Zone-specific cell proliferation during compensatory adrenal growth in rats.

Compensatory adrenal growth after unilateral adrenalectomy (ULA) leads to adrenocortical hyperplasia. Because zonal growth contributions are not clear, we characterized the phenotype of cortical cells that proliferate using immunofluorescence histochemistry and zone-specific cell counting. Rats underwent ULA, sham adrenalectomy (sham), or no surgery and were killed at 2 or 5 days. Adrenals were weighed and sections immunostained for Ki67 (proliferation), cytochrome P-450 aldosterone synthase (P450aldo, glomerulosa), and cytochrome P-450 11beta-hydroxylase (P45011beta, fasciculata). Unbiased stereology was used to count proliferating glomerulosa and fasciculata cells. Adrenal weight increased after ULA compared with sham and no surgery at both time points, and there was no difference between sham and no surgery. However, either ULA or sham increased Ki67-positive cells in the outer fasciculata at both time points compared with no surgery. Outer fasciculata-restricted proliferation is thus associated with adrenal weight gain in ULA but not sham. Experiment repetition using proliferating cell nuclear antigen and bromodeoxyuridine showed similar results. After ULA, adrenal DNA, RNA, and protein increased at both time points, whereas after sham, only adrenal DNA increased at 2 days. Compensatory growth thus results from hyperplasia and hypertrophy, whereas sham induces only a transient adrenal hyperplasia. Dexamethasone pretreatment prevented the increase in adrenal weight after ULA and blocked Ki67 labeling in the outer fasciculata but not zona glomerulosa in all groups. These results clearly show that the outer fasciculata is the primary adrenal zone responsible for compensatory growth, responding to steroid-suppressible stress signals that alone are ineffective in increasing adrenal mass.

SF-1, DAX-1, and acd: molecular determinants of adrenocortical growth and steroidogenesis.

The formation of the adrenal cortex in humans is notable for the presence of two discrete zones, the fetal zone (FZ) which regresses soon after birth and the definitive zone (DZ) which gives rise to the classic steroidogenic zones of the adult cortex. Mice possess an analogous structure to the FZ referred to as the X-zone (XZ) which regresses at puberty in the male and during the first pregnancy in the female. Similar to the human FZ in X-linked Congenital Adrenal Hypoplasia caused by loss of function mutations in DAX-1 (Dosage-sensitive sex reversal-Adrenal hypoplasia congenita critical region on the X chromosome), the mouse XZ does not regress when DAX-1 is mutated. Only in humans with DAX-1 mutations, however, is the DZ small and hypofunctional. Patients and mice with SF-1 mutations have complete adrenal aplasia with absence of both the DZ and FZ/XZ. Lastly, the phenotype of the Autosomal Recessive Adrenocortical Dysplasia (acd) mouse is strikingly similar to human Miniature Adult Congenital Adrenal Hypoplasia, lacking an XZ/FZ and possessing a dysfunctional DZ. Current work has addressed the regulation of SF-1 and DAX-1 dependent adrenocortical growth and steroidogenesis in vivo utilizing mouse models of simple and combined SF-1 and DAX-1 deficiency. In addition, the model of compensatory adrenal growth in SF-1 haplo-insufficient mice has been applied to evaluate the potential role of SF-1 in adrenocortical proliferation. Additional efforts aim to positionally clone the acd gene, predicated on the hypothesis that it is a critical component of the adrenal developmental cascade.

ACTH inhibits the capsaicin-evoked release of CGRP from rat adrenal afferent nerves.

