Nonelectrolyte permeability of liposomes of hydroxyfatty acid-containing phosphatidylcholines.

Two phosphatidylcholines containing hydroxylated fatty acids, 1-palmitoyl-2-[5-hydroxy-6,8,11,14-eicosatetraenoyl]-sn-glycero-3- phosphocholine (1-palm-2-5HETE PC) and 1-palmitoyl-2-[15(S)-hydroxy-5,8,11,13- eicosatetraenoyl]-sn-glycero-3-phosphocholine (1-palm-2-15HETE PC), and one phosphatidylcholine containing nonhydroxylated fatty acids, 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (1-palm-2-arach PC) were synthesized. Permeation of small nonelectrolytes (glycerol, 1,2-propanediol, urea, methylurea, propionamide and dimethylformamide) was assessed in multilamellar liposomes containing these synthetic PCs plus egg yolk phosphatidycholine (EPC) in the presence and absence of cholesterol. In liposomes containing 23% cholesterol, 69.3% EPC and 7.7% of either 1-palm-2-5HETE PC or 1-palm-2-15HETE PC the permeability to small nonelectrolytes was 60 to 400% greater than in liposomes containing 23% cholesterol and 77% EPC. The HETE-containing PCs also increased permeability in liposomes without cholesterol but the effects were less striking. Addition of the synthetic PCs did not affect the energy of activation of permeation.

Doubleblind evaluation of the antimanic properties of carbamazepine as a comedication to haloperidol.

1. Today carbamazepine is the most important alternative to neuroleptic drugs for the treatment of manic psychoses. Often carbamazepine is administered as a comedication to a neuroleptic. 2. A doubleblind study with 20 patients suffering from manic or schizomanic psychoses was performed to determine whether carbamazepine and haloperidol in comedication are more effective than haloperidol alone. 3. Under the tested conditions (24 mg haloperidol p.d.) only the smaller amount of additional medication with levomepromazine in the experimental group gave evidence for the antimanic effect of carbamazepine in combination with haloperidol. 4. Especially the patients with pure manic psychoses seem to benefit from carbamazepine as an adjunct to haloperidol.

A controlled study of the efficacy and safety of mianserin and maprotiline in outpatients with major depression.

Efficacy and tolerability of mianserin (60-90 mg/day) or maprotiline (100-150 mg/day) were tested in a 4-week double-blind control group study on 317 depressive outpatients. The patients had to fulfill the criteria of a major depression according to DSM-III. The study was performed by psychiatrists after special rater training. Standardized rating procedures were applied to evaluate depressive symptoms and unwanted effects at three measurement points. A significant improvement was found in both groups, without any statistical differences between mianserin and maprotiline. According to the Clinical Global Impressions (CGI) 65% of the patients in both groups were judged as responders. A good tolerability of both drugs were demonstrated. With respect to anticholinergic side-effects there was a certain advantage in favor to mianserin.

COX-1(+/-)COX-2(-/-) genotype in mice is associated with shortened time to carotid artery occlusion through increased PAI-1.

BACKGROUND: We found a high incidence of thrombotic deaths in COX-1(+/-)COX-2(-/-) mice and sought to define the mechanism of these events. The cyclooxygenase products thromboxane A(2) and prostacyclin are important in the regulation of coagulation but their role in fibrinolysis is largely unexplored. PAI-1 blocks fibrinolysis by inhibiting plasminogen activator. AIM: Our objective was to explain the mechanism of increased thrombosis associated with the COX-1(+/-)COX-2(-/-) genotype. METHODS: Carotid artery occlusion times were measured after photochemical injury. PAI-1 levels were measured in the plasma by ELISA. PAI-1 levels in the aorta were measured by RT-PCR and Western blotting. Urinary metabolites of Thromboxane A(2) and prostacyclin were measured by ELISA. RESULTS: The COX-1(+/-)COX-2(-/-) genotype is associated with a decreased time to occlusion in the carotid artery thrombosis model (30 ± 5 minutes vs 60 ± minutes in wild type, p<.001). The COX-1(-/-)COX-2(+/+), COX-1(+/-)COX-2(+/-) and COX-1(+/-)COX-2(+/+) all had occlusion times similar to wild type. COX-1(+/+)COX-2(-/-) had a prolonged occlusion time. COX-1(+/-)COX-2(-/-) had increased PAI-1 levels in the plasma and aorta and with a prolonged euglobulin lysis time (37.4 ± 10.2 hours vs 15.6 ± 9.8 hours in wild type, p<.004). The decreased time to occlusion in the COX-1(+/-)COX2(-/-) mice was normalized by an inhibitory antibody to PAI-1 whereas the antibody had no effect on the time to occlusion in wild type mice. CONCLUSION: The COX-1(+/-)COX-2(-/-) genotype is associated with a shortened time to occlusion in the carotid thrombosis model and the shortened time to occlusion is mediated through increased PAI-1 levels resulting in decreased fibrinolysis.

