Characterisation of the muscarinic receptor subtype M3 in the bovine zona fasciculata-reticularis cells by receptor binding, mRNA and functional studies.
In the present work we have investigated which muscarinic (M) receptor subtype is responsible for the steroidogenic effect of muscarinic agonists in bovine zona fasciculata-reticularis (ZFR) cells in culture. Radioligand binding studies using the muscarinic antagonist [(3)H]quinuclidinyl benzilate ([(3)H]QNB) demonstrated binding sites of high affinity (K(d)=0.45 nM) and low capacity ( approximately 8000 sites/cell). Pharmacological characterisation of muscarinic receptors was assessed by evaluating the effects of the M(3)>M(1)>>M(2) antagonist 4-DAMP (4-diphenylacetyl-N-methylpiperidine) and the M(1)=M(4)> M(3)>>M(2) antagonist pirenzepine on the binding of [(3)H]QNB and carbachol-induced cortisol production. For both parameters, the potency of 4-DAMP was about two orders of magnitude higher than that of pirenzepine. Reverse transcriptase (RT)-PCR analysis of bovine ZFR mRNAs using specific primers for M(2), M(3) and M(4) receptors revealed the expression of only M(3) mRNA. Moreover, carbachol significantly stimulated inositol phosphate accumulation, but had no inhibitory effect on basal or ACTH-induced cAMP production. Indeed, carbachol potentiated ACTH-induced cAMP production and this effect was, in part, mediated through protein kinase C. Lastly, neomycin, an inhibitor of phosphoinositide turnover, significantly attenuated carbachol-evoked cortisol production. Thus, pharmacological, biochemical and mRNA studies indicate that the M(3) receptor subtype is responsible for the biological effects of muscarinic agonists in bovine ZFR cells. (+info
Maturational changes in CYP2D16 expression and xenobiotic metabolism in adrenal glands from male and female guinea pigs.
CYP2D16 is expressed at high levels in the zona reticularis (ZR) of guinea pig adrenal glands and contributes to adrenal metabolism of xenobiotics. Studies were done to evaluate the effects of age and gender on adrenal CYP2D16 expression and xenobiotic metabolism. In both male and female guinea pigs at 1, 7, 14, or 30 weeks of age, in situ hybridization and immunohistochemistry confirmed that CYP2D16 was highly localized to the ZR of the adrenal gland. The steroidogenic P450 isozyme, CYP17, by contrast, was expressed in both the zona fasciculata and ZR. The intensity of CYP2D16 staining was not age- or gender-dependent. However, the proportion of each adrenal gland comprised by ZR and thus expressing CYP2D16 increased with aging in both sexes and was greater in males than in females. The rates of metabolism of bufuralol, a CYP2D-selective substrate, by adrenal microsomal preparations generally correlated with the amount of ZR (and CYP2D16) in the gland. Thus, adrenal xenobiotic-metabolizing activities were greater in males than in females at all ages and increased with aging in males. However, the rates of bufuralol metabolism declined in sexually mature females (14 weeks) from the levels found in prepubertal females (7 weeks) and then increased markedly in retired breeders (30 weeks), suggesting an inhibitory effect of estrogens on enzyme activity. The results indicate that the age and gender differences in adrenal CYP2D16 content are largely determined by differences in the size of the ZR rather than the concentrations of CYP2D16 within cells of the ZR. However, adrenal xenobiotic-metabolizing activities in females seem to be further modulated by an inhibitory effect of estrogens. (+info
Gap junction proteins and cell-cell communication in the three functional zones of the adrenal gland.
Mouse and monkey adrenal glands were used to study the relationships between gap junction protein expression, intercellular communication and adrenal zonation. Dye communication patterns were determined by incubating freshly excised and hemisected adrenal glands in Lucifer yellow, a gap junction permeable fluorescent dye. Immunohistochemical techniques were used to localize adrenal gap junction proteins. The combination of these two techniques permitted the correlation of gap junction proteins with dye transfer and hormone responses in specialized regions of the adrenal cortex. Lucifer yellow dye communication was most pronounced in the inner glucocorticoid/androgen-producing regions (zona fasciculata/zona reticularis), but was virtually absent in the outer mainly mineralocorticoid-producing region (zona glomerulosa). This pattern of dye communication was coincident with immunohistochemical localization of the gap junction protein, alpha(1)Cx43. The variations in communication and alpha(1)Cx43 expression within the adrenal cortex are thought to be relevant to normal physiological regulation of the adrenal gland. (+info
The undifferentiated cell zone is a stem cell zone in adult rat adrenal cortex.