The adrenal cortex is innervated by afferent fibers that have been implicated in affecting cortical steroidogenesis. Modulation of neurotransmitter release from afferents may represent a regulatory system for the control of adrenal cortical function. The present studies validate an in vitro superfusion technique for adrenal capsules employing the drug capsaicin, which activates a subset of afferent fibers and induces the release of calcitonin gene-related peptide (CGRP). Capsaicin-evoked CGRP release from adrenal afferents was blocked by capsazepine, a competitive antagonist for the capsaicin receptor, or by removal of extracellular calcium. Exogenous ACTH prevented capsaicin-evoked CGRP release, elevated basal aldosterone release, and prevented capsaicin-induced reduction in aldosterone release. Immunolabeling for the recently cloned capsaicin vanilloid receptor 1 demonstrated its presence in adrenal nerves. These results show that in vitro superfusion of adrenal capsules can be used to characterize factors that modulate neurotransmitter release from adrenal afferents. Furthermore, the results suggest that activation of adrenal afferents in vivo may attenuate aldosterone steroidogenesis and that high levels of ACTH may prevent this phenomenon.

The effect of fetal hypoxia on adrenocortical function in the 7-day-old rat.

Fetal hypoxia in late gestation is a common cause of postnatal morbidity. The purpose of the present study was to evaluate adrenal function in vivo and in vitro in 7-d-old rat pups previously exposed to normoxia or hypoxia (12% O2) during the last 2-3 d of gestation. Seven-day-old rats exposed to fetal hypoxia had a small, but significant decrease in plasma aldosterone despite no decreases in plasma ACTH or renin activity. There was a small (approx 20%) but significant decrease in the aldosterone and corticosterone response to cAMP in vitro in dispersed cells from 7-d-old pups exposed to fetal hypoxia. The aldosterone, corticosterone, and cAMP response to ACTH, however, was not altered by prior fetal hypoxia. There was also no effect of fetal hypoxia on steroidogenic enzyme expression or zonal dimension in 7-d-old rats. We conclude that fetal hypoxia in late gestation results in a subtle decrease in cAMP-stimulated steroidogenesis. Fetal hypoxia appears to have minimal effects on subsequent adrenal function in the neonatal rat.

Rat adrenal transplants are reinnervated: an invalid model of denervated adrenal cortical tissue.

Adrenal autotransplantation is a widely used approach to investigate the potential for neural modulation of adrenal cortical function. It is believed that regenerating adrenal transplants are not reinnervated, thereby providing a model to investigate adrenal function in the absence of neural modulation. However, the hypothesis that adrenal transplants become reinnervated has not been directly tested. The purpose of the present study was to characterize the time course, extent, and nature of the reinnervation of the regenerating adrenal transplant and to assess whether the recovery of steroidogenic function and enzyme expression correlates temporally with the presence of innervation. Using immunohistofluorescent detection of tyrosine hydroxylase (TH), neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), and vasoactive intestinal peptide (VIP), the innervation of regenerating adrenals was assessed 14-30 days after transplantation of adrenal capsules beneath the kidney capsule in rats. Extensive reinnervation by TH-, NPY-, and VIP-positive fibres was present by 14 days after transplantation including regions of the adrenal capsule and cortex, with only minimal reinnervation by CGRP-positive fibres up to 30 days. TH- and NPY-positive chromaffin cells were also observed in the regenerating transplants. In addition, there was marked recovery of steroidogenic function and steroidogenic enzyme expression up to 30 days. The finding that nerve fibres are present in the transplants during the re-establishment of steroidogenic function and enzyme expression suggests that innervation may modulate the regeneration and functional recovery of adrenal transplants. In an attempt to prevent reinnervation of transplants, adrenal capsules were autotransplanted to denervated kidneys. Immunohistochemical analysis showed that, despite extensive denervation of the kidney tissue, the reinnervation and regeneration of the adrenal transplants still occurred. These data demonstrate the marked capacity of the regenerating adrenal to become reinnervated and reinforces the conclusion that adrenal transplants are an invalid model of denervated adrenal cortical tissue.

Hyperinnervation during adrenal regeneration influences the rate of functional recovery.

