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H W Harris

Publications and source records attributed to H W Harris.

At least 55 records · Page 3Linked to original sources
PubMed1996

Ontogeny of the extracellular calcium-sensing receptor in rat kidney.

We recently cloned extracellular Ca(2+)-sensing receptors (CaRs) from bovine parathyroid and rat kidney that play key roles in Ca2+ homeostasis. Inactivating mutations of the CaR in the inherited human disorder, familial hypocalciuric hypercalcemia, cause reduced responsiveness of the parathyroid to extracellular Ca2+ (Cao2+), as well as abnormally avid renal tubular reabsorption of both Ca2+ and Mg2+ in the distal tubule, suggesting an important role for the CaR in regulating parathyroid hormone (PTH) secretion and renal handling of divalent cations. High Cao2+ also inhibits vasopressinstimulated adenosine 3',5'-cyclic monophosphate accumulation in the medullary thick ascending limb (MTAL) and water reabsorption in the collecting duct (CD) and modulates various other aspects of renal function. The relevance of the CaR to these processes, however, is uncertain. Reduced responsiveness of vasopressin-and PTH-mediated actions on the kidney have been described in the newborn that could potentially reflect effects of the CaR on these aspects of renal function. To define further the role of the CaR in regulating renal function, including the above-mentioned changes during the perinatal period, therefore, we have studied its ontogeny in rat kidney. Northern and Western blot analyses, as well as immunohistochemistry with CaR-specific probes, demonstrate that there is little prenatal expression of the extracellular Ca(2+)-sensing receptor, except in large tubules and branching ureteric buds of developing nephrons. Postnatally, CaR mRNA and protein increase markedly during the 1st wk, related principally to expression of the receptor in the developing TAL and, to a lesser extent, in the CD. The level of expression of the receptor remains nearly constant after postnatal day 14. These results demonstrate that the perinatal increases in expression of CaR mRNA and protein parallel its tissue-specific renal expression. Furthermore, it is possible that some of the previously described changes in renal handling of divalent cations and water in the perinatal and immediate postnatal period are related, in part, to the increasing levels of expression of the CaR and resultant inhibitory effects on the actions of PTH and antidiuretic hormone on the developing nephron.

Aging↗
PubMed1995

Rat kidney papilla contains abundant synaptobrevin protein that participates in the fusion of antidiuretic hormone-regulated water channel-containing endosomes in vitro.

Antidiuretic hormone (ADH) regulates renal water excretion by altering the permeability of the collecting duct to water. ADH-responsive epithelial cells are the major cell type lining kidney tubules in the inner medulla and papilla. ADH modulates apical membrane water permeability by the insertion and removal of vesicles containing aquaporin collecting duct water channel protein (now termed AQP-2). To identify and characterize proteins responsible for trafficking of AQP-2-containing vesicles, we utilized antibody and cDNA probes to synaptobrevin b (also termed VAMP-2, for vesicle-associated membrane protein 2), a protein that mediates synaptic vesicle exocytosis in the brain and whose structural homologs are now considered to be components of a complex responsible for intracellular vesicle fusion in all cells. We now report that rat kidney inner medulla and papilla contain abundant synaptobrevin protein. Only light endosomes, one of two types of purified papillary AQP-2-containing endosomes, possess synaptobrevin. Light endosomes fuse in vitro by means of an ATP-dependent process that is significantly inhibited when endosomes are preincubated with either anti-synaptobrevin antibody or tetanus toxin. These data define a functional role for a synaptobrevin protein in the fusion of endosomes in vitro. The presence of abundant synaptobrevin proteins in endosomes containing AQP-2 water channels, as well as insulin-sensitive glucose transporters [Cain, C. C., Trimble, W. S. & Lienhard, G. E. (1992) J. Biol. Chem. 267, 11681-11684], and in cells of Malpighian tubules responsible for urine formation in insects [Chin, A. S., Burgess, R. W., Wong, B. R., Schwartz, T. L. & Scheller, R. H. (1993) Gene 131, 175-181] suggests a specialized role for synaptobrevin in vesicle-mediated membrane transport modulated by peptide hormones.

Animals↗
PubMed1995

Extracellular signal-regulated protein kinases (ERKs) and ERK kinase (MEK) in brain: regional distribution and regulation by chronic morphine.

