Dietary linoleic acid-induced changes in respiratory beta-adrenergic receptor function and the form of arrhenius plots of isoprenaline- and prostaglandin E2-stimulated adenylate cyclase activity in a model for atopy.

Varying dietary linoleic acid altered lung membrane fatty acid composition with linoleic acid content increasing from approximately 6% total in those on 3 en% diet to approximately 14% total fatty acid in those on a 12 en% diet. Accompanying this were two- to three-fold increases in the levels of the elongation products of linoleic acid, namely 20:2 (n-6) and 22:5 (n-6) and a decrease in 18:1 oleic acid from approximately 26% to approximately 19% total. Administration of Haemophilus influenzae, to animals on 6 en% linoleic acid, serving as a model for atopy, effected a small increase in the levels of 22:5 (n-3) and doubled those of 22:6 (n-3). beta-Adrenergic-induced tracheal relaxation and stimulation of lung adenylate cyclase were elevated by increasing dietary linoleic acid from 3 to 6 en%, although such differences were abolished in the atopic model and when dietary linoleic acid was increased to 12 en%. Arrhenius plots of NaF-stimulated lung adenylate cyclase activities exhibited a break (t1) at approximately 26 degrees C in all dietary groups with unchanged activation energies and activity. In contrast, whilst both isoprenaline and PGE2-stimulated adenylate cyclase activities showed similar break-points in their Arrhenius plots, dietary linoleic acid manipulation markedly altered their form. As with NaF-stimulated activities then, irrespective of dietary manipulation and induction of atopy, these plots showed an invariant break occurring at approximately 26 degrees C. But, for animals on 3 and 6 en% diets, a second break was apparent at approximately 15 degrees C, which was slightly decreased to approximately 12 degrees C upon induction of atopy and completely abolished on increasing dietary linoleic acid to 12 en%. Accompanying such changes were marked alterations in activation energies. We suggest that profound changes in lung plasma membrane bilayer properties occur upon both altering dietary linoleic acid levels and in atopy. These selectively perturb adenylate cyclase activity when it is receptor-stimulated but not when it is activated by direct G-protein stimulation with NaF. We suggest that atopy and dietary challenge elicit an asymmetric perturbation of the plasma membrane that predominantly affects the outer half of the lipid bilayer.

Mitochondrial effects of the guanidino group-containing cytostatic drugs, m-iodobenzylguanidine and methylglyoxal bis (guanylhydrazone).

The involvement of mitochondrial damage in the antiproliferative effects of m-iodobenzylguanidine [MIBG] and methylglyoxal bis (guanylhydrazone) [methylGAG] was studied in human neuroblastoma SK-N-SH, mouse neuroblastoma N1E115 and mouse lymphosarcoma S49 cells. Proliferation of SK-N-SH cells was insensitive to MIBG (100 microM gave 15% inhibition), but sensitive to methylGAG (IC50 = 50 microM). MIBG and methylGAG were approximately equitoxic to N1E115 cells (IC50 of 92 and 87 microM, respectively). S49 cells were most sensitive to both MIBG (IC50 = 11 microM) and methylGAG (IC50 = 5 microM). In isolated sonicated mitochondria, MIBG inhibited respiration a complex I of the respiratory chain (EC50 = 0.5 mM), whereas methylGAG was much less effective (EC50 greater than 15 mM). In intact cells, MIBG at 31 microM impaired mitochondrial respiration and stimulated the glycolytic flux. In contrast, equimolar concentrations of methylGAG had no effect on oxygen consumption, ATP content, glucose consumption and lactate production. MethylGAG significantly increased putrescine levels in N1E115 and S49 cells within 12 hr via inhibition of S-adenosylmethionine decarboxylase. No such effects were seen in SK-N-SH cells for up to 48 hr. Equimolar concentrations of MIBG had no effect on the putrescine levels in the various cell lines, suggesting that MIBG did not inhibit S-adenosylmethionine decarboxylase. It is concluded that the antiproliferative mechanisms of the guanidino compounds are essentially different. MIBG inhibited mitochondrial respiration at complex I with concomitant stimulation of the glycolytic flux but was essentially without effect on polyamine levels. On the other hand, cytotoxicity of methylGAG was not associated with mitochondrial dysfunction.

