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Biomedical subjects

J D Laskin

Publications and source records attributed to J D Laskin.

At least 55 records · Page 3Linked to original sources
PubMed1994

Inhibition of skin tumorigenesis by rosemary and its constituents carnosol and ursolic acid.

A methanol extract of the leaves of the plant Rosmarinus officinalis L. (rosemary) was evaluated for its effects on tumor initiation and promotion in mouse skin. Application of rosemary to mouse skin inhibited the covalent binding of benzo(a)pyrene [B(a)P] to epidermal DNA and inhibited tumor initiation by B(a)P and 7,12-dimethylbenz[a]anthracene (DMBA). Topical application of 20 nmol B(a)P to the backs of mice once weekly for 10 weeks, followed 1 week later by promotion with 15 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) twice weekly for 21 weeks, resulted in the formation of 7.1 tumors per mouse. In a parallel group of animals that were treated topically with 1.2 or 3.6 mg of rosemary 5 min prior to each application of B(a)P, the number of tumors per mouse was decreased by 54 or 64%, respectively. Application of rosemary to mouse skin also inhibited TPA-induced ornithine decarboxylase activity, TPA-induced inflammation, arachidonic acid-induced inflammation, TPA-induced hyperplasia, and TPA-induced tumor promotion. Mice initiated with 200 nmol DMBA and promoted with 5 nmol TPA twice weekly for 19 weeks developed an average of 17.2 skin tumors per mouse. Treatment of the DMBA-initiated mice with 0.4, 1.2, or 3.6 mg of rosemary together with 5 nmol TPA twice weekly for 19 weeks inhibited the number of TPA-induced skin tumors per mouse by 40, 68, or 99%, respectively. Topical application of carnosol or ursolic acid isolated from rosemary inhibited TPA-induced ear inflammation, ornithine decarboxylase activity, and tumor promotion. Topical application of 1, 3, or 10 mumol carnosol together with 5 nmol TPA twice weekly for 20 weeks to the backs of mice previously initiated with DMBA inhibited the number of skin tumors per mouse by 38, 63, or 78%, respectively. Topical application of 0.1, 0.3, 1, or 2 mumol ursolic acid together with 5 nmol TPA twice weekly for 20 weeks to DMBA-initiated mice inhibited the number of tumors per mouse by 45-61%.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1994

Osteopontin inhibits induction of nitric oxide synthase gene expression by inflammatory mediators in mouse kidney epithelial cells.

We report that osteopontin (OPN), a secreted, Arg-Gly-Asp-containing phosphoprotein expressed at high levels in the kidney, suppresses nitric oxide (NO) synthesis induced by the inflammatory mediators gamma-interferon and lipopolysaccharide in primary mouse kidney proximal tubule epithelial cells. Northern blot and immunofluorescence analyses of inducible nitric oxide synthase (iNOS) expression revealed that the inflammatory mediators increased iNOS mRNA and protein levels. Recombinant human OPN (purified from both mammalian cells and from Escherichia coli) inhibited this response by a process that was blocked by anti-OPN antiserum and by the peptide GRGDS, but not GRGES. The data suggest that inhibition of NO synthesis by OPN in these kidney cells is mediated by an integrin, possibly the alpha v beta 3 integrin, which is known to be an OPN receptor. NO is believed to control blood flow through the glomerulus, regulating salt and water balance, and to be important as a defense against tumor cells and infecting microorganisms. The ability of OPN to inhibit the induction of iNOS suggests that OPN may be an important regulator of the NO signaling pathway and NO-mediated cytotoxic processes.

Amino Acid Oxidoreductases↗
PubMed1994

Induction of hepatic Ito cell nitric oxide production after acute endotoxemia.