Mechanisms of transit of lipid mediators of inflammation and bacterial peptides across intestinal epithelia.

The transit of two lipid mediators of inflammation, leukotriene B4 (LTB4) and prostaglandin E2 (PGE2), and a formylated peptide produced by intestinal bacteria, N-formylmethionylleucylphenylalanine (FMLP), across Caco-2 cell monolayers was characterized and compared with the transit of mannitol, a hexose known to cross epithelial monolayers by paracellular pathways. The permeability of less mature low-resistance ( < 200 ohm.cm2) monolayers to all four test compounds was similar, but as monolayers matured and the transmonolayer resistance increased, the transit of LTB4, PGE2, FMLP, and mannitol decreased to different degrees, resulting in a selectivity of permeability to the four test compounds in the order LTB4 > PGE2 > mannitol > FMLP. The transit of all four test compounds across Caco-2 cell monolayers was bidirectional, nonsaturable, and energy independent. A small portion of the added LTB4 was incorporated into the cells, whereas the other three compounds were not. Thus the transit of PGE2, mannitol, and FMLP across Caco-2 monolayers appears to be solely by the paracellular pathway, whereas the transit of LTB4 also involves the paracellular pathway but may also involve diffusion through the cell membrane and around tight junctions.

Platelet-activating factor acetylhydrolases in Caco-2 cells and epithelium of normal and ulcerative colitis patients.

BACKGROUND & AIMS: Platelet-activating factor (PAF) is a potent inflammatory mediator implicated in the pathogenesis of inflammatory bowel disease and necrotizing enterocolitis. Metabolism by platelet-activating factor acetylhydrolase (PAF-AH) is the major pathway for platelet-activating factor degradation. The aim of this study was to investigate the possible role of intestinal epithelium as a source of PAF-AH. METHODS: Intracellular and secreted PAF-AHs were characterized in human colonic epithelial cells isolated from histologically normal mucosa and inflamed mucosa from patients with ulcerative colitis and in the human intestinal epithelial cell line Caco-2 by measuring the metabolism of [3H]-PAF to [3H]lysoPAF. RESULTS: Human colonic epithelial cells and Caco-2 cells synthesize and secrete PAF-AH as shown by in vitro hydrolysis of [3H]PAF to [3H]-lysoPAF in cell lysates and conditioned media. Both intracellular and secreted PAF-AHs are calcium-independent and substrate-specific for phospholipids similar to PAF. Epithelial cells from involved areas of resections for ulcerative colitis had increased levels of secreted PAF-AH and decreased levels of intracellular PAF-AH compared with epithelial cells from histologically normal areas. CONCLUSIONS: Human colonic epithelial cells and Caco-2 cells produce intracellular and secreted PAF-AHs, which are distinct proteins. This is the first demonstration of PAF-AH production by epithelial cells.

Physiological Studies on Pea Tendrils : XIV. Effects of Mechanical Perturbation, Light, and 2-Deoxy-d-Glucose on Callose Deposition and Tendril Coiling.