The adrenal cortex of mammals has been known to consist of three morphologically and functionally distinct zones, i.e. the zona glomerulosa (zG), the zona fasciculata (zF) and the zona reticularis (zR), each of which secretes a specific corticosteroid different from those produced by the other two zones. We found previously, however, that an additional zone existed between zG and zF of adult rat adrenal cortex and that the cells in that zone were in a functionally undifferentiated state as an adrenocortical cell [Endocrinology 135, (1994) 431]: they were incapable of synthesizing highly active forms of corticosteroids, such as aldosterone and corticosterone, although they could produce their precursors. Hence, we named the zone as the undifferentiated cell zone (zU) of the adrenal cortex. Here we show that zU and its surroundings, i.e. the innermost portion of zG and the outermost portion of zF are the sites for cell replication in adult rat adrenal cortex and that the cells raised there migrate to other regions. Such cell replications in this region occur regardless of physiological conditions, such as the rise and fall of hormonal stimuli and circadian fluctuation of adrenocortical activities. On the bases of these and other findings previously described, we propose that zU is the stem cell zone of the adult rat adrenal cortex. Our recent success in isolating novel cell lines, which display an undifferentiated phenotype similar to that of zU cells, could facilitate the exploration of molecular mechanisms for the differentiation and development of the adrenocortical cells. (+info
Zonal expression of dickkopf-3 and components of the Wnt signalling pathways in the human adrenal cortex.
The mechanisms underlying the differentiation of the adrenal cortex into zones are unclear. Microarray studies on RNA from microdissected zona reticularis (ZR) and zona fasciculata/zona glomerulosa (ZF/ZG) derived from adult human adrenal glands showed that a gene of the dickkopf family (DKK), DKK3, is differentially expressed in the zones. The Dickkopf proteins are morphogens involved in Wnt signalling. Northern blotting showed higher DKK3 transcript levels in ZF/ZG than ZR samples. In situ hybridization on adult human adrenal gland sections showed that DKK3 expression was much higher in the ZG than in the ZF or ZR. DKK3 expression was also higher in the medulla. We screened for expression of other members of the DKK family and the related Wingless-type mouse mammary tumor virus integration site gene family (WNT), frizzled (FZD), and dishevelled (DVL) gene families. Among dickkopf family members, only DKK3 was expressed at a detectable level in both human and mouse adrenocortical RNA samples. Consistent with previously published data on the effects of Wnt4 gene disruption in the mouse, we found only WNT4 expression within the WNT family in both human and mouse RNA. Northern blotting showed that WNT4 was expressed at a higher level in ZF/ZG cells than in ZR. The higher level of DKK3 and WNT4 expression in ZF/ZG cells was confirmed by real-time PCR. In the frizzled and dishevelled families we found FZD1, FZD2 and DVL3 transcripts in human adrenocortical RNA, and FZD2 and DVL3 in mouse adrenocortical RNA. These data show that a variety of genes of the Wnt signalling pathways are expressed in the adrenal cortex. The zonal distribution of DKK3 expression suggests that it could be involved in zonal differentiation or growth. (+info
Expression of activin/inhibin signaling components in the human adrenal gland and the effects of activins and inhibins on adrenocortical steroidogenesis and apoptosis.
Activins and inhibins are structurally related glycoprotein hormones modulating pituitary FSH secretion and gonadal steroidogenesis. Activins and inhibins are also produced in the adrenal cortex where their physiological role is poorly known. Hormonally active human adrenocortical tumors express and secrete inhibins, while in mice adrenal inhibins may function as tumor suppressors. To clarify the significance of adrenal activins and inhibins we investigated the localization of activin/inhibin signaling components in the adrenal gland, and the effects of activins and inhibins on adrenocortical steroidogenesis and apoptosis. Activin receptor type II/IIB and IB, activin signal transduction proteins Smad2/3, and inhibin receptor betaglycan were expressed throughout the adrenal cortex, whereas Smad4 expression was seen mainly in the zona reticularis and the innermost zona fasciculata as evaluated by immunohistochemistry. Treatment of cultured adrenocortical carcinoma NCI-H295R cells with activin A inhibited steroidogenic acute regulatory protein and 17alpha-hydroxylase/17,20-lyase mRNA accumulation as evaluated by the Northern blot technique, and decreased cortisol, androstenedione, dehydroepiandrosterone and dehydroepiandrosterone sulfate secretion as determined by specific enzyme immunoassays. Activin A increased apoptosis as measured by a terminal deoxynucleotidyl transferase in situ apoptosis detection method. Inhibins had no effect on steroidogenesis or apoptosis. In summary, activin/inhibin signaling components are coexpressed in the zona reticularis and the innermost zona fasciculata indicating full signaling potential for adrenal activins and inhibins in these layers. Activin inhibits steroidogenic enzyme gene expression and steroid secretion, and increases apoptosis in human adrenocortical cells. Thus, the activin-inhibin system may have a significant role in the regulation of glucocorticoid and androgen production and apoptotic cell death in the human adrenal cortex. (+info
The morphological and functional basis of the new conception about the suprarenal cortex regeneration.
