endocrine methodologies chapter 4 9-1.2012.pdf

Endocrine Methodologies

ome of the methods presently used in endocrinological studies were developed for more practical purposes In many cultures castration was practiced as a form of punishment In the Middle and Far East castration was performed to provide

servants (eunuchs) for harems in Italy castrated young boys were trained to be adult sopranos Castration is presently used to improve the flavor ofmeat from some domes-tic animals (eg castrated chickens produce capons) These practical operations were the forerunners of the gonadectomies of present-day endocrinological studies

The idea that glands contained humors or substances that could act as replacement therapy for a lost function ofa gland was first el1lertained seriously by a French physi-cian Charles Brown-Sequard He injected himself with extracts from dog guinea pig and rabbit testicles and proclaimed that the extracts had remarkable rejuvenating effects Brown-Sequard even recommended that extracts be obtained from the mature calf to give to men the vigor ofhorses and other larger animals It is now believed that these extracts had only a placebo effect N evertheless glandular extraction purifica-tion and injection into animals have become important methods in elucidating the hormonal role of particular tissues and organs

Although hormones control a large variety of physiological evenls their basic function is to act at the level of the cell to stimulate cellular physioLogicaL processes inherent in the cell M eLanocytes for exampLe synthesize the pigment meLanin Melanocyte-stimuLating hormone (MSH) enhances melanin formation by increasing the activity of the rate-limiting enzyme tyrosinase Cortisol is a hormone produced by adrenal steroidogenic tissue Corticotropin (ACTI-I) activation of adrenocortical enzyme activity leads to eLevated cortisol biosynthesis Thus effects of hormones Clre reflected in physioLogical processes in the many cell types present within an organism This chapter summarizes some of the methods employed by endocrinoLogists to study endocrine glands and the target cells and tissues that they regulate

nsi rations The methods now employed to study endocrine systems are highly diverse [8929] lassical in vivo and in vitro methods include surgical manipulations tissue extract preparation for hor-mone isolation and identification histological methods for the localiza tion of hormones and numerous hormone and receptor assay methods Modern enducrine research utilizes virtually any of the latest molecular cellular physiological behavioral and gene tic approaches-often in combination with the classical techniques-to further our understanding of hormone synthesis secretion physiological roles and mechanisms of action The techniques described in what follows will be mentioned in more detail in later chapters

68 Chapter 4 Endocrine Methodologies

o Endocrinologists to bout orllo Following discovery of a hormone endocrine studies usually focus on the following investi-gations (not necessarily in the order delineated)

1 Source The distribution of the hormone will be determined T he hormone may be fo und in more tban a single organ tiss ue or cellular source For example several gastrointestina l bormones are also found within the central nervo us system

2 Structure determin(ion and synthesis Depending on the type of hormon e the struc-ture will be determined by any of a variety of methods In the case of a peptide hor-mone the pri mary (amino acid) sequence will be determined From this in forma tion the peptide will then be synthesized and its biological acti vity compared to the purified extract will be determined This will estab lish the substance and structure as the authen tic hormone

3 Biosynthesis The biosyntbetic pathway of hormone production should be delineated Knowledge of the primary structure of a peptide hormone can be used to predict the cOITesponding sequence of the gene that encodes it the gene can be cloned and the cDNA struc ture corresponding to the hormone m R NA can be used to predict the prohormone structure and possible enzymol ogical even ts rel ated to production of the mature horm one

4 Controlosecretion The extrinsic or intrinsic factor(s) regulating the control of hormone secretion must then be determined E ndogenous stimuli may involve negative or positive feedback by other hormones or the circulating products (other hormones metabolic sub-stra tes) or consequences (blood volume water andor electrolyte composition of the blood) of its actions The nervous system may directly or indirectly regulate hormone secretion from endocrine organs or tissues

5 Cellular mechanisms of secretion O nce the first messenge rs regulating hormone secre-tion have been determined it is then necessary to detelmine the nature of the second messengers and structural elements (ion channels cytoplLLltmic organelles) that participate in the process of hormone release (secretion)

6 Circulation and metabolism It is importa nt to determine the half-life of the hormone in the systemic circulation Ste roid or peptide hormones may be noncovalently bound to circulating proteins Fluctuations in the levels of the binding proteins may affect the total amount of hormone present in the blood an d therefore availabl e for hormone action The half-life of the hormone in the circulation may be affected by degradation or other alterations of the hormone by serum enzymes The retention time of the hormone in the circulation may also be affected by processes of filtra tion by the kidney

7 Biological actions and roles Removal of the hormone fro m the body by one or more methods usuall y will result in physiological effects in the animal that will predict one or more functi ons for the hormo ne Administration of the hormone (rep lacement therapy) or related ana log to the ani mal sh ou ld confirm one or more roles for the hormone Studies on othe r species of animals may suggest add iti on al or alternate roles for the hormone

8 Mechan isms 0 Clclion Following administration of the hormone in vivo and in vitro ce llular changes in biochemical processes and products should indicate one or more second messengers involved in hormone action The receptor and signal transd uction mech an isms involved in hormone action should define the temporal aspects between receptor activation and cellular response Structure-activity studi es will determine the esse nti al features of the hormone required for hormonc action in the case of a peptide the message seq uence

9 Pathophysiological aspects The existence of a hormone is someti mes glea ned from the symptoms of a human pathophysiological condition In other cases the discovery of a hormone in experimenta l animals provides a new explanation for a previ ously misun-derstood clinical condition In either casc it is important to study the re lations hip be tween the normal biology of a horm one and the clinical consequences of alterations in synthesis secreti on and actions of that hormone These studies take place in animal models of huma n diseases a nd to the extent that is ethically possible in human clinical popu lations

Hormon R pI c m nlTh r 69

ad

10 Comparative endocrinology Some bormones are evolutionarily conserved in struc-ture andor function in organisms ranging from the primitive to tbe most reccntly evolved The structures and biological roles of other hormones however have clearly evolved and become specialiied for particular adaptations in variolls species In some cases the responses to the same hormone molecule are specialized by the types of receptors and signaling mechanisms that ale present in the respol1sive cells In o ther

variations in the molecular structure of the bormone itself have arisen through gene mutatiol1s and these can alter andor expand the biological activities of a particu-lar molecular variant and even increase the number 01 diffe rent molecular variants produced within the same org1nism Comparative endocrinologists attempl to track the evolution of hormone structure and function al1c1 determine the adaptational significance of these evolutionary relationships for the physiological ecology of different animal species

urgca Surgical removal of a putative endocrine gland or ti ssue from lt1n animal is followed by sub-sequent assessment of physiological alterations A change in functional activity of suspected target organs (ie atrophy) suggests an endocrlne function for the extirpated tissue O ne might also monitor chang s in blood or urinary levels o[ certain meta bolites or electrolytes Removal of the adrenals for example would result in lowercd circula ting levels of adrenal steroids and and a reduction in the plasma I a concentration Transplanting an organ back into the same or a different animal also provides information on the func-tional role of the organ In mammals transplantation of an orgltln from a donor to a host is usually done in genetically related (inbred) strains of animals 50 that the transplant is not rejected by immunological proctsses Endocrine organs can be transplanted to an ectopic (abnormal) site in the animaL usually beneath the kidney capsu le or within the eye where rapid vascularization often occurs Removal of the pituitary is referred to as a hYJophyseclOmy rlne pituitary target organs of hypox animals become atrophic due to the absence of hormonal stimulation In a fevv examples endocrine ti ssue is normally under a tonic inhibitory controL the tissue then becomes hypertrophic when transplanted to an ectopic site within the animal or when incubated in vltro

Removal of botl1 members of paired (bilateral) target organs (eg adrenal glands or gonads) usually leads to complete loss of dependent tis5ud organ functions [f only one of the pair (unilateral) is removed the remaining organ usually undergoes compensatory hypertro-phy In otber words there is an increase in cell size and number in the remaining organ to compensate functionally for the loss of hormone secretion [10111 the ablated organ (Fig 4 I) In endocrine systems in which one endocrine organ may control secretions by anothcr the feedback mechanisms that may operate are often studied by assess ing the effects of organ removal and hormone replacement on the putative feedback targe t

Other examples of endocrine include pinealectomy (epiphysectomy) adrena-lectomy tl1yroidectomy anc thymectomy The nature of sLich endocrine tissues as the gastrointestinal hormone-secreting cells prevents such (] surgical procedure Tne pancrelt1tic islets are similarly impossible to remove without removing concomitantly the exocrine tissue that comprises most of the mass of the pancreas lslel ablation however can be accom-plished by the use of alloxan or streptozotocin drugs that specifically destroy the insulin-secreting cells of the islets Cobalt chloride is similarly effective in the elimination of the glucagon-secreting cells of the pancreatic islets In brain tissucs localized electrolytlc lesions can be made to e liminate specific brain nuclei that contain cells of interest and the conse-quences of these ablations can be functionally assesscoci

e ce t The The undesirable effects of hormone loss following surgical ablation or loss cue to certain disease states often can be adequately reverse c by administration of the needed hormone or related analog At menopaLise for example many women experience bone mineral loss (osteoporosis) clue to declining levels of ovarian estrogens Estrogenprogesterone

71 Hts 0 m 1Cmiddot

bovine (ca ttle) ovine (sheep )porcine (pig) or even equine (horse) origin Often these foreign proteins are immunologically neutralized within the body follo wi ng administration therefore necessitating a change in the type (source) of the hormone used teroid hormones are com-monly extracted from the urine of large domestic animals for example horse and us d in th e preparation of hormone replacement therapies such as the estrogen-containing treatments taken by some postmenopausal women

Before the advent of modern molecular biological techniques the structures of most protein hormones were identified by painstaking purification of the source tissue followed by meticulous biochemical analysis of the hormones amino acid sequence A purified ho rmone was chemicaJly identified and then subjected to a simple analysis of the percentage of car-bon hydrogen oxygen nitrogen sulfur or other atoms present thus providing an empirical formula Further analysis of the protein then indicated the number and nature of amino acids present (an amino acid analysis) This was followed by a determination of the primary sequence (exact amino acid sequence) of the peptide or protein under study This is nol easy if a large protein hormone is being studied Nevertheless one might obtain information on the -terminal or C-terminal sequences of these large protein hormones

