George Sayers

Isolated adrenal cells: Steroidogenesis and cyclic AMP accumulation in response to ACTH

Abstract After the addition of 1000 μU of ACTH to isolated adrenal cells, accumulation of cyclic AMP preceded corticosterone production. This observation supports the thesis that cyclic AMP mediates the steroidogenic action of ACTH on the adrenal cortex. Alterations in the concentration of cyclic AMP and of corticosterone in cell suspensions exposed to various doses of ACTH may be described as follows: (1) low doses of ACTH (5–25 μU) stimulated steroidogenesis without causing detectable changes in the concentration of cyclic AMP; (2) intermediate doses of ACTH (50–250 μU) induced parallel increases in cyclic AMP and corticosterone accumulation; and (3) large doses of ACTH (250 – 10,000 μU) caused additional increases in the concentration of cyclic AMP without causing further increases in corticosterone accumulation. The bearing of these results on the second messenger theory is discussed.

Isolated adrenal cortex cells: ACTH agonists, partial agonists, antagonists; cyclic AMP and corticosterone production☆

Abstract Isolated adrenal cortex cells respond to the addition of ACTH1–39 or analogs with increased production of cyclic AMP and corticosterone. It is estimated that cyclic AMP production need proceed at less than 20% of maximum to induce maximum corticosterone production. ACTH1–24, [Lys17, Lys18]ACTH1–8 amide, and ACTH1–16 amide induce a maximum rate of cyclic AMP and of corticosterone production equal to those of ACTH1–39. The relative potencies as determined by cyclic AMP and by corticosterone production are in excellent agreement. The analog, ACTH5–24, induces maximum cyclic AMP production equal to 45% of that of the natural hormone, but as predicted, induces maximum corticosterone production equal to that of ACTH1–39. The derivative, [Trp(Nps)9]ACTH1–39 induces 77% of maximum corticosterone production and less than 1% of maximum cyclic AMP production. The fragment ACTH11–24 is a competitive antagonist of ACTH1–39 for both cyclic AMP and corticosterone production. The observations on agonists, a partial agonist and a competitive antagonist are in harmony with the “second messenger” role assigned to cyclic AMP. A provisional model, based on the fit of the experimental observations to a set of equations, provides expressions of “intrinsic activity,” “receptor reserve”, “sensitivity”, and “amplification” in terms of maximum cyclic AMP production, concentration of ACTH which induces 1 2 maximum cyclic AMP production and concentration of cyclic AMP which induces 1 2 maximum corticosterone production.

Isolated adrenal cells: Log dose response curves for steroidogenesis induced by ACTH1–24, ACTH1–10, ACTH4–10 and ACTH5–10

ACTHr-39 * increases the production of corticosterone when added to suspensions of isolated rat adrenal cells [ 1 ] . Response and dose are related by the expression BIB,, = A/(A +AsO) where B is the rate of corticosterone production, B,,, is the maximum rate of corticosterone production, A is the dose of ACTH and A,, is the dose required to induce i B max [2] _ According to the model proposed by Ariens [3l,A,,, an apparent dissociation constant, is the reciprocal of the affinity of the hormone for its receptor, and B,,, is a measure of the effectiveness with which the hormone interacts with its receptor (“intrinsic activity”). In contrast to conventional methods of assay [4-61 , the isolated adrenal cell technique provides measures of both affinity and “intrinsic activity”, information of some importance to the understanding of the relation of structure to biological action among members of a series of polypeptides related to ACTH.

Isolated adrenal cells: ACTH11–24, a competitive antagonist of ACTH1–39 and ACTH1–10

ACTH4_,0 * has-been demonstrated to be a weak agonist in the isolated adrenal cell system [l] Although low in potency, this fragment of the ACTH molecule, when given in sufficiently large doses induces the same maximum rate of corticosterone production (Bmw) as t a c h t h aracteristic of a strong agonist (ACTH,_39 or ACTH,-24). It appears that the full complement of amino acids involved in activa. tion of receptor is in the region 4 to 10 of the ACTH molecule; amino acids in the region 1 l-24 are not involved in activation but rather provide affinity of ACTH for receptor. In support of this suggestion are the observations of this report to the effect that ACTH, 1--24 is a competitive antagonist of ACTH 1 _-39.

