Recent Progress in Hormone Research (eBook)

Proceedings of the 1974 Laurentian Hormone Conference

Roy O. Greep (Herausgeber)

eBook Download: EPUB

2013 | 1. Auflage
646 Seiten
Elsevier Science (Verlag)
978-1-4832-1951-6 (ISBN)

Infos

Recent Progress in Hormone Research, Volume 31 covers the proceedings of the 1974 Laurentian Hormone Conference held in Mount Tremblant, Quebec, Canada, on August 25-30, 1974. The book discusses the relationship between catecholamines and other hormones; the hormone receptor complexes and their modulation of membrane function; and receptors for insulin, NSILA-s, and growth hormone. The text also describes the mechanism of action of pituitary growth hormone; hormonal regulation of ovalbumin synthesis in the chick oviduct; and studies on the hepatic glucocorticoid receptor and on the hormonal modulation of specific mRNA levels during enzyme induction. The endocrine neurons; the formation of estrogens by central neuroendocrine tissues; and the operating characteristics of the hypothalamic-pituitary system during the menstrual cycle and observations of biological action of somatostatin are also considered. The book further tackles somatostatin; the relationship of sleep and sleep stages to neuroendocrine secretion and biological rhythms in human; and the genetic approaches to the study of the regulation and actions of vasopressin. The identification and actions of gastric inhibitory polypeptide; the studies on the pathogenesis of Graves' ophthalmopathy, and qualitative and quantitative gonad-pituitary feedback is also looked into.

Textauszug

Hormone Receptor Complexes and Their Modulation of Membrane Function

PEDRO CUATRECASAS, MORLEY D. HOLLENBERG, KWEN-JEN CHANG and VANN BENNETT,     Department of Pharmacology and Experimental Therapeutics and Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland

Publisher Summary

This chapter discusses the problems in the study of hormone receptors and their modulation of membrane function. Because physiological membrane receptors are present in extremely small quantity in membranes, the hormone must be labeled to very high specific activity without destroying the biological activity of the hormone. Virtually, all chemical compounds used as binding ligands exhibit some nonspecific adsorptive or binding properties to a variety of inert and non-receptor biological materials, and such binding may be of extremely high affinity and the number of such nonspecific binding sites may greatly exceed that of specific receptors, therefore, great difficulties may be encountered in detecting such specific receptors. The general approach in these studies has been to measure the interaction, binding, of a radioactively labeled hormone with intact target cells or with isolated membrane preparations derived from such cells. The binding is presumed to reflect specific receptor interactions if it demonstrates the following characteristics: (1) strict structural and steric specificity, (2) saturability that indicates a finite and limited number of binding sites, (3) tissue specificity in accord with biological target cell sensitivity, (4) high affinity in harmony with the physiological concentrations of the hormone, and (5) reversibility that is kinetically consistent with the reversal of the physiological effects observed upon removal of the hormone from the medium.

I Introduction

During the past few years there has been a large burst of interest in membrane receptors for hormones, and considerable progress has been made in the identification and study of receptors for such peptide hormones as insulin, glucagon, adrenocorticotropin, thyrotropin, angiotensin, calcitonin, growth hormone, prolactin, follicle-stimulating hormone, luteinizing hormone, chorionic gonadotropin, oxytocin, and vasopressin, as well as nonpeptide hormones such as catecholamines, prostaglandins, and acetylcholine (reviewed in Cuatrecasas, 1974a,b; Birnbaumer, 1973). The general approach in these studies has been to measure the interaction (binding) of a radioactively labeled hormone with intact target cells or with isolated membrane preparations derived from such cells. The binding is surmised to reflect “specific” receptor interactions if it demonstrates (a) strict structural and steric specificity; (b) saturability, which indicates a finite and limited number of binding sites; (c) tissue specificity in accord with biological target cell sensitivity; (d) high affinity, in harmony with the physiological concentrations of the hormone; and (e) reversibility which is kinetically consistent with the reversal of the physiological effects observed upon removal of the hormone from the medium. In addition, it is of considerable help if specific chemical or enzymic perturbations of the cells or of the hormone result in changes in the biological activity which parallel closely similar changes in binding.

It should be realized that the term “receptor” in all these studies is a general term used for convenience, and its use implies a lack of knowledge of the discrete or unique chemical structures involved in the interactions. As the history of the study of drugs has shown, the use of the term “receptor” is in effect a reflection of ignorance of the molecular locus of action of the drug; when this locus is known (e.g., whether a specific enzyme such as a kinase for cyclic AMP, or hemoglobin for a vitamin or certain drugs), we no longer refer to it as the receptor, but rather by its chemical nature. “Receptor” in the context used currently in hormonal studies is defined operationally as those molecules that specifically recognize and bind the hormone and, as a consequence of this recognition, can lead to other changes (or series of changes) which ultimately result in the biological response. This is done in analogy with classical enzyme–substrate systems, in which substrate binding and catalysis are separate and discrete but sequential processes which can be studied independently. As in the case of competitive inhibitors of enzymes, hormone analogs (e.g., as known for glucagon, ACTH, angiotensin, and oxytocin) may in certain cases bind to “receptors” but fail to trigger biological responses, thus serving as relative or absolute inhibitors depending on the quantitative comparison of their intrinsic activity relative to their binding affinity.

