Pollination context alters female advantage in gynodioecious Silene vulgaris.

Gynodioecy, the co-occurrence of females and hermaphrodites, is arguably the most common angiosperm gender polymorphism in many florae. Females' ability to invade and persist among hermaphrodites depends, in part, on pollinators providing adequate pollination to females. We directly measured diurnal and nocturnal pollinators' contributions to female and hermaphrodite seed production in artificial populations of gynodioecious Silene vulgaris by experimentally restricting pollinator access. We found that female relative seed production in this system depended strongly on pollination context: females produced more than twice as many seeds as hermaphrodites in the context of abundant, nectar-collecting moths. Conversely, females showed no seed production advantage in the context of pollen-collecting syrphid flies and bees due to acutely hermaphrodite-biased visitation. We infer that variation in pollinator type, behaviour and abundance may be important for achieving the female relative fitness thresholds necessary for the maintenance of gynodioecy. Generally, our study illustrates how pollinator-mediated mechanisms may influence the evolution of breeding systems and associated suites of floral traits. Segments of a pollinator community may facilitate gynodioecy by selecting for plant characteristics that increase the attractiveness of both sexes to pollinators, such as nectar rewards. Conversely, discriminating visitors in search of pollen may restrict gynodioecy in associated plant lineages by reducing male steriles' fitness below threshold levels.

The EMG of conventional abdominal exercise and exercise with a semi-upright commercial device: comparative effects and technique considerations.

AIM: The purpose of this study was to measure and compare the muscle activity of the rectus abdominis (RA) and external obliques (EO) with conventional exercises and while using an upright commercial abdominal training device (the CoreMaster). It was hypothesized that the upright device would elicit higher electromyography (EMG) values compared to conventional abdominal exercises. METHODS: Fifteen subjects (8 males, 7 females) participated in this study. Each subject performed 10 repetitions for 5 exercises: truck lift (TL); trunk rotation to opposite knee (TROK); trunk lift on the CoreMaster (TLCM); trunk rotation on the CoreMaster (TRCM); and trunk rotation with a leg lift on the CoreMaster (TRLLCM). Muscle activity was measured for the RA and EO using surface EMG. A Biopac system (Goleta, CA, USA) processed the EMG signals. A repeated measures analysis of variance (ANOVA) determined any difference in the root mean square values and Bonferroni comparisons were used to clarify the order of differences (P<0.05). RESULTS: For the RA, all exercises on the CoreMaster produced significantly higher EMG values compared to the conventional TL. For the EO, TRCM elicited the highest EMG values. However, no significant difference was found for EO between TROK and TRLLCM. CONCLUSION: The CoreMaster elicited a greater challenge to the RA. For the EO, the CoreMaster yielded optimal effects for exercises that required pronounced rotation.

Fine scale spatial structuring of sex and mitochondria in Silene vulgaris.

Fine scale spatial structure (FSSS) of cytoplasmic genes in plants is thought to be generated via founder events and can be amplified when seeds germinate close to their mother. In gynodioecious species these processes are expected to generate FSSS in sex ratio because maternally inherited cytoplasmic male sterility genes partially influence sex expression. Here we document a striking example of FSSS in both mitochondrial genetic markers and sex in roadside populations of Silene vulgaris. We show that in one population FSSS of sexes influences relative fruit production of females compared to hermaphrodites. Furthermore, FSSS in sex ratio is expected to persist into future generations because offspring sex ratios from females are female-biased whereas offspring sex ratios from hermaphrodites are hermaphrodite-biased. Earlier studies indicated that pollen limitation is the most likely mechanism underlying negative frequency dependent fitness of females. Our results support the theoretical predictions that FSSS in sex ratio can reduce female fitness by decreasing the frequency at which females experience hermaphrodites. We argue that the influence of FSSS on female fitness is complementary to the influence of larger scale population structure on female fitness, and that population structure at both scales will act to decrease female frequencies in gynodioecious species. Better comprehension of the spatial structure of genders and genes controlling sex expression at a local scale is required for future progress toward understanding sex ratio evolution in gynodioecious plants.

Associations among cytoplasmic molecular markers, gender, and components of fitness in Silene vulgaris, a gynodioecious plant.

