Differentiation of rabbit adipocyte precursor cells in a serum-free medium.

A serum-free, hormone-supplemented medium containing insulin, transferrin, and triiodothyronine (ITT medium), able to support differentiation of rat adipose precursor cells, has been used to study the regulation of the development of adipocytes in the rabbit. Adipose conversion was assessed by the appearance of glycerol-3-phosphate dehydrogenase activity. Stromal-vascular cells from rabbit perirenal adipose tissue differentiated to a very low extent or not at all in ITT medium. Supplementation of ITT medium with growth hormone or fibroblast growth factor did not increase the proportion of differentiated cells. In contrast, rabbit stromal-vascular cells were able to differentiate in ITT medium supplemented with glucocorticoids (dexamethasone, corticosterone) whereas sex steroids (beta-estradiol, testosterone, progesterone) did not affect the differentiation process. In the presence of both dexamethasone and insulin, 20 to 50% of rabbit stromal-vascular cells differentiated into adipocytes within 2 wk of culture. The stimulatory actions of dexamethasone or insulin were dose-dependent. Insulin-like growth Factor-I (IGF-I), did not replace insulin under our culture conditions and had only a slight effect when added along with dexamethasone (100 nM) and insulin (1.7 nM). The results suggest that glucocorticoids, in association with insulin, may play an important role in the development of adipocytes from rabbit precursor cells.

Platelet-derived growth factor, epidermal growth factor, and insulin-like growth factor I regulate specific cell-cycle parameters of human diploid fibroblasts in serum-free culture.

The growth regulation of human diploid fibroblasts by platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor I (IGF-I) (somatomedin C), dexamethasone, and transferrin was investigated in a serum-free, chemically defined culture system. Cell-cycle kinetic parameters were determined using 5'-bromodeoxyuridine (BrdU) incorporation and flow cytometric analysis with the DNA-specific dye Hoechst 33258. We found that PDGF and EGF regulate the proportion of cells capable of entering the cell cycle from the quiescent state, with smaller effects upon the rate of cell transition from G1 into S phase. IGF-1, on the other hand, regulates the rate of cell exit from G1 without affecting the cycling fraction. Transferrin and dexamethasone showed less effect upon the cell-cycle kinetics under these culture conditions. The data provide functional evidence that PDGF and EGF regulate similar cell-kinetic parameters in human fibroblast cultures. IGF-I is functionally distinct from both PDGF and EGF in its role of regulating G1 exit rate without affecting the cycling fraction. These observations made by BrdU-Hoechst flow cytometric techniques provide a novel perspective on the regulatory effects exerted by different classes of growth factors, and suggest a mode of interdependence of these mitogens in regulating the net growth rate which could be a feature of growth regulation in vivo. These data also provide a different perspective on the regulation of the growth of fibroblast-like cells than that of the "competence/progression" cell-cycle model.

Expression of sodium pump activity and of transepithelial voltage induced by hormones in cultured cortical collecting tubule cells.

Primary monolayer cultures of polarized epithelia were derived from single cortical collecting tubules (CCT) of the mammalian kidney (rabbit). The expression of Na-K-ATPase activity (mol 10(-15)/min.cell) and of the transepithelial voltage (VT) in monolayers on membranes was measured in response to defined hormonal supplements of the nephron culture medium (NCM). Hormones were: triiodothyronine (T3; 10(-11) M), dexamethasone (10(-8) M), and aldosterone (10(-9) M). Control was NCM plus fetal calf serum (FCS; 3% v/v). Data are mean (SEM). Na-K-ATPase activity was 10.4 (6.2) in control (n = 27); 18.8 (5.2) in T3 (n = 35); 21.2 (5.5) in dexamethasone (n = 26), and 30.9 (6.5) in aldosterone (n = 24). VT (mV) was 4.2 (3.2) in control (n = 12); 12.7 (3.7) in T3 (n = 12); 16.8 (2.5) in dexamethasone (n = 12), and 28.4 (3.6) in aldosterone (n = 14). Data are evidence that thyroid and corticosteroid hormones selectively induce the postmitotic expression of the sodium carrier activity and other functions related to vectorial solute transport in this polarized epithelium.

Glucocorticoid- and nerve growth factor-induced changes in chromogranin A expression define two different neuronal phenotypes in PC12 cells.

