The action of adrenocortcotrophic hormone (ACTH) upon the rate of corticosteroid biosynthesis by the isolated perfused bovine adrenal
Macchi, Italo Alden
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Introduction Tile action of adrenocorticotrophic hormone (CTH) in promoting corticosteroid biosynthesis has been demonstrated both in vivo and in vitro. An in vitro action of ACTH in corticosteroidogenesis was first demonstrated in the isolated bovine adrenal perfused with homologous citrated blood. This effect was obtained initially with glands perfused by cannulation of the aorta. Subsequently, it was discovered that this preparation could transform certain corticosteroids and respond to ACTH stimulation more efficiently by perfusing through the adrenal vein and lacerating the surface of the gland (This latter preparation is referred to as the V-L adrenal gland). Furthermore, it was demonstrated that under the influence of large concentrations of a crude Armour protein ACTH preparation the rate of corticosteroid output of the V-L gland was not proportional to concentration. Since the concentrations employed may have stimulated maximal rates of corticosteroid biosynthesis, significant differences in the dose-response relationship might be apparent only at lower dose levels. In the studies to be reported, the bovine V-L adrenal preparation has been used to investigate and extend studies to quantitate various aspects of ACTH action in corticosteroid biosynthesis. Specifically, the purpose of these investigations has been as follows: 1. To evaluate the rate of corticosteroid biosynthesis of the V-L perfused bovine adrenal under conditions in which ACTH concentration was not a limiting factor. 2. To demonstrate the action of various small ACTH concentrations upon the rate of corticosteroid output. 3. To demonstrate the duration of ACTH action in corticosteroidogenesis after its withdrawal from the circulation. 4. To compare the relative corticosteroidogenic activities and adrenal ascorbic acid depleting activities of various ACTH preparations of different degrees of purity and of growth hormone. Methods In these studies V-L bovine adrenal glands were perfused with homologous citrated blood containing added antibiotics at a temperature of 38°C and a flow rate of 1 liter per hour independent of gland weight using nonpulsatile pressures. An initial perfusion period of 48 minutes with blood not containing added ACTH was employed to stabilize the preparation and to obtain the level of corticosteroid output in the absence of added hormone. Thereafter, each gland was perfused continuously for varying periods of time with or without added ACTH, and perfusate samples were collected separately during successive 18 minute intervals for corticosteroid analysis. Fresh ACTH dissolved in physiological saline was added to warmed blood at 18 minute intervals in an attempt to maintain a constant supply at or near the desired concentration since ACTH may be partially inactivated in blood with time. Corticosteroids were dialyzed from 20 mL. perfusate aliquots and were measured chemically as formaldehydogenic substance (F.S.) liberated upon periodic acid oxidation of unpurified dried dialysate chloroform extracts. Corticosteroid output has been expressed as micrograms DOC equivalent/gram adrenal/hour perfusion in excess of that obtained in the absence of added ACTH. These data were corrected for variable recovery by dialysis of cortisone added to bovine blood in the same approximate range of concentrations as the corticosteroids in adrenal perfusate samples only when absolute values were compared. Rate of Corticosteroidogenesis With Maximal Concentrations Of ACTH Under conditions in which ACTH concentration was not a limiting factor, the efficiency of the V-L adrenal gland for studying ACTH action as well as the influence of certain factors other than concentration upon the rate of corticosteroid biosynthesis were evaluated. Using ACTH concentrations known to produce maximal rates of corticosteroidogenesis, the output of cow glands was 38736.2 ug./gm./hour as compared to a value of 376.55 ug./gm./hour in the absence of added ACTE. This represents a 10 fold increase in corticosteroid output rate as the result of ACTH stimulation. Under these conditions there appeared to be no significant correlation at the 5% level of confidence between corticosteroid output and flow rate in the range from 0.5 to 1.5 ml./min./gm. or adrenal weight in the range from 10 to 28 gms. Under identical conditions of perfusion, steer glands exhibited a corticosteroid output inferior to that obtained with cow glands while the output of bull glands appeared to be intermediate. The difference observed with steer glands was statistically significant(P<0.005), but with bull glands it was not possible to evaluate the available, limited data statistically. In accord with other in vitro findings which have shown that the 17-hydroxylating enzyme activity demonstrable in cow adrenal tissue is reduced or absent in steer glands, these data suggest that the differences in corticosteroid output observed between cow and steer glands may reflect basic differences in enzyme systems involved in corticosteroid biosynthesis. Finally, a comparison with other in vitro studies shows the V-L gland to be superior consistently in magnitude of corticosteroid output and in the increased response attained with maximal ACTH stimulation. This is not the case consistently in the absence of ACTH. Effect of ACTH Concentration To demonstrate the effect of ACTH concentration upon the rate of corticosteroid biosynthesis in the V-L adrenal preparation as well as to determine the output rate obtained during consecutive 18 