Ernest E. McCoy

Methods for the study of purine metabolism in human cells in vitro

Methods are described by which the apparent activities of 23 enzymes of purine metabolism can be measured in small quantities of intact human erythrocytes, leukocytes, and cultured human lymphoblasts and fibroblasts. Normal values of these apparent enzyme activities are reported for erythrocytes and leukocytes. Normal values of adenine and hypoxanthine-guanine phosphoribosyltransferase activities in extracts of mixed leukocytes are also reported. The apparent activities of the enzymes of adenine, hypoxanthine and guanine metabolism in intact mixed leukocytes are not appreciably different from those in separated lymphocytes and granulocytes. Neither these apparent activities in intact mixed leukocytes and erythrocytes nor the purine phosphoribosyl-transferase activities in extracts of these cells were significantly affected by high-purine or low-purine diets. The apparent activity of hypoxanthine-guanine phosphoribosyltransferase was 14 to 17% of normal in erythrocytes from patients with the Lesch-Nyhan syndrome, and 20 to 70% of normal in their leukocytes.

Sodium transport ouabain binding, and (Na+/K+)-ATPase activity in Down's syndrome platelets.

Summary: The mechanism of low serotonin content in Downs syndrome (DS) platelets was studied by comparing the activity of (Na+/K+)-ATPase, ouabain binding, and rate of outward movement of sodium in platelets from DS and control subjects.Ouabain inhibited (Na+/K+)-ATPase activity in sodium iodide-extracted platelet membranes from 10 normal subjects had an average value of 2.87 ± 0.20 μmol Pi./hr/109 platelets whereas the activity from DS platelet membranes was 2.13 ± 0.19 μmol Pi/hr/109 platelets (P < 0.02). Total ATPase activity in normal platelet membranes was 3.58 ± 0.33 μmol Pi/hr/109 platelets whereas that from DS membranes was 2.49 ± 0.35 μmol Pi/hr/109 platelets (P < 0.02). (Na+/K+)-ATPase activity is decreased in DS platelets.Ouabain binds specifically to (Na+/K+)-ATPase sites on membranes. The amount of ouabain bound by DS platelets is less than that by control platelets. The average amount of ouabain bound was 1.08 ± 0.04 × 10-15 mol/109 platelets for controls and 0.86 ± 0.02 × 10-15 mol/109 platelets for DS (P < 0.001). There appear to be fewer (Na+/K+)-ATPase sites on DS platelets compared to normal.The outward movement of Na+ in platelets is catalyzed by the enzyme (Na+K+)-ATPase. The average ouabain-sensitive outward movement of sodium in normal platelets was 9.03 ± 0.46 μmol/hr/109 platelets. For DS subjects the average outward movement was 50% of normal, 4.41 ± 0.14 /μmol/hr/109 platelets. The rate of outward Na+ transport is significantly less (P < 0.001) in DS compared to normal platelets.Decreased serotonin content of DS platelets may be secondary to decreased (Na+/K+)-ATPase activity, which results in decreased rate of outward Na+ transport and increased Na+ content. As serotonin is cotransported with Na+, the decreased rate of serotonin uptake may be secondary to increased Na+ content of DS platelets.Speculation: If decreased (Na+/K+)-ATPase activity, decreased rate of Na+ outward transport, increased Na+ content, and decreased rate of serotonin uptake were also present in synaptosomes of DS patients, decrease in amine content might occur and result in altered ncurotransmission.

Decreased ATPase and Increased Sodium Content of Platelets in Down's Syndrome

Abstract ATPase activity and sodium and potassium concentrations were studied in platelets from normal subjects and patients with Downs syndrome. Platelet ATPase activity of the patients was significantly decreased (p < 0.001 )-136.8 ± 5.2 (±S.E.M.) nm per hour per 108 platelets as compared to 173.2 ± 7.3 nm per hour per 108 normal platelets. Platelet potassium content of the patients was also decreased, 13.12 ± 0.48 μg per 109 platelets as compared to 19.78 ± 0.72 μg per 109 normal platelets (p < 0.001). Platelet sodium content of the patients was increased, 6.79 ± 0.40 μg per 109 platelets as compared to 3.32 ± 0.21 μg per 109 normal platelets (p < 0.001). The potassium-sodium ratios in the platelets of the patients had no overlap with values obtained in the normal controls. Decreased platelet ATPase activity of patients with Downs syndrome could cause decreased outward sodium transport, resulting in an increased platelet sodium content. Since the transport of serotonin into platelets is coupled to in...

