Biomedical subjects

P B Gordon

Publications and source records attributed to P B Gordon.

At least 37 records · Page 2Linked to original sources

PubMed1991

Hepatocytic autophagy.

Autophagy is a non-selective bulk process for degradation of cytoplasm, as indicated by ultrastructural evidence and by the similarity in autophagic sequestration rates of various cytosolic enzymes with different half-lifes. The initial autophagic sequestration step is subject to feedback inhibition by amino acids, an effect which is potentiated by insulin and antagonized by glucagon. Epinephrine and other adrenergic agonists inhibit autophagic sequestration through a prazosin-sensitive, alpha 1-adrenergic mechanism. The sequestration is also inhibited by cAMP and by protein phosphorylation as indicated by the effects of cyclic nucleotide analogues, phosphodiesterase inhibitors and okadaic acid. Asparagine specifically inhibits autophagic-lysosomal fusion without having any significant effects on autophagic sequestration, intralysosomal degradation or on the endocytic pathway. Autophaged material that accumulates in prelysosomal vacuoles in the presence of asparagine is accessible to endocytosed enzymes, revealing the existence of an amphifunctional organelle, the amphisome. Evidence from several cell types suggests that endocytosis may be coupled to autophagy in a differential (ligand-dependent) manner, and that amphisomes may play a central role as collecting stations for material destined for lysosomal degradation.

Amino Acids↗

PubMed1991

Role of cyclic nucleotides in the control of hepatic autophagy.

Using isolated hepatocytes as a model system we have investigated whether the cyclic nucleotides cAMP and cGMP are involved in the regulation of the autophagic process. The dibutyryl-cyclic nucleotide analogues db-cAMP and db-cGMP both inhibited autophagic sequestration, suggesting that cAMP and cGMP may be of significance for this step. The adenylate cyclase stimulator deacetyl-forskolin both raised the level of intracellular cAMP and reduced sequestration markedly. In contrast, the guanylate cyclase stimulating agent atriopeptin did not affect sequestration although, it effectively elevated, the level of cGMP. Several inhibitors of cyclic nucleotide phosphodiesterases strongly suppressed autophagy and elevated the level of both cAMP and cGMP. However, one inhibitor, milrinone, raised the cAMP level 3-4 x while having no significant effect on cGMP. These results suggest that cAMP may be involved in the control of hepatic autophagy, whereas the role of cGMP, if any, remains unclear.

Animals↗

PubMed1991

Effects of adrenergic agonists and antagonists on autophagic activity in isolated rat liver cells.

The effect of various adrenergic agonists on autophagic sequestration--measured as the transfer of electroinjected [3H]raffinose from cytosol to vacuoles of the autophagic pathway--was investigated. Epinephrine and other agonists with alpha-effects inhibited sequestration through a specific alpha 1-adrenergic, i.e. prazosin-sensitive, mechanism. The beta-adrenergic agonist isoproterenol also inhibited sequestration, but by a non-beta-specific (propranolol-insensitive) mechanism. All sequestration-inhibitory agents suppressed overall autophagic-lysosomal proteolysis. The inhibitory action of the adrenergic agonists on protein metabolism was not specific to the autophagic pathway since protein synthesis was suppressed as well. However, intracellular levels of ATP were not adversely affected, ruling out the possibility that the agonists might be generally cytotoxic.

Adenosine Triphosphate↗

PubMed1990

Extracellular matrix derived from Trypanosoma cruzi infected endothelial cells directs phenotypic expression.

Infection of confluent human umbilical vein endothelial cells by the parasite Trypanosoma cruzi results in the appearance of an altered heparan sulfate proteoglycan (HSPG) isolated from the extracellular matrix of infected endothelial cells (ECMi). HSPG from ECMi differed from HSPG obtained from the extracellular matrix of uninfected endothelial cells (ECMu) by virtue of an 8-10-fold increase in sulfation and a different elution pattern using DEAE Sepharose chromatography. Analysis of the HSPG that binds to acidic fibroblast growth factor (aFGF) revealed that infection increased the proportion of HSPG that binds to aFGF by 35%. Heparitinase and alkaline borohydride treatment of aFGF-binding HSPG and chromatographic resolution on Sepharose CL4B column revealed an infection-associated 10-fold increase in sulfation of the GAG side chain with no significant change in the migration of the core protein. In addition, the aFGF binding HSPG isolated from ECMi demonstrated a markedly attenuated synergistic mitogenic activity with aFGF in a cell proliferation assay. All of the infection associated changes in HSPG could be demonstrated in HSPG obtained from uninfected endothelial cell cultures grown on ECMi. Hence, the ECMi is associated with signals capable of modulating the ECM associated metabolism of uninfected endothelial cells. This facility of ECMi was also shown to extend to patterns of Gs protein synthesis as revealed by Western blot analysis. The observation that the ECM produced by infected endothelial cells can direct the synthetic patterns of uninfected endothelial cells in a manner uniquely observed in infected endothelial cells suggests a plausible pathway by which infection of only a few cells can ultimately result in the coordinate responses of neighboring uninfected cells.

