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H W Harris

Publications and source records attributed to H W Harris.

At least 91 records · Page 5Linked to original sources
PubMed1990

Apical membrane vesicles of ADH-stimulated toad bladder are highly water permeable.

Antidiuretic hormone (ADH) stimulation of the toad urinary bladder causes intracellular vesicles called aggrephores to fuse with the apical plasma membrane of granular cells. Aggrephore membranes contain particle aggregates. Particle aggregates are believed to be water channels that cause large increases in the water permeability (PF) of the granular cell apical membrane. Removal of ADH causes the retrieval of particle aggregate-containing apical membrane via endocytosis and a decline in PF. We have previously shown that fluid phase markers are sequestered in these particle aggregate-containing vesicles during retrieval of the apical membrane and that these vesicles can be recovered in cell homogenates. We have now loaded these vesicles with the self-quenching fluorophore carboxyfluorescein (CF) to measure and compare their PF with that of CF-loaded resealed human erythrocyte ghosts. The membranes of these retrieved vesicles have a very high water permeability. The minimum PF of 99% of these vesicles is 4.5 X 10(-2) cm/s. This PF is comparable with that of erythrocyte ghosts (5.4 X 10(-2) cm/s) measured under identical conditions. We conclude that these vesicles are highly permeable to water, and this is consistent with their postulated function of retrieving water channels that have been inserted into the apical membrane in response to ADH.

Animals↗
PubMed1990

High proton flux through membranes containing antidiuretic hormone water channels.

Antidiuretic hormone (ADH) stimulation of toad urinary bladder granular cells causes simultaneous increases in transepithelial water and H+ permeabilities (PF and PH+, respectively), suggesting that ADH-elicited water channels inserted into granular cell apical membranes might be permeable to both water and H+. We have previously used self-quenching fluorophores entrapped within endocytic vesicles selectively retrieved from water-permeable apical membranes to measure vesicle PF. The membranes of these vesicles possess an extremely high PF such that our measurements provide only minimum estimates of vesicle PF and have limited our ability to quantitate the properties of ADH water channels. We therefore quantitated vesicle PH+ using similar rapid mixing techniques. Vesicle PH+ was 5.1 +/- 0.5 x 10(-3) cm/s. Activation energy of this process was 3.6 +/- 0.6 kcal/mol, indicative of H+ flux through an aqueous channel. The mercurial reagent, para-chloromercuribenzenesulfonate (PCMBS), which inhibits ADH-stimulated transepithelial PF in intact bladders by 50-60%, inhibited vesicle PH+ by 55%. N-Ethylmaleimide and phloretin, which do not alter ADH-stimulated PF, did not affect vesicle PH+. We conclude that membranes containing ADH water channels possess substantial PH+ that likely reflects proton flux through water channels. The apparent high PH+ of the ADH water channel may have important implications for intracellular trafficking of these water channels in ADH-responsive epithelial cells.

4-Chloromercuribenzenesulfonate↗
PubMed1990

Human very low density lipoproteins and chylomicrons can protect against endotoxin-induced death in mice.

Endotoxemia stimulates many physiologic responses including disturbances in lipid metabolism. We hypothesized that this lipemia may be part of a defensive mechanism by which the body combats the toxic effects of circulating endotoxin. We tested the effects of mixtures of endotoxin, lipoproteins, and lipoprotein-free plasma and determined the ability of varying concentrations of human very low density lipoproteins (VLDL) and chylomicrons, as well as low density lipoproteins (LDL) and high density lipoproteins (HDL), and of the synthetic lipid emulsion SOYACAL to prevent endotoxin-induced death in mice. This study demonstrates that the triglyceride-rich VLDL and chylomicrons, as well as cholesterol-rich LDL and HDL, and cholesterol-free SOYACAL can protect against endotoxin-induced death. Protection required small amounts of lipoprotein-free plasma, and depended on the incubation time and the concentration of lipoprotein lipid. Despite stringent techniques to prevent exogenous endotoxin contamination eight of ten duplicate VLDL preparations contained endotoxin (5,755 +/- 3,514 ng endotoxin/mg triglyceride, mean +/- SEM) making the isolation of endotoxin-free VLDL difficult. In contrast, simultaneous preparations of LDL and HDL were relatively free of endotoxin contamination (3 +/- 3 and 320 +/- 319 ng/mg total cholesterol, respectively), suggesting that the contamination of VLDL occurs in vivo and not during the isolation procedure. These observations suggest a possible role for increased triglyceride-rich lipoproteins in the host's defense against endotoxemia and infection.

