Reversible Hypothermia in a Drug-naive Inpatient with Alzheimer's Disease Receiving Pipamperone.

Hypothermia is a potentially life-threatening side effect of antipsychotic drugs, especially those with strong 5-HT2 antagonist properties. However, the exact underlying mechanism is still under debate. We discuss a case of hypothermia following pipamperone treatment in an elderly female inpatient with Alzheimer's disease, which occurred at day 4 after medication onset and vanished after dose reduction. Thus, this case demonstrates 1) the importance of monitoring body temperature even in low-potency antipsychotics, at least in the elderly, and 2) that in some cases, dose reduction may be a sufficient countermeasure.

Evidence for a role of the multidrug resistance protein (MRP) in the outward translocation of NBD-phospholipids in the erythrocyte membrane.

Phosphatidylserine (PS) containing a 7-nitrobenz-2-oxa-1, 3-diazol-4-yl- (NBD-) hexanoyl residue, like native PS, preferentially distributes into the inner membrane leaflet of human erythrocytes. In the case of NBD-PS, this preference results from two opposite active processes, an inward translocation mediated by the aminophospholipid flippase and an outward translocation mediated by an ill-defined floppase. Selective inhibition of this floppase by alkylating reagents or cationic and anionic drugs increases the extent of accumulation of NBD-PS in the inner membrane leaflet from about 70% in control cells to about 90%. Different inhibitor sensitivities of the flippase and the floppase strongly suggest that both represent different entities. The floppase was characterized in further detail by comparing inhibitory effects of various compounds on this translocase with their effects on known primary active transport systems for amphiphilic compounds. The inhibitory effects of various drugs, glutathione conjugates and GSSG on the floppase activity closely correlate with those reported for the active transport by the multidrug resistance protein (MRP) while only poorly going parallel with those for the active transport by the low affinity pump for glutathione conjugates and the multidrug resistance MDR1 P-glycoprotein. The NBD-phospholipid floppase activity of the erythrocyte is thus probably a function of MRP.

Conserved sequence patterns in phages Mu and lambda DNA.

The genetic maps of bacteriophages Mu and lambda can be aligned with respect to the functions of their genes. We were interested to ascertain whether the congruence of gene order is reflected at the nucleotide sequence level. A sliding window analysis of sequences from the early regions of both phages revealed a substantial degree of similarity. Equally high scores, however, were found when the early region of Mu was compared to the late region of lambda and in self-comparisons of either Mu or lambda. Hence, the similarity is due to a common pattern of nucleotides rather than to sequence similarities between functionally related genes. Employing degenerated scoring matrices we could show that primarily adenine and thymine residues contribute to the high scores and that a specific clustering of these residues is the basis for the conserved pattern. Since such a similarity was not observed with control sequences of other phages. Escherichia coli or eukaryotic viruses, the data support the notion that Mu and lambda have diverged from a common phage module. In general, our approach could offer a simple and sensitive way to trace distant relationships.

Penetration of 2,4,5-trinitrobenzenesulfonate into human erythrocytes. Consequences for studies on phospholipid asymmetry.

The glutathione content of human erythrocytes rapidly diminishes when cells are exposed to 2,4,6-trinitrobenzenesulfonate (20 mumol/l cells) at 37 degrees C. Even at 0 degrees C a slow decrease in glutathione content is observed. The uptake of trinitrobenzenesulfonate by the cells is retarded by inhibitors of the inorganic anion exchange system, indicating that trinitrobenzenesulfonate enters the cells by this pathway. The disappearance of glutathione most probably results from the reaction: 2 GSH + trinitrobenzenesulfonate leads to GSSG + aminodinitrobenzenesulfonate. The reaction of trinitrobenzenesulfonate with glutathione occurs prior to its covalent binding to amino groups of hemoglobin which makes this reaction a more sensitive method of detection of penetration of trinitrobenzenesulfonate into erythrocytes. Results of studies on the asymmetric distribution of phospholipids using trinitrobenzenesulfonate as the only probe should be reconsidered in the light of these new data.