When excised tendrils of pea (Pisum sativum L. cv Alaska) are mechanically perturbed there is an immediate and transient increase in callose deposition in the sieve cells. Mechanical perturbation (MP) results in a coiling response in light-grown tendrils and in dark-adapted tendrils, provided, in the latter case, that they receive adequate illumination within a limited period of time after MP. In nonperturbed tendrils the number of callose deposits decreases to some minimum with increasing time in the dark, and their ability to coil in the dark in response to MP diminishes with time in the dark. The transient increase of callose deposition due to MP, however, occurs whether or not tendrils are dark adapted, and whether they receive light or are retained in the dark after MP. This indicates that if callose is directly involved in tendril coiling, then it exerts its effect on the sensory perception of the mechanical stimulus. In the present investigation, there is never tendril coiling without the transient increase in callose, and the time after MP at which the peak of callose deposition occurs precedes the time of the peak amount of coiling.An inhibitor of callose formation, 2-deoxy-d-glucose (DDG), is equally effective at inhibiting tendril coiling and MP-induced callose deposition, indicating, within the limitations of the specificity of DDG, that callose deposition may be required in order for the coiling response to occur. Alternatively, DDG may prevent the availability of some other factor necessary for tendril coiling.

Metabolism of 15-hydroxyeicosatetraenoic acid by Caco-2 cells.

Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated with [1-14C]15-hydroxyeicosatetraenoic acid (15-HETE), a lipid mediator of inflammation, and [1-14C]arachidonic acid. Both fatty acids were taken up readily and metabolized by Caco-2 cells. [1-14C]Arachidonic acid was directly esterified in cellular phospholipids and, to a lesser extent, in triglycerides. When [1-14C]15-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, about 10% was directly esterified into cellular lipids but most (55%) was beta-oxidized to ketone bodies, CO2, and acetate, with very little accumulation of shorter carbon chain products of partial beta-oxidation. The radiolabeled acetate generated from beta-oxidation of [1-14C]15-hydroxyeicosatetraenoic acid was incorporated into the synthesis of new fatty acids, primarily [14C]palmitate, which in turn was esterified into cellular phospholipids, with lesser amounts in triglycerides. Caco-2 cells were also incubated with [5,6,8,9,11,12,14,15-3H]15-hydroxyeicosatetraenoic acid; most of the radiolabel was recovered either in ketone bodies or in [3H]palmitate esterified in phospholipids and triglycerides, demonstrating that most of the [3H]15-hydroxyeicosatetraenoic acid underwent several cycles of beta-oxidation. The binding of both 15-hydroxyeicosatetraenoic acid and arachidonic acid to hepatic fatty acid binding protein, the only fatty acid binding protein in Caco-2 cells, was measured. The Kd (6.0 microM) for 15-HETE was three-fold higher than that for arachidonate (2.1 microM).

Metabolism of oxygenated derivatives of arachidonic acid by Caco-2 cells.

Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated separately with 3H8-labeled preparations of three different lipid mediators of inflammation: 5-hydroxyeicosatetraenoic acid, 12-hydroxyeicosatetraenoic acid, and leukotriene B4. Both [3H8]5-hydroxyeicosatetraenoic and [3H8]12-hydroxyeicosatetraenoic acids were taken up and metabolized by Caco-2 cells, but [3H]leukotriene B4 remained unmetabolized in the incubation medium. [3H]5-hydroxyeicosatetraenoic acid was esterified into cellular phospholipids (15%) and triglycerides (4%) but did not undergo beta-oxidation. When [3H]12-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, 14% underwent two cycles of beta-oxidation to form [3H]8-hydroxyhexadecatrienoic acid, and 3% underwent three cycles of beta-oxidation to form [3H]6-hydroxytetradecadienoic acid, both of which were released into the media. [3H]12-Hydroxyeicosatetraenoic acid was also esterified into cellular phospholipids (13%), but none was esterified into cellular triglycerides.

Regulation of paracellular permeability in Caco-2 cell monolayers by protein kinase C.

