Thyroid hormone control of erythrocyte 2,3-diphosphoglyceric acid concentrations.

A biphasic thyroid hormonal effect has been shown on 2,3-diphosphoglyceric acid synthesis in a crude enzyme, hemoglobin-free preparation from normal human erythrocytes.

Mechanism of action of thyroid hormones on erythrocyte 2,3-diphosphoglyceric acid synthesis.

Normal erythrocytes, when incubated with thyroid hormone, were found to have increased levels of 2,3-diphosphoglyceric acid. In addition, a partially purified enzyme preparation, when incubated with either a 1,3-diphosphoglyceric acid generating system or 1,3-diphosphoglyceric acid directly, showed increased levels of 2,3-diphosphoglyceric acid when exposed to thyroid hormone. The hormonal effect was biphasic and was witnessed after 5 min of incubation. Substitution on the 3 and 5 positions of the basic thyronine molecule was essential for hormonal effect. It appears that thyroid hormone acts by directly stimulating the diphosphoglycerate mutase enzyme. The hormonal effect on 2,3-DPG synthesis may offer a biochemical explanation for the shift in the oxyhemoglobin dissociation curve observed in thyroid disorders.

The absence of 2,3-diphosphoglycerate from myocytes, hepatocytes and adipocytes.

Myocytes, hepatocytes and adipocytes were prepared from heart, liver and epididymal fat pad of the rat. No detectable level of 2,3-diphosphoglycerate was found. Evidence is also presented which indicates the absence from these cells of 2,3-diphosphoglycerate mutase and 2,3-diphosphoglycerate phosphatase. Previous findings by others of the presence of 2,3-diphosphoglycerate and 2,3-diphosphoglycerate mutase probably resulted from erythrocytes sequestered in the tissue.

Effect of fasting on insulin binding to hepatocytes and liver plasma membranes from rats.

Binding of [125I]iodoinsulin to isolated hepatocytes from fed and fasted rats was measured. Hepatocytes from fed rats bound significanlty more [125I]iodoinsulin at insulin concentrations ranging from 0.15--100 nM. Scatchard and average affinity profile data analyses showed no significant difference in the affinities of the binding sites but indicated a decrease in the receptor concentration of the hepatocytes from fasted rats. In contrast to hepatocytes, liver plasma membranes of fasted rats showed increased insulin binding at all insulin concentrations, except the highest level of 100 nM. Analysis of the data indicated a significant increase in the affinity of the receptors but no change in the receptor concentration. Evidence is presented that the differences in the results obtained with hepatocytes and membranes were not due to the differences in the recovery of the plasma membranes. The results suggest that insulin binding by intact hepatocytes does not necessarily reflect the receptor protein content of the cell membrane and that the liver cell modulates its response to insulin by changes in the architecture of the plasma membrane.

Amino acid stimulation of oxygen and substrate utilization by cardiac myocytes.

The inclusion of plasma levels of the natural amino acids plus 2.5 mM glutamate and 2.5 mM malate (PAAGM) raised the oxygen consumption and glucose oxidation of isolated cardiac myocytes in phosphate buffered saline. The addition of calcium (1.25 mM) and magnesium (0.66 mM) potentiated the stimulatory effect of PAAGM on glucose oxidation and oxygen consumption, PAAGM did not alter the shape of the dose-response curve for glucose oxidation by the isolated cardiac myocyte preparation. It did increase the amount of glucose oxidation at any given media glucose concentration up to 20 mM. PAAGM also increased the rate of lactate oxidation by the isolated cardiac myocyte preparation. PAAGM did not stimulate the oxidation of octanoate unless there was glucose present in the incubation media as well. Measurements of the concentrations of free amino acids indicated higher levels in myocytes incubated in PAAGM than in myocytes incubated in phosphate buffered saline. The data suggest that substrate metabolism by the isolated cardiac myocyte preparation can be influenced by the presence of plasma constituents that would be available to the myocardium in vivo.

Effect of ouabain on exogenous substrate oxidation by isolated cardiac myocytes.

Ouabain (10(-4) M) stimulated glucose oxidation and inhibited octanoate oxidation by isolated cardiac myocytes. Omission of potassium or calcium from the buffer abolished the ouabain stimulation of glucose oxidation. The inhibition of octanoate oxidation by ouabain persisted in the calcium deficient media.

Hexose monophosphate shunt in isolated cardiac myocytes from normal rats.

The activity of the hexose monophosphate shunt was studied in myocytes obtained from the ventricles of normal, adult, male rats. When myocytes were incubated in buffer containing either 1-14C- or 6-14C-labeled glucose the ratio of C-1/C-6 14CO2 evolved was essentially unity. The addition of plasma levels of amino acids did not alter this finding. If, however, a competitive substrate (pyruvate, octanoate, acetate, or lactate) was present, in sufficient quantity to lower the oxidation of glucose to approximately 20% of the control, the C-1/C-6 14CO2 ratio rose to values between 1.3 and 2.1. This ratio was dependent on the concentration of the competitive substrate, which was dependent on the buffer system. The data indicates that the hexose monophosphate shunt is active in the heart because it can be demonstrated when a substrate, which competes with glucose for oxidation, is present. The presence of competing substrates parallels the situation occuring in vivo.

Separation of beating cardiac myocytes from suspensions of heart cells.

Heart cells were obtained in suspension after incubation with collagenase and hyaluronidase in Saline A. Cardiac myocytes were separated by isopycnic centrifugation in 88.6 to 92.4% purity from other heart cells with different densities, and by velocity or rate-zonal sedimentation, in 92.8 to 97.4% purity from heart cells with different diameters. A previously described computer integration of the differential sedimentation equation was used to determine the centrifugal force, duration of centrifugation and gradient design, which would permit the separation of cardiac myocytes from other heart cells by velocity sedimentation. The myocytes continued to contract rhythmically after being recovered from the density gradients. Velocity sedimentation was superior to isopycnic sedimentation for the separation of cardiac myocytes from heart cell suspensions because it gave the most highly purified myocytes, resulted in recovery of the largest proportion of myocytes in purified fractions from the gradient and required lower centrifugal forces for shorter periods of time. The potential significance of the availability of pure cardiac myocytes is discsused.