MUSCULATURE AND NEUROTRANSMITTERS IN THE DIGESTIVE SYSTEM OF TREMATODES

In this paper we analyzed the results of our own and published data concerning the presence of muscle elements in various parts of the digestive system in adult and larval forms of trematodes. The data on the localization of the circular and longitudinal muscle fibers in the pharynx, esophagus, and intestine of various representatives of trematodes are presented. The results of immunocytochemical studies indicate the presence the serotonergic and peptidergic (FMRFamidergic) nerve elements in the parts of the digestive system of trematodes. The available literature date is supplemented by the studies conducted on Prodistomum alaskense, a representative of the family Lepocreadiidae, an intestinal parasite of deep-sea fish (Zaprora silenus and Aptocyclus ventricosus). The localization of the serotoninergic and FMRFamidergic nervous structures was identified using immunocytochemical methods and the confocal scanning laser microscopy. For musculature staining the TRITC (tetramethylrhodamine isothiocyanate) – conjugated phalloidin was used. The preparations were examined using a fluorescence microscope and a confocal scanning laser microscope. The analysis of the data obtained and the information available in the literature suggests that the muscular system of the digestive tract is well developed in trematodes of various taxonomic groups. The musculature of the digestive system of trematodes is innervated by serotonergic and peptidergic (FMRFamidergic) nerve elements, which are involved in the regulation of the contractile activity of various parts of the digestive system of trematodes.

In vivo biodistribution of carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles in rats

Carboxymethylchitosan/poly(amidoamine) (CMCht/PAMAM) dendrimer nanoparticles, comprised of a PAMAM dendrimer core grafted with chains of CMCht, have recently been proposed for intracellular drug delivery. In previous reports, these nanoparticles had lower levels of cytotoxicity when compared with traditional dendrimers. In this study, the short-term in vivo biodistribution of fluorescein isothiocyanate (FITC)-labeled CMCht/PAMAM dendrimer nanoparticles after intravenous (IV) injections in Wistar Han rats was determined. The brain, liver, kidney, and lung were collected at 24, 48, and 72 h after injection and stained with phalloidin–tetramethylrhodamine isothiocyanate (TRITC, red) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, blue) to trace the nanoparticles within these tissues. The liver, kidney, and lung were also stained for hematoxylin and eosin to assess any morphological alterations of these organs. CMCht/PAMAM dendrimer nanoparticles were observed within the vascular space and parenchyma of liver, kidney, and lung and in the choroid plexus, after each injection period. No particles were observed in the brain parenchyma, nor any apparent deleterious histological changes were observed within these organs. The CMCht/PAMAM dendrimer nanoparticles were stable in circulation for a period of up to 72 h, targeting the main organs/systems through internalization by the cells present in their parenchyma. These results provide positive indicators to their potential use in the future as intracellular drug delivery systems.

Tumor necrosis factor-α-induced leukocyte adhesion and microvessel permeability

The objective of this study was to investigate whether leukocyte adhesion and/or emigration are critical steps in increased microvessel permeability during acute inflammation. To conduct this study, we combined autologous blood perfusion with a single microvessel perfusion technique, which allows microvessel permeability to be measured precisely after the endothelium has interacted with blood-borne stimuli. Experiments were carried out in intact venular microvessels in rat mesenteries. Firm attachment of leukocytes to endothelial cells was induced by intravenous injection of TNF-α (3.5 μg/kg) and resuming autoperfusion in a precannulated microvessel. Leukocyte emigration was facilitated by superfusion of formyl-Met-Leu-Phe-OH. Microvessel permeability was measured as hydraulic conductivity ( Lp) or the solute permeability coefficient to tetramethylrhodamine isothiocyanate-labeled α-lactalbumin before and after leukocyte adhesion and emigration in individually perfused microvessels. We found that perfusion of a microvessel with TNF-α did not affect basal microvessel permeability, but intravenous injection of TNF-α caused significant leukocyte adhesion. However, the significant leukocyte adhesion and emigration did not cause corresponding increases in either Lpor solute permeability. Thus our results suggest that leukocyte adhesion and emigration do not necessarily increase microvessel permeability and the mechanisms that regulate the adhesion process act independently from mechanisms that regulate permeability. In addition, silver staining of endothelial boundaries demonstrated that leukocytes preferentially adhere at the junctions of endothelial cells. The appearance of the silver lines indicates that the TNF-α-induced firm adhesion of leukocyte to microvessel walls did not involve apparent changes in the junctional structure of endothelial cells, which is consistent with the results of permeability measurements.

Effects of deletion of muscle LIM protein on myocyte function

Muscle LIM protein (MLP) may serve as a scaffold protein on the actin-based cytoskeleton, and mice deficient in this protein (MLPKO) have been recently reported to develop dilated cardiomyopathy. To determine the causes of depressed contractility in this model, we measured intracellular Ca2+ concentration ([Ca2+]i) transients (fluo 3), cell shortening, L-type Ca2+ channel current ( I Ca,L), Na/Ca exchanger current ( I Na/Ca), and sarcoplasmic reticulum (SR) Ca content in left ventricular MLPKO myocytes. I Ca,L-voltage relationships, I Na/Ca density, and membrane capacitance did not differ between wild-type (WT) and MLPKO myocytes. The peak systolic [Ca2+]i was significantly increased in MLPKO myocytes (603 ± 54 vs. 349 ± 18 nM in WT myocytes). The decline of [Ca2+]i transients was accelerated in MLPKO myocytes, and SR Ca2+ content was increased by 21%, indicating that SR Ca2+-ATPase function is normal or enhanced in MLPKO myocytes. Confocal imaging of actin filaments stained with tetramethylrhodamine isothiocyanate-labeled phalloidin showed disorganization of myofibrils and abnormal alignment of Z bands, and fractional shortening was significantly diminished in MLPKO myocytes compared with that in WT myocytes at comparable peak [Ca2+]i. Thus a reduced [Ca2+]-induced shortening may be involved in the pathogenesis of myocardial dysfunction in this genetic model of heart failure.

