Biomedical subjects

R N Pinckard

Publications and source records attributed to R N Pinckard.

At least 37 records · Page 2Linked to original sources

PubMed1987

Ontogeny of the responsiveness to intravenous platelet-activating factor.

Neonatal and young animals fail to develop antigen-induced, lethal, systemic anaphylactic reactions. Recent evidence has documented that, in the adult rabbit, an unusual phospholipid autacoid, platelet-activating factor, induces almost all of the physiologic events associated with IgE-induced anaphylaxis. Thus, in the present study, the intravascular alterations after intravenous infusion of synthetic platelet-activating factor (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine; AGEPC) into young rabbits were examined. In comparison to 13-week-old, adult rabbits, the intravascular infusion of greater than 1.0 micrograms/kg AGEPC was not lethal in rabbits of 8 weeks of age or less. In dose-response studies, the amount of AGEPC required to induce a lethal response in 50% of the animals tested (LD50) was found to inversely correlate with age. In contrast, AGEPC-induced platelet aggregation in vitro was not affected by the age of the donor animal. Consistent with age-independent platelet responsiveness in vitro, AGEPC-induced thrombocytopenia and intravascular accumulation of platelet factor 4 and thromboxane B2 were also unaffected by animal age. Neutropenia and basopenia, as well as platelet and neutrophil sequestration in the pulmonary microvasculature after intravenous AGEPC infusion also were similarly unaffected by animal age. Although the mechanisms which modulate the profound and lethal physiologic responses following AGEPC infusion in the adult rabbit remain to be established, the current study clearly documents an age-dependent acquisition of systemic physiologic sensitivity to AGEPC and/or other mediators released as a result of intravascular AGEPC administration.

Aging↗

PubMed1986

Characterization of inflammatory mediator release from purified human lung mast cells.

The release of inflammatory mediators (histamine, PGD2, TxB2, and LTC4) from purified human lung mast cells was characterized by kinetic and anti-IgE dose-response parameters. The relative rate of mediator release was histamine greater than PGD2 = TxB2 greater than LTC4, with one half maximal release occurring at approximately 2, 5, and 10 min, respectively. In 2 experiments, stimulation with anti-IgE caused significant quantities of platelet-activating factor (PAF) to appear rapidly (2 min) in the cell pellet; cell-associated PAF declined to low levels by 45 min. The optimal concentration of anti-IgE for the release of the arachidonate cyclooxygenase metabolites PGD2 and TxB2 (0.3 microgram/ml) was 10- to 30-fold less than that required for the release of histamine and LTC4 (3 to 10 micrograms/ml), suggesting that these release processes may have differential IgE Fc receptor cross-linking requirements. At optimal histamine release, the magnitude of the release of each arachidonate metabolite was found to correspond to the magnitude of histamine release, however, suggesting that the 2 processes are linked either in series or in parallel.

Antibodies, Anti-Idiotypic↗

PubMed1986

Age-dependent differential development of leukotactic and vasoactive responsiveness to acute inflammatory mediators.

Age-associated development of increased cutaneous vascular permeability and neutrophil accumulation in response to several inflammatory mediators was examined in young rabbits. The development of increased cutaneous vascular permeability was mediator-, age- and dose-dependent and was potentiated by prostaglandin E2 (PGE2). While 4-week-old rabbits responded to bradykinin, they did not develop increased vascular permeability in response to histamine, acetyl glyceryl ether phosphocholine (AGEPC), N-formyl-methionyl-leucyl-phenylalanine, or zymosan-activated plasma at doses which induced significant increases in vascular permeability in 13-week-old rabbits. Dose-dependent increases in vascular permeability after intracutaneous injection of AGEPC and histamine were observed by 6 weeks of age although fewer animals responded to histamine as compared to AGEPC. In addition, coadministration of PGE2 allowed the expression of increased vascular permeability to AGEPC, N-formyl-methionyl-leucyl-phenylalanine and zymosan-activated plasma in all 4-week-old animals. Interstitial neutrophil accumulation as well as increased vascular permeability was also age-, time-, mediator- and dose-dependent. Compared to 13-week-old rabbits, fewer numbers of interstitial neutrophils were observed in 4-week-old animals for up to 4 hours after intracutaneous injection of AGEPC. PGE2 potentiated the neutrophil response to N-formyl-methionyl-leucyl-phenylalanine and zymosan-activated plasma in both young and adult rabbits; nevertheless, the response in young animals was never as extensive as in the adult animal with the exception of the neutrophil infiltrative response after administration of AGEPC and PGE2. These results suggest that PGE2 synthesis and/or reactivity in young animals may be significantly different from adult animals and in part may account for the lowered reactivity to acute inflammatory mediators in the young animal.

