Retinoylation Reaction of Proteins in Leydig (TM-3) Cells

The covalent incorporation of [³H]all-trans-retinoic acid into proteins has been studied in Leydig (TM-3) cells. The maximum retinoylation activity of Leydig cells proteins was 570± 27 fmoles/8×10⁴ cells at 37C. About 95% of [³H]retinoic acid was trichloroacetic acid-soluble after proteinase-K digestion or after hydrolysis with hydroxylamine. Thus, retinoic acid is most probably linked to proteins as a thiol ester. The retinoylation process was inhibited by 13-cis-retinoic acid and 9-cis-retinoic acid with IC₅₀ values of 0.6 and 1.2 M respectively. Dibutyryl-cAMP and forskolin increased the retinoylation activity by 75 and 81% at 500 and 25 M respectively. Also hCG increased the retinoylation binding activity of 110% at 250 ng/mL. After cycloheximide treatment of the Leydig cells the binding activity of [³H]RA was about the same that in the control, suggesting that the bond occurs on proteins in pre-existing cells. Retinoylation was not inhibited by high concentrations of palmitic or myristic acids (500 M); on the contrary, there was an increase of the binding activity of about 60 and 50% respectively.This paper is dedicated to the memory of Prof. J. A. Olson.     

Retinoylation of the type II cAMP-binding regulatory subunit of cAMP-dependent protein kinase is increased in psoriatic human fibroblasts

Previously, we have reported a defect in the cAMP-dependent protein kinases (cAMP-PK) in psoriatic cells (i.e., a decrease in 8-azido-[³²P]cAMP binding to the regulatory subunits and a decrease in phosphotransferase activity) which is rapidly reversed with retinoic acid (RA) treatment of these cells. This led us to examine a possible direct interaction between retinoids and the RI and RII regulatory subunits through retinoylation. Retinoylation of RI and RII present in normal and psoriatic human fibroblasts was analysed by [³H]RA treatment of these cells, followed either by chromatographic separation of the regulatory subunits or by their specific immunoprecipitation. These studies indicated that RI and RII can be retinoylated. [³H]RA labeling of the RII subunit was significantly (P < 0.005) greater in psoriatic fibroblasts (nine subjects; mean 7.47 relative units ± 1.37 SEM) compared to normal fibroblasts (eight subjects; mean 2.46 relative units ± 0.49 SEM). [³H]RA labeling of and the increase in 8-azido-[³²P]-binding to the RI and RII subunit in psoriatic fibroblasts showed a similar time course. This suggests that the rapid effect of retinoic acid treatment to enhance 8-azido-[³²P]-cAMP binding to the RI and RII in psoriatic fibroblasts may be due, in part, to covalent modification of the regulatory subunits by retinoylation. © 1996 Wiley-Liss, Inc.     

Synergistic anti-proliferative effects of vitamin D derivatives and 9-cis retinoic acid in SH-SY5Y human neuroblastoma cells

This study examines the effect of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 9-cis retinoic acid and all-trans retinoic acid on proliferation of SH-SY5Y human neuroblastoma cells, after treatment for 7 days. Cell number did not change when the cells were incubated with 1, 10 or 100 nM 1,25(OH)₂D₃ or its derivatives, but significantly decreased in the presence of the two retinoids (0.001–10 μM final concentration). A synergistic inhibition was observed, when SH-SY5Y cells were treated combining 0.1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060, and 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM EB 1089. Acetylcholinesterase activity showed a significant increase, in comparison with controls, after treatment of the cells for 7 days with 0.1 or 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060 or 10 nM EB 1089. This increase was synergistic, combining 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or EB 1089. The levels of the c-myc encoded protein remarkably decreased after treatment of SH-SY5Y cells for 1, 3, 7 days with 0.1 and 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060 or 10 nM EB 1089. In particular, the association of 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM EB 1089 resulted in a synergistic c-myc inhibition, in comparison with that obtained in the presence of the retinoid alone. These findings may have therapeutic implications in human neuroblastoma.     

