Action Of Adrenocorticotropic Hormone Research Articles

Adenosine 3′: 5′-Monophosphate Binding Activity and Guanosine 3′: 5′-Monophosphate Binding Activity during Adrenocorticotropic Hormone Stimulation in Rat Adrenal Gland

The effect of adrenocorticotropic hormone (ACTH) on the change in adenosine 3′: 5′-monophosphate (cyclic AMP) binding activity and guanosine 3′: 5′-monophosphate (cyclic GMP) binding activity in adrenal glands of hypophysectomized rats was examined by measuring cyclic [3H]AMP-binding and cyclic [3H]GMP-binding, respectively. When ACTH1–24-retard (200 μg/100 g body weight/day) was injected into rats for 3 days, the increase in cyclic AMP-binding activity (activity/100 g body weight) found in the supernatants, mitochondria, and microsomes of adrenal glands was greater than the growth of adrenal glands, whereas the increase in cyclic GMP-binding activity (activity/100 g body weight) was almost equal to that of adrenal size. Administration of less than 200 μg ACTH1–24-retard did not effectively stimulate both activities. During 3 days, a mixture of ACTH1–24-retard and -immediate (200 μg and 5 U/100 g body weight/day) induced increases of 2.8-fold in adrenal weight, 6.0-, 1.5-, and 5.8-fold in cyclic AMP-binding activity and 1.3-, 1.3-, and 2.1-fold in cyclic GMP-binding activity in the supernatants, microsomes, and mitochondria, respectively. In regard to the effect of the ACTH1–24-mixture on the specific activity (activity/μg protein), the activity of the cyclic AMP-binding protein increased in supernatants and mitochondria but not in microsomes, whereas that of the cyclic GMP-binding protein decreased in all three subcellular fractions. The effect of administration of the ACTH1–24-mixture for 9 days was marked on the stimulation of cyclic AMP-binding activity, while the specific activity was not significantly affected. These results indicated the presence of a differential effect on the cyclic AMP- and cyclic GMP-binding activities during ACTH action, and suggested that both cyclic AMP- and cyclic GMP-binding proteins may be involved in ACTH action and play an important role in hormonal control of steroidogenesis through regulation of the adrenal cortex.

On the Neurochemical Mechanism of Action of ACTH

Publisher Summary This chapter attempts to outline the three most currently proposed neurochernical mechanisms by which peptides may affect brain function. First of all, neuropeptides may act transsynaptically in peptidergic synapses. Secondly, neuropeptides may exert their action by modulation of ongoing transmission in specific neuronal networks and, finally, they can be considered to act as effectors (neurohormones) in a classical endocrine view with the brain as the target tissue for circulating neurohormones. Emphasis is given to the behaviorally active fragments of adrenocorticotropic hormone (ACTH) and their modulation of phosphorylation of synaptic membrane constituents (proteins, lipids), supporting the view that such peptides may act as neuromodulators. In view of the wide variety of behavioral, neurophysiological and neurochemical effects on record, one should reserve judgment regarding the unifying central mechanism of action of ACTH. Therefore, for the moment, it seems appropriate to presume multiple sites and mechanisms of action of behaviorally active neuropeptides chemically related to ACTH.

Influence of adrenocorticotropin on transport of a cholesteryl linoleate-low density lipoprotein complex into adrenal tumor cells.

The action of adrenocorticotropin (ACTH) on the specific (receptor-mediated) uptake of cholesteryl linoleate . low density lipoprotein complexes was examined in Y-1 mouse adrenal tumor cells. High affinity binding (KA 4.1 X 10(8) M) was observed with ACTH; lower affinity was seen in the absence of ACTH. The effect of ACTH was observed within 10 min at physiological concentrations of low density lipoprotein (100 microgram/ml). Binding was followed by uptake (internalization) of the ester . lipoprotein complex which was transported to lysosomes. The site of action of ACTH was localized to the uptake process (internalization) since no effect of ACTH was observed on binding to the cell membrane nor on movement of internalized complex to lysosomes. ACTH increases the transport of cholesterol derived from cholesterol ester to the mitochondria. This cholesterol is converted to 20 alpha-hydroxypregn-4-en-3-one and this conversion is accelerated by ACTH. Dibutyryl cyclic AMP (but not butyrate) also stimulates uptake of cholesteryl linoleate . low density lipoprotein. The process stimulated by ACTH and dibutyryl cyclic AMP is specific for low density (as opposed to high density) lipoprotein and for ACTH as distinct from other peptide hormones. The possible physiological importance of this response is considered.

