Concentration-dependent Enhancement Research Articles

The chondroprotective drugs, arteparon and sodium pentosan polysulphate, increase collagenase activity and inhibit stromelysin activity in vitro

The effects of the chondroprotective drugs, sodium pentosan polysulphate (SP54) and Arteparon (glycosaminoglycan polysulphate), on the in vitro activities of the purified matrix metalloproteinases interstitial collagenase (matrix metalloproteinase 1, MMP1) and stromelysin (MMP3) were examined. Both drugs produced concentration-dependent enhancement of the degradation of type I collagen fibrils by purified human fibroblast collagenase and rat tumour collagenase. Rat collagenase activity was increased by drug concentrations above 0.5 μg/mL, whereas human collagenase activity was only increased by higher drug concentrations, a μg/mL. The concentration dependence of the increase in rat collagenase activity was similar for both drugs, with a maximal 3-fold increase at 50 μg/mL. In contrast, human collagenase activity was increased to a greater extent by SP 54 compared to Arteparon, with maximal increases at 5000 μg/mL of 6-fold and 2–4-fold, respectively. Both drugs produced concentration-dependent inhibition of the proteoglycan-degrading activity of both human fibroblast stromelysin and rat tumour stromelysin. Rat and human stromelysin activities were inhibited at drug concentrations above 0.005 μg/mL, with a similar concentration dependence for both drugs. Fifty percent inhibition of rat stromelysin was produced by concentrations of each drug in the 0.5–5 μg/ mL range. The pattern of inhibition of human stromelysin was similar, except that drug concentrations in the 500–5000 μg/mL range produced 50% inhibition. The possible modes of action for these drug effects and their possible pharmacological significance are discussed.

Possible involvement of both N- and L-type voltage-dependent Ca channels in adrenergic neurotransmission of canine saphenous veins in low Ca2+ plus tetraethylammonium medium.

The involvement of N- and L-type voltage-dependent Ca channels (VDCCs) in adrenergic neurotransmission under the superfusion with 0.25 mM Ca2+ + 20 mM tetraethylammonium (low Ca2+ + TEA) medium has been studied by examining the effects of omega-conotoxin GVIA (omega-CTX) and dihydropyridine antagonists and agonist on transmural nerve stimulation (TNS)-evoked 3H overflow from canine saphenous veins preloaded with [3H]-noradrenaline. Nisoldipine (10 and 30 microM) and nifedipine (30 microM) reduced significantly the TNS-evoked 3H overflow in low Ca2+ + TEA medium, while the two dihydropyridine antagonists failed to suppress it in normal Krebs medium. Bay K 8644 (30 and 100 nM) produced a significant and concentration-dependent enhancement of the TNS-evoked 3H overflow in low Ca2+ + TEA medium. The enhancing effects of Bay K 8644 were antagonized by both 3 microM nisoldipine and 10 microM nifedipine. omega-CTX inhibited markedly the TNS-evoked 3H overflow in both normal Krebs and low Ca2+ + TEA media, the inhibition by omega-CTX being ten times more potent in low Ca2+ + TEA medium. Nisoldipine (30 microM), when combined with 1 nM omega-CTX, produced a further significant inhibition of the TNS-evoked 3H overflow in low Ca2+ + TEA medium. However, no additional inhibition by 30 microM nisoldipine was observed when omega-CTX concentration was raised to 2 nM. In the veins superfused with normal Krebs medium, nisoldipine (30 microM) did not affect the inhibitory effect of 10 nM omega-CTX on the evoked 3H overflow.(ABSTRACT TRUNCATED AT 250 WORDS)

Guanine nucleotides stimulate car☐yl methylation of kidney cytosolic proteins

We studied the effect of guanine nucleotides on the car☐yl methylation catalyzed by class II protein car☐ylmethyltransferases (PCMT). Addition of guanosine 5′- O-(γ-thio)triphosphate (GTPγ-S) promoted a time- and concentration-dependent enhancement of protein methylation in the cytosolic fraction isolated from kidney cortex. GTPγS affected the kinetics of the methylation reaction, as reflected by alterations of both apparent K m and V max of the methyltransferase. This effect was specific for guanine nucleotides and was completely abolished by addition of S-adenosyl- l-homocysteine, a well-known inhibitor of methyltransferase-catalyzed reactions. No GTPγS stimulation of methylation was found in cytosolic extracts from any of the other tissues studied, including brain, testis, spleen, and liver, nor in brush-border membranes isolated from the kidney cortex. The methylated proteins were highly sensitive to moderately alkaline conditions, suggesting that the methyl esters were formed on l-isoaspartyl residues and thus methylated by a class II PCMT. These results suggest that class-II-associated protein methylation activity from the soluble fraction of the kidney can be regulated by guanine nucleotides.

