Selective effect of levocabastine on histamine receptor and histamine release from human leukocytes and guinea pig isolated tissue.

BACKGROUND: Levocabastine is a potent histamine H1 receptor antagonist used topically in the treatment of patients with allergic rhinitis. It has been suggested that antihistamines also have anti-inflammatory properties. OBJECTIVE: The present study was performed to investigate whether levocabastine, in addition to the anti-H1 receptor activity, has anti-inflammatory properties and thus is able to modulate the release of histamine and cytokines, such as interleukin 5 from human leukocytes and isolated tissues. METHODS: Leukocytes suspensions were prepared by dextran sedimentation of peripheral venous blood drawn from allergic and healthy volunteers. Leukocytes obtained from allergic volunteers were preincubated for 30 minutes with levocabastine (doses 10(-8) M to 10(-6) M) and thereafter incubated with allergen. Leukocytes obtained from healthy volunteers were incubated for zero to three hours with levocabastine (doses 10(-14) M to 10(-3) M). Histamine release was measured by an automated fluorometric method. Interleukin-5 release was measured by enzyme linked immunoassay. Contractile responses to histamine on guinea pig trachea and lung parenchyma as well as the release of histamine and interleukin-5 by the tissues were investigated in the absence or presence of levocabastine and/or the histamine H2 receptor antagonist cimetidine. RESULTS: Levocabastine did not influence allergen-induced histamine release from leukocytes obtained from allergic volunteers. High concentrations (10(-4)and 10(-3) M) of levocabastine, however, caused release of histamine from leukocytes obtained from healthy volunteers as well as guinea pig airway smooth muscle tissues. Pretreatment with levocabastine dose-dependently decreased the contractile response to histamine, showing an irreversible competitive mechanism. Interleukin 5 release from human leukocytes and by guinea pig airway smooth muscle was not detectable. CONCLUSIONS: These findings indicate that the H1 receptor blocker, levocabastine, has probably no anti-inflammatory properties, measured as histamine release, and that the histamine release from both human leukocytes and guinea pig trachea and lung parenchyma is significantly increased by the drug only at high concentrations.

Effect of topical levocabastine on nasal response to allergen challenge and nasal hyperreactivity in perennial rhinitis.

BACKGROUND: It has been demonstrated that some oral antihistamines reduce nasal nonspecific reactivity and that topical levocabastine reduces cellular influx after nasal allergen challenge. This suggests that antihistamines possess other properties besides classical H1-receptor antagonism. OBJECTIVE: To evaluate the effect of 1 week's treatment with topical levocabastine on the nasal clinical response, inflammatory mediators, and nasal hyperreactivity. METHODS: In a double-blind, placebo-controlled, 2-period, 2-treatment, crossover study, 21 rhinitic patients allergic to house dust mite participated. After each treatment period patients were challenged with house dust mite extract. Symptom scores and nasal lavages were collected for nine and one-half hours after challenge. Allergen-induced nasal hyperreactivity was determined by nasal methacholine challenge 24 hours after allergen challenge. A nasal histamine challenge was performed as well. RESULTS: Patients showed only an immediate nasal response. Levocabastine significantly reduced the symptom score after 100 (P = .0063), 1000 (P = .0035), and 10,000 biological units (BU)/mL (P = .0013) of house dust mite extract. Albumin influx and tryptase release were not significantly reduced by levocabastine. No release of histamine and eosinophil cationic protein was seen. Levocabastine did not reduce nasal response to methacholine. Active treatment significantly reduced histamine-induced nasal secretion (P = .0009) and the number of sneezes (P = .0001). CONCLUSION: A significant effect of levocabastine was shown on the immediate clinical response to house dust mite and to histamine challenge only. Our findings suggest that levocabastine is an effective H1-receptor antagonist without anti-inflammatory properties.

Relationship between photodynamically induced damage to various cellular parameters and loss of clonogenicity in different cell types with hematoporphyrin derivative as sensitizer.