The role of cyclic AMP in aldosterone production by isolated zona glomerulosa cells.

The role of cyclic AMP in the regulation of aldosterone production by adrenocorticotropic hormone (ACTH), angiotensin II (A II), potassium, and serotonin was examined in collagenase-dispersed adrenal glomerulosa cells. The ability of 8-bromo cyclic AMP and choleragen to stimulate maximum aldosterone production indicated that cyclic AMP could act as second messenger for certain of the aldosterone-stimulating factors. The actions of ACTH and choleragen on aldosterone and cyclic AMP production were correlated in dog and rat cells, and a similar relation was seen during stimulation of rat cells by serotonin. In contrast, A II and potassium did not cause changes in cyclic AMP formation while stimulating aldosterone production. Intracellular and receptor-bound cyclic AMP were increased 3-fold by 10(-7) M ACTH but not by A II. Addition of a phosphodiesterase inhibitor increased the magnitude of the cyclic AMP response to ACTH but did not change the lack of stimulation by A II or potassium. In dog cells, the effects of A II and potassium on aldosterone production were partially additive to those of ACTH, choleragen, and 8-bromo cyclic AMP. In contrast, no additivity was observed between A II and potassium, or between combinations of the cyclic AMP-dependent stimuli. These results indicate that the actions of ACTH on aldosterone secretion are mediated by cyclic AMP formation, whereas A II and potassium stimulate aldosterone production through an independent mechanism. The lack of additivity between steroid responses to A II and potassium suggests that these factors could share a common mode of action on steroidogenesis in zona glomerulosa cells.

Adrenocorticotropin action in isolated adrenal cells. The intermediate role of cyclic AMP in stimulation of corticosterone synthesis.

Adrenocorticotropin action in isolated adrenal cells. The intermediate role of cyclic AMP in stimulation of corticosterone synthesis.

Effect of corticotropin treatment in vivo on the synthesis of a specific adrenal cytosolic protein. Characterization by dual-labelling technique and polyacrylamide-gel electrophoresis

The effect of corticotropin in vivo on total and specific protein synthesis in the adrenal was studied. Adrenal slices from control and corticotropin-treated animals were incubated with [14C]- and [3H]-leucine respectively, followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of subcellular components. With this sensitive dual-labelling technique the following results were obtained. There was a general trophic effect on most adrenal proteins, but corticotropin produced a marked stimulation of a specific adrenal cytosolic protein. This protein has mol.wt. approx. 30 000 and pI 5.5. Corticotropin increased the incorporation of labelled leucine into proteins within 4 h, but no effect was observed before 2 h and after 16 h there was no further increase. These data suggest that this protein is not involved in the corticosteroidogenic action of corticotropin, but rather in the trophic action of this hormone.

Mechanism of corticotropin action in rat adrenal cells I. The effects of inhibitors of protein synthesis and of microfilament formation on corticosterone synthesis

The contribution of protein synthesis and formation of microtubules and microfilaments to corticotropin-stimulated steriodogenesis in rat adrenal cell suspensions has been assessed by use of a series of inhibitors to each function. Five inhibitors of protein synthesis (cycloheximide, puromycin, blastocidin S, anisomycin, and trichodermin) each exhibited time-dependent inhibition of corticotropin-stimulated steroidogenesis. For the first 30 min, steroidegenesis was more extensively inhibited than protein synthesis, after which the effectiveness of the inhibitors diminished on steroidegenesis but not on protein synthesis. The reversal effects was not observed at high levels of inhibitors. One inhibitor of microfilament fromation (cytochalasin B) and four inhinitors of microtubule formation (colchicine, podophyllotoxin, vinblastine sulfate and griseofulvin) inhibited steroidogenesis without inhibiting protein synthesis and without any reversal effect with prolonged incubation. The actions of all ten inhinitors were shown to be fully reversible. Cell superfusion of adrenal cells showed that the decay of steroidogenesis upon addition of all the protein synthesis inhibitors was similar to decay upon removal of corticotropin from the medium ( t 1 2 = 4–6 min ). Recoveries from inhibition upon removal of the inhibitors were similar to each other and comparable to initial corticotropin stimulation of the cells (lag of 3–5 min, f 1 2 = 7–9 min ). Similar kinetics of inhibition and recovery were observed for vinblastine sulfate while a direct inhbition of cytochrome P-450 sec by an aminoglutethimide was complete within 1 min and was rapidly reversed. Injection of each inhibitors (all classes) into hypophysectomized rats inhibited the elevation of plasma corticosterone by corticotropin. The extent of cholesterol combination with cytochrome P-450 sec in adrenal mitochondria isolated from these rats was also decreased by all inhbitors. Decreases in plasma corticosterone correlated directly with decreases in cholesterol combination with cytochrome P-450 sec ( r = 0.94). It is concluded that protein synthesis and steroidogenesis must be intimately coupled propbably due to the requirement of a labile protein for cholesterol transport to cytochrome P-450 sec. An involvement of microtubules and microfilaments in this process is clearly indicated.