In the more recent past hormone structures have been determined by sc reening of cD A libraries cloning cDNAs representing candidate hormone genes and det rmining the pre-dicted amino acid sequence of the putative hormone In some cases the gene encoding the hormone was identified using forward genetic methods (from phenotype to genotype) these approaches generally involve the genetic analysis of mutant mouse strains that exhibit phenotypic evidence for disruption of a gene of interest For example the gene encoding the adipocyte-derived hormone leptin was positionally cloned by analyzing genomic markers and deducing the genomic location of the heritable DNA sequence linked to the ob sc phenotype in the mutant (obob) mouse [41] With the sequencing of the human mouse and other animal genomes it has also become possible to screen sequence data banks and identify putative hormone and receptor nucleotide and amino acid sequences based on the presence of characteristic structural motifs All of the foregoing methods have similarly been used to characterize hormone receptors and their corresponding genes

From the primary hormone structure the secondary (a-helical or j3-pleated sheet) and tertiary (folding intrachain bonding) structures may often be implied from the distribution of basic or acidic amino acids or the presence of sulfhydryl (-SH) groups within the protein Some peptide hormones such as insulin possess a quaternary structure that is the hor-mone is made up of two peptide chains folded together into a three-dimensional structure (conformation) A number of the larger peptide hormones tor example follicle-stimulating hormone (FSH) luteinizing hormone (LH ) and thyroid-stimulating hormone (TS H ) are composed of two chains the so-called a and j3 subunits of their structures

Chemical analysis may indicate that proteins are modified through sulfation or conjugation to carbohydrate moieties The pituitary hormones FSH LH and TSH are examples of glycoproreins (Chap 5) Determination of steroid or other hormone structures requires differ-ent chemical and physical methods of analysis After determination of the putative hormone structure it is necessary to synthesize the proposed structure and demonstrate that the na tural and synthetic structures are identical with respect to chemical physical and biological charac-teristics One can then synthesize related structural analogs of the hormone to determine the structural basis for the biological activity of the hormone Table 41 provides the abbreviations used to symbouze the amino acids present within the primary structure of a peptide hormone (See eg Figs 58 and 1lS)

5

The early endocrine studies were as expected anatomical and purely descriptive in nature The light microscope was used to determine the histological nature of the endocrine glands Many early observations were made on these tissues even before their endocrine role was suspected Much of this work was accomplished by the great German Italian and other

TABLE 4 Abbreviations used for amino acids

A

72 Chapter 4 ldocrln Mothodologles

ysteinc cys C Aspartic acid asp D G lutamic acid glu E Phenylalaninc phe F G lycine gly G H istadinc his H Isoleucine ile I Lysine Iys K Leucine leu L Methionine met M Asparagine asn N Proline pro P G lutamine gIn Q Argi nine arg R Serine SCI S 1111eOn ille thr T Valine val V Tryptophan trp W Tyrosine tyr Y

E uropean cytologists of the nin eteenth century Although light microscopic me thods are still important the electron microscope is now the tool in the investigation of cellular function at the ultras tructural level The scanning electron microscope bridges the gap be tween light and electron microscopy

Gross observations of endocrine tissue provide only general details of anatomicallocaliza-tion organ size vascularization and innervation Severe alterations from the normal in organ size may provide clues to an underlying pathophysiology The thyroid gland for example may become enlarged (goitrous) under certain conditions of hyperstimulation (see Fig 1311) At the histological level one is ab le to discern the cytological characteristics of endocrine tissues The cells may be hypertrophic (enlarged) or atrophic (diminished) depending on whether they are hyperactive or hypoactive respectively Hypertrophic cells contain an abund ance of endoplasmic reticulum and Golgi bodies as these organelles function in many cellular syn-thetic processes A trophic cells on the other hand lack this synthetic machine ry and contain a much di minished cytoplasmic mass E ndocrine cell hypertrophy is usually accompanied by hyperplasia an increase in cell number

Histological stains are available to provide further information on chemical components of cells H ematoxylin and eosin are two popular dyes for staining cells for routine histological observation H ematoxylin a basic dye interacts with acidic components of the cell such as the phosphoric acid of DNA and RNA These components are said to be basophilic or to exhibit basophilia On the other hand eosin an acidic dye interacts with the basic components of the cell which are then sa id to be acidophilic or to exhibit acidophilia Used in combination hema-toxylin and eosin usually stain the nucleus and cytoplasm blue and pink respectively

By the use of a battery of histological stains one can characterize each cell present in the pituitary With hematoxylin and eosin it is only possible to separate the basophils from the acidophils and chromophobic (nonstaining) cells (chromophobes) Other histological stains are needed to further differentiate the several types of basophils Cer tain stains can be used to demonstrate the presence of specific organic constituents such as glycoproteins in gonadotrophs of the pituitary or glycogen in hepatocytes [29]

Hem

immunological methods have been used for many years to characterize and analyze the expres-sion of hormones in endocrine cells nley are also used to characterize the specific distribution of receptors cell signaling molecules transcription factors and other proteins that mediate

Principle )f immunohistofluores-ence methods for the nicroscopic detection of l protein of interest in ndividual cells

Fix Section

Incubate tissue 4- wittl primary antibody A Target r y cr 0

0 A (

Wasil mount on microscope slide view under fluorescent microscope

Incubate with reagents

H 01 Cdl C middottol qlCal Studt 73

hormone actions in target tissues Immunohistochemistry refers to the use of spccific antibodies to detect and label specific antigens (molecular targ ts of immune mediators) for example hor-mones in a tissue of interest This term is often used intercha ngeably with immunocytochemistry which is a narrower and perhaps more accurate description of those techniques that a llow iden-tification of a particular antigen at the level of indi vidual cells or even within specific subcellular compartments (Fig 42) These powerful procedures make usc of antibodies that specifically rec-ognize and bind to a specific epitope (portion of a particular molecule) thereby allowing one to visualize and identify which cells in a tissue contain that specific mol eule E ither polycfonal or monoclonal an tibodies can be used in immunohistochemical procedures Polyclonal antisera are generated by immunizing animals with a suspension containing the target molecule and subse-quently obtaining the hyperimmune serum from those animals that mount a robust and specific immune reaction to the antigen The polyclonal antise rum as its name implies contains several different clonal strai ns of immunoglobulins that may contribute to varying degrees to th total antibody titer Monoclonal antibodies are produced by immunizing a mouse with a specific antigen obtaining spleen cells from the animal and fusing these ells with chemically selectable myeloma cells to produce hybridomas Each hybridoma is capable of expanding to produce a homogeneous clona l line of ce lls that produce chemically and immunologically homogeneous antibodies Most importantly the selected clonal cell line produces large quantities of a single antibody species that recognizes and binds to a single antigenic determinant

Irrespective of the type of antibody utilized the success of any immunohistochemical approach depends upon the specificity and the affini ty of the antibody for a particular antigen] n a version of immunohistochemistry referred to as immunohistoflllorescence antibodies to pep-tide or protein hormones are conjugated to a fluorescent dye and used to identify cells tha t pro-duce the molecule of interest The tissue to be examined is first treated with a fixative such as glutaraldehyde and sectioned into thin slices After the tissue is placed on a microscope slide a solution of the antibody conjugate is added to the tissue and the excess solution is then rinsed from the slide several times Microscopic observations usually revea l that the fluorescent anti-body is bound only in those cells that produce the hormone the antigen (see Fig 108) Specificity is determined by prior incubation with a blocking peptide a synthetic peptide that is identical to the portion of the target hormone or receptor that is known to be bound by the antibodies The

bull

74 Chapter 4 Ildocnne Methodoloql

blocking peptide interacts with the antibody and thus prevents it from binding the endogenous antigen TIle absence of fluorescent labeling of tissue following preincubation with the blocking peptide confirms the specificity of the antibody for the intended antigen target

In immunoenzyme histochemistry an antibody to a hormone is conjugated to an enzyme such as peroxidaseThe antibody-enzyme conjugate is then allowed to interact with the tissue slice When substrate for the enzyme is added the conjugated enzyme catalyzes very localized reactions that yield a color or opaque product in the vicinity of the antigen (the hormone) By this immunoperoxidase method and use of the electron microscope it is even possible to localize the site of the enzyme activity to the secretory vesiclesgranules of a cell which pro-vides strong evidence that these organelles contain the hormone under investigation

In double-labeling immunocytochemical procedures two antibodies can be used to simulta-neously examine the expression of two antigens in a single tissue section TIle two antibodies possess specificity for the two different antigens respectively and they are linked to two differ-ent labels for differential visualization under the microscope The utility of these procedures is far-ranging and includes the ability to characterize the expression of specific hormone recep-tors to specific subsets of potential target cells in a complex tissue such as the brain

Immunohistochemical methods have also proven to be useful in functional studies of hormone secretion and action The activation of a neurohormone-secreting cell for exam-ple is often accompanied by the expression of immediate early genes such as those encod-ing the proto-oncogene c-Fos [11] With immunocytochemical methods cells that express this gene product can be identified as those that are activated under certain experimental conditions For example brain cells that are activated by physical or behavioral stress have been mapped by means of c-Fos immunocytochemical methods Similarly some hor-mone actions are mediated by intracellular messengers that become phosphorylated upon activation antibodies can be developed that specifically recognize the phosphorylated form of signaling molecule and thus they can be used to immunocytochemically map the cell groups in which hormone treatments activate that particular signaling pathway

s e E ndocrine studies utilize all the modern tools of physiology molecular biology and genetics to understand hormone function Below are considered those quantitative methods that have proven to be particularly important in analyzing the synthesis secretion and actions of specific hormones

1 e a ctivi The physiological activity of a hormone was originally determined by bioassay Although some assays have been replaced by more modern methods in some instances the only method available is bioassay In a bioassay the activity of a hormone is studied on living cells tissues or organs Physiological responses such as muscle contraction and relaxation or glan-dular secretion are monitored TIle assay may be performed in vitro or in vivo (in situ) depending on the assay used Usually the tissue or organ selected is naturally responsive to the hormone These biological preparations are usually responsive to hormones in the nanomolar (10-9 M) to picomolar (10 - ]2 M) range [25] Other hormones may also affect these tissues but usually in pharmacological (micromolar 10-6 M) doses