A comparison of the serotonin and ATP content in platelets from subjects with Down's syndrome

Abstract Studies of serotonin content and uptake; ATP, ADP, and AMP content and ATP, ADP, and AMP synthesis from 14C-adenine were carried out in platelets obtained from Downs syndrome (D.S.) and control subjects. Serotonin content was decreased 60%, the Vmax for serotonin uptake less, and Km for serotonin greater in D.S. platelets compared to controls. ATP, ADP, and AMP content were each decreased 30% in D.S. platelets. ATP synthesis was greater, ADP synthesis similar and AMP synthesis less in D.S. platelets. The ATP/serotonin ratio was significantly higher in D.S. platelets indicating that in relation to serotonin, there was adequate ATP available.

A STUDY OF γ‐AMINOBUTYRIC ACID UPTAKE IN NORMAL AND DOWN'S SYNDROME PLATELETS

1 Initial uptake rates of γ‐aminobutyric acid (GABA) were compared in Downs syndrome (D.S.) and normal platelets. GABA uptake was decreased in D.S. platelets (1.10 ± 0.10 nmol h−1 10−9) compared to uptake by normal platelets (1.89 ± 0.21 nmol h−1 10−9), P < 0.005. 2 The effect of varying the Na+ concentration was similar on D.S. and normal platelets. Increasing the media Na+ concentration resulted in increased rates of GABA uptake in both D.S. and normal platelets. 3 GABA uptake in the presence of 2,4‐dinitrophenol or at 2°C is approximately 56% of the uptake at 37°C for both D.S. and normal platelets. 4 Extrapolation of a reciprocal plot indicates a two affinity uptake system: a high affinity and a low affinity mechanism. 5 A significant defect in GABA uptake exists in D.S. platelets.

ENHANCEMENT OF IN VITRO CELLULAR IMMUNE RESPONSE BY L-TETRAMISOLE

L-tetramisole, a potent antihelminthic, has recently been shown to enhance the ability of anergic cancer patients to both become sensitized and respond to the contact sensitizing agent 2, 4-DNCB (New Engl. J. Med. 289: 354, 1973). L-tetramisole is hypothesized to stimulate the T-cell system (ibid, 375), and we have tested this hypothesis by examining the effects of L-tetramisole on the response of normal human lymphocytes to T-cell stimulating antigens. Following ficollhypaque isolation, 2×106 lymphocytes were cultured in triplicate for 5 or 8 days, with or without specific antigens (candida, measles virus, or PPD), in the presence or absence of L-tetramisole (1 mg/culture, optimal dose), in 4 ml RPMI 1640 containing 20% autologous serum. Each culture received 2 μc 3H-thymidine 24 hours prior to harvesting, and in vitro cellular immune response was measured as 3H uptake. Cells cultured in the presence of L-tetramisole showed significantly greater response to each of the three antigens than those cultured without the drug (p<0.01). Cells from donors with pre-existing antigen immunity responded within 5 days, but those with mild or absent pre-existing sensitivity responded after 8 days. Our results clearly indicate an in vitro immunostimulant activity of L-tetramisole, and suggest the potential importance of this drug both in the treatment of patients with primary cellular immunity defects and in the management of cancer.

Potassium uptake by platelets from Down's syndrome and normal subjects

Abstract Potassium uptake was studied in Downs syndrome (D.S.) platelets to determine if the Na + /K + ATPase mediated movement of this ion was decreased compared to normal platelets. Total uptake of 42 K was 1.58±0.16 μmoles/hr/10 9 normal platelets but was decreased to 1.06±0.06 μmoles/hr/10 9 D.S. platelets (p + /K + ATPase mediated (ouabain sensitive) K + uptake was 0.87±0.05 μmoles/hr/10 9 normal platelets but only 0.54±0.04 μmoles/hr/10 9 in D.S. platelets (p + /K + ATPase mediated outward movement of Na + is decreased in D.S. platelets, the present work demonstrates that bidirectional functional imparrment of the Na + /K + ATPase pump is present in D.S. platelets.