Impaired mitochondrial respiration and stimulated glycolysis by m-iodobenzylguanidine (MIBG).

m-Iodobenzylguanidine (MIBG) is a functional analogue of the neurotransmitter norepinephrine. Radio-iodinated 131I-MIBG is used clinically as a tumor-targeted radiopharmaceutical agent in the diagnosis and treatment of adrenergic tumors. Native MIBG has previously been demonstrated to be cytotoxic in cultured cells and to produce anti-tumor responses in animals when non-toxic schedules are used. In this study the effect of MIBG was investigated on isolated rat liver mitochondria and on various tumor cell lines (human neuroblastoma SK-N-SH, mouse neuroblastoma N1E115 and mouse lymphosarcoma S49). Results revealed that MIBG inhibits respiration of isolated liver mitochondria at complex I of the respiratory chain, without affecting F1 ATP-ase. In cell lines, impairment of the mitochondrial respiration was evident from reduced oxygen consumption and decreased intracellular ATP levels. In response to this effect, the glycolytic flux was stimulated as shown by increased glucose consumption and lactic acid production. Cytotoxicity of MIBG was proportional to drug-induced alterations in glucose metabolism.

Intracellular inhibition of mono(ADP-ribosylation) by meta-iodobenzylguanidine: specificity, intracellular concentration and effects on glucocorticoid-mediated cell lysis.

meta-Iodobenzylguanidine (MIBG) is a high-affinity substrate for mono(ADP-ribosyl)transferase of cholera toxin and turkey erythrocyte membranes (Loesberg, C., Van Rooij, H. and Smets, L.A.(1990) Biochim. Biophys. Acta 1037, 92-99). In the present study the drug was investigated as a potential inhibitor of intracellular ribosyltransferases by competition with endogenous acceptors. To this end, MIBG was compared with the conventional ADP-ribosylation inhibitors nicotinamide and 3-aminobenzamide in cell-free ribosylation systems and in intact L1210 leukemia cells. Poly(ADP-ribose)polymerase (poly-ADPRP) was assayed by the DNAse-I-induced incorporation of [14C]NAD in nuclei of permeabilized L1210 cells. Mono(ADP-ribosyl)transferase (mono-ADPRT) was assayed as NAD linkage to [125I]iodoguanyltyramine catalysed by turkey erythrocyte membranes or activated cholera toxin. Poly-ADPRP was inhibited by nicotinamide (IC50 = 0.03 mM) and by 3-aminobenzamide (IC50 less than or equal to 0.03 mM) but was insensitive to MIBG. Conversely, mono-ADPRT was inhibited by MIBG (IC50 = approx. 0.1 mM) but not by 3-aminobenzamide and only weakly so by nicotinamide in high concentration (10 mM). In L1210 cells, intracellular levels of nicotinamide equilibrated at 60-70% of the extracellular drug concentrations assayed at 1 and 10 mM. In contrast, MIBG was concentrated 15-fold by nonspecific uptake. The preferential interference of the drugs with endogenous mono- or poly-ADP ribosylations, predicted from inhibitory capacity in vitro and intracellular concentrations, was confirmed by their effect on dexamethasone-induced lysis of L1210 cell lines. Inhibition of endogenous mono-ADPRT with 0.03 mM MIBG or 10 mM nicotinamide induced sensitivity to glucocorticoids in refractory L1210-wt cells. In contrast, inhibition of poly-ADPRP by 3-aminobenzamide or nicotinamide (1 mM each) did not confer susceptibility to refractory cells but enhanced the lytic process in the sensitive subline L1210-H7 or in L1210-wt cells sensitized by MIBG. These results indicate that MIBG is the first substrate for guanidino-specific mono-ADPRT which accumulates in intact mammalian cells and effectively competes with intracellular acceptors for endogenous enzymes.

