Inhibition of Dopamine Agonist-Induced Phosphoinositide Hydrolysis by Concomitant Stimulation of Cyclic AMP Formation in Brain Slices

Abstract: We examined the effects of cyclic AMP on dopamine receptor-coupled activation of phosphoinositide hydrolysis in rat striatal slices. Forskolin, dibutyryl cyclic AMP, and the protein kinase A activator Sp -cyclic adenosine monophosphothioate ( Sp -cAMPS) significantly inhibited inositol phosphate formation stimulated by the dopamine D₁ receptor agonist SKF 38393. Conversely, the protein kinase A antagonist Rp -cyclic adenosine monophosphothioate ( Rp -cAMPS) dose-dependently potentiated the SKF 38393 effect. In the presence of 200 µ M Rp -cAMPS, the dose-response curves of the dopamine D₁ receptor agonists SKF 38393 and fenoldopam were shifted to the left and maximal agonist responses were markedly increased. The agonist EC₅₀ values, however, were not significantly altered by protein kinase A inhibition. Neither Sp -cAMPS nor Rp -cAMPS significantly affected basal inositol phosphate accumulation. These findings demonstrate that dopaminergic stimulation of phosphoinositide hydrolysis is inhibited by elevations in intracellular cyclic AMP. Dopamine receptor agonists that stimulate adenylyl cyclase could suppress their activation of phosphoinositide hydrolysis by concomitantly stimulating the formation of cyclic AMP in striatal tissue. The interaction between dopamine D₁ receptor-stimulated elevations in cyclic AMP and dopaminergic stimulation of inositol phosphate formation suggests a cellular colocalization of these dopamine-coupled transduction pathways in at least some cells of the rat striatum.     

Carbon allocation in developing spruce needles. Enzymes and intermediates of sucrose metabolism

In lyophilized needles of Norway spruce ( Picea abies [L.] Karsten) and starting from bud break, we determined enzyme activities (sucrose phosphate synthase [SPS; EC 2.4,1.14]. sucrose synthase [SS; EC 2.4,1.13]. acid invertase [AI; EC 3.2,1.26]) and intermediates (starch, sucrose, glucose, fructose; fructose 6-phosphate, fructose 2.6-bisphosphate [F26BP]) of carbohydrate metabolism together with needle weight, shoot length, chlorophyll and protein. For up to 110 days after bud break, samples were taken twice a week from about 25-year-old trees under field conditions. At least three periods can be distinguished during needle maturation. During the first period (up to 45 days after bud break) Al showed the highest extractable activity. This coincided with very high levels of F26BP (up to 11 pmol [mg dry weight]⁻¹) and a transient increase of starch in parallel to a decrease of sucrose. The interval between 45 and 70 days after bud break was characterized by high SS activity (ratio of fructose/glucose >1), much decreased levels of F26BP (down to below 1 pmol [mg dry weight]⁻¹), and a pronounced increase in the dry weight/fresh weight ratio. In parallel, starch declined and soluble carbohydrates increased. Finally, needle maturation was characterized by decreasing SS and continuously increasing SPS activities, so that the ratio of SPS/SS increased more than 6-fold. AI. however, did not decline with maturation. Changes in pool sizes of metabolites and enzyme activities (AI. SPS) are consistent with current concepts on sink/source transition. SS is obviously important with regard to the synthesis of structural polysaccharides.     

The role of acid phosphatases in plant phosphorus metabolism

Hydrolysis of phosphate esters is a critical process in the energy metabolism and metabolic regulation of plant cells. This review summarizes the characteristics and putative roles of plant acid phosphatase (APase). Although immunologically closely related, plant APases display remarkable heterogeneity with regards to their kinetic and molecular properties, and subcellular location. The secreted APases of roots and cell cultures are relatively non-specific enzymes that appear to be important in the hydrolysis and mobilization of P_i from extracellular phosphomonoesters for plant nutrition. Intracellular APases are undoubtedly involved in the routine utilization of P_i reserves or other P_i-containing compounds. A special class of intracellular APase exists that demonstrate a clear-cut (but generally nonabsolute) substrate selectivity. These APases are hypothesized to have distinct metabolic functions and include: phytase, phosphoglycolate phosphatase, 3-phosphoglycerate phosphatase, phosphoenolpyruvate phosphatase, and phosphotyrosyl-protein phosphatase. APase expression is regulated by a variety of developmental and environmental factors. P_i starvation induces de novo synthesis of extra- and intracellular APases in cell cultures as well as in whole plants. Recommendations are made to achieve uniformity in the analyses of the different APase isoforms normally encountered within and between different plant tissues.     

Effects of vanadate on the ATP content,ATPase activity and phosphate absorption capacity of maize roots

Although the sensitivity of the plasma membrane H⁺-ATPase to vanadate is well known, the metabolic response of plant cells to vanadate is less well characterised in vivo and its use as an inhibitor in whole plant experiments has had mixed success. Experiments with maize (Zea mays, L.) roots and with purified plasma membrane fractions from the same tissues showed that exposure to vanadate caused: (i) a reduction in the capacity for phosphate uptake; (ii) a reduction in the extractable ATPase activity from the tissue; and (iii) a significant increase in the ATP level. The measurements on the extractable ATPase activity and the ATP level showed that the effect of vanadate developed slowly, apparently reflecting the slow accumulation of intracellular vanadate. The marked effect of vanadate on the ATP level-exposure to 500 M vanadate for 5 h doubled the ATP content of the roots tips-indicates that there is no stringent control over the ATP level in the roots and that the plasma membrane H⁺-ATPase activity is likely to have a significant role in determining the ATP level under normal conditions.     