Animals↗
PubMed1989

Saphenous vein bypass to pedal arteries. An aggressive strategy for foot salvage.

Patients with forefoot ischemia and severe tibial artery disease present a major challenge to revascularization and foot preservation. Encouraged by the success of saphenous vein bypass to the more proximal arteries of the lower leg, we extended this technique to the pedal arteries. Between February 1986 and September 1988, we performed 26 bypasses in 24 men (mean age, 66 years) with critical foot ischemia. Sixty-three percent of the patients had diabetes mellitus, 50% had hypertension, and 71% were actively smoking. Angiography invariably revealed multiple tibial artery occlusions with reconstitution of the pedal arteries. The foot salvage rate was 83% (mean survival, 14 months), primary patency was 83% (mean survival, 9 months), and survival was 86% (mean, 12 months). There were 11 wound complications (42%); two resulted in disruptions of the distal anastomosis and eventual graft failure. Bypass to the pedal arteries yields a high rate of foot salvage and is comparable with more proximal bypass procedures; however, wound complications are common and require special technical considerations.

Aged↗
PubMed1989

Interleukin 1 regulates synthesis of amyloid beta-protein precursor mRNA in human endothelial cells.

We have analyzed the modulation of amyloid beta-protein precursor (APP) gene expression in human umbilical vein endothelial cells (HUVEC). The level of the APP mRNA transcripts increased as HUVEC reached confluency. In confluent culture the half-life of the APP mRNA was 4 hr. Treatment of the cells with human-recombinant interleukin 1 (IL-1), phorbol 12-myristate 13-acetate, or heparin-binding growth factor 1 enhanced the expression of APP gene in these cells, but calcium ionophore A23187 and dexamethasone did not. The protein kinase C inhibitor 1-(isoquinolinsulfonyl)-2-methylpiperazine (H7) inhibited IL-1-mediated increase of the level of APP transcripts. To map IL-1-responsive elements of the APP promoter, truncated portions of the APP promoter were fused to the human growth hormone reporter gene. The recombinant plasmids were transfected into mouse neuroblastoma cells, and the cell medium was assayed for the human growth hormone. A 180-base-pair region of the APP promoter located between position -485 and -305 upstream from the transcription start site was necessary for IL-1-mediated induction of the reporter gene. This region contains the upstream transcription factor AP-1 binding site. These results suggest that IL-1 upregulates APP gene expression in HUVEC through a pathway mediated by protein kinase C, utilizing the upstream AP-1 binding site of the APP promoter.

Amyloid↗
PubMed1989

Regulation of class I antigen expression in the rat.

We have investigated the regulation of expression of class I transplantation antigens encoded by the A and E loci in the rat with regard to resting expression level, induction by interferon, and antigen turnover. Flow cytometric measurements showed that the expression of the A locus products is significantly greater than that of the E locus product. Comparisons among a number of haplotypes indicated that the expression of A locus products varies only marginally (+/- 20%) among strains and that expression of the E locus product is approximately one-fifth that of the A locus products. The influence of nonMHC genes on the expression of class I antigens was evaluated by comparing their expression in inbred and congenic strains: it was not significant in this system. Stimulation of lymphocytes with alpha or gamma interferon resulted in proportional increases in the expression of the antigens encoded by both the A and the E loci, and these increases in cell surface antigen expression were reflected in similar increases in the levels of class I mRNA and in the rate of antigen synthesis. Measurement of turnover kinetics showed that the turnover of the A locus antigens is substantially more rapid than that of the E locus antigen, and the difference in half-lives is 3-4-fold. The addition of a broadly reactive anti-class I monoclonal antibody resulted in significant dose-dependent inhibition of the proliferative response of lymph node lymphocytes stimulated with concanavalin A. The inhibition was maximal when the antibody was added during the early stages of activation.