The frog skin bioassay for melanocyte-stimulating hormone (MSH) is simple specific and exquisitely sensitive M elanin granules within me lanophores of frog skin disperse in response to the hormone causing the skin to turn from light green to dark brown (Chap 8) T his change can be measured in a number of ways but one objective method is to monitor changes in light reflectance off the surface of the sk in l25] TIle toad bladder and such epithelial structures as frog skin and the mammalian renal nephron have been used in vitro to study the actions of vasopressin on transport of wa te l and other components such as Na + and urea across these organs Studies of ion transport in the toad bladd er have contributed deta iled information on the mechanisms involved in vasopressin action (Chap 7)

111e steroid-primed uterus of the rat has been used in vitro to study the mechanisms of oxytocin-induced contraction of this organ Mammary ti ssue from the lactating mouse is used in vitro to determine milk-ejecting potency of oxytocin and related analogs Other bioassays include in vitro adrenal steroidogenesis and secretion in response to ACTH prostate and

M thodolO 1 75

seminal vesicle growth in vivo in response to testosterone in the castrated male rode nt measurement of iodide uptake by the thyroid after exogenous TSH injections measurement of epiphyseal plate (cartilage) growth in the young rat in response to groWLh ho rmone Many investigators now use primary cell culture systems or immortalized cell lines rather than intact animals or removal of organs from such animals

Bioassays determine whether a putative hormone or hormone analog possesses biological activity Structure-activity studies determine the amount of activity usually compared with some standard hormone The minimal effective dose (MED) and maximal activities within the particular assay are usually determined first Then intermediate concentr ations of the hormone between the two extremes (maximum and minimum) can be utilized Lo provide a dose-response curve Usually the half-maximal activity of the hormone is compared with a similar half-maximal control response (eg to the parent hormone or to an analog) and expressed as a potency ratio The hormone or hormone analog may possess one or more times greater or lesser activity (potency) than the native hormone (see F ig 818)

io s ys ut genesis t eli n I

Although the primary sequence of many hormone receptors is known how the receptors function cannot be readily discerned from the amino acid sequence One would like to know the sequence(s) responsible for ligand (hormone) binding and the sequence(s) responsible for sig-nal transduction leading to a cellular response A mutant receptor can be synthesized fro m the original normal receptor cDNA and the mutated DNA introduced into immortalized (usually cancer) cells For example single base changes multiple base changes additions or deletions (subtracting amino acids) can be made Then a functional response to a hormone by these cells containing mutated DNA can be measured [26] or example one can measure changes in levels of a second messenger such as cAMP following addition of the hormone to the cells

Ie Lev s of

[H J + [HJ + [Ab] lt--- [H) [Ab] + [H] [Ab] koff

The development of the radioimmunoassay (RIA) has allowed the detection of hormones in minute concentrations and with a high degree of specificity [40) RI A has provided greatly increased diagnostic accuracy of pathological states characterized by hormonal excess or defi-ciency These assays are routinely employed to de termine the concentration of hormones and other molecules in blood or other body fluids As with the immunohistochemical methods the success of RIA procedures is dependent upon the use of antibodies that specifically recognize and bind the hormone with high affinity Both polyclonal and monoclonal antisera can be used in an RIA The second important reagent for an RIA is radiolabeled hormone In most RIA the hormone is iodinated or tritiated in peptide hormones or in other proteins the tyrosine moi-ety incorporates iodide he typical RI A procedure depends upon the binding of the specific antibody to the radiolabeled antigen (eg hormone) with the same kinetics as described for receptor binding to ligand in the previous chapter

kon [H] + [Ab] lt--- [H ) [Ab]

koff

where [H] = radiolabeled hormone [Ab] = antibody [H ) [Ab] = hormone bound by anti-body and kon and koff are association and dissociation rate constants respectively Like the binding of ligand with receptor this is a reversible reaction in which kon is greater than Koff-At a fixed concentration of [H] and [Ab] the reaction equilibrates at a maximum Ie el of [H) [Ab] This is referred to as the maximum binding or Bo The principle of the R[A is that the unlabeled hormone [H) when it is added to such a reaction mixture will compele with the radiolabeled hormone [H] for binding to the antibody [Ab]

kOl

76 Chapter 4 ldocrme Mcthodologle

Maximum Standard Standard Standard Standard Unknown Unknown etc Binding 1 2 3 4 X y

A I nj J

Antibody + + + + + + + Hormone + + + + + + + Hormone + ++ +++ ++++ Unknown

E I Unknown X 0 ()

ltlJ c o E I Unknown Y

3 Schematic ( -repre sentation of a

typical RIA p rocedure with hypothetical standard curve Log [Hormone]

Since the amount of radiolabeled hormone and antibody is fixed then increasing leve ls of unlabe led hormone will increasingly bind to the antibOdy and displace labeled hormone from doing so Thus the higher the concentration of [Ii ] in the reaction solution the more the lHl [A b] and the less the [Ab] will be formed T he R IA is therefore considered a compelitive inhibition assay rn practice the R IA is executed by adding reagents to a series of test lubes as depicted in Fig 43 An initial set of tubes constitutes the standard curve in whieh increasing sta ndard (known) amounts of unlabeled hormone are added to the fixed amoun t of radiolabeled hormone and antibody Remaining tubes contain the same amounts of [11 ] and [Ab] as do tubes in the standard curve but in place of a standard amount of hormone a given amo unt of the fiuid containing unknown amounts of the hormone (eg serum) is added T he reaction is allowed to proceed to equilibrium In some RIA pro-cedures a second antibody is then added that recognizes and binds to the first antibody creating a larger protein complex that is more easily separated by the final ste p which is centrifugation to separate [H] [Ab] from unbound [H] The amount of [H ] [Ab] in the pe llet followi ng centrifugation is determined by monitoring the radioactivity emitted by the tubes in a gamma radiation counter and plotted versus the standard amounts of [H] added to yield a standa rd curvilinear plot (Fig 42) The amount of radioactivity from [H] [AbJ in the sample (unknown) tubes is then used to extrapolate the value for the unknown from the standard curve In addition to protein hormones cyclic nucleotides and many other nonpro-tein substances are also measured by these RI A procedures with remarkable sensitivity precision an d specificity The non-proteinaceous substances are however usually conju-gated to an antigenic carrier protein to produce antibodies for their assay

idcly Us

llle avai labil ity of monoclonal antibodies was a major enabling step in the deve lopment of ne ve r im munoassays many of which can be perform ed without the use of radiolabeled reagen ts Immu1ometric assays are those which utilize chemically labeled monoclonal anti-bodies to directl y measure hormone levels in a non-isotopic procedure Th ese assays usually utilize two diffe re nt monoclonal anti bodies tha t recognize two separate d epitopes on the same target ho rmone molecule The fir st antibody is attached to a solid phase (eg the surface of a we ll in a test p late) and it is used to capture the hormone by binding to its orrespond ing epit ope A second signal antibody is chemically attached to a fiuorescent

chemiluminesccnt or enzymatic label When this signal antibody is subsequently added to th e incuhation it a ttaches to a second epitope on the captured hormone The ch emical signal

bstrate

Primary Antibody 2

Antigen)

AUbody

Capture Assay Sandwich

Principle of typical immunometric andwich assay

An I 1 01 Harmon Acuor 77

provided by the signal antibody can then be read as a direct re flection of the amount of bound hormone Figure 44 depicts the principle of this type of immunometric assay which is sometimes referred to as a sandwich immunoassay Many other variations of immunomet-ric assays have been developed and their ease of use and sensitivity has prompted their widespread use in research and in medical diagnostics Many of the home pregnancy tests for example make use of all immunometric assay principle to detect the earl pregnancy associated hormone human chorionic gonadotropin (hCG ) in urine

al si of Hormone Action u oJ or

A number of endocrine methods use the radioactive isotopes of various elements (cg 125L 45Ca 35S 32p 2JNa 14C 3H) to determine physiological and biochemical responses within a cell The half-life of a hormone for example can be ascertained by radiolabeling the hor-mone and then determining its distribution excretion rate and metabolism within the body of an animal Iodide [12Sr] is taken up by thyroid cells and incorporated into th yroid hor-mones (T4 and T3) Radiometric methods can be used to measure uptake of radioactive iodide atoms by the thyroid Information obtained often reflects the biosynthetic activity of the thyroid gland and this method is also used to localize hyperplastic thyroid tissues

The radioisotope of carbon [14C] can be incorporated synthetically into the structure of a molecule such as glucose Subsequent metabolism of glucose with the concomilant evolution of radioactive carbon dioxide C4C] 02 is a measure of the metabolic activity of the cell Insulin for example increases release of radioactive CO2 by stimulating the uptake of radi-olabeled glucose into diaphragl11 muscle where it is metabolized This is also a good example of a bioassay Tritium a radioisotope of hydrogen CHJ is widely used in autoradiography and in enzyme assays The tritium isotope is generally used as a component of some organic structures such as an amino acid or nucleotide (eg thymidine) The sodium radioisotope [23Na] is most often used to measure Na + uptake into nerve or muscle cells during studies on transmembrane potential changes in response to chemical messengers or other stimuli Radioactive calcium [45Ca] is often used to measure uptake of this divalent cation during muscle contraction and relaxation nerve stimulation or cellular secretion Moreover this isotope can localize Ca2+ sequestration within mitochondria or in the sarcoplasmic reticu-lum Incorporation of sulfur eSS] into the amino acid cysteine has been particularly produc-tive in the study of neurohypophysial hormone synthesis because neurohypophysial peptides contain a high percentage of this amino acid Such labeling of newly synthesized neurohypophysial hormones has greatly advanced studies on the movement of these pro-teins by axoplasmic transport down the neuronal secretory axons (C hap 7)