Nitric oxide is a highly reactive mediator released in the liver by hepatocytes, Kupffer cells and endothelial cells during endotoxin-induced inflammation. In this study we determined whether Ito cells also produce nitric oxide after exposure to endotoxin. For induction of endotoxemia, rats were injected intravenously with Escherichia coli lipopolysaccharide (2.5 mg/kg). Ito cells were isolated from the animals 48 hr later by means of in situ perfusion of the liver with protease and collagenase followed by purification on an arabinogalactan gradient. Ito cells from untreated and endotoxemic rats were found to produce low levels of nitric oxide in response to interferon-gamma. In both cell types, this response depended on L-arginine and was blocked by NG-monomethyl-L-arginine, a specific nitric oxide synthase inhibitor. Cells from rats treated with endotoxin produced significantly more nitric oxide than did cells from untreated animals; this was due, at least in part, to increased expression of protein for an inducible form of nitric oxide synthase. These cells also responded to stimulation with lipopolysaccharide in vitro, as well as the combination of interferon-gamma and lipopolysaccharide, which was synergistic in stimulating nitric oxide production. Tumor necrosis factor-alpha and macrophage colony-stimulating factor were also found to stimulate nitric oxide production by Ito cells from endotoxemic rats. In addition, in these cells, tumor necrosis factor-alpha synergized with interferon-gamma in inducing nitric oxide production. The combination of interferon-gamma and lipopolysaccharide was also found to inhibit Ito cell DNA synthesis, as measured on the basis of [3H]-thymidine uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1994

Age and development-related changes in osteopontin and nitric oxide synthase mRNA levels in human kidney proximal tubule epithelial cells: contrasting responses to hypoxia and reoxygenation.

Osteopontin (OPN) encodes a secreted glycosylated phosphoprotein containing a GRGDS motif that can mediate cell attachment through the alpha v beta 3 integrin, and has recently been shown to down-regulate nitric oxide synthase (NOS) expression. We report here that primary cultures of renal proximal tubule epithelial (PTE) cells prepared from human kidneys of different developmental stages and ages show a positive correlation between developmental age and the expression, at the mRNA level, of both OPN and constitutive NOS. However, OPN and NOS responded in different manners, as assessed by mRNA measurements, to hypoxia-reoxygenation injury. The OPN mRNA level, assessed by Northern blotting, increased slightly during 60 min of hypoxia and more substantially during subsequent reoxygenation of primary PTE cells derived from the kidneys of young but not of aged donors. The abundance of NOS mRNA, measured using a cDNA probe to the constitutive form of the enzyme, was enhanced during hypoxia in kidneys derived from humans of all ages, and then decreased during reoxygenation--possibly as the result of increased OPN expression. PTE cells from aged kidneys are more susceptible to cell death under hypoxic conditions that PTE cells from young kidneys. An investigation of the effect of an oxidant on OPN and NOS mRNA levels revealed that within 30 min of exposure to H2O2, NOS mRNA levels decreased simultaneously with an increase in OPN mRNA levels. Nitric oxide (NO), the product of NOS, is at low levels an important signal transduction molecule participating in the regulation of vascular tone and renal reabsorption; at high levels it is cytotoxic. We suggest that the diminished ability of cells from old kidneys to down-regulate NO production and to increase OPN expression after hypoxia-reoxygenation may contribute to their increased susceptibility to oxidant injury.

Aging↗
PubMed1994

Production of nitric oxide by differentiated LSTRA cells is associated with expression of macrophage-inducible nitric oxide synthase.

LSTRA is a mouse lymphoma cell line overexpressing the src-related oncogene product and T cell marker p56lck. We have discovered that LSTRA cells, like HL60 and U937 promyelocytic leukemia cells, can be induced to differentiate toward macrophages by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) or granulocytes by the cyclic nucleotide analogue dibutyryl cAMP (dbcAMP). One property of mature macrophages and granulocytes is the ability to produce nitric oxide, a highly reactive free radical that is important in non-specific host defense. Nitric oxide production by mature macrophages is stimulated by inflammatory mediators via an inducible form of the enzyme, nitric oxide synthase. We report that LSTRA cells acquire the ability to produce nitric oxide following differentiation induced by TPA and, to a lesser extent, by dbcAMP. Nitric oxide production by the cells is dependent on L-arginine and blocked by several inhibitors of nitric oxide synthase, including N-monomethyl-L-arginine, L-canavanine, L-arginine benzyl ester, and L-arginine methyl ester. In macrophage-differentiated LSTRA cells, the inflammatory cytokine interferon-gamma was found to be a potent inducer of nitric oxide production. This was correlated with a marked increase in nitric oxide synthase activity in the cells that was due to interferon-gamma-induced expression of the macrophage-inducible form of nitric oxide synthase protein as well as mRNA. Differentiated LSTRA cells also expressed increased amounts of a constitutive form of nitric oxide synthase that was also present in undifferentiated cells. Taken together with previous findings, these results support the model that LSTRA cells have the capacity to differentiate toward mature macrophages.