Topology of membrane sulfhydryl groups in the human erythrocyte. Demonstration of a non-reactive population in intrinsic proteins.

A major fraction of the protein sulfhydryl groups of human erythrocyte membranes can be oxidized to disulfide bonds by the lipid soluble reagent, diamide, and the hydrophilic reagent, tetrathionate. Furthermore, the same fraction also reacts with the monofunctional reagent, N-ethylmaleimide. About 20% of the SH groups, however, do not react with any of these agents even upon prolonged treatment and increased concentrations. These 'non-reacting' SH groups were now localized by a procedure involving blockage of the accessible SH groups by non-labeled N-ethylmaleimide or by diamide, subsequent isolation and solubilization of the membranes in SDS and labelling of the now accessible, residual SH groups with N-[ethyl-2-3H]ethylmaleimide. The distribution of the radioactivity over the peptide fractions shows that the non-reacting SH groups are mainly localized in the intrinsic proteins, while essentially all of the SH groups of the extrinsic protein, spectrin, are reactive. After solubilization of the membranes with Triton X-100 the non-reacting SH groups became reactive towards N-ethylmaleimide. It is proposed that lack of reaction of SH groups in the native membranes is due to their localization within the hydrophobic core of the membrane.

Pathways for flip-flop of mono- and di-anionic phospholipids in the erythrocyte membrane.

The inward translocations (flip), from the outer to the inner membrane leaflet of human erythrocytes, of di-anionic NBD-labeled phospholipids containing as a head group phosphate esters of glycolate, butyrate and hydroxyethanesulfonate are slow processes (k = 0.005-0.008 h-1, 37 degrees C) at pH 7.4. A decrease of pH highly stimulates the flip. A major role of the anion exchanger (AE1), band 3, in this flip is indicated by (a) the strong inhibition of the flip (55-85%) by stilbene disulfonates and other inhibitors of anion transport, (b) the stimulation and loss of pH dependence of the flip after modification of band 3 by Woodward's reagent K and NaBH4, and (c) the stimulation of the flip after proteolytic cleavage of band 3 by papain. The flip of mono-anionic NBD-phospholipids with phosphate esters of glycerol, glycol, methanol, butanol and benzyl alcohol is much faster than that of their dianionic analogs (k = 0.04 to > 3.0 h-1, 37 degrees C). It is inhibited by stilbene disulfonates to a decreasing extent (35 to 0%) and is not affected by several reversible inhibitors of anion exchange. This indicates a minor component of band-3-mediated flip and a major component of nonmediated flip. The outward translocations (flop), from the inner to outer membrane leaflet, of both mono- and di-anionic phospholipids are very fast (1.0-5.9 h-1), ATP-dependent and inhibitable by vanadate, fluoride, SH-reagents or Mg(2+)-depletion of cells and thereby likely to be largely mediated by a 'floppase'. The stationary distributions of the NBD-labeled anionic phospholipids are asymmetric to an extent (outer to inner leaflet ratio 2-9) correlating with the ratio of the rates of the outward and the inward translocation. Thus, asymmetry is largely abolished by blockage of the floppase-mediated translocation.

Transbilayer reorientation of phospholipid probes in the human erythrocyte membrane. Lessons from studies on electroporated and resealed cells.

In order to characterize in more detail the previously observed (Dressler et al. (1983) Biochim. Biophys. Acta 732, 304-307) increases in transbilayer mobility of phospholipids in the erythrocyte membrane following electroporation at 0 degrees C and subsequent resealing at 37 degrees C of the cells, we have studied rates of flip and flop as well as steady state distributions of the fluorescent N-(NBD)-aminohexanoyl-analogues of the four major membrane phospholipids. Measurements comprised the passive non-mediated components as well as those mediated by specific translocases (flippase and floppase). The major new findings and insights can be summarized as follows. (1) The enhancement of passive transbilayer mobility which increases with the strength, duration, and number of field pulses at 0 degrees C, cannot be fully reversed by subsequent resealing at 37 degrees C. Flip-flop remains considerably elevated relative to the original values.(2) Enhanced mobilities induced by electroporation differ for the probes studied in the sequence SM <<< PS << PC < PE. Other membrane perturbations going along with enhanced flip-flop share only in part this pattern. (3) Mediated, ATP-dependent components of flip and flop of the probes are suppressed in electroporated/resealed cells, partly due to loss of cellular Mg2+, partly - in case of flippase - due to competition by externalized endogenous PS. (4) Electroporated/resealed cells provide an elegant means to demonstrate the contribution of various components of flip and flop to the steady state transbilayer distribution of phospholipids, in particular the role of passive mobility. The new, detailed information on the displacements of phospholipid between the two leaflets of the membrane bilayer in porated/resealed cells will help to understand erythrocyte shape changes following poration and during resealing (Henszen et al. (1993) Biol. Chem. Hoppe-Seyler 374, 114).