Differential effects of divalent cations on fibrin structure

Calcium, a divalent cation, serves as a cofactor for several reactions of the haemostatic cascade and causes concentration-dependent enhancement of fibrin fibre size and fibrin structure. We have evaluated the ability of magnesium and zinc to influence fibrin assembly and structure. 20 mM magnesium shortened the lag phase prior to turbidity development and increased the fibre mass/length ratio (0.28 to 1.23 x 10(13) Da/cm) in a purified fibrin-thrombin system. In plasma clotted with thrombin, 20 mM magnesium increased fibre mass/length ratios from 0.60 to 1.65 x 10(13) Da/cm. Fibrinopeptide B release was not required for expression of magnesium's effect since lag phase shortening and increased fibre mass/length ratios occurred when clotting was initiated with batroxobin. In all cases, magnesium effects were similar to effects seen with calcium. In the purified system, zinc exerted effects at concentrations as low as 5 microM. At 10 microM, zinc shortened the period prior to turbidity rise from 15.3 to 2.3 s. The final fibre size increased from 0.30 to 1.16 x 10(13) Da/cm over the same concentration range. Zinc caused similar alterations in gels clotted with batroxobin. The lag phase shortened from 38.3 to 15.3 s, and the fibre mass/length ratio increased from 0.49 to 1.47 x 10(13) Da/cm as zinc concentration increased from 0 to 10 microM. The addition of zinc to citrated plasma at concentrations as high as 100 microM had no appreciable impact on fibrin assembly or structure. To avoid possible effects of citrate, zinc was added to fresh non-anticoagulated plasma. At concentrations as high as 100 microM, zinc had no effect on thrombin-induced clot formation or structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a ceramide-activated protein kinase: stimulation by tumor necrosis factor alpha.

Recent investigations have identified a signal-transduction system involving sphingomyelin and derivatives. In this paradigm, sphingomyelin hydrolysis by a sphingomyelinase generates ceramide, which may be converted to the protein kinase C inhibitor sphingosine or to ceramide 1-phosphate. Ceramide may have second-messenger function because it induces epidermal growth factor receptor phosphorylation, presumably on Thr-669 in A-431 cells. The present studies describe a kinase that may mediate ceramide action. With a 19-amino acid epidermal growth factor receptor peptide containing Thr-669, a membrane-bound activity that phosphorylated the peptide was detected in A-431 cells. Activity was linearly related to ATP (0.3-300 microM) and peptide concentration (0.02-1 mg/ml), possessed a physiologic pH optimum (pH 7.0-7.4), and was Mg(2+)-dependent. Other cations--Ca2+, Mn2+, and Zn(2+)--were ineffective. Natural and synthetic ceramide induced time- and concentration-dependent enhancement of kinase activity. Ceramide (0.5 microM) increased kinase activity 2-fold by 30 s, and activity remained elevated for at least 15 min. As little as 0.001 microM ceramide was effective, and 1 microM ceramide induced maximal phosphorylation. Sphingosine was similarly effective. Because tumor necrosis factor (TNF) alpha rapidly induces sphingomyelin hydrolysis to ceramide during monocytic differentiation of HL-60 cells, its effects on kinase activity were assessed. Kinase activity was increased 1.5-fold at 5 min and 2-fold at 2 hr in membranes derived from TNF-stimulated cells. The effective concentration range was 3 pM-30 nM TNF. Exogenous ceramide induced a similar effect. In sum, these studies demonstrate the existence of an unusual Mg(2+)-dependent ceramide-activated protein kinase that may mediate some aspects of TNF-alpha function.