Caco-2 cells are an enterocyte-like cell line derived from a human colonic adenocarcinoma. Paracellular permeability was assessed in monolayers of these cells by transmonolayer resistance and by the permeation of [3H]mannitol across the monolayer. Paracellular permeability was increased by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (50 nM), carbachol (500 microM), and the combination of carbachol (50 microM) and monolein (100 microM), an inhibitor of diacylglycerol kinase, as manifested by a decrease in transmonolayer resistance and an increase in mannitol permeation. The effects of all of these stimuli on transmonolayer resistance were inhibited by staurosporine (3 nM), an inhibitor of PKC. The effects of carbachol plus monolein were also inhibited by atropine (0.1 microM), a muscarinic antagonist. Treatment of the monolayers with each of the stimuli was associated with translocation of PKC activity from cytosol to a membrane-associated state. Stimulation of Caco-2 cell monolayers with phorbol myristate acetate or with the combination of carbachol and monolein was also associated with phosphorylation of the MARCKS protein, an endogenous substrate of PKC. These data support the hypothesis that intestinal paracellular permeability is regulated by the activity of enterocyte PKC and demonstrate that the increase in paracellular permeability induced by binding of carbachol to the muscarinic receptor is mediated by activation of PKC.

Amebic infection in the human colon induces cyclooxygenase-2.

We sought to determine if infection of the colon with Entamoeba histolytica induces the expression of cyclooxygenase-2 and, if it does, to determine the contribution of prostaglandins produced through cyclooxygenase-2 to the host response to amebic infection. Human fetal intestinal xenografts were implanted subcutaneously in mice with severe combined immunodeficiency and allowed to grow; the xenografts were then infected with E. histolytica trophozoites. Infection with E. histolytica resulted in the expression of cyclooxygenase-2 in epithelial cells and lamina propria macrophages. Infection with E. histolytica increased prostaglandin E(2) (PGE2) levels 10-fold in the xenografts and resulted in neutrophil infiltration, as manifested by an 18-fold increase in myeloperoxidase activity. Amebic infection also induced an 18-fold increase in interleukin 8 (IL-8) production and a >100-fold increase in epithelial permeability. Treatment of the host mouse with indomethacin, an inhibitor of cyclooxygenase-1 and cyclooxygenase-2, or with NS-398, a selective inhibitor of cyclooxygenase-2, resulted in (i) decreased PGE(2) levels, (ii) a decrease in neutrophil infiltration, (iii) a decrease in IL-8 production, and (iv) a decrease in the enhanced epithelial permeability seen with amebic infection. These results indicate that amebic infection in the colon induces the expression of cyclooxygenase-2 in epithelial cells and macrophages. Moreover, prostaglandins produced through cyclooxygenase-2 participate in the mediation of the neutrophil response to infection and enhance epithelial permeability.

Efficacy and tolerability of a new antipsychotic compound (risperidone): results of a pilot study.

Risperidone is a new benzisoxazole derivative displaying a very potent serotonin antagonism and a potent dopamine antagonism in pharmacological studies. These properties suggest the hypothesis that risperidone may exert antipsychotic effects and be superior to classic neuroleptics in its beneficial effects on negative and affective symptoms and its low extrapyramidal side-effect propensity. In an open pilot study 13 patients suffering from acute schizophrenic psychosis were treated with risperidone within an individually adapted dose range from 1 to 10 mg per day. A good antipsychotic efficacy could be demonstrated in 6 of the 8 patients who completed the trial. Risperidone was very well tolerated. The substance possesses a low EPS-inducing profile. Future research has to test the suggested advantage of risperidone over other neuroleptic drugs and its performance in the treatment of chronic schizophrenic patients.

Lipopolysaccharide is radioprotective in the mouse intestine through a prostaglandin-mediated mechanism.

