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Clinical proteomics 11(1), 37 (2014). doi:10.1186/1559-0275-11-37, Published by Humana Press, Totowa, NJ

The gastrointestinal tract is the largest endocrine organ and was the site where the first hormones were discovered: secretin in 1902 and gastrin in 1905. Discovery of these gut peptides led to the concept of blood-borne communication between cells. Gut peptide hormones and neurotransmitters regulate the complex processes of digestion, motility, epithelial growth, and integrity. Investigation of this complex endocrine organ has depended on the development of well-characterized radioimmunoassays. Radioimmunoassays have increased our understanding of appetite regulation and of pathological processes affecting the gut, including gastroenteropancreatic neuroendocrine tumors. The object of this chapter is to provide an overview of measuring gastrointestinal hormones and neuropeptides by radioimmunoassay.

Current knowledge of the relationship between the HPA axis and physical exercise in normal and highly trained individuals highlights the importance of adequate preparation for exercise. The activation of the HPA (hypothalamic–pituitary–adrenal) axis in response to physical activity in humans has been abundantly reported. The HPA axis consists of three structurally independent components including the hypothalamus, the anterior pituitary, and the adrenal cortex. These structures are intimately interacting through the release of neuroendocrine messengers and the activation or the inhibition of the nervous system influencing the functions of most organs and tissues in the body. Such interactions imply among others peptide hormones CRF (corticotrophin-releasing factor), ACTH (adrenocorticotropic hormone), and arginine vasopressin (AVP) and their specific receptors, as well as smaller molecular species such as corticosteroids. The information summarized in this chapter points out that the HPA axis importantly influences stress-induced functions and that the intensity of HPA axis activation is intimately related to the type of training and the intensity at which it is performed. Altogether, these effects indicate the influences of the activation of the HPA axis in physical activity.

Mass spectrometry methods have the potential to measure different hormones during the same analysis and have improved specificity and a wide analytical range compared with many immunoassay methods. Increasingly in clinical laboratories liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays are replacing immunoassays for the routine measurement of testosterone, 17-hydroxyprogesterone, and other steroid hormones. Reference LC-MS/MS methods for steroid, thyroid, and peptide hormones are being used for assessment of the performance and calibration of commercial immunoassays. In this chapter, the general principles of tandem mass spectrometry and examples of hormone assays are described.

When I became a physician and an endocrinologist in the early 1960s, peptide hormone sequencing was still in its infancy; it was also far removed from my immediate interests. Through chance encounters with prominent teachers and mentors, I later became increasingly convinced that elucidation of the primary sequence of peptide hormones is key to understanding their production as well as their functions in human health and disease. My interest for pituitary hormones led me to discover that the sequence of β-melanocyte-stimulating hormone was contained within that γ and β-lipotropins and could be released from the latter by limited endoproteolysis. This prohormone theory became the leitmotiv of my career as a clinician/scientist. Through serendipity and the efforts of many laboratories including mine, this theory has now been widely confirmed, extended to various precursor proteins and implicated in many diseases. It has led to our discovery of the proprotein convertases.

Tumors originating in the diffuse neuroendocrine system are characterized by a propensity to secrete a variety of peptide hormones, biogenic amines, and other vasoactive substances. These circulating factors can give rise to a remarkable array of clinical signs and symptoms including flushing, diarrhea, diabetes, hypoglycemia, rash, bronchospasm, and valvular heart disease. Neuroendocrine tumors which produce pathologically elevated hormone levels associated with a corresponding clinical syndrome are termed “functioning” whereas tumors lacking any association with a hormonal syndrome are considered “nonfunctioning.”