Arachidonylethanolamide (anandamide) binds with low affinity to dihydropyridine binding sites in brain membranes

The purpose of this study was to explore the hypothesis that the dihydropyridine (DHP) binding site of the L-type calcium channel is a high affinity binding site for the cannabimimetic arachidonylethanolamide (AEA). Binding affinities were determined from competition isotherms using the DHP analog [³H]PN-200. AEA competed for [³H]PN-200 binding with a K₁ of 40 ± 4 μM. Inclusion of phenylmethylsulfonyl fluoride to inhibit an amidohydrolase that converts AEA to arachidonic acid had little effect on the K₁ of AEA (48 ± 6 μM). Arachidonic acid had a slightly higher K₁ (120 ± 11 μM) and other N-acylethanolamides examined (linolenylethanolamide, dihomo-γ-linolenylethanolamide, docosatetraenylethanolamide, and palmitoylethanolamide) had no effect on [³H]PN-200 binding at concentrations as high as 10 μM. Our conclusions are that AEA binds to the DHP binding site with relatively low affinity and its conversion to arachidonic acid is not required for binding.     

l-serine and GABA uptake by synaptosomes during postnatal development of rat

Postnatal development changes in mechanisms of synaptosomal amino acid transport have been studied in rat cerebral cortex. Specific uptake of radiolabeled l-serine was examined and compared with that of radiolabeled GABA using synaptosomes-enriched fractions freshly prepared from cerebral cortex at different postnatal days from the birth to young adulthood. The preparations were incubated with 10 nM of [³H]l-serine and 10 nM of [³H]-GABA in either the presence or absence of NaCl, KCl or choline chloride, at 2 and 30 °C, for different periods up to 30 min. The uptake of [³H]l-serine was temperature dependent in synaptosomal fractions prepared from cerebral cortex of rats in postnatal days 5, 7, 13 and 21, but stronger dependence was observed in adult brain, irrespective of the presence of Na⁺, K⁺ or choline ions. At all postnatal ages studied, [³H]-GABA uptake showed a high activity in the presence of Na⁺ ions and at 30 °C. The values of K_m were 90–489 μM in l-serine uptake. However, in the uptake of GABA the values of K_m were 80–150 μM. The highest values of V_max were obtained at 5 and 21 postnatal days for both transport systems. These results indicate that the uptake of l-serine and GABA are regulated differentially during postnatal development.     

Geranylgeraniol Promotes Entry of UT-2 Cells into the Cell Cycle in the Absence of Mevalonate

Although UT-2 cells, a mutant clone of Chinese hamster ovary cells, have been shown to require mevalonate for growth due to a deficiency in 3-hydroxy-3-methylglutaryl-CoA reductase, the precise mevalonate-derived product(s) essential for proliferation has not been identified. These studies show that UT-2 cells proliferate in the presence of free geranylgeraniol (GG-OH), as well as mevalonate. Cell growth was optimal when the culture medium was supplemented with 5–10 μMGG-OH. Under these growth conditions [³H]GG-OH is actively incorporated into UT-2 proteins. Prominent [³H]geranylgeranylated polypeptides in the size range (19–27 kDa) of the small GTP-binding proteins are observed by SDS–PAGE. Analysis of the butanol-soluble products released from the metabolically labeled proteins by digestion with Pronase E reveals that the proteins contain [³H]geranylgeranylated cysteine residues. Even though [³H]farnesol is also incorporated into cysteinyl residues of a different set of UT-2 proteins, farnesol added at 10 μMdid not satisfy the mevalonate requirement for cell growth. These results show that UT-2 cells divide in the presence of exogenously supplied GG-OH, providing evidence that one or more geranylgeranylated proteins are essential for entry of UT-2 cells, and probably other mammalian cells, into the cell cycle.     

