IgE-mediated food allergy--diagnosis and management in New Zealand children.

AIM: To summarise the diagnosis and management of IgE-mediated food allergy (FA) in New Zealand children. METHOD: A review of the scientific literature and subsequent consensus development. RESULTS: FA is a common problem in New Zealand children with management necessitating accurate diagnosis, appropriate risk management, and reassessment over time. CONCLUSION: This paper highlights the importance of a structured approach to diagnosis and management of FA in New Zealand children, guided by appropriately skilled health professionals.

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.

Outcome of primary vesicoureteric reflux detected following fetal renal pelvic dilatation.

OBJECTIVE: Postnatal investigation of mild degrees of fetal hydronephrosis has allowed subsequent detection of infants with vesicoureteric reflux (VUR). This study was designed to provide short to medium term information on such infants who had primary VUR, the rates of renal damage and progression over time, the risk factors for such damage and to compare the characteristics of those who had mild dilatation of the fetal renal pelvis (4-9 mm) with those who had moderate-severe dilatation (> or = 10 mm). METHODOLOGY: Since June 1989, infants whose antenatal sonography had identified a fetal renal pelvis with an anteroposterior diameter of > 4 mm were investigated postnatally with renal ultrasonography and micturating cystourethrogram (MCU), and placed on antimicrobial prophylaxis. Those with VUR received 99mTc-dimercaptosuccinic acid (DMSA) scintigraphy. Infants were followed until discharge based on resolution of VUR, surgery, or low grade VUR. A 5.5 year cohort between June 1989 and December 1994 formed the study population. A review of notes and clinical review (if still under follow up) was undertaken. Vesicoureteric reflux on MCU was regraded according to the International Classification, and reflux nephropathy on DMSA scans was regraded according to criteria proposed by Goldraich. Regression analysis was used to assess risk factors for renal damage. RESULTS: There were 69 infants (37 girls, 32 boys) who were identified with primary VUR, with 37/69 having bilateral reflux. Eight had a urinary tract infection during the follow-up period. There was a broad distribution of grades of reflux detected (Grades I-3, Grades II-23, Grades III-19, Grades IV - 17, Grades V-7). 99m-Tc-dimercaptosuccinic acid scans on 57/69 (83%) demonstrated renal damage in eight infants (14%). This was predominantly global contraction of function. No progression of renal damage was seen over 2-7 years. Regression analysis showed a strong association between Grades IV, V reflux and the presence of renal damage (P < 0.001). Review of the degrees of fetal renal pelvic dilatation showed that 60/69 infants were detected because of mild (4-9 mm) dilatation. The majority (43/60) had lower grades of reflux (Grades I, II, 3), but there was no obvious cut-off between 4 and 9 mm that could predict high grade VUR (Grades IV, V). CONCLUSIONS: The use of 4 mm to define an abnormal fetal renal pelvis allows a much larger group of infants with high grade primary VUR to be detected than if a higher cut-off measurement is used. Although it also detects many more infants with low grade primary VUR, there is no obvious cut-off point at which this effect predominates. Progressive renal damage was not seen in follow up of up to 7 years of age. Renal damage on DMSA scanning in this group is almost exclusively a pattern of global contraction of function. The presence of high-grade VUR appears to be the only important factor in predicting the presence of renal damage.

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.

Drugs that influence tubulin polymerization modulate thyrotropin-releasing hormone receptor number in AtT-20 cells.

In order to examine the role of cytoskeleton in modulating the cell surface receptors, AtT-20 cells (stably expressing thyrotropin-releasing hormone receptors) were incubated with drugs that are known to modify the tubulin-microtubule system. The binding of [3H]methyl thyrotropin-releasing hormone ([3H]mTRH) to intact cells increased as a function of time, and was linear from 1.25 x 10(6) to 6.25 x 10(6) cells/ml. Cells incubated with colchicine, vinblastine, and taxol for 16 hr were harvested and the cell concentration was determined using a haemocytometer. Because the drugs inhibited the cell proliferation at 100 nM, it was decided to examine the effect of 100 nM of each of the three drugs on the ability of [3H]mTRH to bind cell surface receptors. Cells were incubated with the drugs for 16 hr at 37 degrees. After the incubation, cells (5 x 10(6) cells/ml) from each group were assayed for [3H]mTRH binding. Colchicine, vinblastine, and taxol stimulated [3H]mTRH binding by up to 27, 27, and 21%, respectively, without altering the Ka of the ligand to the receptor. These results suggest that perturbation of cytosolic microtubules leads to a reorganization of the spatial location of hormone receptors.