Extragranular storage of the neuron blocking agent meta-iodobenzylguanidine (MIBG) in human neuroblastoma cells.

Human SK-N-SH neuroblastoma cells accumulate and store the adrenal imaging agent metaiodobenzylguanidine (MIBG) with minor involvement of specialized cytoplasmic storage granules (Smets LA et al., Active uptake and extravesicular storage of meta-iodo-benzylguanidine in human neuroblastoma SK-N-SH cells. Cancer Res 49: 2941-2944, 1989). In the present study the mechanism of extravesicular MIBG retention was investigated and compared with granular storage of MIBG and norepinephrine (NE) in PC-12 pheochromocytoma cells. SK-N-SH cells concentrated both MIBG and NE by neuron-specific Uptake-1 but long-term retention was only observed with MIBG. Retention of accumulated NE was, however, promoted by inhibition of intracellular catecholamine degradation with pyrogallol. Drug release by controlled cell permeabilization and by KCl-induced exocytosis indicated that MIBG was mainly stored as freely diffusible, cytoplasmic molecules. SK-N-SH cells were depleted from stored MIBG by the Uptake-1 inhibitor imipramine but poorly so by the granule-depleting drug reserpine. Conversely, PC-12 cells were depleted by reserpine but insensitive to imipramine. The data suggest that extravesicular retention of MIBG in SK-N-SH cells is not based on intracellular sequestration but is solely due to efficient re-uptake of accumulated drug after leaking from the cells. The accumulation of MIBG in SK-N-SH cells, reflecting "pure" Uptake-1, appears to be a powerful system for exploring various cellular and molecular aspects of catecholamine uptake.

Meta-iodobenzylguanidine (MIBG), a novel high-affinity substrate for cholera toxin that interferes with cellular mono(ADP-ribosylation).

Meta-iodobenzylguanidine (MIBG) is a guanidine analogue of the neurotransmitter norepinephrine. Radioiodinated [131I]MIBG is clinically used as a tumor-targeted radiopharmaceutical in the diagnosis and treatment of adrenergic tumors. Moreover, non-radiolabelled MIBG exerts several cell-biological effects, tentatively ascribed to interference with cellular mono(ADP-ribosyl) transferases (Smets, L.A., Bout, B. and Wisse, J. (1988) Cancer Chemother. Pharmacol. 21, 9-13; Smets, L.A., Metwally, E.A.G., Knol, E. and Martens, M. (1988) Leukemia Res. 12, 737-743). In the present study it was investigated whether MIBG could serve as an acceptor for the ribosyl transferase activity of cholera toxin and of erythrocyte membranes. MIBG appeared a substrate for the cholera toxin-catalyzed transfer of the ADP-ribose moiety of NAD to arginine-like residues with the highest affinity for this enzyme reported as yet (Km = 6.5 microM). MIBG was also ADP-ribosylated by the mono(ADP-ribosyl)transferase(s) of turkey erythrocyte membranes. Moreover, the drug appeared a potent affector of the ADP-ribose linkage to membrane proteins by these enzymes. Interference by MIBG was stronger than by related guanyltyramine, the monoamine precursors of MIBG, meta-iodobenzylamine had no effect at all. In contrast, the drug failed to affect endogenous, O-linked poly(ADP-ribose) polymerase, induced in nuclei of S49-leukemia cells by deoxyribonuclease. Since MIBG is the first described drug that specifically interferes with the cellular N-linked mono(ADP-ribosyl) transferase reactions, it may be an important tool to elucidate the physiological role of this posttranscriptional protein modification.

Active uptake and extravesicular storage of m-iodobenzylguanidine in human neuroblastoma SK-N-SH cells.