The rat adrenal cortex has the uncommon ability to demonstrate morphological and functional regeneration after injury-induced loss of cortical tissue. Peripheral nerves are involved in tissue regeneration and healing after injury, implying that nerves may also be involved in modulating the regeneration of the adrenal cortex. Studies were initiated to assess changes in adrenal innervation during cortical tissue regeneration subsequent to adrenal enucleation. Innervation of regenerating adrenals was assessed from 3 to 62 days postenucleation by immunohistofluorescent detection of neuronal markers for primary afferent, preganglionic sympathetic, and postganglionic sympathetic fibers. The regenerating adrenal contained few nerves at 3 days postenucleation, but became differentially innervated, with extensive innervation by nerve fibers positive for calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH), neuropeptide Y (NPY), and neuronal nitric oxide synthase (nNOS). In contrast, there was only minimal innervation by nerve fibers positive for vasoactive intestinal peptide. By 14 days postenucleation, the CGRP-, TH-, and NPY-positive innervation included areas of hyperinnervation in the capsule, cortex, and central inflammatory site of the regenerating gland. In addition, many chromaffin cells were present at all time points postenucleation. Quantification of the regenerating gland content of CGRP, norepinephrine, epinephrine, and nNOS verified the immunohistofluorescent observations. The period of extensive innervation correlated temporally with the time (3-30 days) during which the regenerating glands recovered steroidogenic function. Moreover, splanchnic nerve transection at the time of adrenal enucleation decreased the innervation by CGRP-positive and vesicular acetylcholine transporter-positive fibers and delayed regeneration. These results support the hypothesis that adrenal innervation modulates tissue regeneration and functional recovery of the enucleated adrenal gland.

The effect of hypoxia from birth on the regulation of aldosterone in the 7-day-old rat: plasma hormones, steroidogenesis in vitro, and steroidogenic enzyme messenger ribonucleic acid.

Adaptation to hypoxia in the neonate requires an appropriate adrenocortical response. The purpose of this study was to examine the adaptation of the aldosterone pathway in rat pups exposed to hypoxia in vivo from birth to 7 days of age. Neonatal rats (with their lactating dams) were exposed to normoxia (21% O2) or hypoxia (12% O2) continuously for 7 days from birth. Trunk blood was collected, and entire adrenal glands were processed from 7-day-old rats to study the activity of the steroidogenic pathway in dispersed cells and isolated mitochondria, for measurement of expression of the steroidogenic enzyme messenger RNAs (mRNAs) by RT-competitive PCR and in situ hybridization histochemistry, for measurement of zona glomerulosa width by immunohistofluorescent staining for P450c11AS protein, and for measurement of mitochondrial number and distribution by transmission electron microscopy. Exposure to hypoxia for 7 days from birth resulted in a marked increase in plasma ACTH, corticosterone, and aldosterone with no change in PRA. Aldosteronogenesis and P450c11AS activity were both augmented in dispersed cells; this effect was lost in isolated mitochondria (from entire adrenal glands) using a permeable substrate for P450c11AS. There was no significant effect of hypoxia on expression of the steroidogenic enzyme mRNAs measured by RT-competitive PCR or in situ hybridization histochemistry. Finally, hypoxia had no effect on mitochondrial number or stereology as assessed by transmission electron microscopy or on zona glomerulosa width as assessed by staining for P450c11AS protein. We conclude that, as opposed to that in adults, hypoxia in the neonate results in an augmentation of aldosteronogenesis. This effect is not accounted for by a change in steroidogenic enzyme mRNA expression, zona glomerulosa width (i.e. hyperplasia), or mitochondrial number or distribution. This functional augmentation of aldosteronogenesis may be due to a change in mitochondrial permeability to steroid substrates and/or the effect of cytosolic factors that control mitochondrial steroidogenesis.

Changes in the glomerulosa cell phenotype during adrenal regeneration in rats.

