Michael S. Poosch

NMDA receptor subunit gene expression in the rat brain: a quantitative analysis of endogenous mRNA levels of NR1Com, NR2A, NR2B, NR2C, NR2D and NR3A

Electrophysiological recordings have shown NMDA receptors to be heterogenous structures capable of responding to selected antagonists and agonists in multiple ways. This diversity in functional response has led investigators to conclude that these channels are comprised of unique combinations of receptor subunits which determine a cells functional NMDA-signature [H. Meguro, H. Mori, K. Araki, E. Kushiya, T. Kutsuwada, M. Yamazaki, T. Kumanishi, M. Arakawa, K. Sakimura, M. Mishina, Functional characterization of a heteromeric NMDA receptor channel expressed from cloned cDNAs, Nature (London) 357 (1992) 70-74; T. Ishii, K. Moriyoshi, H. Sugihara, K. Sakurada, H. Kadotani, M. Yokoi, C. Akazawa, R. Shigemoto, N. Mizuno, S. Nakanishi, Molecular characterization of the family of the N-methyl-d-aspartate receptor subunits, J. Biol. Chem. 268 (1993) 2836-2843; K.A. Wafford, C.J. Bain, B. Le Bourdelles, P.J. Whiting, J.A. Kemp, Preferential co-assembly of recombinant NMDA receptors composed of three different subunits, NeuroReport 4 (1993) 1347-1349; T. Priestley, P. Laughton, J. Myers, B. Le Bourdelles, J. Kerby, P.J. Whiting, Pharmacological properties of recombinant human N-methyl-d-aspartate receptors comprising NR1a/NR2A and NR1a/NR2B subunit assemblies expressed in permanently transfected mouse fiberblast cells, Mol. Pharmacol. 48 (1995) 841-848; P.H. Seeburg, N. Burnashev, G. Kohr, T. Kuner, R. Sprengel, H. Monyer, The NMDA receptor channel: molecular design of a coincidence detector, Recent Prog. Horm. Res. 50 (1995) 19-34; A.L. Buller, D.T. Monagahan, Pharmacological heterogeneity of NMDA receptors: characterization of NR1a/NR2D heteromers expressed in Xenopus oocytes, Eur. J. Pharmacol. 320 (1997) 87-94]. In situ hybridization and immunocytochemical studies have shown that there is a spatio-temporal level of expression throughout the brain for each of the receptor subunits with some regions showing a strong preference for a particular subunit. Although these studies collectively show that there are regional differences with respect to NMDA receptor subunit expression in the brain, it has not been determined at what level(s) these genes are expressed or whether each region displays a unique NMDA-subunit signature. The present study was undertaken to examine the level of gene expression for the NR1, NR2A, NR2B, NR2C, NR2D and NR3A receptor subunits in isolated regions of rat brain using the nuclease protection assay. Results show that each of the brain regions examined expresses all six NMDA receptor subunits. The level of message expression for NR1 greatly exceeded that of the other subunits combined, with values ranging from 67-88% of the total subunit gene expression. The relative proportions of the other subunits (NR2A-D and NR3A) varied widely, suggesting that NMDA receptor composition is unique to each region of the brain.

Determination of peroxisomal fatty acyl-CoA oxidase activity using a lauroyl-CoA-based fluorometric assay.

A simple, sensitive fluorometric method for the determination of peroxisomal fatty acyl-CoA oxidase (EC 1.3.99.3) activity has been developed. Studies of enzyme activity relative to subcellular distribution and to clofibrate induction indicate that this assay is specific for peroxisomal fatty acyl-CoA oxidase. The lauroyl-CoA-dependent production of H2O2 is quantitated by measuring the oxidation of 4-hydroxyphenyl-acetic acid to a fluorescent product in a horseradish peroxidase-coupled assay. Assays can be performed in either a fixed time or continuous mode. In either mode, H2O2 production is related to a change in fluorescence intensity through use of a standard curve generated with known amounts of H2O2. The use of lauroyl-CoA (12:0), rather than the more generally used substrate palmitoyl-CoA (16:0), provides significant advantages. Much of the substrate inhibition problem associated with palmitoyl-CoA has been avoided, and a greater than 4.5-fold higher specific activity has been achieved compared with a palmitoyl-CoA-based assay. In the fixed-time mode, linearity relative to time and to the amount of enzyme added has been established without resorting to the use of bovine serum albumin as a substrate binding medium. Sensitivity is estimated to be at least equal to that of the most sensitive methods reported, while reliability, versatility and range have been improved. Use of this method should greatly facilitate the study of peroxisomal beta-oxidation regulatory mechanisms in hepatocyte cell culture systems as well as in other circumstances where low activities or small samples must be assayed.