We know neuropeptides now for over 40 years as chemical signals in the brain. The discovery of neuropeptides is founded on groundbreaking research in physiology, endocrinology, and biochemistry during the last century and has been built on three seminal notions: (1) peptide hormones are chemical signals in the endocrine system; (2) neurosecretion of peptides is a general principle in the nervous system; and (3) the nervous system is responsive to peptide signals. These historical lines have contributed to how neuropeptides can be defined today: "Neuropeptides are small proteinaceous substances produced and released by neurons through the regulated secretory route and acting on neural substrates." Thus, neuropeptides are the most diverse class of signaling molecules in the brain engaged in many physiological functions. According to this definition almost 70 genes can be distinguished in the mammalian genome, encoding neuropeptide precursors and a multitude of bioactive neuropeptides. In addition, among cytokines, peptide hormones, and growth factors there are several subfamilies of peptides displaying most of the hallmarks of neuropeptides, for example neural chemokines, cerebellins, neurexophilins, and granins. All classical neuropeptides as well as putative neuropeptides from the latter families are presented as a resource.

The description of the incretin effect was first described in 1964 by Elwick when he observed a greater insulin secretory response if glucose was administered orally compared to intravenously despite similar elevations in plasma glucose. This incretin effect is attributed to the release of peptide hormones from the K and L cells in the intestines in the response to eating, so that approximately 60% of the insulin subsequently secreted is related directly to the effect of incretins [1]. Glucagon like peptide-1 (GLP-1) is secreted by the L cells, suppresses postprandial glucagon secretion, reduces appetite and stimulates insulin secretion in a dose dependent manner.

Acromegaly is usually caused by somatotroph adenomas of the pituitary gland that secrete excess growth hormone (GH). This state of GH excess stimulates IGF-I, a peptide hormone that stimulates cell growth. This chronic stimulation of tissue growth leads to multiple symptoms and signs that comprise the acromegalic condition, characterized by tissue overgrowth leading to significant morbidity. The diagnosis can be confirmed by measuring GH suppression following oral glucose administration and measurement of serum IGF-I concentration. Early diagnosis is facilitated by an awareness of the presenting features of the disease and the availability of high-quality, specific, and sensitive diagnostic tests.

Steroid hormones play an important role in coordinating rapid, as well as sustained, responses of target cells in complex organisms to changes in the internal and external environment. The broad physiologic effects of steroid hormones in the regulation of growth, development, and homeostasis have been known for decades. Often, these hormone actions culminate in altered gene expression, which is preceded many hours earlier by enhanced nutrient uptake, increased flux of critical ions, and other preparatory changes in the synthetic machinery of the cell. Because of certain homologies of molecular structure, specific receptors for steroid hormones, vitamin D, retinoids, and thyroid hormone are often considered a receptor superfamily. The actions of ligands in this superfamily are postulated to be regulated by receptors in the cell nucleus. On binding ligand, nuclear receptors associate with target genes and permit selective transcription. This genomic mechanism is generally slow, often requiring hours or days before the consequences of hormone exposure are evident.

The kidney plays an essential role in the maintenance of life in higher organisms, not only through regulating the blood pressure and body fluid homeostasis and clearing the wastes, but also by acting as a major endocrine organ. The kidney secretes (1) renin, a key enzyme of the renin-angiotensin system (RAS) that leads to the production of a potent pressor hormone angiotensin, and produces the following hormones and humoral factors: (2) kallikreins, a group of serine proteases that act on blood proteins to produce a vasorelaxing peptide bradykinin; (3) erythropoietin (EPO), a peptide hormone essential for red blood cell (RBC) formation by the bone marrow; and (4) 1,25-(OH)2 vitamin D3, the active form of vitamin D essential for calcium homeostasis, which is produced by the proximal tubule cells via the enzyme 1α-hydroxylase.

This chapter describes how adrenocorticotropin (ACTH) can be measured in biological fluids. The principle can be applied to similar peptide hormones. Following an introduction of the principle of the method, details are given on sample collection and storage, and materials required. Detailed descriptions are given for the methodology with a "Notes" section that gives practical tips on the more complex or difficult steps in the analytical process. A brief bibliography lists peer reviewed articles relevant to the development and application of this method.