Endogenous nitric oxide modulates adrenergic neural vasoconstriction in guinea‐pig pulmonary artery

1. Electrical field stimulation (EFS) of guinea-pig isolated pulmonary artery induced a frequency-dependent contraction. This was abolished by tetrodotoxin (1 microM) and prevented by phentolamine and prazosin (both 1 microM), indicating a role for alpha 1-adrenoceptors activated by noradrenaline (NA) released from perivascular adrenergic nerves. 2. L-NG-monomethyl arginine (L-NMMA, 0.3-100 microM) caused a concentration-dependent enhancement of the EFS-induced contraction with a 3.4 +/- 0.5 fold increase at 100 microM n = 6). The augmenting effect of 30 microM L-NMMA on the contraction to EFS was completely reversed by 100-300 microM L-arginine, but not by an identical concentration of D-arginine. 3. The contractile response to exogenous NA was similarly enhanced by 30 microM L-NMMA (2.9 +/- 0.6 fold increase, n = 5). 4. The contractile responses to exogenous phenylephrine and prostaglandin F2 alpha while matched the contraction to EFS (4 Hz) were equally augmented by 30 microM L-NMMA. 5. In vessel rings submaximally contracted with the thromboxane analogue U44069 (2 microM), the selective alpha 2-adrenoceptor agonist UK14304 induced concentration-dependent relaxation, which was abolished by removal of endothelium. NA had little relaxant effect on these precontracted vessel rings unless in the presence of prazosin (1 microM). 6. Indomethacin had no significant effect on the contractile response to EFS or NA, indicating that vasodilator cyclo-oxygenase products such as prostacyclin are not involved in modulating these responses. 7. Our results suggest that endogenous nitric oxide inhibits the contractile response to adrenergic nerve stimulation in the guinea-pig pulmonary artery by a postjunctional mechanism, but release of prostacyclin does not modulate these responses. Basal release of nitric oxide from endothelial cells may account for this inhibition.

Phosphatidic acid as a second messenger in human polymorphonuclear leukocytes. Effects on activation of NADPH oxidase.

Receptor-mediated agonists, such as FMLP, induce an early, phospholipase D (PLD)-mediated accumulation of phosphatidic acid (PA) which may play a role in the activation of NADPH oxidase in human PMN. We have determined the effect of changes in PA production on O2 consumption in intact PMN and the level of NADPH oxidase activity measured in a cell-free assay. Pretreatment of cells with various concentrations of propranolol enhanced (less than or equal to 200 microM) or inhibited (greater than 300 microM) PLD-induced production of PA (mass and radiolabel) in a manner that correlated with enhancement or inhibition of O2 consumption in PMN stimulated with 1 microM FMLP in the absence of cytochalasin B. The concentration-dependent effects of propranolol on FMLP-induced NADPH oxidase activation was confirmed by direct assay of the enzyme in subcellular fractions. In PA extracted from cells pretreated with 200 microM propranolol before stimulation with 1 microM FMLP, phospholipase A1 (PLA1)-digestion for 90 min, followed by quantitation of residual PA, showed that a minimum of 44% of PA in control (undigested) sample was diacyl-PA; alkylacyl-PA remained undigested by PLA1. Propranolol was also observed to have a concentration-dependent enhancement of mass of 1,2-DG formed in PMN stimulated with FMLP. DG levels reached a maximum at 300 microM propranolol and remained unchanged up to 500 microM propranolol. However, in contrast to PA levels, the level of DG produced did not correlate with NADPH oxidase activation. Exogenously added didecanoyl-PA activated NADPH oxidase in a concentration-dependent manner (1-300 microM) in a reconstitution assay using membrane and cytosolic fractions from unstimulated PMN. In addition, PA synergized with SDS for oxidase activation. Taken together, these results indicate that PA plays a second messenger role in the activation of NADPH oxidase in human PMN and that regulation of phospholipase D is a key step in the activation pathway.