The role of the hexose transporter in the chloroplasts of Arabidopsis thaliana L.

The metabolism of wild-type Arabidopsis thaliana L. and its mutant TC265 were compared in order to reveal the role of the chloroplast glucose transporter. Plants were grown in a 12-h photoperiod. From 20 to 40 days after germination, starch per gram fresh weight of shoot in the mutant was four times that in the wild type. The extent of this difference did not alter during this period. Stereological analysis showed that the chloroplasts in the mutant were larger than those in the wild type; the thylakoids appeared to be distorted by the high starch content. [U-¹⁴C]Glucose and [U-¹⁴C]glycerol were supplied, separately, to excised leaves in the dark. [U-¹⁴C]Glucose was a good precursor of sucrose in the wild type and mutant; [U-¹⁴C]glycerol was a poor precursor of sucrose in both. The distribution of ¹⁴C in the wild type was used to calculate that the net flux was from hexose monophosphates to triose phosphates, not vice versa. During the first 4 h of the night the sugar content (75% sucrose, 20% glucose) of the leaves of the mutant dropped sharply, and at all times during the night it was less than that of the wild-type leaves. This drop in sugar coincided with a decrease in the rate of respiration. The growth rate of the mutant was less than that of the wild type. Addition of sucrose restored the rate of respiration at night and increased the rate of growth. It is argued that a major function of the glucose transporter in Arabidopsis chloroplasts is export of the products of starch breakdown that are destined for sucrose synthesis at night.We thank Professor C.R. Somerville for his generous gift of seed of the Arabidopsis mutant TC265. We are also grateful to Mr B. Chapman for assistance with the preparation of the sections for electron microscopy. R.N.T. thanks the Science and Engineering Research Council for a studentship.     

Effect of shock-loading of heavy metals on total organic carbon and phosphate removal in an anaerobic-aerobic activated sludge process

The effect of shock-loading of zinc, copper and cadmium ions on the removal of total organic carbon (TOC) and phosphate in an anaerobic-aerobic activated sludge process was investigated. TOC removal was not sensitive to shock-loading of Zn²⁺ and Cd²⁺ ions, and complete removal was achieved even at 20 mg Zn²⁺/l and 20 mg Cd²⁺/l. However, with over 1 mg Cu²⁺/1 TOC removal efficiency decreased. PO _inf4 ^sup3- removal, in contrast, was extremely sensitive to these metal ions, with the threshold being 1 mg Zn²⁺/l and 1 mg Cd²⁺/l. Higher concentrations adversely affected PO _inf4 ^sup3- removal. Copper again proved detrimental; no PO _inf4 ^sup3- removal was achieved even at 1 mg Cu/l. These results highlight the sensitivity of the removal efficiencies of TOC and PO _inf4 ^sup3- to shock loadings of these heavy metals.Y.P. Ting is with the Department of Chemical Engineering, National University of Singapore, Kent Ridge, 0511, Singapore; H. Imai and S. Kinoshita are with the Department of Chemical Process Engineering, Hokkaido University, Sapporo 060, Japan.     

Similarity between pyridoxal/pyridoxamine phosphate-dependent enzymes involved in dideoxy and deoxyaminosugar biosynthesis and other pyridoxal phosphate enzymes.

A multiple sequence alignment among aspartate aminotransferase, dialkylglycine decarboxylase, and serine hydroxymethyltransferase (DAS) was used for profile databank search. The DAS profile could detect similarities to other pyridoxal or pyridoxamine phosphate-dependent enzymes, like several gene products involved in dideoxysugar and deoxyaminosugar synthesis. The alignment among DAS and such gene products shows the conservation of aspartate 222 and lysine 258, which, in aspartate aminotransferase, interacts with the N1 of the coenzyme pyridine ring and forms the internal Schiff base, respectively. The lysine is replaced by histidine in the pyridoxamine phosphate-dependent gene products. The alignment indicates also that the region encompassing the coenzyme binding site is the most conserved.     

Dissociation of the plasticity of 5-HT1A sites and 5-HT transporter sites

To study the early effects of neonatal 5,7-dihydroxytryptamine lesions on 5-hydroxytryptamine_1A (5-HT_1A) receptors, we measured regional [³H]8-OH-DPAT-labeled 5-HT_1A sites in binding assays and compared them to our previous studies of [³H]paroxetine-labeled 5-HT transporter sites during the first month in the same rats. While there were significant time- and dose-dependent effects of 5,7-DHT on 5-HT transporter sites, there were no significant changes in 5-HT_1A sites in cortex, hippocampus, diencephalon, brainstem, cerebellum, or spinal cord. 5,7-DHT lesions also did not alter the K_i of Gpp(NH)p at brainstem 5-HT_1A sites or the K_i of 5-HT in cortex or brainstem in the presence or absence of GTPS or Gpp(NH)p. There were significant regional differences between the density of 5-HT_1A sites and 5-HT transporter sites. The ontogeny of brainstem 5-HT_1A sites was a pattern of increases until three weeks postnatal, and 5,7-DHT lesions did not alter the ontogeny of 5-HT_1A sites. These data suggest differential plasticity of 5-HT_1A and 5-HT transporter binding sites during the first month after neonatal 5,7-DHT lesions.