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Passive (non-aggressive)

Striving

Control Noradrenaline t Corticosterone Gonadotrophinsf Testosterone f Oxytocin t

Striving

Adrenaline f Prolactin t p endorphins f Renin f Fatty acids t Glycogenosis t

Loss of control ACTHt

Corticosterone f Catecholamines** Gonadotrophinsf Testosterone | Pepsin f

FIGURE 10-8 Defense reaction is activated when the organism is challenged but remains in control. With the loss of control there is activation of the hypothalamopituitary-adrenal axis, and the go-nadotrophic species preservative system shuts down. Visceral fat accumulates with a Cushingoid distribution, and there is a shift from active defense to a passive nonaggressive coping style. Reproduced with permission from J. P. Henry, (1993). Biological basis of the stress response. NIPS 8:69-73.

as loss of control is perceived. A chronic emotional reaction of passivity and defeat to a stressful situation can produce dire consequences as the adrenal hypertrophies and levels of Cortisol continue to increase. This can generate a Cushingoid-like bodily reaction in which visceral fat accumulates (Figure 10-8), blood pressure becomes elevated, and arteriosclerosis and type II diabetes eventually develop. Sequential episodes of elevated glucocorticoids cause sufficient repression of glucose uptake in peripheral cells to involve insulin release from the /6-cells of the pancreas. Eventually, chronic repetitions of this scenario could lead to exhaustion of the /3-cells' ability to produce and secrete insulin. The metabolic changes in the organism induced by elevated levels of glucocorticoids, such as Cortisol, are mediated by the amount of available glucocorticoid receptor proteins located in the cytoplasm of target tissues. Very important organs are liver, lymphocytes (including thymus cells), adipose cells, kidney, anterior pituitary, and various parts of the brain. Some of the effects of Cortisol on tissues of normal or adrenalectomized rats are shown in Table 10-1.

Thus, as will be detailed later, the glucocorticoid hormone affects the transcription and stimulates the production of certain mRNAs, which are subsequently translated into proteins whose actions culminate in an anabolic (liver and kidney) or a catabolic (lymphatic and other tissues) shift in cellular activity. This anabolic effect has been shown clearly for glucocorticoid action on tyrosine aminotransferase in hepatoma cells. The changes in metabolism in each cell containing the hor-

TABLE 10-1 Effects of Glucocorticoids in Various Tissues"

Time after glucocorticoid administration

Sequence of responses

2 min

Liver uptake of hormone, accumulation of unmetabolized hormone in liver Binding to liver cytosol macromolecules Cortisol metabolized to anions in liver, conversion of rough endoplasmic reticulum to smooth endoplasmic reticulum Feedback inhibition of CRH and ACTH Steroid-receptor complex in liver nucleus Glycogen deposition

Nuclear RNA polymerase activity increased Increased RNA synthesis, increased fatty acid release from adipose, decreased glucose utilization in many sensitive tissues peripheral to liver

Protein breakdown in peripheral tissues: decreased glucose utilization, nucleic acid synthesis and protein synthesis decreased, ornithine decarboxylase activity increased in liver, tyrosine aminotransferase activity peaks in liver, tryptophan oxygenase activity peaks in liver, polysome aggregation Increased general liver protein synthesis, decreased glucose utilization, increased hepatic gluconeogenesis, lympholysis Small increase in threonine dehydrase activity (liver), alanine aminotransferase activity increases (liver), many glycolytic enzyme activities increase (liver, kidney), urea cycle enzymes increase: arginine synthetase system; arginine succinase; arginase

" Some of the data of this table are reproduced from Shulster, D., Burstein, S., and Cooke, B. A. (1976). "Molecular Endocrinology of the Steroid Hormones," p. 275. Wiley, New York.

8-20 hr

4 hr to days mone receptor are the culmination of the hormonal effect at the level of the cell. The summation of metabolic changes in different tissues constitutes the adaptation of the organism to stress at this level. Antiinflammatory and antipain effects and Na+ movements contribute as major alterations produced by glucocorticoids. Not all of these biochemical changes are understood. For example, the stress signals mediated by the brain and the feedback effects of Cortisol on elements of the brain remain to be elucidated clearly. Behavioral modifications, if any, to stress may be clarified by future research in this direction. Elaboration of /3-endorphin by the anterior pituitary in response to stress is bound to have important stress adaptation effects mediated by the central nervous system.

B. Corticotropin-Releasing Hormone (CRH)

The 41-amino acid primary sequence of CRH peptide is given in Figure 3-8. Figure 10-7 shows that the response of the hypothalamus to signals from the limbic system is the secretion and subsequent resynthesis of the CRH. CRH is released from specific cells in the hypothalamus. CRH activity appears to be present in other tissues, such as cerebral cortex and liver. It appears to be produced by the placenta and fetal membranes throughout gestation, and a myometrial CRH receptor whose affinity increases during pregnancy has been reported. CRH appears in several tissues as a result of arthritic inflammation.

