Long-term dietary lipid regimen alters adrenocortical function at the cellular level.

Evidence indicates that dietary lipids influence adrenocortical function. In the present study, weanling rats were fed isocaloric synthetic diets for 6 and 12 months that contained 10% of one of the selected fatty acids as the predominant lipid: butter fat (high saturated, low polyunsaturated fat); olive oil (monounsaturated); corn oil (polyunsaturated); omega-3 ethyl ester mixture (long-chain polyunsaturates); elevated eicosapentaenoic acid; elevated docosahexaenoic acid. Adrenocortical cells derived from individual rats were evaluated for corticosterone and aldosterone responses to adrenocorticotropic hormone (ACTH). All comparisons were to the butter fat diet. Adrenocortical cell sensitivity to ACTH was not affected by the diets. However, there were differences in basal and maximal ACTH-induced corticosteroid production. Compared to the butter fat diet, the other diets variably decreased cellular corticosteroid production. Corticosterone and aldosterone production were affected similarly. The greatest decrease was most often seen with the omega-3 mixture diet (about -67%). At 6 months, the docosahexaenoic acid-elevated diet had selective suppressive actions on adrenocortical function whereas at 12 months, both docosahexaenoic and eicosahexaenoic acid-elevated diets had similar suppressive efficacies. The data indicate that a diet rich in high saturated, low polyunsaturated fat augments adrenocortical function and increasing the representation of long-chain unsaturated fatty acids suppresses adrenocortical function.

Lack of effects of atrazine on estrogen-responsive organs and circulating hormone concentrations in sexually immature female Japanese quail (Coturnix coturnix japonica).

The widely used herbicide, atrazine, has been reported to exhibit reproductive toxicity in rats and amphibians. The present studies investigate toxicity of atrazine in Japanese quail and its ability to influence reproduction in sexually immature females. Atrazine was administered in the diet at concentrations from 0.001 to 1000 ppm (approximately 109 mg kg-1 per day) or systemically via daily subcutaneous injections (1 and 10 mg kg-1) or Silastic implants. Atrazine did not cause overt toxicity in sexually immature female quail (no effects on change in body weight, feed intake, mortality or on circulating concentrations of the stress hormone, corticosterone). It was hypothesized that if atrazine were to have estrogenic activity or to enhance endogenous estrogen production, there would be marked increases in the weights of estrogen sensitive tissues including the oviduct, the liver and the ovary together with changes in gonadotropin secretion. However, atrazine had no effect on either liver or ovary weights. Atrazine in the diet increased oviduct weights at 0.1 and 1 ppm in some studies. These effects were not consistently observed and were not significant when data from studies were combined. Systemic administration of atrazine had no effect on oviduct weights. Dietary (concentrations from 0.001 to 1000 ppm) and systemically administered atrazine had no effect on circulating concentrations of luteinizing hormone (LH). The present studies provide evidence for a lack of general or reproductive toxicity of atrazine in birds.

Dietary protein restriction stress in the domestic chicken (Gallus gallus domesticus) induces remodeling of adrenal steroidogenic tissue that supports hyperfunction.

The stress of dietary protein restriction in the immature domestic turkey (Meleagris gallopavo) induces adrenal steroidogenic hypofunction that is associated with an alteration in the proportion of density-dependent subpopulations of steroidogenic cells within the adrenal gland. In contrast, when imposed on immature chickens, this nutritional stressor induces long-term enhancement of adrenal steroidogenic function. However, whether this alteration in function is accompanied by a remodeling of chicken adrenal steroidogenic tissue as in the turkey is not known. To address this question, immature cockerels (2 weeks old) were fed established isocaloric synthetic diets containing either 20% (control) or 8% (restriction) soy protein for 4 weeks. Adrenal glands were processed for the isolation of defined, density-separable, adrenal steroidogenic cell subpopulations: three low-density adrenal steroidogenic cell subpopulations [LDAC-1 (rho = 1.0285-1.0430 g/ml), LDAC-2 (rho = 1. 0430-1.0485 g/ml), and LDAC-3 (rho = 1.0485-1.0500 g/ml)] and one high-density subpopulation [HDAC (rho = 1.0510-1.0840 g/ml)]. The steroidogenic function of these cell subpopulations was assessed. Protein restriction consistently, but differentially, enhanced maximal ACTH-induced corticosterone production by the subpopulations: values of LDAC-1, -2, and -3 and HDAC from protein restricted birds were, respectively, 116, 43, 33, and 20% greater than those of corresponding cell subpopulations from control birds. However, it had contrasting influences on maximal ACTH-induced aldosterone production by the cell subpopulations. Whereas the value of LDAC-1 from protein-restricted birds was 70% greater than that from control birds, the values for LDAC-2 and -3 were not different from those of the control, and the value for HDAC was 22% less than that of the control. Protein restriction also altered the cell subpopulation composition of the adrenal gland: compared to control, it increased the proportion of LDAC-1 by 46% and decreased the proportion of LDAC-3 and HDAC by 34 and 20%, respectively. Thus, dietary protein restriction increased the proportion of cells (i.e., LDAC-1) having the greatest enhancement in corticosteroid production. This pattern of remodeling of chicken adrenal steroidogenic tissue in response to dietary protein restriction contrasts sharply with the pattern that occurs in another galliform species, the domestic turkey.