Radio-iodinated m-iodobenzylguanidine (MIBG), an analogue of the neurotransmitter norepinephrine (NE), is increasingly used in the diagnosis and treatment of neural crest tumors. Active uptake and subsequent retention of MIBG and NE was studied in human neuroblastoma SK-N-SH cells. Neuron-specific uptake of [125I]MIBG and [3H]NE saturated at extracellular concentration of 10(-6) M and exceeded by 20-30-fold that by passive diffusion alone. A minimum of 50% of accumulated MIBG remained permanently stored but the SK-N-SH cells were incapable of retaining recaptured [3H]NE. [125I]MIBG was displaced from intracellular binding sites by unlabeled MIBG with 10-fold higher potency than by unlabeled NE. MIBG stored in SK-N-SH cells was insensitive to depletion by the inhibitor of granular uptake reserpine (RSP) and was not precipitated in a granular fraction by differential centrifugation. Only few electron-dense granules were found in these cells by electron microscopy. In contrast, MIBG storage in PC-12 pheochromocytoma cells which contained many storage granules, was sensitive to RSP and part of accumulated drug was recovered in a granular fraction. Accordingly, storage of MIBG in the SK-N-SH neuroblastoma cells is predominantly extravesicular and thus essentially different from that of biogenic amines in normal adrenomedullary tissue or in pheochromocytoma tumors, while sharing with these tissues a common mechanism of active uptake.

Membrane fluidity of guinea pig lymphocytes and the dysfunction of the respiratory airway and lymphocyte beta-adrenergic systems of the guinea pig.

The beta-adrenergic receptor responsiveness of isolated guinea pig tracheal spirals can be negatively affected by intraperitoneal administration of the Gram-negative bacterium Haemophilus influenzae, four days prior to the experiment. The reduction in tracheal relaxation is accompanied by a decrease in beta-adrenergic receptor binding sites in splenic lymphocyte membranes and by a decrease in the fluidity of these membranes. The H. influenzae-induced dysfunction of both the respiratory airway and lymphocyte beta-adrenergic systems can be mimicked by modulating the amount of linoleic acid in the diet. This linoleic acid induced dysfunction of the beta-adrenergic system is also accompanied by a decrease in the plasma membrane fluidity of the splenic lymphocyte membranes of the guinea pigs. The role for plasma membrane fluidity in asthma is discussed in relation to current concepts for atopy.

Inverse relationship between superoxide anion production of guinea pig alveolar macrophages and tracheal beta-adrenergic receptor function; influence of dietary polyunsaturated fatty acids.

It was shown recently that dietary linoleic acid could modulate beta-adrenergic receptor function in guinea pig tracheal spirals (Loesberg, Folkerts & Nijkamp. Prostagland. Leukotr. essen. Fatty Acids, in press). In the present study the relationship between beta-adrenergic receptor function and oxygen radical production was examined by analyzing the effect of dietary linoleic acid (3, 6 and 12 energy%) on superoxide anion production of guinea pig alveolar macrophages and on relaxation of tracheal spirals upon beta-adrenergic receptor stimulation. An additional dietary group receiving fish oil (3 energy%) in addition to 3 energy% linoleic acid was also included in this study. The zymosan-stimulated (but not the basal) superoxide anion production of the alveolar macrophages differed between the dietary groups. Highest superoxide production was found in those diets that showed the greatest deterioration of beta-adrenergic receptor function. Moreover, a highly significant positive correlation was demonstrated between the amount of generated superoxide and the severity of the attenuation of the beta-adrenergic receptor function in the individual animals. These data suggest that optimal dietary conditions could lead to an improved lung function possibly via modulation of alveolar macrophage radical production.

Effects of dietary linoleic acid on beta-adrenergic responsiveness of the guinea pig respiratory system.

Respiratory autonomic beta-adrenergic receptor function was investigated in isolated tracheal spirals of guinea pigs receiving different diets. Comparison was made between control and Haemophilus influenzae treated animals; this latter group serving as animal model for atopy. The different semi-synthetic diets (35 energy%) varying in their linoleic acid contents (5.85, 11.25 and 22.05 en%), exerted profound effects on membrane fatty acid composition. No influence of these diets on either food intake or growth could be detected. Isoprenaline induced relaxation of guinea pig tracheal spirals was maximal in the dietary group receiving moderate linoleic acid (11.25 en%). Both the addition and the withdrawal of linoleic acid to this diet resulted in a significant impairment of beta-adrenergic receptor function, to the same extent as can be induced by Haemophilus influenzae. The results are discussed in view of current concepts for atopy.

