September 5, 2001

-	Control of hormonal action
Regulation of gene expression
Regulation of hormone secretion

-  Assessment of hormonal actions

control of hormonal action

1.  Control of hormone formation
2.  Control of hormone secretion
3.  Control of hormone transport/plasma levels of free hormone
4.  Control of receptor and post-receptor events
5.  Control of hormone elimination

CONTROL OF HORMONE FORMATION:  REGULATION OF GENE EXPRESSION

Chromosomes:  	DNA + histones ( = chromatin)

Gene:  	a DNA sequence that is transcribed as a single unit and encodes one set of closely related polypeptide chains  

Human genome:  	50,000 – 100,000 genes

Each nucleated somatic cell contains the full genetic message.

Is the complete genetic message expressed in each somatic cell?  IT IS NOT.  Gene expression is regulated.  There are factors that determine 
a)	if a cell is capable of producing a certain protein or not and 
b)	if it is capable of producing that protein then how much will be produced  (i.e., a cell can change the expression of its genes in response to various stimuli)

The synthesis of all hormones requires the biosynthesis of proteins.  
These proteins may be:
-	secreted peptide hormones or
-	enzymes involved in the biosynthetic process of non-peptide hormones

I.  structure of the gene

Regulatory (promoter) region	Transcription unit (CODING region)	

1.  Core promoter; binds
-	RNA polymerase II
-	TATA binding protein
-	Core transcription factors

2.  Enhancer site(s) of the gene; binds activators

3.  Silencer site(s) of the gene;  binds repressors

Activators and enhancers are also called “DNA-sequence specific transcription factors” (e.g., c-fos, c-jun, etc.)

“Adapter proteins”:  a.  connect activators and repressors
b. integrate the signals from activators and repressors
c. forward the integrated signal to core transcription factors	
1.  Exons:  coding blocks
 separate functional domains

2.  Introns:  non-coding sequences

3.  5’ and 3’-flanking regions 

Splicing - alternative splicing

Genetic rearrangement by recombination

II. Peptide hormones:  regulation of gene expression

1.   Tissue-specific gene expression:  tissue-specific silencer/enhancer

2.    Induction or suppression of genes normally expressed
At several levels:
1.  Amount of DNA:  cell growth/division (hyperplasia, e.g., parathyroid)
2.  Transcription
3.  Posttranscriptional processing of mRNA (RNA stability, alternative splicing)
4.  Translation
5.  Posttranslational processing

DNA

   transcription (by RNA polymerase)

Pre-messenger RNA

  post-transcriptional processing (RNA cleavage, excision of introns, rejoining of exons, etc.)

mRNA

  translation (ribosomes, nucleotide triplets-codons, translated between START and STOP codons by transfer RNA)

Pre-prohormone (or prehormone):  N-terminal signal sequence, transport across the membrane of rER

•	posttranslational processing:  - proteolytic cleavages (e.g., signal sequence)
- derivatization of amino acids, e.g., gglycosylation, phosphorylation, acetylation, sulfatation, carboxylation and hydroxylation

Prohormone:  (multiple) bioactive peptides; spacer sequences; “cryptic peptides”

Hormone

CONTROL OF HORMONE FORMATION:  REGULATION OF HORMONE SECRETION

Steroid hormones are released as soon as synthesized  the release process is not directly coupled with the biosynthesis of the peptide hormones and amino acid derivatives.

I.  Biosynthesis of peptide hormones

1.  Rough ER

Proteins secreted into the lumen of rER

carried to Golgi complex by transfer elements

2. Golgi complex

Hormones packed into immature secretory granules

mature s.g. (condensation of protein, second membrane)

II. Hormone release from secretory granules

1.  Regulated pathway of secretion:  exocytosis of mature granules in response to specific extracellular stimuli, e.g., -  plasma electrolytes  ADH 
-  calcium  PTH
-  plasma glucose   insulin  and glucagon  
-  tropic hormones

2.  Constitutive pathway of secretion:  exocytosis of immature granules and transfer elements (secretory vesicles);  rapid secretion of newly synthesized peptides

