Effects of sulfhydryl reagents on nitroglycerin-induced relaxation of bovine coronary artery

The mechanism whereby nitroglycerin relaxes vascular smooth muscle remains uncertain. A current hypothesis suggests that nitroglycerin reacts with critical cellular sulfhydryl groups to form an intermediate, which activates guanylate cyclase, resulting in cGMP accumulation and relaxation. This study investigated further the potential involvement of sulfhydryls in nitroglycerin-induced vascular smooth muscle relaxation by evaluating effects of a variety of sulfhydryl alkylating and reducing agents on responses to nitroglycerin and other relaxants in bovine coronary arterial strips submaximally contracted using 30 mM K. Whereas 10(-4) M 5,5'-dithiobis-(2-nitrobenzoic acid), 10(-5) MN-ethylmaleimide, and 10(-4) MN-naphthylmaleimide did not affect nitroglycerin-induced relaxation, 10(-4) MN-ethylmaleimide and 10(-4) M ethacrynic acid significantly inhibited relaxation induced by nitroglycerin. Both ethacrynic acid and N-ethylmaleimide at 10(-4) M also inhibited relaxation induced by sodium nitroprusside. N-ethylmaleimide, but not ethacrynic acid, inhibited relaxation induced by isoproterenol and forskolin. Ethacrynic acid significantly reduced both relaxation and cGMP elevation induced by both 10(-7) M nitroglycerin and 10(-7) M sodium nitroprusside. Ethacrynic acid, but not N-ethylmaleimide, significantly reduced relaxation induced by 8-Br-cGMP. Pretreatment with the sulfhydryl-containing agents N-acetylcysteine, 2-mercaptoethanol, or dithiothreitol, at 10(-3) M did not affect nitroglycerin-induced relaxation in nontolerant arteries. Similarly, N-acetylcysteine and dithiothreitol did not alter the depressed responses to nitroglycerin in arteries in which tolerance to nitroglycerin was induced in vitro. A slight but statistically significant reversal of nitroglycerin-tolerance occurred after treatment of tolerant arteries with 2-mercaptoethanol.(ABSTRACT TRUNCATED AT 250 WORDS)     

New Developments and Applications of the MP2RAGE Sequence - Focusing the Contrast and High Spatial Resolution R1 Mapping

MR structural T1-weighted imaging using high field systems (>3T) is severely hampered by the existing large transmit field inhomogeneities. New sequences have been developed to better cope with such nuisances. In this work we show the potential of a recently proposed sequence, the MP2RAGE, to obtain improved grey white matter contrast with respect to conventional T1-w protocols, allowing for a better visualization of thalamic nuclei and different white matter bundles in the brain stem. Furthermore, the possibility to obtain high spatial resolution (0.65 mm isotropic) R₁ maps fully independent of the transmit field inhomogeneities in clinical acceptable time is demonstrated. In this high resolution R₁ maps it was possible to clearly observe varying properties of cortical grey matter throughout the cortex and observe different hippocampus fields with variations of intensity that correlate with known myelin concentration variations.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Glutamate 47 in 1-aminocyclopropane-1-carboxylate synthase is a major specificity determinant

Glutamate 47 is conserved in 1-aminocyclopropane-1-carboxylate (ACC) synthases and is positioned near the sulfonium pole of (S,S)-S-adenosyl-L-methionine (SAM) in the modeled pyridoxal phosphate quinonoid complex with SAM. E47Q and E47D constructs of ACC synthase were made to investigate a putative ionic interaction between Glu47 and SAM. The k(cat)/K(m) values for the conversion of (S,S)-SAM to ACC and methylthioadenosine (MTA) are depressed 630- and 25-fold for the E47Q and E47D enzymes, respectively. The decreases in the specificity constants are due to reductions in k(cat) for both mutant enzymes, and a 5-fold increase in K(m) for the E47Q enzyme. Importantly, much smaller effects were observed for the kinetic parameters of reactions with the alternate substrates L-vinylglycine (L-VG) (deamination to form alpha-ketobutyrate and ammonia) and L-alanine (transamination to form pyruvate), which have uncharged side chains. L-VG is both a substrate and a mechanism-based inactivator of the enzyme [Feng, L., and Kirsch, J. F. (2000) Biochemistry 39, 2436-2444], but the partition ratio, k(cat)/k(inact), is unaffected by the Glu47 mutations. ACC synthase primarily catalyzes the beta,gamma-elimination of MTA from the (R,S) diastereomer of SAM to produce L-VG [Satoh, S., and Yang, S. F. (1989) Arch.Biochem. Biophys. 271, 107-112], but catalyzes the formation of ACC to a lesser extent via alpha,gamma-elimination of MTA. The partition ratios for (alpha,gamma/beta,gamma)-elimination on (R,S)-SAM are 0.4, < or =0.014, and < or =0.08 for the wild-type, E47Q, and E47D enzymes, respectively. The results of these experiments strongly support a role for Glu47 as an anchor for the sulfonium pole of (S,S)-SAM, and consequently a role as an active site determinant of reaction specificity.     

