EFFECT OF Ni2+ ON THE TESTOSTERONE PRODUCTION OF MOUSE PRIMARY LEYDIG CELL CULTURE

This study evaluated the effects of Ni2+ on testosterone (T) production of mouse Leydig cells in vitro following an in vivo or in vitro exposure. CFLP mice were subjected to repeated exposure (4 treatments, subcutaneously, every 3 d) to 10, 20 or 40 mg/ kg body weight of NiSO4 or 1.0 ml of 0.9% NaCl solution. Depressed human chorionic gonadotropin (hCG)-stimulated T response was seen over a 48-h culture of testicular interstitial cells obtained from the animals exposed to 20 mg/ kg or higher dose of NiSO4, while the basal T production remained unaltered. There were no Ni2+-related changes in the body weights or in the weights of testes, epididymides, adrenals, and kidneys. No histopathological alteration was found in the examined organs of NiSO4 treated groups except the dose-dependent tubular lesions in kidney as a result of a specific rather than a general cytotoxic action. To assess the direct effect of Ni2+ on Leydigcell T production, testicular interstitial cells were cultured with Ni2+ (62.5 to 1000 mu M) for 48 h in the presence or absence of maximally stimulating concentration of hCG. Dose-dependent depression in hCG-stimulated T production was seen at 125 mu M or higher dose of Ni2+, while basal T production was unaffected. In order to evaluate the time dependency of this effect the cells were cultured for various times in the presence or absence of 250 and 1000 mu M Ni2+. Decreased hCG-stimulated T production was found in the cultures maintained at least for 4 h in the presence of 1000 mu M Ni2+, whereas at 250 mu M at least 16 h was required to elicit the depression. Cell viability was assessed by a metabolic activity (MTT) assay. The viability of cells was unaltered by 250 mu M Ni2+, and only a slight decrease was found even at the end of the 48-h culture period in the presence of 1000 mu M Ni2+. Our results show a dose-related depression in stimulated T production of mouse Leydig cells in culture following either in vivo or in vitro Ni2+ treatment at a dose that does not induce any general toxic or significant cytotoxic action. The data of the time-course study indicate that the effect of Ni2+ on Leydigcell T production is both time and concentration dependent, and not due to cytotoxicity.     

Effect of cadmium and other metal cations on in vitro Leydig cell testosterone production.

In vivo assessment of toxicant action on Leydig cell function is subject to homeostatic mechanisms which make it difficult to determine whether any changes seen in serum testosterone (T) concentration are due to extragonadal endocrine alterations or to a direct effect on the Leydig cell. For example, metal cations administered in vivo have been shown to depress serum T concentration and alter serum concentrations of pituitary hormones in laboratory animals. The studies reported here use a testicular cell culture technique to evaluate Leydig cell testosterone biosynthesis in the presence of several metal cations. To determine the site of toxic action, the Leydig cells were stimulated to produce testosterone by using human chorionic gonadotrophin (hCG), dibutyl cyclic adenosine monophosphate (db-cAMP), or several substrates required for the biosynthesis of testosterone. hCG was chosen because resultant T production requires an intact membrane receptor and db-cAMP was used to test for post LH receptor defects caused by the metals. The other substrates were chosen to isolate the effect of metals on enzymatic pathways. Collagenase dispersed testicular cells (15% Leydig cells) were incubated with metal cations (1 to 5000 microM) for 3 hr in the absence and presence of maximally stimulating concentrations of hCG, db-cAMP, 20 alpha-hydroxycholesterol (HCHOL), or pregnenolone (PREG), and T concentration was determined by radioimmunoassay. In one separate experiment we also tested the effect of the substrates progesterone, 17 alpha-hydroxy-progesterone, and androstenedione on Cd2(+)-treated Leydig cells. The results show no change in Leydig cell viability with any metal cation treatment during the 3-hr incubation. Ca2+, Cr3+, Fe3+, Mg2+, Na+, or Pb2+ had no effect on stimulated testosterone. Dose-response depression in both hCG- and db-cAMP-stimulated T production were seen with Cd2+, Co2+, Cu2+, Hg2+, Ni2+, and Zn2+ treatment. Surprisingly, Cd2+, Co2+, Ni2+, and Zn2+, which caused a depression in hCG- and db-cAMP-stimulated T production, caused significant increases in HCHOL- and PREG-stimulated T production over untreated and similarly stimulated cultures. This indicates that these cations may act at multiple sites within the Leydig cell.     