The regulation of chromogranin A mRNA was examined in PC12 cells after treatment with nerve growth factor, dexamethasone, or a combination of the two agents. PC12 cells have low levels of chromogranin A mRNA, and this does not change upon treatment with nerve growth factor. Dexamethasone treatment of these cells results in a 4-fold increase in the amount of chromogranin A mRNA. The dexamethasone-stimulated increase in chromogranin A mRNA is not apparent until at least 16 h after the addition of the drug and is maintained only with continuous culture in the presence of the drug. Dexamethasone and nerve growth factor together increase chromogranin A mRNA to the level seen with dexamethasone alone. Immunohistochemistry shows a similar pattern of protein accumulation within individual cells. Chromogranin B mRNA levels are unaltered by any of the drug treatments described. Treatment with dexamethasone plus NGF seems to be required for full expression of the adrenergic, neuronal phenotype in PC12 cells. Measurement of chromogranin A mRNA provides more specific delineation of neural differentiation and how it is influenced by hormones and growth factors.

Glucocorticoids regulate adrenal opiate peptides.

Although glucocorticoids and impulse activity are well-recognized mediators of adrenal catecholamine biosynthesis, the effects of these signals on the colocalized opiate peptide system is only presently emerging. Since it is generally agreed that impulse activity regulates adrenal opiate peptides, in the present report we sought to determine whether adrenal opiates are also subject to hormonal control. Pharmacological destruction of the adrenal cortex resulted in a decrease in baseline Leu-enkephalin levels in vivo. This suggested a tonic regulatory effect of adrenal cortical steroids on enkephalin pathways. To further examine the role of glucocorticoid hormones in regulating enkephalin biosynthesis in a more dynamic system, medullae were grown as explants where peptide levels typically rise 30- to 50-fold above baseline. Explanted medullae required medium supplemented with dexamethasone or corticosterone to achieve maximal levels of Leu-enkephalin in a dose-dependent fashion. The effects of glucocorticoid treatment were blocked by specific glucocorticoid receptor antagonists or by inhibition of receptor translocation to the nucleus. Since enkephalin levels rose in cultured medullae (even in the absence of added glucocorticoids), glucocorticoid-independent regulatory mechanisms may also play a role in this model. Based on this and previous results, it appears that adrenal opiate peptides, like catecholamines, are subject to dual hormonal and transsynaptic regulatory influences. The interaction of these two regulatory mechanisms may serve an adaptive role in modulating complex biochemical and behavioral responses with exquisite precision.

Properties and dynamics of glucocorticoid binding capacity in rat skin.

[3H]Dexamethasone binding was detected in cytosol prepared from rat skin only in the presence of dithiothreitol (DTT). Simultaneous supplement of sodium molybdate (Mo) induced synergistic enhancement of the binding. In the presence of DTT and Mo the dissociation constant was approximately 1 nM, the number of maximum binding sites was approximately 100-200 fmol/mg protein, and only steroids that possessed glucocorticoid activity competed with [3H]dexamethasone binding. [3H]Dexamethasone-receptor complexes in dermal cytosol were able to bind to DNA-cellulose after brief heating, and receptors were eluted from DEAE-cellulose with 0.2 M KCl. These observations showed that [3H]dexamethasone binding sites observed in dermal cytosols have similar binding characteristics to glucocorticoid receptors in other glucocorticoid target tissues. Binding capacity in dermal cytosols was depleted after in vivo administration of all 4 glucocorticoids used in the present study. Both the extent and the duration of depletion were dose-dependent in all instances. Non-fluorinated glucocorticoids required higher doses to induce a profound depletion than did fluorinated ones and the duration of depletion induced by the former was shorter than by the latter. Since fluorinated glucocorticoids usually have higher anti-inflammatory potency than non-fluorinated ones, we concluded that the pattern of depletion and replenishment of the dermal cytosol binding capacity was correlated with glucocorticoid biopotency.

Role of adrenal hormones in regulating distal nephron structure and ion transport.