High-performance liquid chromatography (HPLC) is a refinement of traditional column chromatographic techniques. The speed of analysis and the resolution are increased with new column-packing materials and eluant pumped through the column at high pressure. The potential for achieving measurements of hormones in small volumes of plasma or urine is limited, both in normal and pathological situations. Using HPLC with ultraviolet absorption, the detection limit is only nanogram amounts of hormones per milliliter of blood serum. The applications of the technique to specific hormones from recent and older literature will be used throughout this chapter to illustrate aspects of the technology.

The natriuretic peptide family consists of a group of structurally similar, but genetically distinct, peptide hormones that play a major role in the regulation of cardiovascular, endocrine, and renal homeostasis. Since the discovery of atrial natriuretic peptide almost 25 yr ago, remarkable progress has been made in the field of natriuretic peptide research. This chapter reviews current knowledge in biology of the natriuretic peptides, including mechanisms of synthesis and release, biological effects, and clearance.

Oxytocin (OT) and arginine vasopressin (AVP) were among the first peptide hormones to be isolated and sequenced (1). Both OT and AVP consist of nine amino acids that form a ring structure from disulfide bonds, bridging two cysteine residues. Although the two hormones are similar in structure, differing at only two positions, their functions appear to be quite distinct. Oxytocin has been implicated in the induction of labor during parturition, milk ejection during lactation (2), and the control of reproductive and maternal behaviors (3). Arginine vasopressin, also known as antidiuretic hormone, plays a crucial role in maintaining osmotic homeostasis and vascular tone, and has more recently been implicated in cognition and social behaviors (4–6).

The side chain groups of amino acid residues in polypeptide hormones, neurotransmitters, growth factors, substrates, antigens, and other bioactive peptides have been demonstrated to be extremely important pharmacophores for receptor/acceptor binding and for signal transduction (1). Therefore, complete understanding of the stereochemical requirements of side chain groups important in peptide ligand-receptor/acceptor interactions plays a crucial role in the rational design of bioactive peptides and nonpeptide mimetics. Topographical considerations provide a major approach to explore the structure-activity relationships of the side chains of the amino acid residues in the bioactive peptide analogues (2). This approach can be realized by incorporation of conformationally constrained unusual amino acids into a peptide or nonpeptide template. Thus, design and synthesis of unusual amino acids with specific biased side chain conformations have tremendous impact on the exploration of the mechanism of peptide ligand-receptor recognition and of activating signal transduction. Our laboratory has been engaged in the design and synthesis of conformationally constrained amino acids for almost a decade. One representative family among the special amino acids we have designed is the β-substituted unusual amino acids, in which substitution of the diastereotopic β-hydrogens of natural α-amino acids provides an approach to topographic control of side chain conformation (3). Incorporation of these β-substituted unusual amino acids into bioactive peptide hormones has produced highly selective and potent peptide hormone analogues and provided new insights into the stereochemical requirements in peptide-receptor interactions (4).

Knowledge of the pathophysiological roles of gastrointestinal (GI) peptides has progressed with the advances in peptide chemistry. However, a decisive step has been the development of potent and specific peptide receptor antagonists, which not only became important experimental tools for elucidating the physiological roles of peptide hormones, but also in defining the contribution of peptide hormones to disease states, but they may even ultimately prove to be important as therapeutic agents. Among all GI peptides, nonpeptidic molecules with potent and selective antagonistic properties have been made available for gastrin and cholecystokinin (CCK). Comprehensive reviews on the biological actions of CCK (Mutt, 1980; Morley, 1982; Crawley and Vanderhaeghen, 1985; Albus, 1988; Brawley 1991; Hokfelt et al., 1991; Moran and McHugh, 1991; Smith and Gibbs, 1992; Crawley and Corvin 1994; Walsh, 1994a, 1994b) and on CCK receptors (Dourish and Hill, 1987; Jensen et al., 1990; Woodruff and Hughes, 1991; Silvente-Poirot et al., 1993; Williams and Blevins, 1993; D’ Amato et al., 1994; Rasmussen, 1995; Wang, 1995) are available. Moreover, Adler and Beglinger (1991), provide comprehensive coverage of the role of CCK in the control of GI functions with the use of CCK antagonists.