Acceleration of phospholipid flip-flop in the erythrocyte membrane by detergents differing in polar head group and alkyl chain length.

The detergents, alkyltrimethylammonium bromide, N-alkyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (zwittergent), alkane sulfonate, alkylsulfate, alkyl-beta-D-glucopyranoside, alkyl-beta-D-maltoside, dodecanoyl-N-methylglucamide, polyethylene glycol monoalkyl ether and Triton X-100, all produce a concentration-dependent acceleration of the slow passive transbilayer movement of NBD-labeled phosphatidylcholine in the human erythrocyte membrane. Above a threshold concentration, which was well below the CMC and characteristic for each detergent, the flip rate increases exponentially upon an increase of the detergent concentration in the medium. The detergent-induced flip correlates with reported membrane-expanding effects of the detergents at antihemolytic concentrations. From the dependence of the detergent concentration required for a defined flip acceleration on the estimated membrane volume, membrane/water partition coefficients for the detergents could be determined and effective detergent concentrations in the membrane calculated. The effective membrane concentrations are similar for most types of detergents but are 10-fold lower for octaethylene glycol monoalkyl ether and Triton X-100. The effectiveness of a given type of detergent is rather independent of its alkyl chain length. Since detergents do not reduce the high temperature dependence of the flip process the detergent-induced flip is proposed to be due to an enhanced probability of formation of transient hydrophobic structural defects in the membrane barrier which may result from perturbation of the interfacial region of the bilayer by inserted detergent molecules.

Spectrin as a stabilizer of the phospholipid asymmetry in the human erythrocyte membrane.

After treatment of intact human erythrocytes with SH-oxidizing agents (e.g. tetrathionate and diamide) phospholipase A2 cleaves approx. 30% of the phosphatidylserine and 50% of the phosphatidylethanolamine without causing hemolysis (Haest, C.W.M. and Deuticke, B (1976) Biochim. Biophys. Acta 436, 353--365). These phospholipids are scarcely hydrolysed in fresh erythrocytes and are assumed to be located in the inner lipid layer of the membrane (Verkleij, A.J., Zwaal, R.F.A., Roelofsen, B., Comfurius, P., Kastelijn, D. and van Deenen, L.L.M. (1973) Biochim. Biophys Acta 323, 178--193). The enhancement of the phospholipid cleavage is now shown to be accompanied by a 50% decrease of the membrane SH-groups and a cross-linking of spectrin, located at the inner surface of the membrane, to oligomers of less than 10(6) dalton. Blocking approx. 10% of the membrane SH groups with N-ethylmaleimide suppresses both the polymerization of spectrin and the enhancement of the phospholipid cleavage. N-Ethylmaleimide, under these conditions, reacts with three SH groups per molecule of spectrin, 0.7 SH groups per major intrinsic 100 000 dalton protein (band 3) and 1.1 SH groups per molecule of an extrinsic protein of 72 000 daltons (band 4.2). Blocking studies with iodoacetamide demonstrate that the SH groups of the 100 000-dalton protein are not involved in the effects of the SH-oxidizing agents. It is suggested that a release of constraints imposed by spectrin enables phosphatidylserine and phosphatidylethanolamine to move from the inner to the outer lipid layer of the erythrocyte membrane and that spectrin, in the native erythrocyte, stabilizes the orientation of these phospholipids to the inner surface of the membrane.

