Protein Malnutrition in the Domestic Fowl Induces Alterations in Adrenocortical Cell Adrenocorticotropin Receptors*

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Abstract
Our previous work suggests that persistent protein malnutrition in immature domestic fowl (Gallus gallus domesticus) alters ACTH-adrenocortical cell interaction, possibly including ACTH receptors. To investigate this possibility, we measured some ACTH receptor parameters in isolated adrenocortical cells from normal and dietary protein-restricted domestic fowl. White Leghorn cockerels (2 weeks old) were fed isocaloric semipurified diets containing either 8% [low (L)] or 20% [normal (N)] soy protein for 4 weeks ad libitum. Cockerels were quickly killed by decapitation and exsanguination, and adrenal glands were removed and prepared for cell isolation. Highly enriched (> 80% pure) adrenocortical cells (collagenase isolated, followed by separation on a Percoll continuous density gradient) were evaluated for ACTH receptors using pharmacological and radioligand approaches. In a pharmacological approach, we measured the influence of the complete, competitive antagonist, human (h) ACTH-(7-38) on hACTH-(1-39)-induced corticosterone production by adrenocortical cells from L and N cockerels. Inhibitor constants of hACTH-(7-38), calculated from Schild plots, were 3.16 .times. 10-7 and 9.82 .times. 10-7 M for L and N cockerel cells, respectively, thus suggesting differences in ACTH receptor function between the two treatment groups. To characterize ACTH receptors directly, we measured the binding of a monoiodinated ACTH analog [125I-Tyr23]hACTH-(1-39) to domestic fowl adrenocortical cells. Binding was linear with cell concentration, highly specific (only ACTH peptides caused significant displacement), rapid (maximal binding by 1h), reversible (half-time of dissociation, .apprx. 40 min), and saturable. Curvilinear Scatchard plots were obtained, and vectorial analysis resolved both high and low affinity sites. The concentrations (femtomoles per 50 .mu.g DNA) and dissociation constants (Kd) of both classes of sites were different between N and L bird cells. Values of these receptor parameters for N and L cockerel cells were, respectively, as follows: concentrations of low affinity sites, 7.45 and 11.60; concentrations of high affinity sites, 3.16 and 5.50; kd of low affinity sites, 2.05 .times. 10-8 and 2.58 .times. 10-9 M; Kd of high affinity sites, 1.01 .times. 10-9 and 1.27 .times. 10-10 M. Thus, the overall binding capacity of L bird cells was 65% greater than that of N bird cells. In addition, the overall affinity (1/Kd) of sites of L bird cells was 9 times that of sites of N bird cells. These data indicate that persistent protein malnutrition in the domestic fowl increased both the number and affinity of adrenocortical cell ACTH receptors.