Antagonism by essential divalent metals and amino acids of nickel(II)-DNA binding in vitro

In vitro binding of nickel(II) to DNA and the effects of divalent essential metals calcium, magnesium, manganese, copper, and zinc, and of amino acids histidine, cysteine, glutamine, arginine, lysine, alanine, and glycine upon that binding were investigated. Samples of 0.156 mg of calf thymus DNA (0.078 mg/ml in 5 mM ammonium acetate buffer, pH = 7.4) were incubated for 1 hr at 24 degrees C with various concentrations of nickel(II)acetate labeled with 63Ni (0.1 to 250 microM) in the absence or presence of 50 microM concentrations of the essential metal acetates, or with 100 microM concentrations of the amino acids. Free and DNA-bound nickel(II) fractions were separated by gel filtration on Sephadex G-25 and quantified by liquid scintillation counting. Scatchard analysis revealed more than two types of nickel(II)-binding sites and a positive cooperativity of binding at the bound-Ni concentrations below 0.35 microM. The high-affinity nickel(II)-binding sites at DNA were identified as phosphate groups. Their binding capacity equalled 0.043 mumol/mg DNA (approx. 1 mol Ni/70 mol of DNA bases). The apparent dissociation constant of nickel(II) from the high-affinity sites was 5.35 microM. Double reciprocal plots showed the essential divalent metals to be competitive antagonists of nickel(II)-binding to the high-affinity sites, ranking Mg(II) greater than or equal to Mn(II) greater than or equal to Ca(II) greater than or equal to Cu(II) = Zn(II). Similarly, the amino acids antagonized nickel binding to DNA with a relative strength of His greater than Gln greater than or equal to His/Cys greater than Arg greater than Cys greater than or equal to Gly = Ala greater than or equal to Lys. The strongest inhibitors of nickel(II)-DNA binding in vitro appear to be magnesium and manganese, i.e., the same metals that are capable of attenuating nickel carcinogenicity in vivo.     

Biphasic effect of cadmium in non-cytotoxic conditions on the secretion of nitric oxide from peritoneal macrophages

The effects of cadmium (Cd) in non-cytotoxic conditions on the nitric oxide (NO) production in peritoneal macrophages (pM) were studied. Peritoneal macrophages from Balb/c mice were incubated over 18 h with 5, 10, 20, or 25 microM Cd2+ (as CdCl2 21:2 H2O) in the culture medium. Concentrations of 20 microM Cd2+ and over had cytotoxic effects, measured by MTT assay. Cell viability with 10 microM Cd2+ in the medium was above 90% after 18 h of incubation, and above 80% after 72 h. At this same Cd2+ concentration, NO production increased from 6 to 18 h. At 24 h production decreased but was still above control levels. At 48 h production NO was near control levels, and continued to decrease until the end of the experiment (72 h). NO levels produced with Cd2+ concentrations of 5, 10 and 20 microM in the medium were above the control at 18 h. NO production and lipoperoxidation increased simultaneously after 18 h with 10 microM of Cd in the medium. Amounts of inducible nitric oxide synthase (iNOS) protein and iNOS activity also increased. At a concentration of 10 microM Cd has a biphasic effect on NO production over time.     