In vivo 13C NMR assessment of brain glycogen concentration and turnover in the awake rat

Brain glycogen metabolism was recently observed in vivo and found to be very slow in the lightly alpha-chloralose anesthetized rat [J. Neurochem. 73 (1999) 1300]. Based on that slow turnover, the total glycogen content in the awake rat brain and its turnover time were assessed after administering 13C-labeled glucose for 48 h. Label incorporation into glycogen, glucose, amino acid, and N-acetyl-aspartate (NAA) resonances was observed. The amount of 13C label incorporated into glycogen was variable and did not correlate with that in glutamate (r=-0.1, P>0.86). However, the amount of 13C label incorporated into glycogen was very similar to that in NAA (r=0.93), implying similar turnover times between brain glycogen and NAA (approximately 10 h). Absolute quantification of the total concentration of brain glycogen in the awake, normoglycemic rat yielded 3.3+/-0.8 micromol/g (n=6, mean+/-S.D.).     

A mathematical model of compartmentalized neurotransmitter metabolism in the human brain

After administration of enriched [1-13C]glucose, the rate of 13C label incorporation into glutamate C4, C3, and C2, glutamine C4, C3, and C2, and aspartate C2 and C3 was simultaneously measured in six normal subjects by 13C NMR at 4 Tesla in 45-ml volumes encompassing the visual cortex. The resulting eight time courses were simultaneously fitted to a mathematical model. The rate of (neuronal) tricarboxylic acid cycle flux (V(PDH)), 0.57 +/- 0.06 micromol. g(-1). min(-1), was comparable to the exchange rate between (mitochondrial) 2-oxoglutarate and (cytosolic) glutamate (Vx), 0.57 +/- 0.19 micromol. g(-1). min(-1)), which may reflect to a large extent malate-aspartate shuttle activity. At rest, oxidative glucose consumption [CMR(Glc(ox))] was 0.41 +/- 0.03 miccromol. g(-1). min(-1), and (glial) pyruvate carboxylation (VPC) was 0.09 +/- 0.02 micromol. g(-1). min(-1). The flux through glutamine synthetase (Vsyn) was 0.26 +/- 0.06 micromol. g(-1). min(-1). A fraction of Vsyn was attributed to be from (neuronal) glutamate, and the corresponding rate of apparent glutamatergic neurotransmission (VNT) was 0.17 +/- 0.05 micromol. g(-1). min(-1). The ratio [VNT/CMR(Glcox)] was 0.41 +/- 0.14 and thus clearly different from a 1:1 stoichiometry, consistent with a significant fraction (approximately 90%) of ATP generated in astrocytes being oxidative. The study underlines the importance of assumptions made in modeling 13C labeling data in brain.     

Compartmentalized neurotransmitter and amino acid synthesis in vivo studied by high field MRS

There is strong evidence that the brain can use multiple substrates for energy including glucose, lactate, ketone bodies, glutamate and glutamine. Competition studies show that certain substrates are preferentially used for energy by synaptic terminals even when other substrates are available. It has recently been shown that synaptosomes can use both glutamine and glutamate for energy and synthesis of amino acids; however, these substrates yield very different patterns of 13C-labelling of end products. These findings provide evidence of differential compartmentalisation of the metabolism of glutamate taken up from the extracellular milieu as compared to the glutamate produced from glutamine within synaptic terminals. This compartmentalisation is related to the specific role(s) of glutamate vs. glutamine in synaptic terminals as well as the metabolism of these amino acids in either partial or complete TCA cycles for energy. The presence of glucose, which provides a source of acetyl-CoA, can greatly modulate both the metabolic fate of other substrates and the pool size of amino acids such as glutamate and GABA. The differential localization of the enzymes glutamate dehydrogenase and aspartate aminotransferase contribute to this compartmentalisation as does the necessity that synaptic terminals balance their energy needs with the requirement to synthesize neurotransmitters.     

In vivo quantification of neuro‐glial metabolism and glial glutamate concentration using 1H‐[13C] MRS at 14.1T

Astrocytes have recently become a major center of interest in neurochemistry with the discoveries on their major role in brain energy metabolism. An interesting way to probe this glial contribution is given by in vivo ¹³C NMR spectroscopy coupled with the infusion labeled glial‐specific substrate, such as acetate. In this study, we infused alpha‐chloralose anesthetized rats with [2‐¹³C]acetate and followed the dynamics of the fractional enrichment (FE) in the positions C4 and C3 of glutamate and glutamine with high sensitivity, using ¹H‐[¹³C] magnetic resonance spectroscopy (MRS) at 14.1T. Applying a two‐compartment mathematical model to the measured time courses yielded a glial tricarboxylic acid (TCA) cycle rate (V_g) of 0.27 ± 0.02 μmol/g/min and a glutamatergic neurotransmission rate (V_NT) of 0.15 ± 0.01 μmol/g/min. Glial oxidative ATP metabolism thus accounts for 38% of total oxidative metabolism measured by NMR. Pyruvate carboxylase (V_PC) was 0.09 ± 0.01 μmol/g/min, corresponding to 37% of the glial glutamine synthesis rate. The glial and neuronal transmitochondrial fluxes (V_x^g and V_xⁿ) were of the same order of magnitude as the respective TCA cycle fluxes. In addition, we estimated a glial glutamate pool size of 0.6 ± 0.1 μmol/g. The effect of spectral data quality on the fluxes estimates was analyzed by Monte Carlo simulations.