The present article is intended not as a review of the pertinent literature, but rather as a presentation of special selected topics and ideas which have been of interest and concern in this laboratory.

II Problems in the Study of Hormone Receptors

A number of problems and pitfalls can be encountered in the kinds of studies described above, and considerable caution must be exercised in the interpretation of data, especially when detailed mechanistic inferences are made or when extrapolating to complex in vivo biological systems or disease states. Some examples of such actual or potential problems relating to different aspects of hormone-binding studies will be presented here.

A THE PROBLEM OF “NONSPECIFIC” BINDING AND RELATED TECHNICAL PROBLEMS

Because physiological membrane receptors are present in extremely small quantity in membranes, the hormone must be labeled to very high specific activity (e.g., with 125I or 131I at 1–2 Ci/μmole) without destroying the biological activity of the hormone. Since virtually all chemical compounds used as binding ligands (hormones) exhibit some nonspecific adsorptive or binding properties to a variety of inert as well as nonreceptor biological materials, since such “binding” may be of extremely high affinity, and since the number (“infinite,” by definition) of such nonspecific binding sites may greatly exceed that of specific receptors, great difficulties may be encountered in detecting such specific receptors. The “background binding” of labeled hormone to a filter or apparatus used to separate membrane-bound from free hormone, or to the tissue material itself, may contribute very significantly to the total amount of hormone bound.

Of particular importance is the fact that such nonspecific binding can appear as “specific” by many criteria, such as saturability, specificity, reversibility, which are generally used to define receptors. We have observed many instances where this has been a troublesome complication. For instance, it has been observed that, in the absence of cellular material, vigorous shaking in glass but not plastic tubes of insulin-125I solutions in Hanks’ or Krebs-Ringer-bicarbonate buffers in the presence and in the absence of native insulin yields large quantities of spurious “displaceable counts” which are trapped on Millipore filters (Table I). This artifactual specific binding apparently does not represent binding to silica or glass particles released from the surface of the tubes, since shaking before measurement of binding does not produce these effects (Table II). This insulin binding system is also demonstrably “saturable” in concentration-dependence studies, and the apparent affinity (about 6 × 10−10 M) as measured in such studies is relatively high (Fig. 1). The explanation for this artifact has not been elucidated, but the observation itself should sound a cautionary note.

TABLE I

“Specific” Binding of Insulin in the Absence of Tissue by Vigorous Shaking of Assay Mixturesa

aIncubation media (0.2 ml) consisting of Hanks’ buffer–0.1% albumin were incubated with and without vigorous agitation at 24° for 40 minutes in the presence of 125I-labeled insulin (2 × 105 cpm, 1.1 Ci/μmole) and with and without native insulin. Glass or plastic tubes (12 × 75 mm) were used as indicated, and binding was determined (Cuatrecasas, 1971) by filtration over EA cellulose acetate Millipore filters.

bValues are expressed as counts per minute.

TABLE II

“Specific” Insulin Binding in the Absence of Tissue by Vigorous Mixing of Assay Mixture in Glass Tubesa,b

aIncubation media (0.2 ml) consisting of Krebs-Ringer-bicarbonate buffer–0.1% albumin (in 12 × 75 mm disposable glass tubes) were shaken vigorously for 50 minutes at 24° in the absence (“before”) or presence (“during”) of various concentrations of native insulin and 2.8 × 105 cpm 125I-labeled insulin (1.6 Ci/μmole). The samples were then assayed for binding (“during”), or native and 125I-labeled insulin were added and binding was assayed after incubation (without shaking) for another 50-minute period at 24° (“before”). Binding was determined...

Produkt-Details

• Erscheint lt. Verlag: 22.10.2013
• Sprache: englisch
• Themenwelt: Informatik ► Weitere Themen ► Bioinformatik
• Themenwelt: Medizinische Fachgebiete ► Innere Medizin ► Endokrinologie
• Themenwelt: Studium ► 1. Studienabschnitt (Vorklinik) ► Biochemie / Molekularbiologie
• Themenwelt: Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
• ISBN-10: 1-4832-1951-8 / 1483219518
• ISBN-13: 978-1-4832-1951-6 / 9781483219516