Tolerance to high Zn in the metallophyte Erica andevalensis Cabezudo & Rivera

The tolerance to high Zn was studied in the metallophyte Erica andevalensis Cabezudo & Rivera grown in nutrient solutions at different Zn concentrations (5, 500, 1,000, 1,500 and 2,000?μM Zn). Plant growth and nutrient uptake were determined. Metabolic changes were assessed by the analysis of peroxidase activity, organic metabolites related to metal chelation (amino acids, organic acids (malate, citrate) or protection (polyamines). While plants tolerated up to 1,500?μM Zn, despite presenting of low growth rates, the concentration of 2,000?μM Zn was toxic producing high mortality rates. Roots accumulated high Zn concentration (11,971?mg/kg) at 1,500?μM external Zn) apparently avoiding metal transfer into shoots. After 30?days of treatment with high Zn (1,000 and 1,500?μM Zn), the leaves accumulated high levels of glutamine. Short-term treatment with 500?μM Zn, significantly increased the concentration of asparagine and glutamine in roots. Citrate concentration was also considerably increased when exposing roots to Zn excess. Metal immobilization in the root system, low interference with the uptake of nutrients and an increased production of putative organic ligands (amino acids, citrate) might have provided the Zn tolerance displayed by Erica andevalensis.     

Differential effects of octylphenol, 17beta-estradiol, endosulfan, or bisphenol A on the steroidogenic competence of cultured adult rat Leydig cells.

In the current studies, we evaluated the effects of 4-tert-octylphenol (OP), endosulfan, bisphenol A (BPA), and 17beta-estradiol on basal or hCG-stimulated testosterone formation by cultured Leydig cells from young adult male rats. Exposure of Leydig cells to increasing concentrations of OP (1 to 2000 nM), 17beta-estradiol (1 to 1000 nM), endosulfan (1 to 1000 nM) or BPA (1 to 1000 nM), alone or with 10 mIU/mL hCG for 4 or 24 h, did not lower ambient testosterone levels, although cells exposed to higher OP concentrations + hCG for 24 h often had modest declines in testosterone (10 to 20%). Of interest, exposure to the highest concentration OP (2000 nM) alone for 4 or 24 h increased testosterone levels (approximately 2-fold in 4-h exposed cells). Whether prior exposure to OP + hCG for 24 h affects the subsequent conversion of steroid substrates to testosterone over 4 h was evaluated. Progressive declines in 1 microM 22(R) hydroxycholesterol, 1 microM pregnenolone, or 1 microM progesterone conversion to testosterone was observed beginning at 100 to 500 nM OP exposure (maximal declines of 40 to 12% of controls were observed); however, the conversion of 1 microM androstenedione to testosterone was not affected by OP. These results suggested that 24-h exposure to OP + hCG has no effect on 17beta-hydroxysteroid dehydrogenase, which converts androstenedione to testosterone, but that it inhibits the 17alpha-hydroxylase/C17-20 lyase step, which converts progesterone to androstenedione. In addition, potentially, OP could inhibit cholesterol side/chain cleavage activity, which converts cholesterol to pregnenolone, and/or 3beta-hydroxysteroid dehydrogenase, which converts pregnenolone to progesterone. Of interest, exposure to increasing concentrations of 17beta-estradiol (1 to 1000 nM), endosulfan (1 to 1000 nM), or BPA (1 to 1000 nM) + hCG for 24 h had no effect on subsequent conversion of 22(R)hydroxycholesterol to testosterone. Furthermore, the inhibiting effects of OP + hCG exposure on subsequent conversion of progesterone to testosterone was unaffected by concomitant exposure to the pure estrogen antagonist, ICI 182,780, or the antioxidants, ascorbate or dimethyl sulfoxide, suggesting that the actions of OP are not mediated through binding to estrogen receptor alpha or beta or by free radical induced damage to steroidogenic enzymes, respectively. These results demonstrate that direct exposure of adult Leydig cells to OP may have subtle effects on their ability to produce testosterone, which may not be detected by measuring ambient androgen levels. In addition, the effects of OP on Leydig cell testosterone formation appear to be different from those of the native estrogen, 17beta-estradiol, and from other reported weak xenoestrogens such as endosulfan and BPA.     