The effect of ethanol on muscarinic receptor-G protein coupling in the rat cortex.

To understand the mechanism of ethanol action on G protein-mediated signal transduction pathway, the effect of ethanol on muscarinic receptor-G protein coupling in the rat cerebral cortex was examined. Acetylcholine (ACh)-stimulated G protein GTPase activity was used as an index of receptor-G protein coupling. ACh stimulation of G protein GTPase activity was time- and concentration-dependent, and atropine-sensitive. Rats injected with ethanol (3 g/kg body weight) were sacrificed after 4 hr, and the cerebral cortices removed. The ability of ACh to stimulate GTPase activity was similar in cortical cell membranes obtained from control and ethanol-treated rats; ACh maximally stimulated the enzymatic activity by 22% in membranes from both groups of rats. Next, in cortical cell membranes obtained from control rats (i.e., not injected with ethanol) the ability of ACh to stimulate GTPase activity in the presence of ethanol was examined. ACh stimulated GTPase activity in a concentration-dependent manner; the activity was 12.3 +/- 0.1, 14.5 +/- 0.64, 15.7 +/- 0.54, and 16.1 +/- 0.33 Pi pmol/min/mg protein, at 0, 0.01, 0.1, and 1 mM ACh, respectively (P < 0.05). In the presence of 100 mM ethanol ACh-stimulated GTPase activity was significantly inhibited. The IC50 value of ethanol inhibition of ACh-stimulated GTPase activity was approximately 50 mM. These results suggest that: 1) in vitro, ethanol impairs ACh-stimulated G protein GTPase activity in the rat cortical cell membranes, and 2) in vivo, the acute effects of alcohol on G protein function may be transient and reversible.

Radiation mucositis: a new approach to prevention and treatment.

Mucosal reactions in patients receiving radiation treatment for head and neck cancer are regarded as unavoidable side-effects. The degree of mucositis experienced is determined by the treatment dose, radiation field size and fractionation schedules prescribed for individual patients. This article reviews a double-blind placebo-controlled trial of an antibiotic pastille which was aimed at reducing the more extreme effects of radiation mucositis. It identifies the role of oral care and hygiene, oral assessment and nursing support within a clinical trial setting. The results showed that the active pastille had a beneficial effect on the degree of mucositis experienced. Patients had a reduction in their weight loss in the active arm of the trial and the amount of yeast bacteria present in the oral cavity of those patients also diminished. It is worth noting that those patients in the placebo arm of the trial received both physical and psychological support from the nursing staff which may not have been available as frequently if they had not been in the trial.

The reduction of radiation mucositis by selective decontamination antibiotic pastilles: a placebo-controlled double-blind trial.

The aim of this study was to see if antibiotic pastilles could reduce radiation mucositis, pain, dysphagia and weight loss in patients undergoing radical radiotherapy for head and neck cancer. A total of 275 patients with T1-T4 tumours entered the study; 136 were allocated to suck four times daily a pastille containing amphotericin, polymyxin and tobramycin. The remaining 139 patients received an identical placebo. In all, 54 patients were unevaluable (24 active, 30 placebo). Bacteriological monitoring was carried out before and twice weekly during treatment. Both arms of the study were well balanced for T and N stage, age, sex and radiation dose (60 Gy). There was a slight imbalance in the site of disease which had no substantive effect on the results. The primary study end point was the percentage of patients who developed intermediate or thick pseudomembranes. No statistically significant difference was found in this end point, with 36% of patients in the active arm developing this type of membrane compared with 48% in the placebo arm (P = 0.118). The estimated odds ratio (placebo/active) of developing an intermediate or thick pseudomembrane was 1.59 (95% CI 0.89-2.82). However a more sensitive test comparing the worst recorded mucositis grade between the two arms was statistically significant (P = 0.009). This indicated that the active pastilles had a beneficial effect, but the magnitude was probably smaller than the trial was designed to detect. There was a reduction in mucositis distribution (P = 0.002), mucositis area (P = 0.028), dysphagia (P = 0.006) and weight loss (P = 0.009) in the active arm. There was a clear tendency for patients with positive cultures for aerobic Gram-negative bacteria (AGNB) (P = 0.003) and yeasts (P = 0.026) during treatment to have more severe mucositis. The active pastilles reduced the percentage of patients with yeast cultures (P = 0.003) but had less effect on AGNB. The benefit derived from the pastilles should materially increase patient tolerance to radical radiotherapy for head and neck cancer.