Radioactive phosphorus e2p] in the form of phosphate can be used to monitor protein phosphorylation as induced for example by hormonal stimulation of cells (see Fig 317) A variety of assays use radioisotopes to determine the concentration of a hormone in an endocrine tissue in the blood or in the urine Radioisotope assays can also be used to deter-mine enzyme activation or inhibition by a hormone In each of these assays specialized equipment capable of measuring the decay of the isotope is used (eg liquid scintillation counters or gamma counters)

adioliq gu1 lion

The actions of hormones in target cells may be subject to physiological regulation such as up-or down-regulation and endocrine researchers use a variety of methodologies to investigate how these adjustments occurln many cases it has been shown that alteration in the respon-siveness of a tissue to a hormone may occur as a result of alterations in the number or affinity of active receptors that are present in the tissue Chronic hyperinsuJinemia for example can induce a pronounced reduction in plasma membrane insulin receptor number a phenomenon that results from a high rate of ligand binding and subsequent internalization and degradation of receptors in target cells The reduced number of insulin receptors in turn is associated with a reduction in the responsiveness of the target tissues to insulin Radioligand binding assays can be used to document changes such as these in receptor number or affinity for ligand

Figure 46 Radioisotope enzyme assay tyrosinase assay

AnalySIS of Gen Expres Ion In Endocnn Sys 79

HO H Hb-I I H -NH2 - Tyrosinase-7HO II r-r Ho-L gt-T--NH WOH H COOH H COOH

[3H]-Tyrosine Dihydroxyphenylalanine C [3H]-Water Tyrosinase Assay

grown A sample of ti ssueorgan [rom the anim al or the ce ll cu lture is then placed on a microscope slide as is done for routine microscopy T11 the darkroom tbe slide is now immersed in a photographic emulsion and kept in the dark fo r a period of time Decay of the isotope (usually tritium) leads to reduction of the ilve r bromide in the overlyi ng ernulsion to silver crystals Because the beta particle released by the tritium tra ve l only a short dis-tance the silver grains d irectly over the emulsion are prefe rentially exposed since they are closest to the source o[ isotope decay T he tissue with emuls ion is then placed in developer and stained if desired By this method for example one can determine the incorporation of tritium-labeled thymidine into D A [14] This nucleotide is generally used to de termine the mitotic activity of cells After orchidectomy for example ther enhanced hypothalamic activation of pituitary gonadotrophs (due to the lack of nega tive feedb Llck in hihition by testosterone) This leads to increased incorporation of radio labclcd thymid inc into the DNA of these cells which can be visualized by autoradiographic me thods

The action of tc roid hormones involves interaction with nuclear DNA Au toradiogra phy using tritium-labeled steroids provides a method for demonstra ti ng the ce llular site of steroid action (Fig 47) Radiolabeled peptide can be used to de te rmine the topo-graphical localization of hormone recep tors in an organ or tissue (sec Fig 77) Some hor-mones mediate their effects through stimulation of RNA synthesi s This is d c mo llSlr lt1 ted by the use of tritium-labeled uracil which is incorporated specifically into RNA rather than D A Using giant chromosomes of the dipteran fly (midgc) Chironomus for example it is possible to localize tritium upta ke to specific Ba lbiani rings ( large p uffs) of thc chromo-somes [2] sites known to be responsible for very active RIA syn tl1c is (F ig 413 )

nal sis GeneE ression in Endocrine stems The processes by which the DNA sequence encoding a pro tein is decoded leading to the production of that protein are collectively re ferred to a gene express ion Hormones often exert their effects by inducing alterations in the gene expre ssion of specific proteins in a target tissue Moreover hormone genes themselves are ty pically subject to physi ological

19b e (a) Heavily labeled cells in the medial p reop tic nucleus of the mouse after inj e ction with eHJestradiol x 850 (b) Labeled neurons at a higher magnification showing the concentration of silver grains over the cell nuclei x 1360 (c) Guinea pig anterior pituitary ce1ls radioactivity is re tained in nuclei of certain cells after injection of e HJestradiol x 1360 (From Warembourg [36J with permission)

f ldocrtne MethodologIes 80 Chapter 4

Pattern of eHJuridine incorporation into RNA produced by larval salivary chromo-somes of a dipteran fly Note intense labeling of Balbiani rings 1 and 6 (From Beermann [2] with permission)

regulation In many endocrine studies therefore the analysis of changes in the expression of specific genes provides critically important information about hormon e synthesis and action There are several potential ways in which gene expression may be regulated including the stimulation or inhibition of (1) transcription of D NA to RNA (2) processing of RNA to mRNA (3) rate of m RNA turnover (conversely its stability) (4) translation of mRNA to protein andor (5) post-translational processing of the translated protein Some methods permit assessment of the rate of transcription while others rely on steady-state mRNA lev-els as a summary indicator of processes 1- 3 Measurements of mature protein accumulation can give an estimate of the overall rate of synthesis although these values may be difficult to interpret if the rate of degradation or release of the protein is also regulated independently of synthesis

p Irvels a a Hybridization or annealing involves pamng of complementary strands of nucleic acids H ybrids can form between D NA -DNA DNA-R NA or RNA-RNA strands DNA is analyzed on a solid support such as a membrane (Southern hybridization or Southern blot) The oldest and most common method for the analysis of R NA is the Northern blot In this procedure RNA is isolated from a group of cells or a tissue electrophoretically fractionated by size in an agarose gel and then transferred to a nylon or nitrocellulose membrane A radiolabeled cDNA probe corresponding to the mRNA of interest is then hybridized to the R NA on the blot The bands of hybridization are then detected via exposure of apposed X-ray film and the magnitude of the specific autoradiographic band thus serves as a valid index of the amount of the specific RNA sequence present in the sample [33] Another common method for mR NA analysis is the ribonuclease protection assay in which a radiolabeled RNA probe and the cor-responding target mRNA are hybridized in a solution and then treated with a ribonuclease that digests single-stranded but not double-stranded RNA The remaining probe- mRNA hybrids are then electrophoresed and autoradiographically quan tiiied [21] (Fig 1215)

The most methods for measurement of mRNAs include reverse transcriptase-polymerase chain reaction (RT-peR) and real-time polymerase chain reaction (real-time peR) techniques Both approaches provide powerful alternatives for the measurement of mRNAs Ihat are expressed in low abundance such as those that encode many hormone receptors or intracellular signaling molecules A reverse transcriptase enzyme is first used to reverse transcribe the mRNA contained within a sample cDNA and then the sequence of in terest is amplified (copied thousands of times) by the polymerase chain reaction The p e R makes use of a pair of -20-mer Oligonucleotide primers that are designed to hybridize

An yst of Gene ExPI Ion m Endocnn Sys 81

Total RNA1Reverse Transcriptase

Fi Schematic representation of the principle of the polymerase chain reaction (peR)

dsDNA

Primer A Denature 93-99C

3 Annealing 37-59degC

3

dNTPs Taq Polymerase

Primer B

Add Primers A amp B 1

Extension 72degC 1 kbmin

lRepeating Cycles of Denaturation Annealing ExtenSion

Multiple copies of dsDNA of the same size as the distance

between primers A and B

with nucleotide sequences that flank the D NA sequence to be amplified (Fig 49) The reaction mixture contains the samp le cD A_ specific oligonucleotide primer pair a DNA polymerase enzyme and deoxynucleotide triphosphate (d TP ) precursors and is subjected to repeated cycles of heating and cooling in a thermocycler apparatus During each cycle the following occurs (1) heating causes the dou ble-s tranded 0 A to be de nct tured ie rendered single-stranded (2) the prime rs anneal with the corresponding flanking sequences on each 0 A strand (3) the primer sequences are e longated by the actions of D A polymerase which incorporates the dNTPs into the extensions thus producing (4) two double-stranded D NA molecules that correspond to the original double-stranded D A These cycles of denaturing primer ann e tlling and extension are repeated to yield two-fold amplification of the peR product or amplimer per peR cycle Thus thirty cycles of p eR would be expected to increase the amount of DNA corresponding to the sequence between the two primers by 230 or -1 billion times For semi-quantitative RT-PCR the amplified product is electrophoresed and either stained or autoradiographi-cally analyzed for the extent of radiolabeled dNTP incorporation The amount of the ampli-fied product is then considered to be proportional to the amount of the specifi c cDNA sequence originally present in the sa mple Often the results are normalized to those obtained from the amplification of an mRNA sequence of unvarying abundance for example a housekeepi ng gene as an internal procedural control

More accurate quantitation of mRNA can now be achieved using real-time PCR proce-dures Several versions of thi s basic method have been developed but all use a fluorescence-sensitive apparatus that monitors in real-time the progress of a PCR that emits fluorescence signal in proportion to the amplification of product Often the relative amounts of the target sequence present in a sample are indexed as a function of the number of PCR cycles or cycle times (C t ) required to reach a minimum threshold of fluorescence

82 Chapter 4 ndocrine MelhodolOgies

C A T

In situ hyb ridizationThe probe (RNA or DNA) is labeled with a radioactive or nonradioactive d etection system The DNA probe in this example (5-CATG-3) is hybridiz-ing with the messenger RNA associated with the rough endoplasmic reticulum After hybridization to ce lls or tissue sections the RNA or DNA of interest can b e detected by autoradiograp hy or non-isotopic methods Advantages of in situ hybridization include more precise intracellular localization of the RNA or DNA of interest and direct visualization of positive cells in relationship to surrounding cells and tissues (From Lloyd with permission Endocrine Pathophysiology 464-72 1993)