Chagas Disease↗

PubMed1990

Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes.

Seven cytosolic enzymes with varying half-lives (ornithine decarboxylase, 0.9 h; tyrosine aminotransferase, 3.1 h; tryptophan oxygenase, 3.3 h; serine dehydratase, 10.3 h; glucokinase, 12.7 h; lactate dehydrogenase, 17.0 h; aldolase, 17.4 h) were found to be autophagically sequestered at the same rate (3.5%/h) in isolated rat hepatocytes. Autophagy was measured as the accumulation of enzyme activity in the sedimentable organelles (mostly lysosomes) of electrodisrupted cells in the presence of the proteinase inhibitor leupeptin. Inhibitors of lysosomal fusion processes (vinblastine and asparagine) allowed accumulation of catalytically active enzyme (in prelysosomal vacuoles) even in the absence of proteolytic inhibition, showing that no inactivation step took place before lysosomal proteolysis. The completeness of protection by leupeptin indicates, furthermore, that a lysosomal cysteine proteinase is obligatorily required for the initial proteolytic attack upon autophagocytosed proteins. The experiments suggest that sequestration and degradation of normal cytosolic proteins by the autophagic-lysosomal pathway is a nonselective bulk process, and that nonautophagic mechanisms must be invoked to account for differential enzyme turnover.

Animals↗

PubMed1990

Non-selective autophagy.

Autophagy is the major process by which cells degrade their own cytoplasm. Autophagy begins with the sequestration of a portion of the cytoplasm by a membraneous organelle called a phagophore. The resulting vacuole (autophagosome) can fuse with an endocytic vacuole to form am amphisome, which subsequently fuses with a lysosome to have its mixed autophagic/endocytic content degraded by lysosomal enzymes. Autophagy is a non-selective bulk process as indicated by the fact that hepatocytic cytosol enzymes with widely different half-lives are sequestered at the same rate. Regulation of autophagy is exerted at the sequestration step by amino acids, purines, ATP-depleting metabolites, cyclic nucleotides, phosphorylation, and hormones like insulin, glucagon and alpha-adrenergic agonists.

Amino Acids↗

PubMed1989

Energy dependence of different steps in the autophagic-lysosomal pathway.

The energy dependence of the autophagic-lysosomal pathway was investigated in isolated rat hepatocytes, using electroinjected [14C]lactose as an autophagy probe and atractyloside to alter intracellular ATP levels. Since autophagocytosed lactose is hydrolyzed in lysosomes, several steps in the pathway could be analyzed. The following observations were made. 1) The overall autophagic degradation of electroinjected [14C]lactose was strongly energy-dependent. More than 85% inhibition was obtained when the ATP content decreased from the control value of 10 mumol/g dry weight to 4 mumol/g dry weight. 2) The initial step, i.e. the autophagic sequestration of [14C]lactose, measured in the presence of vinblastine to prevent transfer of lactose to lysosomes, was as sensitive to small changes in ATP as was the overall lactose degradation. 3) The steady state level of sequestered [14C]lactose remained constant as ATP decreased from 10 to 4 mumol/g dry weight, indicating that the sequestration step and some postsequestrational process were inhibited to a similar extent by ATP depletion. 4) The final step in the pathway, intralysosomal hydrolysis, was measured by allowing [14C]lactose to preaccumulate intralysosomally in the presence of the reversible lysosome inhibitor propylamine. Following propylamine removal and inhibition of further sequestration by 3-methyladenine, ATP-dependent hydrolysis of the intralysosomal [14C]lactose could be demonstrated. However, this hydrolysis step was not as sensitive to small changes in ATP as was the sequestration step or the overall autophagic lactose degradation. Control of the autophagic-lysosomal pathway in response to energy deprivation would therefore not seem to occur at the lysosomal level, but may be exerted both at the sequestration step and at a postsequestrational, prelysosomal step.