Effects of dietary poly-unsaturated fatty acids on tracheal histaminergic and cholinergic responsiveness in experimental models of bronchial hypersensitivity and hyperreactivity.

Respiratory histaminergic and cholinergic receptor function was investigated in isolated tracheal spirals of guinea pigs receiving different diets. Comparison was made between saline treated (controls) and Haemophilus influenzae treated animals in non sensitized conditions, the latter being a model for bronchial hyperreactivity, and in sensitized conditions, being a model for allergen induced bronchial hypersensitivity. The different semi-synthetic diets (35 energy% fat), varying in linoleic acid content (5.85, 11.25 and 22.05 en% fat) and one diet with low linoleic acid (3.55 en%) in which linolenic acid was added additionally (5.30 en%), exerted profound effects on tracheal reactivity to histamine. In sensitized animals the maximal induced histamine contraction was significantly diminished in the dietary group receiving 5.85 en% linoleic acid as compared with the other dietary groups (35% decrease in the H. influenzae-treated, 20-30% decrease in saline treated animals). Results in non-sensitized animals were similar, though less pronounced. No effect on food intake or growth of the animals could be demonstrated during the six week experimental periods. The carbachol induced contraction of the tracheal spirals of sensitized animals receiving low linoleic acid was also significantly decreased as compared to the other dietary groups (30% for saline treated and 20-30% for H. influenzae-treated animals). No difference in carbachol responsiveness could be detected between the different dietary groups under non-sensitized conditions. The results are discussed in view of the current concepts for bronchial hyperreactivity, especially in relation to eicosanoid involvement.

Cell cycle-dependent inhibition of human vascular smooth muscle cell proliferation by prostaglandin E1.

We examined the influence of prostaglandins on the initiation of proliferation of growth-arrested human adult aortic and fetal smooth muscle cells. Prostaglandins of the E series (25 nM) exerted a significant (p less than or equal to 0.05) inhibitory effect on DNA synthesis. Inhibition was observed when PGE1 was added in the G1 phase of the cell cycle. PGE1 had no effect when added once DNA synthesis had started. Thus prostaglandins of the E series may inhibit the responsiveness of smooth muscle cells to the mitogenic action of critical growth factors, such as PGDF. This inhibitory response is cell-cycle dependent. Once smooth muscle cells have entered S phase, PGE1 is no longer effective. Our data also suggest that cAMP is involved in the PGE1-induced growth inhibition, since concomitant with PGE1 addition, cAMP levels rose rapidly; addition of the cAMP analogue db-cAMP resulted in a cell-cycle-dependent inhibition pattern comparable to that observed with PGE1.

Thrombin-induced release of von Willebrand factor from endothelial cells is mediated by phospholipid methylation. Prostacyclin synthesis is independent of phospholipid methylation.

The biochemical events that lead to thrombin-stimulated release of von Willebrand factor and prostacyclin synthesis in cultured endothelial cells are examined. Treatment of human umbilical vein endothelial cells with thrombin results in an instantaneous increase in phospholipid methylation which can be blocked by 3-deazaadenosine, a methyltransferase inhibitor. 3-Deazaadenosine also blocks the thrombin-induced Ca2+ influx into endothelial cells and the release of von Willebrand factor, indicating that these processes are coupled. The phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) and the Ca2+ ionophore A23187 both bypass the phospholipid methylation and directly stimulate Ca2+ influx and von Willebrand factor release. In contrast to the stimulus-induced von Willebrand factor release, the thrombin-induced prostacyclin synthesis cannot be blocked by 3-deazaadenosine. Similarly, incubation of endothelial cells with EDTA has no influence on the thrombin-induced prostacyclin synthesis, and PMA has no stimulatory effect on prostacyclin synthesis. These observations indicate that thrombin induces different metabolic responses in endothelial cells: phospholipid methylation followed by a Ca2+ influx, which subsequently leads to release of von Willebrand factor, and liberation of arachidonic acid from phospholipids for prostacyclin formation, which is independent of phospholipid methylation and Ca2+ influx.

