Effects of DIDS, a disulfonic stilbene derivative, on chloride movements in toad skeletal muscles.

In order to investigate the characteristics of the movement of Cl- ions in toad skeletal muscles we decided to study the relative membrane permeabilities of chloride and nitrate and the effects of DIDS (4,4'-diisothyocyanatostilbene-2,2'-disulphonate) upon the hyperpolarizations produced in muscle fibers when chloride or nitrate ions rapidly replace impermeant sulphate ions in the external solution. For experiments where membrane potential changes were recorded in response to sudden changes in extracellular solutions, small bundles from the semitendinosus muscles were used. We showed that DIDS reduced in a reversible manner the Cl- permeability (pCl) in toad skeletal muscle fibers. The results supporting this conclusion were the following. First, a diminished hyperpolarization in response to a sudden exposure of the fibers to a solution containing Cl-. In these experiments DIDS reduced the pCl/pK ratio to 5.5 from a control value of 12. Second, a smaller transient of the resting potential when [Cl]o was changed from 120 to 30 mM and vice versa.     

Inhibition of complement-mediated functions of human neutrophils by impermeant stilbene disulfonic acids

The impermeant labeling reagents 4,4'-diisothiocyanostilbene-2-2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid (SITS) inhibited in a concentration-related manner the enhanced generation of superoxide radicals (O2) by human neutrophils engaged in the phagocytosis of zymosan that had been opsonized in fresh serum, without altering the O2 generation by neutrophils exposed to zymosan opsonized in heat-decomplemented serum or to phorbol myristate acetate (PMA). That the stimulus specificity of the suppression of O2 generation by SITS and DIDS is predominantly attributable to an action on neutrophil plasma membrane receptors for complement was suggested by the similarity of the concentration dependence of the inhibition of the expression of neutrophil C3b receptors, as assessed by a rosetting assay. Washing neutrophils that had been pretreated with the covalent label DIDS failed to reverse either the suppression of C3b-dependent rosetting or the inhibition of O2 generation stimulated by opsonized zymosan. In contrast, pretreatment with DIDS and washing or erythrocytes bearing C3b and of opsonized zymosan did not inhibit their capacity to form rosettes and to stimulate O2 generation by neutrophils, respectively. In the same rosetting assay, the expression of IgG-Fc receptors was unaffected by SITS and DIDS. The rapid and apparently selective inhibition of the expression of neutrophil C3b receptors by noncytotoxic concentrations of the impermeant stilbene disulfonic acids may provide a means to analyze the complement dependence of other neutrophil effector functions.     

Sodium regulation of alpha 2-adrenoreceptors of human platelets: inactivation by 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS)

In the present study, we investigated the effect of DIDS on Na+ regulation of alpha 2-adrenoreceptors in human platelets. Pretreatment of platelet membranes at 23 degrees C for 60 min with DIDS produced a reduction in the affinity of the receptors for both antagonist and agonist in a concentration related manner; however, there was a marked difference in the degree of reduction in the affinity of the receptors for antagonist and agonist. Thus, at 1 mM concentration of DIDS, the affinity of the receptors for antagonist was reduced by 2-fold while the affinity of the receptors for agonist was reduced by 14-fold. Furthermore, this concentration of DIDS abolished the ability of Na+ in reducing the affinity of the receptors for agonist. We suggest that the effect of DIDS is via Na+ binding component of the alpha 2-adrenoreceptor-adenylate cyclase complex.     

Effect of disulfonic stilbene anion-channel blockers on the guinea-pig myocardium

The role of anions in the maintenance of tension in electrically driven left atria isolated from guinea pigs has been examined. The disulfonic stilbene anion-channel blockers SITS (4-acetamido-4'-isothiocyanostilbene 2'-disulfonate) and DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate) decreased the contractile force developed in a time- and concentration-dependent manner. As in the red cell anion channel, DIDS was more potent than SITS, but the maximal inhibition of tension produced by N-(4-azido-2-nitrophenyl)-2-aminoethyl sulfonate (NAP-taurine) was considerably lower than the near maximal inhibition produced by SITS and DIDS. The inhibition by SITS and DIDS was irreversible, suggesting a covalent interaction, and could not be overcome by increasing the calcium concentration or the frequency of stimulation. Consistent with a requirement for chloride anion, substitution of chloride and bicarbonate by the impermeant anion gluconate did not support contraction, while only partial tension was maintained with the lipophilic anions acetate and thiocyanate. Incubation of atria with 400 microM SITS blocked both 36Cl and 45Ca uptake to a similar extent, whereas the efflux of both these ions was not affected by incubation of the atria with SITS. The blockade by disulfonic stilbene anion-channel blockers of the contraction of the guinea pig myocardium may result from impairment of excitation-contraction coupling.     