The exhaustion of main intracellular and cellular reserves of the regeneration in suprarenal cortex under the influence of exogenous hyperthermia activates the supplementary sources of cellular regeneration. First type of low differentiated cortical cells appears under the outer connective tissue capsule on approximately the 7(th) day of hyperthermia. These cells proliferate and some of them migrate along the cortical blood capillaries towards the deep portion of zona fasciculate. They differentiate into the typical cortical cells (spongiocytes) of zona glomerulosa and zona fasciculate according to their location. Second type of low differentiated cortical cells appears through one circadian-septal period (during 7 days) near the inner connective tissue capsule that separate cortex from the medulla. This cell type gradually differentiates into the typical cortical cells of zona reticularis. Stem cortical cells were found in suprarenal cortex. They did not have specific morphological signs and could synthesize nucleus DNA. (+info
Inhibitory effects of digoxin and digitoxin on corticosterone production in rat zona fasciculata-reticularis cells.
The aim of the present study was to investigate the direct effects and action mechanisms of digitalis on the production of corticosterone in rat adrenocortical cells. Male rats were challenged with digoxin (1 microg ml(-1) kg(-1)) in the presence or absence of adrenocorticotropin (ACTH, 5 microg ml(-1) kg(-1)) administered by intravenous injection to the right jugular vein. Blood samples were collected at 0, 30, 60, and 120 min following the challenge. The concentration of corticosterone in the rat plasma samples was measured by radioimmunoassay. Zona fasciculata-reticularis (ZFR) cells in male rats were prepared and then incubated with or without digoxin or digitoxin in the presence or absence of ACTH (10(-9) m), forskolin (10(-7) m), 8-bromo-cyclic 3' : 5'-adenosine monophosphate (10(-4) m), cyclopiazonic acid (CPA, 10(-5) m), trilostane (10(-6) m), 25-OH-cholesterol (10(-5) m), pregnenolone (10(-5) m), progesterone (10(-5) m), or deoxycorticosterone (10(-5) m) at 37 degrees C for 1 h before collection of the media. Corticosterone or pregnenolone levels were measured by radioimmunoassay. A single injection of digoxin did not alter the basal level of plasma corticosterone, but did inhibit the level of plasma corticosterone released in response to ACTH in vivo. Administration of digoxin or digitoxin decreased both spontaneous and ACTH-stimulated release of corticosterone in vitro. Digoxin (10(-7)-10(-5) m) and digitoxin (10(-7)-10(-5) m), but not ouabain (10(-7)-10(-5) m), dose-dependently inhibited corticosterone production in response to forskolin and 8-Br-cyclic AMP in rat ZFR cells. Both digoxin (10(-6)-10(-5) m) and digitoxin (10(-6)-10(-5) m) attenuated corticosterone production in response to CPA. Digoxin (10(-5) m) or digitoxin (10(-5) m) inhibited cytochrome P450 side-chain cleavage enzyme (cytochrome P450scc) activity (catalyses conversion of cholesterol to pregnenolone in the presence of trilostane) in rat ZFR cells. The enzyme activity of 11 beta-hydroxylase (catalyses conversion of deoxycorticosterone to corticosterone) in ZFR cells was also inhibited by the administration of digoxin (10(-5) m) or digitoxin (10(-5) m).10 These results together suggest that digoxin and digitoxin decrease the release of corticosterone by acting directly on ZFR cells via a Na+, K+-ATPase-independent mechanism involving the inhibition of the activities of adenylyl cyclase, cytochrome P450scc and 11 beta-hydroxylase, as well as the functioning of cyclic AMP and intracellular calcium. (+info