Dean A. Handley

Effect of platelet activating factor on endothelial permeability to plasma macromolecules

We have examined the effect of intrajugular administration of platelet activating factor (PAF-C16) on vascular permeability in the guinea pig. To examine the loss of selective endothelial permeability, the extravasative effect of PAF was assessed by monitoring hemoconcentration and the plasma loss of 125I-albumin (6.7 nm), 125I-low density lipoproteins (22.0 nm) or 125I-very low density lipoproteins (62.1 nm). Extravasation was dose-dependent and began 1 min after PAF administration, continuing for 5-7 min. During extravasation, there was no evidence for selective plasma retention of any of the labeled plasma tracers, as measured by plasma radioactivity. These results suggest that PAF-induced extravasation is dose-dependent, with increases in vascular permeability sufficient to permit similar plasma efflux rates of albumin, low density lipoproteins and very low density lipoproteins.

The design and use of a simple device for rapid quench-freezing of biological samples

The detailed design of a simple device for rapid quench‐freezing of biological samples under reproducible conditions is presented. With spring‐augmented descent, sample immersion velocity of 10 m s−1 into a cryogenic liquid is achieved. Biological samples, loaded in Balzers planchets, Denton holders, or a newly designed ‘titanium envelope’, are suitable for rapid‐freezing with this device. Using 4 μm titanium foil, light weight (1 mg) streamlined holders can easily be made to enclose cell suspensions or tissue samples. The foil envelope is designed for efficient heat dissipation while protecting the sample from possible impact or flow distortions occurring from spring‐augmented immersion. Human erythrocytes, quench‐frozen in the titanium envelope, were prepared for electron microscopy by the freeze‐substitution technique. Two opposing 25–30 μm surface zones were frozen in the apparent absence of ice. The extended depth of cryofixation is attributed to the advantages of thin foil in the titanium envelope design and the use of rapid‐immersion technique.

Inhibition and reversal of endotoxin-, aggregated IgG- and paf-induced hypotension in the rat by SRI 63-072, a paf receptor antagonist

Platelet activating factor (paf) given intravenously produces systemic hypotension in the rat. Similar effects can be induced using endotoxin or heat-aggregated IgG challenges, which are thought to involve endogenous paf release. Extending this concept, we have examined the ability of the paf antagonist SRI 63-072 to inhibit or reverse systemic hypotension induced with paf, heat-aggregated IgG or endotoxin 0111-B4 in rats. At 100 ng kg-1 paf, there occurred a 38.6 +/- 5.1% decrease in carotid mean arterial pressure (MAP) followed by a 3.2 +/- 0.7 min recovery period (RP) to return to normal pressure values. The ED50 of SRI 63-072 was 0.16 mg kg-1 i.v. (MAP) and 0.25 mg kg-1 (RP) when given 1-5 min before the paf challenge. Endotoxin (15 mg kg-1 i.v.) produced a hypotensive response (54 +/- 8% decrease in MAP) and a corresponding 80% decrease in mesenteric artery blood flow. When given 2-8 min after endotoxin, 1.0 mg kg-1 i.v. SRI 63-072 totally restored blood pressure and artery blood flow. SRI 63-072 similarly reversed heat-aggregated IgG (10 mg kg-1) induced reduction of MAP, with an ED50 of 0.05 mg kg-1 i.v. The observations that SRI 63-072 can inhibit or reverse systemic vascular effects produced from paf and other provocators of endogenous paf release strongly implicates paf as a common final mediator of hypotension and shock. As SRI 63-072 is a competitive receptor antagonist, the hypotensive effects of these provocators appear to be mediated by vascular receptors for paf.

Prevention of non‐specific airway hyperreactivity after allergen challenge in guinea‐pigs by the PAF receptor antagonist SDZ 64–412

1 Allergen challenge by aerosol in sensitized guinea‐pigs elicited non‐specific airway hyperreactivity assessed by reactivity to i.v. histamine or acetylcholine. Airway hyperreactivity to histamine persisted for at least 48 h and was accompanied by pulmonary eosinophilia as determined by bronchoalveolar lavage cell analysis. 2 Airway hyperreactivity was independent of vagal reflex mechanisms since it was not abrogated by bilateral vagotomy. 3 The novel platelet‐activating factor (PAF) receptor antagonist SDZ 64–412 inhibited the development of airway hyperreactivity, as measured 24 h after aerosol allergen challenge, when given as a single treatment orally 2 h before allergen challenge. The PAF receptor antagonist WEB 2086 as well as methylprednisolone and ketotifen also showed efficacy in preventing development of airway hyperreactivity. 4 Neither the two PAF antagonists nor ketotifen had any effect on bronchoalveolar lavage (BAL) eosinophil numbers. Methylprednisolone was the only substance which readily prevented eosinophil recruitment in addition to airway hyperreactivity. 5 We conclude that allergen‐induced airway hyperreactivity in guinea‐pigs is inhibited by prophylactic anti‐asthma drugs and specific PAF receptor antagonists, thus demonstrating a pivotal role of PAF in this response. There was a lack of correlation between airway hyperreactivity and the presence of BAL eosinophils.

Antitumor activity of SRI 62-834, a cyclic ether analog of ET-18-OCH3

SRI 62-834, an analog of the antitumor agent ET-18-OCH3 in which the oxygen atom at carbon atom 2 has been incorporated into a five-membered heterocycle, has been prepared and evaluated as an antitumor agent. The compound exhibited good cytotoxicity in vitro against a variety of tumor cell lines and was as effective as ET-18-OCH3 given orally in the mouse Meth A sarcoma model. SRI 62-834 was shown to be an inhibitor of platelet-derived growth factor (PDGF), possibly at the receptor level, and platelet-activating factor (PAF) at the receptor level.