Amino Acid Oxidoreductases↗
PubMed1994

Cellular depletion of p56lck during thymocyte apoptosis.

The src-related protein tyrosine kinase p56lck is thought to be important in regulating maturation and functional responsiveness of T cells and thymocytes. In the present studies we report that expression of p56lck is suppressed during apoptosis. Using primary cultures of rat thymocytes, we found that agents that are effective in inducing apoptosis, including okadaic acid, dexamethasone, and antibodies to the CD3 receptor, also deplete cells of p56lck. This process is rapid, occurring within 24 h, and is not due to cytotoxicity. Inhibition of DNA fragmentation in apoptotic cells with the endonuclease inhibitor ZnCl2 failed to prevent depletion of p56lck, suggesting that it was not a consequence of the DNA degradation process. Using the thymic lymphoma cell line LSTRA, apoptosis was also associated with cellular depletion of p56lck. In contrast to thymocytes, this process required 48-72 h possibly because these cells overexpress p56lck. Although at this time we are uncertain as to the precise role of p56lck in the process of apoptosis, our results indicate that changes in the expression of this protein in thymocytes is an important marker of programmed cell death.

Animals↗
PubMed1994

Distinct patterns of nitric oxide production in hepatic macrophages and endothelial cells following acute exposure of rats to endotoxin.

Hepatic macrophages and endothelial cells play an important role in the clearance of endotoxin from the portal circulation. These cells are activated by endotoxin to release reactive mediators including superoxide anion, hydrogen peroxide, and nitric oxide, which have been implicated in hepatic inflammation and tissue injury. In the present studies we analyzed mechanisms regulating the production of nitric oxide by hepatic macrophages and endothelial cells following in vivo exposure to endotoxin. Rats were injected intravenously with Escherichia coli lipopolysaccharide (LPS, 5 mg/kg). Cells were isolated from the animals 48 h later by in situ perfusion of the liver with collagenase and pronase followed by differential centrifugation and centrifugal elutriation. We found that macrophages and endothelial cells from both untreated and endotoxin-treated rats readily synthesized nitric oxide following in vitro stimulation with interferon-gamma (IFN-gamma) and LPS alone and in combination. This response was dependent on l-arginine and was blocked by two nitric oxide synthase inhibitors, NG-monomethyl-l-arginine and l-canavanine. Macrophages produced more nitric oxide in response to LPS or LPS plus IFN-gamma than endothelial cells. In addition, nitric oxide production by both cell types in response to LPS plus IFN-gamma was increased after treatment of rats with endotoxin. Macrophages appeared to be more sensitive than endothelial cells to the in vivo effects of this inflammatory stimulus. Northern and Western blot analysis demonstrated that nitric oxide production by macrophages and endothelial cells in response to LPS plus IFN-gamma was due to increased expression of an inducible form of nitric oxide synthase (iNOS) mRNA and protein. Using fluorescence image analysis, iNOS protein was found to be localized in the cytoplasm of the cells. Treatment of rats with endotoxin was associated with increased expression of iNOS protein in the macrophages. The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also stimulated nitric oxide production by macrophages and endothelial cells from endotoxin-treated rats, although not as effectively as LPS and IFN-gamma. Macrophages were more responsive than endothelial cells to TPA. Furthermore, depletion of the cells of glutathione using buthionine sulfoximine had no major effect on nitric oxide production by macrophages but resulted in small but significant inhibition in endothelial cells. This suggests that this sulfhydryl-containing tripeptide does not regulate intracellular levels of reactive nitrogen intermediates in activated macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Oxidoreductases↗
PubMed1994

Production of nitric oxide and peroxynitrite in the lung during acute endotoxemia.