TiSSUt- d Cell-Specific RNA ExpressionPaltern e Analyzed y h ft t lion

llle nucleotides can also be localized within cells and tissues by in situ hybridization (ISH) [20323839] (Fig 410) This powerful technique used to localize mRNA involved in hormone synthesis is beautifully demonstrated in Fig 85 Most ISH methods utilize radiolabeled cDNA or cR NA probes to hybridize to an mRNA sequence present in the cells of a slide-mounted tissue slice The location of the hybridized probe is indicated by development of apposed photo-graphic fi lm or more precisely by exposure of radiographic emulsion coating the mounted tissue TIl US the identity and location of cells expressing the mRNA of interest are indicated by the appearan ce of si lver grains in the overlying emulsion The number of silver grains that develop over an individual cell is proportional to the number of mRNAs encoding the protein of interest that hybridized with the radiolabeled probe The ISH method can therefore permit semi-quantj tation of ill RNA expression the principal advantage of the ISH technique is that it permi ts both quantitation and cellular resolution of mRNA expression patterns- an advantage not shared by total RNA hybridiza tion methods With in situ hybridization it is also possible to address qllestions regarding differential gene regulation within different nuclei in a given brain region or within different cell populati()ns in a specific nucleus This level of resolution can reveal subtleties of gene regulation that cannot be detected with Northem blots or RNase pro-tection assay methods Moreover it is also possi ble to determine if specific colocal ized glnes are coordinatLly regula ted in brain nuclei in which some or all of the neurons display such colocal-ization The principal disadvantages of in situ hybridization methods are that they are time- and labor-intensive ilnd th at absolute cannot be ensured [6]

Hor Uee e es ion b fill I 11 NA Micoarramiddot Many horm()nes exert their actions on a particular targe t tissue by simultaneously altering the expression of ma ny different genes The development of DNA microarray technologies has enabled endocrine researchers to identify hormone-responsive genes among thousands of candidatc$ in single experiments Several ve rsions of these technologies have been devel-()ped In general however either oligonucleo tides or cDNA probes corresponding to large nu mbers of genes-in some cases encompassing the great majority of genes in a particular genome - are covalently attached to specified sites in a micronized grid on glass chips The RNA sample is converted to eRN A or eDNA labeled with a fluorescent marker and hybridized to the immobilized probes on the chip A specialized chip reader measures the in tensity of the fl uorescence at each probe positi on on the chip reflecting the level of the corresponding I11RNA Computer analysis and comparison of the data among chips then permits ick ntifica tion of those gen es that are expressed to a different extent in hormone-stimulated versus untrea ted conditions [27]

J pl)rtfr cts The techni ques of molecular biology also ena ble endocrine researchers to analyze the mech-(l nisms controlling transcription of genes that encode hormones receptors and proteins that are regu lated by hormone action in target tissues Often the objective of these studies is to iden tify promoter regions of a particular gene that regulate its transcription and analyze regions within the promoter that are targets of specific transcription al regulators Reporter gene constructs are often used in these studies to obtain a read-out of promoter activity Such i construct might he made by cloning the putative promoter region of a gene and fusing it to a common reporter gene such as the firefly luciferase gene The promoter-reporter con-struct can be transfected (chemically in troduced) into cells in culture and the transcriptional ac tivity of the promoter is monitored by measurement of reporter gene expression In the case of the lucifc rase reporter construct transcriptional activity is reported by luciferase enzyme activity this is easily qu antitated by lysing the cells adding luciferin and the luciferase substrate ATP and moni toring the lucife rase-cata lyzed luminescence with a luminometer E ndocrine researchers often study the fea tures () f a promoter by bashing it making trun-cat ions or spicific sequence mutations or deletions in the promoter sequence and then assessing cbanges to its basal act iv ity andor responses to putative transcriptional regulators A scyuence that normally mediates a positive or negative regulation of transcription can thus be identified by the loss o r gain of transcriptiona l activi ty in its absence

83 PhaIInacolog1C tho

Most cells are excitable that is they will respond to st imuli by becoming depolarized or hyperpolarized which results for example in relaxation or contraction respectively of smooth muscle These potenti al transmembrane changes can be monito red b in tracellular or extracellular microelectrodes The respo nse of the cells to hormonal or oth r stimuli therefore can be detected Most stimuli depolarize cells but some chemical messengers-for example gamma-aminobutyric acid (GABA ) a central nervous system neurotrans-mitter hyperpolarize cells Chemical messengers can be applied to the surface of a cell by microionlophoresis and resulting transmembrane potential cha nges recorded downstre am by microelectrode recordings E lectrophysiological methods have been important in stud y-ing electrical properties of the neurosecretory neurons of the neurohypophysis (see Fig 73) and the response of pituitary cells to hormones (see Fig 619)

Pharmaco d Many exogenous substances (eg drugs) interac t with molecular components of cells and they therefore can be used to study the physiological activity of cells or the mechanism by which chemical messengers stimulate or inhibit such activity [10] For example a t the level of the plasmalemma cardiac glycosides such as ouabain are used to inhib it the Na + j K + pump Certai n hormone secretions are enhanced or inhibited by ouabain and th is suggests that active transport (Na + jK + ATPase) systems may be involved in the secretion mecha nism of these hormones

Many agents are inhibitors of in tracellular enzyme activity Of particular inte rest are the methylxanthines such as theophylline (from tea) and caffeine (from coffee) which are phos-phodiesterase inhibitors These drugs mimic the actions of many hormones because they elevate intrace llular cAMP levels (see Fig 37)

There are a number of metabolic inhibitors that affect util ization of substrates for ATP formation Iodoacetic acid blocks glycolysis dinitrophenol (D P) uncouples oxida ti ve phos-phorylation and oligomycin prevents mitochondrial phosphorylation of ADP to ATP The false substrate 2-deoxyglucose can be used to slow down glucose utiliza tion with in cells TIlese inhibitors provide information on the degree to which the glycolytic (Embden-Meyerhof) pathway or the citric acid cycle (Krebs cycle) contributes to cellular function

Colchicine a plant alkaloid inhibits microtubule assembly by binding with tubulin the pro te in subunit of these intrace llular filaments Inhibition of hormone action by colchicine or related micro tubule inhibitors (vincristine and vinblastine) might S Lggest therefore that the hormone works through a cellular mechanism in which microtubules a re involved Insulin secretion is inhibited by colchicine suggesting that microtubules may function in the secretion of this hormone Cytochalasin B is a fungal metabolite that specifically inte rferes with microfilament function and is without effect on microtubule integrity Cytochalasin B is inhibitory to the secretion of a number of hormones suggesting that these filamentous organelles may be involved in the secretory process of these hormones

At the leve l of the nucleus actinomycin D inhibits RNA producti on Puromycin and cycloheximide on the other hand inhibit protein synthesis Thus inhibition of hormone action by actinomycin D implies that the hormone works through a transcriptional process of RNA synthesls Inhibition of hormone action by puromycin or cycloheximide in contrast is interpreted to implicate a translational process of protein synthesis in the action mecha-nism of the chemical messenger

Rather specific cation ionophores have been discovered and synthesized These organic cation transport molecules specifically incorporate certain cations into the ir structure They cross biological membranes and carry ions into cells Ionop hore A23l87 for examp le is pe-cific for Ca2+ and because many hormones activate cells by a calcium ion mechanism this ionophore is in some systems hormone-mimetic Valinomycin is an ionophore with relatively high specificity for K+ transport Some agents on the othe r hand are specific inhibitors of ion transport into cells Verapamil a Ca2+ channel antagonist blocks Calmiddot entry into cells Usee concomitantly with a hormone verapamil s inhibitory action may suggest t ha t a partic-ular hormone action is Ca2+ -dependent Local anesthe tics such as procaine and te tracaine may also block the action of some hormones by their antagonism of Ca2+ flux into cells

R omblii nl DNA T hruqu 85

TULE 1 Some pharmaceutical and other agents used in cell physiology studies

Actinomycin D Alloxan Chlorpromazine Cobalt chloride Colchicine Cycloheximide Cyproterone acetate Cytochalasin B 2-DeoxY-D-glucose Dexamethasone Dinitrophenol Ergot alkaloids (eg ergocryptine) lodoacteic acid (or iodoacteamide) [allophore A23 187 Liposomes Local aneslhctics (procaine tetracaine) Methylxanthines (theophyline caffeine) Oligomycin Puromycin Saralasin Spironolactone Thiouracil [3H]Thymidine [ H]Uracil Valinomycin Verapamil

proceptive (eg ho pping and

Inhibits RNA syn thesis (transc ri ption) Destroys beta (insulin-secreting) ce lls of pancrea tic islets Dopamine receptor antagonist Destroys alpha (gl ucagon- ecrering) cells of pancreatic islets Disrupts microtubules 1nhibits protein synthsis Tes tosterone rece p tor an tagonist Disrupts microfilam cnts Metabolic inhibi to r inhibits glucose uptake and utilization Synthetic glococorticoid agonist Metabolic inhibitor uncouples oxidati ve phosphorylation D opam ine receptor agonist (inhibit middot prolactin secre tion) Me tabolic inhibito r inhibits glycolysis Calcium (CalT ) transport carrier (ionophore) Drug carriers Irthibit ce llular uptake of Cal Phosphodiesterasc inhibitors (elevate cAMP) Metabolic inhibitor inhibits phosphorylation of ADP Lo ATP Inhibits prote in synthesis (translation) Angiotc nsin II receptor antagonist Aldosterone receptor antagoni t Inhi bits lilyroid iod ide up ta ke and T-TJ synthe is Studies on D A synthesis S tudies on RNA syn thes is Potassium transport carrier (ionophore) Inhibits cellular uptake of Cah

darting in females) as we ll as consummatory behaviors (eg mounting intromission and ejaculation by the male or lordotic behavior of the female) Specific behavioral assay methods a re co nsidered in subsequent chapters as they are used to study hormone effects on specific behavi rs Se nsitive and sophisticatcd behav-ioral assays have been developed to assess the effects of hormones on feeding drinking sexual behaviors

R ecombinant DNA technologies are now used to cx press recombinant pro te ins in a varie ty of in vitro expression systems In some cases the purpose of the recombinant protein expres-sion is to a nalyze its biological activi ty or its structure- func tion relatio nships in a biologica l co ntext In other circumstances larger amounts of purified pro te in are required fo r biophys-ical studies of its molecular structure Express ion and p urificat ion at the larg st scale and perhaps posing some of the biggest technical ha ll nges is req uireJ ror the product ion of hormones for medical andor commercial pu rposes

H ormones play important Toles in regulati ng the activity o t most cells of the body indeed some hormones are necessary for life Parath ormone and a ldosterone two hormones that regula te th e electrolyte composition of the body arc examples G rO til ho rmone (GH) is necessary for growth and its absence in the young animal r suits in short stature Fail ure of the pancreas to produce insulin results in diab tes mell itus In middotul in replac ment therapy requires obtaining these hormones from other an imal so urces but some individuals develop antibodies to these nonhuman sources of the hormone [n the cas( of GHo nly pitu-itaries obtained from human cadavers or primates can be lIsed since GH from other animals is ineffective

