William A. Peck

Study of calcium absorption in man: a kinetic analysis and physiologic model

A physical model of calcium absorption was developed from analysis of data obtained on 23 subjects, including 13 patients having a variety of abnormalities of calcium metabolism. The model was tested and found consistent in all subjects studied. This technique provides a quantitative description of the rate of entry of oral dose of (47)Ca into the circulation as a function of time by analysis of serum or forearm radioactivity in response to intravenous and oral administration of (47)Ca. The kinetics of the absorption process as proposed by the model are characterized by an initial delay phase of 15-20 min, by a maximal rate of absorption at 40-60 min after ingestion, and by 95% completion of the absorption within 2(1/2) hr. Partial identification of the physiological counterparts of the model was possible by introduction of the isotope at various levels of the gut. Although the region of the duodenum was found to have the greatest rate of absorption per unit length in normal subjects, it was least responsive to stimulation by parathyroid hormone and suppression by calcium loading. Furthermore, the response of the gut to parathyroid hormone was delayed, whereas the suppression of absorption by intravenous or oral calcium loading was rapid and dramatic. The implications of these observations are discussed.

Lipid metabolism in bone and bone cells I. The in vitro incorporation of [14C]glycerol and [14C]glucose into lipids of bone and bone cell cultures

Abstract 1. 1. To study lipid synthesis in mineralizing tissues, calvaria of new-born rats were incubated in buffer containing radioactive glycerol or glucose. The effects of non-labeled ATP, CTP, and glucose on lipid synthesis were investigated. Lipid synthesis by bone cells grown in primary cell culture was also studied. 2. 2. Calvaria and bone cells were found to contain cholesterol, cholesterol esters, mono-, di- and triglycerides, free fatty acids, and various phospholipids. The phospholipids included lecithin, lysolecithin, phosphatidyl ethanolamine, lysophosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, cardiolipin, sphingomyelin, and various unidentified components. Appreciable quantities of phosphatidyl serine were not extractable from calvaria until after demineralization with EDTA. 3. 3. The incubation of calvaria or bone cell cultures with [14C]glycerol and [14C]glucose gave rise to lipids labeled in the glycerol moiety of the various glycerides. Triglycerides and lecithin were the two most heavily labeled lipids. Phosphatidyl serine, cholesterol, sphingomyelin, and fatty acids were not labeled with these two labeled precursors. ATP generally stimulated neutral lipid synthesis but retarded phosphohpid synthesis from [14C]glycerol in a medium containing a low level of unlabeled glucose. An elevated glucose level generally decreased labeling of lipids from [14C]glycerol, probably by increasing intracellular glycerol phosphate concentrations. At higher levels of ATP and non-radioactive glucose, varied results were obtained which suggest that critical levels of both substances are required for optimal lipogenesis. CTP stimulated the synthesis of diglycerides, triglycerides, and lecithin, but inhibited the labeling of phosphatidyl ethanolamine. The synthesis of lipid from [14C]glucose was generally inhibited by ATP, but the simple effect of ATP on phosphofructokinase does not totally account for this inhibition.

Alpha-aminoisobutyric acid transport in human leukemic lymphocytes: in vitro characteristics and inhibition by cortisol and cycloheximide

We have studied the transport of alpha-aminoisobutyric acid (AIB)-3-(14)C and its response to cortisol and cycloheximide in vitro in blood lymphocytes from untreated patients with chronic lymphocytic leukemia. The accumulation of AIB-3-(14)C increased in a linear fashion for 60 min, and reached an apparent steady state in 120 min. The initial rate of AIB accumulation (V(o)) varied from 1.1 to 10.2 mumoles/kg cell H(2)O per min in cells from 16 different patients; however, V(o) was reproducible in cells from five of six patients which were studied repeatedly over 1-9 months, and correlated positively with the lymphocyte count (r = 0.51, P = < 0.01). Virtually total inhibition of protein synthesis with cycloheximide was found to decrease the accumulation of AIB in cells from four patients which had high rates of AIB transport, but had no effect on transport in cells from four patients which accumulated AIB more slowly. These results indicate that active transport depends, in part, upon the presence of labile protein with a turnover rate which varies among different cell populations. Treatment with 10 muM cortisol for 240 min in vitro reduced the initial rate of AIB-3-(14)C accumulation (V(o)) by 43.4+/-4.1% (SE) (range, 9-66%) in cells from 16 patients. The degree of inhibition did not vary appreciably over a 9 month period in four of five patients. The effect of cortisol was proportional to its starting concentration, and developed at low concentrations (0.1-1.0 muM). Cortisol appears to decrease AIB accumulation by inhibiting active uptake, since it neither enhanced the exodus of AIB, nor inhibited apparently nonsaturable transport. Inhibition was noncompetitive in type, suggesting that cortisol decreases the total capacity of the active transport mechanism.Cortisol inhibited AIB transport indirectly by a process which involved de novo protein synthesis, since inhibition (a) appeared only after 60 min of treatment, (b) was present in treated cells which were subsequently incubated for 60 min in cortisol-free medium, and (c) failed to develop during simultaneous blockade of protein synthesis with cycloheximide, even when cycloheximide itself did not decrease AIB transport.