Decreased Polyamine Content of Concanavalin A Stimulated Lymphocytes in Down's Syndrome Subjects

Summary: Increased polyamine content is associated with increased rates of cell growth. Several Downs syndrome (D.S.) tissues have been shown to have decreased growth rates. Studies were undertaken to determine if the polyamine content of stimulated D.S. lymphocytes was similar to that of stimulated normal cells. Lymphocytes were isolated and cultured in the presence of Concanavalin A for 4 or 5 days. Polyamines were then extracted and quantitated. After 4 days spermidine content for normal cells was 930.9 ± 127 and for D.S. cells 489.2 ± 113.1 nmoles/109 cells (P < 0.025). Spermine content of normal cells was 1152.8 ± 157.4 and for D.S. cells 533.9 ± 82.0 (P < 0.005). After 5 days in culture spermidine content of normal cells was 803.0 ± 75.9 and for D.S. cells 446.2 ± 76.5 nmoles/109 cells (P < 0.005). Spermine content was 1155.7 ± 121.9 for normal cells and 555.1 ± 68.4 nmoles/109 for D.S. cells. Decreased content of polyamines in D.S.-stimulated lymphocytes is most probably due to decreased rate of polyamine synthesis. Decreased content of polyamines in response to stimulation may be a factor in decreased growth rates and altered immune function seen in D.S. patients.Speculation: Polyamine content is associated with increased rates of DNA and cell replication. If decreased polyamine synthesis occurred in multiple tissues in Downs syndrome, it could result in a total decrease in cell number and small stature present in Downs syndrome patients.

Phospholipid composition of Down's syndrome and normal platelets

1. Total and individual phospholipids were quantitated in platelets isolated from normal and Downs syndrome (D.S.) subjects. Phospholipids were extracted from isolated platelets, separated by thin layer chromatography and quantitated by measurement of fluorescence of the compounds using a thin layer chromatogram scanner. The total amount of phospholipid was similar in D.S. and control subjects. The amount and percent composition of phosphatidyl serine, phosphatidyl ethanolamine, phosphatidgl inositol, phosphatidyl choline and sphingomyelin was also similar in D.S. and normal platelets. 2. The studies were undertaken to determine if the decrease in Na+/K+ AtPase activity observed in D.S. platelets was associated with alteration in phospholipid composition. The present report would indicate that no major differences in phospholipid composition are present in D.S. platelets that would account for the observed decrease in Na+/K+ AtPase activity.

Sodium and serotonin uptake into Na+ depleted platelets from Down's syndrome and normal subjects.

Abstract The mechanism of decreased serotonin (5HT) uptake in D.S. platelets was studied by determining 5HT and Na+ uptake in Na+-depleted and non-Na+-depleted platelets. 5HT uptake was decreased in D.S. compared to normal platelets. 5HT uptake studied during Na+ repletion was significantly increased compared to the rate of non-Na+-depleted D.S. and normal platelets. However, the increased rate was still significantly less than that of normal platelets. 5HT uptake studied in Na+-free buffers was decreased to a greater degree in D.S. compared to normal platelets. This suggests a greater Na+ dependence for 5HT uptake in D.S. platelets. The rate of sodium uptake was increased in both non-Na+-depleted and Na+-depleted D.S. platelets. Although the content of non-Na+-depleted platelets is increased in D.S., the Na+ repletion was not increased compared to normal platelets. The results were interpreted to mean that decreased 5HT uptake is not due to a decreased rate of Na+ uptake in D.S. platelets. Although the rate of 5HT uptake can be increased by depleting the platelets of Na+, the rate of increase is less than that of normal platelets. The results favor a defect in 5HT transport involving the transport site or 5HT binding protein.