Endocrine Methodologies

1
Endocrine Methodologies

• Following discovery of a hormone, endocrine
  studies focus on the following
• Source distribution of the hormone
• Structure will be determined and then synthesized
  and bioactivity compared to purified extract.
• Biosynthetic pathway should be determined by cDNA
  and mRNA analysis.

2
Endocrine Methodologies

• Control of secretion is determined by what
  intrinsic and extrinsic factors regulate
  secretion.
• Cellular mechanisms of secretion involving
  discovery of 1o and 2o messengers.
• Circulation metabolism to determine ½ life.
• Biological actions determined by
  ablation/replacement.
• Mechanism of action to determine changes in
  biochemical processes and products after
  administration.

3
Histological Studies

• Early endocrine studies were anatomical and
  descriptive.
• Gross observations of endocrine tissue only
  provides general details of anatomical
  localization, size, vascularization and
  innervation.
• Histological observations will show hypertropic
  or atrophic cells. Hypertrophic cells have
  abundant ER, Glogi. Atrophic cells lack these
  and have a diminished cytoplasm.

4
Histological Studies

• Hypertropy is usually accompanied by hyperplasia.
• Hematoxylin is a basic dye that interacts with
  acidic residues like DNA and RNA. These are
  termed basophilic. Eosin is an acidic dye that
  interacts with basic components. Hematoxylin
  stains the nucleus blue and eosin stains the
  cytoplasm pink

5
Immunocytochemistry

• Antibodies to protein hormones or peptides are
  conjugated to a flourescent dye and used to
  identify hormone producing cells.
• Tissue is put on a slide, AB-conjugate is added
  to tissue and xs washed away. Flourescence
  appears only in cells producing the hormone.

6
Immunocytochemistry

• In immunoenzyme histochemistry and AB to a
  hormone is conjugated to an enzyme, like
  peroxidase. AB-enzyme conjugate interacts with
  tissue and then substrate is added and the
  conjugated enzyme catalyzes very localized
  reactions.
• This allows visualization of secretory vesicle or
  granules of a cell.

7
Surgical Methods

• Surgical removal of a putative endocrine organ is
  followed by assessment of physiological
  alterations.
• Monitor things like changes in blood and/or
  urinary levels of metabolites or electrolytes.
• Transplanting the organ back into the same or
  different animal also provides info on the
  functional role. This is usually done in inbred
  strains to prevent rejection.

8
Surgical Methods

• Endocrine organs can be transplanted to an
  ectopic site such as the kidney capsule or eye
  where vascularization takes place.
• Removal of both member of bilateral target organs
  leads to complete loss of dependent tissue/organ
  functions. If removed unilaterally, the
  remaining organ undergoes compensatory-hypertrophy
  , an increase in cell size and number in the
  remaining organ.

9
Surgical Methods

• Animals can be sutured together and their
  vascular systems anastomosed. This is termed
  parabioses.
• Other examples are pinelaectomy, adrenalectomy,
  thyroidectomy, thymectomy.
• Pancreatic islets are impossible to remove but
  streptozotocin or alloxan can be used to kill the
  b cells. CoCl2 can be used to eliminate the
  glucagon secreting cells.

10
Hormone Replacement Therapy

• Administration of a hormone or analog can be used
  following surgical removal.
• AB can be used against most peptide hormones to
  immunoneutralize the biological effect of
  endogenous hormones.
• Crude extracts of endocrine or other tissues were
  used early on to determine if these materials
  could replace the endocrine tissues.

11
Hormone Replacement Therapy

• Replacement therapy with purified substances can
  fully restore target organ function or even lead
  to hypertrophy.
• Insulin of bovine, ovine, porcine and equine
  origin used to be used but the species had to be
  changed because they were neutralized in the
  body.

12
Bioassays

• The activity of a hormone is studied on living
  cells, tissues, or organs.
• Usually the tissue or organ selected is naturally
  responsive to the hormone in the nM to pM range.
  Other hormones may affect these tissues, but at
  pharmacological doses (mM).
• Frog skin bioassay for MSH is an example, toad
  bladder bioassay for vasopressin.
• Steroid-primed rat uterus has been used to study
  mechanisms of oxytocin induced contraction of the
  organ.

13
Structure Activity Studies

• Structure activity studies determine the amount
  of activity compared with some standard hormone.
• MED and maximal activities within a particular
  assay are determined first. Then intermediate
  doses are used to generate a dose-response curve.
• Potency can then be expressed as a ratio of the
  parent hormone to the analog.