Polypeptide hormone receptors were first identified using direct binding techniques in which radiolabeled ligands of high purity were incubated with tissue preparations, cells, or subcellular membrane fractions in the presence and absence of unlabeled hormone (Cuatrecasas et al., 1971; Freychet et al., 1971). In this manner the key receptor properties of specificity and high affinity were characterized (Roth, 1973; Posner et al., 1974). Using this relatively simple approach peptide hormone receptors were found in tissues not previously regarded as targets for the hormones (Gavin et al., 1972; Posner et al., 1973; Kelly et al., 1974). Thus insulin receptors were observed in brain and placenta in addition to being present in the classical targets of muscle, liver, and adipose tissue (Posner et al., 1973, 1974; Kelly et al., 1974). It was also soon shown that tissue peptide hormone receptor levels were modulated, and that the hormone itself played a role in both down-regulating (Gavin et al., 1974) and up-regulating (Posner et al., 1975) receptors, in part depending on ambient ligand concentration (Barash et al., 1988). Using receptor preparations it was possible to measure hormone levels in analogous fashion to radioimmunoassay except that a higher degree of hormonal integrity was assessed in radioreceptor assays (Posner et al., 1980).

α-Melanotropin (α-melanocyte stimulating hormone, α-MSH), Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2, is one of the first peptide hormones isolated from the pituitary gland. The biologic functions of α-MSH and structure-activity relationships of α-MSH analogues before 1993 have been extensively researched and reviewed (1–12). This hormone plays a well-known and principal role in pigmentation. Other effects ascribed to this hormone include: 1. Regulation of the release of pituitary and peripheral hormones, such as somatostatin and corticotropin 2. Sebotrophic effects, adrenal steroidogenesis, immune response, and cardiovascular and metabolic effects 3. Important roles in the nervous system, particularly in the central nervous system (CNS)

Among the many hormones and growth factors that collaborate to allow normal growth and development, growth hormone (GH) is one of the most important and extensively studied. GH has captured the interest of basic and clinical endocrinologists for many reasons. GH was recognized early in the history of modern endocrinology as a pituitary-derived peptide hormone that is a major positive regulator of growth. As a more sophisticated appreciation of hormonal physiology emerged, an important theory of hormone action, termed the “somatomedin hypothesis,” was developed to explain the indirect growth promoting effects of GH; this concept spawned a burgeoning interest in insulin-like growth factors (IGFs) and related hormones.

The insulin-like growth factor (IGF) system is a multilayered network of molecules involved in the regulation of cell growth and death in many organs and tissues, normal and pathologic. The IGF system also participates in the modulation of important cell- and tissue-specific physiological and metabolic functions, via endocrine, paracrine, and autocrine interactions. The IGFs, IGF-receptors (IGF-R), IGF binding proteins (IGFBPs), and the IGFBP-receptors and proteases all feature prominently in the healthy prostate and in the common conditions of abnormal prostate growth, benign prostatic hypertrophy (BPH), and prostatic carcinoma (CaP). The IGF system similarly participates in the proliferation and hypertrophy of the bladder, both in congenital obstructive conditions and in acquired diseases of the urinary system. In the testis, the IGF system interacts with gonodatropins, androgens, and multiple other peptide hormones in the coordination of growth, spermatogenesis, and steroidogenesis.

