Reverse Haemolytic Plaque Assay Study of Corticotropin-Releasing Hormone and Arginine Vasopressin Interaction in Ovine Corticotropes

Corticotropin-releasing hormone and arginine vasopressin are known to interact in stimulating secretion of adrenocorticotropin-related peptides from corticotropes. However, the mechanism mediating this interaction is uncertain. Recently, evidence has been provided using a reverse haemolytic plaque assay that in rat pituitary cells, arginine vasopressin potentiates the effects of corticotropin-releasing hormone by increasing the percentage of target cells that secrete adrenocorticotropin. To determine whether a similar mechanism also operates in the sheep corticotrope, which is reportedly more sensitive to arginine vasopressin than that of the rat, a reverse haemolytic plaque assay for β-endorphin secretion was used to study the response of ovine corticotropes to stimulation by increasing doses of corticotropin-releasing hormone or arginine vasopressin (0.1 nM to 10.0 nM) alone or in combination. In the reverse haemolytic plaque assay, β-endorphin antiserum at 1:50 and complement at 1:10 were found to be optimal dilutions for plaque formation. A concentration-dependent response curve to corticotropin-releasing hormone was obtained with a significant increase in plaque area from basal to reach maximal levels at 1.0 nM. Arginine vasopressin also stimulated an increase in plaque area, however, plaques formed were significantly smaller than those caused by corticotropin-releasing hormone. Since in the reverse haemolytic plaque assay, plaque area is related to the amount of hormone secreted by the cell, results demonstrate that although corticotropin-releasing hormone and arginine vasopressin both stimulate β-endorphin secretion from ovine corticotropes, corticotropin-releasing hormone is a more potent secretagogue than arginine vasopressin in that it causes the formation of significantly larger plaques. The addition of arginine vasopressin to low concentrations of corticotropin-releasing hormone caused plaque areas to reach maximal levels at 0.1 nM whereas these levels were only attained at 1.0 nM when corticotropin-releasing hormone was used alone. Therefore, arginine vasopressin interacts with corticotropin-releasing hormone to increase corticotrope responses by increasing their secretory response to corticotropin-releasing hormone. These data are consistent with previous work suggesting that arginine vasopressin increases the expression of corticotropin-releasing hormone receptors on the corticotrope cell surface. However, no significant increase in the percentage of plaque-forming cells was seen with either corticotropin-releasing hormone or arginine vasopressin alone or in combination implying that there was no recruitment of previously non-secreting cells.