Enhancement of methylmercury toxicity by L-cystine in cultured mouse blastocysts

Expanded mouse blastocysts incubated with 1 to 2 microM methylmercury (MeHg) in modified Eagle's basal medium (BME + AA), which contains amino acids, collapsed and degenerated within 24 h. In contrast, blastocysts incubated with the same concentration of MeHg in egg culture medium (ECM), which does not contain amino acids, survived and remained expanded as control embryos did. By systematically omitting each BME amino acid from BME + AA and adding each BME amino acid to egg culture medium, we determined that L-cystine (0.5 mM in BME + AA) was the component of BME + AA that was responsible for the enhancement of the toxicity of MeHg. The shortest incubation time during which the cystine-enhanced MeHg toxicity became irreversible was 2 h, and the addition of any of the neutral BME amino acids (except threonine) or non-BME neutral amino acids (alanine, glycine, or serine) during the 2 h incubation eliminated or reduced the cystine-enhanced MeHg toxicity. Basic amino acids (except histidine) were less effective in protecting embryos: Glutamine and lysine reduced the toxic effect only slightly, and arginine had no effect. DL-buthionine sulfoximine (7.5 mM), a specific inhibitor of glutathione, also reduced cystine-enhanced MeHg toxicity. It therefore appears that cystine enhances MeHg toxicity indirectly, at least in part, by stimulating the synthesis of cellular glutathione, which may in turn enhance MeHg transport. In the absence of cystine, 10 microM MeHg (2 h incubation) was necessary to cause the collapse and degeneration of all blastocysts treated.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro/in vivo effects of ethane dimethanesulfonate on Leydig cells of adult rats.

Although ethane dimethanesulfonate (EDS) is well recognized as a Leydig cell toxicant, the dose responsiveness of Leydig cells to EDS, both in vitro and in vivo, is not well established. In addition, the cellular site of action of EDS during Leydig cell toxicity and the status of Leydig cell viability during the affected period remain controversial. We determined the in vitro EC50 (370 microM) and in vivo ED50 (60 mg/kg) for human chorionic gonadotropin (hCG)-stimulated testosterone (T) production using both highly purified (98%) and interstitial (14%) Leydig cell preparations, respectively. Leydig cells were recovered in approximately equal numbers following all in vivo and in vitro EDS exposures. The Leydig cells in these preparations were viable and steroidogenically active (3 beta-HSD positive) subsequent to all exposures, both before and after incubations to stimulate T biosynthesis. When hCG-stimulated T production was decreased 50% following in vivo or in vitro exposures, the morphological integrity of the Leydig cells appeared normal, with no discernible lesion at either the light or the electron microscope level. We used stimulants of various reactions in the pathway of T biosynthesis (20 alpha-hydroxycholesterol and pregnenolone) to determine the site of action impaired when T biosynthesis was decreased. Our results indicate that when Leydig cells are exposed to EDS either in vitro or in vivo, the biosynthesis of T is compromised between the cyclic adenosine monophosphate activation of protein kinase and the cholesterol side chain cleavage enzyme.     

Effects of divalent cations and La3+ on contractility and ecto-ATPase activity in the guinea-pig urinary bladder.

1. Several cations (Ba2+, Cd2+, Co2+, Cu2+, Mn2+, Ni2+, Zn2+ and La3+, all as chloride salts, 1-1000 microM) were tested in the guinea-pig urinary bladder for their ability to: (i) modify contractile responses to electrical field stimulation (EFS), ATP, alpha,beta-methylene ATP (alpha,beta-meATP), carbachol (CCh), and KCl; (ii) affect ecto-ATPase activity. 2. Ba2+ (10-1000 microM) concentration-dependently potentiated contractile responses evoked by EFS (4-16 Hz), ATP (100 microM), alpha,beta-meATP (1 microM), CCh (0.5 microM), and KCl (30 mM). Ni2+ at concentrations of 1-100 microM also potentiated contractility of the urinary bladder, but at concentrations tested its effect was not concentration-dependent. Cu2+ at a concentration of 10 microM and Cd2+ at a concentration of 1 microM potentiated responses to all stimuli, except KCl. Ni2+ at a concentration of 1000 microM and Cd2+ at a concentration of 100 microM inhibited contractions evoked by all stimuli, and at a concentration of 1000 microM Cd2+ abolished any contractions. Responses to ATP and alpha,beta-meATP were selectively inhibited by Cu2+, Zn2+ or La3+, each at a concentration of 1 mM. 3. Cu2+, Ni2+, Zn2+ and La3+ (100-1000 microM) concentration-dependently inhibited ecto-ATPase activity in the urinary bladder smooth muscle preparations, while Ba2+ and Mn2+ were without effect, and Cd2+ and Co2+ caused significant inhibition only at a concentration of 1000 microM. 4. There was no correlation between the extent of ecto-ATPase inhibition and the effect on contractile activity of any of the cations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Covalent modification of amino acid nucleophiles by the lipid peroxidation products 4-hydroxy-2-nonenal and 4-oxo-2-nonenal