Selective alteration of erythrocyte deformabiliby by SH-reagents: evidence for an involvement of spectrin in membrane shear elasticity.

In order to elucidate the molecular basis of membrane shear elasticity, the effect of membrane protein modification by SH-reaents on the deformability of human erythrocytes was studied. Deformability was quuantified by measuring the elongation of erythrocytes subjected to viscometric flow in a transparent cone plate viscometer. Impermeable SH-reagents proved to have no mechanical effect. Many, but not all, permeable SH-reagents markedly decreased the elongation. Among these, bifunctional SH-reagents (e.g. diamide, tetrathionate and N, N' -p-phenylenedimaleimide) able to cross-link membrane SH-groups were more effective than monofunctional SH-reagents (e.g. N-ethylmaleimide and ethacrynic acid). The bifunctional SH-reagents produced a 50% decrease of elongation after modification of less than 5% of the membrane SH-groups. In contrast, for a comparable effect, more than 20% of the SH-groups had to be modified by the monofunctional reagents. The effect of SH-oxidizing agents was fully reversible after treatment with disulfide-reducing agents. All bifunctional SH-reagents induced a dimerization of a small fraction of spectrin. Anaalysis of the distribution of the diamide-induced disulfide bonds among the various membrane protein fractions showed that this agent preferentially acts on the spectrin polypeptides. The results provide direct experimental evidence that the native arrangement of spectrin is essential for the shear resistance of the erythrocyte membrane and that introduction of small numbers of intermolecular cross-links as well as modification within the molecule lead to a rapid loss of this function.

Intra- and intermolecular cross-linking of membrane proteins in intact erythrocytes and ghosts by SH-oxidizing agents.

In intact human erythrocytes, SH-oxidizing agents exclusively cross-link spectrin via disulfide bonds. In ghosts, additional dimerization of the major intrinsic protein, band 3, is observed. After blockade of intracellular GSH the agents dimerize band 3 in the intact cell too, indicating that GSH may prevent band 3 dimerization under physiological conditions. The oxidizing agents reversibly oxidize 80% of the membrane SH-groups, suggesting that these groups are arranged close enough to each other to form disulfide bonds. This arrangement may protect other cell cell structures against free radicals or oxidative stress.

Asbestos-induced injury to cultured human pulmonary epithelial-like cells: role of neutrophil elastase.

The mechanisms responsible for asbestos-induced pulmonary epithelial cell cytotoxicity, especially oxidant-independent mechanisms, are not established. We determined whether human polymorphonuclear leukocyte (PMN) proteases contribute to asbestos-induced damage to human pulmonary epithelial-like cells (PECs) assessed using an in vitro chromium-51 release assay. Serine antiproteases, phenylmethylsulfonyl fluoride and alpha 1-antitrypsin, each ameliorated PEC injury induced by amosite asbestos and PMNs. A role for a specific proteinase, human neutrophil elastase (HNE), is supported by the facts that (1) asbestos increased HNE release assessed by an enzyme-linked immunosorbent assay technique (1.7 +/- 0.5 vs. 2.8 +/- 0.5 micrograms/ml; P < .025), (2) purified HNE or porcine pancreatic elastase (PPE) each alone caused PEC detachment, (3) asbestos plus either HNE or PPE caused PEC lysis similar to that mediated by asbestos and PMNs, and (4) cationic agents released from PMNs were unlikely to be involved because polyanions did not ameliorate injury resulting from asbestos and PMNs. Compared to elastase, cathepsin G caused less PEC detachment and negligible augmentation in asbestos-induced PEC lysis. Asbestos increased the association of 125I-labeled elastase with PECs nearly 50-fold compared with PPE alone (14.4% vs. 0.3%, respectively; P < .01) and nearly 10-fold compared with another particle, opsonized zymosan. We conclude that PMN-derived proteases, especially elastase, may contribute to asbestos-induced lung damage by augmenting pulmonary epithelial cell injury.