Aging↗

PubMed1985

Fast atom bombardment-mass spectrometric identification of molecular species of platelet-activating factor produced by stimulated human polymorphonuclear leukocytes.

Fast atom bombardment mass spectrometry was used to identify molecular species of platelet-activating factor (PAF) produced by stimulated human neutrophilic polymorphonuclear leukocytes. Normal and reverse-phase high performance liquid chromatography were employed to separate the individual regions with PAF activity prior to mass spectrometric analysis. The following alkyl chain homologs of acetyl glyceryl ether phosphorylcholine (AGEPC) were found: C16:0, C17:0, C18:0 and C18:1. There was also evidence for the presence of the C15:0 homolog, as well as other species which have not yet been identified.

Chromatography, High Pressure Liquid↗

PubMed1985

Modulation of platelet-activating factor (PAF) synthesis and release from human polymorphonuclear leukocytes (PMN): role of extracellular albumin.

Human neutrophilic polymorphonuclear leukocytes (PMN) stimulated with N'-formyl-methionyl-leucyl-phenylalanine (FMLP) in the presence of cytochalasin B but in the absence of human serum albumin (HSA) synthesized only small amounts of platelet-activating factor (PAF) that attained maximum levels within 60-120 s after stimulation; in addition, no release of PAF occurred. However, in the presence of 2.5 mg HSA/ml, there was a threefold increase in PAF synthesis, 30-40% of which was released within 5 min after FMLP stimulation. In the presence of 50 mg HSA/ml there was at least a fourfold increase in PAF synthesis and release, with maximal synthesis occurring 10-20 min after stimulation. Thus, the presence of HSA during PMN stimulation not only induced an albumin dose-dependent increase in PAF release but significantly augmented the synthesis of PAF. In contrast to PAF synthesis and release, the presence or absence of HSA had no effect upon lysosomal enzyme secretion from FMLP-stimulated PMN, which was maximal within 30-60s after stimulation. These results demonstrate that HSA plays an essential role in vitro in the synthesis and release of PAF from human PMN, and support the hypothesis that there is a cyclic PAF synthesis-release coupling mechanism in the stimulated human PMN.

Calcium↗

PubMed1985

The role of complement in the pathogenesis of postischemic acute renal failure.

One hour occlusion of total renal blood flow results in oliguric acute renal failure as defined by an abrupt and severe diminution in glomerular filtration rate. In rats, after 1-2 h of such ischemia, the acute renal failure which follows is characterized by decreased renal blood flow and by intratubular obstruction with necrotic cellular debris. The present study has examined the possible role of the complement system in the development of this model of acute renal failure. Immunoreactive C3 was extensively localized within necrotic tubular epithelial cells and the walls of small muscular arteries in reperfused kidneys after 1 h of total renal ischemia. Depletion of complement by the administration of cobra venom factor 18 h prior to the induction of ischemia abrogated C3 localization and significantly attenuated the subsequent fall in renal blood flow following reperfusion but did not alter the oliguria or marked fall in glomerular filtration.

Acute Kidney Injury↗

PubMed1985

Kinetics of acetyl glyceryl ether phosphorylcholine (AGEPC)-induced acute lung alterations in the rabbit.