It has been suggested that the dynamics of chloroplast DNA (cpDNA) or mitochondrial DNA (mtDNA) genetic markers used in studies of plant populations could be influenced by natural selection acting elsewhere in the genome. This could be particularly true in gynodioecious plants if cpDNA or mtDNA genetic markers are in gametic disequilibrium with genes responsible for sex expression. In order to investigate this possibility, a natural population of the gynodioecious plant Silene vulgaris was used to study associations among mtDNA haplotype, cpDNA haplotype, sex and some components of fitness through seed. Individuals were sampled for mtDNA and cpDNA haplotype as determined using restriction fragment length polymorphism (RFLP) methods, sex (female or hermaphrodite), fruit number, fruit set, seeds/fruit and seed germination. The sex of surviving germinating seeds was also noted. All individuals in the population fell into one of two cytoplasmic categories, designated haplotypes f and g by a unique electrophoretic signature in both the mtDNA and cpDNA. The subset of the population carrying haplotype g included a significantly higher proportion of females when compared with the sex ratio of the subset carrying the f haplotype. Haplotype g had a significantly higher fitness when measured by fruit number, fruit set and seeds/fruit, whereas haplotype f had significantly higher fitness when measured by seed germination. Offspring of individuals carrying haplotype g included a significantly greater proportion of females when compared with offspring of individuals carrying the f haplotype. Other studies of gynodioecious plants have shown that females generally have higher fitness through seed than hermaphrodites, but in this study not all fitness differences between haplotypes could be predicted from differences in haplotype-specific sex ratio alone. Rather, some differences in haplotype-specific fitness were due to differences in fitness between individuals of the same sex, but carrying different haplotypes. The results are discussed with regard to the potential for hitchhiking selection to influence the dynamics of the noncoding regions used to designate the cpDNA and mtDNA haplotypes.

Down-regulation and recovery of endothelin-1 binding and signaling in rat cardiomyocytes.

Down-regulation and recovery of endothelin (ET) receptors and of ET-dependent phosphoinositide-specific phospholipase C (PI-PLC) signaling was examined in cultured cardiomyocytes from neonatal rats. Three hours treatment with 5 nM ET-1 decreased surface receptors to 30%, and transduction to 19%, of their respective time-zero values. After extensive washing and a 3 h recovery period surface receptors returned to 74% of the time-zero value, with concomitant recovery of signal transduction to 75% of the time-zero value. The recovery of PI-PLC signaling in these cells is in contrast with a previous report, but consistent with recovery of the receptor complement.

A quantitative genetic analysis of nuclear-cytoplasmic male sterility in structured populations of Silene vulgaris.

Gynodioecy, the coexistence of functionally female and hermaphroditic morphs within plant populations, often has a complicated genetic basis involving several cytoplasmic male-sterility factors and nuclear restorers. This complexity has made it difficult to study the genetics and evolution of gynodioecy in natural populations. We use a quantitative genetic analysis of crosses within and among populations of Silene vulgaris to partition genetic variance for sex expression into nuclear and cytoplasmic components. We also use mitochondrial markers to determine whether cytoplasmic effects on sex expression can be traced to mitochondrial variance. Cytoplasmic variation and epistatic interactions between nuclear and cytoplasmic loci accounted for a significant portion of the variation in sex expression among the crosses. Source population also accounted for a significant portion of the sex ratio variation. Crosses among populations greatly enhanced the dam (cytoplasmic) effect, indicating that most among-population variance was at cytoplasmic loci. This is supported by the large among-population variance in the frequency of mitochondrial haplotypes, which also accounted for a significant portion of the sex ratio variance in our data. We discuss the similarities between the population structure we observed at loci that influence sex expression and previous work on putatively neutral loci, as well as the implications this has for what mechanisms may create and maintain population structure at loci that are influenced by natural selection.

Asymmetrical crossing barriers in angiosperms.

