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Biomedical subjects

J Kowal

Publications and source records attributed to J Kowal.

At least 55 records · Page 3Linked to original sources
PubMed1981

Normal mammary cells in long term culture. I. development of hormone-dependent functional monolayer cultures and assay of alpha-lactalbumin production.

Mammary cells from normal tissue of virginal, pregnant, and lactating rats have been adapted to long term monolayer culture in plastic culture dishes with retention of hormone-responsive functional activity. The addition of PRL, insulin, and corticosterone resulted in an increase in the proportion of epithelial cells. the development of intracellular lipid droplets, and the ordered aggregations of these cells. In the presence of these hormones, the milk protein, alpha-lactalbumin (a-LA), was secreted into the growth medium at rates of 20-100 ng/mg cellular protein . 24 h. A double antibody RIA for a-LA capable of measuring 0.1 ng a-LA/100 microliter growth medium was developed in our laboratory for these studies. Both intracellular and extracellular a-LA fell below detectability within 2-3 weeks after hormone withdrawal. Intracellular a-LA accounted for less than 3% of the total a-LA accumulated in each culture in 24 h. the production rate of cells continuously given hormones increased 4- to 7-fold over a period of several months in culture, and their output was greater than 100-fold above that of cells not given hormones. These cells were obtained by overnight digestion and dispersion of tissue using selected batches of collagenase in the presence of 5% fetal calf serum. Plating densities of at least 3 X 10(4) cells/cm2 in Minimum Essential Medium supplemented with 14% fetal calf serum were required for optimal functional activity. Despite several months without added hormones, these cultures can retain their hormone responsiveness, since subsequent hormone addition resulted in detectable a-LA production beginning within 7-14 days. Our studies demonstrate for the first time that normal mammary cells can be maintained in a functional hormone-responsive state for extended periods in primary cell culture. These long term cell cultures provide a system with which the effects of these and other hormones on milk production and cell differentiation can be assessed under conditions which minimize the influence of the prior in vivo hormonal millieu. (Endocrinology 108: 573, 1981)

Animals↗
PubMed1981

Glucocorticoid regulation of alpha-lactalbumin production in long-term cultures of normal rat mammary cells.

Cultures of normal rat mammary cells have been developed which produce alpha-lactalbumin (a-LA) for several months in response to physiological levels of corticosterone whereas none is measureable without glucocorticoid. In contrast to recent studies in short-term organ cultures, these long-term cell cultures derived from virginal or mid-pregnant rats show no inhibition of a-LA production at high glucocorticoid concentrations. Comparable a-LA production was observed after a few months whether cells were initially cultured into media with various corticosterone levels or initially into high levels of corticosterone for 3 weeks prior to the shift to various lower doses.

Animals↗
PubMed1980

Acute steroidogenic stimulation of cultured adrenocortical tumor cells: an electron microscopic analysis.

The addition of ACTH to primary cultures of functional mouse adrenocortical tumor cells results in a marked increase in the production and release of steroids by these cells. The steroidogenic response to ACTH is detectable within minutes and is associated with a 'rounding-up' of the cells. The effects of ACTH were analyzed by scanning, conventional transmission and stereoscopic high voltage electron microscopy. Cell morphology was studied in unstimulated cells, in cells stimulated for 10-15 min in which only partial rounding had occurred, and after 2 hr of continuous stimulation when most of the cells had fully rounded. In unstimulated cultures, bundles of microfilaments (stress fibers) were prominent in cell extensions, subjacent to coated regions of the plasma membrane and frequently in proximity to microtubules and clusters of lysosome-like organelles. As the rounding process commenced, stress fibers disappeared from the cell edges. In 2 hr stimulated cells, Golgi complexes were hypertrophied. There were more microprojections from the plasma membrane than in control cells, as well as evidence of increased pinocytotic activity. There was an apparent polymerization of microtubules in elongated processes extending outward from the bases of the rounded cells and a concentration of lysosome-like organelles in these formations. The possible significance of these changes with respect to the differentiated function of adrenal cells is under continuing investigation in our laboratory.

Adrenal Cortex Neoplasms↗
PubMed1980

Adrenocorticotropic hormone increases specific proteins of the mitochondrial fraction that are translated inside or outside this organelle in cultured adrenal tumor cells.

In addition to its stimulatory effects on steroidogenesis, adrenocorticotropic hormone (ACTH) also has a trophic action on the adrenal cell. This is manifested in part by increases in the levels of key mitochondrial steroidogenic enzymes. The mechanism by which this trophic action of ACTH occurs has been studied in monolayer cultures of mouse adrenal cortical tumor cells. ACTH treatment of these cells stimulates the relative incorporation of amino acids into at least eight specific proteins in mitochondrial preparations. Two of these ACTH-responsive proteins are among the nine major adrenal polypeptides that fulfill the criteria of mitochondrial translation products: (i) their synthesis in intact cells is specifically resistant to inhibition by cycloheximide yet uniquely sensitive to chloramphenicol and (ii) they are synthesized in vitro by isolated mitochondria. The other six ACTH-responsive proteins are within the much larger category of mitochondrial proteins that are synthesized on cytoplasmic ribosomes. One of the proteins synthesized in the cytoplasm electrophoretically comigrates with purified beef adrenodoxin reductase and another with beef adrenodoxin. These findings indicate that ACTH regulates the synthesis (and turnover, or both) of specific mitochondrial proteins that are synthesized inside as well as outside the mitochondria of these adrenal cells.