In situ hybridization was used to examine cellular differentiation during rat adrenal regeneration, defining zona glomerulosa [cytochrome P-450 aldosterone synthase (P-450aldo) mRNA positive], zona fasciculata [cytochrome P-450 11beta-hydroxylase (P-45011beta) mRNA positive], or zona intermedia [negative for both but 3beta-hydroxysteroid dehydrogenase (3beta-HSD) mRNA positive]. After unilateral adrenal enucleation with contralateral adrenalectomy (ULE/ULA), the expression of all mRNA was reduced at 2 days. From 5 to 10 days, P-45011beta and 3beta-HSD mRNA increased while P-450aldo remained low; at 20 days, all mRNA were increased. From 2 to 10 days, cells adjacent to the capsule showed intermedia cell differentiation; by 20 days, the subcapsular glomerulosa cells reappeared. This suggests that after enucleation the glomerulosa dedifferentiates to zona intermedia. The experiment was repeated in rats where the postenucleation ACTH rise was prevented. Rats underwent ULE with sham ULA (ULE/SULA) or ULE/SULA with ACTH treatment. Adrenals from ULE/SULA rats expressed increased P-450aldo mRNA at 10 days and reduced P-45011beta mRNA and adrenal weight at 30 days. ACTH treatment reversed the pattern toward that seen in ULE/ULA. These findings show that the enucleation-induced dedifferentitation of the glomerulosa cell may result in part from elevated plasma ACTH and that prevention of dedifferentiation may result in impaired regeneration.

Development of adrenal zonation in fetal rats defined by expression of aldosterone synthase and 11beta-hydroxylase.

The adult rat adrenal cortex is comprised of three concentric steroidogenic zones that are morphologically and functionally distinguishable: the zona glomerulosa, zona intermedia, and the zona fasciculata/reticularis. Expression of the zone-specific steroidogenic enzymes, cytochrome P450 aldosterone synthase (P450aldo), and P450 11beta hydroxylase (P45011beta), produced by the zona glomerulosa and zona fasciculata/reticularis, respectively, can be used to define the adrenal cortical cell phenotype of these two zones. In this study, immunohistochemistry and in situ hybridization were used to determine the ontogeny of expression of P450aldo and P45011beta to monitor the pattern of development of the rat adrenal cortex. RIA was used to measure adrenal content of aldosterone and corticosterone, the resulting products of the two enzymatic pathways. Double immunofluorescent staining for both enzymes at gestational day 16 (E16) showed P45011beta protein expressed in cells distributed throughout most of the adrenal intermixed with a separate, but smaller, population of cells expressing P450aldo protein. Whereas expression of P45011beta protein retained a similar pattern of distribution from E16 to adulthood (ignoring distribution of SA-1 positive, presumptive medullary cells), P450aldo protein changed its pattern of distribution by E19, becoming localized in a discontinuous ring of cells adjacent to the capsule. By postnatal day 1, P450aldo protein distribution was similar to that observed in adult glands; P450aldo-positive cells formed a continuous zone underlying the capsule. In situ hybridization showed that the pattern of P45011beta messenger RNA expression paralleled protein expression at all times, whereas P450aldo messenger RNA paralleled protein at E19 and after, but was undetectable before E19. However, adrenal aldosterone and corticosterone, as measured by RIA, were detected by E16, supporting the functional capacity of both phenotypes for all ages studied. These data suggest that the development of the adrenal zona glomerulosa occurs in two distinct phases; initial expression of the glomerulosa phenotype in scattered cells of the inner cortex before E17, followed by a change in distribution to the outer cortex between E17 and E19. It is hypothesized that this change in distribution occurs via cell differentiation, rather than cell migration, and that a possible regulator of these events is the fetal renin-angiotensin system.

Functional innervation of the adrenal cortex by the splanchnic nerve.