Acetyl glyceryl ether phosphorylcholine (AGEPC; 1-O-hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) was infused intravenously into rabbits (0.5 micrograms/kg); subsequently, temporal pulmonary alterations were assessed histologically. Within 30 seconds after AGEPC infusion, widespread platelet and neutrophil aggregates were distributed throughout the pulmonary microvasculature. Concomitantly, small muscular arteries and bronchioles throughout the lungs were contracted. Five minutes after AGEPC infusion, intravascular pulmonary platelet aggregates were less frequent and smaller than those observed at 30 seconds after infusion; however, AGEPC-induced pulmonary neutrophil sequestration persisted. Moreover, at this time, large mononucleated cells and damaged endothelial cells were prevalent throughout the pulmonary microvasculature. Sixty minutes after infusion, neither platelet aggregates nor arterial or bronchiolar constriction was observed. However, in most animals, neutrophils and large mononucleated cells were still abundant, and focal endothelial cell alterations persisted. In addition, discrete areas of interstitial hemorrhage around small and medium-sized arteries were present. These studies suggest that the intravascular release of AGEPC could initiate significant pulmonary injury and therefore could be an important etiologic factor in the development of inflammatory lung diseases.

Animals↗

PubMed1984

Molecular heterogeneity of platelet-activating factor produced by stimulated human polymorphonuclear leukocytes.

The molecular heterogeneity of platelet-activating factor (PAF) produced by stimulated human neutrophilic polymorphonuclear leukocytes (PMN) was assessed by both normal and reverse phase high performance liquid chromatography (HPLC). As detected by rabbit platelet stimulation, at least 5 PAF molecules were separated by HPLC. Fast atom bombardment (FAB) mass spectrometry revealed one of these PAFs was acetyl glyceryl ether phosphorylcholine (AGEPC) with a C16:0 alkyl chain in the sn-1 position. Although the structures of the remaining PAFs are unknown, two of the peaks of PAF activity had the same retention times on reverse phase HPLC as the C15- and C18-saturated alkyl chain AGEPC homologues. These studies indicate that the human PMN produces multiple molecular species of PAF.

Amino Acid Sequence↗

PubMed1984

Modulation of platelet-activating factor (PAF) synthesis and release from human polymorphonuclear leukocytes (PMN): role of extracellular Ca2+.

Extracellular Ca2+ regulated the synthesis and release of platelet-activating factor (PAF) from human polymorphonuclear leukocytes (PMN) stimulated with N'-formyl-methionyl-leucyl-phenylalanine (FMLP) in the presence of cytochalasin B. Maximum PAF synthesis and release required the presence of 0.14 mM Ca2+ whereas 1.4 mM Ca2+ was necessary for maximum lysosomal enzyme secretion. The synthesis of PAF occurred within 2.5 min after PMN stimulation in the presence of 1.4 mM Ca2+; however, PAF release did not occur until 5 min after stimulation. Peak PAF release occurred by 7.5 min but accounted for only 30-40% of the total amount of PAF synthesized, the remainder being retained on or within the PMN. Stimulation of PMN in the presence of 0.01 M EDTA or EGTA decreased PAF synthesis and release by greater than 95%. In the absence of extracellular Ca2+, stimulated PMN synthesized PAF in amounts that were 10-30% of maximum, but there was no release of the newly synthesized PAF. At Ca2+ concentrations greater than 0.01 mM, there was a dose-dependent (up to 0.14 mM) increase in PAF synthesis that was associated with the initiation and concomitant increase in the amount of PAF released. These data suggest the presence of a PAF synthesis-release coupling mechanism in which the extracellular Ca2+-dependent release of PAF stimulates additional PAF synthesis.

Adult↗

PubMed1984

Observations on the critical micellar concentration of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine and a series of its homologs and analogs.