Lipid peroxidation yields the aldehydes 4-hydroxynonenal (4HNE) and 4-oxononenal (4ONE). Protein adduction by 4HNE is thought to be involved in the pathogenesis of several diseases. Currently, the reactivity of 4ONE toward proteins is unknown. The purpose of this study was to identify amino acids that react with 4HNE and 4ONE, characterize the chemical structure of the adduct, and determine the preference for amino acid modification. Model peptides containing one or more nucleophilic residues (i.e., Arg, Cys, His, Met, and Lys) were reacted with 4HNE and 4ONE and analyzed using matrix-assisted laser desorption/ionization mass spectrometry. Post-source decay analysis was used to confirm peptide modification. The bimolecular rate constant for adduction of amino acids and peptides by 4HNE and 4ONE was measured. Results of this work indicate that Cys, His, and Lys are modified by 4HNE and 4ONE. In addition, Arg was adducted by 4ONE. The predominant adduct resulting from modification of peptides by 4HNE or 4ONE had a mass of 156 or 154 Da (respectively), indicating that adduction occurs via Michael addition. Reactivity of amino acids toward 4HNE and 4ONE was found to have the following order: Cys > His > Lys (> Arg for 4ONE). The presence of an Arg on a Cys-containing peptide increased the reaction rate with 4HNE and 4ONE by a factor of approximately 5-6 compared to the Cys nucleophile alone. Rate constants determined for the modification of Cys by the lipid aldehydes demonstrated a >100-fold difference in reactivity between 4HNE and 4ONE toward Cys. Results of the present study indicate that both 4HNE and 4ONE modify amino acid nucleophiles; however, the reactivity between these two lipid aldehydes differs both qualitatively and quantitatively.     

Specificity and formation of unusual amino acids of an amide ligation strategy for unprotected peptides

An important step in the recently developed ligation strategy known as domain ligation strategy to link unprotected peptide segments without activation is the ring formation between the C-terminal ester aldehyde and the N-terminal amino acid bearing a 8-thiol or 8-hydroxide. A new method was developed to define the specificity of this reaction using a dye-labeled alanyl ester aldehyde to react with libraries of 400 dipeptides which contained all dipeptide combinations of the 20 genetically coded amino acids. Three different ester aldehydes of the dye-labeled alanine: α-formylmethyl (FM), β-formylethyl (FE), and β,β,β-dimethyl and formylethyl esters (DFE), were examined. The DFE ester was overly hindered and reacted with N-terminal Cys dipeptides (Cys-X). Interestingly, it also reacted slowly with the sequences of X-Gly where Gly was the second amino acid and the X-Gly amide bond participated in the ring formation. Although the FE ester reacted similarly as the FM ester in the ring formation, the subsequent O,N-acyl transfer was at least 30-fold slower than those of the FM-ester. The FM α-formyl methyl ester was the most suitable ester and was reactive with dipeptides of six N-terminal amino acids: Cys, Thr, Trp, Ser, His and Asn. The order and extent of their reactivity were highly dependent on pH, solvent and neighboring participation by the adjacent amino acid. In general, they could be divided into three categories. (1) N-Terminal Cys and Thr were the most reactive. Cys reacted very rapidly and completely within 0.5 h to form thiazolidine in both aqueous and high content of water-miscible organic solvents. Thr reacted to form oxazolidine slowly in aqueous buffer (t₁/2 > 300 h) but rapidly and completely within 20 h in organic-water solvents. (2) N-Terminal Trp, His and Ser were comparatively much less reactive than Cys or Thr. Trp reacted slowly and completely in aqueous buffer but significantly more slowly and incompletely in water-organic solvents. Both His and Ser reacted very slowly and incompletely in both solvent systems. (3) Finally, Asn reacted nearly insignificantly in both solvent systems. The significant rate enhancement by the water-miscible organic solvent on Thr was particularly important to allow the synthesis of disulfide-rich protein domains. Furthermore, the ring formation with N-terminal Trp, His and Asn provided a convenient route to prepare their bicyclic and unusual heterocyclic derivatives for structure-activity study.     