A selective effect of protein kinase C activators on noradrenaline release compared with subsequent contraction in canine isolated saphenous veins

1. Effects of protein kinase C (PKC) activators and inhibitors on both tritium overflow and subsequent contraction evoked by transmural nerve stimulation (TNS) were investigated in canine saphenous veins prelabelled with [3H]-noradrenaline. 2. Activation of PKC by stepwise increasing concentrations (0.01 nM-1 microM) of 12-O-tetradecanoylphorbol 13-acetate (TPA), phorbol 12,13-dibutyrate (PDBu) or mezerein caused a significant and concentration-dependent enhancement of the tritium overflow evoked by TNS, while the activators failed to affect the corresponding contraction except with the highest concentration of PDBu when the contraction was significantly reduced. Phorbol, which is inactive on PKC, had no effects on the tritium overflow and contraction induced by TNS. 3. PKC inhibitors, polymyxin B (1 and 10 microM) and the isoquinolinesulphonamide, H-7 (1 microM), inhibited significantly the phorbol ester-potentiated tritium overflow evoked by TNS with no effects on the contraction. H-7 and the related inhibitor H-8 at 10 microM reduced significantly both responses to TNS in the presence of TPA, while they suppressed only the TNS-induced contraction in the absence of TPA. 4. None of the PKC activators or inhibitors affected the spontaneous tritium overflow. 5. PDBu (0.01 and 0.1 microM) elevated resting tension of the veins more effectively than TPA and mezerein. 6. These results suggest that PKC may modulate electrically stimulated noradrenaline release from adrenergic nerve endings of the canine saphenous veins and the PKC activators may act more selectively on presynaptic than postsynaptic sites, but have no apparent effect on postjunctional noradrenergic mechanisms.

Dictyostelium discoideumLipids Modulate Cell-Cell Cohesion and Cyclic AMP Signaling

During Dictyostelium discoideum development, cell-cell communication is mediated through cyclic AMP (cAMP)-induced cAMP synthesis and secretion (cAMP signaling) and cell-cell contact. Cell-cell contact elicits cAMP secretion and modulates the magnitude of a subsequent cAMP signaling response (D. R. Fontana and P. L. Price, Differentiation 41:184-192, 1989), demonstrating that cell-cell contact and cAMP signaling are not independent events. To identify components involved in the contact-mediated modulation of cAMP signaling, amoebal membranes were added to aggregation-competent amoebae in suspension. The membranes from aggregation-competent amoebae inhibited cAMP signaling at all concentrations tested, while the membranes from vegetative amoebae exhibited a concentration-dependent enhancement or inhibition of cAMP signaling. Membrane lipids inhibited cAMP signaling at all concentrations tested. The lipids abolished cAMP signaling by blocking cAMP-induced adenylyl cyclase activation. The membrane lipids also inhibited amoeba-amoeba cohesion at concentrations comparable to those which inhibited cAMP signaling. The phospholipids and neutral lipids decreased cohesion and inhibited the cAMP signaling response. The glycolipid/sulfolipid fraction enhanced cohesion and cAMP signaling. Caffeine, a known inhibitor of cAMP-induced adenylyl cyclase activation, inhibited amoeba-amoeba cohesion. These studies demonstrate that endogenous lipids are capable of modulating amoeba-amoeba cohesion and cAMP-induced activation of the adenylyl cyclase. These results suggest that cohesion may modulate cAMP-induced adenylyl cyclase activation. Because the complete elimination of cohesion is accompanied by the complete elimination of cAMP signaling, these results further suggest that cohesion may be necessary for cAMP-induced adenylyl cyclase activation in D. discoideum.

Dictyostelium discoideum lipids modulate cell-cell cohesion and cyclic AMP signaling.

