Rat glomerulosa cells in primary culture and E. coli lipopolysaccharide action
Introduction
The adrenal gland is a stress responding organ that undergoes structural and functional changes under different pathophysiological conditions; the adrenal gland plays an important role in the response to stressors, such as infections and sepsis [1]. Lipopolysaccharide (LPS or endotoxin), a major component of the outer cell wall of all Gram-negative bacteria, is considered the main initiator factor of endotoxic or septic shock. In endotoxic shock a dysfunction of the adrenal gland has been observed. During endotoxemia the hypothalamic–pituitary–adrenal axis is stimulated and plasma ACTH and glucocorticoid levels are markedly increased [2], [3], [4]; however a blunted cortisol response to ACTH stimulation has been related to a poor prognosis [5]. A small percentage of patients with sepsis develop an adrenocortical insufficiency [6], but a lack of adrenal reserve defined by less response to ACTH administration appears to be common and is associated with increased mortality [7]. We have reported that adrenocortical cells isolated from adrenal glands of endotoxemic rats show impairment in the steroidogenic response to ACTH [3] and that LPS in vitro inhibits ACTH-induced corticosterone secretion in fasciculata-reticularis cells [8], [9]. In septic shock, the zona glomerulosa of the adrenal cortex is altered too; there is an increase in aldosterone secretion in response to hypotension, but plasma aldosterone levels are disproportionately low despite hyperreninemia [10]. It has also been described that atrial natriuretic peptide (ANP) concentrations are increased during endotoxic shock in rats [11] and it is known that ANP inhibits aldosterone secretion [12].
In previous studies, we have demonstrated that bacterial lipopolysaccharide (LPS) binds to fasciculata-reticularis cells [13] inducing increases in [Ca2+]i and in the production of reactive oxygen species [14]. The interaction of LPS with specific binding sites in fasciculata-reticularis cells produces a diminution in ACTH-induced corticosterone secretion both in isolated cells and in primary cultures [8], [9]. Since LPS also binds to glomerulosa cells [13], the purpose of the present study was to evaluate the direct effect of lipopolysaccharide from Escherichia coli 0111:B4 on glomerulosa cells in primary cultures.
Section snippets
Reagents and media
Lipopolysaccharide (LPS) was from E. coli serotype 0111:B4, obtained by phenol–water extraction and was purchased from Sigma (St. Louis, USA). Dulbecco’s modified Eagle’s medium (DMEM), HEPES, angiotensin II, basic fibroblastic growth factor (bFGF) and methylene blue were also from Sigma. Collagenase A and DNase were obtained from Boehringer-Mannheim Diagnostica (Germany). ACTH was from Ciba-Geigy (Switzerland).
Animals
Adult male Wistar rats (Charles River, Spain) weighing 200–250 g were used in all the
Glomerulosa cells in primary culture: effect of LPS
Glomerulosa cells were cultured in DMEM+20% FBS, since serum lipoproteins are necessary for the maintenance of steroidogenesis. Glomerulosa cells were cultured for 2 days in these basal conditions, then several agents were added and the cultures were maintained for another 4 and 6 days (6 and 8 days in culture). Cell proliferation was determined using methylene blue assay and DNA amount measurement.
The results of methylene blue assay are shown in Fig. 1. Cell density is expressed as staining
Discussion
The present study examines the in vitro effect of LPS on cell proliferation and on steroid secretion by rat glomerulosa cell in culture. Glomerulosa cells that were initially epithelial, after 4 days in culture under our basal conditions (DMEM+20% FBS), lost much of their cytoplasmic lipids, formed monolayers of flat elongated cells, underwent significant proliferation and round cells began to emerge from the underlying monolayer. These features are analogous to that described by Roskelly and
Acknowledgements
This work was supported by grants from DGICYT (MEC PB94-0244) and Multidisciplinar Grant from Universidad Complutense (PR 218/94-5677). AES was supported by a predoctoral fellowship from UCM. The authors are particularly grateful to A. Mayo Ph.D. (Department of Statistic and Operation Research, Universidad de Valladolid, Spain) for his technical assistance in the statistical analysis.
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Present address: Ocular Surface Group, IOBA, Faculty of Medicine, Universidad de Valladolid, Valladolid, Spain.