Patterns of reproductive isolation between species may provide insight into the mechanisms and evolution of barriers to interspecific gene exchange. We used data from published interspecific hybridization experiments from 14 genera of angiosperms in order to test for the presence of asymmetrical barriers to gene exchange. Reproductive isolation was examined at three life-history stages: the ability of interspecific crosses to produce seeds, the viability of F1 hybrids, and the fertility of F1 hybrids. Statistically significant asymmetries in the strength of reproductive isolation between species were detected in all genera and at each of the three life-history stages. Asymmetries in seed production may be caused by a variety of mechanisms including differences in stigma/style lengths, self compatibility and differential fruit abortion. Asymmetries in post-zygotic isolation are probably caused by nuclear-cytoplasmic interactions. Asymmetrical reproductive isolation between plant taxa may have important implications for the dynamics of hybrid zones, the direction of genetic introgression and the probability of reinforcement.

End-of-life decision making by adolescents, parents, and healthcare providers in pediatric oncology: research to evidence-based practice guidelines.

Participating in end-of-life decisions is life altering for adolescents with incurable cancer, their families, and their healthcare providers. However, no empirically developed and validated guidelines to assist patients, parents, and healthcare providers in making these decisions exist. The purpose of the work reported here was to use three sources (the findings of three studies on decision making in pediatric oncology, published literature, and recommendations from professional associations) to develop guidelines for end-of-life decision making in pediatric oncology. The study designs include a retrospective, descriptive design (Study 1); a prospective, descriptive design (Study 2); and a cross-sectional, descriptive design (Study 3). Settings for the pediatric oncology studies included a pediatric catastrophic illness research hospital located in the Midsouth (Studies 1 and 2); and that setting plus a children's hospital in Australia and one in Hong Kong (Study 3). Study samples included 39 guardians and 21 healthcare providers (Study 1); 52 parents, 10 adolescents, and 22 physicians (Study 2); and 43 parents (Study 3). All participants in the studies responded to six open-ended questions. A semantic content analysis technique was used to analyze all interview data. Four nurses independently coded each interview; interrater reliability per code ranged from 68% to 100% across studies. The most frequently reported influencing factors were "information on the health and disease status of the patient," "all curative options having been attempted," "trusting the healthcare team," and "feeling support from the healthcare provider." The agreement across studies regarding influencing factors provides the basis for the research-based guidelines for end-of-life decision making in pediatric oncology. The guidelines offer assistance with end-of-life decision making in a structured manner that can be formally evaluated and individualized to meet patient and family needs.

A23187 causes release of inositol phosphates from cultured rat Kupffer cells.

The Ca2+ ionophore A23187 is routinely used to illustrate the extracellular Ca2+-dependence of a variety of cellular reactions. We found that A23187-induced hydrolysis of phosphoinositides to various inositol phosphates in rat Kupffer cells was accompanied by their release from the cells. The synthesis and release of inositol phosphates was A23187 concentration-dependent (0.5-10 microM), and was apparent at the lowest concentration tested. A23187-induced release of inositol phosphates increased time-dependently, was apparent at 5 s of stimulation and maximal at 20 min. The effects of A23187 were reversed by EGTA. The integrity of the cells was not affected by A23187 treatment as indicated by their exclusion of trypan blue and the lack of release of lactate dehydrogenase. We propose that such effects should be considered while evaluating the Ca2+-dependence of biological processes based on the actions of A23187.

Comparison of three techniques to measure unsaturated-zone air permeability at Picatinny Arsenal, NJ.