Remodeling of turkey adrenal steroidogenic tissue induced by dietary protein restriction: the potential role of cell death.

The present study focused on the cellular remodeling of steroidogenic tissue in the domestic turkey (Meleagris gallopavo) adrenal gland in response to dietary protein restriction stress. Immature male turkeys (1 week old) were fed isocaloric synthetic diets containing either 28% (control) or 8% (restriction) soy protein for 4 weeks. Adrenal glands were processed for the isolation of density- separable, visibly distinct adrenal steroidogenic cell subpopulations: three low-density subpopulations [LDAC-1 (rho = 1. 0350-1.0490 g/ml), LDAC-2 (rho = 1.0490-1.0570 g/ml), and LDAC-3 (rho = 1.0570-1.0585 g/ml)] and one high-density subpopulation [HDAC (rho = 1.0590-1.0720 g/ml)]. Dietary protein restriction increased the proportion of LDAC-3 and HDAC by 98 and 350%, respectively, and decreased LDAC-2 by 46%. LDAC-1 also showed signs of proportional decrement. To determine the role of cell death in this process, the potential for apoptosis was assessed in adrenal tissue and isolated adrenal steroidogenic cells using short-term culture followed by analysis of oligonucleosome formation. Basal, culture-triggered oligonucleosome formation of tissue and cells derived from protein-restricted birds was 80% greater than that of tissue and cells derived from control birds. This differential in apoptotic potential persisted with a variety of treatments, in vitro. Apoptotic potential was suppressed by human adrenocorticotropin and enhanced by angiotensin II (Ang II). The proapoptotic effect of Ang II (100 nM) with adrenal fragments was inhibited by the Ang II receptor antagonist [Sar(1), Ile(8)]ang II (10 microM) to below basal values (by about 60%), but the inhibition was surmountable by high concentrations (10 and 100 microM) of Ang II. The antagonist also attenuated basal, culture-triggered DNA fragmentation of tissue and cells, suggesting that at least part of the basal DNA fragmentation was due to intrinsically generated Ang II. Differences in apoptotic potential were also apparent with cell subpopulations. Compared to control subpopulations, protein restriction enhanced basal oligonucleosome formation in LDAC-1 and -2 by 38 and 122%, respectively, and reduced it in LDAC-3 and HDAC by 53 and 70%, respectively. These data suggest a role for apoptotic cell death in the remodeling of turkey adrenal steroidogenic tissue induced by dietary protein restriction. In addition, other data suggest that Ang II is an important regulator of adrenal steroidogenic cell turnover in the avian adrenal gland.

Concentration-dependent, biphasic effect of prostaglandins on avian corticosteroidogenesis in vitro.

Previous work with mammalian and frog adrenocortical tissue and cells indicates that prostaglandins (PGs) can directly stimulate corticosteroidogenesis. However, work with avian adrenal preparations is absent. Therefore, the present studies with isolated chicken (Gallus gallus domesticus) and turkey (Meleagris gallopavo) adrenal steroidogenic cells were conducted to determine whether PGs can directly influence avian corticosteroidogenesis as well. Cells (1 x 10(5) cells/ml) were incubated with a wide range of concentrations of PGs in the presence of indomethacin (1 microg/ml) (to attenuate endogenous PG production) and 1-methyl-3-isobutylxanthine (0.5 mM) [to preserve cyclic AMP (cAMP)] for 2 h. Corticosterone and cAMP production were measured by highly specific radioimmunoassay. PGI2 was without effect. With the exception of PGF2alpha, which had a slight stimulation in chicken but not in turkey cells, the influence of the other PGs on corticosterone production was biphasic. For the stimulatory phase (up to a concentration of 5 x 10(-5) M), there were prostanoid structural and avian species differences in both potency and efficacy of PGs. Overall, PGs were 11 times more potent in turkey cells than in chicken cells. However, the order of potency for stimulation was similar for both chicken and turkey cells: for chicken cells the order was PGE2 > PGE1 > PGA1 > PGB2 > PGB1 > PGF2alpha and for turkey cells it was PGE2 > PGE1 > PGA1 > PGB2 = PGB1. In contrast, PG efficacy for stimulation was greater for chicken cells. In addition, the orders of efficacy were different from the orders of potency. In chicken cells, the order of efficacy was PGE2 = PGA1 > PGE1 > PGB2 > PGB1 > PGF2alpha and for turkey cells it was PGB2 = PGE2 > PGA1 > PGE1 > PGB1. Because of the greater maximal corticosterone response over basal production of chicken cells to PGs, they were used to assess the interaction of PGs with ACTH and to examine more fully the inhibitory phase of PGs. Cells were incubated with PGs in the presence of threshold (2.5 x 10(-11) M), half-maximal (1 x 10(-10) M), and maximal (1 x 10(-7) M) steroidogenic concentrations of ACTH. With the exception of PGF2alpha, the average efficacy of PGs to elevate corticosterone was increased 55% by a threshold steroidogenic concentration of ACTH. However, with higher concentrations of ACTH, this enhancement of efficacy disappeared as did the stimulatory effect of some PGs. The results suggest that the steroidogenic actions of PGs and ACTH converge on the same pool of steroidogenic enzymes leading to corticosterone. At concentrations greater than 5 x 10(-5) M, several PGs (notably PGA1, PGA2, PGB1, and PGB2) inhibited both ACTH-induced and basal corticosterone production. PGA1 and PGA2 were the most potent inhibitors. Corticosterone and cAMP production were closely associated in the biphasic action of PGs, suggesting that the effect of PGs was mediated by the changing levels of intracellular cAMP. Collectively, these data suggest that PGs may be important modulators of corticosteroidogenesis in the avian adrenal gland.