Secretion of steroid hormones by the adrenal cortex is required to maintain whole body homeostasis; that is the ability to maintain blood pressure and volume, carbohydrate, protein and fat metabolism and immune and nervous system function within normal limits is dependent on adrenocortical hormones. The premise of this report is that autonomic-endocrine interactions occurring in the adrenal cortex are required for normal control of steroid secretion. Under non-stress conditions when reduced steroid secretion is required, splanchnic neural activity appears to be inhibitory, whereas during stress conditions when elevated steroid secretion is necessary, neural activity is excitatory. The capacity for innervation to produce both inhibitory and excitatory effects suggests that neural input must be encoded differentially; encoding could be dependent on the neurotransmitter released or on the intra-adrenal target affected. Neural input could act directly at the adrenal cell to affect steroidogenesis or act indirectly by changing adrenal blood flow. An index of the role of innervation has been obtained by assessing adrenal corticosteroid secretion after splanchnicectomy, severing the thoracic splanchnic nerve which is the major source of innervation of the adrenal gland. This approach has resulted in alterations in corticosteroid secretion under non-stress and stress conditions, but in many cases has demonstrated no profound effect on in vivo steroidogenesis. It is likely that splanchnicectomy results in variable secretory responses in part due to the multiplicity of adrenal neurotransmitter systems that are regulated by the splanchnic nerve. Splanchnicectomy alters multiple neurotransmitters at different adrenal sites. Splanchnic innervation acts as an extra-ACTH mechanism in the control of adrenal corticosteroid secretion, yet further elucidation of the physiological conditions under which splanchnic neural activity affects function is clearly warranted.

Ontogeny of innervation of rat and ovine fetal adrenals.

The formation of adrenocortical zonation occurs in rats during late gestation. Since adult cortical function is modulated by neural mediators, it is possible that the development of differentiated function is dependent on cortical innervation. The goal of this study was to compare the pattern and timing of rodent and ovine adrenal innervation during late organogenesis by staining with antibodies directed against the neuropeptides vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and neuropeptide tyrosine (NPY) and the catecholamine biosynthetic enzyme, tyrosine hydroxylase (TOH). Rat adrenals were collected from fetal days 17-21 (term=21 days) and ovine adrenals from fetal days 101-136 (term=145 days). Adrenals were fixed, cryosectioned at 100 microns and immunostained using Cy3-conjugated secondary antibodies. In both species, staining of VIP, CGRP, NPY and TOH fibers was observed in the capsule and subcapsular layers of the cortex during gestation. In late gestation, VIP- and NPY-positive ganglions cells were observed near the medulla extending processes toward the outer cortex; in ovine adrenals, fibers from ganglion cells appeared to surround nests of outer cortical (presumably, zona glomerulosa) cells. These data show that phenotypically distinct neural elements appear at different stages of adrenocortical development. The presence of neural elements in contact with adrenal cortical cells supports the possibility for neural control of adrenocortical development.

Insulin is degraded extracellularly in wounds by insulin-degrading enzyme (EC 3.4.24.56).

The exact mechanism by which insulin reverses impaired wound healing is unknown. Previous investigators have shown that insulin is degraded in experimental wounds, suggesting that the action of insulin may be locally modified. The following study corroborates these findings and identifies the major proteinase responsible for insulin degradation in wound fluid (WF). Adult male Fisher rats were wounded by subcutaneous implantation of polyvinyl alcohol sponges while under pentobarbital sodium anesthesia. WF and serum were collected on 1, 5, 10, and 14 days postinjury. Decreased insulin concentration in late WF correlated with an increased insulin-degrading activity. Multiple proteinases appear to participate in the overall degradation of insulin in WF. However, the primary enzyme responsible for insulin degradation in WF was characterized by immunoprecipitation and immunoblotting and identified as the neutral thiol-dependent metalloproteinase, insulin-degrading enzyme (EC 3.4.24.56). Exogenous steroid administration caused a decrease in WF insulin-degrading activity. Glucagon and adrenocorticotrophin degradation was also observed, whereas minimal degradation of insulin-like growth factors I and II and epidermal growth factor was detected in WF. The ability to extracellularly degrade insulin may represent a unique mechanism for the regulation of this hormone's role in healing wounds.

