upward or downward deviations from some predetermined set point, but chang- ing environmental demands often require temporary deviation from constancy. This can be accomplished in some cases by adjusting the set point and in other cases by a signal that overrides the set point. For example, epinephrine secreted by the adrenal medulla in response to some emergency inhibits insulin secretion and increases glucagon secretion even though the concentration of glucose in the blood may already be high.Whether the set point is changed or overridden, devi- ation from constancy is achieved by the intervention of some additional signal from outside the negative feedback system. In most cases that additional signal originates with the nervous system. Hormones also initiate or regulate processes that are not limited to steady or constant conditions.Virtually all of these processes are self-limiting, and their control resembles negative feedback, but of the open-loop type. For example, oxytocin is a hormone that is secreted by hypothalamic nerve cells, the axons of which terminate in the posterior pituitary gland. Its secretion is necessary for the extrusion of milk from the lumen of the mammary alveolus into secretory ducts so that the infant suckling at the nipple can receive milk.In this case, sensory nerve endings in the nip- ple detect the signal and convey afferent information to the central nervous system, which in turn signals release of oxytocin from axon terminals in the pituitary gland. Oxytocin causes contraction of myoepithelial cells, resulting in delivery of milk to the infant.When the infant is satisfied, the suckling stimulus at the nipple ceases. P OSITIVE FEEDBACK Positive feedback means that some consequence of hormonal secretion acts on the secretory cells to provide augmented drive for secretion. Rather than being self-limiting, as with negative feedback, the drive for secretion becomes Regulation of Hormone Secretion 43 al p ha cells beta cells liver glucose (+) (-) (+) (-) hormone secretion rate blood g lucose concentration insulin 100 200 normal range glucagon glucagon insulin Figure 26 Negative feedback regulation of blood glucose concentration by insulin and glucagon. (−), Inhibition; (+) stimulation. progressively more intense. Positive feedback systems are unusual in biology because they terminate with some cataclysmic, explosive event.A good example of a positive feedback system involves oxytocin and its other effect: causing con- traction of uterine muscle during childbirth (Figure 27). In this case the stimulus for oxytocin secretion is dilation of the uterine cervix. On receipt of this infor- mation through sensory nerves, the brain signals the release of oxytocin from nerve endings in the posterior pituitary gland. Enhanced uterine contraction in response to oxytocin results in greater dilation of the cervix, which strengthens the signal for oxytocin release, and so on until the infant is expelled from the uterine cavity. 44 Chapter 1. Introduction CNS posterior pituitary 1 2 3 4 5 (+) (+) (+) (+) (+) (+) Figure 27 Positive feedback regulation of oxytocin secretion. (1) Uterine contractions at the onset of parturition apply mild stretch to the cervix. (2) In response to sensory input from the cervix, oxy- tocin is secreted from the posterior pituitary gland, and stimulates (+) further contraction of the uterus, which in turn stimulates secretion of more oxytocin (3), leading to further stretching of the cervix, and even more oxytocin secretion (4), until the fetus is expelled (5). FEED FORWARD Feed forward controls can be considered as anticipatory or preemptive and prepare the body for an impending change or demand. For example, following a meal rich in glucose, secretory cells in the mucosa of the gastrointestinal tract secrete a hormone that signals the pancreas to secrete insulin (see Chapter 5). Having increased insulin present in the blood by the time the glucose is absorbed thus moderates the change in blood glucose that might otherwise occur if insulin were secreted after the blood glucose concentrations started to increase. Unlike feedback systems, feed forward systems are unaffected by the consequences of the changes they evoke, and simply are shut off when the stimulus disappears. MEASUREMENT OF HORMONES Whether it is for the purpose of diagnosing a patient’s disease or for research to gain understanding of normal physiology, it is often necessary to measure how much hormone is present in some biological fluid. Chemical detection of hor- mones in blood is difficult.With the exception of the thyroid hormones, which contain large amounts of iodine, there is no unique chemistry that sets hormones apart from other bodily constituents. Furthermore, hormones circulate in blood at minute concentrations, which further complicates the problem of their detection. Consequently, the earliest methods developed for measuring hormones are bioas- says and depend on the ability of a hormone to produce a characteristic biological response. For example, induction of ovulation in the rabbit in response to an injec- tion of urine from a pregnant woman is an indication of