Isolated Pituitary Cells: CRF-Like Activity of Neurohypophysial and Related Polypeptides

Summary Neurohypophysial and structurally related compounds elicit secretion of ACTH from suspensions of isolated pituitary cells. However, the maximum secretion of ACTH in response to the polypeptides is significantly less than that for an extract of rat hypothalamic median eminence (HME) tissue. When added in combination with appropriate doses of HME extract, argvasopressin inhibits ACTH secretion. These findings suggest that the neurohypophysial polypeptides may be partial agonists of corticotropin secretion. The authors are grateful to Beth Wiblin-Portanova and James Larry Simmons for their technical assistance.

Biological half-life of endogenous ACTH.

Summary The biological half-life of endogenous ACTH in blood of the adrenalectomized rat is approximately one minute (0.95 and 1.25 minutes for 2 experiments). No appreciable loss of biological activity occurs when adrenalectomized rat blood is incubated in vitro for one minute at 37°C. The rapid disappearance of ACTH from the blood of the adrenalectomized rat one minute after removal of the adenohypophysis cannot be attributed to inactivation of the hormone by blood itself.

A Technic for the Preparation of Isolated Rat Adrenal Cells

Summary A simple and reproducible technic for the preparation of active isolated rat adrenal cells has been described. Quartered glands are incubated at 37° with mechanical agitation in the presence of trypsin (0.25%). The dispersed cells are collected by centrifugation and suspended in Krebs-Ringer bicarbonate buffer to which has been added glucose (0.2%), albumin (3.0%) and trypsin inhibitor (2 mg/ml). Aliquots of the suspension are incubated at 37° for 2 hr. ACTH in concentrations of 10, 40, and 160 μU increases corticosterone production by the isolated cells. The regression line relating corticosterone production and log dose of ACTH is straight.

A Trypsin Technic for the Preparation of Isolated Rat Anterior Pituitary Cells

Summary Isolated rat anterior pituitary cells have been prepared by mechanical agitation of quartered glands in the presence of trypsin. The cells appear morphologically and functionally intact and contain large amounts of ACTH. The medium separated from cells incubated at 37° for 20 min contains small but significant amounts of ACTH. Acetic acid extracts of rat HME or cerebral cortical tissue and arginine vasopressin increase the ACTH content of the medium. The authors acknowledge the technical assistance of Mary Vegh and Beth Wiblin.

BIOASSAY OF ACTH USING ISOLATED CORTEX CELLS APPLICATIONS: STRUCTURE ACTIVITY RELATIONSHIP FOR ACTH AND ANALOGUES, ASSAY OF CORTICOTROPIN‐RELEASING FACTOR, AND ASSAY OF PLASMA ACTH*

Suspensions of isolated cells from the adrenal cortex respond to the addition of adrenocorticotropic hormone (ACTH) with increased production of cortico~teroids.~-~ The isolated cell system is of particular advantage in assay work. Interanimal variation is eliminated, and the accuracy is that of a chemical analysis rather than a bioassay. Dispersed cells retain the functional capacity of cells in vivo; most importantly they are free from diffusion barriers inherent in adrenal quarters. Sensitivity is such that a dose of one picogram of ACTH induces a significant response. Specificity is excellent; of a large number of substances tested only ACTH and structurally related polypeptides are active. Recently, we have found that suspensions of cells prepared from adrenals of hypophysectomized rats exhibit a remarkably high degree of sensitivity to ACTH as compared to cells prepared from adrenals of intact rats. This phenomenon has been exploited in the development of a bioassay with increased sensitivity. In this report, we shall describe the techniques for dispersion of rat adrenal tissue and for incubation of aliquots of a suspension of isolated adrenal cortex cells. Log dose-response curves relating corticosterone production to dose of ACTH will be displayed and methods for estimation of potency discussed. Accuracy, specificity, sensitivity, and the number of samples that can be analyzed in one day will be compared to other bioassays. We shall describe the relationship of molecular structure to biologic activity, as revealed by the assay of analogues of ACTH. Next, we shall discuss the application of the technic to the assay of the corticotropin releasing factor (CRF) . Finally, we shall describe our experience with the isolated adrenal cortex cell system in the assay of ACTH in the plasma of rat and man.

Corticotropin releasing activity of a pepsin labile factor in the hypothalamus.

Summary Ability of acid extracts of calf hypothalamus (stalk-median eminence area) to induce adrenal ascorbic acid depletion in the median eminence lesioned rat is attributable in large measure to a pepsin labile factor distinct from ACTH and from vasopressin.