Adrenocorticotropic Hormone↗
PubMed1980

Vasoactive intestinal peptide- and adrenocorticotropin-stimulated adenyl cyclase in cultured adrenal tumor cells: evidence for a specific vasoactive intestinal peptide receptor.

Vasoactive intestinal peptide (VIP) has been shown to be steroidogenic in monolayer cultures of a murine adrenal tumor but must be present at concentrations about 100-fold greater than ACTH to elicit the same degree of stimulation. Both peptides enhanced cAMP synthesis, although there was again a difference of 2 orders of magnitude in the dose-response curves. In contrast, VIP stimulated adenyl cyclase activity of tumor membranes in the same concentration range as ACTH. Maximum activity with VIP was less than that with ACTH in the absence or presence of a saturating amount of guanylyl imido-diphosphate. Saturating amounts of both peptides stimulated activity to levels greater than that with either ACTH or VIP alone, but the activity was not fully additive. An o-nitrophenyl sulfenyl derivative of ACTH inhibited ACTH-stimulated adenyl cyclase activity but not VIP-stimulated activity. Low concentrations of calcium potentiated the ability of submaximal doses of ACTH to stimulate adenyl cyclase but had no effect on the response to VIP. Concentrations of secretin or glucagon comparable to that of VIP did not stimulate steroidogenesis in intact cells. These data suggest that VIP may bind to a unique receptor which may be distinct from that of ACTH.

Adenylyl Cyclases↗
PubMed1978

The ultrastructure of functional mouse adrenal cortical tumor cells in vitro.

Cells derived from a transplantable mouse adrenal cortical tumor maintain their differentiated function in vitro and secrete steroids in response to ACTH and other stimulatory agents. The cell line has been widely employed for various biochemical investigations but there have been few attempts to correlate this work with morphologic data. This communication describes the electron microscopic appearance of the tumor transplant in vivo and primary cultures derived from it at various intervals after the cells are placed in culture. Tumor cells in vivo bear considerable resemblance to normal adult mouse adrenal cortical cells. Organelles generally considered to be directly involved in steroid biosynthesis (mitochondria, smooth endoplasmic reticulum and lipid droplets) are not drastically altered. Certain modifications of the vasculature and cell membrane, seemingly related to steroidogenesis, are present in both the tumor and normal adrenal cortex. Within 2 days after the tumor cells are introduced to culture, their cytoplasm assumes a more simplified appearance. Smooth endoplasmic reticulum is less conspicuous and free ribosomes and polysomes are very abundant. Mitchondrial inner membranes are reorganized from a saccular arrangement in the cells in vivo into distinct lamellar cristae. The tumor cells now resemble undifferentiated embryonic adrenal cells, or cultured adrenal cells from various mammalian sources which have dedifferentiated in the absence of ACTH. In their morphologically unspecialized state, the normal cells are incapable of functional responses to ACTH. In contrast, the cultured, dedifferentiated tumor cells respond within minutes to this hormone and can demonstrate 5-20 fold increases in their basal steroid output. These data suggest that substantial steroidogenic activity can occur although the characteristic appearance of adrenal mitochondria is absent.

Adrenal Cortex↗
PubMed1975

Adrenal cells in tissue culture the effects of choleragen and ACTH on steroid and cyclic-AMP metabolism.

Primary cultures of mouse adrenocortical tumors provide a sensitive system for investigating the effects of the enterotoxin of the V. cholerae (choleragen) on cyclic-AMP metabolism in the intact cell. Like ACTH, the toxin stimulates the synthesis and release of steroids from these cells but its mode of action differs from that of ACTH. The steroidogenic response to ACTH is immediate and of limited duration. The initial rate of steroidogenesis is the highest. In contrast, the steroidogenic response to choleragen is preceded by a 30-240 minute lag period which is inversely related to the concentration of the toxin. Whereas prolongation of the response to a single dose of ACTH requires hormone concentrations above those producing maximal initial steroidogenic activity, persistent steroidogenesis is induced at all levels of the toxin. Steroidogenic responses are detectable with 10 pg/ml of choleragen or less. The respective effects of ACTH and choleragen on cyclic-AMP synthesis and release into the medium parallel those on steroidogenesis. Intracellular cyclic-AMP levels in ACTH-treated cells reach a peak within 20-30 minutes and decline to normal levels within 2-4 hours. In choleragen-treated cells, after the lage period, the levels of intracellular cyclic-AMP remain above control levels indefinitely. The effects of ACTH and choleragen on cyclic-AMP biosynthesis are additive at all levels of the two compounds. The effects of choleragen are blocked by prior treatment of the toxin with a five-fold molar excess of ganglioside GM1, a presumed constituent of the toxin-binding site.

Adrenal Cortex↗