DuP 753, the selective angiotensin II receptor blocker, is a competitive antagonist to human platelet thromboxane A2/prostaglandin H2 (TP) receptors

DuP 753 is a potent, selective angiotensin II type 1 (AT1) receptor antagonist. The possibility was investigated that DuP 753 may crossreact with thromboxane A₂/prostaglandin H₂ (TP) receptors. DuP 753 inhibited the specific binding of the TP receptor antagonist [³H]SQ 29,548 (5 nM) in human platelets with k_d/slope factor values of 9.6±1.4 μM/1.1±0.02. The AT2-selective angiotensin receptor ligand, PD 123,177 was a very weak inhibitor of specific [³H]SQ 29,548 binding in platelets (K_d/slope factor:200 μM/0.86). [³H]SQ 29,548 saturation binding in the absence and presence of DuP 753 resulted in an increase in equilibrium affinity constant (K_d: 9.3, 22, 33 nM, respectively) without a concentration-dependent reduction in binding site maxima (B_max: 3597, 4597, 3109 fmol/mg protein, respectively). Platelet aggregation induced by the TP receptor agonist U 46,619 was concentration-dependently inhibited by DuP 753 (IC₅₀=46 μM). These data indicate for the first time that DuP 753 is a weak but competitive antagonist at human platelet TP receptors.     

Identification of α2 adrenergic receptors in human frontal cortex membranes by binding of [H]RX 821002, the 2-methoxy analog of [H]idazoxan

The antagonist [³H]idazoxan binds with comparable affinity to α₂ adrenergic receptors and to phentolamine-displaceable non-stereoselective sites in human frontal cortex membranes. In contrast, idazoxan analogs possessing alkyl and alkoxy substituents at the 2-position of the benzodioxan moiety (i.e. RX 821002: 2-methoxy-1,4-[6,7-³H]benzodioxan-2-yl-2-imidazolin HCl, 43.8 Ci/mmol) possess 300–1200 times lower affinity for the non-stereoselective sites. Their affinity for the α₂ receptors is increased as well, resulting in more than a 1000-fold selectivity towards the receptors as compared to the non-stereoselective sites. [³H]RX 821002, the 2-methoxy analog of idazoxan possesses an approx. 10-fold higher affinity for the α₂ receptors (K_D = 2.8 nM than [³H]idazoxan (K_D = 24 nM) and about equal affinity as [³H]rauwolscine (K_D = 3.6 nM).[³H]Rauwolscine binds with comparable affinity to α₂ receptors and to 5-HT_1A receptors, and competition studies indicate that the K_i value of unlabelled RX 821002 for the 5-HT_1A receptors (30 nM) is about one order in magnitude above its K_i value for the α₂ receptors (4.1 nM). Labelling of the 5-HT_1A receptors by [³H]RX 821002 and by [³H]rauwolscine can be prevented by selective masking with 8-OH-DPAT (30 nM) or 5-HT (0.3 μM). Under these conditions, specific binding of [³H]RX 821002 to the α₂ receptors represents 84% of total binding (at its K_D), as compared to 77% for [³H]rauwolscine and 20% for [³H]idazoxan.[³H]RX 821002 labels the α₂ receptors as a single class of non-cooperative sites. Association and dissociation kinetics are very fast at 37°C. Antagonist competition curves are steep with Hill coefficients close to one and the agonist curves can be analysed in terms of two affinity sites, confirming the antagonistic properties of [³H]RX821002. About 60% of the α₂ receptors possess high agonist affinity.     

Enhancement of glycosylation of cellular glycoconjugates in the squamous carcinoma cell line MDA886Ln by β-all-trans retinoic acid