Dietary protein restriction stress in the domestic fowl (Gallus gallus domesticus) alters adrenocorticotropin-transmembranous signaling and corticosterone negative feedback in adrenal steroidogenic cells.

Previous work with growing chickens (Gallus gallus domesticus) indicates that transient dietary protein restriction induces long-term enhancement of adrenal steroidogenic function in response to adrenocorticotropin (ACTH). The present study investigated two possible cellular functions mediating this enhanced response: (a) ACTH signal transduction and dissemination and (b) short-loop feedback inhibition of ACTH-induced corticosterone production by exogenous corticosterone. Cockerels (2 weeks old) were fed isocaloric synthetic diets containing either 20% (control) or 8% (restriction) soy protein for 4 weeks. Adrenal glands were processed for the isolation of adrenal steroidogenic cells nearly devoid of chromaffin cells ( approximately 90% adrenal steroidogenic cells). Results of experiments to assess signal transduction and dissemination indicated that protein restriction selectively enhanced ACTH-induced corticosterone production mediated by the cyclic AMP (cAMP)-dependent pathway. In addition, protein restriction substantially counteracted exogenous corticosterone-dependent inhibition of acute ACTH-induced corticosterone production (by 40.7% vs control). The proximal portion of the cAMP pathway seemed most affected by this stressor. Protein-restricted cells exhibited enhanced homologous sensitization to ACTH (136% greater than that of control cells) which appeared to be localized at a step(s) prior to or at the formation to cAMP. Also, maximal ACTH-induced cAMP production and sensitivity to ACTH in terms of cAMP production by protein-restricted cells were, respectively, 2.2 and 15.8 times those of control cells. However, variable results were obtained from other experiments designed to pinpoint the altered early steps in ACTH-transmembranous signaling. For example, with intact cells, cAMP responses to cholera toxin (CT) and forskolin (FSK) did not corroborate the results suggesting an augmentation of ACTH-signal transduction induced by protein restriction. Furthermore, basal and stimulatable (by ACTH, CT, FSK, and NaF) adenylyl cyclase activities from membranes from protein-restricted cells were, respectively, 47.2 and 40.2% less than those from control cells (normalized to 10(7) cell equivalents of crude membranes). Collectively, these findings suggest that protein restriction stress potentiates ACTH-induced corticosterone secretion by chicken adrenal steroidogenic cells in at least two ways: (1) on the proximal end, by modulating unknown factors which enhance cellular sensitivity to ACTH, ACTH receptor-adenylyl cyclase coupling, and adenylyl cyclase activity, and (2) on the distal end, by suppressing end-product corticosterone negative feedback, thus facilitating an increase in net corticosterone secretion.

Dietary protein restriction stress in the domestic turkey (Meleagris gallopavo) induces hypofunction and remodeling of adrenal steroidogenic tissue.

In the present study, we investigated the influence of dietary protein restriction stress on adrenal steroidogenic function of the domestic turkey. Immature male turkeys (2 weeks old) were fed isocaloric synthetic diets containing either 28% (control) or 8% (restriction) soy protein for 4 weeks. Trunk plasma was processed for the determination of adrenocorticotropin (ACTH), corticosterone, aldosterone, and total 3, 5, 3'-triiodothyronine (T3). In addition, adrenal glands were processed for the isolation of defined, density-separable, adrenal steroidogenic cell subpopulations: three low-density adrenal steroidogenic cell subpopulations [LDAC-1 (rho = 1.0350-1.0490 g/ml). LDAC-2 (rho = 1.0490-1.0570 g/ml), and LDAC 3 (rho = 1.0370-1.0585 g/ml)] and a high-density subpopulation [HDAC (rho = 1.0590-1.0720 g/ml)], and the steroidogenic function of these cell subpopulations was evaluated. Protein restriction did not influence plasma ACTH However, it increased relative adrenal weight (mg/100 g body wt) (+37.8%) and plasma corticosterone (+317%). By contrast, it depressed plasma aldosterone (-51.2%). In addition, it caused a modest depression in plasma T3 (-25.9%). At the cellular level, protein restriction induced panhypofunction. Basal corticosteroid (aldosterone and corticosterone) production values of LDAC-1, -2, and -3 and HDAC from protein-restricted birds were, respectively, 42.9, 47.9, 30.8, and 57.5% less than those of corresponding cell subpopulations from control birds. In addition, maximal corticosteroid production values of LDAC-1, -2, and -3 and HDAC from protein-restricted birds, in response to ACTH, angiotensin II (AngII), and 25-hydroxycholesterol support, were depressed by 56.8, 55.1, 22.7, and 42.9%, respectively. Interestingly, LDAC-3 was relatively refractory to the influence of this stressor. By contrast, there was the lack of a concentration-dependent aldosterone response of LDAC-1 and -2 to AngII with protein restriction. This was not due to a failure in cell function since aldosterone responses of these cell subpopulations to ACTH and to 25-hydroxycholesterol support were apparent. In addition, the concentration of AngII receptors of cell subpopulations from protein-restricted turkeys, if anything, was greater than that of cell subpopulations from control turkeys. Protein restriction also altered the cell subpopulation composition of the adrenal gland: compared to control, it decreased the proportion of LDAC-2 by 42.3% and increased the proportion of LDAC-3 and HDAC by 68.7 and 302%, respectively. Thus, dietary protein restriction induces adrenal steroidogenic hypofunction in turkeys. In addition, the present study suggests that this nutritional stressor induces marked remodeling of the steroidogenic tissue in the turkey adrenal gland.