Two essential amino acids, L-lysine and L-histidine, in five types of experimental seizures

L-Lysine (250-2,000 mg/kg) and L-histidine (1,000-2,000 mg/kg) significantly raised the electroconvulsive threshold. D-Histidine (1,000 mg/kg) was completely ineffective in this regard. Both amino acids were generally inactive in pentetrazole-, picrotoxin- and aminophylline-induced seizures, though L-histidine (2,500 mg/kg) significantly reduced the number of mice with clonic convulsions in the pentetrazole test. Also, L-lysine (2,500 and 3,000 mg/kg) significantly diminished mortality rate in aminophylline-induced seizures. In addition, L-lysine (2,500-3,000 mg/kg) and L-histidine (2,000-2,500 mg/kg) delayed the onset of aminophylline- and picrotoxin-evoked convulsions. L-Lysine and L-histidine (both up to 1,000 mg/kg) did not affect amygdala-kindled seizures in rats. The results indicate that some of indispensable amino acids may play a role in the inhibitory transmission in the central nervous system. A possibility arises that appropriate diet may be an important supportive factor in the treatment of some epileptic patients, probably suffering from generalized tonic-clonic seizures.     

Effects of bismuth citrate on the viability and function of Leydig cells and testicular macrophages

Bismuth is present in several popular over-the-counter drugs for nausea and diarrhea and is occasionally abused by patients with chronic gastrointestinal disorders. The most common consequence of bismuth overdose is neurological dysfunction. In experimental animals, bismuth overdose results in lowered serum testosterone levels, suggesting that reproductive dysfunction may be an additional component of bismuth toxicity. Although the precise mechanisms responsible for the lowered testosterone levels are unknown, it has been shown that bismuth accumulates within testicular macrophages. This may be important because these cells, which are commonly found in direct contact with Leydig cells, are known to exert paracrine influences on the Leydig cells for local control of testosterone production. However, bismuth may also exert direct effects on Leydig cells because it passes by these cells on its way to the phagocytic macrophages. The purpose of the present studies was to isolate both testicular macrophages and Leydig cells from rat testis and study the direct effects of bismuth on these cells with regard to their viability and function. We found that when Leydig cells were treated for 24 h with bismuth (1-100 microM) no change in viability or secretion of testosterone was observed. However, when testicular macrophages were similarly treated with bismuth a significant effect on viability was observed with as little as 6.25 microM bismuth, with near-complete cell death at 50 microM after 24 h. However, bismuth had no effect on the viability on testicular macrophages at 50 microM up to 8 h, therefore, we studied the secretion of tumor necrosis factor alpha (TNF-alpha) after 4 h of exposure to 50 microM bismuth and found no influence on the production of TNF-alpha. Taken together, it seems likely that bismuth has no direct effects on Leydig cells but, rather, lowers testosterone levels by killing testicular macrophages, thereby interrupting their local paracrine influence on Leydig cells through factors other than TNF-alpha.     

Renal function in the freshwater rainbow trout (Oncorhynchus mykiss) following acute and prolonged exposure to waterborne nickel

Renal function was investigated in adult rainbow trout following acute and prolonged exposure to waterborne Ni in moderately hard Lake Ontario water (approximately 140 mgL(-1) as CaCO3). Fish were exposed for 36 days to a sublethal concentration of 442 microg Ni L(-1), followed by 96 h of exposure to 12,850 microg Ni L(-1) (approximately 33% of the 96 h LC50). Prolonged exposure markedly affected only the renal handling of Ni, with no substantial effect on the plasma concentration, urinary excretion rate (UER) or clearance ratio (CR) of Na+, Cl-, K+, Ca2+, Mg2+, inorganic phosphate (P(i)), glucose, lactate, total ammonia (T(amm)), protein and free amino acids (FAA). Glomerular filtration rate (GFR) was reduced by 75% over 96 h of acute Ni challenge in both fish previously exposed to Ni and naive fish, with no significant change in urine flow rate (UFR), suggesting a substantial reduction in water reabsorption to maintain urine flow and water balance. Renal Mg2+ handling was specifically impaired by acute Ni challenge, leading to a significantly increased UER(Mg2+) and significantly decreased plasma [Mg2+] only in naive fish. Previously-exposed fish were well-protected against Ni-induced Mg2+ antagonism, indicating true acclimation to Ni. Only in naive, acutely challenged fish was there an increased UER of titratable acidity (TA-HCO3), net acidic equivalents, P(i), T(amm) and K+. Again, all of these parameters were well-conserved in previously-exposed fish during acute Ni exposure, strongly suggesting that prolonged, sublethal exposure protected against acute Ni-induced respiratory toxicity.     