minute intervals of stimulation, cow adrenals were perfused separately with ACTH concentrations of 0.001, 0.01, O.1, and 10 I.U./liter of blood. The results obtained demonstrate that a minimal concentration of ACTH is required to evoke a consistently positive response, that maximal adrenal response is attained with concentrations betvveen 0.01 and 0.1 I.U./1iter, and that independent of concentration peak response is achieved only after 18 to 36 or 36 to 54 minutes after initiation of ACTH treatment. The magnitude of corticosteroid output appears to be a function of the log concentration of ACTH within the range of 0.001 to 0.1 I.U./liter but is not proportional to concentration. Differences in output observed between consecutive dosage levels within this range were statistically significant. A depression of output observed with concentrations of 10 I.U./liter was not significant statistically. While extended perfusion with blood for 2.5 hours before ACTH treatment does not eliminate significantly the latent period required to achieve peak output, the responsiveness of the isolated V-L cow adrenal to ACTH stimulation is fully maintained for this period of time. The minimal concentration required to achieve maximal response in the V-L perfused cow adrenal as determined in these studies is in accord with that calculated from in vivo data in man to stimulate effectively the human adrenal but is 400 to 1000 times less than the required concentration reported by others to produce maximal or near maximal response in incubated adrenal tissue. Duration of ACTH Action To demonstrate whether maintaining an effective concentration of circulating ACTH is the sole factor required to increase corticosteroidogenesis or whether other factors are also involved, the corticosteroid output of V-L cow adrenals initially stimulated with ACTH was measured for a period of time after withdrawal of the hormone from the circulation. Adrenals were first perfused for 18 minutes with an ACTH concentration of 1 I.U./liter. After stimulation the gland was perfused with 2.4 liters of blood not containing added ACTH over a period of 144 minutes, and corticosteroid output was measured during separate 18 minute intervals. In approximate accord with other findings, the results obtained demonstrate consistently that increased corticosteroid production attained during ACTH stimulation declined exponentially to 45% at 18 to 36 minutes and to 18% at 126 to 144 minutes after withdrawal of ACTH from the circulation. It is suggested that this persistence of ACTH activity might occur as a result of (a) adsorption and binding of ACTH in the medium by adrenal tissue or(b) stimulation by ACTH of a reaction involved in corticosteroid biosynthesis which once initiated proceeds for a limited period of time in the absence of ACTH by accumulation of corticosteroid intermediaries. On the basis of these suggestions, the following are possible explanations for the removal of ACTH activity with time: (a ) ACTH is removed from a bound tissue complex by washing and/or by enzymatic destruction, or (b) the postulated accumulation of intermediaries might be exhausted with time. If ACTH is bound in adrenal tissue, prolonged perfusion with minimal effective ACTH concentrations might lead in time to accumulation of sufficient ACTH by the adrenal to increase corticosteroid output. This possibility was tested by the continuous perfusion of a minimal effective concentration of ACTH (0.001 I.U./liter) over a period of 2 hours during which time corticosteroid output was measured at 18 minute intervals. The results indicate that after a small initial increase obtained during the first 72 minutes of perfusion, there is no further increase with time despite continued ACTH treatment. It is concluded that if ACTH is accumulated by the perfused adrenal, the amount retained at this concentration is insufficient to activate corticosteroid biosynthesis. However, these findings do not preclude the possibility of accumulation. Comparative Corticosteroidogenic Activities of Various ACTH Preparations, Growth Hormone, and Insulin Studies were undertaken to compare the corticosteroidogenic activities of a variety of protein hormone preparations administered in successively increasing concentrations. In addition to the standard Armour protein ACTH, a preparation purified by oxycellulose treatment of glacial acetic acid pituitary extracts and an ACTH polypeptide mixture were tested. A purified growth hormone preparation was tested since it has been suggested that this hormone stimulates adrenal production of mineralocorticoid activity. Insulin was tested also since it is a hormone without known direct influence upon corticosteroid biosynthesis. A sigmoid log dose-response curve was obtained only with the hypophyseal preparations. These dose-response curves are not considered to represent the absolute effect of ACTH concentration since factors other than ACTH concentration have been shown in these studies to influence the rate of corticosteroid biosynthesis in this type in vitro preparation. The corticosteroidogenic activity of the grovuth hormone has been attributed tentatively to an ACTH contaminant rather than to the growth hormone moiety. Independent of concentration, insulin did not increase corticosteroid output above that attained with minimally effective concentrations of ACTH. Relative to the Armour preparation, a comparison of the concentration required to evoke 50% of the maximal corticosteroid output rate and the adrenal ascorbic acid depleting activities of each of the hypophyseal preparations shows a good degree of correlation.
Thesis (Ph.D.)--Boston University
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