Retinoids have been shown to inhibit the growth and modulate the glycosylation of head and neck squamous cell carcinoma (HNSCC) cells including the MDA886Ln cells. To examine the effects of -all-trans retinoic acid (RA) on glycoconjugates in HNSCC MDA886Ln cells, the cells were grown in the absence or presence of 1 µM RA and then labeled with tritiated monosaccharides, extracted and analysed by polyacrylamide gel electrophoresis and fluorography. RA increased markedly the incorporation of [³H]-glucosamine, [³H]-galactose, and [³H]-mannose into numerous cellular glycoconjugates, however, the incorporation of [³H]-fucose and [³H]-leucine was almost unaffected by RA. RA increased the incorporation of glucosamine and galactose but not mannose into high molecular weight (HMW) glycoconjugates of about 220 and 500–600 kDa. To analyse the steady state level of glycoconjugates by lectin blotting, extracts of unlabeled cells were separated by gel electrophoresis and the gels were probed with¹²⁵I-labeled wheat germ agglutinin (WGA) andMaackia amurensis (MA) agglutinin. Both lectins were found to bind to numerous glycoconjugates including the HMW glycoconjugates, whereas¹²⁵I-peanut agglutinin bound only to the HMW glycoconjugates. RA treatment increased the binding of all three lectins to the HMW glycoconjugates. These findings demonstrate that RA enhanced the incorporation of specific monosaccharides into a variety of glycoconjugates and in particular into HMW mucin-like glycoconjugates. This effect of RA may be the result of induction of a more normal differentiation state of the HNSCC cells.     

Identification and characterization of pipecolic acid binding sites in mouse brain

Pipecolic acid (PA, piperidine-2-carboxylic acid) is the major product of lysine metabolism in the mammalian brain (Giacobini et al., 1980). In this study we have characterized the binding of [³H]PA to P₂ fraction membranes and its distribution in the mouse brain. The binding was found to be saturable (70 nM), temperature and Na⁺ and Cl^– dependent. A high affinity binding site with an apparentK _D of 33.2 nM and aB _max of 0.2 pmol/mg protein was demonstrated. The regional distribution of [³H]PA specific binding in mouse brain showed the highest concentration in cerebral cortex, thalamus and olfactory bulb. Unlabeled PA (10^–3–10^–11M) displaced specific binding of [³H]PA in a concentration dependent manner. Out of several substances tested, only proline showed a similar pattern of displacement. Pre-incubation of the membrane preparation with GABA (10^–3–10^–11M) resulted in either an increase or decrease of [³H]PA binding depending on the concentrations of GABA and PA. These results suggest a modulatory action of GABA on PA binding sites. The postnatal development of [³H]PA specific binding was studied in the whole brain of the mouse. [³H]Pipecolic acid binding increased progressively (8-fold) from one day after birth to 16 days. Following this developmental peak, the binding decreased gradually to 30 days at which age, adult values were attained.     

PGJ2 and ΔPGJ2 inhibit cell growth accompanied with inhibition of phosphoinositide turnover in human astrocytoma cells

PGJ₂ and Δ¹²PGJ₂ (1 μM to 30 μm) inhibited the growth of human astrocytoma cells (1321N1) in a time-dependent manner within 48 hrs, determined by [³H]thymidine incorporation into acid-insoluble fraction or amounts of protein. The EC₅₀ values for PGJ₂ and Δ¹²PGJ₂ were approximately 8 μM and 6 μM, respectively. [³H]Thymidine incorporation to acid insoluble fraction was inhibited by these PGs within 1 hr, indicating that these PGs rapidly affect cell functions. Although it has been reported that an increase in cyclic AMP inhibits cell growth, PGJ₂ and Δ¹²PGJ₂, but not PGE₁, reduced isoproterenol (10 μM)-induced accumulation of cyclic AMP, suggesting that PGJ₂ and Δ¹²PGJ₂ may disturb adenylate cyclase system, which might be independent on cell growth. On the other hand, these PGs inhibited the incorporation of [³H]inositol into phospholipid fraction within 6 hrs. Furthermore, PGJ₂ and Δ¹²PGJ₂ inhibited carbachol- and/or histamine-induced accumulation of inositol phosphates with a similar dose-dependency to their inhibitions of cell growth. In membrane preparations, however, PGJ₂ and Δ¹²PGJ₂ failed to inhibit GTPγS (10 μM)- nor Ca²⁺ (1mM)-induced accumulation of inositol phosphate. The site of PGJ₂ or Δ¹²PGJ₂ in inhibition of inositol phosphate accumulation would not be phospholipase C nor a putative GTP binding protein involved in activation of phospholipase C. The present results indicate that PGJ₂ and Δ¹²PGJ₂ inhibit cell growth in human astrocytoma cells and the inhibition of phosphoinositide turnover by these PGs might be involved in the inhibition of cell growth.     