The effects of anaesthetics on the uptake and release of amino acid neurotransmitters in thalamic slices

1 The effect of thiopentone, methohexitone, urethane and ketamine on the uptake and release of gamma-aminobutyric acid (GABA) and D-aspartate by rat thalamic slices has been investigated. 2 A high, supra-anaesthetic concentration of methohexitone increased the uptake of both D-aspartate and GABA. 3 None of the anaesthetics used had any detectable effect upon the spontaneous release of either amino acid. 4 Urethane and ketamine had no effect upon the K+-stimulated release of either amino acid. 5 Methohexitone and thiopentone produced a biphasic dose-response on the K+-stimulated release of both amino acids; low concentrations enhanced release, high concentrations depressed release. 6 Bicuculline hydrochloride and picrotoxin both significantly reduced the barbiturate-induced enhancement of K+-stimulated amino acid release, but did not significantly alter the depression of K+-stimulated release at higher barbiturate concentrations. 7 Baclofen, either alone (1 microM to 1 mM), or tested against the barbiturates, had no detectable effect.     

REACTION OF OSMIUM REAGENTS WITH AMINO ACIDS AND PROTEINS: Reactivity of Amino Acid Residues and Peptide Bond Cleavage

We report a study of the relative reactivity of the common amino acids and of their residues in lysozyme with osmium tetroxide, the osmium tetroxide-pyridine reagent, and with the oxo-osmium(VI)-pyridine reagent. With free amino acids, the osmium(VIII) reagents are most reactive with Met, Cys, His, Thr, Ser, Trp, Lys, and Pro; the osmium(VI) reagent only reacts significantly with His, Met, Cys, Thr, and Ser. In lysozyme, only Cys, Met, and Trp react extensively with the osmium(VIII) reagents; with the osmium (VI) reagent, Cys and Met are most reactive. We also note evidence both for cross-linking of proteins and for peptide bond cleavage, which appears to have considerable specificity for tryptophanyl residues.     

The effects of nitrogen mustards on the proliferative and Ig synthetic response of human peripheral blood lymphocytes

Maximal inhibition of pokeweed mitogen-stimulated Ig production and [3H]thymidine incorporation was shown to occur when unfractionated human peripheral blood mononuclear cells were cultured with concentrations of the nitrogen mustards melphalan, mechlorethamine or chlorambucil in the 20-100-microM range, whereas concentrations of microsome-activated cyclophosphamide (A-Cy) in the 2-mM range were required for equivalent inhibition. Around 400 microM A-Cy, IgM secretion was not inhibited, but secretion of IgA and IgG was. The [3H]thymidine incorporation of enriched populations of both large and small B and T cells all showed about 20-50-fold greater sensitivity to melphalan than to A-Cy, despite a difference of only 6-fold in alkylating activity between these drugs. Large (250 micron 3) B and T cells were only marginally more sensitive to melphalan and A-Cy than small (210 micron 3) T and B cells. Kinetic studies showed that IgG and IgA secreted by day 7 could be maximally inhibited by melphalan added as late as day 3, and IgM synthesis as late as day 2. In contrast, inhibition of Ig production by A-Cy steadily declined after the first day, especially IgM, which was no longer inhibitable by A-Cy on day 3. Inhibition of cumulative Ig production did not occur when A-Cy or melphalan was added on day 5 or later. Cell recombination experiments performed with drug pulsed and untreated monocytes plus B cells and irradiated T cells showed that inhibition of [3H]thymidine or Ig production was most striking when monocytes + B cells (rather than T cells) were exposed to melphalan in the first 16 h. When A-Cy was used in the first 16 h, inhibition of Ig production was partial and inconsistent, and inhibition of monocytes + B cell or T cell [3H]thymidine incorporation was not evident. We conclude that the nitrogen mustards melphalan and A-Cy can inhibit pokeweed mitogen-stimulated DNA synthesis by human T or B cells and Ig production in vitro, but that their mechanisms of action differ.     