C. Mode of Action of Releasing Hormones (e.g., CRH)

Following signals from the limbic system to the hypothalamus (Figure 10-7), CRH is released into a closed portal circulation intimately connected with the anterior pituitary (Figure 3-2). Detailed information on anatomical considerations and modes of action of hypothalamic releasing hormones may be found in Chapters 3 and 5.

D. Mode of Action of ACTH

ACTH is the peptide hormone secreted from the anterior pituitary in response to CRH. ACTH is a 39-amino acid peptide; the amino acid sequence is presented in Figure 5-11. The common N-terminal sequence of residues 1-13 of ACTH and residues 1-13 of a-MSH constitutes an important homologous structure. Also, sequence 11-17 of /3-MSH and residues 4-10 of ACTH are homologous. These homologies in amino acid sequence are suggestive of a close relationship or of a common precursor between ACTH and MSH. However, in humans, only ACTH, /3-lipotropin, and /3-endorphin appear to be secreted by corticotrophic cells of the anterior pituitary. Normally, a-MSH derives from the intermediate pituitary cells. Thus, in corticotropic cells of the anterior pituitary, the precursor protein proopiomelanocortin is cleaved by proteases, ultimately to /3-lipotropin with some further cleavage to ACTH, to y-LPH and /3-endorphin together with a 16,000 fragment (big y-MSH) and a smaller fragment. Thus, the main products in corticotropes are ACTH, /3-LTH, y-LPH, and /3-endorphin. In neuroin-termediary cells (and in certain extra pituitary tissues), proopiomelanocortin is cleaved to the same products mentioned for corticotropic cells, but the processing of these products is continued. ACTH is further broken down into a-MSH and CLIP (corticotropic-like intermediary peptide); y-LPH is further cleaved to /3-MSH and /3-endorphin could be cleaved further (into enkephalins?). Also, big y-MSH is further cleaved into two or three smaller products.

In the anterior pituitary corticotroph, endopeptidase cleavages occur at Lys-Arg and Arg-Arg sites; however, Arg-Lys, Lys-Lys, and Lys-Lys-Arg-Arg sites are not cleaved. The specificity apparently resides in the enzymes because the resistant sites in the corticotroph are susceptible sites in the melanotroph.

In the intermediary pituitary cells, ACTH is processed extensively into a-MSH and CLIP. This occurs by endopeptidase and exopeptidase cleavage together with acetylation and amidation. The major products of these cells are a-MSH, CLIP, and N-acetyl-/3-endorphin. In diseases characterized by altered secretion of ACTH, skin pigmentation (theoretically due to MSH action) can occur. It is not completely clear how MSH is secreted in this case; possibly in certain diseases ACTH is degraded to a-MSH. Thus, hyperpig-mentation occurs in diseases of negative feedback by reduced Cortisol levels, and the level of ACTH rises. However, other work suggests that ACTH may be more important than MSH in regulating melanocyte pigmentation. Consequently, the elevated levels of circulating ACTH in Addison's disease may be directly related to changes in skin pigmentation and MSH may not be a critical factor.

It is known that ACTH, MSH, and /3-lipotropin are all coded for by a single gene. The gene gives rise to an mRNA that is translated to a preprotein called the opiocortin precursor or proopiomelanocortin. This is represented diagrammatically in Figure 5-10. Not all of the information in the preprotein sequence is known, as depicted by the blank spaces in the parent peptide chain.

Following the secretion of ACTH into the blood circulation after stimulation by CRH from the hypothalamus, ACTH molecules bind to a specific receptor on the outer cell membranes of all three layers of cells of the adrenal cortex, the zona glomerulosa, the zona fasciculata, and the zona reticularis. The results of many experiments appear in Figure 10-9 in the form of a speculative mechanism of action.

Molecules of ACTH bind to an outer cell membrane receptor. Formation of this complex activates adenylate cyclase of the inner membrane and increases the affinity of the enzyme for ATP. This could be accomplished by a mechanism described in Chapter 1. The cytoplasmic level of cyclic AMP is increased and inactive protein kinases are converted to active catalytic sub-units and cyclic AMP-bound regulatory subunits (see Chapter 1). The active catalytic subunits phosphorylate proteins, which increase the rates of hydrolysis of cholesteryl esters to free cholesterol. The rate of transport of free cholesterol into the mitochondria is in-