Nitric oxide is a short-lived cytotoxic mediator that has been implicated in the pathogenesis of endotoxin-induced tissue injury and septic shock. In the present studies we determined whether this mediator is produced in the lung during acute endotoxemia. We found that intravenous injection of rats with bacterially derived lipopolysaccharide (LPS), a condition that induces acute endotoxemia, caused a time-dependent increase in inducible nitric oxide synthase (iNOS) mRNA expression in the lung, which reached a maximum after 24 h. This was correlated with nitric oxide production in the lung as measured by electron paramagnetic spin trapping, which was detectable within 6 h. Alveolar macrophages (AMs) and interstitial macrophages (IMs) isolated from rats 6-12 h after induction of acute endotoxemia were also found to exhibit increased nitric oxide production in response to in vitro stimulation with interferon-gamma (IFN-gamma) and LPS measured by nitrite accumulation in the culture medium. The effects of acute endotoxemia on nitric oxide production by these cells were, however, transient and returned to control levels by 24 h in AMs and 36 h in IMs. Interestingly, although nitrite accumulation in the culture medium of IMs isolated 48 h after induction of acute endotoxemia and stimulated with low concentrations of IFN-gamma and LPS was reduced, when compared with cells from control animals, these cells, as well as AMs, continued to express high levels of iNOS protein and mRNA. This was correlated with increased peroxynitrite production by the cells. Peroxynitrite has been shown to act as a nitrating agent and can generate nitrotyrosine residues in proteins. Using a specific antibody and immunohistochemistry, we found evidence of nitrotyrosine residues in sections of lungs 48 h after treatment of rats with endotoxin. These data suggest that nitric oxide produced by IMs and AMs can react with superoxide anion to form peroxynitrite. Taken together, the present studies demonstrate that AMs and IMs are activated following acute endotoxemia to produce reactive nitrogen intermediates and that both cell types contribute to inflammatory responses in the lung.

Amino Acid Oxidoreductases↗
PubMed1994

Cellular and molecular mechanisms in photochemical sensitization: studies on the mechanism of action of psoralens.

The interaction of chemicals and light to induce sensitization reactions in the skin is a complex multistep process resulting in physiological changes in both the dermal and epidermal cell layers as well as characteristic inflammatory reactions. It is becoming increasingly apparent that an array of growth factors and cytokines acting on different components of the skin are involved in the regulation of these processes. One of the best characterized classes of chemical photosensitizers are the psoralens, a group of compounds that must be activated by UV light in wavelengths ranging from 320 to 400 nm (UVA) to initiate their biological actions. Recent evidence suggests that the ability of the psoralens to induce sensitization reactions, which include alterations in epidermal cell growth and differentiation, is highly specific and due to interactions with the epidermal growth factor (EGF) receptor. Specific receptor proteins for the psoralens have been identified in cytoplasmic and membrane fractions of responsive cells. Binding of psoralens to these proteins is of high affinity and reversible. UVA light causes psoralens to photoalkylate their receptors, a process thought to activate the receptor. One early biochemical event at the cell surface membrane linked to psoralen-receptor activation is the inhibition of EGF binding and alterations in the structure and function of the EGF receptor. These findings suggest that the cell surface membrane is an important target for chemical photosensitizers such as the psoralens. In addition, since photoactivated psoralens modulate epidermal cell growth and differentiation, the ability of these compounds to modify the function of the EGF receptor may underlie their biological activity as chemical photosensitizers.

Animals↗
PubMed1994

Production of nitric oxide by rat type II pneumocytes: increased expression of inducible nitric oxide synthase following inhalation of a pulmonary irritant.