Vesicle distribution in the arterial endothelium determined with ruthenium red as an extracellular marker.

The numerical densities of free and attached vesicles have been determined in the peripheral zone of canine arterial endothelium with the use of ruthenium red as an extracellular marker. Ruthenium red only penetrates pinocytotic invaginations that are continuous with the extracellular space, allowing the distinction of truly free vesicles from apparently free vesicles which are actually attached to the plasma membrane in a different section plane. Attached vesicles are distributed bimodally, with density maxima 20–40 nm from the luminal and abluminal cell membranes. Free vesicles show a nearly uniform distribution across the cytoplasm. Approximately three-fourths of the apparently free vesicles are attached, and only one-fourth of these are actually free. The ratio of attached vesicles to free vesicles obtained with the use of the ruthenium red is 11.29 ± 0.63 (mean ± SD). This ratio tended to be underestimated in studies in which an extracellular marker was not employed. These data on vesicle distribution are valuable in understanding the dynamics of vesicle attachment and for predicting the possible effects of enhanced vesicle diffusion on macromolecular transport.

Biological properties of the antagonist SRI 63–441 in the PAF and endotoxin models of hypotension in the rat and dog

Intravenous administration of platelet-activating factor (PAF) produces dose-dependent hypotension in several species. We have evaluated a recently developed PAF antagonist, SRI 63-441, for its ability to inhibit the hypotensive effect of PAF in the rat and dog. In the rat, 100 ng/kg PAF produced a 38.6 +/- 5.1% decrease in carotid mean arterial pressure (MAP), followed by a 3.2 +/- 0.7 min recovery period for MAP to return to baseline values. SRI 63-441 reduced the hypotension response in the rat, where the ED50 values for inhibition of MAP were 0.16 mg/kg i.v. and 0.19 mg/kg i.v. for the recovery period. Dogs challenged with 1.5 micrograms/kg PAF i.v. demonstrated a 52 +/- 8% decrease in MAP that persisted for at least 15 min. The ED50 for inhibition of MAP by SRI 63-441 was 0.20 mg/kg i.v. Following injection of tritium-labeled SRI 63-441, 56.8 +/- 2.4% of the dose was recovered in the urine and 43.2 +/- 8.9% in the feces in the rats while in dogs 38.7 +/- 5.6% and 60.9 +/- 23.5% of the dose was excreted in the urine and feces, respectively. In the rat model of endotoxin-induced hypotension, SRI 63-441 given 1 min after a 5 mg/kg endotoxin challenge (which produced a 52 +/- 7% decrease in MAP), reversed the systemic effects, with an ED50 of 0.18 mg/kg i.v. The ED50 for reversal 6 min after endotoxin injection was 0.01 mg/kg. These results of inhibition and reversal by SRI 63-441 strongly implicate PAF as a pivotal mediator of hypotension and shock.

Vascular responses of platelet-activating factor in the Cebus apella primate and inhibitory profiles of antagonists SRI 63-072 and SRI 63-119

We have evaluated several effects of intravenous administration of synthetic platelet-activating factor (PAF) in the non-human primate Cebus apella. Parameters measured were hemoconcentration (monitored by changes in hematocrit), thrombocytopenia (platelet counts), leukopenia (loss of buffy coat), bronchoconstriction (increased airway resistance to fixed airway ventilation), thromboxane A2 production (radioimmunoassay to thromboxane B2) and in vitro aggregation responses of platelets in platelet-rich plasma. Cebus platelets were refractory to PAF-induced aggregation (up to 50 microM) and there was no evidence of thrombocytopenia, elevated thromboxane B2 levels, loss of buffy coat or bronchoconstriction following systemic PAF injection. Animals exhibited reproducible but varying sensitivities to PAF-induced hemoconcentration, where 3.5-30 micrograms/kg PAF (6.6-57 nmol/kg) was required to produce 28-32% increased hematocrit range for the colony. Hemoconcentration induced by PAF in baboons and rhesus occurred at similar doses, suggesting comparable sensitivity. Prior administration of PAF receptor antagonists SRI 63-072 or SRI 63-119 at 3 mg/kg inhibited cebus hemoconcentration responses to 3.5 micrograms/kg PAF by 96% and 100%, respectively. The ED50 values were 0.95 and 0.60 mg/kg, respectively. These results suggest that the cebus exhibits a reproducible hemoconcentration effect to PAF and that these vascular responses can be inhibited by a PAF receptor antagonist.

Colloidal Gold: A Pluripotent Receptor Probe

Abstract Colloidal gold is an electron-dense, lyophobic colloid that readily forms a stable electrostatic interaction with a variety of macromolecules. Monodispersed colloids ranging from 3-150 nm in diameter can be produced to provide the researcher with flexibility in selecting the optimally sized probe. Gold labeling of antibodies and lectins has been extensively used to study surface antigens and cell components. Recently, the use of gold labeling has been extended to study receptor-ligand binding, enzyme-substrate reactions, and transcellular pathways. Published applications include gold labeling of metabolites (low-density lipoproteins), enzymes (DNAase and RNAase, RNA polymerase, thrombin, collagenase, elastase), hormones (insulin, epidermal growth factor, glucagon), circulating plasma proteins (asialoglycoprotein, α2-macroglobulin, factor VIII-von Willebrand factor), and endotoxins (tetanus toxin, cholera toxin). This broad spectrum of applications emphasizes the versatility and usefulness of colloidal gold as a probe in areas of cell biology related to receptors, endocytosis, transport, and functions of proteins.

Butvar B-98 as a thin support film.

Support films prepared from butvar B-98 resin are mechanically stable, electron transparent, and possess minimum intrinsic structure. A simple procedure for routine preparation of support films using this resin is provided.