In a study designed to explore the physical chemical characteristics of platelet activating factor (PAF), or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, the critical micellar concentration of this compound, as well as the propionyl, butyryl and hexanoyl homologs was determined. In addition, an analogous series of compounds, in which the 1-O-alkyl was replaced by a 1-O-hexadecanoyl or a 1-O-octadecanoyl group, were examined for their critical micellar concentration. A variety of analytical techniques including NMR, gas liquid chromatography, infra-red spectrometry, thin layer chromatography, phosphorus, choline, glyceryl ether and fatty acid analyses were used to confirm the high purity of the individual derivatives. A dye binding assay and a surface tension technique were compared as to their suitability for determination of the critical micellar concentration of these compounds. Whereas the dye binding method proved highly variable, the surface tension procedure proved to be a facile, reproducible technique and was the assay of choice. The critical micellar concentration of the 1-O-alkyl and the 1-O-acyl derivatives showed comparable values for each short chain substituent at carbon 2, with values, in microM, ranging from 1.3 +/- 0.03 for 1-O-hexadecanoyl-2-acetyl-sn-glycero-3-phosphocholine and 1.1 +/- 0.10 for 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine to 0.22 +/- 0.01 for 1-O-hexadecanoyl-2-hexanoyl-sn-glycero-3-phosphocholine and 0.18 +/- 0.03 for 1-O-hexadecyl-2-hexanoyl-sn-glycero-3-phosphocholine. The data show that at the molar concentration usually employed in biological studies with these compounds, i.e., 1 X 10(-7) to 10(-11) M, one can assume that they will be present as monomolecular species. Thus, it seems unlikely that the widely diverse biological activities of these compounds can be explained by this physical parameter.

Acylation↗

PubMed1984

Acetyl glyceryl ether phosphorylcholine (AGEPC). A putative mediator of cardiac anaphylaxis in the guinea pig.

Platelet-activating factor is a novel phospholipid that has been implicated as an important mediator of acute allergic reactions. The intravenous administration of acetyl glyceryl ether phosphorylcholine, a pure, synthetic platelet-activating factor, causes electrocardiographic changes in the rabbit similar to those which are characteristic manifestations of systemic anaphylaxis. To determine whether platelet-activating factor contributes to anaphylactic cardiac dysfunction, we measured platelet-activating factor release from the sensitized guinea pig heart challenged in vitro with specific antigen and compared the resulting cardiac dysfunction with that induced by the injection of acetyl glyceryl ether phosphorylcholine into nonsensitized hearts. The results of these studies document that, during anaphylaxis in the isolated guinea pig heart, a platelet-activating factor is released into the coronary effluent that has physicochemical and functional properties similar to those of acetyl glyceryl ether phosphorylcholine. The intracardiac administration of acetyl glyceryl ether phosphorylcholine (10(-14) to 3 X 10(-9) mol) induced dose-related decreases in left ventricular contractile force (-5 to -85%) and coronary flow (-5 to -85%), as well as impaired atrioventricular conduction. The negative inotropic effect of acetyl glyceryl ether phosphorylcholine also was present in hearts perfused at constant flow. Although, in these hearts, acetyl glyceryl ether phosphorylcholine increased coronary resistance, which may have caused regional shunting and ischemia, it is unlikely that the negative inotropic effect of acetyl glyceryl ether phosphorylcholine was secondary to changes in coronary flow, since acetyl glyceryl ether phosphorylcholine also caused a dose-dependent negative inotropic effect in the electrically paced, noncoronary-perfused left atrium and right ventricular papillary muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Anaphylaxis↗

PubMed1984

High performance liquid chromatography of platelet-activating factors.

Silica and C18 reverse phase high performance liquid chromatography (HPLC) were used to fractionate synthetic molecular species of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) and semi-synthetic platelet-activating factor (PAF) synthesized from beef heart plasmalogens. A single coincident peak from silica HPLC was observed for either a mixture of synthetic AGEPC's with alkyl chain lengths from C12 to C18 or for beef heart-derived PAF. This peak was well separated from other classes of phospholipid standards including 2-lysophosphatidylcholine and 3H-labeled lyso-PAF. Subsequently, the synthetic AGEPC mixture or beef heart PAF was separated into individual species on a C18 reverse phase column. Beef heart-derived PAF was fractionated into at least four molecular species of PAF activity which had similar retention times as the radioactivity of 3H-labeled beef heart PAF. Approximately 56% of the radioactivity of 3H-labeled PAF was found in the fraction with a similar retention time as 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, 10% as 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine, 11% as 1-O-pentadecyl-2-acetyl-sn-glycero-3-phosphocholine, and 13% in an unidentified fraction which eluted after C-16-AGEPC. The unidentified fraction did not correspond to any of the homologous series of synthetic AGEPCs with saturated alkyl chain lengths from C12 to C18. Recoveries of radioactive phospholipids from silica or reverse phase columns were greater than 95%.