the presence of the pla- cental hormone chorionic gonadotropin and is the basis for the rabbit test, which was used for many years as an indicator of early pregnancy. Before hormones were identified chemically they were quantitated in units of the biological responses they produced.For example, a unit of insulin is defined as one-third of the amount needed to lower blood sugar in a 2-kg rabbit to convulsive levels within 3 hours. Although bioassays are now seldom used, some hormones, including insulin, are still standardized in terms of biological units. Terms such as milliunits and microunits are still in use. I MMUNOASSAYS As knowledge of hormone structure increased, it became evident that peptide hormones are not identical in all species. Small differences in amino acid sequence, which may not affect the biological activity of a hormone, were found to produce antibody reactions with prolonged administration. Regulation of Hormone Secretion 45 Hormones isolated from one species were recognized as foreign substances in recipient animals of another species, which often produced antibodies to the foreign hormone. Antibodies are exquisitely sensitive and can recognize and react with tiny amounts of the foreign material (antigens) that evoked their production, even in the presence of large amounts of other substances that may be similar or different. Techniques have been devised to exploit this characteristic of antibodies for the measurement of hormones, and to detect antibody–antigen reactions even when minute quantities of antigen (hormone) are involved. Radioimmunoassay Reaction of a hormone with an antibody results in a complex with altered properties such that it is precipitated out of solution or behaves differently when subjected to electrophoresis or adsorption to charcoal or other substances. A typical radioimmunoassay takes advantage of the fact that iodine of high specific radioactivity can be incorporated readily into tyrosine residues of peptides and pro- teins and thereby permits detection and quantitation of tiny amounts of hormone. Hormones present in biological fluids are not radioactive, but can compete with radioactive hormone for a limited number of antibody binding sites.To perform a radioimmunoassay, a sample of plasma containing an unknown amount of hor- mone is mixed in a test tube with a known amount of antibody and a known amount of radioactive iodinated hormone.The unlabeled hormone present in the plasma competes with the iodine-labeled hormone for binding to the antibody. The more hormone present in the plasma sample, the less iodinated hormone can bind to the antibody. Antibody-bound radioactive iodine is then separated from unbound iodinated hormone by any of a variety of physicochemical means, and the ratio of bound to unbound radioactivity is determined. The amount of hormone present in plasma can be estimated by comparison with a standard curve constructed using known amounts of unlabeled hormone instead of the biological fluid samples (Figure 28). Although this procedure was originally devised for protein hormones, radioimmunoassays are now available for all of the known hormones. Production of specific antibodies to nonprotein hormones can be induced by first attaching these compounds to some protein, e.g., serum albumin. For hormones that lack a site capable of incorporating iodine, such as the steroids, another radioactive label can be used or a chemical tail containing tyrosine can be added. Methods are even available to replace the radioactive iodine with fluorescent tags or other labels that can be detected with great sensitivity. The major limitation of radioimmunoassays is that immunological rather than biological activity is measured by these tests, because the portion of the hor- mone molecule recognized by the antibody probably is not the same as the portion 46 Chapter 1. Introduction Measurement of Hormones 47 H-Ab H-Ab H-Ab H-Ab H-Ab H-Ab Ab Ab Ab Ab H H H H H H H H H + H H-Ab H-Ab H-Ab H-Ab H-Ab H-Ab Ab Ab Ab Ab H H H H H H H H H + H H H H H H H H-Ab H-Ab H-Ab H-Ab H-Ab H-Ab H-Ab Ab Ab Ab Ab H H H H H H + H Ab Ab Ab Ab Ab Ab Ab Ab Ab Ab Ab Ab Ab Ab Ab A 10 0 101 0 20 40 60 80 100 550330 hormone concentration ( n g /ml ) B 1 2 3 B/F (%) Figure 28 (A) Competing reactions that form the basis of the radioimmunoassay. Labeled hormone (H), shown in blue, competes with hormone in the biological sample (shown in black) for a limiting amount of antibodies (Ab).As the concentration of hormone in the biological sample rises (rows 1, 2, and 3) decreasing amounts of the labeled hormone appear in the hormone–antibody (H–Ab) complex and the ratio of bound/free labeled hormone (B/F) decreases. (B) A typical standard curve used to estimate the amount of hormone in the biological sample.A B/F ratio of 70% corresponds to 5 ng/ml in this example. recognized by the hormone receptor. Thus a protein hormone that may be biologically inactive may retain all of its immunological activity. For example, the biologically active portion of parathyroid hormone resides in the amino-terminal one-third of the molecule, but the carboxyl-terminal portion formed by partial