Lipoic acid stimulates cAMP production in T lymphocytes and NK cells

The anti-oxidant lipoic acid (LA) potently suppresses clinical and pathologic disease in the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis, by inhibiting the migration of pathogenic T cells to the spinal cord. The mechanism by which this occurs is largely unknown. In this report we demonstrate that LA induces increases in cyclic AMP, a known immunosuppressant, in human T cells. The increase in cAMP is associated with increased adenylyl cyclase activity and is partially blocked by prostanoid receptor antagonists. We present evidence that LA also stimulates cAMP production in natural killer (NK) cells. This novel mechanism of action is highly relevant to the immunomodulatory effects of LA and provides further support for the study of LA as a therapeutic agent for multiple sclerosis and other autoimmune diseases.     

Complement subcomponent C1q stimulates Ig production by human B lymphocytes

The regulation of Ig production by human B lymphocytes is a complex process involving interactions among B cells, APC, T lymphocytes and soluble factors including activation, growth, and differentiation factors. Components of the complement system, including C3a, C3b, C3d, and C5a, have been shown to influence various stages in this process. In this study, we demonstrate that the C1q subcomponent of complement binds to both small resting and large activated B cells and stimulates immunoglobulin production by Staphylococcus aureus Cowan-activated tonsillar B lymphocytes. This effect is present whether C1q is added to the B cells either at the beginning or near the end of a 7-day culture period and is not associated with enhancement of proliferation. The C1q stimulation of Ig production is, however, associated with increased steady state levels of mRNA for the mu Ig H chain. Furthermore, C1q stimulated IgM production by the human B cell line SKW 6.4, which is capable of secreting IgM in response to B cell differentiation factors (BCDF). SLE is a disorder frequently associated with polyclonal activation of B lymphocytes. We studied the effect of C1q on B cells from two patients with this disorder and one with an SLE-like illness, all selected for the predominance of either IgM or IgG in serum. Spontaneous or BCDF-stimulated Ig secretion was of the isotype predominant in vivo, whereas C1q selectively stimulated B cells to produce the other isotype (IgG vs IgM). Thus, C1q interacts with B lymphocytes in a manner distinct from that of BCDF found in mixed lymphocyte supernatants. C1q may be an important factor influencing the production of Ig by B lymphocytes in normal individuals and in patients with abnormalities of B cell activity.     

Lysozyme stimulates immunoglobulin production by human-human hybridoma and human peripheral blood lymphocytes

Lysozyme [EC 3.2.1.17] derived from hen egg white stimulated immunoglobulin production by human-human hybridoma, HB4C5 cells producing human lung cancer specific monoclonal IgM. IgM production by HB4C5 cells was enhanced more than 13-fold by the addition of lysozyme at 380 μg/ml in a serum-free medium. The immunoglobulin production stimulating effect of lysozyme was observed immediately after inoculation and maintained for 5 days. Lysozyme enhanced immunoglobulin production by the hybridoma line without growth promotion. This enzyme also accelerated IgM and IgG production of human peripheral blood lymphocytes 5.3-fold and 2.3-fold, respectively. These results suggest that lysozyme stimulates immunoglobuling production of not only specific hybridoma line, but also non-specific immunoglobulin producers. However, although the enzymatic activity of lysozyme was almost lost by heat-treatment at 100 °C for 30 min, the IPSF activity was retained. This fact suggests that IPSF activity of lysozyme does not come from its enzymatic activity or reaction products. All these findings clearly indicate that lysozyme has a novel function as an immunoglobulin production stimulating factor. GAPDH - glyceraldehyde-3-phosphate dehydrogenase; Ig - immunoglobulin; IPSF - immunoglobulin production stimulating factor; PBL - peripheral blood lymphocytes; HPLC - high-performance liquid chromatography. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of an anion channel blocker, 4, 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), on human neutrophil function

We investigated the capacity of DIDS to influence degranulation and superoxide anion (O-2) generation by human neutrophils exposed to soluble, surface-active stimuli. DIDS caused a concentration-related (25-200 microM) inhibition of myeloperoxidase (MPO) release from N-formyl-methionyl-leucyl-phenylalanine (FMLP), 5(S), 12(R)-dihydroxy-6, 14-cis-8, 10-trans-eicosatetraenoic acid (LTB4), 1-0-hexadecyl/octadecyl-2-0-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) and phorbol myristate acetate (PMA)-stimulated neutrophils. In contrast, DIDS had no effect on O-2 production by FMLP and PMA activated cells, whereas it enhanced LTB4 and AGEPC-elicited O-2 generation. The respective effects of DIDS on these neutrophil activities were reversed by washing the cells prior to exposure to stimulus. Thus, DIDS represents a useful pharmacologic probe for investigating the role(s) of anions in the mechanisms of neutrophil activation with structurally and chemically dissimilar stimuli.     