During Dictyostelium discoideum development, cell-cell communication is mediated through cyclic AMP (cAMP)-induced cAMP synthesis and secretion (cAMP signaling) and cell-cell contact. Cell-cell contact elicits cAMP secretion and modulates the magnitude of a subsequent cAMP signaling response (D. R. Fontana and P. L. Price, Differentiation 41:184-192, 1989), demonstrating that cell-cell contact and cAMP signaling are not independent events. To identify components involved in the contact-mediated modulation of cAMP signaling, amoebal membranes were added to aggregation-competent amoebae in suspension. The membranes from aggregation-competent amoebae inhibited cAMP signaling at all concentrations tested, while the membranes from vegetative amoebae exhibited a concentration-dependent enhancement or inhibition of cAMP signaling. Membrane lipids inhibited cAMP signaling at all concentrations tested. The lipids abolished cAMP signaling by blocking cAMP-induced adenylyl cyclase activation. The membrane lipids also inhibited amoeba-amoeba cohesion at concentrations comparable to those which inhibited cAMP signaling. The phospholipids and neutral lipids decreased cohesion and inhibited the cAMP signaling response. The glycolipid/sulfolipid fraction enhanced cohesion and cAMP signaling. Caffeine, a known inhibitor of cAMP-induced adenylyl cyclase activation, inhibited amoeba-amoeba cohesion. These studies demonstrate that endogenous lipids are capable of modulating amoeba-amoeba cohesion and cAMP-induced activation of the adenylyl cyclase. These results suggest that cohesion may modulate cAMP-induced adenylyl cyclase activation. Because the complete elimination of cohesion is accompanied by the complete elimination of cAMP signaling, these results further suggest that cohesion may be necessary for cAMP-induced adenylyl cyclase activation in D. discoideum.

Role of cyclic AMP in the prejunctional α2-adrenoceptor modulation of noradrenaline release from the rat tail artery

Experiments were designed to evaluate the effect of cyclic AMP on the electrically-induced release of noradrenaline from vascular sympathetic nerve terminals. The possible implication of the inhibition of adenylate cyclase in the negative feed-back control by prejunctional alpha 2-adrenoceptors of neurotransmitter release was also investigated. Rat isolated tail arteries were preincubated with [3H]-noradrenaline; the preparations were subsequently perfused/superfused with [3H )-noradrenaline-free medium and their perivascular nerves were field stimulated with 24 pulses at 0.4 Hz (0.3 ms, 200 mA). 2 compounds known to enhance the intracellular concentration of cyclic AMP, namely the membrane permeant analogue 8-Br-cAMP (10-300 mumol/l) and forskolin (0.3-10 mumol/l), an activator of adenylate cyclase, concentration-dependently enhanced the stimulation-evoked tritium overflow. The 1,9-dideoxy derivative of forskolin, which does not stimulate adenylate cyclase, was ineffective. Exposure to the cyclic AMP phosphodiesterase inhibitor rolipram 30 mumol/l produced a moderate increase (about 20%) in tritium overflow. However, in the presence of rolipram the facilitatory effect of forskolin was significantly more pronounced than in its absence. Whereas 8-Br-cAMP produced a slight concentration-dependent enhancement of the stimulation-induced vasoconstriction, forskolin and rolipram depressed it. The alpha 2-adrenoceptor agonist B-HT 933 (3-30 mumol/l) concentration-dependently inhibited the tritium overflow. The effect of B-HT 933 30 mumol/l was slightly, but significantly reduced in the presence of 8-Br-cAMP 100 and 300 mumol/l, but was not changed in the presence of forskolin 3 mumol/l. The facilitatory effect of rauwolscine 1 mumol/l was enhanced in the presence of 8-Br-cAMP 100 mumol/l. During perfusion with 8-Br-cAMP 100 mumol/l, the current strength and frequency were decreased to 150 mA and 0.2 Hz, respectively in order to obtain similar amounts of tritium overflow to those observed in the absence of the cyclic AMP analogue with the initial stimulation parameters. Under these conditions, the inhibition of the overflow by B-HT 933 30 mumol/l and the facilitation by the alpha 2-adrenoceptor antagonist rauwolscine 1 mumol/l were unaltered as compared to controls under initial stimulation conditions. It is concluded that, in the rat tail artery, the terminals of perivascular sympathetic nerves are endowed with an adenylate cyclase system. Cyclic AMP is able to modulate noradrenaline release, but does not appear to play a role in the initiation of the release process itself. In addition, the results do not support the hypothesis that prejunctional alpha 2-adrenoceptors depress noradrenaline release through the inhibition of adenylate cyclase.