The effect of all-trans-retinoic acid on the synthesis of epidermal cell-surface-associated carbohydrates

1. all-trans-Retinoic acid at concentrations greater than 10⁻⁷m stimulated the incorporation of d-[³H]glucosamine into 8m-urea/5% (w/v) sodium dodecyl sulphate extracts of 1m-CaCl₂-separated epidermis from pig ear skin slices cultured for 18h. The incorporation of ³⁵SO₄²⁻, l-[¹⁴C]fucose and U-¹⁴C-labelled l-amino acids was not significantly affected. 2. Electrophoresis of the solubilized epidermis showed increased incorporation of d-[³H]glucosamine into a high-molecular-weight glycosaminoglycan-containing peak when skin slices were cultured in the presence of 10⁻⁵m-all-trans-retinoic acid. The labelling of other epidermal components with d-[³H]glucosamine, ³⁵SO₄²⁻, l-[¹⁴C]fucose and U-¹⁴C-labelled l-amino acids was not significantly affected by 10⁻⁵m-all-trans-retinoic acid. 3. Trypsinization dispersed the epidermal cells and released 75–85% of the total d-[³H]glucosamine-labelled material in the glycosaminoglycan peak. Thus most of this material was extracellular in both control and 10⁻⁵m-all-trans-retinoic acid-treated epidermis. 4. Increased labelling of extracellular epidermal glycosaminoglycans was also observed when human skin slices were treated with all-trans-retinoic acid, indicating a similar mechanism in both tissues. Increased labelling was also found when the epidermis was cultured in the absence of the dermis, suggesting a direct effect of all-trans-retinoic acid on the epidermis. 5. Increased incorporation of d-[³H]-glucosamine into extracellular epidermal glycosaminoglycans in 10⁻⁵m-all-trans-retinoic acid-treated skin slices was apparent after 4–8h in culture and continued up to 48h. all-trans-Retinoic acid (10⁻⁵m) did not affect the rate of degradation of this material in cultures `chased' with 5mm-unlabelled glucosamine after 4 or 18h. 6. Cellulose acetate electrophoresis at pH7.2 revealed that hyaluronic acid was the major labelled glycosaminoglycan (80–90%) in both control and 10⁻⁵m-all-trans-retinoic acid-treated epidermis. 7. The labelling of epidermal plasma membranes isolated from d-[³H]glucosamine-labelled skin slices by sucrose density gradient centrifugation was similar in control and 10⁻⁵m-all-trans-retinoic acid-treated tissue. 8. The results indicate that increased synthesis of mainly extracellular glycosaminoglycans (largely hyaluronic acid) may be the first response of the epidermis to excess all-trans-retinoic acid.     

Antimalarial activity of anthothecol derived from Khaya anthotheca (Meliaceae)

Antimalarial activity of anthothecol, a limonoid of Khaya anthotheca (Meliaceae) against Plasmodium falciparum was tested using a [³H]-hypoxanthine and 48 h culture assay in vitro. Anthotechol showed potent antimalarial activity against malaria parasites with IC₅₀ values of 1.4 and 0.17 μM using two different assays. Also, gedunin had antimalarial activity with IC₅₀ values of 3.1 and 0.14 μM. However, the citrus limonoids, limonin and obacunone did not show any antimalarial activity. The antimalarial activities were compared with the three currently used antimalarial medicines quinine, chloroquinine and artemisinin.     

