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H L Leffert

Publications and source records attributed to H L Leffert.

At least 55 records · Page 3Linked to original sources
PubMed1983

Monoclonal antibodies to rat Na+,K+-ATPase block enzymatic activity.

A panel of nine mouse monoclonal antibodies has been prepared against purified preparations of rat kidney Na+,K+-ATPase (EC 3.6.1.3). Selection for specific antibody was based upon the ability of crude hybridoma fluids to inhibit Na+-ATPase activity (using luciferase-linked ATPase assays) and upon antibody binding to both the purified kidney membrane enzyme and to glutaraldehyde-fixed hepatocytes by using standard enzyme-linked immunoadsorbent assays. After immunoaffinity purification, two of the antibodies (both of the IgG1 subclass) fully inhibit kidney and liver membrane Na+,K+-ATPase activity with Ki (apparent) values of 30 nM ("9-A5") and 600 nM ("9-B1"). Immunoblots demonstrate directly that three different 125I-labeled antibodies (6-4, 9-A5, and 9-B1) bind predominantly to a 94,000 Mr protein that comigrates in NaDodSO4/polyacrylamide gels with the fluorescein isothiocyanate-labeled alpha subunit of the Na+,K+-ATPase. Indirect immunofluorescence studies with these antibodies on paraformaldehyde-fixed liver slices reveal staining patterns congruent with bile canalicular membrane domains. These results together suggest that the antibodies exert inhibitory effects by recognizing alpha subunits of both liver and kidney Na+ pumps in their native conformations.

Animals↗
PubMed1983

Selective effects of portal blood diversion and glucagon on rat hepatocyte rates of S-phase entry and deoxyribonucleic acid synthesis.

Hepatocyte growth transition constants were measured during liver regeneration induced by 70% hepatectomy in portacaval shunted rats and in pair-fed sham-operated controls. Portal blood diversion 48 h before 70% hepatectomy coordinately reduced DNA synthetic and mitotic responses 60-70%. Both responses reflected preferential changes in overall rates of S- and M-phase entry (S delta and M delta, respectively); marked differences were not seen between experimental or control DNA synthesis and mitotic onset times (St = approximately 12 h; and Mt = approximately 20 h, for each group). Proliferative defects were manifested progressively across the liver lobule, from portal (approximately 44% of control) to midzonal (approximately 20%) to central areas (approximately 5%). In addition, radioautography and studies of [3H]thymidine uptake into nuclear DNA showed that in shunted rats, average DNA synthesis rates per hepatocyte fell 44-86% between 16-30 h. RIAs of glucagon and insulin in portal and aortic plasma obtained from 0-72 h showed overall reductions of 37-41% in hepatic hormone gradients of shunted rats. The greatest decreases occurred 8-24 h after 70% hepatectomy. Early (0-2 h) or late prereplicative (8-10 h) administration of insulin (0.02-1 mg kg-1) failed to restore [3H]thymidine uptake rates at 22-24 h to normal levels. However, exogenous glucagon (20 micrograms kg-1) given at 8-10 h increased these rates to 50% of control values. Higher doses (0.2-1 mg glucagon kg-1) were inhibitory. By contrast, no effects on DNA synthesis were found when glucagon was injected between 0-2 h. These findings suggest that hepatocyte growth transition constants are modulated selectively by different mitogens. Glucagon may control S delta and average hepatocellular DNA synthesis rates apart from other endocrine factors that regulate St.

Animals↗
PubMed1981

The effect of amiloride on hormonal regulation of amino acid transport in isolated and cultured adult rat hepatocytes.

Insulin and glucagon stimulate amino acid transport in isolated rat hepatocytes. Amiloride, a specific Na+-influx inhibitor, completely inhibited the hormonal (glucagon or insulin) stimulation of alpha-aminoisobutyric acid influx by preventing the emergence of a high-affinity transport component. The drug also inhibited [14C]valine incorporation into hepatocyte protein. The half-maximal concentration of amiloride for inhibition of protein synthesis was similar to that required for inhibition of hormone-stimulated amino acid transport (approx. 0.1 mM). In primary cultured rat hepatocytes, amiloride markedly depressed the stimulation of alpha-aminoisobutyric acid transport by glucagon, or a mixture of glucagon, insulin and epidermal growth factor. These results suggest that amiloride inhibits the hormonal stimulation of hepatocyte amino acid transport by preventing the synthesis of high-affinity transport proteins. They also suggest that the hormonal stimulation of hepatocyte amino acid transport is dependent, at least partly, on Na+ influx.

Amiloride↗
PubMed1981

Autoradiography of "oval cells" appearing rapidly in the livers of rats fed N-2-fluorenylacetamide in a choline devoid diet.