The possible causal relationship between various forms of photodynamically inflicted damage and reproductive cell death of cultivated cells was evaluated according to three criteria. The probability for the existence of such a relationship is high, when the particular form of cellular damage (i) exhibits a dose-effect curve, comparable to the dose-effect curve of loss of clonogenicity, (ii) is not readily repairable during further incubation of the treated cells and (iii) varies in a way comparable to the loss of clonogenicity under varying experimental conditions. According to these criteria it could be shown that many forms of photodynamically inflicted cellular damage are presumably not directly involved in loss of clonogenicity. Only for a few kinds of cellular damage studied in the present investigations was the probability for a causal relationship with reproductive cell death much higher. For L929 fibroblasts this is either an inhibition of the Na+/K(+)-ATPase activity, or a relatively slight DNA damage combined with a strong inhibition of DNA excision repair. For T24 human bladder carcinoma cells the kinds of cellular damage that may be causally related to reproductive cell death are again inhibition of Na+/K(+)-ATPase activity, inhibition of amino-acid (AIB and glycine) transport activity or impairment of mitochondrial function. Finally, for CHO cells, inhibition of leucine and phenylalanine transport and impairment of mitochondrial function may be crucial for loss of clonogenicity. These results indicate that the pathways leading to photodynamically induced reproductive cell death may be quite different for different cell types.

Preferential uptake of cytotoxic porphyrins from hematoporphyrin derivative in murine L929 fibroblasts and Chinese hamster ovary K1 epithelial cells.

Photodynamically induced loss of clonogenicity of murine L929 fibroblasts and Chinese hamster ovary K1 epithelial cells was determined with two different assays. It appeared that the loss of clonogenicity was much higher when 20 cells/cm2 were incubated with hematoporphyrin derivative (HPD) and illuminated, than when confluent cell layers were incubated with the same amount of HPD and illuminated prior to plating out. This dependency of cell killing on the experimental protocol was also observed when protoporphyrin (90-95% pure) was used as photosensitizer, but not when the cells were photodynamically treated with rose bengal or exposed to mitomycin C. Further, when cell layers were incubated with the residual solution that remained after the previous incubation of a confluent cell layer with HPD, illumination of these layers appeared to be almost non-toxic, although the overall porphyrin concentration in the residual solution was only slightly lower than in HPD. These results indicate that the porphyrins, responsible for loss of clonogenicity, are present in relatively small amounts in HPD and unpurified protoporphyrin and are preferentially taken up by the cells. Although 2-aminoisobutyric acid transport and DNA synthesis are among the most photosensitive targets with HPD, photodynamic treatment of L929 cells with the residual solution did not result in inhibition of the transport system and DNA synthesis. In contrast, the K+ content of the cells still decreased considerably, when utilizing the porphyrins, remaining in the residual solution as sensitizer. This indicates that under the present experimental conditions the disturbance of the membrane barrier function does not contribute to loss of clonogenicity of these cells and, moreover, that the photodynamically induced K+ leakage is caused by a component of HPD other than inhibition of 2-aminoisobutyric acid transport and DNA synthesis.

Damaging action of photodynamic treatment in combination with hyperthermia on transmembrane transport in murine L929 fibroblasts.

Photodynamic treatment of murine L929 fibroblasts with hematoporphyrin derivative caused inhibition of the 2-aminoisobutyric acid transport system. This was reflected by an increase in the apparent Km with a constant Vmax, indicating impairment of the carrier function rather than a decrease of the number of transport sites. Hyperthermic treatment of these cells resulted in a moderate decrease of the activity of the 2-aminoisobutyric acid transport system. Overall protein synthesis was severely inhibited both by photodynamic treatment and by hyperthermia. Hyperthermia subsequent to photodynamic treatment resulted in an additive inhibition of 2-aminoisobutyric acid transport and of protein synthesis. After photodynamic treatment both 2-aminoisobutyric acid transport and protein synthesis were repaired. The repair of 2-aminoisobutyric acid transport depended on protein synthesis, as shown by the virtually complete blockage of repair by anisomycin. After hyperthermia (either alone or subsequent to photodynamic treatment), no recovery of 2-aminoisobutyric acid transport was observed, although protein synthesis was restored to the initial level. Apparently, hyperthermia subsequent to photodynamic treatment blocks the repair of photodynamically induced damage of this transport system. The experimental results further indicate that protein synthesis is not the rate-determining step for the repair of 2-aminoisobutyric acid transport, although it is necessary in this process. Cell survival was decreased both by photodynamic treatment and by hyperthermia. The combined effects of these two treatments were additive. It is discussed that these results indicate that photodynamic inhibition of 2-aminoisobutyric acid transport is not causally related to loss of clonogenicity, contrary to earlier suggestions.