Mechanism and Mechanism-Based Inactivation of 4-Hydroxyphenylpyruvate Dioxygenase

Six substrate analogs of 4-hydroxyphenylpyruvate, specifically pentafluorophenylpyruvate, 4-hydroxytetrafluorophenylpyruvate,2-thienylpyruvate, 3-thienylpyruvate, thiophenol oxalate, and p-thiocresoloxalate were synthesized and their interactions with porcine liver 4-hydroxyphenylpyruvate dioxygenase investigated. Both pentafluorophenylpyruvate and thiophenol oxalate are competitive inhibitors of the enzyme with K_I values of 14 and 150 μM, respectively, but p-thiocresol oxalate has no effect on the enzymic activity. The other three substrate analogs are both substrates and mechanism-based inactivators of the enzyme with the following kinetic characteristics (compound, K_m, V_max, k_inact, K′, partition ratio) at pH 6.0, 37°C, and an air atmosphere: 4-hydroxytetrafluorophenylpyruvate, 50 μM, 1.9 mkat/kg, 1.5/min, 70 μM 4.2; 2-thienylpyruvate, 500 μM, 7.8 mkat/kg, 0.6/min, 400 μM, 41; 3-thienylpymvate, 250 μM, 2 9 mkat/kg, 0.6/min, 300 μM, 22. When inactivated, the dioxygenase was found to contain per mole of active enzyme, 0.78 mol of label from 3-thienyl-3[³H]pyruvate and 0.85 mol of label from 4-hydroxytetrafluorophenyl-3 [³H]pyruvate. The product formed from the enzyme-catalyzed oxidation of 3-thienylpyruvate was determined to be 3-carboxymethyl-3-thiolene-2-one. The implication of these results to the mechanism of the dioxygenase is considered,     

Intracellular acetylation of desacetyl αMSH in the xenopus laevis neurointermediate lobe

High performance liquid chromatography (HPLC) followed by radioimmunoassay (RIA) of the chromatographic fractions were used to separate and quantify, respectively, the αMSH-like peptides stored in the neurointermediate lobe (NIL) of the Xenopus laevis (X. laevis) pituitary gland and released from the X. laevis NIL, in vitro. Immunoreactive (IR) material eluting with a similar HPLC retention time as desacetyl αMSH was the major IR peptide in the NIL. Material with a retention time similar to αMSH and immunological properties equivalent to αMSH was also present in the NIL. However, the retention times of the X. laevis and mammalian αMSH-like peptides were not identical, suggesting species difference in these peptides. Following incubation of NILs in the presence of [³H]-acetyl CoA, the X. laevis variant of αMSH was the major [³H]-labeled, immunoprecipitable material present. Following an incubation of NILs in the presence of [³H]-amino acids for 21 hours, immunoprecipitable [³H]-αMSH was detected in the NILs and the ratio of [³H]-desacetyl αMSH to [³H]-αMSH was similar to the ratio of IR-desacetyl αMSH to IR-αMSH. The X. laevis variant of αMSH was the major αMSH-like peptide released from the NILs into the incubation medium. Dopamine (50 μM) significantly inhibited the release of IR-αMSH but not IR-desacetyl αMSH. No net increase in total αMSH (sum of release and NIL content) was observed in the actively secreting (control) NIL group versus the dopaminetreated group. These results indicate that acetylation of desacetyl αMSH occurs intracellularly.     

Dictyostelium discoideum chemotaxis: Threshold for directed motion

The chemotactic response of Dictyostelium discoideum cells to stationary, linear gradients of cyclic adenosine 3′,5′-monophosphate (cAMP) was studied using microfluidic devices. In shallow gradients of less than 10⁻³ nM/μm, the cells showed no directional response and exhibited a constant basal motility. In steeper gradients, cells moved up the gradient on average. The chemotactic speed and the motility increased with increasing steepness up to a plateau at around 10⁻¹ nM/μm. In very steep gradients, above 10 nM/μm, the cells lost directionality and the motility returned to the sub-threshold level. In the regime of optimal response the difference in receptor occupancy at the front and back of the cell is estimated to be only about 100 molecules.     