Nitric oxide is a highly reactive molecule that has been implicated in host defense and tissue injury. In the present studies, we determined whether rat type II alveolar epithelial cells have the capacity to produce this mediator. We found that type II cells synthesize significant quantities of nitric oxide after treatment with the inflammatory cytokines, interferon-gamma (IFN-gamma) and/or interleukin-1 beta (IL-1 beta), or with the combination of IFN-gamma and tumor necrosis factor-alpha. In contrast to rat alveolar macrophages, type II cells were unresponsive to lipopolysaccharide. Production of nitric oxide by type II cells in response to IFN-gamma was dose dependent, reaching a maximum at 100 U/ml, and blocked by NG-monomethyl-L-arginine (L-NMA), a nitric oxide synthase inhibitor. Northern blot analysis demonstrated that nitric oxide production by type II cells was due to expression of mRNA for an inducible form of nitric oxide synthase (iNOS). Following brief exposure of rats to irritant-inducing doses of ozone (2 ppm, 3 h), type II cells were found to produce significantly more nitric oxide than were cells from control animals. This was due to increased expression of iNOS mRNA. Cells from ozone-treated rats were also sensitized to produce more nitric oxide in response to IFN-gamma and IL-1 beta. This was associated with a marked increase in expression of iNOS mRNA and enzyme protein in the cells. We also found that ozone inhalation caused enhanced production of hydrogen peroxide, as well as spontaneous and IFN-gamma-induced cytostasis of type II cells toward P815 mouse mastocytoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Inhalation↗
PubMed1994

Enhanced production of interleukin-1, tumor necrosis factor-alpha, and fibronectin by rat lung phagocytes following inhalation of a pulmonary irritant.

Interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and fibronectin are macrophage-derived mediators thought to be important in the pathogenesis of lung injury, inflammation, and fibrosis. In the present studies, we examined the effects of acute exposure of rats to the pulmonary irritant, ozone (O3), on production of these mediators by lung phagocytes. Cells were isolated from lungs 48 h after exposure of rats to air or O3 (2 ppm, 3 h). We found that cells from O3-exposed rats released 2- to 3-fold more IL-1 and TNF-alpha into the culture medium than did cells from air-exposed rats. These effects were time dependent, reaching a maximum at 2 and 24 h for IL-1, and 2 to 4 h for TNF-alpha. We also found that alveolar macrophages from O3-treated rats produced increased amounts of fibronectin, both alone and in response to transforming growth factor-beta, lipopolysaccharide, and interferon-gamma when compared with cells from control rats. Examination of immunohistochemically stained tissue sections indicated increased IL-1, TNF-alpha, and fibronectin in lungs from O3-exposed animals when compared with control animals. IL-1 and TNF-alpha were localized in lung macrophages, whereas fibronectin was associated with blood vessel walls and the lung interstitium. These results demonstrate that lung phagocyte production of these inflammatory mediators is elevated following O3 exposure and suggest that they may play a role in oxidant-induced pulmonary inflammation and injury.

Administration, Inhalation↗
PubMed1994

Pulmonary and hepatic effects of inhaled ozone in rats.

Nitric oxide is a highly reactive molecule that has been implicated in host defense and in tissue injury. In the present studies we analyzed the effects of brief exposure of rats to inhaled ozone on production of this mediator by lung macrophages and type II epithelial cells. We found that ozone exposure (1-2 ppm, 3 hr) induced a marked increase in spontaneous nitric oxide production by alveolar (AM) and interstitial macrophages, as well as type II cells. These effects were apparently due to increased expression of inducible nitric oxide synthase protein and mRNA, which was evident in vitro in isolated cells and in situ in histologic sections. Macrophages and epithelial cells from ozone-treated rats were also sensitized to produce increased amounts of nitric oxide in response to inflammatory cytokines such as interferon-gamma, a response that was also mediated by inducible nitric oxide synthase. Unexpectedly, we also discovered that brief inhalation of ozone caused dramatic effects on the liver, including increased production of nitric oxide by hepatocytes and enhanced protein synthesis. These data suggest that this inhaled irritant induces an acute phase response. Additional studies indicate that AM from ozone-treated rats produced significantly more tumor necrosis factor-alpha and interleukin-1 than did cells from control animals. Elevated levels of tumor necrosis factor-alpha were also noted immunohistochemically in both lung and liver tissue. These results indicate that the extrapulmonary effects of ozone may be mediated by inflammatory cytokines released by activated lung macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Inhalation↗
PubMed1994

Alterations in expression of p56lck during myeloid differentiation of LSTRA cells.