The influence of porphyrins on iron-catalysed generation of hydroxyl radicals.

Uroporphyrin I, haematoporphyrin and haematoporphyrin derivative had no effect on O2-. generation during oxidation of hypoxanthine by xanthine oxidase and on the formation of hydroxyl radicals (OH.) in the hypoxanthine/xanthine oxidase/Fe3+-EDTA/deoxyribose system. On the other hand, these porphyrins strongly inhibited O2-. formation in a horseradish peroxidase/H2O2/NADPH mixture, whereas they augmented OH. generation in this system after addition of Fe3+-EDTA. Experimental evidence suggests that these observations should be ascribed to the formation of a porphyrin anion radical in the horseradish peroxidase/NADPH system. The formation of this anion radical was confirmed by e.s.r. spectroscopy. This radical is apparently unable to reduce cytochrome c, but it can replace O2-. in the OH.-generating Haber-Weiss reaction.

Photodynamic effects of hematoporphyrin-derivative on enzyme activities of murine L929 fibroblasts.

Photodynamic treatment of murine L929 fibroblasts with hematoporphyrin-derivative resulted in the inactivation of cytosolic, mitochondrial and lysosomal enzymes and in a decrease in cellular adenosine triphosphate and reduced glutathione concentrations. Comparison of these results with those of previous studies revealed that transmembrane transport systems and DNA repair enzymes are inactivated after much shorter illumination periods than are intracellular enzymes. Although the pattern of photodynamic damage altered by varying the protocol of preincubation with hematoporphyrin-derivative and washing, it appeared that under all experimental conditions the plasma membrane was much more sensitive to photodynamic damage than were the intracellular enzymes. Lysosomal membrane disruption with subsequent detrimental release of lysosomal enzymes has been implicated previously in certain forms of porphyrin-induced photodynamic cell destruction. Cytochemical studies on enzyme localization virtually exclude such a mechanism in hematoporphyrin-derivative-induced cell inactivation in L929 fibroblasts.

Photodynamic effects of haematoporphyrin derivative on DNA repair in murine L929 fibroblasts.

Illumination with red light of murine L929 fibroblasts that had been sensitized with haematoporphyrin derivative caused DNA single-strand breaks after a lag time of about 20 min, as revealed by alkaline elution. The cells appeared not to be capable of recovering from this damage. The photodynamic effect of haematoporphyrin derivative on DNA repair was assessed by monitoring the repair kinetics of DNA damage induced by either X-rays, u.v. light (254 nm) or methyl methanesulphonate treatment subsequent to a non-DNA-damaging photodynamic treatment with haematoporphyrin derivative. On 'post-incubation', the normally rapid repair of X-ray-induced DNA strand breaks did not occur, whereas with u.v. light and methyl methanesulphonate treatment after photodynamic treatment prolonged post-incubation resulted in an increase in the number of strand breaks rather than the normally observed decrease. This clearly shows that, after a photodynamic treatment with haematoporphyrin derivative that itself did not cause strand breaks, excision repair in L929 cells is severely inhibited at a stage beyond the incision step.

The mechanism of potentiation of horseradish peroxidase-catalysed oxidation of NADPH by porphyrins.