Recombinant DNA techniques offer the hope tha t a number of hum an hormones can be synthesized by bacteria in large amounts and at reasonable prices Recombinant DNA

86 Chapter 4 tndocrtnc Melhodolooles

Recombin ant DNA technique for the microbial p rod uction of insulin (From Rig gs et al [23] with permission )

Photograph of gigantism produced in the mouse by injection of metallothionein-growth hormone fusion genes into the pronucleus of the fertilized egg followed by implantation within the uterus of a recipient mother Giant mice (left) became almost twice as heavy as littermates (not bearing the GH gene) (Reprinted by permission from Palmiter et al [18] Nature 300 [5893] 611-l5copy1982 by Macmillan Journals Limited)

E coli

fJ-Gal A

t

Galactosidase A Chain

1 1 Partial Purification 2 Cleavage with CNBr 3 Purification of

Insulin Chains Insulin Insulin

A Chai n otfXX) B Chain y

1 cxxxxo S S S S

Active Insulin

E coli

fJ-Gal B

(

t Beta B Chain

Galactosidase1

techniques are essentially in vitro extensions of natural phenomena These methods involve purification or synthesis of genetic material and its insertion into a bacterial host Several bacterial strains have been constructed that can produce human hormones and genetically engineered bacteria now provide important sources of peptide hormones [23] Two methods have bee n used to provide the genetic material to be cloned For shorter peptides (eg insulin somatostatin) DNA synthesis is used to make a gene of the desired hormone In the case of the two chains of insulin the 21-amino-acid A chain and the 30-amino-acid B chain are made in separate bacterial strains as tails on a larger precursor protein the enzyme

The individual insulin chains are then clipped from the precursor proteins and after purification of the separate insulin chains they are joined by air oxidation (Fig 411) The method used for larger proteins utilizes mRNA and reverse transcriptase to make a D NA copy that is then inserted into the bacterial genome and cloned For example cDNA fo r proinsuJin is inserted into Esch erichia coli cells which are then grown by fermen-tation to produce proinsulin 1l1e connecting peptide is then cleaved enzymatically from proinsulin to produce human insulin

The functions of hormones and their receptors are now routinely studied using the powerful and remarkably informative approaches afforded by genetic engineering These methods are commonly used to produce a desired genetic modification in a mouse and observe the pheno-typic consequences of the genetic alteration Most often genetic engineering is used to generate animals in which either a foreign gene is expressed (a transgenic animal) or a specific gene is inactivated (a gene knockout animal)

In the early 1980s endocrine researchers first injected foreign genes into mammalian embryos and found that this transgenesis approach could provide a powerful tool for use in endocrine research Foreign DNA can be introduced into the mammalian genome by microinjection of DNA molecules of interest into pronuclei of fertilized cells followed by the implantation of the eggs into the reproductive tracts of recipient mothers The integration of the fo reign DNA into one of the host chromosomes at an early stage of embryonic develop-ment results in the development of a transgenic animal [1132234 35] In the earlier exper-iments microinjection of the stru ctural gene for rat growth hormone into the pronuclei of fertili zed mouse eggs resulted in the development of some offspring that grew significantly larger than their litlermates (Fig 412) This methodology has important implications for

EnQln nn 87

studying the biological effects of growth hormone as a way to accelerate animal growth as a model for gigantism and possibly as a means of correct ing genetic disease In the years following these original studies transgenesis has been used in my riad ways to study hormone and receptor function Transgene expression can be targeted such that the transoene is only active in a desired ceIl or tissue This can be accomplished by placin g the coding region of lhe transgene under the control of a promoter sequence that is aclive only in the desired target cells In some cases this is done to assess the consequences of overexpression of a hormone or receptor by the specific targeted cell group in other circumstance the transgenic protein is designed to be functionally inactive and serve as a dominant negative (inhibitory) modulator of its endogenous counterpart in a targeted cell group Transgenesis is also used to label cells of a certain phenotype such that they may be visualized in the living sta te For example the promoter sequence for the hypothalamic neurohormone gonadotropin-releasing hormone (Gn RH) can be fused to a gene encoding the jellyfish green fluorescent p rotein (GFP) to cre-ate transgenic mice in which the transgene and hence the fluorescen t JFP molecule are only expressed in GnRH neurons This particular animal model has been used to permit visualiza-tion of these few ne urons in living brain tissu $ fo r electrophysiological experiments [28]

Transgenic technologies have been studied in several commercially important animal species with emphasis on growth enhancement as a strategy to shorten long production cycles For the production of transgenic Pacific salmon an all-salmon genetic construct consisting of a metal-10thionein-f3 promoter fused with the full-length type I GH gene was developed D1e pO n-MTGHI DNA was injected into coho salmon eggs with extraordinary result s (Fig 413) On average the transgenic salmon were more than 11-fold heavier than the nontransgenic controls Most interes ting was the observation th at the transgenic fish precociously developed a silver body coloration typical of salmon undergoing the physiological preadaptation (smoltification) necessary for the spring migration from freshwater to the marine environment

Gene knockout animals have provided a wealth of vital information about the physio-logical roles of hormones and their receptors Indeed the loss of function in a gene dele-tion mutant animal can confirm the essential role of a protein in a specific endocrine process or even recapitulate a disease state that is believed to arise from an analogous gene mutation in humans The first step in the gene targe ting procedure is the illtroduction of a disrupted allele of the target gene into cultured e mbryonic stem cells The targeting construct is fashioned to contain a selectable marker such as the gene encoding a protein that confers resistance to a cytotoxic drug O nly those cells in which the homologous recombination event occurs will therefore survive growth in media containing the toxic drug Using this process the cells heterozygous for the targe ting construct are selected grown in culture and the n injected into mouse blastocysts T hese embryos are then

Nontransgenic (left) and transgenic (right) coho salmon siblings at 14 months old showing size difference and silver appearance of transgenic individuals indicative of transformation to seawater adaptability Length of top large fish (fork length) 418 cm (Used by permission from Devlin et al Extraordinary salmon growth Nature 371209-101994)

88 Chapter 4 docnne Methodotoqtes

surgically transferred into the uteri of pseudopregnant femal e mice The resultant progeny are chimeric for the targeted cells Breeding of th ese founder animals produces some mice carrying a single copy of the disrupted gene in all cells and thus subsequen t breeding of two heterozygous mice will produce some mice that are homozygous for the inactivated gene sequence These homozygous knockouts if they are physiologically viable can then be studied to assess the potential loss of function that accompanies the loss of th e specific hormone receptor or other protein of in teres t

In some cases mice carrying a germline mutation in a vital gene have lethal defects tha t prec lude their use To circumvent this probl em gen e ticists have developed methods that c rmit cell-specific gene targeting in which the gene mutation can be induced in a specifically targeted subset of cells in the body The 10xP-Cre recombination system is one such method This strategy involves the generation of mice bea ring site-specific recombi-na tion sites call ed loxP sites in the intronic sequences that flank an ess ential exon of the target ge ne A second line of mice is produced that harbor a transgene construct contain-ing a gene promoter fused to the Cre recombinase gene importantly the promoter is known to be active only in the cells of interest Mating of the two mouse lines results in the express ion of C re protei n only in the targeted cells where it acts at the 10xP sites to delete th e exo n of the targeted gene Thus the specific gene deletion only occurs in the target ce lls avoiding the lethal complications of total germline gene deletion Many strik ing examples of the successful use of thi s system are found among studies of the tissue-specific actions of insulin Conditional disruption of the insulin receptor gene in brain for exam ple recently provided unambiguous confirmation th a t insulin acts centrally to regu late feeding behavior and energy homeostasis [4]

Both vertebrate and invertebrate animals are used as model sys tems for endocrine research A few examples of vertebrate models are discussed

These most primitive vertebrates which include the hagfish and lampreys have not been extensively stud ied These eel-shaped animals spend their lives in both marine and freshwa-ter tnv ironments and therefore most likely possess a complex endocrine system as in other more evolved (higher) vertebrates

TIlese ancestors of the bony fishes include sharks skates and rays and although prevalent in numbers and readily available they have not been the subject of much endocrine research

These fish represent the largest and most diverse group of vertebrates They have evolved adaptive features that enable them to survive in a multitude of ecological niches Many of their specialized physiological functions are under endocrine regulation [20] Migration to and from the sea by some species requires unique roles for some hormones Prolactin for example is essential for survival in some fish transferred from saltwater to freshwater (as happens in salmon migrations) The control of color change in fish is much more complex than in other ver-tebrates and may in some species use a recently discovered melanin-concentrating hormone whose function may be limited to more recent (teleost) fishes (Chap 8) The corpuscles of Stan-nius are also unique to certain bony fi shes and produce a hormone teleocalcin not found in other vertebrate species 1l1e urotensins derived from the urophysis of certain species provide another example of hormones whose roles may be restricted to certain bony fishes (Chap 7)

A mphibians include urodeles (salamanders eg mudpuppies newts) and anurans (frogs and loads) as well as less well-known representatives 111eir ready availability their rela-tive ly sma ll size and the fact that they go through a dramatic metamorphic change from

intrusive me thods of tudy

AnirTt W I ar 89

larval to adult life has made them importa nt models for c ndocrin rese arch O ur under-standing of the roles of the thyroid gla nd and its hormones in endocrine physiology has been greatly influenced by studies on amphibians (Char 1) ) 111e amphibian egg has always been an important model for an understanding of ea rly vertebrat e de e lopme nl T hese eggs have provided evidence for roks of ho rmones (growth factors and ste roid hormones) during the earli est developmental stages

Snakes lizards turtles and torto ises as we ll as the crocodilians and related species compose this vertebrate group The re has been relatively li llIe research done on these animaL but important information o n some unique reproductive and beha io ral stra tegies under endocrine control have been discovered (see Fig 712)