The purpose of this study is to compare three techniques to measure the air permeability of the unsaturated zone at Picatinny Arsenal, NJ and to examine the effects of moisture content and soil heterogeneity on air permeability. Air permeability was measured in three ways: laboratory experiments on intact soil cores, field-scale air pump tests and calibration of air permeability to air pressures measured in the field under natural air pressure conditions using a numerical airflow model. The results obtained from these three methods were compared and found to be similar. Laboratory experiments performed on intact cores measured air permeability values on the order of 10(-14) to 10(-9) m2. Low-permeability cores were found between land surface and a depth of 0.6 m. The soil core data were divided into two layers with composite vertical permeability values of 1.3 x 10(-13) m2 from land surface to a 0.6-m depth and 3.8 x 10(-10) m2 for the lower layer. Analyses of the field-scale pump tests were performed for two scenarios: one in which the entire unsaturated zone was open to the atmosphere and one assuming a cap of low permeability extending 0.6 m below land surface. The vertical air permeability values obtained for the open scenario ranged from 1.2 x 10(-9) to 1.5 x 10(-9) m2, and ranged from 3.6 x 10(-9) to 6.8 x 10(-9) m2 in the lower layer, assuming an upper cap permeability of 6.0 x 10(-14) m2. The results from the open scenario are much higher than expected and the possible reasons for this ambiguity are discussed. The results from the capped scenario matched closely with those from the other methods and indicated that it is important to have background information on the study site to correctly analyze the pump test data. The optimized fit of the natural subsurface air pressure was achieved with an intrinsic permeability value of 3.3 x 10(-14) m2. When the data were refitted to the model assuming two distinct layers of the unsaturated zone, the optimized fit was achieved with intrinsic air permeabilities of 1.6 x 10(-14) m2 for the upper, low-permeability region and 8.4 x 10(-9) m2 for the lower region. Vertical air-permeability predictions from the three methods are similar provided that soil cores are collected from all representative depths to account for heterogeneous layers and that the correct assumptions are made when analyzing field-scale pump test data.

Correlation between male and female reproduction in the subdioecious herb Astilbe biternata (Saxifragaceae).

Genotypic trade-offs between male and female reproduction are commonly assumed in theoretical studies of the evolution of gender specialization. Although these trade-offs are supported by higher seed production of females than hermaphrodites in natural populations of gynodioecious species, comparisons between male and female reproductive allocation among hermaphrodite individuals under controlled conditions are rare. We assessed phenotypic and genotypic correlations between stamen and fruit production in fruiting males of the near-dioecious herb Astilbe biternata. In the field, we found a significant negative phenotypic correlation between stamen production and fruit production within individuals that produced both stamens and fruit as well as higher fruit set in females than fruiting males. The negative correlation between fruit and stamen production that was observed in the field was also apparent across clonally propagated genotypes. These results suggest that negative genetic correlations between male and female reproduction may limit the independent evolution of fruit and stamen production in A. biternata.

Bone morphogenetic protein-9. An autocrine/paracrine cytokine in the liver.

Bone morphogenetic proteins (BMPs) occupy important roles during development serving to direct cells through specific differentiation programs. While several BMPs are essential for embryonic viability, their significance in mediating intercellular communication in the context of adult organ systems remains largely unknown. In the adult rat we characterized the tissue- and cell-specific transcription and translation of BMP-9. Utilizing a ribonuclease protection assay, we determined that in the adult animal, BMP-9 expression occurs predominantly in the liver. Furthermore, we determined that the non-parenchymal cells of the liver, i.e. endothelial, Kupffer, and stellate cells, are the major sources of this message. Western analyses corroborate the ribonuclease protection assay results, confirming that LEC and KC contain an abundance of immunoreactive BMP-9. Using [(125)I]BMP-9, a receptor with specific binding affinity for BMP-9 was characterized in primary cultures of hepatic endothelial cells and Kupffer cells. BMP-9 binding to these cell types was observed to be fully reversible and highly specific for this ligand. Additionally, we demonstrate that BMP-9 is specifically internalized upon binding to its receptor. This may represent a novel BMP receptor and is the first to be characterized in primary cultures of mature liver non-parenchymal cells. Our results depict BMP-9 as a potential autocrine/paracrine mediator in the hepatic reticuloendothelial system.

The expression and localization of plasma platelet-activating factor acetylhydrolase in endotoxemic rats.