Quantitative blot immunolabeling techniques were used to determine the concentrations of ERK1 (M(r) 44 kDa) and ERK2 (M(r) 42 kDa), the two major extracellular signal-regulated protein kinases, in different regions of rat brain. The aggregate ERK concentrations (ERK1 and ERK2) were relatively high in each of the brain regions studied, ranging from approximately 0.35 ng/microgram protein in cerebellum to approximately 1.2 ng/microgram protein in nucleus accumbens. However, differences in the regional distributions of ERK1 and ERK2 resulted in ratios of their relative abundance that differed by close to 10-fold among the regions studied. The ratios of ERK1 protein to ERK2 protein varied along a rostral-caudal gradient from a low of 0.16 in frontal cortex to a high of 1.5 in pons/medulla. In hypotonic homogenates from regions at either extreme of the gradient, ERK1 and ERK2 were both found to be predominantly (> 80%) soluble. In subcellular fractions prepared from sucrose homogenates of frontal cortex and pons/medulla, both ERK1 and ERK2 were enriched in the synaptosomal and cytosolic fractions, whereas ERK2 was also enriched in the microsomal fraction. By contrast, in subfractions containing purified nuclei, levels of ERK1 and ERK2 were about one-third of those seen in homogenates and, in subfractions enriched in mitochondria, both ERK1 and ERK2 were barely detectable. The catalytic activity of the ERKs paralleled their protein levels in all of the brain regions except the hippocampus, in which the activity and phosphotyrosine content were disproportionately high. As a possible explanation for this apparent disparity, the regional distribution of ERK kinase (MEK), which phosphorylates and activates the ERKs, was also investigated. The levels of immunoreactivity of the M(r) 45 kDa ERK kinase band differed by about threefold among the brain regions, with the highest levels being present in nucleus accumbens, hippocampus, substantia nigra, and caudate/putamen. Therefore, a higher concentration of ERK kinase immunoreactivity did not appear to account for the disproportionate levels of ERK activity and phosphotyrosine content in the hippocampus. Potential regulation of ERK and ERK kinase levels was also investigated in rats subjected to chronic morphine treatment. ERK1 and ERK2 levels were increased selectively in locus coeruleus and caudate/putamen after chronic morphine treatment, whereas ERK kinase immunoreactivity remained unchanged in all of the brain regions analyzed. In summary, the regional differences in ERK and ERK kinase expression and the region-specific regulation of ERK expression suggest that ERK-related signaling may play an important role in CNS function and its adaptive responses.

Animals↗
PubMed1994

Characterization of purified endosomes containing the antidiuretic hormone-sensitive water channel from rat renal papilla.

Antidiuretic hormone (ADH) stimulation of renal epithelial cells elicits a large increase in apical membrane osmotic water permeability (Pf) produced by the fusion of water channel containing vesicles with the apical membrane. Removal of ADH stimulation results in retrieval of apical water channels into a specialized non-acidic endosomal compartment. Previous studies (Sabolic, I., Wuarin, F., and Shi, L. B. (1992) J. Cell Biol. 119, 111-122) have shown that water channel containing papillary endosomes labeled with fluorescein-dextran can be isolated from rat renal papilla. We have utilized small particle flow sorting methodology to both monitor and improve upon the purification of these water channel containing endosomes (WCV). Flow cytometry analysis on a vesicle-by-vesicle basis demonstrates that WCV are homogeneous with respect to entrapped fluorescein-dextran, the apical membrane enzyme marker leucine amino peptidase and ultrastructural morphology. WCV do not acidify their luminal contents after addition of Mg-ATP but contain abundant functional water channels (Pf0.28 cm/s at 23 degrees C) as determined by stopped flow fluorimetry. SDS-polyacrylamide gel electrophoresis analysis shows that purified WCV are composed of 20 major protein bands. To determine the identity of WCV water channels, WCV proteins were probed with affinity purified antisera recognizing two renal water channel proteins. These include Aquaporin-CHIP found in the proximal tubule and thin descending limb of Henle and the candidate ADH water channel protein WCH-1 or Aquaporin- (AQP) CD present in the ADH-responsive epithelial cells of the collecting duct. These data reveal that WCV contained little or no AQP-CHIP protein. In contrast, WCV are highly enriched for AQP-CD protein. Together, these data define the protein composition of the papillary WCV and link directly the presence of functional apical membrane water channels with the presence of the AQP-CD protein.

Animals↗
PubMed1994

Permeability properties of rat renal lysosomes.