Models to elucidate the regulation of adrenal cell death.

Hypophysectomy-induced apoptosis in the rat adrenal gland is slow (not apparent until 12-24 h) and in-situ, 3'-end labeling (ISEL) of DNA strand breaks is restricted to a subpopulation of zona reticularis cells. In addition, it is completely blocked by ACTH. By contrast, apoptosis in the intact rat adrenal gland, cultured in the absence of trophic support, is extensive and rapid. Culture-triggered apoptosis (as determined by oligonucleosome formation) is attenuated by ACTH and is largely restricted to the zonae fasciculata and reticularis even at 3 h (as determined by ISEL and DAPI cytochemistry). Thus, this organ culture system may help elucidate factors that can acutely regulate adrenocortical cell survival. Quartered glands have a nearly 2-fold increase in oligonucleosome formation compared to intact glands at 3 h and are resistant to the antiapoptotic action of ACTH. In contrast, ACTH-induced corticosterone secretion is not attenuated. Angiotensin II (Ang II) enhances culture-triggered apoptosis, and its apoptotic action is attenuated by ACTH. These observations suggest that 1) acute hormonal modulation of apoptosis may require some level of gross adrenal structural integrity, and 2) ACTH and Ang II act in an antagonistic fashion to regulate adrenocortical apoptosis. The apoptotic effect of Ang II may be mediated via the type 2 receptor.

The thyroid hormone, 3,5,3'-triiodothyronine, is a negative modulator of domestic fowl (Gallus gallus domesticus) adrenal steroidogenic function.

Previous work with chickens (Gallus gallus domesticus) suggests a relationship between depressed thyroid hormone status and enhanced adrenal steroidogenic function. In addition, in hypophysectomized chickens, replacement of the thyroid hormone, 3,5,3'-triiodothyronine (T3), maintains chicken adrenal steroidogenic cell sensitivity to adrenocorticotropin (ACTH) but decreases steroidogenic capacity further than that due to hypophysectomy alone. The present in vivo and in vitro studies were conducted to determine the influence of thyroid status and T3 per se on avian adrenal steroidogenic function. Chicks (1 day old) were thyroidectomized using combined surgical and chemical (6-propyl-2-thiouracil) treatments and were administered a replacement dose of T3 (0, 1.5, 4.5, 15, and 45 microg/kg body wt/day) for 5 weeks. Whereas thyroidectomy (TX) decreased adrenal weight (-20%), it increased relative adrenal weight (mg/100 g body weight) (+171%), trunk plasma corticosterone (+880%), and aldosterone (+124%). In addition, TX increased basal, maximal ACTH-induced, maximal 8-bromo-cyclic AMP-induced, and maximal 25-hydroxycholesterol-supported corticosterone production (+520, +93, +124, and +195%, respectively) and aldosterone production (+578, +288, +280, and +275%, respectively) by isolated adrenal steroidogenic cells. T3, in a dose-dependent manner, reversed the effects of TX on these in vivo and in vitro parameters of adrenal steroidogenic function. Restoration of most of these parameters to those in the sham-treated control was attained with 4.5-15 microg/kg body wt/day. Although some of the effects of TX and T3 replacement on adrenal steroidogenic function may have been mediated through changes in circulating levels of ACTH, other data suggest a direct effect on adrenal steroidogenic cell function. Adrenal steroidogenic cells from sham-treated and TX birds were preincubated (0, 4, and 12 hr) with various concentrations of T3 (0, 0.3, 3, and 30 nM), washed, and then incubated for an additional 2 hr in medium containing the same respective concentrations of T3, with or without a maximal steroidogenic concentration of ACTH (100 nM). T3 had no acute effects on TX-dependent enhancement of adrenal steroidogenic cell function (2-hr incubation). However, with preincubation (4 and 12 hr), T3 inhibited basal and maximal ACTH-induced corticosterone production in a dose-dependent manner. This concentration-dependent, direct effect of T3 was not observed with cells from sham-treated birds. In addition, the ostensibly inactive thyroid hormone metabolite, 3,3',5'-triiodothyronine [reverse T3; 30 nM], was without effect. Taken collectively, these studies indicate that T3 is a direct negative modulator of avian adrenal steroidogenic function.

Hormonal modulation of apoptosis in the rat adrenal gland in vitro is dependent on structural integrity.