Failure of 1,25 Dihydroxycholecalciferol [1,25-(OH)1-D3] to Modify Cyclic AMP Levels in Parathyroid Hormone-Treated and Untreated Bone Cells (1)

Treatment with 1, 25-(OH) 2-D3 (1 ng/ml-25 ng/ml) for periods ranging from 2.5 min. to 60 min. did not alter cyclic AMP levels in bone cells isolated from periosteum-free rat calvaria, or in cells isolated from rat periosteal tissues. 1, 25-(OH) 2-D3 failed to modify the acute increases in cyclic AMP elicited by PTH (10 ng/ml-1 ug/ml). Two separate 1, 25-(OH) 2-D3 preparations, biologically active in other systems, were ineffective under a wide variety of experimental conditions. These results suggest that 1, 25-(OH) 2-D3 is not an acute modulator of cyclic AMP metabolism in PTH-treated and untreated bone cells.

Cortisol-induced inhibition of amino acid transport in thymic lymphocytes: Kinetic parameters: relation to ATP levels and protein synthesis; and specificity

1. 1. α-Aminoisobutyric acid accumulation in rat thymic lymphocytes increased in linear fashion for 10 min and reached an apparent steady state in 60 min. α-Aminoisobutyric acid was transported by concentrative and non-concentrative processes. Cortisol inhibited only the concentrative process. Inhibition was non-competitive, indicating a decrase in the total capacity of the active transport mechanism. The effect of cortisol on α-aminoisobutyric acid transport was evident only after 60–90 min of treatment but preceeded the appearance of reductions in cellular ATP by an additional 60–90 min, suggesting that cortisol inhibition of α-aminoisobutyric acid transport was not caused by the decrease in ATP. 2. 2. Inhibition of protein synthesis with cycloheximide reduced α-aminoisobutyric acid uptake without decreasing cellular ATP, pointing to the participation of labile proteins in the active transport of α-aminoisobutyric acid which are not related to ATP metabolism. Inhibition of α-aminoisobutyric acid transport caused by cortisol and cycloheximide together was no greater than that caused by each agent alone. Moreover, cortisol failed to decrease cellular ATP levels during simultaneous exopsure of thymic lymphocytes to cycloheximide. Hence, inhibition of α-aminoisobutyric acid transport and reduction of ATP content are separate manifestations of cortisol action, each arising by a process which depends upon de novo protein synthesis. 3. 3. Inhibition of α-aminoisobutyric acid transport by cortisol appeared to be related to its glucocorticoid activity sicne (a) low concentrations (0.1 μM and 1.0 μM) were inhibitory, yet steroids devoid of glucocorticoid activity (progresterone, cortisone and testosterone) were non-inhibitory at the same concentrations, and (b) 11-deoxy-cortisol (cortexolone), which is known to impede the binding of glucocorticoids to cellular receptors, appreciably reduced the degree of inhibition caused by cortisol. High concentrations of progesterone and cortisone (10 μM) inhibited α-aminoisobutyric acid transport slightly suggesting that the decrease in α-aminoisobutyric acid uptake caused by 10 μM cortisol might reflect both specific (glucocorticoid-related) and non-specific effects.

Lipid metabolism in bone and bone cells. II. The in vitro incorporation of [32P] orthophosphate and [14C] serine into lipids of bone and bone cell cultures.

Abstract 1. 1. Calvaria of new-born rats or bone cell cultures were incubated with buffer containing [ 32 P]orthophosphate or [ 14 C]serine to study lipid synthesis in mineralizing tissues. 2. 2. [ 32 P]Orthophosphate was readily incorporated into phospholipids by calvaria and bone cell cultures. As with [ 14 C]glucose or [ 14 C]glycerol (ref. 1), lecithin was more heavily labeled than the other phospholipids. The labeling of sphingomyelin and phosphatidyl serine could not be demonstrated from radioactive phosphate. ATP decreased the incorporation of this isotope into lipids. 3. 3. [ 14 C]Serine gave rise to a variety of radioactive lipids. The synthesis of phosphatidyl serine was enhanced by the addition of both CTP and ATP whereas the labeling of sphingomyelin and phosphatidyl ethanolamine was little affected by these two co-factors.