ARL 17477, a potent and selective neuronal NOS inhibitor decreases infarct volume after transient middle cerebral artery occlusion in rats.

We tested the effects of administration of a selective neuronal nitric oxide synthase (nNOS) inhibitor, ARL 17477, on ischemic cell damage and regional cerebral blood flow (rCBF), in rats subjected to transient (2 h) middle cerebral artery (MCA) occlusion and 166 h of reperfusion (n = 48) and in rats without MCA occlusion (n = 25), respectively. Animals were administered ARL 17477 (i.v.): 10 mg/kg; 1 mg/kg; 3mg/kg; N-nitro-L-arginine (L-NA) 10 mg/kg L-NA 1 mg/kg; and Vehicle. Administration of ARL 17477 1 mg/kg, 3 mg/kg and 10 mg/kg reduced ischemic infarct volume by 53 (p < 0.05), 23, and 6.5%, respectively. L-NA 1 mg/kg and 10 mg/kg increased infarct volume by 2 and 15%, respectively (p > 0.05). Administration of ARL 17477 (10 mg/kg) significantly (p < 0.05) decreased rCBF by 27 +/- 5.3 and 24 +/- 14.08% and cortical NOS activity by 86 +/- 14.9 and 91 +/- 8.9% at 10 min or 3 h, respectively, and did not alter mean arterial blood pressure (MABP). L-NA (10 mg/kg) significantly reduced rCBF by 23 +/- 9.8% and NOS activity by 81 +/- 7% and significantly (p < 0.05) increased MABP. Treatment with 3 mg/kg and 1 mg/kg ARL 17477 reduced rCBF by only 2.4 +/- 4.5 and 0%, respectively, even when NOS activity was reduced by 63 +/- 13.4 and 45 +/- 15.7% at 3 h, respectively, (p < 0.05). The data demonstrate that ARL 17477 inhibits nNOS in the rat brain and causes a dose-dependent reduction in infarct volume after transient MCA occlusion.

The role of free radicals in asbestos-induced diseases.

Asbestos exposure causes pulmonary fibrosis and malignant neoplasms by mechanisms that remain uncertain. In this review, we explore the evidence supporting the hypothesis that free radicals and other reactive oxygen species (ROS) are an important mechanism by which asbestos mediates tissue damage. There appears to be at least two principal mechanisms by which asbestos can induce ROS production; one operates in cell-free systems and the other involves mediation by phagocytic cells. Asbestos and other synthetic mineral fibers can generate free radicals in cell-free systems containing atmospheric oxygen. In particular, the hydroxyl radical often appears to be involved, and the iron content of the fibers has an important role in the generation of this reactive radical. However, asbestos also appears to catalyze electron transfer reactions that do not require iron. Iron chelators either inhibit or augment asbestos-catalyzed generation of the hydroxyl radical and/or pathological changes, depending on the chelator and the nature of the asbestos sample used. The second principal mechanism for asbestos-induced ROS generation involves the activation of phagocytic cells. A variety of mineral fibers have been shown to augment the release of reactive oxygen intermediates from phagocytic cells such as neutrophils and alveolar macrophages. The molecular mechanisms involved are unclear but may involve incomplete phagocytosis with subsequent oxidant release, stimulation of the phospholipase C pathway, and/or IgG-fragment receptor activation. Reactive oxygen species are important mediators of asbestos-induced toxicity to a number of pulmonary cells including alveolar macrophages, epithelial cells, mesothelial cells, and endothelial cells. Reactive oxygen species may contribute to the well-known synergistic effects of asbestos and cigarette smoke on the lung, and the reasons for this synergy are discussed. We conclude that there is strong evidence supporting the premise that reactive oxygen species and/or free radicals contribute to asbestos-induced and cigarette smoke/asbestos-induced lung injury and that strategies aimed at reducing the oxidant stress on pulmonary cells may attenuate the deleterious effects of asbestos.

The sequence of the hemoregulatory peptide is present in Gi alpha proteins.