The intact rat adrenal gland in short-term (3-h) organ culture may be amenable for the identification of factors involved in regulating adrenal cell apoptosis under defined conditions. In this model, culturing in the absence of trophic support (basal; control) triggered apoptosis in the intact rat adrenal gland; oligonucleosome formation, a measure of apoptosis, was 56.4-fold greater than that of glands snap-frozen at the start of incubation. Angiotensin II (Ang II) (100 nM) enhanced apoptosis by 67% over control. By contrast, adrenocorticotropin (ACTH) (100 nM) attenuated basal apoptosis by 59% and antagonized the enhanced apoptosis induced by Ang II back to the control level. Quartering of the glands enhanced basal oligonucleosome formation 182.2% greater than that of intact glands. Interestingly, quartering of the glands abolished the influences of Ang II and ACTH on apoptotic DNA fragmentation, but did not alter ACTH-induced corticosterone secretion. These data suggest that some level of gross adrenal structural information or compartmentalization, sufficiently disrupted by quartering, is required for the hormonal modulation of adrenal cell survival.

Effect of acid or aluminum on growth and adrenal function in young chickens.

Acid precipitation can have a harmful effect on aquatic birds, due in part to increases in aluminum availability. Young rapidly growing (broiler strain) chickens were used as a model to examine the effects of aluminum and acid on growth and circulating concentrations of adrenocorticol hormones. Two concentrations of acid (sulfuric acid) or aluminum (aluminum sulfate) or sodium sulfate were administered to a heavy (broiler) strain of chickens for 10 days (Days 4 to 14 of age). Additional treatment groups included a control diet either fed ad libitum or pair-fed relative to the chicks on the acid or aluminum diets. Compared with the chicks receiving the control diet ad libitum, growth (body weight) was reduced in chicks on the aluminum (high and low level), acid (high level), and sodium sulfate (high level) treatments and the respective pair-fed groups. Circulating concentrations of corticosterone (B) were elevated in the chicks receiving the high dose of aluminum and the respective pair-fed control when compared with the chicks which had free access to the control diet. Thus, the increase in plasma B appears to be linked to the low food intake and not to the A1 per se. Circulating concentrations of aldosterone were increased in the chicks receiving either the high dose of aluminum or the acid relative to chicks fed the control diet (both ad libitum or pair-fed controls). However, circulating concentrations of aldosterone were unaffected by either dose of sodium sulfate employed. Thus, the increase in plasma aldosterone appears to be specific to the metabolic acidosis created by A1 or acid. It is concluded that environmental acid may either directly or indirectly influence adrenocortical function. Moreover, the present study provides evidence for the independent control of circulating concentrations of corticosterone and aldosterone in the chicken.

Apoptotic cell death in the rat adrenal gland: an in vivo and in vitro investigation.

Adrenocortical cell apoptosis was studied by using an established in vivo model, the hypophysectomized rat, and an in vitro model, viz., rat adrenal glands in short-term organ culture. In vivo, apoptosis (biochemical autoradiographic analysis of internucleosomal DNA cleavage) was weak and not apparent until 12-24 h after hypophysectomy. In situ histochemical localization of 3'-end DNA strand breaks revealed that apoptosis in vivo occurred nearly exclusively in subpopulations of zona reticularis cells. Adrenocorticotropic hormone (ACTH) maintenance completely blocked these indices of apoptosis. By contrast, apoptosis (DNA fragmentation) in cultured rat adrenal glands without ACTH was extensive and relatively rapid, being apparent after 1 h and increasing with the duration of incubation. ACTH attenuated (by 44%) but did not completely block apoptosis in vitro. Thus, ACTH appears to be the sole pituitary hormone that forestalls apoptosis of terminally differentiated adrenocortical (zona reticularis) cells. However, the discrepancy between in vitro and in vivo models in terms of the magnitude and rate of DNA fragmentation suggests that, in vivo, other factors finely regulate the magnitude of adrenocortical apoptotic cell death.

Lead alters growth and reduces angiotensin II receptor density of rat aortic smooth muscle cells.

Environmental lead (Pb2+) contributes a small but significant risk to human hypertension. It is postulated that the hypertensinogenic action of Pb2+ may be due, in part, to its direct action on vascular smooth muscle cells. To investigate this hypothesis, freshly isolated rat aortic smooth muscle (RASM) cells were propagated in defined media containing one of two Centers for Disease Control-based concentrations of Pb2+ (as lead citrate): 100 and 500 micrograms Pb2+/l (i.e., equivalent to 5.5 and 27.5 micrograms Pb2+/dl blood; designated 100-RASM and 500-RASM). Control (CON-RASM) cells received sodium citrate. 500-RASM cells exhibited suppressed propagation and fell out of propagation synchrony with CON-RASM cells: when CON-RASM cell approached confluence (approximately 90%), 500-RASM cell density was 6.4% that of CON-RASM cell density. By contrast, 100-RASM cells exhibited marked hyperplasia albeit this was not apparent until passage 3 (p3). Overall, when p3-p6 CON-RASM cells approached confluence, 100-RASM cell density was 107.6% greater than CON-RASM cell density. The protein content of CON-RASM and 100-RASM was not different, whereas that of 500-RASM cells was 29% greater than that of CON-RASM and 100-RASM cells. Phase-contrast microscopy revealed that 100 micrograms Pb2+/l converted normal spindle-shaped/ribbon-shaped RASM cells into less spread, cobblestone-shaped, neointimal-like cells. Immunocytochemical analysis revealed that 100-RASM cells lacked or had markedly fewer actin cables, characteristic of rapidly dividing cells. In addition, Pb(2+)-treated RASM cells exhibited altered membrane fatty acyl composition with a trend towards an increase (by as much as 50%) in membrane arachidonic acid. Interestingly, hyperplastic 100-RASM cells exhibited a 70.6% reduction in angiotensin II (Ang II) receptor concentration whereas the concentrations of alpha 1- and beta-adrenergic and atrial natriuretic peptide (ANP) receptors were not affected. In addition, in experiments designed to control for Pb(2+)-associated differences in RASM cell propagation, there was a concentration-dependent decrease in Ang II receptor concentration: for 100 and 500 micrograms Pb2+/l, Ang II receptor concentration was decreased 39.6% and 65.5%, respectively. Thus, although Pb2+, depending on its concentration, had contrasting effects on RASM cell propagation, it had a consistent, concentration-dependent inhibitory effect on Ang II receptor concentration. Recovery (r) from Pb2+ required at least two additional passages. At p71r the enhanced propagation (+54%) and reduced Ang II receptor concentration (-49%) of 100r-RASM cells persisted.(ABSTRACT TRUNCATED AT 400 WORDS)