Adenine↗

PubMed1989

Extracellular matrix heparan sulfate proteoglycans modulate the mitogenic capacity of acidic fibroblast growth factor.

Confluent cultures of human endothelial cells deposit into extracellular matrix (ECM) distinct heparan sulfate proteoglycans (HSPG) which modulate acidic fibroblast growth factor's (aFGF) ability to stimulate human endothelial cell mitogenic capacity. Extracellular matrix 35S-HSPG were isolated from cultures metabolically labelled with Na235SO4 by DEAE-Sepharose, Sepharose CL-4B, and aFGF-Affi-Gel 15 column chromatography and identified by resistance to chondroitinase ABC and sensitivity to nitrous acid. Fifty to sixty percent of the 35S-HSPG deposited into ECM do not bind aFGF. The bound 35S-HSGP (40-50% of the total counts applied) eluted from the aFGF-Affi-Gel column after the addition of buffer containing 2 M NaCl. aFGF-binding and aFGF-nonbinding 35S-HSPG were individually pooled and further purified by Sepharose CL-4B column chromatography. 35S-HSPG which bind aFGF, designated HSPGP, were 100-fold superior to heparin in augmenting the mitogenic efficacy of aFGF in sparse proliferating cultures. In contrast, however, 35S-HSPG, which did not bind aFGF, designated HSPG1, inhibited aFGF-stimulated proliferation in both sparse and subconfluent endothelial cell cultures. The majority of the biological activity of both aFGF-potentiating HSPGP and aFGF-inhibitory HSPG1 was contained in the glycosaminoglycan chains released by alkaline borohydride treatment of intact HSPGP or HSPG1, respectively. 3H-Core protein derived from HSPGP or HSPG1 contained only minor biological activity. The ability of heparitinase or heparinase (Flavobacterium heparinum) to abolish biological activity differed, depending upon the HSPG tested, also suggested that these are two distinct HSPGs.

Cell Division↗

PubMed1989

Experimental characterization of the autophagic-lysosomal pathway in isolated rat hepatocytes.

When isolated rat hepatocytes are incubated under amino-acid-free conditions autophagy is maximally activated and overall lysosomal proteolysis is greatly enhanced. To facilitate this, cytoplasmic material is sequestered by autophagic membranes, and the resulting autophagosomes enter the lysosomes by a fusion process to have their contents degraded. The various steps in the autophagic-lysosomal pathway have been investigated by utilizing reversible electropermeabilization to introduce radioactive sugar probes into the hepatocyte cytosol. The inert disaccharide sucrose and the inert trisaccharide raffinose have been used as sequestration probes to study the sequestrational step of autophagy, while the hydrolysable disaccharide lactose has been used to study the fusion and intralysosomal hydrolysis steps. Sucrose and lactose have been used to map the extent of convergence between the autophagic and endocytic (heterophagic) pathways. Sequestration of native cytosolic enzymes has been measured to investigate the question of selectivity in the autophagic process. Finally, the effect of insulin on the sequestrational step has been evaluated.

Adenine↗

PubMed1988

Prelysosomal convergence of autophagic and endocytic pathways.

[14C]Lactose, electroinjected into the cytosol of isolated rat hepatocytes, was sequestered by autophagy, transferred to lysosomes and eventually hydrolysed. Asparagine prevented the fusion between prelysosomal autophagic vacuoles and lysosomes, and caused lactose to accumulate in the former. However, if the hepatocytes were simultaneously allowed to endocytose added beta-galactosidase, no lactose accumulation occurred. These results suggest that autophagically sequestered lactose and endocytosed beta-galactosidase were delivered to the same prelysosomal vacuole, where the lactose was hydrolysed by the enzyme. The name amphisome is suggested for this new functional compartment, common to the autophagic and endocytic pathways.

Animals↗

PubMed1988

Early hydronephrosis following aortic bifurcation graft surgery: a prospective study.