Splanchnicotomy increases adrenal sensitivity to ACTH in nonstressed rats.

Awake rats demonstrate an ultradian rhythm in adrenal secretion of corticosterone. Splanchnic denervation in unstressed rats increases the frequency of corticosterone pulses, revealing an inhibitory function of adrenal innervation. In contrast, one day after surgical stress, adrenal denervation reduces adrenal pulsatility, suggesting a stimulatory function of adrenal innervation. To test whether neural modulation of pulsatile secretion was due to a direct effect of the splanchnic nerve on adrenal sensitivity to adrenocorticotropic hormone (ACTH), rats treated with dexamethasone were administered repetitive pulses of ACTH, and the amplitude of corticosterone responses was determined. Intact or control (C) and splanchnicotomized (SPLNX) rats were tested at 2 or 5 days after surgery. Five days after surgery, adrenal responsiveness in C animals was reduced compared with SPLNX animals. However, no differences were seen 2 days after surgery. To determine whether the reduction in adrenal responsiveness involved a cellular or organ level mechanism, dispersed adrenal cortical cells isolated from intact or denervated adrenal glands were stimulated with ACTH, and corticosterone secretion was determined. Consistent with in vivo results, denervation increased the responsiveness of adrenal cells obtained 5, but not 1 or 2, days after surgery. These findings support a neurally mediated inhibition of adrenal sensitivity to ACTH in unstressed rats.

Differential gene expression of cytochrome P450 11beta-hydroxylase in rat adrenal cortex after in vivo activation.

In situ hybridization histochemistry was used to monitor the expression of 3beta-hydroxysteroid dehydrogenase, delta4-isomerase (3betaHSD) and cytochrome P450 11beta-hydroxylase (P45011beta) messenger RNA (mRNA) in adult rat adrenals after stimulation in vivo. In Exp 1, adrenals were collected from rats injected with saline or ACTH for 1, 2, 3, or 4 days. Adrenal sections from saline-treated rats showed uniform expression of 3betaHSD mRNA that extended from the adrenal capsule to the medullary border. In contrast, P45011beta mRNA showed high levels in the outer fasciculata and low levels in the inner fasciculata/reticularis. In response to ACTH, the integrated density of 3betaHSD hybridization did not increase until 4 days. The integrated density of P45011beta hybridization increased in ACTH-treated rats between 1-4 days due to increased hybridization in the inner fasciculata/reticularis. In Exp 2, rats were treated with ACTH or saline, and adrenals were harvested at 4, 8, or 24 h. The hybridization density of 3betaHSD did not change after ACTH or saline injection. Increased expression of P45011beta mRNA was observed at 4 and 8 h, but not 24 h post-ACTH. In Exp 3, to determine the response to acute stress, adrenals were collected from rats 24 h after surgical laparotomy. The integrated density of 3betaHSD labeling did not change, whereas both hybridization area and mean density of P45011beta increased. Increased expression of P45011beta mRNA was observed in the inner fasciculata similar to that observed after ACTH injection. In addition, adrenal cells were more responsive to ACTH in vitro after surgical stress. These results suggest that the rat adrenal cortex can respond to acute stress by up-regulation of the expression of steroidogenic enzyme genes and that this occurs in part by increasing the number of cells actively expressing P45011beta mRNA. The adrenal response after stress most likely results at least in part from stimulation by ACTH. These findings suggest that changes in adrenal steroidogenesis in response to ACTH may result from recruitment of steroidogenic cells to synthesize and secrete corticosteroids.

Water deprivation and rat adrenal mRNAs for tyrosine hydroxylase and the norepinephrine transporter.