Inhibitory effect of antidepressants on the NMDA-evoked [H]noradrenaline release from rat hippocampal slices

We have previously shown that monoamine uptake blocker-type antidepressants with different chemical structure and selectivity are able to inhibit neuronal nicotinic acetylcholine receptors (nAChRs) in concentrations observed during antidepressant treatment. The mechanism of action of these drugs is similar to that of mecamylamine, a channel blocker-type antagonist of nAChRs. Since mecamylamine has been shown to block also NMDA receptors, our aim was to investigate whether the monoamine uptake blockers may affect the function of these ionotropic glutamate receptors.We studied, therefore the effect of the two most potent nicotinic antagonist antidepressants, the tricyclic desipramine and the selective serotonin reuptake inhibitor fluoxetine on the NMDA-induced [³H]noradrenaline ([³H]NA) release from rat hippocampal slices. The NMDA-induced hippocampal [³H]NA release was effectively blocked by the selective, non-competitive NMDA antagonist MK-801 (IC₅₀ = 0.54 μM), indicating that the [³H]NA release was mediated through NMDA receptors. This response was also dose-dependently inhibited by desipramine (IC₅₀ = 14.57 μM) and fluoxetine (IC₅₀ = 41.06 μM). The Na⁺-channel blocker TTX equally inhibited both the electrical stimulation- and the NMDA-evoked [³H]NA release (the IC₅₀ was 55 nM and 66 nM, respectively), whereas the antidepressants inhibited only the NMDA-evoked response. These data suggest that the inhibitory effect of fluoxetine and desipramine on the NMDA-evoked [³H]NA release is exerted directly on NMDA receptors rather than indirectly on Na⁺-channels.Due to accumulation processes the concentration of desipramine and fluoxetine in the brain might be in the same range as the observed IC₅₀ values, thus our data indicate that monoamine uptake blocker-type antidepressants are able to influence the function of NMDA receptors during antidepressant treatment, and the inhibitory effect on NMDA receptors might contribute to the therapeutic effects of these drugs.     

Allopregnanolone increase in striatal N-methyl-D-aspartic acid evoked [3H]dopamine release is estrogen and progesterone dependent

1. The neurosteroids are compounds derived from steroid hormones and synthesized in the nervous system. They can modulate different neurotransmitter pathways. In previous work we demonstrated that progesterone modulates dopamine release induced by the glutamatergic agonist N-methyl-D-aspartic acid (NMDA).2. The aim of this work was to evaluate a possible modulatory role of the progesterone metabolite allopregnanolone on NMDA-evoked [³H]dopamine release from corpus striatum slices obtained from cycling and ovariectomized female rats.3. We used a dynamic superfusion method to evaluate the release of [³H]dopamine. Allopregnanolone at 50–600 nM was added to the superfusion buffer (Krebs–Ringer–bicarbonate–glucose, pH 7.4, with constant O₂/CO₂ gassing). The results are expressed as a percentage over basal [³H]dopamine loaded by the tissue.4. Allopregnanolone (50 and 100 nM) increased the NMDA-evoked[³H]dopamine release from estrus rats. The remaining doses did not show significant changes in the pattern of release. This effect was not observed in diestrus rats. The ovariectomy abolished the facilitatory effect of allopregnanolone on NMDA-evoked 2 [³H]dopamine release.5. Subcutaneous administration of exogenous estrogen (25 mg/rat) and progesterone (1 mg/rat) restored the facilitatory effect on dopaminergic input.6. These results suggest that allopregnanolone is a neurosteroid able to modulate dopamine release in an ovarian-hormone-fluctuation-dependent manner and provide further support for a role of allopregnanolone as a modulator of glutamatergic–dopaminergic interaction in the corpus striatum.     