The true incidence and natural history of ureteral obstruction following reconstructive vascular surgery have not been determined previously. A series of 101 patients undergoing aortofemoral and aortoiliac reconstructive surgery were studied prospectively to determine the frequency of hydronephrosis in the first postoperative year. Serial real-time ultrasound examinations were performed preoperatively as well as at 1 week, 3 months and 1 year postoperatively. Ninety-three patients completed the study, with a total of 181 kidneys at risk. Hydronephrosis of mild to moderate degree developed in 15 kidneys (8 per cent) in 11 patients (12 per cent). All patients were asymptomatic, and the obstruction resolved spontaneously in 10 of 11 patients, including 9 within 3 months of onset. Only a single case persisted at 1 year. This study confirms that the hydronephrosis that occurs within the first year after aortic bifurcation graft surgery is not uncommon but it is rarely of clinical significance in the asymptomatic patient.

Aorta, Abdominal↗

PubMed1988

Sonographic appearance of normal intramammary lymph nodes.

Sonographic breast examinations were performed on 1030 patients with palpable or mammographically detected masses. In five cases, the lesions were normal intramammary lymph nodes. The ultrasonographic characteristics of this entity were confirmed by a sonographic/pathologic correlation study carried out on a cadaver axilla. Normal lymph nodes appeared as well-defined echo-poor masses with echogenic centers. In the absence of other suspicious lesions or proven malignant tumors, these nodes should be considered benign and not biopsied, but followed by repeat examinations.

Axilla↗

PubMed1987

Temperature dependence of protein degradation, autophagic sequestration and mitochondrial sugar uptake in rat hepatocytes.

Lysosomal (propylamine-sensitive) protein degradation as well as the energy-dependent (chymostatin-sensitive) part of the non-lysosomal protein degradation was found to be strongly affected by temperature in isolated rat hepatocytes, the activation energy (Ea) being about 25 kcal/mol for both processes. In contrast, the energy-independent (chymostatin-resistant) part of the non-lysosomal degradation had an Ea of approx. 10 kcal/mol only. Sequestration of electroinjected [14C]sucrose into sedimentable organelles showed a pronounced temperature dependence. By means of digitonin extraction it was possible to distinguish between a moderately temperature-sensitive mitochondrial sugar uptake (Ea approx. 12 kcal/mol) and a strongly temperature-dependent autophagic sequestration (Ea approx. 22 kcal/mol). There was no significant autophagic sequestration below 20 degrees C. The sequestration process is more temperature-sensitive than, for example, the early steps of endocytosis, and is likely to represent the major controlling step in the overall autophagic-lysosomal pathway.

Animals↗

PubMed1987

Sugar uptake by fluid-phase pinocytosis and diffusion in isolated rat hepatocytes.

Measurements of sugar pinocytosis (fluid-phase endocytosis of radiolabelled sucrose, lactose and raffinose) in freshly isolated rat hepatocytes are disturbed by sugar diffusing into the cells through plasma-membrane blebs. Non-pinocytic entry may be even more pronounced at 0 degrees C, and is a major contributor to 'background' radioactivity. By electrodisruption of the plasma membrane, a distinction can be made between pinocytotically sequestered sugar and free sugar that has entered the cytosol by diffusion. Pinocytosis proceeds at a rate of 2%/h (relative to the intracellular fluid volume), whereas the rate of sucrose entry by diffusion is more than twice as high. Three pinocytotic compartments are distinguishable in isolated hepatocytes: (1) a rapidly recycling compartment, which is completely destroyed by electrodisruption, and which may represent pinocytic channels continuous with the plasma membrane; (2) a non-recycling (or very slowly recycling) electrodisruption-resistant compartment, which allows accumulation of the lysosomally hydrolysable sugar lactose, and which therefore must represent non-lysosomal vacuoles (endosomes?); (3) a lysosomal compartment (non-recycling, electrodisruption-resistant), which accumulates raffinose and sucrose, but which hydrolyses lactose. The last two compartments can be partially resolved in metrizamide/sucrose density gradients by the use of different sugar probes.

Animals↗

PubMed1987

Energy dependence of autophagic protein degradation in isolated rat hepatocytes.