CHANGES IN PREJUNCTIONAL POTENCY OF B‐HT 933 DURING AGING IN THE RAT VAS DEFERENS

1. Age-related changes in prejunctional alpha 2-adrenoceptors were examined in the rat vas deferens using pharmacological techniques. 2. B-HT 933 (1 x 10(-8) - 1 x 10(-6) mol/L) caused a concentration-dependent inhibition of isometric contractions (tetrodotoxin-sensitive) induced by stimulation with single field-stimulus pulses, in both the epididymal and prostatic regions of rat vas deferens. The concentration-response curve to B-HT 933 was shifted to the right with age in the prostatic regions of the vas deferens. 3. In high concentrations (10(-6) - 3 x 10(-4) mol/L), B-HT 933 caused concentration-dependent enhancement of the contractile response to stimulation and evoked spontaneous contractile activity. No significant difference in this postjunctional activity occurred with age in either the prostatic or epididymal regions of the vas deferens. 4. Schild analysis revealed no significant differences in pA2 values for the antagonisms of the prejunctional inhibitory effect of B-HT 933 by rauwolscine in either the prostatic or epididymal regions of vas deferens between young and old rats. 5. These results could be interpreted as a decrease in alpha 2-adrenoceptor number with age. The more marked decrease in the prejunctional inhibitor potency of B-HT 933 in prostatic regions of vas deferens with aging may be due to a smaller receptor reserve in this region of the vas deferens.

Gastrin secretion from human antral G cells in culture

Receptor-dependent an -independent regulation of gastrin secretion from cultured human antral G cells was investigated. Human antral mucosal cell preparations that were enriched for G cells were obtained by sequential incubations with collagenase and ethylenediaminetetraacetic acid, centrifugal elutriation, and short-term culture. After a 2-day incubation period, gastrin- and somatostatin-containing cells accounted for 15% and 5%, respectively, of the total adhered-cell population. Forskolin, A23187, and β-phorbol 12 myristate 13-acetate stimulated basal gastrin secretion from cultured human G cells in a concentration-dependent fashion. These results indicate that gastrin release could be mediated by elevations in cytosolic cyclic adenosine monophosphate levels, calcium influx, or activation of protein kinase C. A direct stimulatory role for bombesin- and gastrin-releasing peptide was supported by experiments showing concentration-dependent enhancement of gastrin release by bombesin from 0.01 fmol/L to 10 nmol/L. The putative bombesin antagonist [Leu13-ψ-CH2NH-Leu14] bombesin augmented basal gastrin levels by itself and produced weak inhibition of bombesin-induced gastrin secretion from human antral G cells. Somatostatin potently suppressed forskolin- and bombesin-mediated gastrin release but did not significantly alter basal gastrin levels. These results suggest that bombesin and somatostatin directly activate and inhibit G-cell function via specific and sensitive receptors. Furthermore, the adenylate cyclase and phosphatidyl inositide second messenger systems seem to be intracellular mediators of gastrin secretion from human antral G cells.

Proliferating or interleukin 1-activated human vascular smooth muscle cells secrete copious interleukin 6.

The cells that make up blood vessel walls appear to participate actively in local immune and inflammatory responses, as well as in certain vascular diseases. We tested here whether smooth muscle cells (SMC) can produce the important inflammatory mediator IL6. Unstimulated SMC in vitro elaborated 5 X 10(3) pg recIL6/24h (i.e., biological activity equivalent to 5 X 10(3) pg recombinant IL6 (recIL6), as determined in B9-assay with a recIL6 standard). Several pathophysiologically relevant factors augmented IL6 release from SMC including 10 micrograms LPS/ml (10(4) pg recIL6), 10 ng tumor necrosis factor/ml (4 X 10(4) pg recIL6), and most notably 10 ng IL1/ml (greater than or equal to 3.2 X 10(5) pg recIL6). Production of IL6 activity corresponded to IL6 mRNA accumulation and de novo synthesis. SMC released newly synthesized IL6 rapidly, as little metabolically labeled material remained cell-associated. In supernatants of IL1-stimulated SMC, IL6 accounted for as much as 4% of the secreted proteins. In normal vessels SMC seldom divide, but SMC proliferation can occur in hypertension or during atherogenesis. We therefore tested the relationship between IL6 production and SMC proliferation in response to platelet-derived growth factor (PDGF) in vitro. Quiescent SMC released scant IL6 activity, whereas PDGF (1-100 ng/ml) produced concentration-dependent and coordinate enhancement of SMC proliferation and IL6 release (linear regression of growth vs. IL6 release yielded r greater than 0.9). IL6 itself neither stimulated nor inhibited SMC growth or IL6 production. Intact medial strips studied in short-term organoid culture produced large quantities of IL6, similar to the results obtained with cultured SMC. These findings illustrate a new function of vascular SMC by which these cells might participate in local immunoregulation and in the pathogenesis of various important vascular diseases as well as in inflammatory responses generally.