Quantitation, cellular localization and regulation of neurokinin receptor gene expression within the rat substantia nigra

The diverse biological effects of substance P and related peptides are mediated by multiple neurokinin receptors. The CNS sites of neurokinin receptor biosynthesis have not been fully elucidated and little is known about the regulation of neurokinin receptor gene expression. In the present study, the abundance of neurokinin-1, neurokinin-2 and neurokinin-3 receptor messenger RNAs in various rat brain regions was quantitated using a sensitive solution hybridization assay. Midbrain neurokinin receptor gene expression was then examined in detail. In situ hybridization experiments localized high levels of neurokinin-3 receptor messenger RNA to presumptive dopamine neurons, as evidenced by sensitivity to 6-hydroxydopamine lesions and the presence of tyrosine hydroxylase messenger RNA in serial sections. Lesions of nigral afferent (including substance P-containing) pathways from the caudate-putamen increased both nigral neurokinin-3 and neurokinin-1 receptor messenger RNA levels two- to three-fold. These data provide the anatomical substrate for physiological data suggesting that substance P (released from striatonigral neurons) may act on nigral cells through neurokinin-1 receptors, while the substance P co-transmitter neurokinin A may act preferentially on dopamine neurons through neurokinin-3 receptors. The magnitude of denervation-induced changes in neurokinin receptor messenger RNAs suggests significant plasticity of neurokinin receptor gene expression.

Neurokinin-3 receptors modulate dopamine cell function and alter the effects of 6-hydroxydopamine.

Neurokinin-3 receptor expression within rat midbrain dopamine neurons was demonstrated using a combination of in situ hybridization and receptor autoradiographic techniques. Continuous intranigral infusion of the neurokinin-3 receptor agonist senktide selectively increased striatal dopamine metabolism over a period of several days, followed by apparent development of tolerance. In contrast, in animals with moderate unilateral 6-hydroxydopamine-induced lesions of nigrostriatal dopamine cells, intranigral senktide infusion increased dopamine turnover in the surviving dopamine neurons and reduced functional dopamine asymmetry (reflected by spontaneous rotations) over the 2-week period tested. Thus nigral neurokinin receptors can modulate normal dopamine cell activity and may provide a therapeutic target in the treatment of Parkinsons disease.

Preprotachykinin gene expression in the human basal ganglia: characterization of mRNAs and pre-mRNAs produced by alternate RNA splicing

The nature and distribution of preprotachykinin (PPT, i.e. substance P/neurokinin A-encoding) gene expression in human basal ganglia was determined. Northern blot analysis visualized a single band of approximately 1300 bases, confirming the postmortem stability of PPT mRNA. Gross anatomical analysis indicated that PPT gene expression was relatively evenly distributed throughout the human caudate and putamen, but absent in the globus pallidus and substantia nigra. Nuclease protection analysis of these tissues established that human PPT mRNA consisted of approximately 80-85% beta-PPT (exon 1-7 derived) mRNA and 15-20% gamma-PPT (minus exon 4), with no alpha-PPT (minus exon 6) mRNA detected; these data contrast with the proportions of PPT mRNAs seen in non-human species. The incompletely spliced PPT RNA species detected in basal ganglia accounted for approximately 8% of total human PPT RNA and suggested a fixed order of exon splicing. Since various PPT mRNAs encode different combinations of tachykinin peptides with distinct biological activities, the markedly different proportions of PPT mRNAs seen in human basal ganglia compared to non-human tissues may be of physiological significance.

Noradrenaline transport and transporter mRNA of rat chromaffin cells are controlled by dexamethasone and nerve growth factor.