Collagen synthesis by isolated bone cells: stimulation by ascorbic acid in vitro.

Abstract Bone cells isolated from rat calvaria were cultured on a flat surface, and collagen synthesis was determined by the incorporation of radioactive proline into peptide hydroxyproline. Addition of ascorbic acid to the incubation medium markedly increased the radioactivity of hydroxyproline in cold water-soluble and -insoluble protein fractions, without increasing the radioactivity of peptide-bound proline or the DNA content of the cell cultures. Stimulation was demonstrated with low concentrations of ascorbic acid (3 μM) and was directly proportional to the starting concentration. Hydroxylation of radioactive proline that had previously been incorporated into cell protein was stimulated despite puromycin- or cycloheximide-induced inhibition of protein synthesis. These results indicate that ascorbic acid promotes collagen synthesis in isolated bone cells by directly stimulating the hydroxylation of a proline-rich peptide. Inhibitors of protein synthesis consistently increased the radioactivity of peptide hydroxyproline when added to pulse-labeled cultures in the absence of ascorbic acid. The data suggest that they prevented the dilution of radioactive, unhydroxylated collagen precursor with non-radioactive precursor, thus providing substrate of higher specific activity for hydroxylation.

Specificity and Sensitivity of Cortisol-Induced Changes in Alpha Aminoisobutyric Acid Transport in Human Leukemic Small Lymphocytes and Leukemic Myeloblasts

Abstract We have examined the in vitro effect of glucocorticoid and nonglucocorticoid steroids on the transport of [3-14C]alpha aminoisobutyric acid (AIB) in lymphocytes from patients with chronic lymphocytic leukemia (CLL), and myeloblasts from patients with acute granulocytic leukemia (AGL). AIB uptake by CLL lymphocytes was markedly inhibited at 1.0 μM (52±2.1%) and slightly inhibited at 0.1 μM (17±3.0%) cortisol. A similar degree of inhibition developed at 50-fold lower concentrations of dexamethasone, indicating that the effect of these steroids on AIB accumulation parallels their glucocorticoid activity in vivo. In contrast, minimal or no inhibition was observed with steroids devoid of glucocorticoid activity (progesterone, testosterone, cortisone). 11-deoxycortisol, a nonglucocorticoid known to impede the binding of cortisol to cellular receptors in animal lymphocytes, failed to inhibit AIB uptake by CLL lymphocytes appreciably, but reduced the effect of cortisol to a statistically significant degree. Hence, cortisol-induced inhibition of AIB transport in CLL lymphocytes is related to its glucocorticoid activity and appears to require initial interaction with glucocorticoid-specific cellular receptors. In contrast, 1.0 μM cortisol enhanced the accumulation of AIB in AGL myeloblasts from each of five patients studied (mean = 19%, range 7-43%). Neither cortisone nor 11-deoxycortisol stimulated AIB uptake, and cortisol-mediated stimulation was not seen during simultaneous treatment with 11-deoxycortisol, suggesting that this effect of cortisol also represents a specific glucocorticoid effect. The divergent effects of cortisol on amino acid transport in CLL lymphocytes and AGL myeloblasts may explain, in part, the contrasting clinical effects of glucocorticoids administered to patients with these lymphoid and granulocytic hematopoietic malignancies.

Inhibition of Time-dependent Enhancement of Amino Acid Transport by Leukemic Leukocytes: A Possible Index of the Sensitivity of Cells to Drugs

Leukemic leukocytes increase their rates of alpha aminoisobutyric acid (AIB) accumulation when incubated for prolonged periods in amino acid deficient media. The time-dependent increase was prevented by concurrent exposure of cells to cycloheximide or actinomycin D in vitro. In addition, the increase in AIB uptake was not present in leukemic blasts studied in vitro when the cells were obtained from subjects with acute myeloblastic leukemia who had received antileukemic therapy. Cortisol added to cell suspensions in vitro inhibited the development of time-dependent increases in AIB uptake in lymphoid cells, but accentuated the process slightly in myeloblasts. Cortisol administered to a subject with CLL by infusion reduced the time-dependent increase in AIB uptake by CLL cells subsequently studied in vitro. These data indicate that the time-dependent increase in AIB uptake may be a means of testing the sensitivity of leukemic cells to drugs.