Several porphyrins, including HpD (haematoporphyrin derivative), potentiate the oxidation of NADPH by horseradish peroxidase/H2O2. To elucidate the mechanism of potentiation, the following observations are relevant. During peroxidase-catalysed NADPH oxidation, O2-.(superoxide radical) is generated, as judged from superoxide dismutase-inhibitable cytochrome c reduction. This generation of O2-. is suppressed by HpD. Peroxidase-catalysed NADPH oxidation is stimulated by superoxide dismutase and by anaerobic conditions. Under anaerobic conditions HpD has no influence on peroxide-catalysed NADPH oxidation. Previous studies have shown that horseradish peroxidase is inhibited by O2-.. Thus the experimental results indicate that the potentiating effect of HpD can be explained by its ability to inhibit O2-. generation in the horseradish peroxidase/H2O2/NADPH system.

The influence of cupric ions on porphyrin-induced photodynamic membrane damage in human red blood cells.

Photooxidation of various susceptible substrates with hematoporphyrin derivative (photofrin) as sensitizer was strongly inhibited by simultaneous addition of cupric acetate to the reaction mixture. With sulfhydryl-containing compounds, however, an increased rate of photooxidation was observed under these experimental conditions. Preincubation of photofrin and cupric acetate at equimolar concentrations for 24 h at room temperature yielded a stable photofrin-Cu2+ complex. This complex did not act as photosensitizer with histidine, tryptophan, tyrosine, methionine or guanosine as substrates. With dithiothreitol, however, the photofrin-Cu2+ complex still acted as a photosensitizer, with an efficiency of about 30% as compared to free photofrin. Also in red blood cell membranes only sulfhydryl groups were photooxidized with the photofrin-Cu2+ complex as sensitizer. Illumination of intact erythrocytes in the presence of the photofrin-Cu2+ complex resulted in K+ leakage and, ultimately, photohemolysis. Pretreatment of the cells with N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) inhibited this photodynamically induced K+ loss. Considering recent studies on the reactivity of distinct membrane SH-groups with various sulfhydryl reagents this suggests that a sulfhydryl group, located in the 17 kDa membrane-bound fragment of band 3, is involved in photodynamic K+ leakage with the photofrin-Cu2+ complex as sensitizer.

A study on post-synthetic modifications in alfa-alfa enolase (EC 4.2.1.11) brought about by a human serum protein.

Human alfa-alfa enolase is modified in the blood circulation by a serum protein for which the name 'modifying protein' is proposed. The protein occurs in every human serum tested and appears to be the same protein that is responsible for the post-synthetic modification in the M subunit of creatine kinase. Three alfa-alfa enolase forms, the original one plus two modified forms can be found in serum both in vivo and after in vitro incubation. The original alfa-alfa enolase is modified completely within a few hours in vitro in a pH-controlled human serum matrix at 37 degrees C. As the modification also takes place in vivo it is theoretically possible to acquire information about the activity of a disease process by doing one single determination of the amount of circulating alfa-alfa 3 enolase. A mechanism is proposed for the modification, where at first one of the two alfa chains is modified resulting in the alfa-alfa 2 form. Ultimately the second alfa chain is also modified. The alfa-alfa 1 form seems to be the completely modified alfa-alfa form. The three enolase forms differ in their isoelectric points but have similar Michaelis-Menten constants.

The sphingomyelin pools in the outer and inner layer of the human erythrocyte membrane are composed of different molecular species.

Analyses of the fatty acid composition of the outer and inner pools of sphingomyelin in the human erythrocyte membrane revealed significant differences in molecular species composition of these two pools. The sphingomyelin in the inner monolayer, representing 15-20% of the total sphingomyelin content of this membrane, is characterized by a relatively high content (73%) of fatty acids, which have less than 20 carbon atoms, whereas these account for only 31% of the total fatty acids in the sphingomyelin in the outer leaflet. On the other hand, the ratio saturated/unsaturated fatty acids in the two pools is similar. Significant differences are also observed for the fatty acid composition of the sphingomyelin in human serum when compared to that in the outer monolayer of the corresponding red cell. These results are interpreted to indicate an (almost) complete absence of transbilayer movements of sphingomyelin molecules in the human erythrocyte membrane, whereas an exchange of this phospholipid between the red cell membrane and serum is either virtually absent, or affects only a minor fraction of the sphingomyelin in the outer membrane layer.