Autoradiographic analysis of liver sections from rats fed the hepatocarcinogen N-2-fluorenylacetamide (FAA) in a choline devoid (CD) diet suggests that proliferating small "oval" cells arise from a few small portally-situated cells, and spread rapidly across the entire liver lobule. Small cells with detectable grains are first located where liver plates meet the portal areas. This cell type gradually increases in number over a 10-12 day period, then proliferates rapidly. After 28 days, microscopic nodules consisting of heavily labeled large eosinophilic cells appear, whereas residual hepatocytes are not labeled. Combined immunofluorescent and autoradiographic labeling studies reveal that many of the small cells contain AFP; approximately half of the alpha-fetoprotein-containing cells are labeled with [3H]thymidine (dT). Feeding CD-FAA diets to rats with hepatocytes prelabeled with [3H]dT after 70% hepatectomy 7 weeks earlier provides data which suggest that small "oval" cells do not arise from prelabeled hepatocytes but, instead, infiltrate the prelabeled hepatocytes during the diet induced proliferative phase. We conclude that "oval" cells arise from a small number of portal cells, not from hepatocytes. Exact identification of the oval cell precursor is not possible, but it could be a "stem" cell. Although hepatocyte-like properties are found in small cells (e.g., albumin staining), there is no evidence that they differentiate into normally functioning hepatocytes.

2-Acetylaminofluorene↗
PubMed1981

Rapid development of large numbers of alpha-fetoprotein-containing "oval" cells in the liver of rats fed N-2-fluorenylacetamide in a choline-devoid diet.

Fischer rats, fed 0.05% w/w N-2-fluorenylacetamide in a choline-devoid diet for 2 weeks, develop a massive infiltration of the liver by small "oval" cells. This occurs rapidly one week after feeding the diet for two weeks. All rats fed choline-devoid diet die within 5 weeks, with massive oval cell infiltration of the liver. Although similar changes occur in rats fed N-2 fluorenylacetamide in a choline-supplemented diet, their degree is much less. In rats fed a choline-devoid diet without N-2-fluorenylacetamide, proliferation of hepatocytes, but not of oval cells, is observed. Because the carcinogen-enhancing effects of choline-devoid diets seem to exceed those of partial hepatectomy, such diets may work by causing changes distinct from those induced by partial hepatectomy. Many oval cells contain alpha-fetoprotein, and the rapid oval cell increase is associated with an exponential increase in serum alpha-fetoprotein concentration. These observations suggest that a cellular change, not an alteration of gene expression in parenchymal cells, is the primary cause of hyper-alphafetoproteinemia during the course of chemical carcinogenesis in rats.

2-Acetylaminofluorene↗
PubMed1979

Increased sodium ion influx is necessary to initiate rat hepatocyte proliferation.

Serum-free media containing 10-50 ng insulin, glucagon and epidermal growth factor (EGF) ml-1 stimulate adult rat hepatocyte proliferation in 10-15 day old primary liver cell cultures. The kinetics of this response simulate hepatocellular transitions that accompnay liver regeneration after 67% hepatectomy. Amiloride, a Na+ influx inhibitor, reversibly blocks these transitions in vitro (ID50 approximately 0.02 mM) and in vivo (ID50 approximately 25 mg kg-1). Inhibition is observed with other cation flux modulators, including ouabain (ID50 approximately 0.2 mM), 0.2 microM monensin and 0.2 microM nigericin, but not with 0.3 mM furosemide or tetrodotoxin. The prereplicative interval in culture (0-12 hr) is characterized by preferential cellular responsiveness to EGF (0-3 hr) followed by insulin plus glucagon (3-12 hr). Parallel culture and animal studies show that the amiloride-sensitive and prereplicative intervals coincide. In culture, a "burst" of 22Na+ influx, stimulated by peptide-supplemented media within 1 min but decreased later at 12 hr, is retarded by amiloride. This drug also blocks delayed prereplicative events involving increased amino acid "A" transport system function at 4-8 hr, and 3H-uridine and 3H-leucine incorporation into RNA and protein, respectively, at 8-12 hr. These findings suggest that at least two time-ordered processes are necessary to initiate hepatic growth fully: first, activation of Na+ flux systems by peptides similar or identical to EGF; and second, potentiation of these and subsequent cellular events by the combined action of insulin plus glucagon. [Amiloride: N-amidino-3,5-diamino-6-chloropyrazinecarboxamide; furosemide: 4-chloro-N-furfuryl-5-sulfamoylanthranilic acid; AIB: alpha-aminoisobutyric acid; ID50: administered dose giving 50% inhibition of a maximal response; dFBS: dialyzed fetal bovine serum; L.I.: 3H-dT nuclear labeling index.]

Amiloride↗
PubMed1979

Liver cells.

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Animals↗