Behavioral influences of the neuropeptides ACTH and MSH: A methodological review

During the past 20 years, there has been repidly accumulating evidence that polypeptide hormones of pituitary origin are important mediators of behavior. The research has been focused primarily upon the actions of adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (MSH). A large number of methodological difficulties have developed and are in need of careful review. Important pharmacological and behavioral variables are reviewed both in general and as they relate to current research on the actions of ACTH and MSH. In addition, suggestions are given for control and/or study of these variables. Finally, the main integrating hypotheses, fear, memory and attention, are reviewed and evaluated.

Glucocorticoid Modulation of Adrenocorticotropin-Induced Melanogenesis in the Cloudman S-91 Melanoma <i>in vitro</i>

The acute in vitro action of adrenocorticotropin (ACTH) and corticosterone alone and in combination were determined in the Cloudman S-91 melanoma grown in vivo. Hormone-treated melanoma dice (5-240 min) were analyzed for tyrosinase activity (EC 1.14.18.1), cyclic AMP (cAMP) and cyclic GMP (cGMP). ACTH elevated cAMP levels in the S-91 melanoma. However, these increases in cAMP were not accompanied by increased tyrosinase activity. Corticosterone depressed cAMP levels while stimulating tyrosinase activity. ACTH plus corticosterone produced an early cAMP peak followed by depression. ACTH plus corticosterone stimulated tyrosine activity coincident with the early cAMP peak followed by a drop in tyrosinase activity which was subsequently elevated. cGMP levels were not altered by any hormone treatment. The results indicate that cAMP is not the sole modulator of tyrosinase activity and suggest the interaction of ACTH, corticosterone and cAMP in the regulation of melanoma tyrosinase activity.

Interaction of ACTH, corticosterone and cyclic nucleotides in harding-passey melanoma malanogenesis

The acute in vitro action of adrenocorticotropin (ACTH) and corticosterone alone and in combination were determined in the Harding-Passey (HP) melanoma grown in vivo. Hormone treated melanoma dice (5--240 min) were analyzed for tyrosinase activity, cyclic AMP (cAMP) and cyclic GMP (cGMP). ACTH elevated cAMP and cGMP levels 20- and 13-fold, respectively, in the HP melanoma. However, these large increases in cyclic nucleotide levels were accompanied by only a 49% increase in tyrosinase activity. Corticosterone elicited a similar response. ACTH plus corticosterone produced an early cAMP and cGMP peak followed by depression. ACTH plus corticosterone stimulated tyrosinase activity coincident with the early cyclic nucleotide peak followed by a drop in tyrosinase activity which was subsequently elevated. The results indicate that neither cAMP nor cGMP are the sole modulators of tyrosinase activity and suggest the interaction of ACTH, corticosterone and cyclic nucleotides in the regulation of melanoma tyrosinase activity.

Purification and characterization of high-molecular-weight forms of adrenocorticotropic hormone of ovine pituitary glands

A highly purified preparation of high-molecular-weight adrenocorticotropic hormone (ACTH) was prepared from ovine pituitary glands by dilute acetic acid extraction, oxycellulose fractionation. Sephadex gel filtration, and affinity chromatography on immobilized alphap(1-39)ACTH antibodies. Two ACTH peptides of molecular weights of 24 000 and 34 000 were detected by sodium dodecyl sulfate-acrylamide gel electrophoresis in this preparation. It appeared that the immobilized antibodies adsorbed two forms equally well and could not distinguish between them under the conditions used. These two ACTH peptides were found to be present in crude extracts of ovine pituitary glands, indicating that they were not artifacts produced by the purification procedure. The high-molecular-weight forms of ACTH were found to be susceptible to degradation by tissue enzymes. They could be easily destroyed during the extraction, if precautions were not taken. Moreover, they were poorly adsorbed by oxycellulose which had been used for the adsorption of ACTH activity from crude preparations by most investigators. These properties probably accounted for the fact that high-molecular-weight forms of ACTH remained undetected until very recently.