Atrial natriuretic peptide stimulates aldosterone production by turkey (Meleagris gallopavo) adrenal steroidogenic cells.

The inhibitory action of atrial natriuretic peptides (ANPs) on mammalian aldosterone synthesis is well documented. In addition, other work indicates that ANP and an analogue of its second messenger, 8-Br-cGMP, inhibit aldosterone production by chicken adrenal steroidogenic cells. However, the interaction between angiotensin II (AII) and ANP in the regulation of avian aldosterone production is poorly understood because chicken adrenal steroidogenic cells, the commonly used in vitro avian model, are comparatively unresponsive to AII. By contrast, turkey (Meleagris gallopavo) adrenal steroidogenic cells are sensitive to AII. Thus, in the present study, the action of ANPs and related peptides and their interaction with other stimulators of aldosterone production were investigated using freshly isolated and briefly cultured turkey adrenal steroidogenic cells. Surprisingly, several ANPs [rat (r), human (h), chicken (c)], and rat brain natriuretic peptide (rBNP) were as efficacious as [Ile5]AII for stimulating aldosterone production (2 hr) in freshly isolated cell suspensions but were less potent than [Ile5]AII (ED50 of ANPs approximately 5-10 nM; [Ile5]AII ED50 approximately 0.1 nM). In addition, chicken ANP enhanced maximal aldosterone production induced by [Ile5]AII (1 nM), K+ (25 mM), and hACTH-(1-39) (ACTH) (1 nM): maximal enhancement of the action of these secretagogues was +49%, +137% and +15%, respectively (P < 0.05; n = 3). Furthermore, other ANPs and related peptides [rBNP and bovine aldosterone secretion inhibiting factor (bASIF)] enhanced maximal [Ile5]AII-induced aldosterone production: the order of maximal enhancement was rBNP (+180%) > hANP/rANP (+50%) > bASIF (+25%) (P < 0.05; n = 3).(ABSTRACT TRUNCATED AT 250 WORDS)

Dissociation of increases in intracellular calcium and aldosterone production induced by angiotensin II (AII): evidence for regulation by distinct AII receptor subtypes or isomorphs.

In mammalian zona glomerulosa cells, angiotensin II (AII)-induced increases in intracellular Ca2+ ([Ca2+]i) and AII-induced aldosterone production seem to be inextricably linked. However, in avian adrenal steroidogenic (adrenocortical) cells studied thus far, inducible aldosterone production seems to be insensitive to alterations in the mobilization of cellular Ca2+. This raises the hypothesis that alternative signal transduction pathways are implemented to induce aldosterone production in avian adrenocortical cells. In the present study, this hypothesis was investigated by using isolated turkey (Meleagris gallopavo) adrenocortical cells that are known to be three times more sensitive to AII than to ACTH for aldosterone production. In isolated turkey adrenocortical cells, the mammalian AII receptor antagonist, [Sar1,Ile8]AII, was as efficacious as [Ile5]AII in stimulating aldosterone production, albeit it had about 1/150 the potency of [Ile5]AII. The actions of both analogs required extracellular K+, suggesting a voltage-sensitive event. However, a maximal aldosteronogenic concentration of [Sar1,Ile8]AII not only failed to increase [Ca2+]i but also completely blocked maximal (10(-8) M)[Ile5]AII-induced increases in [Ca2+]i when added before [Ile5]AII and partially dampened (approximately 50%) maximal [Ile5]AII-induced increases in [Ca2+]i when added after (3 min) [Ile5]AII. This blockade in [Ca2+]i elevation was surmounted by high concentrations of [Ile5]AII (> 10(-6) M). By contrast, [Sar1,Ile8]AII did not alter maximal aldosterone production induced by [Ile5]AII and vice versa, thus suggesting that the action of both analogs converged on the same aldosteronogenic pathway, and that AII-induced aldosterone production was not coupled to elevations in [Ca2+]i. Detailed homologous-heterologous ligand-binding analyses supported the presence of two AII-binding sites that were discriminated by [Sar1,Ile8]AII (dissociation constants, 4.2 +/- 1.4 and 21.9 +/- 2.2 nM; concentration distribution, approximately 40% and approximately 60%, respectively; mean +/- SE, n = 4) but not by [Ile5]AII (dissociation constant, 2.1 +/- 0.1 nM for both sites). In addition, [Sar1,Ile8]AII- and [Ile5]AII-binding sites exhibited different physicochemical and pharmacological properties. The sensitivity of [Sar1,Ile8]AII-binding sites was about twice that of [Ile5]AII-binding sites to dithiothreitol. In addition, whereas both the high- and low-affinity sites detected by [Sar1,Ile8] AII exhibited equivalent competitive sensitivities to the type-1 receptor, the nonpeptidic antagonist, losartan (DuP 753), the sensitivity of the low-affinity site was 2.7 times that of the high-affinity site to the type-2 receptor, nonpeptidic antagonist, PD123319. Taken collectively, the data suggest that in turkey adrenocortical cells, elevations in [Ca2+]i and aldosterone production are dissociable events regulated by distinct AII receptor subtypes or isomorphs.