The effect of small changes in intracellular ATP on autophagic flux was studied in isolated rat hepatocytes by using inhibitors of ATP production or by varying the metabolic conditions. The following observations were made. There was a linear relationship between endogenous protein degradation and intracellular ATP, the rate of proteolysis declining with decreasing ATP concentrations. 15% of the maximal proteolysis is either independent of ATP or has a very high affinity for this metabolite. There was a linear relationship between the autophagic sequestration of cytosolic [14C]sucrose and intracellular ATP, the sequestration rate decreasing with decreasing ATP concentrations. ATP depletion did not cause release of [14C]sucrose previously sequestered in autophagosomes and lysosomes at high ATP levels. Intracellular accumulation of chloroquine, used as an indicator of the pH inside lysosomes and other acidic cell compartments, diminished with decreasing cellular ATP content. Amino acids inhibited proteolysis without affecting ATP levels or chloroquine accumulation. We conclude from the high sensitivity of autophagy towards relatively small changes in the concentration of intracellular ATP that, besides amino acids, ATP is a very important factor in controlling the rate of autophagy in rat hepatocytes.

Adenosine Triphosphate↗

PubMed1986

Extracellular matrix proteoglycans and cell-substratum adhesion of human endothelial cells: the effect of methyl beta-D-xylopyranoside.

The influence of methyl beta-D-xylopyranoside on human endothelial cell proteoglycans isolated from the medium and extracellular matrix was investigated. Confluent cultures of human endothelial cells incorporate significant amounts of heparan sulfate (78%), chondroitin sulfate (10%), and dermatan sulfate (12%) into the extracellular matrix. Chondroitin sulfate (35%) and dermatan sulfate (37%) were the major glycosaminoglycans present in the medium. In the presence of methyl beta-D-xylopyranoside, incorporation of labeled proteoglycans into extracellular matrix was diminished by approximately 70%. Heparan sulfate comprised the major proteoglycan present in extracellular matrix (89%) in cells grown in the presence of methyl beta-D-xylopyranoside. In contrast to the incorporation of proteoglycan into extracellular matrix, methyl beta-D-xylopyranoside stimulated the secretion of labeled glycosaminoglycan chains into the medium 2.5-fold. In the presence of methyl beta-D-xylopyranoside, secretion of chondroitin sulfate into the medium was markedly stimulated, with a slight increase in secretion of heparan sulfate. Chondroitin sulfate (62%) and heparan sulfate (34%) were the major labeled glycosaminoglycans present in medium from methyl beta-D-xylopyranoside-treated cultures. The effect of methyl beta-D-xylopyranoside on cell adhesion and detachment was investigated. Cell detachment from extracellular matrix depleted of proteoglycan was significantly faster than detachment from normal matrix. Conversely, human endothelial cells adhered faster to normal matrix than to matrix depleted of proteoglycan.

Cell Adhesion↗

PubMed1986

Isolation and partial characterization of endothelial cell extracellular complexes.

Human endothelial cells release components into the growth medium that stimulate cell-substratum adhesion. Several macromolecular components were isolated by ultracentrifugation of the endothelial cell conditioned medium. The components were heterogeneous, consisting of several sizes when examined by sedimentation velocity and gel filtration. When the extracellular components were evaluated by electron microscopy, structurally discrete particles were observed. The extracellular components and the complexes mediated cell-substratum adhesion to both human umbilical and arterial endothelial cells. The majority of the extracellular components that promote endothelial cell adhesion were pelleted by ultracentrifugation. Although the complexes contained fibronectin, antibodies to fibronectin did not inhibit cell adhesion to the complexes. Significant inhibition of endothelial cell adhesion was observed in the presence of heparin and heparan sulfate. The supernatant fraction following ultracentrifugation of the growth medium contained a component that suppressed endothelial cell adhesion to culture dishes coated with fibronectin, type I collagen, and endothelial cell complexes. SDS-polyacrylamide gel electrophoresis indicated that the complexes contained several components, and the majority of the large-molecular-weight components were pelleted by ultracentrifugation. The conditioned medium from human endothelial cells contains specific complexes that promote cell-substratum adhesion and components that suppress cell-substratum adhesion.

Antigens, Surface↗

PubMed1986

Use of [3H]raffinose as a specific probe of autophagic sequestration.

The trisaccharide [3H]raffinose, introduced into the cytosol of isolated rat hepatocytes by means of electropermeabilization, was sequestered autophagically and accumulated in lysosomes and pre-lysosomal vacuoles. In contrast to the disaccharide [14C]sucrose previously used as a sequestration probe, raffinose was not taken up by the mitochondria. The sequestration of raffinose was completely inhibited by the autophagy suppressor 3-methyladenine, stressing its potential utility as a specific probe of hepatocytic autophagy.

Adenine↗