Multiple pituitary hormone deficiency (MPHD) denotes subnormal production of growth hormone (GH) and one or more of the five other peptide hormones that are normally produced by the anterior pituitary. Synonyms for MPHD include combined pituitary hormone deficiency (CPHD) and panhypopituitarism. The latter term can include deficiency of antidiuretic hormone (ADH), which is produced in hypothalamic nuclei and released from the posterior pituitary.

Gastrin was recognized as a hormone that stimulates gastric secretion almost 60 yr ago. Its existence was proved by Komarov (1) in 1938. It was first isolated and characterized by Gregory and Tracy (2,3) in 1961, and since then became a subject of intensive study. It is one of the most well studied peptide hormones (4). Nevertheless, in spite of an enormous amount of research that was already conducted on gastrin, it might be just a beginning of an exciting story. New findings suggest that gastrin has many more important functions than had been recognized in the past, and that it has additional, until recently, unknown cellular and molecular targets that yet need to be characterized. Gastrin is produced mainly by G-cells of antral mucosa and duodenum (5). Low levels of gastrin expression were detected in the pancreas of adult mammals (high in fetal and neonatal rats), in anterior and intermediate pituitary lobes, human spermatogenic cells, bronchial mucosa, vagal neurons, hypothalamo-hypophyseal neurons, and some endocrine cells of the small intestine (6). Gastrin is synthesized in a form of a 101 amino acid residues precursor (7). The major forms of gastrin in circulation are the heptadecapeptide (G-17) and the tetratriaconpeptide (G-34) (8). They are formed from the precursor as a result of removal of the signal peptide, C- and N-terminal extensions and processing of glycine extended intermediates to amidated forms of the hormone (9). Gastrin in mature form shares the same carboxiamidated C-terminus, -Trp-Met-Asp-Phe-NH2 with cholecystokinin (CCK), which is expressed predominantly in duodenal and jejunal mucosa and in the cerebral cortex. This sequence constitutes the active site of both hormones and is highly preserved during evolution (10). Gastrin and CCK have many overlapping activities, because their structures are similar. Though a major role of gastrin is still considered to be in the stimulation of gastric secretion, recent data suggest that the hormone is indeed multifunctional. It was shown to increase blood flow through the stomach mucosa (11), it is involved in contraction of the stomach muscle (12), it has trophic effects on gastric and duodenal mucosa and the pancreas (13–18), it stimulates fractional sodium excretion in kidneys, and increases renal plasma flow (19), and may play a role in regulation of pancreatic secretion (20).

Gross cystic disease of the breast (GCD) is a well-defined clinical and pathologic entity, commonly occurring in the premenopausal decade. Its prevalence was estimated to be about 7% in western countries (1). Although the cyst itself is not considered a premalignant lesion, several studies with long-term follow-up indicate that bearers have a two- to fourfold increased risk of developing cancer, in the same or in the contralateral breast (1–7). Studies on the composition of the aspirated fluid (breast cyst fluid [BCF]) have demonstrated that this “biochemical space” has a peculiar composition with unusual profiles of many constituents, including ions, steroid and peptide hormones, growth factors, enzymes, immunoglobulins, cytokines, and other proteins (8–11). Furthermore, certain constituents show not only an enormous range of cyst fluid levels, but also a distribution of values consistent with the existence of distinct subpopulations. In particular, two major subgroups of cysts have been identified on the basis of the electrolyte composition and more specifically on the K+/Na+ ratio (12–14). Cationic categorization of cysts has gained wide acceptance as an useful tool to evaluate individual patients in relation to the natural history of cystic disease and could be of significance in predicting the breast cancer risk (6–9,14).

The field of gut peptides is constantly evolving, as new members are being discovered and knowledge of existing members increases. This review is by nature selective, and the description of each peptide and the bibliography reflects that. The peptide growth factors and cytokines produced by the gut were not included. A very good book reviewing the gut peptides that includes these substances appeared in 1994 (1).