The production of platelet-activating factor (PAF) and PAF-like phospholipids that also bind the PAF receptor are implicated in numerous pathological situations including bacterial endotoxemia and injury-induced oxidative damage. PAF and PAF-like phospholipids are hydrolyzed and inactivated by the enzyme PAF acetylhydrolase. In the intact rat, infusion of lipopolysaccharide (LPS) into a mesenteric vein served as an acute, liver-focused model of endotoxemia. We determined that the liver responds to LPS exposure with the production of plasma-type PAF acetylhydrolase mRNA and protein expression specifically in the resident macrophages of the liver. Liver macrophages, defined immunohistochemically using antibodies against ED1, present in livers from saline-treated animals contained no detectable PAF acetylhydrolase. Twenty-four hours following in vivo LPS administration, immunohistochemistry detected a slight increase in the number of ED1 staining cells and the ED1-positive cells now contained an abundance of PAF acetylhydrolase. The systemic administration of LPS resulted in increased expression of PAF acetylhydrolase in several tissues. Of the tissues examined, the greatest increase in PAF acetylhydrolase expression was observed in lung followed by increases in spleen, liver, kidney, and thymus. Additionally, the expression of PAF acetylhydrolase mRNA increased in circulating leukocytes and in peritoneal macrophages in response to systemic exposure to LPS. We examined the regulation of PAF acetylhydrolase expression and demonstrated the administration of the PAF receptor antagonists, BN 50739 and WEB 2170, inhibited by 50% the increase in PAF acetylhydrolase expression in response to LPS. The up-regulation of the plasma-type PAF acetylhydrolase expression constitutes an important mechanism for elevating the local and systemic ability to inactivate PAF and oxidized phospholipids in order to minimize PAF-mediated pathophysiology consequent from exposure to endotoxin. The abundance of PAF acetylhydrolase production in the liver lobule likely limits endotoxin-mediated tissue damage due to PAF synthesis.

Linkage disequilibrium and phylogenetic congruence between chloroplast and mitochondrial haplotypes in Silene vulgaris.

Both the chloroplast and mitochondrial genomes are used extensively in studies of plant population genetics and systematics. In the majority of angiosperms, the chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) are each primarily transmitted maternally, but rare biparental transmission is possible. The extent to which the cpDNA and mtDNA are in linkage disequilibrium is argued to be dependent on the fidelity of co-transmission and the population structure. This study reports complete linkage disequilibrium between cpDNA and mtDNA haplotypes in 86 individuals from 17 populations of Silene vulgaris, a gynodioecious plant species. Phylogenetic analysis of cpDNA and mtDNA haplotypes within 14 individuals supports a hypothesis that the evolutionary histories of the chloroplasts and mitochondria are congruent within S. vulgaris, as might be expected if this association persists for long periods. This provides the first documentation of the evolutionary consequences of long-term associations between chloroplast and mitochondrial genomes within a species. Factors that contribute to the phylogenetic and linkage associations, as well as the potential for intergenomic hitchhiking resulting from selection on genes in one organellar genome are discussed.

Expression of osteogenic protein-1 mRNA in cultured kidney cells.

Osteogenic protein-1 (OP-1 or BMP-7) is a multifunctional cytokine that regulates the development of several tissues during embryogenesis, including the skeleton, eye, and kidney. In postnatal life, OP-1 expression is most abundant in the kidney, although the cellular localization of this expression has not been described. In this study, we utilized a cell culture approach to localize OP-1 mRNA expression in various renal cell types and to determine potential target cells for OP-1 effects. OP-1 mRNA expression was demonstrated in several glomerular cell types, such as mesangial, epithelial, and endothelial cells. Distal tubule MDCK cells also expressed OP-1 mRNA but human proximal tubule HK-2 cells did not. Multiple OP-1 transcripts, which ranged in size from 1.6 to 3.8 kb, were observed in both glomerular and tubule cells. Interestingly, the pattern of expression varied among the different cell types, suggesting cell-specific expression of OP-1 mRNA. Analysis of OP-1 receptor expression revealed transcripts for BMP receptors type IA and IB in HK-2 cells and transcripts for BMPR-IA and ALK-2 in mesangial cells. Treatment of HK-2 cells with OP-1 (300 ng/ml) for 24-48 hr increased cellular proliferation whereas treatment of cells with transforming growth factor-beta had no effect. Mesangial cell proliferation was not affected by OP-1. The results suggest that OP-1 is produced in the renal glomerulus and then travels to the proximal tubule to regulate the proliferation of cells in this region of the nephron.

Suppression of lipopolysaccharide-induced nitric oxide synthase expression by platelet-activating factor receptor antagonists in the rat liver and cultured rat Kupffer cells.

