Identification of a phosphorylation site for calcium/calmodulindependent protein kinase II in the NR2B subunit of the N-methyl-D-aspartate receptor.

The N-methyl-D-aspartate (NMDA) subtype of excitatory glutamate receptors plays critical roles in embryonic and adult synaptic plasticity in the central nervous system. The receptor is a heteromultimer of core subunits, NR1, and one or more regulatory subunits, NR2A-D. Protein phosphorylation can regulate NMDA receptor function (Lieberman, D. N., and Mody, I. (1994) Nature 369, 235-239; Wang, Y. T., and Salter, M. W. (1994) Nature 369, 233-235; Wang, L. -Y., Orser, B. A., Brautigan, D. L., and MacDonald, J. F. (1994) Nature 369, 230-232). Here we identify a major phosphorylation site on subunit NR2B that is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), an abundant protein kinase located at postsynaptic sites in glutamatergic synapses. For the initial identification of the site, we constructed a recombinant fusion protein containing 334 amino acids of the C terminus of the NR2B subunit and phosphorylated it with CaM kinase II in vitro. By peptide mapping, automated sequencing, and mass spectrometry, we identified the major site of phosphorylation on the fusion protein as Ser-383, corresponding to Ser-1303 of full-length NR2B. The Km for phosphorylation of this site in the fusion protein was approximately 50 nM, much lower than that of other known substrates for CaM kinase II, suggesting that the receptor is a high affinity substrate. We show that serine 1303 in the full-length NR2B and/or the cognate site in NR2A is a major site of phosphorylation of the receptor both in the postsynaptic density fraction and in living hippocampal neurons.

Age-related decline in rat striatal dopamine metabolism is regionally homogeneous.

Previous research has established that the age-related decrease in rat striatal D2 sites occurs predominantly in the posterior ventral caudate-putamen, and the present work was undertaken to determine whether a corresponding preferential reduction in dopamine, its metabolites, or its synthesis rate occurs in this region. Male F344 rats 4-8 or 25-27 months old were used for regional HPLC electrochemical determinations of 1) dopamine, homovanillic acid (HVA), or dihydroxyphenylacetic acid (DOPAC) obtained from striatal micropunch samples, or 2) 3,4-dihydroxyphenylalanine (DOPA) concentrations in these same micropunch regions 30 minutes after treatment with the aromatic amino decarboxylase inhibitor, NSD-1015 (100 mg/kg, IP). Aged rats had significantly less dopamine, HVA, and DOPAC in their striatal samples than did young adult controls, as well as having less DOPA accumulation after NSD-1015. However, for none of these measures was the age x region interaction significant, suggesting that the decline in these markers of presynaptic dopaminergic function occurs uniformly throughout the striatum. The results provide evidence that the effects of aging on striatal dopamine receptors are dissociable from the influences on the dopaminergic innervation of this structure, suggesting independent control of pre- and postsynaptic elements of these synapses during the lifespan.

Dopamine high-affinity transport site topography in rat brain: major differences between dorsal and ventral striatum.

Investigations were conducted to determine the topography of the high-affinity dopamine uptake process within the rat striatum. [3H]Dopamine uptake into crude synaptosomes prepared from micropunch samples was found to be two- to three-fold higher in dorsal caudate-putamen relative to nucleus accumbens septi. In contrast, the concentrations of dopamine in the two regions were equivalent. The recognition site associated with high-affinity dopamine uptake was labeled using [3H]mazindol, and the binding of this ligand was also found to be two- to three-fold higher in homogenates from dorsal caudate-putamen samples relative to nucleus accumbens septi. Regional differences in uptake of [3H]dopamine or binding of [3H]mazindol were shown to be due to variations in Vmax or Bmax, not to differences in apparent affinity. Autoradiography of [3H]mazindol binding in rat striatum revealed a decreasing density of the site along the dorsal-to-ventral axis, with the highest binding occurring in the dorsolateral caudate-putamen, lower binding in the ventral caudate-putamen, and lowest levels in the septal pole of the nucleus accumbens septi. Quantification showed that the extent of this gradient was two-fold. Further autoradiographic studies revealed less striatal heterogeneity in the pattern of binding of [3H]ketanserin, another radioligand associated with the striatal dopaminergic innervation but not linked to the dopamine uptake process of the plasma membrane. The findings suggest that the dopaminergic fibers of the ventral striatum, especially the medial nucleus accumbens septi, may be relatively lacking in their capacity for dopamine uptake following its release. This organization may result in regional differences in the time-course of of extraneuronal dopamine following transmitter release and may render the dopamine-containing terminals of the ventral striatum less susceptible to the degenerative influences of neurotoxins that are incorporated by the high-affinity dopamine uptake process.

Inherited depression of arterial lipoamide dehydrogenase activity associated with susceptibility to atherosclerosis in pigeons.

The activity of lipoamide dehydorgenase (E.C.1.6.4.3) was measured in arterial homogenates from very young pigeons (5-8 weeks old) known to differ in their susceptibility to atherosclerosis. The activity of the arterial enzyme was significantly lower in the atherosclerosis-susceptible White Carneau pigeons than it was in the atherosclerosis-resistant Show Racer pigeons. Lipoamide dehydrogenase is a component of the pyruvate dehydrogenase and alpha-ketoglutarate multienzyme complexes. The first complex catalyzes the conversion of pyruvate to oxaloacetate via acetyl-CoA, and this reaction represents a crucial link between glycolysis and the Krebs cycle. The second complex is essential for the oxidative breakdown of carbohydrates, fats, and amino acids via the Krebs cycle. Reduced activity of these complexes, resulting from low activity of lipoamide dehydrogenase, favors reduction of pyruvate to lactate and a shift to glycolysis. This situation is in accord with other results obtained in avian and human arteries which appear to indicate a higher rate of glycolysis in atherosclerosis-susceptible and atherosclerotic arteries. It appears that the increased dependence of the White Carneau arteries on glycolysis, suggested by the reduced lipoamide dehydrogenase activity, facilitates the development of atherosclerosis in this pigeon strain.

Elevation of arterial phosphorfuctokinase activity associated with susceptibility to atherosclerosis in pigeons.

The activity of phosphofructokinase (PFK, 2.7.1.11) was measured in arteries of very young (5-8 week old) pigeons known to differ in susceptibility to atherosclerosis. The activity of the arterial enzyme was significantly higher in the atherosclerosis-susceptible White Carneau (WC) pigeons than in the resistant Show Racers (SR). The difference was significant whether enzyme activity was calculated on the basis of extract protein, DNA content or fat-free dry weight. In the White Carneau arteries the activity of the enzyme was higher in the female than the male pigeons. PFK is a key regulatory enzyme of glycolysis and is subject to fine control adjustments. A low ATP/ADP ratio and a fall in citrate concentration, as for example, induced by hypoxia, are meditors of a feedback mechanism leading to a rise in PFK activity and enhancement of glycolysis for energy production. This mechanism appears to be the cause of the higher PFK activity in the WC arteries, because related studies indicate impaired Krebs cycle activity in these vessels. It is suggested that the increased dependence of the WC arteries on glycolysis facilitates the development of atherosclerosis in this pigeon strain and that the mechanism is similar to the mechanism by which tissue hypoxia causes lipid accumulation and connective tissue alterations in the arterial wall.