Endocrine disorders are typically caused by excessive or insufficient production of an otherwise normal hormone. However, in recent years, several endocrine disorders were shown to be caused by other defects, i.e., mutations in peptide hormones, their cognate receptors, G proteins, or downstream effectors (1 ). Although such defects are extremely uncommon and thus provide only a rare diagnostic challenge, their definition at the molecular level often provided important novel insights into the biological significance of the affected protein and/or its functional properties. This is illustrated by the discovery that Jansen’s metaphyseal chondrodysplasia, a rare genetic disorder characterized by short-limbed dwarfism and severe hormone-independent hypercal-cemia, is caused by activating mutations in the parathyroid hormone (PTH) PTH-related peptide (PTHrP) receptor. These findings are likely to have significant implications for understanding the biological importance of the PTH/PTHrP receptor in addition to its role in regulating mineral ion homeostasis.

A common regulatory mechanism in the endocrine and neuroendocrine system is the coupling of secretion with the biosynthesis of a hormone. Factors that regulate hormone release also regulate its biosynthesis. This provides a cellular mechanism to ensure a ready supply of the protein or peptide. Thus, for any cell type in which secretion is regulated by dopamine via a dopamine receptor, it might be expected that dopamine would also have a significant effect on expression of the genes encoding the peptide/protein hormones themselves and/or those genes encoding the proteins involved in hormone production. Indeed, this linkage has been demonstrated in the first studies of regulation of prolactin and pro-opiomelanocortin (POMC) gene expression performed in model pituitary cell culture systems. Since these pituitary hormone-encoding genes were some of the first to be cloned in the early days of recombinant DNA technology, their regulation by dopamine has been well characterized but, surprisingly, some aspects of the underlying mechanisms have not been elucidated. In this chapter we discuss what is known about the expression of different dopamine receptor subtypes in the pituitary gland; their mechanisms of intracellular signaling; and processes by which the activation of these receptors interdicts the gene expression of two major pituitary peptide/protein hormone genes, prolactin and POMC.

The control of reproduction includes a series of hormonal interactions between the central nervous system (hypothalamus and pituitary) and gonads. Gonadotropin-releasing hormone (GnRH) is a decapeptide produced by a few hundred hypothalamic neurons. These cells extend termini to the median eminence to secrete GnRH in a pulsatile fashion into the hypophyseal-portal venous system. GnRH is then carried to the anterior pituitary where the gonadotropes (which comprise 5–7% of the total cell population) respond to GnRH via high-affinity cell-surface GnRH receptors by effecting the synthesis and secretion of the gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). LH and FSH are dimeric peptides, comprised of a common α subunit and distinct β subunits, the latter conferring biological activity. Both LH and FSH are released in pulses into the systemic circulation, and act on the gonads in concert, with FSH predominantly regulating gamete development, but also being required to induce hormonal responses to LH. Thus, the combined actions of LH and FSH control both hormonogenesis (estradiol and progesterone from the ovary, testosterone from the testes, and inhibin, activin, and follistatin in both sexes) and gametogenesis. These steroid and peptide hormones have both local effects to promote gamete formation, and exert feedback regulation at the hypothalamus and pituitary.

During the last several decades, research into neuroendocrine control by LHRH and other hypothalamic hormones has been an area of high activity. Since Schally and Guillemin’s Nobel Prize winning work of the early 1970s, the isolation, determination of structure, and synthesis of hypothalamic peptide hormones, as well as the synthesis of their analogs, have revealed radically new approaches to diagnosis and treatment of endocrine and nonendocrine disorders (1–10). Clinical applications of the hypothalamic hormones include: differentiation between hypothalamic and pituitary lesion, stimulation of fertility, birth control, and treatment of precocious puberty, endometriosis, cryptochidism, diabetes mellitus, acromegaly, and acute pancreatitis. The uses of analogs of hypothalamic hormones for hormone-sensitive tumors is relatively more recent, but of major current clinical value and significance, as evidenced by the quick transfer of basic research results to FDA approvals. LHRH agonist analogs are now used for the treatment of endometriosis and precocious puberty, as well as many of the endocrine-dependent tumors found in prostate cancer, breast cancer, and ovarian cancer.