Transport in blood i

Glucocorticoid target cells t

Hormone action

Transport in blood i

Glucocorticoid target cells t

Hormone action

FIGURE 10-9 Action of ACTH on an adrenal fasciculata cell to enhance the production and secretion of Cortisol. Abbreviations: AC, adenylate cyclase; cAMP, cyclic AMP; PKA, protein kinase A; SCC, side chain cleavage system of enzymes. StAR (steroid acutely regulated) protein is a cholesterol transporter functioning between the outer and inner mitochondrial membranes (see Figure 2-33).

creased (possibly by the sterol carrier protein, StAR protein), and, perhaps most importantly, the cholesterol side chain cleavage reaction in the mitochondrion, which appears to be catalyzed by a single protein, is stimulated. A novel adrenal peptide of low molecular weight, about 3000, has been shown to appear after ACTH stimulation. It could arise from a precursor protein or by synthesis de novo. Conversion of a precursor to the active peptide could be under the control of ACTH, operating perhaps by way of cyclic AMP, which would control some step in the system, for example, proteolytic processing. Generation of this peptide in cells of the adrenal cortex stimulates, in some way, the side chain cleavage system of enzymes in the mitochondrion, converting cholesterol to A5-pregnenolone. This system is reminiscent of the hypothetical insulin mediator peptide that is claimed to regulate phosphoprotein phosphatases (see Chapter 7). The StAR (steroid acutely regulated) protein may account for many of the changes required for the increased synthesis of Cortisol (see Figure 2-33).

This peptide of about 3000 molecular weight could be the "steroidogenesis activator polypeptide," which appears to be generated in the adrenal cortex in response to ACTH and which stimulates cholesterol side chain cleavage in adrenal mitochondria obtained from rats treated with cycloheximide. The level of this activator increases through stimulation by ACTH or cyclic AMP, and its appearance is inhibited by cycloheximide. There are two other candidates for this stimulatory factor. However, other work indicates the rapidly turning over protein to be the StAR protein, which is a steroid transporter between the outer and inner mitochondrial membranes. Thus, the central effect of ACTH is to activate PKA, which in turn phosphoryl-ates inactive cholesteryl esterase to produce the active enzyme. This causes cholesteryl esters in the droplet to be broken down to free cholesterol, which then enters the mitochondrion. By this means, the substrate-limited side chain cleavage enzyme can now act with greater velocity. In addition, there is some mechanism that is responsive to ACTH, perhaps through the StAR protein, that accounts for a stimulation in the activity of the side chain cleavage event. These effects generate higher levels of Cortisol in the zona fasciculata cells.

Since the side chain cleavage enzyme is rate limiting in the mitochondria at the beginning of Cortisol synthesis, and since this step seems to be substrate limited, the role of ACTH is to produce biochemical changes that increase the substrate cholesterol level in the mitochondrion (Figure 10-9). The intracellular level of Ca2+ may also increase, and Ca2+ has been shown to stimulate the activity of 11 ^-hydroxylase in adrenal mitochondria.

There are longer acting effects of ACTH on adrenal cells than the effects described for increasing the availability of cholesterol to the mitochondria and enhancing the rate of cholesterol side chain cleavage. These relate to increasing the synthetic rate of steroids by ACTH-driven enhancement in the levels of mRNAs of steroid hydroxylases. With bovine adrenocorticol cells in primary culture, ACTH was demonstrated to enhance the levels of message for P450scc, P45011/3, adre-noxin, P45017a, P450C21, and adrenoxin reductase. This probably occurs by increased transcription. The spatial arrangement of some of these proteins in relation to the mitochondrial uptake of cholesterol is shown in Figure 10-10.

E. Sources of Cholesterol and Production of Glucocorticoids

The immediate substrate for steroid hormone biosynthesis is cholesterol (derived from circulating lipoproteins) from intracellular stores of cholesteryl esters or from free cholesterol (Figure 10-9). For a discussion of cholesterol biosynthesis, see Chapter 2, Figures 213-2-15.

Cholesterol is synthesized chiefly by the liver and the intestine and to a smaller extent in other tissues. Cholesterol can be transported to peripheral cells as a component of circulating lipoproteins. Lipoproteins are absorbed by many cells, such as the adrenal cortical cells by first interacting with a membrane receptor (Figure 10-9), and degraded within the cell to avail free cholesterol, which is stored as fatty acid esters in the "lipid droplet." Activation of cholesteryl esterase results from cellular stimulation by ACTH, which elevates levels of cyclic AMP, and the esterase is activated through phosphorylation by protein kinase A.