The hemoregulatory peptide PyroGlu-Glu-Asp-Cys-Lys (HP5b), which inhibits myelopoietic colony formation in vitro, is shown to be a sequence motif which is also part of the effector domain of Gi alpha proteins. Out of 8 synthetic peptides with sequence variations of HP5b, those with the closest similarity to the Gi alpha sequence are biologically active. The inhibitory effect appears to be dependent on the blocked N-terminus. It is postulated that these peptides may interfere with signal transduction mediated by Gi alpha proteins.

Fibroblast growth factor-10 upregulates Na,K-ATPase via the MAPK pathway.

We studied the effects of fibroblast growth factor (FGF-10) on alveolar epithelial cell (AEC) Na,K-ATPase regulation. Within 30 min FGF-10 increased Na,K-ATPase activity and alpha1 protein abundance by 2.5-fold at the AEC plasma membrane. Pretreatment of AEC with the mitogen-activated protein kinase (MAPK) inhibitor U0126, a Grb2-SOS inhibitor (SH3-b-p peptide), or a Ras inhibitor (farnesyl transferase inhibitor (FTI 277)), as well as N17-AEC that express a Ras dominant negative protein each prevented FGF-10-mediated Na,K-ATPase recruitment to the AEC plasma membrane. Accordingly, we provide first evidence that FGF-10 upregulates (short-term) the Na,K-ATPase activity in AEC via the Grb2-SOS/Ras/MAPK pathway.

Cigarette smoke augments asbestos-induced alveolar epithelial cell injury: role of free radicals.

Cigarette smoke augments asbestos-induced bronchogenic carcinoma by mechanisms that are not established. Alveolar epithelial cell (AEC) injury due to oxidant-induced DNA damage and depletion of glutathione (GSH) and adenosine triphosphate (ATP) may be one important mechanism. We previously showed that amosite asbestos-induces hydroxyl radical production and DNA damage to cultured AEC and that phytic acid, an iron chelator, is protective. We hypothesized that whole cigarette smoke extracts (CSE) augment amosite asbestos-induced AEC injury by generating iron-induced free radicals that damage DNA and reduce cellular GSH and ATP levels. Asbestos or CSE each caused dose-dependent toxicity to AEC (WI-26 and rat alveolar type I-like cells) as assessed by 51chromium release. The combination of asbestos (5 microg/cm2) and CSE (0.O1-0.1%) caused synergistic injury whereas higher doses of each agent primarily had an additive toxic effect. Asbestos (5 microg/cm2) augmented CSE-induced (0.01-1.0%) AEC DNA damage over a 4 h exposure period as assessed by an alkaline unwinding, ethidium bromide fluorometric technique. These effects were synergistic in A549 cells and additive in WI-26 cells. Asbestos (5 microg/cm2) and CSE (0.5-1.0%) reduced A549 and WI-26 cell GSH levels as assessed spectrophotometrically and ATP levels as assessed by luciferin/luciferase chemiluminescence but a synergistic interaction was not detected. Phytic acid (500 microM) and catalase (100 microg/ml) each attenuated A549 cell DNA damage and depletion of ATP caused by asbestos and CSE. However, neither agent attenuated WI-26 cell DNA damage nor the reductions in GSH levels in WI-26 and A549 cells exposed to asbestos and CSE. We conclude that CSE enhance asbestos-induced DNA damage in cultured alveolar epithelial cells. These data provide additional support that asbestos and cigarette smoke are genotoxic to relevant target cells in the lung and that iron-induced free radicals may in part cause these effects.

Increased prevalence of gastroesophageal reflux symptoms in patients with COPD.