degradation of the hormone has a long half-life in blood and accounts for nearly 80% of the immunoreactive parathyroid hormone in human plasma. Until this problem was understood and appropriate adjustments were made, radioimmunoassays grossly overestimated the content of parathyroid hormone in plasma (see Chapter 8). Similarly, biologically inactive prohormones may be detected. By and large, discrepancies between biological activity and immunoac- tivity have not presented insurmountable difficulties and in several cases even led to increased understanding. Immunometric Assays Even greater sensitivity in hormone detection has been attained with the development of assays that can take advantage of exquisitely sensitive detectors that 48 Chapter 1. Introduction reporter antibody reporte r enzym e capture antibodies hormone sepharose bead Figure 29 Sandwich-type assay. The first (capture) antibody is linked to a solid support such as an agarose bead.The hormone to be measured is shown below the bead.The second (reporter) antibody is linked to a reporter enzyme, which, on reacting with a test substrate, gives a colored product. In this model, the amount of reported antibody captured is directly proportional to the amount of hormone in the sample being tested. Measurement of Hormones 49 Figure 30 Changes in hormone concentrations in blood may follow different patterns. (A) Daily rhythm in luteinizing hormone (LH) secretion. (From Bremer et al., J. Clin. Endocrinol. Metab. 56, 1278, 1983, by permission of The Endocrine Society.) (B) Hourly rhythm of testosterone secretion. (From Yamaji et al., Endocrinology 90,771,1972,by permission of the author.) (C) Episodic secretion of prolactin. (From Hwang et al., Proc. Natl.Acad. Sci.U.S.A. 68, 1902, 1971, by permission of The Endocrine Society.) can be coupled to antibodies. Such assays require the use of two different antibod- ies that recognize different immunological determinants in the hormone. One antibody is coupled to a solid support such as an agarose bead or is adsorbed onto the plastic of a multiwell culture dish. The biological sample containing the unknown amount of hormone is then added under conditions in which there is a large excess of antibody, so that essentially all of the hormone can be bound by the antibody.The second antibody, linked to a fluorescent probe or an enzyme that can generate a colored product, is than added and allowed to bind to the hormone that is held in place by the first antibody, so that the hormone is sandwiched between the two antibodies and acts to link them together. In this way the amount of antibody-linked detection system that is held to the solid support is directly proportional to the amount of hormone present in the test sample (Figure 29). These assays are sometimes called sandwich assays, or enzyme-linked immuno- sorbent assays (ELISAs), when the second antibody is coupled to an enzyme that converts a substrate to a colored product. HORMONE LEVELS IN BLOOD It is evident now that hormone concentrations in plasma fluctuate from minute to minute and may vary widely in the normal individual over the course of a day. Hormone secretion may be episodic, pulsatile, or follow a daily rhythm (Figure 30). In most cases it is necessary to make multiple serial measurements of hormones before a diagnosis of a hyper- or hypofunctional state can be confirmed. Endocrine disease occurs when the concentration of hormone in blood is inappropriate for the physiological situation rather than because the absolute amounts of hormone in blood are high or low. It is also becoming increasingly evi- dent that the pattern of hormone secretion, rather than the amount secreted, may be of great importance in determining hormone responses.This subject is discussed further in Chapter 11. It is noteworthy that for the endocrine system as well as the nervous system additional information can be transmitted by the frequency of sig- nal production as well as by the signal. SUGGESTED READING Conn, P. M. (ed.) (1999). “Handbook of Physiology, Section 7: Endocrinology, Volume 1: Cellular Endocrinology.” American Physiological Society and Oxford University Press, New York. (This volume provides in-depth coverage of many of the topics considered in this chapter.) Dannies, P. S. (1999). Protein hormone storage in secretory granules: Mechanisms for concentration and sorting. Endocr. Rev. 20, 3–21. Gerber, S. H., and Sidhof, T. C. (2002). Molecular determinants of regulated exocytosis. Diabetes 51 (Suppl. 1), S3–S11. 50 Chapter 1. Introduction Gether, U. (2000). Uncovering molecular mechanisms involved in activation of G protein-coupled receptors. Endocr. Rev. 21, 90–113. McKenna, N., Rainer, J., Lanz, B., and O’Malley, B.W. (1999). Nuclear receptor coregulators: Cellular and molecular biology. Endocr. Rev. 20, 321–344. Pearson, G., Robinson, F., Beers Gibson, T., Xu, B., Karandikar, M., Berman, K., and Cobb, M. H. (2001). Mitogen-activated protein (MAP) kinase pathways: Regulation and physiological functions. Endocr. Rev. 22, 153–183. Pekary, A. E., and Hershman, J. M. (1995). Hormone assays. In “Endocrinology and Metabolism” (P. Felig, J. D. Baxter, and L. A. Frohman, eds.), 3rd Ed., pp. 201–218. McGraw-Hill, New York. Pratt, W. B., and Toft, D. O. (1997). Steroid receptor interactions with heat shock protein and immunophilin chaperones. Endocr. Rev. 18, 306–360. Spiegel,A. M. (2000). G protein defects in signal transduction. Horm. Res. 53 (Suppl. 