Animals↗

PubMed1984

Morphologic basis of increased vascular permeability induced by acetyl glyceryl ether phosphorylcholine.

The potent vasoactive and leukotactic properties of acetyl glyceryl ether phosphorylcholine (AGEPC) were further characterized histologically. After intravenous infusion of colloidal carbon and local injection of AGEPC, microscopic examination of rat cremaster muscle and skin revealed histamine-like vascular labeling restricted to postcapillary venules. Ultrastructural studies demonstrated subendothelial carbon accumulation in labeled venules. In rat skin, vascular labeling with colloidal carbon was an equally sensitive indicator of AGEPC-induced vasoactivity as was Evans blue dye extravasation for the assessment of AGEPC-induced increased vascular permeability (i.e., 1 pmole of AGEPC consistently initiated both vascular labeling and increased vascular permeability). In addition to its potent vasoactive effects in rabbits and rats, concomitant leukocyte emigration was observed in venules within 15 minutes after intradermal injection of AGEPC. In rabbit skin, AGEPC was equally as potent for the induction of leukocyte infiltrates as for the stimulation of increased vascular permeability. However, the vasoactive properties of AGEPC appeared to be neutrophil independent as well as independent of mast cell and platelet stimulation; these data suggest that AGEPC may act upon the microvasculature by direct stimulation of the venular endothelial cells. Thus, the putative role of AGEPC as a potent inflammatory mediator includes both the vasoactive and the leukotactic aspects of the acute inflammatory process.

Animals↗

PubMed1984

A facile route to semi-synthesis of acetyl glycerylether phosphoethanolamine and its choline analogue.

A facile route to the semi-synthesis of acetyl glycerylether phosphoethanolamine and, subsequently, its choline analogue (platelet-activating factor) has been developed. In essence, this technique takes advantage of the fact that the phosphatidylethanolamine fraction of bovine erythrocytes contains 75-80% of a 1-O-alkyl-2 fatty acyl derivative. Isolation of the latter by silicic acid chromatography followed by base-catalyzed methanolysis allowed good recovery (60-70%) of the 1-O-alkyl-(lyso)-sn-glyceryl-3-phosphoethanolamine, which contained a mixture of long chain alkyl ethers. This compound was treated with acetic anhydride in the presence of trace amounts of perchloric acid for 45 sec to give, in excellent yield, 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphoethanolamine (AGEPE). This procedure gave a 70% yield of purified AGEPE, based on the starting component, 1-O-alkyl-(lyso)-sn-glyceryl-3-phosphoethanolamine. Separation of AGEPE into fractions individually enriched in the 16:0, 18:0, and 18:1 alkylether substituents was accomplished by silica gel G combined with silver nitrate-impregnated silica gel H thin-layer chromatography. The AGEPE or its individual molecular species can be converted in high yields to the corresponding 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine (AGEPC) analogues by reaction with methyl iodide in the presence of a crown ether. Characterization of the derivatives was achieved through thin-layer chromatography, infrared spectroscopy, gas-liquid chromatography, and combined gas-liquid chromatography-mass spectrometry. The ability of these analogues to induce irreversible aggregation and secretion of serotonin from washed rabbit platelets was evaluated.

Acetylation↗

PubMed1983

Thromboxane B2 (TxB2) release following acetyl glyceryl ether phosphorylcholine (AGEPC) infusion in the rabbit.