Although lysosomes maintain large pH gradients and may be subjected to significant osmotic gradients in vivo, little is known about their passive permeability properties. In recent studies, vacuolar H(+)-adenosine-triphosphatases (ATPases), such as those found in lysosomes, have been suggested to act as water channels. In addition, the erythrocyte and proximal tubule water channel CHIP28 is present on the plasma membrane of proximal tubule cells and may undergo endocytosis so that it is incorporated in lysosomes. We therefore examined water, proton, and small nonelectrolyte permeabilities in freshly purified lysosomes from rat renal proximal tubule. Lysosomes were purified by differential and Percoll gradient centrifugation. The preparation contained only lysosomes when examined by electron microscopy. Moreover, analysis by flow cytometry showed virtually all particles to be positive for acid phosphatase and cathepsin B activities. Permeabilities were measured on a stopped-flow fluorimeter by monitoring the self-quenching or pH-sensitive quenching of entrapped fluorescein derivatives. Osmotic water permeability (Pf) averaged 0.011 +/- 0.003 cm/s (n = 6), a value similar to that of biological membranes containing water channels. However, Pf was insensitive to the organic mercurial reagent p-chloromercuribenzene-sulfonate and to HgCl2 and exhibited an activation energy of 10.8 +/- 0.8 kcal/mol. These results indicate that water flux in lysosomes occurred via the lipid bilayer, and not via water channels. Addition of ATP led to lysosomal acidification (proton flux = 4.6 +/- 0.8 x 10(-11) mmol H+.s-1.cm-2), which was completely inhibited by 0.1 microM bafilomycin. Pf was insensitive to this agent as was the passive proton permeability (0.36 +/- 0.18 cm/s, n = 4). Permeabilities to small nonelectrolytes varied in proportion to the oil-water partition coefficient, confirming the applicability of Overton's rule to lysosomes. We conclude that proximal tubular lysosomes exhibit high Pf, which occurs via the lipid bilayer and not via vacuolar H(+)-ATPase.

Acids↗
PubMed1994

Water channel vesicles from toad urinary bladder contain a family of proteins present in other tissues.

Antidiuretic hormone (ADH) stimulation causes the fusion and subsequent retrieval of cytoplasmic vesicles containing water channels (WCV) with the apical membrane of toad bladder granular cells. Previously, we showed that purified WCV contain 12 major protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To identify various WCV proteins, we screened a panel of mouse monoclonal antibodies and characterized an immunoglobulin G1 monoclonal antibody, 5E5, that recognizes integral membrane WCV proteins of 38, 33, and 31 kDa. Immunocytochemistry and Western blot analyses show that 5E5 binds to multivesicular body endosomes shown previously to contain ADH water channels. In addition, 5E5 recognizes these proteins in selected cells of the skin, intestine, liver, kidney, spleen, and lung. However, 5E5 does not appear to recognize components of the water channel itself. We conclude that WCV contain several membrane proteins recognized by 5E5 that are present in certain cells of the other organs. Monoclonal 5E5 provides a probe to determine the structure and function of these endosomal proteins as well as their role in the ADH water permeability response.

Animals↗
PubMed1994

Acidification of vasopressin-induced endosomes in toad urinary bladder.

It is well established that water channels (WC) are removed from the apical membrane of vasopressin-sensitive epithelia by endocytosis. The processing and the ultimate fate of endocytosed WC is, however, incompletely understood. In many cells, endosome acidification plays an important role in the processing and sorting of endocytosed proteins. Endosome acidification in the toad urinary bladder was therefore examined in vivo by fluorescence ratio video microscopy after induction of endocytosis by vasopressin removal and transepithelial water flow in the presence of the pH-sensitive fluid phase marker 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-dextran. Fifteen minutes after induction of endocytosis, the majority of endosomes had a neutral or slightly acidic pH. The number of acidic endosomes increased progressively with time. Two hours after endocytosis began, 98% of the endosomes had a pH < 6.0. Bafilomycin completely blocked endosome acidification, indicating that H+ transport is mediated by a vacuolar H(+)-adenosinetriphosphatase. Bafilomycin had no effect on transepithelial water flow in bladders repetitively stimulated by vasopressin. These findings, as well as the work of other investigators, suggest that if WC recycling occurs, it is not dependent on acidification of the endosomal compartment. Acidification of vasopressin-induced endosomes most likely represents a terminal event in the endocytic pathway.

Acids↗
PubMed1993

Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney.