ASSESSMENT of hormonal actions

I.  Measurement of hormone levels

Measured variables:

a.	Baseline hormone levels
-	spontaneous hormonal rhythms (e.g., circadian rhythms, monthly rhythms, circannual rhythms, etc.)
-	plasma half-life

b.	Dynamic tests
-	stimulation tests
-	suppression tests

General requirements:
a.	sensitive
mol > millimol > micromol > nanomol > picomol > femtomol
	 10-3 	10-6	10-9	10-12	10-15  mol

Typical hormone concentration in the 10-7 – 10-12 mol/l range

b.	specific
c.	reliable
d.	simple

1.  Immunologic methods  

a.  Radioimmunoassay (RIA) 

Plasma – body fluids

sensitive, specific, reliable

Requirements:  -  radiolabeled hormone (antigen):  125I, 3H
-  specific antibody (from the serum of immunized animals)
-  known amounts of hormone (for standard curve)
-  separation methods:  precipitation of Ag-Ab complexes with a second Ab or with salting out
-  “unknown” sample

Principle:  competitive inhibition of the binding of labeled hormone to antibodies by unlabelled hormone

Standard curve:  calculating bound/free (B/F) ratio of known concentrations of the hormone

Assay of the “unknown”:  determine B/F ratio of the unknown  corresponding hormone level?

b.  Enzyme-linked immunosorbant assay  (ELISA)

Plasma – body fluids

-    first antibody (against the assayed hormone) immobilized on a solid phase
-	incubation of the Ab-coated surface with hormone-containing sample
-	wash the unbound hormone
-    incubation with a second antibody to which an easily assayed enzyme has been linked
-    wash the unbound Ab-linked enzyme
-    measure enzyme activity

e.g., pregnancy tests (ELISA for hCG)

c.  Immunohistochemistry

Tissue preparation (histology)

Ab linked to fluorescent dye or enzyme

2.  Bioassays

In Vitro	In Vivo	
Incubation of “unknown” sample with 
endocrine tissues, 
membranes, 
primary cultures of dispersed cells or 
permanent cell lines 
in tissue culture medium.

The nature of measured responses, e.g., 
- Measuring the activity of second messenger systems such as cAMP
- Measurement of released hormones in response to the tropic hormone, e.g., steroidogenic response to LH or hCG
-  Mitogenic response (measurement of cell proliferation, e.g., PRL effect in lymphoma cell lines)
-  Cytochemical responses (measuring changes in cytosol composition, e.g., PTH effect in chondrocytes)
	Injection of “unknown” sample into animals

measure the effects of the sample

e.g.,  - hypoglycemia test in mouse for insulin
-  rat tibial growth for GH
-  rat ovarian weight for gonadotropins 

Inaccurate, not very specific, expensive, slow, not sensitive

3.  Radioisotope techniques:  administration of radiolabeled hormone or precursor 

-  Half-life studies (measurement the rate of the elimination of labeled hormone from the body)
-  125I uptake by thyroid (measurement of the accumulation of the labeled precursor in the hormone-producing tissue)

4.  Molecular biology techniques:  measurement of gene expression (measurement of specific mRNA).

-  Measurement of the expression of the hormone
-  Measurement of the expression of transcription factors stimulated by a hormone in a target tissue

Most common methods:
-  In situ hybridization (gene expression can be detected in a single cell under microscope but quantification is difficult)
-  Northern blotting 
-  Reverse transcriptase-polymerase chaiin reaction (RT-PCR) (very sensitive, theoretically a single mRNA molecule can be detected)

II.  Morphological measurements

1.  Gross observation of body, organ, or tissue (e.g., goiter)

2.  Histology (from biopsy)
-  Hypertrophy
-  Hypotrophy
-  Hyperplasia
-  Hypoplasia

III.  measurement of receptor binding and the activity of second messenger systems

IV.  Experimental techniques

1.	Removal of endocrine tissues
2.	Enzyme extractions, purifications
3.	Exogenous administration of the hormone
4.	Inhibition of hormone effects (immunoneutralization, receptor antagonists)
5.	Autoradiography
6.	Structure-activity studies
7.	Genetic engineering
8.	Electrophysiology
9.	Parabiosis