1. The biochemical basis for differences in noradrenaline (NA) transporter function between chromaffin cells in the adrenal medulla and those maintained in primary culture was investigated. 2. Intact adrenal medullae of neonatal rats accumulated small amounts of [3H]NA. In contrast, dissociated chromaffin cells placed in culture for 2‐6 days accumulated 100‐1000 times more [3H]NA. 3. Nerve growth factor (NGF) stimulated, whereas glucocorticoids dose dependently and reversibly inhibited, [3H]NA transport in chromaffin cells maintained in culture up to 6 days. During this period, no change in the morphological or biochemical characteristics of either NGF‐treated or ‐untreated chromaffin cells was evident. 4. A rat NA transporter cDNA clone was isolated for use in the quantification of NA transporter mRNA. Intact adrenal medullae contained 40% less NA transporter mRNA than an equivalent number of chromaffin cells in culture. Furthermore, dexamethasone produced nearly 90% loss and NGF elicited approximately 60% increase in NA transporter mRNA levels in cultured cells. 5. In cultured cells, and possibly in vivo, glucocorticoids inhibit NA transporter function of chromaffin cells at least in part through a decrease in NA transporter mRNA.

Immunocytochemical localization of the NMDA-R2A receptor subunit in the cat retina.

Immunocytochemical studies were performed to determine the distribution and cellular localization of the NMDA-R2A receptor subunit (R2A) in the cat retina. R2A-immunoreactivity (R2A-IR) was noted in all layers of the retina, with specific localizations in the outer segments of red/green and blue cone photoreceptors, B-type horizontal cells, several types of amacrine cells, Müller cells and the majority of cells in the ganglion cell layer. In the inner nuclear layer, 48% of all cells residing in the amacrine cell layer were R2A-IR including a cell resembling the GABAergic A17 amacrine cell. Interestingly, the AII rod amacrine cell was devoid of R2A-IR. Although the localization of the R2A subunit was anticipated in ganglion cells, amacrines and Müller cells, the presence of this receptor subunit to the cells in the outer retina was not expected. Here, both the R2A and the R2B subunits were found to be present in the outer segments of cone photoreceptors and to the tips of rod outer segments. Although the function of these receptor subunits in rod and cone photoreceptors remains to be determined, the fact that both R2A and R2B receptor subunits are localized to cone outer segments suggests a possible alternative pathway for calcium entry into a region where this cation plays such a crucial role in the process of phototransduction. To further classify the cells that display NR2A-IR, we performed dual labeling experiments showing the relationship between R2A-labeled cells with GABA. Results showed that all GABAergic-amacrines and displaced amacrines express the R2A-subunit protein. In addition, approximately 11% of the NR2A-labeled amacrines, did not stain for GABA. These findings support pharmacological data showing that NMDA directly facilitates GABA release in retina and retinal cultures [I.L. Ferreira, C.B. Duarte, P.F. Santos, C.M. Carvalho, A.P. Carvalho, Release of [3H]GABA evoked by glutamate receptor agonist in cultured chick retinal cells: effect of Ca2+, Brain Res. 664 (1994) 252-256; G.D. Zeevalk, W.J. Nicklas, Action of the anti-ischemic agent ifenprodil on N-methyl-d-aspartate and kainate-mediated excitotoxicity, Brain Res. 522 (1990) 135-139; R. Huba, H.D. Hofmann, Transmitter-gated currents of GABAergic amacrine-like cells in chick retinal cultures, Vis. Neurosci. 6 (1991) 303-314; M. Yamashita, R. Huba, H.D. Hofmann, Early in vitro development of voltage- and transmitter-gated currents in GABAergic amacrine cells, Dev. Brain Res. 82 (1994) 95-102; R. Ientile, S. Pedale, V. Picciurro, V. Macaione, C. Fabiano, S. Macaione, Nitric oxide mediates NMDA-evoked [3H]GABA release from chick retina cells, FEBS Lett. 417 (1997) 345-348; R.C. Kubrusly, M.C. deMello, F.G. deMello, Aspartate as a selective NMDA agonist in cultured cells from the avian retina, Neurochem. Intl. 32 (1998) 47-52] or reduction of GABA in vivo [N.N. Osborn, A.J. Herrera, The effect of experimental ischaemia and excitatory amino acid agonist on the GABA and serotonin immunoreactivities in the rabbit retina, Neurosci. 59 (1994) 1071-1081]. Since the majority of GABAergic synapses in the inner retina are onto both rod and cone bipolar axon terminals [R.G. Pourcho, M.T. Owzcarzak, Distribution of GABA immunoreactivity in the cat retina: A light and electron-microscopic study, Vis. Neurosci. 2 (1989) 425-435], we hypothesize that the NMDA-receptor plays a crucial role in providing feedback inhibition onto rod and cone bipolar cells.