Vasopressin (antidiuretic hormone) regulates body water balance by controlling water permeability of the renal collecting ducts. The control mechanisms may involve alterations in the number or unit conductance of water channels in the apical plasma membrane of collecting-duct cells. How this occurs is unknown, but indirect evidence exists for the "shuttle" hypothesis, which states that vasopressin causes exocytic insertion of water channel-laden vesicles from the apical cytosol. To test key aspects of the shuttle hypothesis, we have prepared polyclonal antisera against the recently cloned collecting-duct water channel protein and used the antisera in immunolocalization studies (light and electron microscopic levels) in thin and ultrathin cryosections from rat kidney. Labeling was seen exclusively in collecting-duct principal cells and inner medullary collecting-duct cells. Apical membrane labeling was intense. There was heavy labeling of abundant small subapical vesicles and of membrane structures within multivesicular bodies. In addition, labeling of basolateral plasma membranes in inner medullary collecting ducts was present. Depriving rats of water for 24 or 48 hr markedly increased collecting-duct water-channel protein expression determined by immunoblotting and immunolabeling. These results are compatible with at least two complementary modes of water-channel regulation in collecting-duct cells: (i) control of channel distribution between the apical membrane and a reservoir in subapical vesicles (shuttle hypothesis) and (ii) regulation of the absolute level of expression of water-channel protein.

Amino Acid Sequence↗
PubMed1993

Isolation of highly purified, functional endosomes from toad urinary bladder.

Endosomes are difficult to isolate as they share size and density properties with much more abundant cellular organelles such as mitochondria. In cultured cell lines the tandem use of charge-dependent isolation techniques and differential centrifugation is necessary to isolate endosomes. Endosomal populations of the toad urinary bladder are of special interest because they are thought to contain a water channel. Understanding of the molecular structure of the water channel has been constrained, as there is currently no practical method to isolate functional water-channel-containing vesicles. This study reports the tandem use of charge-dependent techniques and centrifugation to isolate populations of endosomes from the toad urinary bladder. To purify water-channel-containing vesicles aqueous two-phase partition was utilized to fractionate a preparation partially purified by differential centrifugation. Populations of endosomes were analysed by small-particle flow cytometry techniques. A 5-fold enrichment in endosomes, achieved with aqueous two-phase partition, allowed us to identify two populations of endosomes of diverse size in a toad bladder endosomal fraction. Preenrichment also improved the efficiency of flow cytometry sorting, allowing isolation of the two endosomal populations in sufficient quantities for secondary analysis. A population of larger endosomes had very high water permeability, indicating the presence of water channels. The two populations had different SDS/PAGE fingerprints. Electron micrographs of the flow-sorted material shows a uniform population of membrane vesicles devoid of mitochondria and other identifiable cellular organelles. Hence, aqueous two-phase partition and flow cytometry allow identification of two populations of endosomes in the toad urinary bladder which have diverse structural and functional properties. Isolation of functional water-channel-containing vesicles allows co-localization of water-channel function with candidate water-channel proteins.

Animals↗
PubMed1993

The molecular structure of the antidiuretic hormone elicited water channel.

Measurements of osmotic water permeability (Pf) have shown that the plasma membranes of human red cells and certain epithelial cells possess specialized water channels. Although these water channels have been characterized extensively using biophysical techniques, the proteins that compose these unique channels have only recently been identified. Antidiuretic hormone (ADH) stimulation rapidly increases collecting duct epithelial cell Pf by fusion of water channel-containing vesicles (WCV) with their apical membranes. The proteins of WCV from toad bladder and rodent kidney have been characterized. The principal proteins in toad bladder WCV are 55,000 daltons (55 kDa) and 53 kDa and span the lipid bilayer of these vesicles. Polyclonal antisera raised against the 55-kDa and 53-kDa proteins inhibit ADH-stimulated toad bladder Pf by 80% and recognize protein bands of 46, 38 and 30 kDa in mouse kidney. Purification of WCV from rat kidney reveals enrichment of the 46-kDa protein. Recently, a 28-kDa integral membrane protein (called CHIP-28) has been isolated from human red cells. It forms functional water channels in Xenopus oocytes and when reconstituted into proteoliposomes. Large amounts of CHIP-28 protein are present in epithelial cells of the proximal tubule and descending thin limb of Henle's loop. Molecular cloning efforts are underway to elucidate the structure and function of these candidate water channel proteins.

Animals↗
PubMed1993

Water channels.