The nature of the membrane sites controlling anion permeability of human red blood cells as determined by studies with disulfonic stilbene derivatives

Summary The disulfonic acid stilbene derivative SITS reported to be covalently bonded to the membrane of the red blood cell, was found to be largely reversibly bound. Reversal of its specific inhibitory effect on anion permeability was attained by washing the cells with buffer containing albumin. The small fraction of covalently bonded SITS could be increased by prolonging the time of exposure of the cells or by multiple exposures. A series of other disulfonic stilbene derivatives was synthesized. All of them specifically inhibited anion permeability whether or not they are capable of forming covalent bonds. Their inhibitory effectiveness, however, varied over a 5,000-fold range, allowing certain conclusions to be made concerning the chemical architecture of the binding site. Certain of the compounds were almost entirely covalently bonded. One of them was labeled with¹²⁵I and used to determine to which membrane proteins the compound is bound. Over 90% was found in a protein band on acrylamide gels of 95,000 mol wt. The most effective compound against sulfate permeability was equally effective against chloride permeability, producing a maximum inhibition of over 95%. The residual anion fluxes respond differently to pH and temperature than do the fluxes of unmodified cells.This paper is based on work performed in part under contract with the U.S. Atomic Energy Commission at the University of Rochester Atomic Energy Project (Report No. UR 3490-199) and in part by the Medical Research Council of Canada, Grant No. MA-4665. Z. I. Cabantchik was the recipient of an International Atomic Energy Agency (Vienna) Fellowship.     

An oxidized derivative of linoleic acid stimulates dehydroepiandrosterone production by human adrenal cells.

We previously reported that an oxidized derivative of linoleic acid stimulated steroidogenesis in rat adrenal cells. This derivative was also detected in human plasma, and was positively correlated with visceral adiposity and plasma DHEA-S. The present study sought to characterize the effects of this derivative, 12,13-epoxy-9-keto-(10- trans)-octadecenoic acid (EKODE), on steroid production by normal human adrenocortical cells obtained during clinically-indicated adrenalectomy. Cell suspensions were incubated in the presence of varying concentrations of EKODE and ACTH. EKODE (16 microM) significantly increased DHEA production by 28% under basal conditions and by 25% in the presence of a low concentration of ACTH (0.2 ng/ml). The effect on DHEA was absent at a higher ACTH concentration (2.0 ng/ml). EKODE decreased cortisol production by 16% (low ACTH) and 25% (high ACTH), but was without effect on cortisol under basal conditions. The results suggest that EKODE affects adrenal DHEA production in the human, possibly by modulating steroidogenic enzyme activity. We postulate that excess visceral fat delivers fatty acids to the liver, where oxidized derivatives are formed that modulate adrenal steroidogenesis. This may be an important phenomenon in the genesis of changes in adrenal function associated with syndromes of obesity, especially those that include androgen excess.     

The adenosine system selectively inhibits TLR-mediated TNF-alpha production in the human newborn

Human newborns are susceptible to microbial infection and mount poor vaccine responses, yet the mechanisms underlying their susceptibility are incompletely defined. We have previously reported that despite normal basal expression of TLRs and associated signaling intermediates, human neonatal cord blood monocytes demonstrate severe impairment in TNF-alpha production in response to triacylated (TLR 2/1) and diacylated (TLR 2/6) bacterial lipopeptides (BLPs). We now demonstrate that in marked contrast, BLP-induced synthesis of IL-6, a cytokine with anti-inflammatory and Th2-polarizing properties, is actually greater in neonates than adults. Remarkably, newborn blood plasma confers substantially reduced BLP-induced monocyte synthesis of TNF-alpha, while preserving IL-6 synthesis, reflecting the presence in neonatal blood plasma of a soluble, low molecular mass inhibitory factor (<10 kDa) that we identify as adenosine, an endogenous purine metabolite with immunomodulatory properties. The neonatal adenosine system also inhibits TNF-alpha production in response to whole microbial particles known to express TLR2 agonist activity, including Listeria monocytogenes, Escherichia coli (that express BLPs), and zymosan particles. Selective inhibition of neonatal TNF-alpha production is due to the distinct neonatal adenosine system, including relatively high adenosine concentrations in neonatal blood plasma and heightened sensitivity of neonatal mononuclear cells to adenosine A3 receptor-mediated accumulation of cAMP, a second messenger that inhibits TLR-mediated TNF-alpha synthesis but preserves IL-6 production. We conclude that the distinct adenosine system of newborns polarizes TLR-mediated cytokine production during the perinatal period and may thereby modulate their innate and adaptive immune responses.     