In the classical endocrine renin—angiotensin system (RAS), renin secreted from the kidney cleaves hepatic angiotensinogen in the circulation to launch a cascade of angiotensin peptides including angiotensin (A) I, II, and III. AII, the most prominent of these peptide hormones, initiates through specific plasma membrane receptors, a host of physiological actions ranging from vasoconstriction to volume and growth regulation (1-4). Circulating plasma levels of renin and/or AII do not always correlate with their purported functions (5, 6),because of inaccuracies of renin and/or AII measurements, or not considering the sodium status (7). Studies with angiotensin-converting enzyme (ACE) inhibitors, and more recently with specific AII receptor antagonists, have demonstrated that even when the circulating levels are low, AII remains a major regulator of many biological responses, including blood pressure (8).

In the 70 years since Banting and Best extracted insulin from a dog’s pancreas (1), it has become the best studied of the peptide hormones. Insulin was the first protein for which the complete amino acid sequence was determined, and it was the first peptide hormone molecularly cloned. Insulin has been found throughout the animal world. It functions as a hormone in animals as divergent as sponges, flies, and humans, with very little sequence change over the one-and-a-half billion years this divergence represents.

The pituitary gland has neural and endocrine tissues that form its working structure. Both portions of the gland serve a vital role in an organism’s response to stress by secreting multiple peptide hormones. It is a so-called neural-hemal organ: that is a portion of the central nervous system acting as a vascular portal by which the brain communicates trophic and regulatory signals to the body. In the case of the mammalian pituitary, there are a diverse set of functions under regulation: thyroid, adrenal, growth, sex, lactation, skin coloration, renal, and uterine. These functions can be divided by the site of secretory activity with the first six being served by the endodermal-derived anterior portion of the gland and the last two being served by the neuro-ectodermal-derived posterior pituitary. The posterior pituitary is the terminal field of axons derived from the magnocellular hypothalamic neurons that synthesize and release vasopressin and oxytocin. Thus, there are two distinct regions of this gland that share a common vascular territory. It is the goal of this chapter to describe the anatomic and physiologic principles of the circulatory control mechanisms by which the glandular activities of the pituitary take place.

Cholecystokinin (CCK) is a gastrointestinal and neural peptide hormone with physiological activities at multiple target tissues (Mutt, 1980). These include the classical targets of the gallbladder muscularis smooth muscle and the pancreatic acinar cell (for which this hormone was named “cholecystokinin-pancreozymin”) (Ivy and Oldberg, 1928; Jorpes and Mutt, 1966), as well as smooth muscle and some nerves at multiple levels of the digestive tract, including esophagus, stomach, intestine, and colon (Mutt, 1980). In addition, several potential activities have been described for this hormone at the levels of the brain and spinal cord (Dockray, 1982; Yaksh et al., 1982). Thus, there are multiple receptor subtypes from which to choose for CCK receptor purification.

The absolute requirement for copper by many enzymes and biological processes has been well established. The low activity in vitro of many copper-requiring enzymes has been shown to be responsible for symptoms associated with copper deficiency. Among these are altered mental status due to changes in norepinephrine levels (dopamine β-hydroxylase)1, loss of hair pigmentation (tyrosinase)2, and reduced bone and artery competence (lysyl oxidase)3. In addition, the symptoms of copper deficiency include other manifestations of neurological dysfunction.4

