J I Mason

Detection of human 11β-hydroxysteroid dehydrogenase isoforms using reverse-transcriptase-polymerase chain reaction and localization of the type 2 isoform to renal collecting ducts

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD), responsible for the interconversion of hormonally active cortisol to inactive cortisone, dictates specificity for the mineralocorticoid receptor (MR) in the distal nephron and colon. Two isoforms of human 11 beta-HSD have been cloned, an NADP(H)-dependent (type 1) dehydrogenase/oxo-reductase enzyme, and a high-affinity NAD-dependent (type 2) unidirectional dehydrogenase. Using the reverse-transcriptase polymerase chain reaction (RT-PCR) amplification of RNA extracted from human adult tissues, type 1 11 beta-HSD mRNA was found in decidua, placenta, liver, lung, spleen, kidney medulla, cerebellum and pituitary, but was absent in kidney cortex, sigmoid and rectal colon, salivary gland and thyroid. In contrast, type 2 11 beta-HSD mRNA was found only in placenta and in the classical mineralocorticoid target tissues, kidney cortex, kidney medulla, sigmoid and rectal colon, salivary gland, and colonic epithelial cell lines (AAC1 and RGC28). In situ hybridization studies of renal cortex, cortico-medullary junction and medulla using a 35S-labeled antisense cRNA probe for type 2 human 11 beta-HSD, revealed specific localization of type 2 11 beta-HSD mRNA expression exclusively to renal cortical and medullary collecting ducts. Type 1 and type 2 isoforms of human 11 beta-HSD are expressed in a distinct tissue-specific fashion, in keeping with the proposed differences in their physiological roles. Type 2 11 beta-HSD is found predominantly in mineralocorticoid target tissues where it serves to protect the MR in an autocrine fashion.

Effects of transforming growth factor beta on ovine adrenocortical cells

Transforming growth factor beta (TGF beta) is a potent regulator of steroidogenic cell function. However, the mechanisms of the effects are not well understood. We studied the actions of TGF beta on primary cultures of ovine adrenocortical (OAC) cells. OAC cells had high affinity receptors for TGF beta (KD congruent to 7.6 +/- 1.5 X 16(-11) M). In addition, TGF beta inhibited the following markers of adrenocortical function: (1) ACTH, cholera toxin and forskolin acute stimulation of cAMP and steroid production; (2) the acute 8-bromo-cAMP stimulation of corticosteroid and pregnenolone production; and (3) the activity and amount of P-450 17 alpha-hydroxylase protein as well as activities of 11 beta- and 21-hydroxylases. The inhibitory effects of TGF beta on ACTH-induced cAMP and steroid production were time (half inhibition at 6 and 3 h respectively) and dose dependent (ID50 congruent to 10(-12) M). From these data we concluded that TGF beta acted rapidly on sites of OAC cell acute responses to stimulation by ACTH before and after the production of cAMP. Pregnenolone production in these cells was not inhibited by TGF beta when steroid production was stimulated on the addition of the readily permeable cholesterol derivative, 22 R-hydroxycholesterol. Thus, the rapid effect on OAC cells was manifest by TGF beta action on the utilization of cellular pools of cholesterol for the acute stimulation of steroid formation and not by direct action on the cholesterol side-chain cleavage enzyme. In addition, cells stimulated with ACTH in the absence or presence of lipoproteins (for up to 36 h) were susceptible to the inhibitory action of TGF beta. Taken together, these data amplify the pleiotropic actions of TGF beta on adrenocortical cell function and demonstrate that one acute action of TGF beta is on the utilization of endogenous supplies of cholesterol for steroid production.

The effect of cytochalasin D on steroid production and stress fiber organization in cultured bovine adrenocortical cells

The effects of cytochalasin D on basal and adrenocorticotropin (ACTH)-stimulated steroid production as well as stress fiber organization by bovine adrenocortical (BAC) cells grown in monolayer culture have been investigated. Corticosteroid and pregnenolone release was determined by use of radioimmunoassay. The addition of ACTH (1 nM) produced a 30-fold increase in steroid release (principally corticosterone) in a 2 h period. Cytochalasin D (1-10 microM) had no effect on ACTH-stimulated release of corticosteroids. Basal steroid release was elevated by cytochalasin D with lower concentrations being more effective. In addition, hormonal stimulation of pregnenolone formation, the initial step in cholesterol metabolism leading to corticosteroid production, was also unaffected by cytochalasin D (1-50 microM) addition. Observation of stress fibers by fluorescence microscopy using the probe NDB - phalladin revealed that cytochalasin D (10 and 50 microM) caused an aggregation of actin-containing filaments into stellate foci within the cytoplasm. This was confirmed by electron microscopic examination of the cells. ACTH, however, had no observable effect on stress fiber organization or cell morphology. These results differ from the inhibitory effect of cytochalasin on steroid production observed using Y-1 adrenal tumor cells and rat adrenal cells in primary culture. BAC cells in contrast to Y-1 adrenal tumor cells differ in non-esterified cholesterol content and their morphological response to ACTH treatment. We suggest that these differences may influence the lack of inhibition of cytochalasin D on ACTH-stimulated steroid release.

Expression of P-450 enzyme activities in heterologous cells by transfection.

The product of the A gene of simian virus 40 (SV40), T antigen, is synthesized and accumulates in the nucleus of infected cells. This regulartory protein acts to initiate viral DNA replication as well as regulating SV40 gene transcription (Tjian 1981). Following transformation of CV-1 cells with an origin-defective mutant of SV40, the resultant monkey kidney cell line (called COS 1) produces T antigen (Gluzman 1981). Transfection of COS 1 cells with plasmid vectors which contain an SV40 origin of replication leads to replication of the plasmid DNA under the influence of T antigen. Accordingly the number of plasmid vectors is amplified in transfected COS 1 cells and the amplified plasmid DNA can then be transcribed leading to production of relatively large quantities of RNA derived from the plasmid. This RNA can, of course, be translated by the endogenous protein synthetic machinery in the COS 1 cells. When the plasmid vector contains a cDNA insert encoding a specific protein, readily detectable quantities of the protein encoded by the insert can be produced in COS 1 cells. A convenient vector for such expression studies is the pcD vector constructed by Okayama and Berg (1982) which contains an SV40 origin of replication and SV40 promotor sequence. A similar vector (pSVL) can be purchased from Pharmacia. pSVL contains a limited multiple cloning site which facilitates insertion of different cDNAs into this expression vector.

“Designer Membranes”: Construction of a Cell Containing Multiple Membrane-Bound Cytochromes P450

Publisher Summary This chapter discusses designer membranes and its construction of a cell containing multiple membrane-bound cytochromes p450. The techniques of molecular biology offer the opportunity to introduce foreign DNA into naive cells to express enzymatically active proteins that can be tested in situ . Indeed, one is able to introduce simultaneously the DNAs for a number of different soluble and membrane-bound proteins, and thereby construct a functional metabolic pathway in a cell not programmed for such activities. Cytochrome P450 is best characterized as the catalyst for many monooxygenase—mixed function oxidase—reactions. For these reactions, a P450 serves as the central agent for the binding of a molecule of substrate, the acceptance of reducing equivalents provided by electrons transferred from reduced pyridine nucleotide via an abbreviated electron transport chain, and the activation of a molecule of molecular oxygen. The use of heterologous expression of enzymes, in particular membrane-bound enzymes, at present offers the possibility to contribute new types of data needed for the better understanding the functioning of enzymes in the environment of a cell.