamiloride, potent inhibitors of the Na+/H+ antiport, blocked 80% of recovery when [Na+] was 5 mM with IC50 values of 100 nM. However, neither drug blocked recovery in normal [Na+]. 4,4′‐Diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS), an inhibitor of Cl−/HCO3− antiports, blocked recovery of pHi in a dose‐related fashion in the presence of bicarbonate, but not in the presence of HEPES. No inhibition occurred with benzamil, an amiloride congener with high affinity for the Na+ channel, nor with dimethylbenzamil, an inhibitor of Na+/Ca2+ exchange. The carbonic anhydrase inhibitor acetazolamide also did not alter recovery from acidification. In CPE that had been pH‐clamped with nigericin and KCl, the initial rate of 22Na+ uptake was very rapid (227 pmol/μg of DNA/min at pH 6.2), was dependent on external [Na+] with an EC50 value of 8 mM, and was inversely related to the pH of the medium. The maximal inhibition of 22Na+ uptake by hexamethyleneamiloride was 60% with an IC50 value of 76 nM. We conclude that both the Na+/H+ antiport and a DIDS‐sensitive bicarbonate‐dependent antiport are important mechanisms of regulation of the internal pH of rabbit CPE under acidifying conditions. Further, our data suggest that the rabbit choroid plexus Na+/H+ exchanger can be classified as amiloride insensitive, suggesting that this antiport may play a greater role in controlling transport mechanisms than does the pH of the CNS.
Nuclear Calmodulin‐Binding Proteins in Rat Neurons
Authors: Pujol Maria Jesús , Bosser Ramon , Vendrell Montserrat , Serratosa Joan , Bachs Oriol
Published: October 2006

Abstract: By using a 125I‐calmodulin overlay assay, three major high‐affinity calmodulin‐binding proteins, showing apparent molecular masses of 135, 60, and 50 kDa, have been detected in purified nuclear fractions isolated from rat neurons. It has been shown that after extraction of the nuclei with nucleases and high salt, all these proteins remain strongly associated with the nuclear matrix. The 60‐ and 50‐kDa proteins have been previously identified as subunits of the calmodulin‐dependent protein kinase II. We report here the immunoblot identification of the 135‐kDa cal modulin‐binding protein as myosin light chain kinase. We also show that the calmodulin‐dependent protein phosphatase calcineurin is present in the neuronal nuclei and associated with the nuclear matrix. The nuclear localization of both calcineurin and myosin light chain kinase has been confirmed by immunocytochemical studies.
Differing Neurotoxic Potencies of Methamphetamine, Mazindol, and Cocaine in Mesencephalic Cultures
Authors: Bennet Barbara A. , Hyde Christine E. , Pecora Julie R. , Clodfelter Jill E.
Published: October 2006

Abstract: The potent reinforcing effects of methamphetamine and cocaine are thought to be mediated by their interactions with CNS dopamine neurons. Both stimulants share the ability to block dopamine uptake potently, and methamphetamine can release cytoplasmic dopamine as well. There is also abundant evidence demonstrating the neurotoxic effects of methamphetamine. There are, however, limited studies that attempt to discern the neurotoxic mechanisms of these agents. The purpose of the present study was to characterize and compare the chronic in vitro effects of methamphetamine, cocaine, and the dopamine uptake blocker, mazindol, on cultured fetal mesencephalic dopamine neurons. Our studies examined biochemical mechanisms to evaluate the contribution of reuptake blockade versus release of dopamine. Using a dispersed cell preparation of fetal mesencephalon, cultures were treated for 5 days with the three uptake blockers. Dopamine function was assessed by measuring high‐affinity [3H]dopamine uptake and by examining cultures for the presence of tyrosine hydroxylase‐immunopositive neurons. Nonspecific neurotoxicity was assessed by staining for neuron‐specific enolase and measuring lactate dehydrogenase activity. The results indicate that repeated administration of high concentrations of methamphetamine (10−4 and 10−3M) caused a generalized neurotoxicity whereas the effects of 10−5M methamphetamine appeared to be specific to dopamine cells. Likewise, treatment of the cultures with mazindol (10−6M) resulted in reduced dopamine uptake while not significantly affecting neuron‐specific enolase or tyrosine hydroxylase immunostaining. On the other hand, repeated exposure to cocaine (10−5 and 10−4M) did not alter dopaminergic function in these cultures. The different mechanisms of action of these stimulants may explain the differences in neurotoxic potency of these compounds. The results demonstrate that a tissue culture model of fetal mesencephalic dopamine neurons provides a useful tool for the study of dopamine uptake systems and neuronal function.
Purification of Cytosolic Malic Enzyme from Bovine Brain, Generation of Monoclonal Antibodies, and Immunocytochemical Localization of the Enzyme in Glial Cells of Neural Primary Cultures
Authors: Kurz Georg M. , Wiesinger Heinrich , Hamprecht Bernd
Published: October 2006

Abstract: Cytosolic malic enzyme (EC 1.1.1.40) was purified from bovine brain 5,600‐fold to a specific activity of 47 U/mg. The enzyme is a homotetramer with a subunit molecular mass of 60 kDa and an isoelectric point of 6.2. Mouse monoclonal antibodies raised against this enzyme were purified and shown to be monospecific, as indicated by immunoblotting. Immunocytochemical examination of rat astroglia‐rich primary cultures at the light microscopic level revealed colocalization of cytosolic malic enzyme with the astroglial marker glial fibrillary acidic protein. Also, a colocalization with the oligodendroglial marker myelin basic protein was found. Neurons in rat neuron‐rich primary cultures did not show positive staining. The data suggest that cytosolic malic enzyme is a glial enzyme and is lacking in neurons.