Effects of harman and norharman on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells

The effects of harman and norharman on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Harman and norharman at a concentration of 20 microM and 100 microM showed 49.4% and 49.5% inhibition of dopamine content for 48 h, respectively. The IC50 values of harman and norharman were 21.2 microM and 103.3 microM. Dopamine content, tyrosine hydroxylase (TH) activity and TH mRNA levels were decreased during the first 6 h, maintained for up to 48 h and then gradually recovered at 72 h after exposure to 20 microM harman and 100 microM norharman. Under the same conditions, the intracellular cyclic AMP levels and Ca2+ concentrations were also decreased by harman and norharman. In addition, harman and norharman at concentrations higher than 80 microM and 150 microM caused cytotoxicity at 48 h in PC12 cells. Non-cytotoxic ranges of 10-30 microM harman and 50-150 microM norharman inhibited L-DOPA (20-50 microM)-induced increases in dopamine content at 48 h. Harman at 20-150 microM and norharman at 100-300 microM also enhanced L-DOPA (20-100 microM)-induced cytotoxicity at 48 h with an apoptotic process. These results suggest that harman and norharman inhibit dopamine biosynthesis by reducing TH activity and enhance L-DOPA-induced cytotoxicity in PC12 cells.     

The inhibition of N-acetyltransferase activity and gene expression in human bladder cancer cells (T24) by shikonin

Shikonin has the potential to prevent, or be used in the treatment of, bladder transitional cell carcinoma induced by arylamines. We evaluated its effectiveness by measuring the amount of acetylated 2-aminofluorene (AF), AF-DNA adducts, changes of NAT mRNA and the amount of NAT enzyme. T24 human bladder cancer cells were incubated with 30 microM AF with different concentrations of shikonin for various times. T24 cells treated with shikonin (16 microM) were then harvested and used in 2 experiments: 1). T24 cells were incubated with 22.5 microM AF and shikonin (0, 16 microM) (co-treatment) for 6, 12, 18, 24 and 48 h. 2). T24 cells were incubated with various concentrations of AF and shikonin (0, 16 microM) for 24 h. AF and AAF were measured by HPLC. Then in the prepared human T24 cell cytosols different concentrations of AF and shikonin were added to measure the kinetic constants of NAT. Next, AF-DNA adducts in human T24 cells with or without treatment with shikonin were detected and measured. The final two steps included measuring the NAT Ag-Ab complex after treatment with and without shikonin and evaluating the effect of shikonin on the NAT genes. Higher concentrations of shikonin induced decreasing AF acetylation. We found that the longer the culture period, the greater the difference in AF acetylation in the same shikonin concentrations. It was also noted that increase in AAF was proportional to incubation time. In the presence of 16 microM of shikonin, N-acetylation of AF decreased by up to 72-84%. Shikonin decreased the amount of AAF production in human T24 cells in all examined AF doses. Both Km and Vmax values in the cytosolic NAT decreased after the addition of shikonin to the cytosol. Finally, shikonin decreased the amount of AAF production and AF-DNA adducts formation in human 724 cells in all examined AF doses. The percentage of cells stained by antibody was significantly different after treatment with shikonin, especially with the higher shikonin concentrations. The NAT1 mRNA level and the NAT1/beta-actin ratio decreased significantly with higher concentrations (16-24 microM) of shikonin. Shikonin affected NAT activity, gene expression (NAT1 mRNA), AF-DNA adducts formation and formation of NAT Ag-Ab in human bladder tumor T24 cells. Therefore, shikonin should be considered as a candidate agent for the prevention or treatment of transitional cell carcinoma.