STUDY OBJECTIVES: To determine the prevalence of gastroesophageal reflux (GER) symptoms in patients with COPD and the association of GER symptoms with the severity of airways obstruction as assessed by pulmonary function tests (PFTs). DESIGN: Prospective questionnaire-based, cross-sectional analytic survey. SETTING: Outpatient pulmonary and general medicine clinics at a Veterans Administration hospital. PATIENTS: Patients with mild-to-severe COPD (n = 100) were defined based on American Thoracic Society criteria. The control group (n = 51) consisted of patients in the general medicine clinic without respiratory complaints or prior diagnosis of asthma or COPD. INTERVENTION: Both groups completed a modified version of the Mayo Clinic GER questionnaire. RESULTS: Compared to control subjects, a greater proportion of COPD patients had significant GER symptoms defined as heartburn and/or regurgitation once or more per week (19% vs 0%, respectively; p < 0.001), chronic cough (32% vs 16%; p = 0.03), and dysphagia (17% vs 4%; p = 0.02). Among patients with COPD and significant GER symptoms, 26% reported respiratory symptoms associated with reflux events, whereas control subjects denied an association. Significant GER symptoms were more prevalent in COPD patients with FEV(1) < or %, as compared with patients with FEV(1) > 50% of predicted (23% vs 9%, respectively; p = 0.08). In contrast, PFT results were similar among COPD patients with and without GER symptoms. An increased number of patients with COPD utilized antireflux medications, compared to control subjects (50% vs 27%, respectively; p = 0.008). CONCLUSIONS: The questionnaire demonstrated a higher prevalence of weekly GER symptoms in patients with COPD, as compared to control subjects. There was a trend toward higher prevalence of GER symptoms in patients with severe COPD; however, this difference did not reach statistical significance. We speculate that although GER may not worsen pulmonary function, greater expiratory airflow limitation may worsen GER symptoms in patients with COPD.

Contrasting effects of inflammatory stimuli on neutrophil and monocyte adherence to endothelial cells.

Leukocyte adherence to endothelial cells (EC) is an important early event in inflammatory responses, which are often characterized by a predominance of either neutrophils (PMN) or monocytes. However, there is little information concerning the molecular events important in leukocyte adherence to EC. Intracellular activation of protein kinase C and the calcium-second messenger system leads to the stimulation of a number of important functions in PMN and monocytes. We compared the effects of members of these pathways on human PMN and monocyte adherence to cultured bovine aortic EC. We observed that phorbol myristate acetate, phorbol, 12,13-dibutyrate, L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol, and ionomycin each induced significant dose-dependent increases in PMN adherence to EC monolayers. In contrast, similar concentrations of each of these agents induced significant decreases in EC adherence of monocytes enriched by countercurrent centrifugal elutriation. Separate experiments determined that the differences in PMN and monocyte adherence to EC were not related to differences in oxidant production because 1) phorbol myristate acetate and L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol caused similar marked increases in both PMN and monocyte superoxide anion and hydrogen peroxide production and 2) ionomycin, which had opposing effects on PMN and monocyte adherence, had no effect on PMN and monocyte superoxide anion or hydrogen peroxide release. We conclude that activators of protein kinase C and the Ca-second messenger pathway have opposite effects on PMN and monocyte adherence to EC and that these effects are mediated by O2 radical-independent mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor-promoting phorbol esters inhibit monocyte adherence to endothelial cells.

Monocyte adherence to endothelial cells (EC) is an important event in the development of a monocytic inflammatory response, yet the effects of inflammatory mediators on monocyte adherence to EC are not well described. We compared the effects of phorbol esters known to activate protein kinase C, including phorbol myristate acetate (PMA) and phorbol 12,13-dibutyrate (PDA), on monocyte adherence to bovine aortic EC. Human monocytes (purity 90 +/- 1% SE) were isolated by centrifugal elutriation to obtain monocytes not previously exposed to a surface. Kinetic studies revealed that 51Cr-labeled monocyte adherence to EC reached a plateau after a 45-min incubation. Concentrations of PMA between 10 and 1,000 ng/ml significantly decreased monocyte adherence to EC (26 +/- 10 and 35 +/- 8% decrease compared with control, respectively). Concentrations of PDA of 100 and 1,000 ng/ml had a similar inhibitory effect. In contrast, the chemotactic stimulus, zymosan-activated serum, significantly increased monocyte adherence (40 +/- 14% increase compared with control). Thus inflammatory stimuli have different effects on the adhesive interaction of monocytes to EC. This may provide a mechanism to selectively modulate monocyte egress from the circulation into extravascular inflammatory sites.