Animals↗
PubMed1989

Identification of aortic thrombus by magnetic resonance imaging.

The unique properties of magnetic resonance imaging result in the potential to differentiate various components of the diseased arterial wall. In this article four cases are presented in which magnetic resonance imaging showed mural aortic thrombus and its anatomic relationship to the visceral and renal arteries. Once thrombus is identified and localized specific operative strategies can be undertaken to prevent recurrent embolic events and/or avoid perioperative thromboembolic complications.

Aged↗
PubMed1989

Particle size distribution of lipoproteins from human atherosclerotic plaque: a preliminary report.

It is commonly believed that low-density lipoproteins (LDLs) carry cholesterol into the artery wall. In addition, some epidemiologic studies have suggested that triglyceride-rich lipoproteins, such as very-low-density lipoproteins (VLDLs), may be much less important than LDLs in atherogenesis. To determine if VLDLs or their metabolic remnants could have a direct role in the formation of atherosclerotic plaque, we examined lipoproteins isolated from endarterectomy specimens. Atherosclerotic plaque was obtained from eight subjects who underwent aortoiliac endarterectomy (4), aortic aneurysm repair (2), or visceral/renal endarterectomy (2). Plaques were washed extensively, minced, and incubated with a buffered saline solution. Lipoproteins were recovered from this solution via a selected-immunoaffinity column by means of a polyclonal antibody to human LDL (apolipoprotein B-100). Particle sizing from electron photomicrographs of negatively stained specimens indicated that 8% of the lipoprotein particles were the size of plasma VLDL (350 to 800 nm). Thirty-six percent were the size of plasma VLDL remnant particles (250 to 350 nm), and 56% were consistent in size with plasma LDL (175 to 250 nm). We conclude that VLDL- and VLDL remnant-sized particles appear to comprise a significant percentage of the lipoproteins found in human atherosclerotic plaque and could have a direct role in the atherosclerotic process.

Aged↗
PubMed1988

Apical membrane endocytosis via coated pits is stimulated by removal of antidiuretic hormone from isolated, perfused rabbit cortical collecting tubule.

Antidiuretic hormone increases the water permeability of the cortical collecting tubule and causes the appearance of intramembrane particle aggregates in the apical plasma membrane of principal cells. Particle aggregates are located in apical membrane coated pits during stimulation of collecting ducts with ADH in situ. Removal of ADH causes a rapid decline in water permeability. We evaluated apical membrane retrieval associated with removal of ADH by studying the endocytosis of horseradish peroxidase (HRP) from an isotonic solution in the lumen. HRP uptake was quantified enzymatically and its intracellular distribution examined by electron microscopy. When tubules were perfused with HRP for 20 min in the absence of ADH, HRP uptake was 0.5 +/- 0.3 pg/min/micron tubule length (n = 6). The uptake of HRP in tubules exposed continuously to ADH during the 20-min HRP perfusion period was 1.3 +/- 0.8 pg/min/micron (n = 8). HRP uptake increased markedly to 3.2 +/- 1.1 pg/min/micron (n = 14), when the 20-min period of perfusion with HRP began immediately after removal of ADH from the peritubular bath. Endocytosis of HRP occurred in both principal and intercalated cells via apical membrane coated pits. We suggest that the rapid decline in cortical collecting duct water permeability which occurs following removal of ADH is mediated by retrieval of water permeable membrane via coated pits.

Animals↗
PubMed1988

Identification of specific apical membrane polypeptides associated with the antidiuretic hormone-elicited water permeability increase in the toad urinary bladder.