Excessive nitric oxide (NO) generated by hepatic cells in response to lipopolysaccharide (LPS) and inflammatory substances (e.g., platelet-activating factor [PAF]) is a key contributor to the pathophysiological outcomes observed in the liver during sepsis. In rats subjected to liver-focused endotoxemia, inducible nitric oxide synthase (iNOS) levels in the intact liver were elevated by 6 hours; cell-specific expression of iNOS messenger RNA (mRNA) was Kupffer cells (KCs), endothelial cells, and hepatocytes. Elevated serum alanine transaminase (ALT) levels at 6 hours confirmed hepatic damage. Pretreatment of endotoxemic rats with PAF receptor antagonists BN 50739 or WEB 2170 reduced serum ALT and iNOS mRNA levels in the intact liver. Pretreatment of cultured KCs with BN 50739 or WEB 2170 inhibited both LPS and PAF-induced iNOS mRNA formation. In addition, LPS-induced iNOS protein levels in KCs pretreated with BN 50739 or WEB 2170 were decreased. Exposure of KCs to either LPS or PAF caused the translocation of the p65 subunit of nuclear factor kappa B (NF-kappaB) into the nucleus and this process was attenuated by BN 50739 and WEB 2170. There was concomitant inhibition of LPS-dependent degradation of the inhibitory protein IkappaBalpha and increase in intracellular Ca(2+) in KC treated with BN 50739 or WEB 2170. Also, in KCs, LPS was able to induce iNOS mRNA expression independent of CD14. This response was inhibited by pretreatment of KCs with either BN 50739 or WEB 2170. Our findings indicate that PAF receptor antagonists convey protection against hepatocellular injury accompanied by a decrease in nitric oxide (NO) formation in the livers of endotoxemic rats.

Relationship between fire fighting suppression tasks and physical fitness.

This investigation evaluated the relationship between physical fitness and performance of fire suppression tasks. The following mean +/- SD values were associated with 91 fire fighters: age 31.69 +/- 7.39 years, height 177.29 +/- 6.38 cm, weight 83.97 +/- 10.86 kg, % fat 13.78 +/- 4.31, fat free weight (FFW) 71.52 +/- 7.66 kg, pull-ups 9.03 +/- 4.79, push-ups 41.02 +/- 14.08, 1.5 mile run 737.60 +/- 108.11 s, sit and reach 32.00 +/- 8.5 cm, sit-ups 39.88 +/- 7.75, and total grip strength 116.75 +/- 17.67 kg. The physical performance assessment (PPA) consisted of the following: stair climb, hoist, forcible entry, hose advance, and victim rescue. Significant correlations (p < 0.01) were found between the PPA and the following: total grip strength (r = -0.54), FFW (r = -0.47), height (r = -0.40), pull-ups (r = -0.38), push-ups (r = -0.38), 1.5 mile run (r = 0.38), sit-ups (r = -0.32), weight (r = -0.30) and % fat (r = 0.30). Multiple regression analysis indicated that the best multiple predictor of PPA was the 1.5 mile run, FFW, and pull-ups, r = 0.73, p < 0.001. This investigation shows the importance of physical fitness as related to performance of fire suppression job tasks.

Effects of calcium channel antagonists on LPS-induced hepatic iNOS expression.

The onset of liver injury is a pivotal event during endotoxemia. Lipopolysaccharide (LPS) activates the Kupffer cells (KC), the resident macrophages of the liver, to generate an abundance of inflammatory substances, including nitric oxide (NO). Elevated levels of NO are thought to contribute to the propagation of liver injury during sepsis. Calcium, a major second messenger in several cellular signaling events, is required by the KC for the generation of inducible nitric oxide synthase (iNOS). The purpose of this study was to determine whether calcium channel antagonists limit hepatic injury and iNOS expression in vivo following LPS exposure and to evaluate their effects on the regulation of iNOS expression in cultured KC. In rats subjected to LPS for 6 h, the serum alanine aminotransferase (ALT) level was elevated significantly; this response was accompanied by an increase in iNOS mRNA formation in the intact liver. Pretreatment of rats with calcium channel antagonists (i.e., diltiazem, nifedipine, or verapamil) before LPS exposure attenuated the serum ALT level and iNOS mRNA expression in the liver. Pretreatment of cultured KC with calcium channel antagonists for 1 h followed by the addition of LPS markedly repressed iNOS protein and mRNA expression. Time-course studies revealed that calcium channel antagonists were most effective at inhibiting LPS-induced iNOS mRNA formation by KC when added before LPS. Treatment of KC with calcium channel antagonists prior to the addition of LPS decreased nuclear levels of the p65 subunit of nuclear factor-kappaB and prevented the LPS-dependent degradation of the inhibitory protein IkappaBalpha. Thus our findings indicate that under endotoxemic conditions calcium channel antagonists limit hepatocellular injury that is accompanied by an inhibition of LPS-mediated iNOS expression in rat liver KC.