Localization of the metabolic processes affected by calcium during corticotropin action

To define the role of calcium during corticotropin-induced steroidogenesis, adrenal sections were incubated under conditions of varying degrees of calcium depletion. Corticosterone production, [ 14C]leucine incorporation into protein, and tissue cyclic AMP levels were measured concomitantly. Omitting calcium from the incubation inhibited all three processes to variable extents, thus limiting conclusions regarding which process is most dependent on calcium. While calcium was required during the early phase of corticotropin action, it was not required during later phases; rapid induction of calcium defiency did not diminish the heightened rate of steroidogenesis previously induced by corticotropin in the presence of calcium. Thus, while calcium is required for induction of steroidogenesis factor(s), the operation of the latter is not dependent upon calcium in the extracellular fluid.

On the requirement for protein synthesis during corticotropin-induced stimulation of cholesterol side chain cleavage in rat adrenal mitochondrial and solubilized desmolase preparations

Following simple homogenization, significant amounts of mitochondrial-derived, cholesterol side chain cleaving enzyme (desmolase) activity are recovered in rat adrenal 105 000 × g- supernatant fraction. Corticotropin administration enhances soluble desmolase activity, and cycloheximide potentiates this effect. The lipid droplet fraction which has no desmolase activity markedly enhances pregnenolone synthesis in the soluble desmolase preparations, presumably by supplying free cholesterol substrate. Corticotropin, particularly with cycloheximide pretreatment, enhances lipid fraction activity. Thus increased cholesterol availability may largely explain the corticotropin effect on the soluble desmolase system. Since protein synthesis is required for corticotropin activity in intact mitochondria, but not in calcium-swollen mitochondria or the soluble enzyme system, the labile protein apparently required during corticotropin action may function to overcome a “barrier” which exists only in the intact mitochondria and restrains cholesterol side chain cleavage.

Some characteristics of adrenal steroidogenesis and their possible relationships to the action of the adrenocorticotropic hormone

The effects of adrenocorticotropic hormone (ACTH) on the biphasic time curve for rat adrenal mitochondrial pregnenolone synthesis, the ACTH sensitive step of steroidogenesis in the adrenal cortex, have been investigated. The observed curve is characterized by a relatively short primary rate followed by a longer, slower secondary rate. The administration of ACTH in vivo results in an increase in both rates; however, the percentage increase is greater for the secondary rate. An increase in the duration of the primary rate was also observed. Similar results were obtained when the complete steroidogenic sequence of reactions to form corticosterone were investigated in a cell-free system. Mitochondrial pregnenolone, both total and that present in the membranes, is increased by ACTH. These data make a model for ACTH action in which an inhibition by pregnenolone of its own synthesis is controlled by the rate of efflux of pregnenolone from the mitochondria unlikely. Addition of 20,22-dihydroxycholesterol, a precursor of corticosterone, results in a decrease in the duration of the primary rate. Furthermore, the extent of the decrease depends upon the concentration of 20,22-dihydroxycholesterol. This result suggests that possible involvement of an inhibitor on the duration of the primary rate of the observed biphasic rate curve. The bearing of these results on some models for ACTH action is discussed.

Adenylate cyclase activity and steroidogenesis in phenotypic revertants of an ACTH-insensitive adrenal tumour cell line

A LARGE body of evidence implicates adenylate cyclase and cyclic AMP as mediators of adrenocorticotropic hormone (ACTH)-stimulated adrenal steroidogenesis (see ref. 1 for review). Recent observations, however, suggest that ACTH acts at separate sites to increase steroidogenesis and cyclic AMP accumulation, and that cyclic AMP may not be an important component of ACTH-stimulated steroidogenesis2–5. An ACTH-responsive adrenocortical tumour cell line6 (clone Y1) and a derivative clone insensitive to ACTH7 (clone Y6), have been used to examine the role of adenylate cyclase and cyclic AMP in adrenal steroidogenesis1,7. Y6 cells do not respond to ACTH with increased steroidogenesis but do respond to added cyclic AMP7. In addition, these cells have an adenylate cyclase system insensitive to ACTH, probably accounting for their variant phenotype1. Y6 cells, when grown as tumours, regain the ability to respond to ACTH with increased steroidogenesis, and continue to express the ACTH-sensitive phenotype after prolonged periods in culture and extensive dilution7. Y6 cells, after animal passage, provide a system of phenotypic revertants to examine the linkage of different biochemical events to ACTH insensitivity7,8. We have now examined adenylate cyclase activity in phenotypic revertants of Y6 cells induced by animal passage to assess the role of this enzyme in steroidogenesis. After animal passage, Y6 cells regained the ability to respond to ACTH both with increased steroidogenesis and with increased adenylate cyclase activity. These data indicate that the actions of ACTH on adenylate cyclase activity and steroidogenesis are closely linked.