Isolation of a storage and secretory organelle containing Von Willebrand protein from cultured human endothelial cells.

Von Willebrand protein was synthesized and secreted by human endothelial cells in culture. Ca2+ ionophore A23187 and phorbol myristate acetate stimulated the release of Von Willebrand protein from the cultured cells. Stimulated release was accompanied by the disappearance of rod-like structures from the cultured endothelial cells immunostained for Von Willebrand protein, suggesting the existence of a storage organelle for Von Willebrand protein in these cells (Loesberg, C., Gonsalves, M.D., Zandbergen, J., Willems, C., Van Aken, W.G., Stel, H.V., Van Mourik, J.A. and De Groot, P.G. (1983) Biochim. Biophys. Acta 763, 160-168). Cultured human endothelial cells were fractionated on a density gradient of colloidal silica. Von Willebrand protein was found in two organelle populations: a buoyant one sedimenting with a variety of cell organelle marker enzymes, including those of the Golgi apparatus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum and plasma membrane fragments (peak density of this fraction: 1.08 g X ml-1), and a dense one with a peak density of 1.12 g X ml-1. The dense organelles containing Von Willebrand protein were apparently free of other organelles. Stimulating Von Willebrand protein release with phorbol myristate acetate or Ca2+ ionophore A23187 resulted in a decrease or even complete disappearance of Von Willebrand protein from the high-density organelle fraction, implying a role of this organelle in the stimulus-induced release of Von Willebrand protein. The Von Willebrand protein content of the buoyant fraction was lowered to some extent or did not change upon incubation of the cells with ionophore A23187 and phorbol myristate acetate. Restoration of Von Willebrand protein content of the dense organelle fraction after stimulation occurred within 2 days; this was accompanied by recurrence of immunostaining of rod-shaped structures in cells and an increase in cellular Von Willebrand protein. The excretion of restored Von Willebrand protein could be stimulated again.

The effect of calcium on the secretion of factor VIII-related antigen by cultured human endothelial cells.

Cultured human endothelial cells derived from the umbilical cord vein are able to release factor VIII-related antigen into the culture medium. The experiments described in this paper show the presence of two pathways for the secretion of factor VIII-related antigen from endothelial cells. There is a basal release of this antigen, independent of the presence of extracellular calcium ions. This release can be inhibited by cycloheximide and is therefore directly related to de novo protein synthesis. Besides this basal release, there is an extra release of factor VIII-related antigen that can be stimulated by thrombin, the Ca2+-ionophore A23187 or phorbol myristate acetate. As demonstrated by immunofluorescence, the stimulus-inducible release originates from storage granules in the cells. This stimulus-inducible release is dependent on extracellular Ca2+ but independent of intracellular cAMP.

The effects of 3',5' adenosine monophosphate on the proliferation of Reuber H35 rat hepatoma cells in vitro.

3',5' Adenosine monophosphate (cAMP) inhibits the proliferation of Reuber H35 rat hepatoma cells at concentrations higher than 10(-5) M. This inhibitory effect can be demonstrated both in exponentially growing monolayer cultures and in single cell clonal growth. The inhibition of the cell proliferation is due to both a short term (division delay) and a long term effect (cytotoxicity). The short term effect seems to be due to cAMP itself as it is potentiated by the phosphodiesterase inhibitor 1-methyl,3-isobutyl xanthine (MIX). The long term effect probably is due to degradation products of the cyclic nucleotide. It is shown by a combination of time lapse cinematography, autoradiography, and flow cytofluorometry that the division delay is due to a prolongation of the S phase with no apparent changes in the duration of the G1 phase. The possible causes of this prolongation of the S phase by cAMP are discussed.