The src-related tyrosine kinase p56lck is overexpressed in the mouse leukemia cell line LSTRA. Although p56lck is thought to be a specific T-cell marker, we found that LSTRA cells can be induced to differentiate towards macrophages or granulocytes by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate or the cyclic nucleotide analogue, dibutyryl cAMP, respectively. Treatment of LSTRA cells with 12-O-tetradecanoylphorbol-13-acetate resulted in marked alterations in morphology including increased size, adherence, and spreading on culture dishes. These cells also ceased proliferating, accumulated in G0-G1 and expressed nonspecific esterase activity. In contrast, although LSTRA cells treated with dibutyryl cAMP stopped growing and accumulated in G0-G1, these cells expressed functionally active chemotactic peptide receptors and became irregular and granular in appearance. Differentiation of LSTRA cells was also found to be associated with altered expression of p56lck. Thus, while 12-O-tetradecanoylphorbol-13-acetate treatment caused the cells to produce higher molecular weight forms of p56lck, dibutyryl cAMP treatment resulted in increased expression of total p56lck mRNA as well as the more mature type II p56lck mRNA transcript. There were no major alterations in p56lck kinase activity in vitro following differentiation. Phospholipase C gamma and p21rasGAP, two putative substrates for the tyrosine kinase activity of p56lck, were found to be constitutively phosphorylated on tyrosine in LSTRA cells. Tyrosine phosphorylation of these substrates was not altered following differentiation. These results indicate that LSTRA cells are relatively early precursors that have the capacity to develop along the myeloid differentiation pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Differentiation↗
PubMed1993

Enhanced production of nitric oxide by rat alveolar macrophages after inhalation of a pulmonary irritant is associated with increased expression of nitric oxide synthase.

Alveolar macrophages represent the first line of defense of the lung against inhaled environmental agents. These cells release a variety of inflammatory mediators including reactive oxygen and nitrogen intermediates that have been implicated in host defense and in tissue injury. In the present studies we characterized production of these mediators by lung phagocytes after exposure of rats to an inhaled pulmonary irritant. Freshly isolated alveolar macrophages from control rats were found to produce nitric oxide as well as hydrogen peroxide and superoxide anion in response to in vitro treatment with inflammatory mediators such as IFN-gamma or LPS and phorbol esters, respectively. Production of nitric oxide by lung phagocytes was enhanced in the presence of superoxide dismutase. Western blot analysis revealed that production of nitric oxide after treatment of the cells with IFN-gamma and LPS was a result of increased expression of inducible nitric oxide synthase. After brief exposure of rats to ozone (O3, 1 to 2 ppm, 3 h), a pulmonary irritant and inflammatory agent that is rapidly converted to molecular oxygen, lung phagocytes produced significantly increased amounts of nitric oxide when compared with control animals. These cells were also sensitized to produce more nitric oxide in response to in vitro treatment with IFN-gamma and LPS. This was due, at least in part, to increased expression of inducible nitric oxide synthase by the cells, which was evident in protein blots and in immunohistochemically stained sections of lung tissue. In further studies we found that O3 inhalation also caused enhanced production of hydrogen peroxide, but an apparent decrease in release of superoxide anion by lung phagocytes. Taken together, these data demonstrate that acute irritant exposure modifies production of reactive oxygen and nitrogen intermediates by lung phagocytes. These alterations may be important in the pathophysiologic response of the lungs to irritants.

Amino Acid Oxidoreductases↗
PubMed1993

Induction of functionally active platelet-activating factor receptors in rat alveolar macrophages.