Molecular weights of adrenocorticotropic hormone in extracts of anterior and intermediate-posterior lobes of mouse pituitary.

The molecular weight of the adrenocorticotropic hormone (ACTH) activity in extracts of the separated anterior and intermediate-posterior lobes of the mouse pituitary was determined by gel filtration in guanidine-HCl. Following dilution or removal of the guanidine-HCl, ACTH activity was quantitated by both radioimmunoassay and bioassay. Extracts of the intermediate-posterior lobe contain approximately a tenth as much ACTH activity as extracts of the anterior lobe. In extracts of both the anterior and the intermediate-posterior lobes, about half of the immunological ACTH activity is similar in size to porcine ACTH (molecular weight 4000--5500). Two higher molecular weight forms of ACTH account for the remainder of the ACTH activity. About 40% of the immunological ACTH activity in anterior lobe extracts has a molecular weight of 6500--9000. Extracts of both the anterior lobe and the intermediate-posterior lobe contain ACTH activity with a molecular weight of 20,000--30,000. While this 20,000--30,000 molecular weight ACTH accounts for only 5% of the immunological ACTH activity in the anterior lobe extracts, it accounts for half of the immunological ACTH activity in extracts of the intermediate-posterior lobe. Extracts of an ACTH-secreting mouse pituitary tumor cell line (AtT-20/D-16v) contain the same three molecular weight forms of ACTH. Each of the three molecular weight forms of ACTH has a characteristic ratio of biological ACTH activity to immunological ACTH activity, independent of the source of the material (anterior lobe, intermediate-posterior lobe, or mouse pituitary tumor cell line).

High molecular weight forms of adrenocorticotropic hormone in the mouse pituitary and in a mouse pituitary tumor cell line

Denaturing solvents have been used to determine the molecular weight of the adrenocorticotropic hormone (ACTH) activity in mouse pituitary, in an ACTH secreting mouse pituitary tumor cell line (AtT-20/D-16v), and in the tissue culture medium from the pituitary tumor cells. ACTH activity was quantitated by radioimmunoassay and by bioassay. It is possible to utilize guanidine hydrochloride or sodium dodecyl sulfate in characterizing the multiple forms of ACTH because treatment of porcine ACTH (the 39 amino acid polypeptide form of ACTH, alpha(1-39)), pituitary extracts, tumor cell extracts, and tumor cell tissue culture medium with these denaturants does not diminish the immunological ACTH activity. Based on gel filtration in the presence of guanidine hydrocholoride, extracts of the pituitary tumor cells and the mouse pituitary contain three distinct molecular weight classes of ACTH activity. The major form of ACTH has a molecular weight similar to alpha(1-39) (molecular weight 4000-5500), but there are significant amounts of two higher molecular weight forms of ACTH: molecular weight 6500-9000 and molecular weight 20,000-30,000. The 6500-9000 molecular weight form of ACTH is the major form of ACTH in the tissue culture medium; there is no peak of alpha(1-39) size ACTH in the medium. In the radioimmunoasay all three forms of ACTH generate competitive binding curves parallel to that of porcine alpha(1-39); in the bioassay (stimulation of steroidogenesis in a mouse adrenal tumor cell line) the dose response curve for each of the molecular forms of ACTH is parallel to that for porcine alpha(1-39).