3), 17–22. Suggested Reading 51 [...]... conserved residue (F88S) in the homeodomain of PROP-1 J Clin Endocrinol Metab 85, 27 79 27 85 Palkovits, M (1999) Interconnections between the neuroendocrine hypothalamus and the central autonomic system Front Neuroendocrinol 20 , 27 0 29 5 76 Chapter 2 Pituitary Gland Shupnik, M A., Ridgway, E C., and Chin, W W (1989) Molecular biology of thyrotropin Endocr Rev 10, 459–475 Vale, W., Rivier, C., Brown,... β-lipotropin ACTH NH2-terminal peptide JP ACTH NH2-terminal peptide JP α-MSH CLIP β-lipotropin γ-lipotropin β-endorphin corticotrope γ-lipotropin β-endorphin melanotrope Figure 3 Proteolytic processing of proopiomelanocortin (POMC) POMC after removal of the signal peptide is shown on the first line The first cleavage by prohormone convertase 1 releases β-lipotropin.The second cleavage releases ACTH A... of the intermediate lobe and split ACTH into α-melanocyte-stimulating hormone (α-MSH) and the corticotropin-like intermediate lobe peptide (CLIP), and divide β-lipotropin into γ-lipotropin and β-endorphin Some cleavage of β-lipotropin also takes place in the corticotrope Additional posttranslational processing (not shown) includes removal of the carboxyl-terminal amino acid from each of the peptides,... resembles that of ACTH SUGGESTED READING Andersen, B., and Rosenfeld, M G (20 01) POU domain factors in the neuroendocrine system: Lessons from developmental biology provide insights into human disease Endocr Rev 22 , 2 35 Eipper, B A., and Mains, R E (1980) Structure and biosynthesis of Pro-ACTH/endorphin and related peptides Endocr Rev 1, 1 27 Fiddes, J C., and Talmadge, K (1984) Structure, expression, and... pituitary, but because an alternative mode of splicing of the RNA transcript is possible, two GH isoforms are produced The larger form is the 22 -kDa molecule (22 K GH), which is about 10 times more abundant than the smaller, 20 -kDa molecule (20 K GH), which lacks amino acids 32 to 46.The other GH gene (hGH V) appears to be expressed only in the placenta and is the predominant form of GH in the blood of pregnant... factors called prop-1 and pit-1 Appearing transiently early in the development process, prop-1 appears to foretell expression of the pituitary-specific pit-1, and its name derives from “prophet of pit-1.”The transcription factor pit-1 is required not only for differentiation of these cell lineages, but also for continued expression of GH, PRL, and the beta subunit of TSH throughout life; pit-1 also regulates... glands secrete hormones that selectively inhibit secretion of either FSH or LH These complex events are discussed in detail in Chapters 11 and 12 The GnRH gene encodes a 9 2- amino-acid preprohormone that contains the 10-amino-acid GnRH peptide and an adjacent 56-amino-acid GnRHassociated peptide (GAP), which may also have some biological activity GAP is found with GnRH in nerve terminals and may be secreted... produced GHRH is unknown Somatostatin was originally isolated from hypothalamic extracts based on its ability to inhibit GH secretion.The somatostatin gene codes for a 118-amino-acid preprohormone from which either a 14-amino-acid or a 28 -amino-acid form of somatostatin is released by proteolytic cleavage Both forms are similarly active.The remarkable conservation of the amino acid sequence of the somatostatin... and (2) because they all arise from the transcription and translation of the same gene (Figure 3) The gene product is proopiomelanocortin (POMC), which consists of 23 9 amino acids after removal of the signal peptide The molecule contains 10 doublets of basic amino acids (arginine and lysine in various combinations), which are potential sites for cleavage by trypsin-like POMC β-lipotropin ACTH NH2-terminal... human corticotropes are ACTH and β-LPH Because final processing of POMC occurs in the secretory granule, β-LPH is secreted along with ACTH Cleavage of β-LPH also occurs to some extent in human corticotropes, so that some β-endorphin may also be released, particularly when ACTH secretion is brisk The intermediate lobe in some animals gives rise principally to - and β-MSH Because the intermediate lobe . 47 H-Ab H-Ab H-Ab H-Ab H-Ab H-Ab Ab Ab Ab Ab H H H H H H H H H + H H-Ab H-Ab H-Ab H-Ab H-Ab H-Ab Ab Ab Ab Ab H H H H H H H H H + H H H H H H H H-Ab H-Ab H-Ab H-Ab H-Ab H-Ab H-Ab Ab Ab Ab Ab H H H H H H + H Ab Ab Ab Ab Ab Ab Ab Ab Ab Ab Ab Ab Ab Ab Ab A 10 0 101 0 20 40 60 80 100 550330 hormone. transcript is pos- sible, two GH isoforms are produced. The larger form is the 22 -kDa molecule (22 K GH), which is about 10 times more abundant than the smaller, 20 -kDa mol- ecule (20 K GH), which. 61 ACTH β-endorphinCLIPα-MSHJPNH 2 -terminal peptide NH 2 -terminal peptide corticotrope melanotrope γ-lipotropin β-endorphin β-lipotropin β-lipotropin ACTHJP γ-lipotropin POMC Figure 3 Proteolytic