14
Radioisotopes

• The ½ life of a hormone can be determined by
  radiolabelling it and determining its
  distribution, excretion rate, and metabolism
  within the body.
• 14C can be incorporated into glucose and its
  subsequent metabolism to 14CO2 is a measure of
  metabolic activity of the cell.
• 3H can be used in autoradiography and enzyme
  assays. 3H is usually a component of an a.a. or
  nucleotide.
• 23Na can be used to measure Na uptake by nerve
  or muscle cells.
• 32P can be used to study phosphorylation.

15
Radioimmunoassays

• RIAs have allowed detection of minute
  concentrations of hormones with a high degree of
  specificity.
• AB against the antigenic principle are raised in
  rabbits or other animals, then hormone is
  iodinated or tritiated.
• Radiolabeled ligand (hormone) can then be shown
  to combine with a given quantity of AB in a
  dose-related manner to provide a standard curve.
  
• Samples containing native (unlabeled) hormone
  compete with labeled hormone for the AB, and the
  decreased binding of labeled hormone to AB is a
  reflection of the amount of native hormone bound
  to AB.

16
Radioreceptor Assays

• Plasma membranes or intact cells are prepared and
  a dose-related binding of radiolabelled hormone
  to the membranes is then demonstrated.
• Instead of AG-AB reaction, the assay employs
  interaction between hormone and natural membrane
  receptors (antigens).
• Specificity of binding is determined by the
  ability of the cold native hormone to compete
  with radiolabelled hormone for its receptor.
  Other cold hormones can be used to determine
  specificity.

17
Enzyme Assays

• Adenylate cyclase or guanylate cyclase can be
  measured by quantitation of the conversion of
  radiolabeled ATP or GTP to labeled nucleotides.
• This is a true measure of the cyclizing activity
  of membrane fractions since measurements of
  cyclic nucleotide levels in cells fail to
  indicate how the nucleotide levels are altered.

18
Enzyme Assays

• cAMP levels can be altered by increased or
  decreased phosphodiesterase activity which are
  unrelated to cyclase activity and cAMP formation.

19
Autoradiography

• A radioisotope is injected into an animal or
  added to medium of cells.
• A sample of tissue/organ is removed and put on a
  slide. The slide is immersed in a photographic
  emulsion in the dark.
• Decay of the isotope leads to reduction of the
  silver bromide in the emulsion to silver
  crystals. Because b particles travel a short
  distance, the grains directly over the emulsion
  are exposed.

20
Autoradiography

• After gonadectomy there is an increased
  incorporation of radiolabeled thymidine into DNA
  of gondaotrophs due to the loss of negative
  feedback of testosterone.
• Actions of steroid hormones can be studied by
  autoradiography using 3H labeled steroids to see
  cellular site of hormone action.
• Radiolabeled peptide hormones can be used to
  determine topographical localization of
  receptors.
• Some hormones mediate effects through stimulation
  of RNA synthesis which can be studied using 3H
  uracil.

21
Hybridization Studies

• Hybridization or annealing involves pairing of
  complementary strands of nucleic acids.
• Hybrids can be DNA-DNA, DNA-RNA, RNA-RNA. DNA is
  analyzed on a solid support such as a membrane
  (Southern) and RNA in a similar manner
  (Northern).
• Nucleotides can be localized within cells and
  tissues by in situ hybridization. In situ
  provides cellular resolution not seen with total
  RNA hybridization. It allows one to examine
  differential gene regulation within different
  nuclei.

22
Pharmacological Methods

• Many exogenous drugs interact with molecular
  components of cells and can be used to study
  physiological activity of cells or mechanisms of
  chemical messengers that stimulate or inhibit.
• Cardiac glycoside, oubain inhibits Na/K pump
  and certain hormone secretions are enhanced or
  inhibited by oubain suggesting active transport
  systems.
• Methylxanthines such as theophylline, and
  caffeine are phosphodiesterase inhibitors. They
  elevate intracellular cAMP by preventing its
  breakdown.

23
Pharmacological Methods

• Colchicine inhibits microtubule assembly by
  binding with tubulin.
• Cytochlasin B is a fungal metabolite that
  interferes with microtubule function
• Actinomycin D inhibits RNA production.
• Puromycin cyclohexamide inhibit protein
  synthesis at the translational step.