BACKGROUND & AIMS: The bone marrow and the intestine are the major sites of radiation-induced injury. The cellular response to radiation injury in the intestine or bone marrow can be modulated by agents given before irradiation. Lipopolysaccharide is known to be radioprotective in the bone marrow, but its effect on the intestine is not known. We sought to determine if lipopolysaccharide is radioprotective in the intestine and, if so, to determine the mechanism of its radioprotective effects. METHODS: Mice were treated with parenteral lipopolysaccharide or vehicle and then irradiated (14 Gy total body irradiation in a cesium irradiator). The number of surviving intestinal crypts was assessed 3.5 days after irradiation using a clonogenic assay. RESULTS: Parenteral administration of lipopolysaccharide 2-24 hours before irradiation resulted in a 2-fold increase in the number of surviving crypts 3.5 days after irradiation. The radioprotective effects of lipopolysaccharide could be eliminated by coadministration of a selective inhibitor of cyclooxygenase 2. Lipopolysaccharide was radioprotective in wild-type mice but not in mice with a disrupted cyclooxygenase 2. Parenteral administration of lipopolysaccharide resulted in increased production of prostaglandins in the intestine and in the induction of cyclooxygenase 2 expression in subepithelial fibroblasts and in villous, but not crypt, epithelial cells. CONCLUSIONS: Lipopolysaccharide is radioprotective in the mouse intestine through a prostaglandin-dependent pathway.

Cyclooxygenase 2 is induced in colonic epithelial cells in inflammatory bowel disease.

BACKGROUND & AIMS: Prostaglandins are synthesized by cyclooxygenases (COX)-1 and -2. The expression and cellular localization of COX-1 and COX-2 in normal human colon and inflammatory bowel disease (IBD) surgical resections were studied. METHODS: COX-1 and COX-2 protein expression and cellular localization were assessed by Western blotting and immunohistochemistry. RESULTS: COX-1 protein was expressed at equal levels in normal, Crohn's disease, and ulcerative colitis colonic epithelial cells. COX-2 protein was not detected in normal epithelial cells but was detected in Crohn's disease and ulcerative colitis epithelial cells. Immunohistochemistry of normal, Crohn's colitis, and ulcerative colitis tissue showed equivalent COX-1 expression in epithelial cells in the lower half of the colonic crypts. COX-2 expression was absent from normal colon, whereas in Crohn's colitis and ulcerative colitis, COX-2 was observed in apical epithelial cells and in lamina propria mononuclear cells. In Crohn's ileitis, COX-2 was present in the villus epithelial cells. In ulcerative colitis, colonic epithelial cells expressing COX-2 also expressed inducible nitric oxide synthase. CONCLUSIONS: COX-1 was localized in the crypt epithelium of the normal ileum and colon, and its expression was unchanged in IBD. COX-2 was undetectable in normal ileum or colon, but it was induced in apical epithelial cells of inflamed foci in IBD.

Expression of inducible nitric oxide synthase and nitrotyrosine in colonic epithelium in inflammatory bowel disease.

BACKGROUND & AIMS: Inducible nitric oxide synthase (iNOS) is generated in several cell types by treatment with lipopolysaccharides or cytokines. Earlier studies suggested that ulcerative colitis is associated with increased NO produced by iNOS; however, the cellular source of the NO synthesis was not identified. A possible mechanism of NO-induced cellular damage is through its interaction with superoxide to produce peroxynitrite, which reacts with tyrosine to form nitrotyrosine in cellular proteins. METHODS: Using immunoperoxidase microscopy with a new monospecific human iNOS antibody (NO-53), the cellular distribution of iNOS and nitrotyrosine was examined using human colonic mucosa from normal bowel, ulcerative colitis, Crohn's disease, and diverticulitis. RESULTS: Intense focal iNOS labeling was localized to the inflamed colonic epithelium in ulcerative colitis, Crohn's disease, and diverticulitis but was not detectable in the uninflamed epithelium. Nitrotyrosine labeling was also observed in the inflamed colonic epithelium and was associated with nearby iNOS staining; nitrotyrosine was undetectable in normal mucosal epithelium. iNOS and nitrotyrosine were also detected in lamina propria mononuclear cells and neutrophils. CONCLUSIONS: These findings suggest that iNOS is induced in the inflamed human colonic epithelium and is associated with the formation of peroxynitrite and the nitration of cellular proteins.