Cortisol is the main product in the zona fasciculata of human adrenal cortex. Aldosterone is the major steroid of the outer zona glomerulosa. Dehydroepian-drosterone and its sulfate are the principal products of the zona reticularis. These biochemical conversions are shown in Figure 2-21. It is important to realize that the nature of the steroid hormone product is governed by the specificities of the converting enzymes produced in a given cell type. In essence, this specialization constitutes the cell's phenotypic function. For example, genes are expressed in the fasciculata cell encoding information for cytosolic 17a-hydroxylase, 21-hydroxylase, and mitochondrial llß-hydroxylase, whose reactions together with other cellular enzymes produce Cortisol. A zona glomerulosa cell expresses genes for cytosolic 21-hydroxylase, but ^«-hydroxylase is not expressed. Mitochondrial 11/3-hydroxylase and 18-hydroxylase are also expressed, resulting in the

labile protein factor

FIGURE 10-10 Arrangements of the proteins involved in cholesterol side chain cleavage in the inner mitochondrial membrane, showing the postulated site of action of the labile protein factor. Abbreviations. ISP (probably the StAR protein), adrenodoxin; Fp, adrenodoxin reductase; IDH, isocitrate dehydrogenase; ISOCIT, isocitrate; aOG, a-oxoglutarate. Reproduced with permission from Simpson and Waterman (1995). Steroid hormone biosynthesis in the adrenal cortex and its regulation by adrenocorticotropin in Endocrinology; DeGroot, L. J. (ed.) (1995). W.B. Saunders, Philadelphia, PA. 3rd ed., ed. Vol. 2, pp. 1630-1641.

labile protein factor

FIGURE 10-10 Arrangements of the proteins involved in cholesterol side chain cleavage in the inner mitochondrial membrane, showing the postulated site of action of the labile protein factor. Abbreviations. ISP (probably the StAR protein), adrenodoxin; Fp, adrenodoxin reductase; IDH, isocitrate dehydrogenase; ISOCIT, isocitrate; aOG, a-oxoglutarate. Reproduced with permission from Simpson and Waterman (1995). Steroid hormone biosynthesis in the adrenal cortex and its regulation by adrenocorticotropin in Endocrinology; DeGroot, L. J. (ed.) (1995). W.B. Saunders, Philadelphia, PA. 3rd ed., ed. Vol. 2, pp. 1630-1641.

production of aldosterone rather than Cortisol. Note that the side chain cleavage of cholesterol, the rate-limiting step in steroid hormone biosynthesis, occurs in the mitochondria. This is not a simple one-step reaction, but apparently the total reaction is catalyzed by a single protein (Figure 2-21). Specific signals to the cells of the adrenal cortex determine which steroid hormone will be synthesized at an increased rate compared to the unstimulated state. Thus, ACTH stimulates fasciculata and reticularis cells to produce Cortisol and dehydroepiandrosterone. Glomerulosa cells also have an ACTH receptor that plays a role in aldosterone synthesis through the stimulation of protein kinase A. The regulation of aldosterone synthesis will be discussed later.

F. Mechanism of Secretion of Glucocorticoids

Secretion can be said to occur at two levels in the processes of synthesis and release of steroid hormones. Biosynthetic intermediates move between cytoplasmic and mitochondrial compartments and finally to the cytoplasm and cell exterior in the case of Cortisol as the end product. Thus, the removal of A5-pregnenolone from the mitochondria to the cytoplasm for action by the microsomal 3/3-hydroxy-A5-steroid dehydrogenase to produce progesterone is a transportation event. 17«-Hydroxylase is also located on the microsomes in the cytoplasm, as is 21-hydroxylase. The resulting 17a-hydroxydeoxycorticosterone must be transported back to the mitochondria, perhaps by way of a transporting protein, but 11 /3-hydroxylation to form Cortisol occurs on the inner mitochondrial membrane (see Table 2-5). Cortisol comes out of the mitochondrion and moves out of the cell into the extracellular space and into the bloodstream. It is difficult to find evidence of "packaging" of newly synthesized Cortisol, and little is known about its exit from the cell. Consequently, it seems possible that the newly formed steroid simply diffuses into the cell membrane and out into the extracellular space.

Free cholesterol is the substrate for the mitochondrial synthesis of Cortisol. Electron microscopy shows that lipid droplets reside near mitochondria. Chol-esteryl ester hydrolase and cholesterol-transporting protein could be activated by enhanced levels of cyclic AMP produced by ACTH action (if they are active as phosphorylated forms). Values for the normal ranges of corticosteroids produced in a day, plasma levels, protein binding, and urinary excretion are presented in Table 10-2.

G. Transport of Glucocorticoids in the Blood (CBG)

A corticosteroid-binding globulin (CBG) is present in blood. This protein is also referred to as transcortin. It is synthesized in the liver and exported to the

TABLE 10-2 Normal Values in the Investigation of Adrenal Cortex0

Hormones and steroids Normal range Hormones and steroids Normal range

TABLE 10-2 Normal Values in the Investigation of Adrenal Cortex0

Hormones and steroids Normal range Hormones and steroids Normal range

Production rates of major hormones

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