Intravenous infusion of acetyl glyceryl ether phosphorylcholine (AGEPC) into rabbits resulted in an AGEPC dose-dependent elevation of plasma thromboxane B2 (TxB2) levels within 30 seconds. In contrast to AGEPC, the deacetylated derivative, lyso-GEPC (36.8 micrograms), did not increase intravascular TxB2 levels when similarly infused into rabbits. Intravascular TxB2 levels were maximal at 60 seconds after the infusion of 0.61 micrograms AGEPC whereas at higher doses of AGEPC (1.21-2.4 micrograms), plasma TxB2 levels were maximally elevated within 30 seconds after initiation of AGEPC infusion. These acute increases in the intravascular levels of TxB2 were accompanied by the development of thrombocytopenia, neutropenia, and basopenia which occurred concomitantly with AGEPC dose-dependent elevations in the plasma levels of platelet factor 4. Elevations in the plasma TxB2 levels returned to pre-infusion levels within 10-20 minutes after the initiation of AGEPC infusion. Thus, in vivo, one potent phospholipid, AGEPC, stimulates the production of another class of potent lipid mediators, the thromboxanes.

Animals↗

PubMed1983

Platelet-activating factor.

Our ability to chemically characterize and synthesize this autacoid of allergy and inflammation has given us a unique opportunity to study these important molecules in a highly disciplined fashion. It has also opened the door to a vista of research approaches to define PAF's normal physiologic role as well as its actions as an immunopathologic mediator. Recent work is described.

Anaphylaxis↗

PubMed1983

Complement localization in ischemic baboon myocardium.

Complement localization was examined by direct immunoperoxidase procedures on frozen sections of baboon myocardium obtained 24 hours after ligation of the left anterior descending coronary artery. There was extensive localization of C3, C4, and C5 in most infarcted myocardial fibers; however, in these infarcted areas of myocardium, complement components were not found in myocytes immediately adjacent to either the endocardium or epicardium. Although C3, C4, and C5 were all present within the same myocardial fibers as assessed in adjacent serial sections, the light microscopic distribution of these components was dissimilar, i.e., C3 and C5 were present in both a granular and a diffuse pattern within myocytes, whereas C4 was always localized in a diffuse pattern. Complement components C3 and C5, but not C4, were also localized in the walls of small muscular arteries in infarcted myocardium. No complement was observed in myocardial fibers or blood vessels in normal baboon myocardium. Electron microscopic evaluation of C3 localization within infarcted myocardium indicated that C3 was associated with contractile elements of myocytes, as well as with membranes of myocyte nuclei, mitochondria, and sarcoplasmic reticulum. Within vascular smooth muscular cells, C3 was associated with myofilaments and mitochondrial membranes. Thus, the results of this study provide new information regarding the cellular and subcellular distribution of complement components in infarcted baboon myocardium. If this localization of C3, C4, and C5 is a result of their in situ activation within the ischemic myocardium, a variety of complement-derived phlogistic products would be expected to have been produced and to have effected, in part, the subsequent inflammatory response.

Animals↗

PubMed1982

The "new" chemical mediators of inflammation.

Significant progress has been made in the elucidation of the chemistry and biology of the products of arachidonic acid metabolism and the acetylated alkyl phosphoglycerides. While many of their biological properties make them tempting candidates as mediators of inflammation, their precise role in the acute and/or chronic inflammatory process remains to be proven. Thus, at present, it might be best to view these potent lipids not as inflammatory mediators but as autacoids. In the words of Douglas (16): "The very fact that the substances have been classified under the noncommital title of autacoids, is, in a sense, a confession that the evidence does not at present permit a more precise functional classification such as, for example, hormone or neurohormone.... But the core of the matter is that, while the autacoids possess an astonishingly wide range of pharmacologic activities and in vanishing small amounts, there are comparatively few instances where a physiologic role can be stated with assurance." Hopefully, the coming years should prove to be exciting and fruitful for students of inflammation and immunopathology; if so, we may be able to unequivocally elucidate the precise role(s) of these fascinating lipid autacoids in modulating the inflammatory process. At that time, the term autacoid may no longer be appropriate, and a new, functional classification, perhaps mediator, can be instituted with assurance.

Animals↗