Up-regulation of novel intermediate filament proteins in primary fiber cells: an indicator of all vertebrate lens fiber differentiation?

The early embryonic development and expression patterns of the eye lens specific cytoskeletal proteins, CP49 and CP95, were determined for the chick and were found to be similar in both human and mouse. These proteins, as well as their homologs in other species, are obligate polymerization partners which form unique filamentous structures termed “beaded filaments.” CP49 and CP95 appeared as protein products after 3 days of embryonic development in the chick during the elongation of primary fiber cells. Although limited data were obtained for human embryos at these early developmental timepoints, they were consistent with the interpretation that the up‐regulation of these lens specific proteins began only after the initiation of lens vesicle closure. In situ hybridization with the mouse lens confirmed that message levels for beaded filament proteins were greatly elevated in differentiating primary fiber cells. Nuclease protection assays established that mRNA levels for CP49 remained relatively constant while CP95 mRNA levels increased once the process of secondary fiber formation was under way. Although present in relatively low abundance, the mRNA for a unique splice variant of CP49, CP49INS, was also detected early in embryonic development and into adulthood. Peptide‐specific antibodies directed against unique predicted sequences were able to confirm the protein expression of CP49INS in both embryonic and adult chick lens cells. These data present the first detailed study of the expression of CP49 and CP95 during early lens development. They suggest that the up‐regulated expression of CP49 and CP95 could serve as pan‐specific markers for all vertebrate lens fiber development. Anat Rec 258:25–33, 2000.

GABA transporter mRNA : in vitro expression and quantitation in neonatal rat and postmortem human brain

A previously isolated rat cDNA clone encoding the membrane transporter for the neurotransmitter gamma-aminobutyric acid was expressed in transfected COS cells. The resultant transporter protein was characterized kinetically and pharmacologically. The apparent Kt (6.1 microM) and the pharmacological profile of a neuronal-type transporter observed in these mammalian cells were consistent with previous data obtained in Xenopus laevis oocytes. Post-natal levels of gamma-aminobutyric acid transporter mRNA in rat cerebellum, cerebral cortex and striatum (as measured by nuclease protection assay) transiently exceeded levels present in the adult brain. Human gamma-aminobutyric acid transporter mRNA also was measured by nuclease protection assay using as probe a human transporter cDNA homolog obtained by polymerase chain reaction. These studies suggest that quantitation of rat and human gamma-aminobutyric acid transporter mRNAs may provide a useful index of transporter gene expression.

The oxidation of dicarboxylic acid CoA esters via peroxisomal fatty acyl-CoA oxidase.

Evidence supporting a common peroxisomal beta-oxidation pathway for the coenzyme A thioesters of medium-chain-length dicarboxylic acids (DCn-CoA) and monocarboxylic acids (MCn-CoA) has been obtained. Using the mono-CoA esters of dodecanedioic acid (DC12-CoA) and lauroyl-CoA (MC12-CoA) as substrates, parallel inductions of activities and parallel increases in specific activities during purification of peroxisomal fatty acyl-CoA oxidase (EC 1.3.99.3) from rat liver after di(2-ethylhexyl)phthalate treatment were seen. The purified enzyme was used for antiserum production in rabbits; antiserum specificity was verified by immunoblot analysis. Coincident losses of oxidase activities with MC12-CoA and DC12-CoA were found in immunotitration experiments with rat liver homogenates, supporting the hypothesis that peroxisomal fatty acyl-CoA oxidase is solely responsible for the oxidation of medium-chain length dicarboxylic acid substrates. Kinetic studies with purified enzyme using the mono-CoA esters of sebacic (DC10-CoA), suberic (DC8-CoA), and adipic (DC6-CoA) acids along with DC12-CoA revealed substrate inhibition. Although these substrates exhibited similar calculated Vmax values, with decreasing chain length, the combination of increasing Km values and decreasing substrate inhibition constant (Ki) caused the maximum obtainable velocity to decrease. These studies offer an explanation for the previously observed limit of the ability of peroxisomes to chain-shorten dicarboxylates and increased urinary excretion of adipic acid when peroxisomal oxidation of dicarboxylic acids is enhanced.