ミトコンドリア機能賦活作用を有する塩化レボカルニチンのラット心筋ホモジネートにおける低酸素条件下での脂肪酸及びグルコースの酸化に対する作用

The effects of levocarnitine chloride [LC-80, (-)-(R)-(3-carboxy-2-hydroxypropyl)-trimethylammonium chloride] on the oxidation of [U-14C]palmitate, [U-14C]glucose and [2-3H]glucose under hypoxic conditions in homogenates from the rat heart were investigated. LC-80 at concentrations of 0.5 and 1.0 mM caused significant and concentration-dependent increases in the depressed 14CO2 production from [U-14C]palmitate or [U-14C]-glucose oxidation under hypoxia up to 50 min after the addition of LC-80. In contrast, a marked increase in 3H2O production from [2-3H]glucose oxidation under hypoxia was observed, and LC-80 at concentrations of 0.5 and 1.0 mM caused further significant and concentration-dependent enhancement of 3H2O production up to 50 min after the addition of LC-80. Moreover, LC-80 at a concentrations of 1.0 mM significantly restored the marked depression of pyruvate dehydrogenase complex activity and mitochondrial respiratory function under hypoxia. These results suggested that LC-80 has an improving effect on myocardial metabolism under ischemia by enhancing fatty acid and glucose metabolisms.

Effects of the Monoamine Oxidase Inhibitor, Tranylcypromine, on Induction of HL60 Cell Differentiation by Hexamethylene Bisacetamide and N-Acetyl-1,6-Diaminohexane

Hexamethylene bisacetamide (HMBA) is converted by successive deacetylation and oxidation reactions to four major metabolites; in vitro, the initial deacetylated metabolite, N-acetyl-1,6-diaminohexane (NAD-AH), is more potent than HMBA (Synder, S.W.; Egorin, M.J.; Geelhaar, L.A.; Hamburger, A.W.; Callery, P.S. Cancer Res. 48:3613-3616; 1988). We propose that monoamine oxidase (MAO) catalyzed metabolism of NADAH to 6-acetamidohexanoic acid (AcHA) is an inactivation pathway and, therefore, investigated whether blocking such metabolism with the MAO inhibitor, tranylcypromine, would potentiate induction of cell differentiation by HMBA and NADAH. Tranylcypromine, at concentrations up to 30 micrograms/mL, did not inhibit HL60 cell growth and did not induce differentiation of HL60 cells. Tranylcypromine did, however, produce concentration-dependent enhancement of HMBA- and NADAH-induced differentiation. In contrast, 30 micrograms/mL of tranylcypromine did not effect the ability of dimethylsulfoxide, at concentrations between 0.25% and 1.25%, to induce differentiation of HL60 cells. Tranylcypromine, at 30 micrograms/mL, did not change cellular concentrations of HMBA or NADAH but did reduce intracellular concentrations of AcHA, consistent with inhibition of MAO catalyzed conversion of NADAH to AcHA. These results support the hypothesis that MAO catalyzed metabolism of NADH to AcHA is an inactivation pathway and may provide the basis for a clinical trail in which HMBA metabolism is modulated with concurrent tranylcypromine therapy.