INCREASE IN PLASMA TESTOSTERONE CONCENTRATION AFTER INJECTION OF ADRENOCORTICOTROPHIN INTO THE BOAR

Injection of a 'rapid-acting' preparation of porcine adrenocorticotrophic hormone (ACTH) into three boars resulted in a rapid rise in plasma testosterone levels which accompanied the expected rise in plasma corticosteroids. Urinary dehydroepiandrosterone (DHA) levels were measured in one boar and were found to be raised also. The results suggest that the effect involved enhaced testicular steroid activity and was related to the dosage of ACTH employed. This action of ACTH is thought to be mediated through the adrenal cortex since injection of cortisol elecited a rise in testosterone similar to that observed after injection of ACTH. ACTH had no effect on testosterone levels in a castrated boar. When a 'long-acting' preparation of ACTH was administered to two boars twice daily for 5 days, testosterone levels were depressed. It was concluded that ACTH may bring about an increase or a decrease in plasma testosterone levels in the boar depending upon the length of time increased levels of ACTH are present in the circulation.

Cytochrome P-450 of adrenal mitochondria. In vitro and in vivo changes in spin states.

Steroid-induced difference spectra have been used to examine the combination of cholesterol with adrenal mitochondrial cytochrome P-450 which participates in cholesterol side chain cleavage (P-450scc) and the depletion of cholesterol from the cytochrome which results from turnover of the enzyme system. Type I difference spectra-induced by cholest-5-ene-3beta, 25-diol (25-hydroxycholesterol) and cholest-5-ene-3beta, 20 alpha, 22R-triol (20alpha, 22R dihydroxycholesterol) have been used to quantitate binding of cholesterol to two sites (I and II) on cytochrome P-450scc. The action of adrenocorticotropic hormone (ACTH) in vivo and the action of calcium or phosphate ions on isolated mitochondria stimulate the combination of cholesterol with site I but not site II. Cholesterol derived from lecithin-cholesterol micelles, however, binds to both sites. Malate-induced cholesterol depletion occurred at a comparable rate to the transfer of cholesterol from lecithin-cholesterol micelles. However, a residual proportion of cholesterol-cytochrome P-450scc complexes remained, even after 10 min of exposure to malate, and was of similar magnitude in mitochondria from both cycloheximide-treated and stressed rats. It is suggested that this reflects a less reactive form of cholesterol-cytochrome complex. Steroid-induced difference spectra indicate that sites I and II on cytochrome P-450scc are similarly depleted after metabolism of mitochondrial cholesterol in vitro and after inhibition of the action of ACTH in vivo. Anaerobiosis of adrenal cells after excision of the accumulation of cholesterol at cytochrome P-450cc. When anaerobiosis was prevented, cytochrome P-450scc in the freshly isolated mitochondria was apparently essentially free of complexed cholesterol, irrespective of the extent of ACTH action. For 30 min after suspension of the mitochondria in 0.25 M sucrose at 4 degrees, cholesterol combines with cytochrome P-450scc. The extent of this process was not affected by the presence of cycloheximide during ether stress treatment of the rats. It is concluded that there are at least two pools of mitochondrial cholesterol with access to cytochrome P-450scc but that ACTH stimulates only the pool which most readily interacts with the cytochrome.

Evolution and selective reduction of hormonal secretion in the mammotropic pituitary tumor (MtT-F4).

Severe hypertensive vascular disease was produced in 1969 in our laboratory by implantation of the mammotropic tumor MtT-F4, secreting very large amounts of corticotropin, prolactin and growth hormone in Fischer F 344 female rats. The hypertension was caused by adrenal cortical dysfunction with hyperproduction of deoxycorticosterone (DOC), and consequent sodium retention. Ultrastructural changes in the zona fasciculata cells of the adrenal glands were also evident in the tumor-implanted hypertensive rats, thus confirming the impaired adrenal steroidogenesis. The tumor has been maintained in our laboratory by continuous passages from rat to rat or it was being stored frozen for 3 years and then reimplanted. Implants with these new strains of tumor resulted in marked reduction or total loss of its hypertensinogenic ability. In addition to the failure to develop hypertension, the same strains of tumor also showed a reduced secretion of corticotropin, reflected in a smaller increase of the adrenal weight, a smaller reduction of thymic weight and a less marked sodium retention with normal levels of plasma sodium. The less elevated secretion of corticotropin was also indicated by a more normal ultrastructural appearance of the zona fasciculata cells. No alterations in the tumor secretion of growth hormone and prolactin was evident with the continuous passage from rat to rat, at least judging from the effect of the two hormones on their target organs. It is still controversial as to whether a synergistic action of corticotropin, growth hormone and prolactin is necessary in order to cause severe hypertensive vascular disease in the rats implanted with the mammotropic tumor. However, the present experiment has outlined, that among the three hormones secreted, corticotropin definitely plays a major role in the development of such disease.