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.

Differences in the characteristics and distribution of rat luteal receptors for native and deglycosylated human choriogonadotropin.

Previous work has suggested that rat luteal cells have two populations of LH/hCG receptors that are located in different parts of the cell membrane. The possibility that these two receptor pools may have functional differences has been investigated through examination of the binding and action of native and deglycosylated hCG to different membrane fractions. Ovaries from eCG/hCG-primed immature female rats were separated into 1,000 x g (heavy) and 20,000 x g (light) particulate fractions. Increasing concentrations of NaCl had a biphasic effect on the binding of native and deglycosylated hCG to both membrane fractions, causing an increase in binding at low concentrations and a decrease in binding at higher concentrations. The binding of deglycosylated hCG to both membrane preparations and the binding of native hCG to light-membrane preparations was maximal at approximately the same NaCl concentration (50-65 mM). This was higher than the concentration of NaCl necessary for maximal binding of native hCG to the heavy-membrane preparation. In addition, maximal native hCG binding to this preparation occurred over a broader NaCl concentration range (15-65 mM). Equilibrium binding experiments showed differences in hCG binding to both fractions. In light membranes there were significantly more receptor sites for deglycosylated hCG (11.2 +/- 4.8 fmol/mg ovary) than for native hCG (4.8 +/- 0.7 fmol/mg ovary), with no significant different in affinity. In contrast, in heavy membranes the affinity for deglycosylated hCG (6.30 +/- 0.19.10(9) M-1), was significantly higher than that for native hCG (2.60 +/- 0.13.10(9) M-1), with no significant differences in receptor number.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of the lutropin/choriogonadotropin receptor in rat ovary during luteinization.

Molecular analysis of the induction of the lutropin/choriogonadotropin receptor during the process of luteinization of the rat ovary was performed. The appearance of receptor binding activity and an immunological analysis of the receptor using Triton solubilized membrane proteins show little receptor present in luteal tissue through day 3 subsequent to hCG treatment, with some in day 4, and a marked increase by day 5. A similar pattern was found in the analysis of RNA hybridizing to several probes derived from the published cDNA sequence suggesting that receptor induction occurs primarily at the level of transcription.

Effects of cations on binding of human choriogonadotropin.

The effect of various salts on the binding of human choriogonadotropin to rat luteal membranes has been examined. Increasing salt concentrations had biphasic effects, initially increasing binding, then decreasing it. With NaCl, these effects were on both the affinity and the number of receptor sites. The affinity increased with increasing NaCl concentrations, to a maximum at 40 mM, and then decreased. Above 40 mM NaCl, the number of binding sites increased. NaCl also altered the effects of Mg2+ and guanyl nucleotides. At low ionic strength, Mg2+ was necessary to observe binding. Guanine nucleotides modulated this binding by decreasing the affinity. At 40 mM NaCl, Mg2+ increased receptor number without altering affinity. Guanyl nucleotides modulated this binding by reducing the number of sites to that observed in the absence of Mg2+. At 150 mM NaCl, Mg2+ and guanine nucleotides had no effect. The results suggest the presence of two pools of human choriogonadotropin receptor in rat corpus luteum, one coupled to the guanine nucleotide regulatory protein (Ns) and being Mg2+ dependent and guanine nucleotide sensitive, and the other not coupled to Ns and being Mg2+ independent and guanine nucleotide insensitive.