The movement of water across cell membranes has been an active area of research for more than 100 years and is of fundamental importance in the normal water metabolism of all terrestrial animals. The objective of this review is to integrate recent data obtained from the isolation and molecular cloning of water channel proteins, with functional information provided by biophysical measurements of membrane water transport. Whereas the water permeability of most cell membranes can be accounted for by the diffusion of water across the lipid bilayer, other cells, including the erythrocyte as well as certain cells in renal epithelia, possess specialized water channels. Water channels are composed of specialized proteins that create highly selective aqueous pores across cell membranes. Data concerning the distribution, permeability, and function of these various water channels will greatly enhance our knowledge of how water is transported across cell membranes.

Amino Acid Sequence↗
PubMed1993

Chylomicrons enhance endotoxin excretion in bile.

Chylomicrons prevent endotoxin toxicity and increase endotoxin uptake by hepatocytes. As a consequence, less endotoxin is available to activate macrophages, thereby reducing tumor necrosis factor secretion. To determine whether the chylomicron-mediated increase in hepatocellular uptake of endotoxin results in increased endotoxin excretion into bile, we examined bile after endotoxin administration. A sublethal dose (7 micrograms/kg) of 125I-endotoxin was incubated with either rat mesenteric lymph containing nascent chylomicrons (500 mg of chylomicron triglyceride per kg of body weight) or an equal volume of normal saline (controls) for 3 h and then infused into male Sprague-Dawley rats. Bile samples were collected via a common bile duct catheter for 24 h. Infusion of endotoxin incubated with chylomicrons increased biliary excretion of endotoxin by 67% at 3 h (P < or = 0.006) and by 20% at 24 h (P < or = 0.01) compared with infusion of endotoxin incubated in saline. Endotoxin activity, as measured by the Limulus assay, was not detected in the bile of test animals. However, endotoxin activity was detected after hot phenol-water extraction of bile, demonstrating that endotoxin is inactive in the presence of bile but retains bioactivity after hepatic processing. Since the majority of an intravenous endotoxin load has been shown to be cleared by the liver, acceleration of hepatocyte clearance and biliary excretion of endotoxin may represent a component of the mechanism by which chylomicrons protect against endotoxin-induced lethality.

Animals↗
PubMed1993

Fate of antidiuretic hormone water channel proteins after retrieval from apical membrane.

In toad bladder granular cells, antidiuretic hormone (ADH) stimulates insertion of vesicles containing water channels (WCV), markedly increasing apical membrane osmotic water permeability (Pf). After withdrawal of ADH stimulation, WCV are removed from the apical membrane and fluid-phase markers endocytosed from the apical solution appear predominantly in endosomes at 10-15 min and multivesicular bodies at 30-60 min. Although the luminal contents of this endocytic pathway have been well characterized, the fate of membrane proteins, including functional ADH water channels in these vesicles remains unclear. Using electron microscopic, flow cytometric, and stopped-flow fluorescence measurements and characterization of labeled vesicle proteins, we examined the fate of membrane proteins contained within WCV. The protein complements of endosomes harvested after 10, 30, and 60 min of ADH withdrawal were similar. Selective covalent labeling of apical proteins during ADH stimulation followed by ADH reversal for 30 or 60 min showed that apical proteins colocalize with fluid-phase marker-labeled endosomes at all times, and most apically labeled protein bands present in the 10-min fraction were also present in the 30- and 60-min endosome fractions. Endosomes at 10 and 30 min but not at 60 min contained functional water channels revealed by high Pf and proton permeability, low activation energy of Pf, and sensitivity of Pf to mercurial reagents. We conclude that a portion of apically exposed membrane proteins, including candidate water channel proteins, travel together with fluid-phase markers from 10-min endosomes into later endosomal compartments. Functional water channels may be inactivated or some essential protein component selectively sorted away between 30 and 60 min after ADH withdrawal.

Animals↗
PubMed1993

Transport defects of rabbit inner medullary collecting duct cells in obstructive nephropathy.