Transport of Bacterial Lipopolysaccharide to the Golgi Apparatus

Addition of lipopolysaccharide (LPS) to cells in the form of LPS–soluble (s)CD14 complexes induces strong cellular responses. During this process, LPS is delivered from sCD14 to the plasma membrane, and the cell-associated LPS is then rapidly transported to an intracellular site. This transport appears to be important for certain cellular responses to LPS, as drugs that block transport also inhibit signaling and cells from LPS-hyporesponsive C3H/HeJ mice fail to exhibit this transport. To identify the intracellular destination of fluorescently labeled LPS after its delivery from sCD14 into cells, we have made simultaneous observations of different organelles using fluorescent vital dyes or probes. Endosomes, lysosomes, the endoplasmic reticulum, and the Golgi apparatus were labeled using Texas red (TR)–dextran, LysoTracker™ Red DND-99, DiOC6(3), and boron dipyrromethane (BODIPY)–ceramide, respectively. After 30 min, LPS did not colocalize with endosomes, lysosomes, or endoplasmic reticulum in polymorphonuclear leukocytes, although some LPS-positive vesicles overlapped with the endosomal marker, fluorescent dextran. On the other hand, LPS did appear to colocalize with two markers of the Golgi apparatus, BODIPY–ceramide and TRITC (tetramethylrhodamine isothiocyanate)–labeled cholera toxin B subunit. We further confirmed the localization of LPS in the Golgi apparatus using an epithelial cell line, HeLa, which responds to LPS–sCD14 complexes in a CD14-dependent fashion: BODIPY–LPS was internalized and colocalized with fluorescently labeled Golgi apparatus probes in live HeLa cells. Morphological disruption of the Golgi apparatus in brefeldin A–treated HeLa cells caused intracellular redistribution of fluorescent LPS. These results are consistent with the Golgi apparatus being the primary delivery site of monomeric LPS.

F-actin modulates swelling-activated chloride current in cultured chick cardiac myocytes

The integrity of F-actin and its association with the activation of a Cl− current ( I Cl) in cultured chick cardiac myocytes subjected to hyposmotic challenge were monitored by whole cell patch clamp and fluorescence confocal microscopy. Disruption of F-actin by 25 μM cytochalasin B augmented hyposmotic cell swelling by 51% (from a relative volume of 1.54 ± 0.10 in control to 2.33 ± 0.21), whereas stabilization of F-actin by 20 μM phalloidin attenuated swelling by 15% (relative volume of 1.31 ± 0.05). Trace fluorochrome-labeled (fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate) phalloidin revealed an intact F-actin conformation in control cells under hyposmotic conditions despite the considerable changes in cell volume. Sarcoplasmic F-actin was very disorganized and occurred only randomly beneath the sarcolemma in cells treated with cytochalasin B, whereas no changes in F-actin distribution occurred under either isosmotic or hyposmotic conditions in cells treated with phalloidin. Swelling-activated I Cl (68.0 ± 6.0 pA/pF at +60 mV) was suppressed by both cytochalasin B (22.7 ± 5.1 pA/pF) and phalloidin (22.5 ± 3.5 pA/pF). On the basis of these results, we suggest that swelling of cardiac myocytes initiates dynamic changes in the cytoarchitecture of F-actin, which may be involved in the volume transduction processes associated with activation of I Cl.

Basal and adenosine-mediated protein flux from isolated coronary arterioles

We have developed a new method to quantify solute flux per unit surface area and concentration gradient (JS/S delta C) from arterioles isolated from pig hearts. The apparent permeability (Ps) was assessed from measurements of JS/S delta C for two proteins, alpha-lactalbumin (alpha-lactalb) and porcine serum albumin (PSA), labeled with the fluorescent dye tetramethylrhodamine isothiocyanate at a mean hydrostatic pressure of 16 +/- 1 cmH2O. Ps for alpha-lactalb (Ps alpha-lactalb) was 16.5 +/- 4.6 x 10(-7) cm/s (mean +/- SE, N = 8 pigs), a value significantly higher than Ps for PSA (PsPSA) (7.1 +/- 1.4 x 10(-7) cm/s, N = 11 pigs, P < 0.05). Suffusion of the arterioles (44 +/- 10 microns diam; n = 48 arterioles) with 10(-5) M adenosine resulted in a 35% decrease in Ps alpha-lactalb and 29% decrease in PsPSA. Data from the present study are consistent with adenosine altering arteriole Js independently from its ability to change arteriolar caliber. One implication of these results is that changes in coronary exchange capacity reflect not only changes in flow through, but also solute permeation from, the microvasculature.