Evidence for functionally distinct subpopulations of steroidogenic cells in the domestic turkey (Meleagris gallopavo) adrenal gland.

A body of histological and functional evidence supports the hypothesis that there are functionally distinct subpopulations of steroidogenic cells comprising the avian adrenal gland. In the present study, we tested this hypothesis by evaluating the steroidogenic responses of density-dependent subpopulations of adrenal steroidogenic cells isolated from domestic turkeys fed either a high-normal (control) sodium diet (0.4% Na+) or a Na(+)-restricted diet (0.04% Na+) for 8 days, the latter to stimulate the activity or appearance of possible zona glomerulosa-like cells. Subpopulations were visually yet reproducibly determined by their density-dependent separation on a continuous density gradient of Percoll (45%). The subpopulations were arbitrarily ascribed as being either low-density or high-density adrenal steroidogenic cells [LDAC (p = 1.0350-1.0585 g/ml) and HDAC (p = 1.0590-1.0720 g/ml), respectively]. LDAC and HDAC comprised 95.2 and 4.8%, respectively, of the total number of adrenal steroidogenic cells isolated. The LDAC was further subdivided into three visually distinct subpopulations. The functional differences between the LDAC subpopulations is discussed but was less dramatic than the functional distinction between the HDAC subpopulation and the pooled LDAC subpopulations. Basal aldosterone production values between control LDAC and HDAC were equivalent. In addition, there were no differences in maximal aldosterone production between control LDAC and HDAC in response to [Ile5]angiotensin II (AII), the avian equivalent, [Val5]AII, K+ (as KCl), and that supported by exogenous corticosterone. However, maximal aldosterone production in response to human ACTH-(1-39) (ACTH) of the LDAC was 32% greater than that of the HDAC. Na+ restriction enhanced basal aldosterone production of the LDAC by 84% over the control LDAC. In addition, it enhanced maximal aldosterone production of the LDAC in response to AII peptides, K+, ACTH and that supported by corticosterone by 54, 164, 83, and 74%, respectively, over that of the control LDAC. However, Na+ restriction disproportionately enhanced basal aldosterone production of the HDAC by 348% over that of the control HDAC. In addition, with Na+ restriction, maximal aldosterone production of the HDAC in response to AII, K+, and ACTH and that supported by exogenous corticosterone was consistently greater than that of the LDAC. Moreover, with Na+ restriction, maximal aldosterone production of the HDAC in response to AII peptides and K+ was increased over that of the control HDAC to a greater extent than was maximal aldosterone production in response to ACTH and that supported by corticosterone (% enhancement over control was as follows: AII peptides, 502%; K+, 668%; ACTH, 273%; corticosterone, 183%).(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of aldosteronogenesis in domestic turkey (Meleagris gallopavo) adrenal steroidogenic cells.

The hormonal and cationic regulation of aldosterone production by freshly isolated turkey (Meleagris gallopavo) adrenal steroidogenic cells was investigated. Angiotensin II (AII), ACTH [human ACTH-(1-39)], and K+ stimulated aldosterone production in a concentration-dependent manner albeit these agents exhibited considerable differences in lag time for the significant stimulation of aldosterone production over basal production. By contrast, Ca2+ was without effect except at a high concentration (10 mM). Although ACTH was more efficacious than AII, it had about one-third the potency of AII for stimulating aldosterone production. However, ACTH potentiated the maximal aldosterone response to AII [maximal enhancement (+499%) at 3 x 10(-10) M ACTH]. Extracellular K+ was an absolute requirement for AII-induced aldosterone production (threshold concentration = 3 mM), and maximal enhancement (+200%) occurred with 5 mM (a physiological concentration). Although extracellular Ca2+ was not an absolute requirement for inducible aldosterone production, it enhanced AII-induced aldosterone production in a concentration-dependent manner [maximal enhancement (+727%) at 3 mM], albeit it did not alter the half-maximal steroidogenic concentration (EC50) of AII. Ca2+ also enhanced maximal ACTH-induced aldosterone production but to a lesser extent (+96% with 1 mM Ca2+). However, Ca2+ dramatically enhanced ACTH potency (ED50) (nearly 100 times at 1 mM Ca2+). The acute augmentation of AII-induced aldosterone production by ACTH, K+, and Ca2+ was not accompanied by increases in the cellular concentration and affinity of AII receptors, suggesting that the agents acted at intracellular loci distal to the AII receptor. Several aspects of the present study with isolated turkey adrenal steroidogenic cells differ markedly from those of studies with isolated chicken (Gallus gallus domesticus) adrenal steroidogenic cells and mammalian zona glomerulosa cells, thus suggesting interclass and intraclass differences in homeothermic vertebrate adrenal steroidogenic regulation.