Urinary obstruction markedly reduces collecting duct Na+ reabsorption. To define the cellular mechanisms of this derangement in Na+ reabsorption in inner medullary collecting duct (IMCD) of obstructed kidneys, suspensions of intact IMCD cells and inner medulla plasma membranes (IMPM) were prepared from 24 h obstructed and untreated control kidneys. Oxygen consumption (QO2) studies revealed marked reductions in both amiloride-sensitive and ouabain-sensitive QO2 but not ouabain-insensitive QO2 in intact IMCD cells from obstructed, compared with control animals, indicating a reduction in oxygen-dependent transport activities of both the Na+ channel and the Na(+)-K(+)-adenosinetriphosphatase (ATPase). Amiloride-sensitive conductive 22Na+ uptake in intact IMCD cells from obstructed kidneys was significantly decreased by 45% at 10 s, 30 s, and 1-5 min (10 s: 2.42 +/- 0.63 vs. 4.49 +/- 0.64 nmol Na+ flux/mg protein, n = 7, P < 0.05; 1 min: 4.65 +/- 0.7 vs. 8.27 +/- 0.98 nmol Na+ flux/mg protein, n = 7, P < 0.05), indicating decreased activity of amiloride-sensitive Na+ channels in these cells. However, immunoblots of IMPM with antibodies to Na+ channel proteins did not show significant differences in content of Na+ channel proteins between membranes from obstructed and control groups. Ouabain-sensitive Na(+)-K(+)-ATPase activity in IMPM of obstructed kidneys was also reduced (61.1 +/- 18.1 vs. 152.6 +/- 25.8 nmol ATP degradation.min-1.mg protein-1, n = 6, P < 0.02), and immunoblots with monoclonal antibodies against the alpha 1- and beta-subunits of rabbit Na(+)-K(+)-ATPase showed a 51 +/- 7% reduction of both subunits in IMPM from obstructed kidneys (n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Transport defects of rabbit medullary thick ascending limb cells in obstructive nephropathy.

To characterize the sodium transport defect responsible for salt wasting in obstructive nephropathy, the major sodium transporters in the medullary thick ascending limb (mTAL), the apical Na-K-2Cl cotransporter and the basolateral Na-K-ATPase, were studied in fresh suspensions of mTAL cells and outer medulla plasma membranes prepared from obstructed and untreated kidneys. Oxygen consumption (QO2) studies in intact cells revealed marked reductions in the inhibitory effects of both furosemide and ouabain on QO2 in cells from obstructed, as compared with control animals, indicating a reduction in activities of both the Na-K-2Cl cotransporter and the Na-K-ATPase. Saturable [3H]bumetanide binding was reduced in membranes isolated from obstructed kidneys, but the Kd for [3H]bumetanide was unchanged, indicating a decrease in the number of functional luminal Na-K-2Cl cotransporters in obstructed mTAL. Ouabain sensitive Na-K-ATPase activity in plasma membranes was also reduced, and immunoblots using specific monoclonal antibodies directed against the alpha and beta subunits of rabbit Na-K-ATPase showed decreased amounts of both subunits in outer medullas of obstructed kidney. A significant decrease in [3H]bumetanide binding was detected after 4 h of ureteral obstruction, whereas Na-K-ATPase activity at this time was still not different from control. We conclude that ureteral obstruction reduces the amounts of both luminal Na-K-2Cl cotransporter and basolateral Na-K-ATPase in mTAL of obstructed kidney and that these reductions contribute to the salt wasting observed after release of obstruction.

Animals↗
PubMed1993

Chylomicrons alter the fate of endotoxin, decreasing tumor necrosis factor release and preventing death.

The hypertriglyceridemia of infection was traditionally thought to represent the mobilization of substrate to fuel the body's response to the infectious challenge. However, we have previously shown that triglyceride-rich lipoproteins can protect against endotoxin-induced lethality. The current studies examine the mechanism by which this protection occurs. Rats infused with a lethal dose of endotoxin preincubated with chylomicrons had a reduced mortality compared with rats infused with endotoxin alone (15 vs. 76%, P < 0.001). Preincubation with chylomicrons increased the rate of clearance of endotoxin from plasma and doubled the amount of endotoxin cleared by the liver (30 +/- 1 vs. 14 +/- 2% of the total infused radiolabel, P < 0.001). In addition, autoradiographic studies showed that chylomicrons directed more of the endotoxin to hepatocytes and away from hepatic macrophages. Rats infused with endotoxin plus chylomicrons also showed reduced peak serum levels of tumor necrosis factor as compared with controls (14.2 +/- 3.3 vs. 44.9 +/- 9.5 ng/ml, mean +/- SEM, P = 0.014). In separate experiments, chylomicrons (1,000 mg triglyceride/kg) or saline were infused 10 min before the infusion of endotoxin. Chylomicron pretreatment resulted in a reduced mortality compared with rats infused with endotoxin alone (22 vs. 78%, P < 0.005). Therefore, chylomicrons can protect against endotoxin-induced lethality with and without preincubation with endotoxin. The mechanism by which chylomicrons protect against endotoxin appears to involve the shunting of endotoxin to hepatocytes and away from macrophages, thereby decreasing macrophage activation and the secretion of cytokines.

Animals↗