Bottom-up controls on a mixed-species HAB assemblage: A comparison of sympatric Chattonella subsalsa and Heterosigma akashiwo (Raphidophyceae) isolates from the Delaware Inland Bays, USA

Recent novel mixed blooms of several species of toxic raphidophytes have caused fish kills and raised health concerns in the highly eutrophic Inland Bays of Delaware, USA. The factors that control their growth and dominance are not clear, including how these multi-species HAB events can persist without competitive exclusion occurring. We compared and contrasted the relative environmental niches of sympatric Chattonella subsalsa and Heterosigma akashiwo isolates from the bays using classic Monod-type experiments. C. subsalsa grew over a temperature range from 10 to 30 °C and a salinity range of 5–30 psu, with optimal growth occurring from 20 to 30 °C and 15 to 25 psu. H. akashiwo had similar upper temperature and salinity tolerances but also lower limits, with growth occurring from 4 to 30 °C and 5 to 30 psu and optimal growth between 16 and 30 °C and 10 and 30 psu. These culture results were confirmed by field observations of bloom occurrences in the Inland Bays. Maximum nutrient-saturated growth rates (μ_max) for C. subsalsa were 0.6 d⁻¹ and half-saturation concentrations for growth (K_s) were 9 μM for nitrate, 1.5 μM for ammonium, and 0.8 μM for phosphate. μ_max of H. akashiwo (0.7 d⁻¹) was slightly higher than C. subsalsa, but K_s values were nearly an order of magnitude lower at 0.3 μM for nitrate, 0.3 μM for ammonium, and 0.2 μM for phosphate. H. akashiwo is able to grow on urea but C. subsalsa cannot, while both can use glutamic acid. Cell yield experiments at environmentally relevant levels suggested an apparent preference by C. subsalsa for ammonium as a nitrogen source, while H. akashiwo produced more biomass on nitrate. Light intensity affected both species similarly, with the same growth responses for each over a range from 100 to 600 μmol photons m⁻² s⁻¹. Factors not examined here may allow C. subsalsa to persist during multi-species blooms in the bays, despite being competitively inferior to H. akashiwo under most conditions of nutrient availability, temperature, and salinity.     

Nitrogenous preference of toxigenic Pseudo-nitzschia australis (Bacillariophyceae) from field and laboratory experiments

Field and laboratory experiments were designed to determine the differential growth and toxin response to inorganic and organic nitrogen additions in Pseudo-nitzschia spp. Nitrogen enrichments of 50 μM nitrate (KNO₃), 10 μM ammonium (NH₄Cl), 20 μM urea and a control (no addition) were carried out in separate carboys with seawater collected from the mouth of the San Francisco Bay (Bolinas Bay), an area characterized by high concentrations of macronutrients and iron. All treatments showed significant increases in biomass, with chlorophyll a peaking on days 4–5 for all treatments except urea, which maintained exponential growth through the termination of the experiment. Pseudo-nitzschia australis Frenguelli abundance was 10³ cells l⁻¹ at the start of the experiment and increased by an order of magnitude by day 2. Particulate domoic acid (pDA) was initially low but detectable (0.15 μg l⁻¹), and increased throughout exponential and stationary phases across all treatments. At the termination of the experiment, the urea treatment produced more than double the amount of pDA (9.39 μg l⁻¹) than that produced by the nitrate treatment (4.26 μg l⁻¹) and triple that of the control and ammonium treatments (1.36 μg l⁻¹ and 2.64 μg l⁻¹, respectively). The mean specific growth rates, calculated from increases in chlorophyll a and from cellular abundance of P. australis, were statistically similar across all treatments.These field results confirmed laboratory experiments conducted with a P. australis strain isolated from Monterey Bay, CA (isolate AU221-a) grown in artificial seawater enriched with 50 μM nitrate, 50 μM ammonium or 25 μM of urea as the sole nitrogen source. The exponential growth rate of P. australis was significantly slower for cells grown on urea (ca. 0.5 day⁻¹) compared to the cells grown on either nitrate or ammonium (ca. 0.9 day⁻¹). However the urea-grown cells produced more particulate and dissolved domoic acid (DA) than the ammonium- or nitrate-grown cells. The field and laboratory experiments demonstrate that P. australis is able to grow effectively on urea as the primary source of nitrogen and produced more pDA when grown on urea in both natural assemblages and unialgal cultures. These results suggest that the influence of urea from coastal runoff may prove to be more important in the development or maintenance of toxic blooms than previously thought, and that the source of nitrogen may be a determining factor in the relative toxicity of west coast blooms of P. australis.