These descendants of the reptil es include a large var ie ty of birds that occupy a grea t d ive r-sity of ecological niches It is not surprising the re fore that these anim als have evolved some unique endocrine strategies Both wild and domes ticated birds (chicken quail) are available in substantial numbers for experimental studies Birds have provided unique mode ls for unders tanding the nc uroanatomica l substrates controlling behavior (eg bull bi rd song co urtship Chap 16) Th e role of the kidney in vitamin D hormone production has been best studied in birds becaue of the importa nt relationship b tween the hormone and the control of Ca2 -- metabolism necessary for eggshe ll formation (Chap 9)

The mouse rat hamster and guinea pig have been pa rticularl y importan t anima ls for the adva ncement of o ur knowledge of endocrinology Use of the mouse has predominated in molecular genetics and the rat has perhaps proven most effec tive in the study of endocrine physiology The hamster has proved to be a par ti cul arly useful model for und erstanding th e role of the pineal gland and its horm one melatonin in the control of reproductiv( cycles (Chap 20)l11e nude mouse has bee n a useful mode l for supporting the growth of human tumors since it lacks an active immune ystem neces ary to reject the transpl an ts The Bra ttleboro rat lacks the hormone vasopressin and the refo re is a unique model of diabetes insipidus (Chap 7)

Monkeys because of their close evolutionary relatedness to humans have played im portant ro les in und e rstanding the human endocrine system For example our knowledge of the structure and function of the corpus lute um of the ovary has been aided by use of primate models

Most endocrinologists would probably strongly agree tha t studies in animal s have been essen tial for th e progress of endocrinology Surgical rem oval of various rgans has provided key insights into the exis tence and origin of ho rmones Most endocrinologists would also probably agree that animals are still absolutely esse ntial fo r basic studies in endocrinological research Only recently for example an atrial natriuretic faclo r (A F ) was isolated from the heart of rats This a tri al peptide probably plays a pivo tal ro le in the pathophysiology of several disease states Nevertheless most endocrinologists p ro bably would agree that not all ani mals have always been treated humanely and in many cases the numbers of animals used may have been in excess of the actua l numbers needed n le past ind iscre tions are being rem(d ied a t most or all research centers

Molecular biology has now provided a variety of nonanimal model systems wherein the actions of hormones can be studied in vitro using cell cultures r37] Although these methods will not entire ly supplement studies on living animals they will provide some alternate less

90 Chapter 4 rldocrmc Methodologies

CllDC

As in other subdisciplines of biology endocrinological research faces some inte resting ethical Controversies regarding the use of embryonic stem cells or fetal tissues are similar to

those of other bioethical debates but with a novel twist because of the contested status of the fe tus and abortion Fetal tissue transplants hold great hope for many pat ients E xtensive work with animal models has shown that human fetal brain cells transplanted into the substantia nig ra o f monkeys with exogenously produced Parkinson s disease have had beneficial effects R esearchers think that the time has come for expanded clinical trials Cl inica l trials conducted e ithe r abroad or with private funding have shown that fetal tissue implants can significantly improve the symptoms of Parkinsons disease in some individuals Transplantation of feta l brain ce ll s fro m 6- to 8-week-old fetuses into multiple sites on each side of the patients brain resulted in an increase in brain dopamine production and more importantly symptoms were greatly red uced for a considerable period of time [31] EXtJerimenta l evidence is also strong that feta l isk t cell transplants can restore normal insu lin function in diabetics A nd fetal thymus and liver transplants may have utility for blood and immune sys tem disorders M any other examples could be cited to docllment the urgent need of fetal tissues for use in clinical medicine Cnfortu-nate ly respect for the needs of such patients appears to conflict with respect fo r prenatal human life and larger socie tal concerns [24] More recently many of the same bopes and COD-

ce rns ha ve arisen regarding the use of embryonic stem cells 1l1ese cells possess the capacity to be differentiated into any type of ce ll (totipotent) or at least a number of different types of cells (pluripo te nt) E mbryo nic stem ce ll s are potentially de rivable from thousands of unused fertil-ized human eggs stored in the freeze rs of in vitro fertilization clinics Advances in developmen-tal cell biology have opened up a new frontie r of biomedical science in which these ce lls may be induced to differentiate into cells that may be capable of restoring specific tissue function in several devastat ing disease states As of the writing of this ed ition of this textbook the United States Congress is considering legislation to permit the use of federa l funding for research with embryonic stem ce ll lines beyond the limited number that are currently approved R egardless of the legislative outcome concerns on the part of some citizens about the use of these cells m akes ill ike ly 1hat the public debate on this issue will continue into the foreseeable future

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d contrast each of the is How are they related 1 Short stature in children may occur as a normal inherited

trai t or it may result from a clinically identifiable pathol-ogy or e ndocrine pathophysio logy Growth hormone orchide cto myovariectomy te Icostcarti laginous deficiency (GHD) is an example of an endocrin e patllo-b[lsop hi licac idop hil ic (fish es ) physio logy in wh ich the abnorma lly low secre tion of GH atroph ylh y pc rp Iasia hypertrophyl can be the appare nt ca use of short stature in ch ildren Howi111 III unoassa yl hyperpl asic would a clinical e nd ocrinologist de te rmine if serum levels radioi 111m u noa SSH V unilaterallbi la te ra l of G H are abnormally low in a ch ild wit h short statureN orthe rn blotlrea l-timc peR imm unoeyloche mi st ryl What method would he or she use to analyze GH le ve ls inin situ hybridization blood What reagents would be required How does the transgenesi sige ne method work targeting

2 E strogen (ER) and progeste rone (Pg R) receptors are prognostic factors in breast cancer The expression of

ch ofth E Rs and PgRs in breast cancer cell s is a lso commo nly analyzed to guid e dec isions regarding th erapeutic op ti ons to pursue Wha t method would be used to chara cterizeectop ic transplantation binding protein the ce ll-specific express ion of the ER or PgR proteins inaxoplasmic tra nspo rt (rlow) immunocytochemistry breast tumor tissue sections D escribe the procedureatroph v glycoprotein and list the specific reagent and major equ ipment thatsandwich assay hormone an a log wo uld be required immuJl osY Ill patheetomy ion chan ncl

3 G ene targe ti ng methods ha ve bee n used to assess fun c-reverse transcrip tase peR imm unohistofluoresce nce tional consequences of hormone receptor ge ne ablationWestern blot transfee tl o n and to thereby clarify the functional roles o f the targe tedre porte r const ruct gene knocko ut anim al horm one receptor The targeting of th e receptor genecon d itiona l gen targeting ste m cells

91

is pres um ably accompanied by a loss of the receptor protein In stud ies such as th ese it is incumbent upon the inves tigator to demonstrate that the binding o f hormone to the targeted receptor is indeed climinated or at least significant ly diminished in targe t tissues of the gene knockout animals

a Describe a receptor binding d dy me th od that could be used to measure th e number and affinity of reccp-tors in the wild-type (no rmal ) anima ls and those in th e receptor gene knockout animals What reagents would be required How would the rece ptor binding assay procedure be performed

b Assume that yo u have performed the assay me th ods descr ibed in (a) on tissues from the wild-type and (homozygous) receptor gene knock out anima ls as we ll as from a heterozygous group of animals in which the receptor numbers may only be partially reduced but not eliminated by the di srup ti on of on ly one al lele of the receptor gene For each of the three ana lyses drClw approximat ions of the saturation binding curves that you wo uld expect to derive from the bi nding assays Include total specific and non-specific binding curves for each How would they dif-fer amo ng the th ree groups Additionally draw schematic representations of the Scatchard plots of each se t of data on the same graph 1low wo uld they differ How would they be simil ar

4 The decline in es trogcn leve ls in mcnopausal wome n is associated with increased ri sk of excess ive bone demi neralizat ion a co nditi on common ly known as osteoporosis Many investigators are atte mpting to unders tand where and how estrogen normally acts to protec t the ske le ton from bone de mine ra liza tion with the ho pe of developing new dru g therapies for patien ts wi th osteoporosis

a There are known to be two isoforms of the estrogen receptor ERa and that are encoded by separa te genes Which molecular methods could you use to determine the relative amounts of the respective receptor isoform mRNAs that are expressed in bone ce ll s Wh ich method would you use to ascertain which specific types of bone ce ll s eg osteoblasts or ostco-clasts express the mR NAs encoding or

b Consider how you might investigate the effects of es trogen decline on bone mineral ization in an exper ime ntal anim al Wha t surgica l me thod mi ght be used to induce a loss of circ ul ating estrogen in a female rat

c Suppose you have avai lable to you both a specific ERlX ago nist and a specific agonist You wish to determine if one or the other or both of the E R isoforms is more important in med iating the effects o f estrogen on bone de nsity Using the experimen-ta l animal model in (b) above design an experi -ment in which you could use these new dru gs to assess the re lative in volvement of the two recepto rs in mediat ing estroge ns protective effects on the skele ton

5 Insulin activates L11e insulin recep tor (TR) in hepa tic ce lls prompting it to beevlJ1 e phosph orylated on key tyrosi ne residues TIle rece ptor in tuUl is a tyrosine kinase tha t phosphorylates downstream signaling molecules such as the insu lin receptor substrate I (1 R 1) protein the reby activat ing this irllraceUular messenger What method

might you use to assess the degree of IR and IRS I ac ti va -ti on in cult ur of hepati c ce lls For your expe rime nts you have avai lable to you a panel of monoclonal antibod-ies that recogni7e tota l fR phosphotyrosine-l R total IRS l and phosphotyrosine- fRS l res pectively Describe the mcthod that you would usc

6 nabo lic steroid abuse has bce n anecdotally linked to a variety of pa thophysiologics and beha vioral di sordc rs but their molecular actions in the brain rcmain poorly un derstood It is likely th at these and rogens like other steroid hormones C)([t their actions in the brain by simultaneo usly altering the expression of many different ge nes and in seve ral diHerent partS of the brain

a What method cou ld be used to simultaneo usly iden-tify genes whose expression levels arc altered by

in the brains of experimental an imals Describe the c- perimental method and technologies that mi ght be used

b If specific testosterone-responsive genes were identi-fi ed by th is method then what methods could be used to specifically identify which brain ce ll populations exhibit testosterone-induced alterat iow in the expres-sion of thesc genes