Freshly isolated alveolar macrophages from control rats express low numbers of platelet-activating factor (PAF) receptors (4.6 x 10(3) binding sites/cell, Kd = 1.3 nM). These receptors appeared to be functionally inert in that PAF failed to induce intracellular calcium mobilization in the cells. Following brief exposure of rats to ozone (2 ppm, 3 h), a pulmonary irritant and inflammatory agent that is rapidly converted to molecular oxygen, expression of PAF receptors was markedly up-regulated on lung phagocytes (13.1 x 10(3) binding sites/cell), with no significant effect on receptor affinity (Kd = 2.0 nM). In these cells, PAF, but not lyso-PAF, an inactive analog, caused a rapid and transient rise in intracellular calcium demonstrating that the PAF receptors were functionally active. The calcium response to PAF, which was largely due to mobilization of calcium from intracellular stores, was found to be dose-dependent in the nanomolar concentration range and inhibitable by the PAF receptor antagonist triazolam. The results of these studies may be important in elucidating mechanisms of lung inflammation induced by this model irritant.

Animals↗
PubMed1993

Platelet-activating factor-induced calcium mobilization and oxidative metabolism in hepatic macrophages and endothelial cells.

Fluorescence image analysis of the calcium sensitive dye Indo-1 was used to characterize platelet-activating factor (PAF)-induced calcium mobilization in hepatic macrophages and endothelial cells. PAF, but not lyso-PAF, an inactive analog, induced a rapid and transient increase in intracellular levels of calcium that appeared to depend on the presence of extracellular calcium. In both macrophages and endothelial cells, these effects were dose dependent, reaching maximal levels with 10 nM PAF. However, the kinetics of the calcium response to PAF in macrophages and endothelial cells was distinct. The endothelial cells responded more rapidly to PAF than the macrophages (within 1 min vs. 2-3 min, respectively) and displayed longer recovery periods (4 min vs. > 10 min, respectively). In contrast, the magnitude of the response to PAF was greater in the macrophages. In both cell types, triazolam and alprazolam, two PAF antagonists, and the calcium channel blocker verapamil inhibited PAF-induced calcium mobilization. PAF also stimulated superoxide anion production alone and in combination with the macrophage activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in both cell types. Macrophages produced more of this reactive oxygen intermediate in response to PAF and TPA than endothelial cells. Interestingly, in the absence of extracellular calcium or when verapamil was added to the cultures, superoxide anion production by the macrophages, but not the endothelial cells, was significantly reduced. These results demonstrate that, although hepatic macrophages and endothelial cells mobilize calcium in response to PAF, the characteristics of the response in each cell type are different. These differences may underlie, at least in part, distinct functional responses of the cells to PAF.

Alprazolam↗
PubMed1993

Selective inactivation of lymphocytes after psoralen/ultraviolet light (PUVA) treatment without affecting systemic immune responses.

Psoralens, together with ultraviolet light A (PUVA), are used for the treatment of several proliferative diseases. It has been suggested that the effectiveness of this treatment is due to induction of a specific immune response by PUVA-treated lymphocytes that down-regulates untreated cells of the same specificity. The present studies show that the clinically used psoralen analogue 8-methoxypsoralen (8-MOP), as well as 4,8-dimethyl-5'-(pyridiniummethyl)psoralen bromide (4NQ), a derivative that does not cross the cell membrane, when activated by UVA light, render lymphocytes unresponsive to mitogenic, antigenic, or phorbol myristate acetate + ionomycin-induced stimulation. This state of unresponsiveness was not reversed by exogenous recombinant interleukin-2. Treatment of lymphocytes with 4NQ and UVA light had no effect on the viability or the homing pattern of the cells to spleen or liver, whereas 8-MOP induced toxicity in about 50% of cells after 3 days in culture. Using an adoptive transfer mouse model, we found that antigen-specific lymphocytes treated with PUVA (8-MOP or 4NQ) had no effect on the ability of these mice to respond to a subsequent challenge with the same or an irrelevant antigen. This was tested by measuring the T cell proliferative responses of lymph node cells from mice primed with keyhole limpet hemocyanin (KLH) or fowl gamma globulin (FGG) before or after adoptive transfer of large numbers of KLH- or FGG-specific PUVA-treated lymph node cells. No effects of antigen-primed PUVA-treated cells on antigen-primed untreated cells were observed in vitro in mixed lymphocyte cultures responding to the relevant antigen. These results suggest that, in our model system, PUVA induces cell inactivation but does not affect systemic T cell responses.

Animals↗