Hepatic regulation of platelet-activating factor acetylhydrolase and lecithin:cholesterol acyltransferase biliary and plasma output in rats exposed to bacterial lipopolysaccharide.

Normal rat bile contains secretory platelet-activating factor acetylhydrolase (PAF-AH), the enzyme capable of hydrolyzing the inflammatory mediator platelet-activating factor (PAF), and phospholipids containing oxidized truncated fatty acids. Because lecithin:cholesterol acyltransferase (LCAT) possesses intrinsic PAF-AH-like activity, it also may represent a potential anti-inflammatory enzyme. The behavior of PAF-AH and LCAT in hepatobiliary inflammatory responses in vivo has not been characterized. We therefore investigated the biliary and plasma secretion and pharmacological characteristics of these enzymes in rats subjected to intraportal bacterial endotoxin exposure (lipopolysaccharide [LPS], Escherichia coli, 055:B5). Portal vein LPS infusion (1 mg/kg, bolus) resulted in a maximal 4- to 5-fold increase in bile PAF-AH-specific activity with a gradual decline to baseline by 18 hours. Biliary PAF-AH hydrolyzed also the truncated sn-2-succinoyl and sn-2-glutaroyl analogs of PAF, indicating a broader activity of PAF-AH in bile toward byproducts of glycerophospholipid peroxidation. Plasma PAF-AH activity was not altered 5 hours after LPS injection compared with saline injection, but it was significantly elevated 18 hours after endotoxin exposure. The levels of LCAT in bile were low and declined to nearly undetectable values by 5 hours after cannulation in both control and LPS-exposed rats. Plasma LCAT activity was significantly increased after 5 hours and decreased 18 hours after LPS injection. In summary, hepatic exposure to endotoxin results in a rapid increase in biliary secretion of PAF-AH followed by elevation of LCAT and PAF-AH levels in plasma. We propose that biliary secretion of PAF-AH may be involved in the hepatic response to endotoxic insult by counteracting potential inflammatory damage in the biliary tree and gastrointestinal tract, whereas plasma increases in LCAT and PAF-AH may promote elimination of excess PAF and oxidized phospholipids in the circulation.

Localization of the platelet-activating factor receptor to rat pancreatic microvascular endothelial cells.

Platelet-activating factor (PAF) is a potent lipid autocoid involved in numerous inflammatory processes. Although PAF plays a key role as a mediator of inflammation in acute pancreatitis, the site(s) of action of PAF in the pancreas remains unknown. One of the aims of this study was to identify cell types within the pancreas expressing the PAF receptor using immunohistochemical protocols. Additionally, pancreatic microvascular endothelial cells were isolated and examined for the PAF receptor using immunohistochemistry, reverse transcription-polymerase chain reaction, and intracellular calcium responses to PAF exposure. Immunohistochemical analysis of pancreatic slices using an antibody directed toward the N-terminus of the PAF receptor revealed specific localization to the vascular endothelium with no localization to other pancreatic cell types. Reverse transcription-polymerase chain reaction of RNA isolated from cultured pancreatic islet endothelial cells yielded the predicted amplicon for the PAF receptor. Cultured pancreatic islet endothelial cells responded to PAF as measured by a transient increase in intracellular calcium, which was ameliorated in the presence of a PAF receptor antagonist. The results demonstrate the localization of PAF receptors on the pancreatic vascular endothelium. The presence of PAF receptors on the pancreatic vascular endothelium provides a defined, highly localized target for therapeutic intervention.