Effects of trifluoroacetic acid, a halothane metabolite, on C6 glioma cells

Effects of trifluoroacetic acid (TFA) on cell growth, DNA, glycoprotein, and dolichol-linked oligosaccharides synthesis and ribonucleotide triphosphate concentrations were examined in exponentially growing C6 murine glioma cells. One day of treatment with TFA caused a slight concentration-dependent enhancement of cell growth and [3H]thymidine incorporation. Exposure for 1 or 5 d to TFA (0.5-7.0 mM) elevated the [3H]leucine incorporation in a dose- and time-dependent manner. The results suggested that TFA stimulated cell growth and enhanced protein synthesis. TFA also affected [3H]mannose incorporation into glycoproteins and dolichol-linked oligosaccharides in a dose-dependent fashion. In addition, it was found that TFA accelerated lectin-induced cell agglutination. These data suggest that TFA, the principle halothane metabolite, alters plasmalemmal glycoprotein synthesis. These findings should form a basis for further understanding on the mechanism underlying halothane-associated neurotoxicity.

Neuromuscular blockade induced by flunarizine alone and in combination with pancuronium, suxamethonium or neomycin: studies in isolated rat phrenic-hemidiaphragm preparations.

The in vitro effects of flunarizine on indirectly- and directly-elicited contractions in rat phrenic-hemidiaphragm preparations were studied. The interactions of flunarizine with non-depolarizing and depolarizing neuromuscular blocking drugs (pancuronium and suxamethonium) and with an aminoglycoside antibiotic (neomycin) were also evaluated. Flunarizine induced a slowly developing concentration-dependent reduction of indirectly-elicited diaphragm twitch height, but only slightly reduced directly-elicited contractions. Flunarizine 1 and 5 mumol.l-1 produced a concentration-dependent enhancement of pancuronium-induced neuromuscular blockade, whereas suxamethonium blockade was significantly increased by flunarizine 5 mumols.l.-1 only. Moreover, both flunarizine 1 and 5 mumols.l-1 also increased the neuromuscular blockade induced by neomycin. In conclusion, flunarizine induced neuromuscular blockade and enhanced the effects of several neuromuscular blocking agents to varying degrees in vitro.

Effect of 7-chloro kynurenic acid on glycine modulation of the N-methyl-D-aspartate response in guinea-pig myenteric plexus

The glycine modulation of the N-methyl-D-aspartate (NMDA) response in guinea-pig myenteric plexus was investigated by using D-serine and 7-chloro kynurenic acid as a glycine agonist and antagonist, respectively. D-serine caused a concentration-dependent enhancement of the NMDA response, an effect which was competitively inhibited by 7-chloro kynurenic acid (pA 2 = 6.0). In addition, 7-chloro kynurenic acid induced a concentration-dependent, non-competitive inhibition of the NMDA response per se, even in the absence of added D-serine. This inhibition was fully reversed by exogenous D-serine, suggesting that this effect was also due to the occupancy of the glycine site. These results emphasize the usefulness of the guinea-pig myenteric plexus for studying the function of the NMDA receptor complex.

Effects of calcitonin gene-related peptide (CGRP), neurokinin A and neurokinin A (4?10) on the mitogenic response of human peripheral blood mononuclear cells

(1) We have studied the ability of some regulatory peptides to induce a mitogenic (incorporation of tritiated thymidine) response in human peripheral blood mononuclear cells (PBMC) and to modify the response produced by phytohaemagglutinin (PHA), a well known PBMC mitogen. (2) Human calcitonin gene-related peptide (hCGRP), human or salmon calcitonin (hCT, sCT), neurokinin A (NKA) and neurokinin (4-10) (up to 1 microM for each peptide) did not produce per se any significant PBMC stimulation. (3) hCGRP (0.1 nM-1 microM) produced a concentration dependent enhancement of the response to a submaximal concentration of PHA (1 microgram/ml). On the other hand, hCGRP decreased the mitogenic response to a maximal concentration of PHA (25 micrograms/ml). (4) Neither hCT nor sCT (0.1 nM-1 microM) had a significant influence on the response to PHA (1-25 micrograms/ml). (5) Both NKA and NKA (4-10) produced a concentration-dependent (1 fM-10 pM) enhancement of the response to 1 microgram/ml PHA, while these compounds had no effect on the response to 25 micrograms/ml PHA. (6) These findings suggest a potent modulatory action of CGRP and NKA, two peptides present in sensory and other nerves, on immune function which is possibly mediated via C2 receptors for CGRP and NK-2 tachykinin receptors, respectively.