Antidiuretic hormone (ADH) increases the water permeability of the toad urinary bladder. The increase occurs in the apical plasma membrane of granular cells that line the urinary surface of the bladder and is produced by the insertion of water permeability units that have been identified by freeze-fracture electron microscopy as intramembrane particle aggregates. Under water-impermeable conditions, particle aggregates reside in intracellular vesicles called "aggrephores." In response to ADH, the aggrephores fuse with the apical plasma membrane and render it water permeable. When ADH is removed, intramembrane particle aggregates and aggrephores are retrieved from the apical membrane, and it returns to a water-impermeable state. To identify proteins involved in the water permeability response, we used lactoperoxidase/glucose oxidase to 125I-label external apical membrane proteins to compare control and ADH-treated bladders. Several polypeptides were consistently labeled in ADH-treated bladders and not in paired controls. After demonstrating that lactoperoxidase behaves as a fluid-phase marker and is sequestered in aggrephore-like vesicles when ADH is withdrawn, we used the technique of Mellman et al. [Mellman, I.S., Steinman, R. M., Unkeless, J. C. & Cohn, Z. A. (1980) J. Cell Biol. 86, 712-722] to label proteins endocytosed when water permeability declines after ADH is withdrawn to test whether the membrane proteins labeled in ADH-treated bladders behaved like particle aggregates. The internalized membranes contained polypeptides of the same molecular weights (55,000, 17,000-14,000, and 7,000) as those labeled on the apical surface of ADH-treated but not control bladders. These polypeptides are evidently involved in the ADH-stimulated water permeability response and may be components of particle aggregates.

Animals↗
PubMed1987

Characterization and purification of a mammalian osmoregulatory protein, aldose reductase, induced in renal medullary cells by high extracellular NaCl.

GRB-PAP1 is a continuous line of epithelial cells derived from a rabbit renal inner medulla. Elevation of the NaCl concentration in the medium bathing these cells strongly induced the expression of a soluble protein with an apparent molecular mass of 39 kDa. The protein, purified by affinity chromatography with Amicon Matrex Gel Orange A, had enzyme activity characteristic of aldose reductase (alditol:NADPH+ oxidoreductase, EC 1.1.1.21). Goat antiserum against this purified aldose reductase selected the 39-kDa band from immunoblots of cells grown in a medium containing high NaCl. When the osmolality of the medium was increased by adding NaCl, the amount of aldose reductase protein and the aldose reductase activity increased together from very low to sustained high levels over several days. The aldose reductase protein was more than 10% of the soluble cell protein when cells were propagated in medium made hyperosmotic by adding NaCl to increase medium osmolality to 600 mosm.kg-1.

Aldehyde Reductase↗
PubMed1987

Improved synthesis and iodination of a cleavable photoactivated probe.

J. B. Denny and G. Blobel (1984, Proc, Natl. Acad. Sci. USA 81, 5286-5290) have described the synthesis of a water-soluble novel heterobifunctional 125I-labeled photocrosslinking reagent, N-[4-(p-azido-m-[125I]iodophenylazo)benzoyl]-3-aminopropionyl-N'- oxysulfosuccinimide, which contains a cleavable internal azo bond. We report several modifications of their synthesis which greatly increase the yield of a synthetic intermediate, N-[4-(p-aminophenylazo)benzoyl]-3-aminopropionic acid (compound VII). Evidence is presented that direct iodination of compound VII with chloramine-T produces low yields of 125I-labeled compound VII in a reaction which is difficult to control. Alternatively, the yield of 125I-labeled reagent can be greatly improved if the iodination is performed following the derivatization of N-[4-(p-axidophenylazo)benzoyl]-3-aminopropionyl-N'-oxysulfosuc cinimide (compound IX) to a ligand such as dextran. We have demonstrated the transfer of 125I label from the reagent when derivatized to amino-dextran to proteins in solution after photolysis and cleavage of this 125I-labeled reagent.

Chemical Phenomena↗
PubMed1987

Isolation and characterization of specialized regions of toad urinary bladder apical plasma membrane involved in the water permeability response to antidiuretic hormone.