Alcohol dehydrogenase-I from horse liver stimulates immunoglobulin production by human hybridoma and human peripheral blood lymphocytes

Immunoglobulin production stimulating activity of alcohol dehydrogenase[EC 1.1.1.1] was assessed. Alcohol dehydrogenase-I (ADH-I) derived fromhorse liver stimulated IgM production by human-human hybridoma, HB4C5 cellsproducing human lung cancer specific monoclonal IgM. IgM production of HB4C5cells was enhanced more than 6 fold by the addition of ADH-I at 400µg/ml under serum-free condition. However, yeast derived ADHs, such asADH-II and -III were ineffective to accelerate immunoglobulin production ofthe hybridoma line. These results imply that the immunoglobulin productionstimulating effect of ADH-I is irrelevant to its enzymatic function, anddefined as a novel feature of ADH-I. This enzyme also stimulated IgM and IgGproduction by human peripheral blood lymphocytes 2.9 fold and 1.4 fold,respectively . This fact suggests that ADH-I stimulates immunoglobulinproduction not only by specific hybridoma cell line, but also bynon-specific immunoglobulin producers.     

Nicotinamide stimulates repair of DNA damage in human lymphocytes

Nicotinamide stimulates the amount of DNA repair synthesis that occurs when freshly isolated, normal human lymphocytes are treated with UV irradiation, N-methyl-N′-nitro-N-nitroso guanidine, or dimethyl sulfate. Stimulation of DNA repair synthesis is concentration dependent and reaches a maximum between 2 to 5 mM nicotinamide. In contrast, DNA synthesis in cells that have not been subjected to DNA damage is not affected by nicotinamide at concentrations below 2 mM and is inhibited by concentrations between 2 to 5 mM. In the same concentration range, nicotinic acid has no effect on the rate of DNA synthesis in the presence or absence of DNA damage.     

Hemin stimulation of cAMP production in human lymphocytes.

Hemin stimulates cAMP production in human peripheral blood mononuclear cells (PBMC). The kinetics are similar to that of hormone-induced cAMP generation, namely a rapid effect followed by a desensitization phase. Several experimental findings suggest that prostaglandins do not mediate this effect. First, macrophage depleted T and B cells purified by erythrocyte-rosetting were as responsive as unfractionated PBMC to hemin. Second, indomethacin, an inhibitor of prostaglandin synthesis, and meclofenamate, a prostaglandin E2 receptor antagonist, had no effect on hemin stimulated cAMP production. In addition, propranolol, a beta-adrenergic receptor antagonist, had no effect on hemin-stimulated cAMP production. We also examined structural analogues of hemin. Among the metalloporphyrins (Fe, Ni, Co, Zn and Sn) and protoporphyrin IX tested only hemin (Fe-protoporphyrin) was active in stimulating cAMP production. No correlation was found between the ability of metalloporphyrins to stimulate cAMP production and their ability to generate H2O2. The data indicate that hemin stimulates cAMP production by directly affecting lymphocytes and that prostaglandins do not mediate this effect.     

Voltage-dependent chloride conductance of the squid axon membrane and its blockade by some disulfonic stilbene derivatives

When giant axons of squid, Sepioteuthis, were bathed in a 100 mM Ca-salt solution containing tetrodotoxin (TTX) and internally perfused with a solution of 100 mM tetraethylammonium-salt (TEA-salt) or tetramethylammonium-salt (TMA-salt), the membrane potential was found to become sensitive to anions, especially Cl-. Membrane currents recorded from those axons showed practically no time-dependent properties, but they had a strong voltage-dependent characteristic, i.e., outward rectification. Cl- had a strong effect upon the voltage-dependent membrane currents. The nonlinear property of the currents was almost completely suppressed by some disulfonic stilbene derivatives applied intracellularly, such as 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which are blockers of chloride transport. On the basis of these experimental results, it is concluded that a voltage-dependent chloride-permeable channel exists in the squid axon membrane. The chloride permeability (PCl) is a function of voltage, and its value at the resting membrane (Em = -60 mV) is calculated, using the Goldman-Hodgkin-Katz equation, to be 3.0 X 10(-7) cm/s.     

Thrombin stimulates the production of phosphatidylinositol 3,4-bisphosphate in human erythroleukemia cells.

The human erythroleukemic cell line, HEL, which has numerous platelet markers, shows enhanced inositol phosphate production in response to thrombin. We investigated the production of phosphoinositides in HEL cells and showed that thrombin stimulates the turnover of several phosphoinositides including the synthesis of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). Phosphatidylinositol 3-monophosphate is also produced in HEL cells and its synthesis is not stimulated by thrombin. Pretreatment of HEL cells with the stable prostacyclin analog iloprost inhibits the thrombin-induced increase in the production of PtdIns(3,4)P2. 3-Phosphorylated phosphoinositides have been implicated in signal transduction and regulation of cell proliferation in other cells and may be involved in signal transduction in HEL cells.