p-Octopamine (OA) occurs in the nervous systems of many invertebrates at much higher concentrations than in vertebrate tissues (Klemm, 1985). A variety of physiological effects have been ascribed to OA including modulation of the activity of excitable tissues in annelids (Tanaka and Webb, 1983), crustacea (Evans et al, 1987) and insects (Hoyle, 1975; Evans and O’Shea, 1978) and the induction of specific behavioural responses in nematodes (Horitz et al, 1982) and crustacea (Livingston et al, 1980; Bevengut and Clarac, 1982; Kravitz et al, 1981). In insects, OA has also been implicated in excitation of the firefly lantern (Carlson, 1986), stimulation of glycogenolysis in nerve cord and fat body (Robertson and Steele, 1972; Downer, 1979a,b). Stimulation of cardiac contraction (Collins and Miller, 1977), enhancement of substrate utilisation by flight muscle (Goosey and Candy, 1980), release of diacylglycerol from locust fat body (Orchard et al. 1982) and regulation of release of peptide hormones from the corpus cardiacum (Orchard et al., 1983; Downer et al., 1984).

The polypeptide hormones are a diverse group of small proteins, approximately 50 in number, ranging in size from three to 300 amino acids, that regulate the processes of metabolism, reproduction, growth, and development (1). In the broad sense, however, many of these polypeptides can be considered regulatory peptides because they serve not only as hormones in which their actions are mediated on target organs by way of their transport through the bloodstream, but also as local cell-to-cell communicators. The designation of the term hormones to certain of these polypeptides is a consequence of the initial physiologic observations that they are products of specific organs and endocrine glands, and are released into the bloodstream to exert their actions on distant target organs. The products of the actions of the hormones in turn regulate (often inhibit) the further production of the specific hormones.

Glucagon is a peptide hormone of 29 amino acids produced and secreted by the A cells of the pancreatic islets (1). It is a member of a structurally related group of peptides that includes secretin (2), vasoactive intestinal peptide (3), gastric inhibitory peptide (4), and growth hormone releasing hormone (5) (Fig. 1). The secretion of glucagon is regulated by blood levels of glucose (6) and amino acids (7), as well as by a variety of hormonal stimuli (8). The action of glucagon on its target tissues, particularly the liver, is an important factor in protein and carbohydrate metabolism (9,10). Abnormal regulation of glucagon gene expression has been implicated in the pathogenesis of diabetes mellitus (11).

Specific “endocrine” markers (polypeptide hormones and biogenic amines) have for several decades now played a vital role in the clinical evaluation and management of patients with a wide range of endocrine tumors. Some of these hormones, such as ACTH, gastrin, insulin, vasoactive intestinal peptide (VIP), and serotonin are responsible for the production of specific and often dramatic clinical syndromes. Others, such as calcitonin, a highly sensitive marker for the spectrum of thyroid C-cell proliferative disorders, are not associated with any known clinical syndromes. Ectopic (inappropriate) endocrine syndromes in patients with non- endocrine tumors have likewise been recognized for several decades. The earliest appreciation of inappropriate endocrine activity by tumors dates back to the 1920s (Klemperer, 1923; Brown, 1928), and over the ensuing years, numerous examples of inappropriate endocrine syndromes have been noted in patients with non-endocrine tumors (Meador et al., 1962; Lipsett et al., 1964; Liddle et al., 1969; And erson, 1973; Ratcliff and Rees, 1974; Blackman et al., 1978; Trump and Baylin, 1979). In 1948, it was first appreciated that humoral factors might be involved in these endocrine syndromes (Albright and Reifenstein, 1948).

Amphibian skin is a rich source for a variety of peptides with diverse physiological actions (1–4). It is surprising that these skin peptides are virtually always similar or identical to peptide hormones found in mammalian brain or other parts of the nervous system or in the gastrointestinal (GI) tract. This “brain-gut-skin triangle” (5) has intrigued pharmacologists and biochemists for many years. Table 1 shows a list of a few prototypes of skin peptides and their mammalian counterparts. Even after two decades of research, new peptides are still discovered in amphibian skin, as exemplified by dermorphin, a potent opioid peptide with the unusual feature of having one D-amino acid in its sequence (6).