[I] Aguzzi A et al 1994 Transge nic and knockout mice Mode ls of ncurological di sease Brain Pathof 43- 20

[2J Heermann W 1973 Di rect changes in th e pattern of Balbiani ring pu ff ing in ChironOI17I1S Effects of sugar trea tmen t Chromosotlw 4297-326

[31 Black J 1Y89 Drugs from emascul ated hormones The principle or syntropic antagonism In Vitro Cell Develop BioI 253 Ll- 20

(41 I3runing 1 C et al 2000 Role of brai n ins ulin recep tor in control of body weigbt and reproduction Scien ce 2892 12 - -

[5] Burgoyne R 0 and A Morga n 1993 Regulated exocytosi s Biochem J 293305-16

[6] Ca mp P e t al 1992 (iene express ion of peptiJergic signa ls rreets of sle ro id s Nrgturopro tocols 167-76

[7] Chi rguin J M 1990 Molecul ar biology for nonmole-cu lar biologists Diabetes Cale 13188- 97

[8] Davis J R E 1996 Molecular biology techniques in endocri nology Clin L nriocrino l 45 1 25-33

[9] FukamizuA 1993Transgenic animals in endocrinological invcstiga tioni L ndocrinol In vest 1646 1-73

[101 G ilman A G L S Goodman and A Gilman 1990 Go odman and G ilmans the pharmacological basis of th erapelltin ew Yo rk Macmillan

[1 1J Hoffman G M S SmitiJ and J G Vcrbalis 1993 c-Fos and related immeJia te tarly gene products as markers of activ ity in neuroendocrinc systems h ont NeLroen-docrinol 14173- 2 [3

[12] Holzwa rth M A 1 R Sladek Jr and K M Knigge 1976 Monosodi um glutamate induced lesions of the arcuate nucleus Anal Ret 186197- 205

[13] Kudlow 1 E 2002 Transgenic anilJ1 allllodels of pitu itary function In J D Baxte r S Melmed and M 1 C W eds Genetics in endocrinology Modern Endocrinology Se ries (L Ma rtini serlCs cd)- Philadelphia Lippincott Willi ams amp Wilkins pp 27-36

92 Chapter 4 docrlnC Methodologies

[14] Lebl ond C P 199 1 Time dimensi on In cell biology A radioautographic survey of the dynamic features of celis ce ll components and extrace llula r matrix Protoplasm a 1605-38

(15) Mc(ube 9149-67

J T et al 1916 Front N euroendocrino

[16] Mi llar S E et al 1989 Vaccination with Cl synthetic zona pe llucid peptide produces 10Jlgt-term con tracepti on in female mice Science 246935-8

[17) Numby M L et al 1985 Mo noclonal antibo dies as pro bes oj stru cture fun ction and isoenzyme forms of the type II regulatory subunit of cycl ic A MP-depe ndent pro-tein kinase Pharmaco l Ther 2167-87

[18] Palmiter R D e t 81 1982 Dramatic growth o f mice that develop from eggs microinjected with me lallotio ne in-growth hormone fu sio n genes Nature 300611-15

(1 9) Parmentier M e t a1 1989 Molecular clonin g of the tllyro tropin rece ptor Scien ce 246 1620-2

[20] Powe rs D A 246352-8

1989 Fi sh as mod e l sys te ms Science

[2 11 P redigc r E A 200 1 De tecti on a nd m RNAs uSing r ibo nuclease protec tion 1101Bioi 160 495- 505

quantitati on of assays M ethods

[22) R eve ntos J and F M une lL 1997 Transgen ic models in re productive endocrine research Endocrin o 136566-80

a nim al E J

[23] Riggs A D et aL 1980 Synthesi s clonin g and expres-sio n o f hormone genes in E lch erichw coli Rec Prog H om1 Res 3626 1- 76

(24) Robertson 1 A 1988 R ight s symbolism and public pol-icy in feta l ti ssue t ran spl an ts Hastings Crr Rpt 5-12

[25) Sh izu me K A B Le rn e r a nd T B F itzpatrick 1954 In vit ro bioassay lor me la nocyte stimula ting hormone Endocrinology 545 53- 60

[26] Simonsen f l and H F Lodish 1994 Cloning by func-tion express io n cloni ng III mam malian ce ll s Trends Phurnlil co l Sci 15middot137-41

[27] Soulet D and S Rives t 2002 mak e microanav se ri a l a nal ys is

Pe rspec tive how to of gene exp ression

and proteomic re le vant to da y-to-day endocrine problems and phys io logical sys te ms Endocrin ology 143 1995-200l

[28J Spergel D J et al 2001 Using reporter genes to label selected neurona l popula tio ns in tran sge nic mice for gene promoter anatomical and ph ysio logical st udies Prog NeLmniol 63( 6)673-86

[29] Spice r S S 1993 Ad vantages of histochemis try fo r the stud y of cell bio logy Hisrochem J 26531-47

[30] TheoharidesT C and W W Douglas 1978 Secre tion in mast cells induced by calcium entrapped within phos-pholipid vesicles Science 2011143-5

(31) Thompson L 1992 Fetal transplants sho w promise Science 257868-70

[32] Tong Y et al 1990 Glucoco rti coid regulati o n of proo-piomelanocortin mRN A levels in rat arcu ate nuc le us Mol Cell Newmiddotosci 1 78- 83

[331 Tra yhurn P 1996 Northern blotting Proc Nurr S oc 55583- 9

[34) Wall R J D E Ke rr and K R B ondioli 1997 Trans-genic dairy ca ttl e Genetic enginee ring on a large sca le J Dmry Sci 802213-24

[35] Wall R 5 e t al 1997 Tra nsge nic anim a l technology 1 Androl 11236-9

[36 J Waremb o urg M 1976 Detectio n of diffusible su b-stances 1 de Mi croscopie et de Biologie Cellulaire 27277-RO

[371 Weiss J and J L Jameson 1993 Pe rifused pituitary cells as a model fo r stuciies of gonado tropin biosynth esis and secre tio n TEM 4265-70

[38) Wilcox J N 1993 Fund amenta ls of ill si tu hybridiza ti on 1 f-fi stochem Cyrochem 41 1725-33

[39] WoodruffT K 1998 CelluJar localizatio n of mRN A an d protein in situ hybridiza ti on histochemistry and in situ ligand binding Methods Cell Bioi 57333-5 1

[40] Yalow R S 1978 R adioi mmunoassay A probe for the fin e st ructure of bi Ologic systems Science 200 1236-45

[41] Zhang Ye t al 1994 Posi tional clol1lng of the mouse obese gene and its human ho mologue NalLire 372425-32

68 Chapter 4 Endocrine Methodologies

o Endocrinologists to bout orllo Following discovery of a hormone endocrine studies usually focus on the following investi-gations (not necessarily in the order delineated)

1 Source The distribution of the hormone will be determined T he hormone may be fo und in more tban a single organ tiss ue or cellular source For example several gastrointestina l bormones are also found within the central nervo us system

2 Structure determin(ion and synthesis Depending on the type of hormon e the struc-ture will be determined by any of a variety of methods In the case of a peptide hor-mone the pri mary (amino acid) sequence will be determined From this in forma tion the peptide will then be synthesized and its biological acti vity compared to the purified extract will be determined This will estab lish the substance and structure as the authen tic hormone

3 Biosynthesis The biosyntbetic pathway of hormone production should be delineated Knowledge of the primary structure of a peptide hormone can be used to predict the cOITesponding sequence of the gene that encodes it the gene can be cloned and the cDNA struc ture corresponding to the hormone m R NA can be used to predict the prohormone structure and possible enzymol ogical even ts rel ated to production of the mature horm one

4 Controlosecretion The extrinsic or intrinsic factor(s) regulating the control of hormone secretion must then be determined E ndogenous stimuli may involve negative or positive feedback by other hormones or the circulating products (other hormones metabolic sub-stra tes) or consequences (blood volume water andor electrolyte composition of the blood) of its actions The nervous system may directly or indirectly regulate hormone secretion from endocrine organs or tissues

5 Cellular mechanisms of secretion O nce the first messenge rs regulating hormone secre-tion have been determined it is then necessary to detelmine the nature of the second messengers and structural elements (ion channels cytoplLLltmic organelles) that participate in the process of hormone release (secretion)

6 Circulation and metabolism It is importa nt to determine the half-life of the hormone in the systemic circulation Ste roid or peptide hormones may be noncovalently bound to circulating proteins Fluctuations in the levels of the binding proteins may affect the total amount of hormone present in the blood an d therefore availabl e for hormone action The half-life of the hormone in the circulation may be affected by degradation or other alterations of the hormone by serum enzymes The retention time of the hormone in the circulation may also be affected by processes of filtra tion by the kidney

7 Biological actions and roles Removal of the hormone fro m the body by one or more methods usuall y will result in physiological effects in the animal that will predict one or more functi ons for the hormo ne Administration of the hormone (rep lacement therapy) or related ana log to the ani mal sh ou ld confirm one or more roles for the hormone Studies on othe r species of animals may suggest add iti on al or alternate roles for the hormone

8 Mechan isms 0 Clclion Following administration of the hormone in vivo and in vitro ce llular changes in biochemical processes and products should indicate one or more second messengers involved in hormone action The receptor and signal transd uction mech an isms involved in hormone action should define the temporal aspects between receptor activation and cellular response Structure-activity studi es will determine the esse nti al features of the hormone required for hormonc action in the case of a peptide the message seq uence

9 Pathophysiological aspects The existence of a hormone is someti mes glea ned from the symptoms of a human pathophysiological condition In other cases the discovery of a hormone in experimenta l animals provides a new explanation for a previ ously misun-derstood clinical condition In either casc it is important to study the re lations hip be tween the normal biology of a horm one and the clinical consequences of alterations in synthesis secreti on and actions of that hormone These studies take place in animal models of huma n diseases a nd to the extent that is ethically possible in human clinical popu lations


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endocrine methodologies chapter 4 9-1.2012.pdf

Documents

histological methods

important methods

numerous hormone

endocrine studies

hormone msh

endocrine glands

physiological roles

receptor assay methods