Mineralocorticoid levels are an important determinant of membrane area and ion transport in the renal initial (ICT) and cortical (CCT) collecting tubules. Adrenalectomy leads to a dramatic and specific decrease in basolateral membrane area of principal (P) cells and depresses sodium reabsorption and potassium secretion. Although aldosterone replacement for 10 days restores basolateral membrane area and ATPase activity to control levels and dramatically elevates ion transport, glucocorticoids have no effect on basolateral membrane area, ion transport, or ATPase. It is suggested that the aldosterone-induced amplification of membrane area occurs as a mechanism whereby cells increase the number of ATPase pumps in the basolateral membrane. An acute (of 2-3 h) increase in aldosterone, but not dexamethasone, also stimulates potassium transport by the ICT. Future studies will have to establish whether the acute stimulation of transport by aldosterone involves a change in basolateral membrane area. It is concluded that mineralocorticoids, but not glucocorticoids, regulate sodium and potassium transport by P cells of the ICT and CCT, in part, by determining the number of ATPase pumps available for transport.

Response of the rat brain beta-endorphin system to novelty: importance of the fornix connection.

In control rats, a step-down inhibitory avoidance training trial using a 0.8 mA footshock, or simple exposure to the training apparatus without footshock, was followed by a decrease of beta-endorphin-like immunoreactivity measured in the hypothalamus and ventral thalamus. The effect of inhibitory avoidance training was also measured in rats submitted to a brain sham operation, to bilateral transection of the dorsal fornix, to anterior or to posterior hypothalamic deafferentation, to adrenal medullectomy, to an adrenal sham operation, to 16 daily ip injections of 0.2 mg/kg dexamethasone, or to 16 daily ip injections of 1 ml/kg saline. The diencephalic beta-endorphin-like immunoreactivity response to training was abolished by fornix transection and was unaffected by all other treatments. This suggests that the response is not mediated by anterior or posterior neural afferents to the hypothalamus, or by a hypersecretion of epinephrine by the adrenal medullae, or of ACTH by the pituitary gland. The response, instead, appears to require the integrity of the pathway that sends projections from the septo-hippocampal system to the hypothalamus. Previous evidence had suggested that the diencephalic beta-endorphin-like immunoreactivity response to training is a result of novelty, and the septo-hippocampal system has been postulated to play a role in the registration of novelty.

Methods in laboratory investigation. Autoradiographic demonstration of the specific binding and nuclear localization of 3H-dexamethasone in adult mouse lung.

This report describes the first autoradiographic demonstration of specific nuclear localization of 3H-dexamethasone in different cell types of the lung. Adult mouse lung tissue was incubated in vitro for 90 minutes with 17 nM 3H-dexamethasone in the presence or absence of various nonradioactive steroids. After extensive washing to remove any nonspecifically bound ligand, the specimens were processed for autoradiography using the thaw-mount method. In the absence of competing steroids, silver grains were localized in the nuclei of alveolar type II cells, bronchiolar and arteriolar smooth muscle cells, fibroblasts, and endothelial cells of the pulmonary vasculature. No significant nuclear concentration of label was observed in the bronchiolar epithelium, however. The specificity of 3H-dexamethasone labeling was demonstrated by incubating 17 nM 3H-dexamethasone with a 600-fold excess of either unlabeled dexamethasone, estrogen, dihydrotestosterone, or progesterone. These autoradiographic binding and steroid competition studies were confirmed by quantifying with liquid scintillation counting the specific 3H-dexamethasone binding in nuclear and cytosolic fractions prepared from lung tissues that had undergone identical incubation and washing procedures as those for autoradiography. These results demonstrate that many cell types in adult lung are targets for glucocorticoids and may respond to physiologic concentrations of this hormone.

The uptake of 3H-dexamethasone during phagocytosis of Staphylococcus aureus by Human neutrophils.

In the normal human neutrophil under basal resting conditions, cell-associated levels of 3H-dexamethasone (3H-DXM) increased in a linear fashion following addition of the drug. However, during phagocytosis of 14C-labelled Staphylococcus aureus (Staph. aureus), the intracellular 3H-DXM levels rose above basal concentrations in direct relation to the quantity of ingested organisms. The observation that the cell-associated activity of 3H-DSM rises in a parallel fashion to the quantity of ingested Staph. aureus must be recognized and taken into consideration as a variable, when assessing the results between resting and activated phases of neutrophil function in the presence of glucocorticoids.