Experiments were performed in rats to test the hypothesis that adrenal mRNA levels of tyrosine hydroxylase (TH) and the norepinephrine transporter (NET) would be modified by water deprivation via activation of the sympathetic nervous system. TH and NET mRNA levels were measured using the ribonuclease protection assay. Adrenal TH mRNA was higher (P < 0.001) in water-deprived (921 +/- 39 fg/microgram total RNA) compared with the water-replete rats (657 +/- 45 fg/microgram total RNA). In contrast, water deprivation decreased (P < 0.01) adrenal NET mRNA levels (275 +/- 66 vs. 433 +/- 63 fg/microgram total RNA). The dehydration-induced increase in TH mRNA was prevented by prior splanchnicectomy, but the decrease in NET mRNA was produced even in the absence of adrenal nerves. Water deprivation also increased (P < 0.05) plasma adrenocorticotropic hormone (84 +/- 16 vs. 42 +/- 14 pg/ml) and corticosterone (358 +/- 87 vs. 44 +/- 15 ng/ml) levels. Interestingly, the corticosterone response was reduced (P < 0.05) by unilateral adrenal denervation. These results suggest that water deprivation increases both adrenal medullary and adrenocortical activity at least in part by stimulation of sympathetic nerve activity.

Phenotype of proliferating cells stimulated during compensatory adrenal growth.

The phenotype of the proliferating cells during adrenocortical growth has remained controversial although glomerulosa, fasciculata and intermediate zone cells have all been considered possible candidates. This was due in part to the inability to identify specific adrenocortical cell types in comparing different types of growth. In the present studies, using immunocytochemical localization of cytochrome P450 aldosterone synthase (P450aldo) and cytochrome P450 11 beta-hydroxylase (P45011 beta) to identify adrenocortical cell phenotypes as well as Ki-67 to label proliferating cells, we have investigated the phenotype of the proliferating cells in the compensatory adrenal growth response to unilateral adrenalectomy. Between 24 and 96 hrs after unilateral adrenalectomy, most Ki-67(+) nuclei were found in the outermost region of the fasciculata, as defined by P45011 beta immunoreactive cells. Few Ki-67(+) nuclei were found in the glomerulosa, defined by P450aldo cells or in the z intermedia, identified by the absence of both P450aldo and P45011 beta. To test which cell type is activated by unilateral adrenalectomy, we altered the phenotypic configuration of the adrenal cortex; rats were placed on a low Na+ diet for three weeks, resulting in a marked expansion of the number of P450aldo(+) cells. An abundance of proliferating cells was identified primarily in the expanded glomerulosa, but not in the intermedia or fasciculata. In contrast, the proliferation associated with compensatory growth in these low Na+ rats, was localized primarily in the outer P45011 beta(+) zone. These findings suggest that the phenotype of the proliferating cell is specific to the growth promoting stimulus.

Phenotypic changes and proliferation of adrenocortical cells during adrenal regeneration in rats.

Adrenal regeneration after enucleation includes both cell proliferation and differentiation, but the phenotype of the proliferating cell remains controversial. Immunoperoxidase localization of cytochrome P450 aldosterone synthase (P450aldo) and cytochrome P450 11 beta-hydroxylase (P45011 beta) and of Ki-67 was used to identify adrenocortical cell phenotypes and proliferating cells, respectively. Comparisons were made between regenerating and intact adrenals collected from rats on low or normal Na+ diets. During the first week after enucleation, P45011 beta was expressed reflecting the presence of fasciculata cells; however, P450aldo was detected only in adrenals from low Na+ rats. On normal and low Na+, glomerulosa cells were replaced by intermedia cells, whereas on low Na+, glomerulosa cells were replaced by fasciculata cells. Proliferation was observed only in glomerulosa and fasciculata, but not intermedia cells. These findings suggest that the expression of the glomerulosa cell phenotype is decreased in the early stages of adrenal regeneration, that differentiation from a glomerulosa to an intermediate or fasciculata cell phenotype is influenced by low Na+ and that glomerulosa and fasciculata cells proliferate in response to enucleation.