Antidiuretic hormone (ADH) increases the apical (external facing) membrane water permeability of granular cells that line the toad urinary bladder. In response to ADH, cytoplasmic vesicles called aggrephores fuse with the apical plasma membrane and insert particle aggregates which are visualized by freeze-fracture electron microscopy. Aggrephores contain particle aggregates within their limiting membranes. It is generally accepted that particle aggregates are or are related to water channels. High rates of transepithelial water flow during ADH stimulation and subsequent hormone removal decrease water permeability and cause the endocytosis of apical membrane and aggrephores which retrieve particle aggregates. We loaded the particle aggregate-rich endocytic vesicles with horseradish peroxidase (HRP) during ADH stimulation and removal. Epithelial cells were isolated and homogenized, and a subcellular fraction was enriched for sequestered HRP obtained. The HRP-enriched membrane fraction was subjected to a density shifting maneuver (Courtoy et al., J. Cell Biol. 98:870, 1984), which yielded a purified membrane fraction containing vesicles with entrapped HRP. The density shifted vesicles were composed of approximately 20 proteins including prominent species of 55, 17 and 7 kD. Proteins of these molecular weights appear on the apical surface of ADH-stimulated bladders, but not the apical surface of control bladders. Therefore, we believe these density shifted vesicles contain proteins involved in the ADH-stimulated water permeability response, possibly components of particle aggregates and/or water channels.

Animals↗
PubMed1987

Precursors of ribose 5-phosphate suppress expression of glucose-regulated proteins in LLC-PK1 cells.

Withdrawal of glucose from the medium bathing mammalian cells in culture results in cessation of growth and induces the synthesis of two stress proteins (Mr approximately 94-100 kDa and 78-80 kDa) that have been termed glucose-regulated proteins (GRPs). In LLC-PK1 cells, proteins of the same molecular weights, assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography, are fully induced by 24 h in glucose-free medium. The GRPs from LLC-PK1 cells cross-react with antibodies to GRPs of nonpolar cells, confirming their identification. Since glucose is not essential for energy production in cultured cells, but is essential for ribose 5-phosphate and nucleotide biosynthesis (Wice et al. J. Biol. Chem. 256: 7812-7819, 1981), we tested the effect of several precursors of ribose 5-phosphate on the induction of GRPs. The addition of 25 mM fructose or galactose to glucose-free medium suppressed the induction of GRPs. The pyrimidine ribonucleosides uridine and cytidine also suppressed GRP synthesis, ribose and the purine ribonucleosides guanosine and adenosine suppressed partially. The results, coupled with indirect evidence in the literature, lead to the suggestion that cell ribose 5-phosphate or a related metabolite regulates the expression of GRPs.

Adenosine↗
PubMed1987

Visualization of endocytosed markers in freeze-fracture studies of toad urinary bladder.

Antidiuretic hormone (ADH) stimulation increases the apical membrane water permeability of granular cells in toad urinary bladder. This response correlates closely with the fusion of tubular cytoplasmic vesicles with the membrane and delivery of intramembrane particle (IMP) aggregates from the tubules (aggrephores) to the apical membrane. These aggregates are believed to be associated with the channels responsible for the water permeability increase. Removal of ADH triggers apical membrane endocytosis and disappearance of aggregates from the apical membrane. However, it has been unclear whether aggregate disappearance is due to disassembly of aggregates within the apical membrane or to their endocytic retrieval as intact structures. Using colloidal gold and horseradish peroxidase to follow endocytosis from the apical surface after ADH removal, we have directly observed in cross-fractured bladder cells the intramembrane structure of intracellular vesicles that contain these fluid-phase markers. Under these conditions, intact aggregates can be identified in the membrane of tubular endocytosed vesicles. This directly demonstrates that conditions which lower apical membrane water permeability cause the tubular aggrephores to "shuttle" intact aggregates from the apical membrane back into the cytoplasm. An additional population of vesicles with tracer are found which are spherical and display structural features of the apical membrane, as well as occasional aggregates. These vesicles may be responsible for retrieval of aggregates from the surface apical membrane.

Animals↗