Properties of angiotensin II receptors of domestic turkey (Meleagris gallopavo) adrenal steroidogenic cells.

In the present study, the properties of angiotensin II (AII) receptors of intact domestic turkey adrenal steroidogenic cells were characterized. AII (but not ACTH) induced an immediate and sustained increase in intracellular Ca2+. In addition, dithiothreitol inhibition of maximal AII-induced aldosterone production was closely correlated with its inhibition of binding suggesting that these receptors are type 1-like and operate through a non-"spare" receptor mode. Equilibrium-binding analysis revealed a single class of binding sites at a concentration of 63,500 sites/cell and having an apparent dissociation constant (Kd) of 1.21 nM. However, the Kd derived from kinetic analyses, 0.27 nM, was lower. Both empirically determined and model-based calculated distributions of bound hormone indicated that at equilibrium, about 30% of hormone-receptor complexes were internalized whereas 70% remained on the surface. This distribution contrasts sharply with that reported for mammalian (rat) adrenocortical cells. In keeping with recent cloning studies, these avian AII receptors of intact adrenal steroidogenic cells discriminated angiotensins and mammalian peptidic and nonpeptidic antagonists differently from mammalian adrenocortical and duck adrenal receptor preparations. Importantly, turkey adrenal steroidogenic cell AII receptors poorly discriminated the nonpeptide antagonists, losartan (DuP 753) (type-1 specific) and PD123177 (type-2 specific). Thus, AII receptors of freshly isolated, intact turkey adrenal steroidogenic cells are pharmacologically distinct from mammalian adrenocortical type-1 receptors.

Isolation of turkey adrenocortical cell angiotensin II (AII) receptor partial cDNA: evidence for a single-copy gene expressed predominantly in the adrenal gland.

A turkey adrenocortical cell AII receptor cDNA fragment (714 bp) was isolated by RT-PCR using oligonucleotide primers based on rat aortic smooth muscle (RASM) and bovine adrenal type-1 (AT1) receptor cDNA coding sequences as primers. Sequence analysis indicated 73% nucleotide identity and 78% amino acid identity to the RASM AT1 receptor. Notable differences were 1) two additional Cys at positions 92 and 99 (first extracellular loop), 2) deletion of amino acid 168, formation of a triplet Asn sequence (Asn 186, 187, 188) and substitution of Arg192 with Pro (second extracellular loop) and 3) two additional potential protein kinase C phosphorylation sites, Thr221 and Thr233 (third intracellular loop). Southern blot analysis indicated that the receptor is a product of a single-copy gene. Northern blot analysis indicated at least three mRNA transcripts (7.5, 3.5 and 2.0 kb) expressed predominantly in the adrenal gland.

Dietary protein restriction stress and adrenocortical function: evidence for transient and long-term induction of enhanced cellular function.

Previous work has demonstrated that 4-week protein restriction of the domestic fowl (Gallus gallus domesticus) increases both adrenocortical cell sensitivity to ACTH and corticosteroidogenic capacity. In the present study we examined the transience (study 1) and the persistence (study 2) of this effect of protein restriction. In study 1, two strains of domestic fowl were used: a slower-growing White Leghorn strain and a faster-growing Cobb broiler strain. Cockerels (2 weeks old) were fed isocaloric diets containing either low (L; 5% or 8%) or control (C; 20%) levels of soy protein for 2 weeks, and then were either switched to the alternate diet (C-L, L-C) or maintained on the initial diet (C-C, L-L) for an additional 2 weeks. Cockerels were killed at 6 weeks of age. In study 2, White Leghorn cockerels (2 weeks old) were fed either diet for 4 weeks and then switched to or maintained on the control diet for an additional 4 weeks (i.e. C vs. restriction followed by repletion). In this study cockerels were killed at 10 weeks of age. In both studies highly enriched populations of adrenocortical cells were isolated from cockerel adrenal glands, and their steroidogenic properties (basal and maximally induced corticosterone and cAMP production; steroidogenic agent ED50 values) were evaluated in 2-h suspension incubations. In study 1, regardless of strain, the greater the length of the restriction period, the greater the magnitude of maximal cellular corticosterone production induced by ACTH; the average value for 4-week restriction (L-L) was 39.5% greater than that for 2-week restriction (L-C, C-L) and 117.5% greater than that for control (C-C). In addition, the value for restriction from 4-6 weeks of age (C-L) was 34% greater than that for restriction from 2-4 weeks of age (L-C), suggesting that the enhancement of maximal ACTH-induced corticosterone production after a 2-week restriction interval might be transient. Although there were no strain differences in the effect of protein restriction on maximal ACTH-induced corticosterone production, there were strain differences in its effect on cellular sensitivity to ACTH, as indicated by the ACTH ED50 values (the lower the ED50 value, the greater tha cellular sensitivity). With the White Leghorn strain, the greater the duration of protein restriction, the greater the adrenocortical cell sensitivity to ACTH; the sensitivity of L-L cells was 3.0 times the sensitivities of L-C and C-L cells and 4.1 times the sensitivity of C-C cells.(ABSTRACT TRUNCATED AT 400 WORDS)