Biochemistry of the Neuroendocrine System

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Biochemistry of the Neuroendocrine System
The Adrenal Glands
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Learning Objectives
Identify the hormones produced by the different
zones of the adrenal cortex, and describe the
regulation of their secretion. Identify the
biochemical basis why these zones can only
produce the hormones that they do. Describe the
mobilization of cholesterol in response to ACTH
with respect to cholesterol esterase, sterol
carrier protein, and steroidogenic acute
regulatory protein. Describe the general
mechanism of action of cytochrome P-450 enzymes
and their involvement in steroidogenesis. Identif
y the rate-limiting step in steroidogenesis.
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Describe the hormone/G protein-coupled receptor
signal pathway for epinephrine
Describe the hormone/receptor/genome signal
pathway of the adrenal steroid hormones.
Describe the metabolic effects of glucocorticoids
(cortisol). Describe the metabolic effects of
aldosterone. Describe the physiology by which
angiotensin II stimulates aldosterone secretion.
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Adrenal glands
Cross section of sheep adrenal gland
Kidneys
Cortex
Medulla
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Hormone production of the adrenal gland
Adrenal Gland
Cortex (steroid hormones)
Medulla (catecholamines)
Zona glomerulosa
Zona fasciculata Zona reticularis
Epinephrine
Cortisol
Aldosterone
Norepinephrine
Androgens
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Hormones of the adrenal cortex
The adrenal cortex synthesizes dozens of
different steroid molecules. Some 50 steroids
have been isolated and crystallized from adrenal
tissue. Most of these are intermediates, and
only a small number are secreted in significant
amounts. These sort into three classes of steroid
hormones glucocorticoids (cortisol,
corticosterone) mineralocorticoids
(aldosterone) androgens (androstenedione, and
the androgen precursor, dehydroepiandrosterone,
DHEA)
Cortisol and aldosterone are the most important.
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Histological subdivisions of the adrenal cortex
capsule
zona glomerulosa
adrenal cortex
zona fasciculata
zona reticularis
adrenal medulla
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Biochemical subdivisions of the adrenal cortex
The zona glomerulosa produces aldosterone, and
lacks the enzyme 17a-hydroxylase and thus cannot
produce cortisol or androgens.
The two inner zones (zona fasciculata and zona
reticularis) appear to function as a unit they
both produce cortisol and androgens (and small
amounts of estrogens). These zones lack the
enzyme P450aldo (18-hydroxylase) and cannot
produce aldosterone.
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Cytochrome P-450
The most numerous and most complex
monooxygenation reactions are those catalyzed by
a type of heme protein called cytochrome P-450.
(The CO complex of its reduced form absorbs
strongly at 450 nm, hence its name). Several
hundred members of the cytochrome P-450 family
are known, each with a different substrate
specificity. They are involved in the
hydroxylation (and thus detoxification) of
foreign compounds (xenobiotics), particularly if
the foreign substances are hydrophobic and poorly
water soluble. Cytochrome P-450 enzymes are
involved in the hydroxylation of steroids to
yield adrenocortical hormones.
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General mechanism of action of cytochrome P-450
Cytochrome P-450 catalyzes hydroxylation
reactions in which an organic substrate RH is
hydroxylated to ROH, incorporating one atom of
dioxygen. The other atom of oxygen is reduced to
water by reducing equivalents donated by NADPH
but passed to P-450 by an iron-sulfur protein.
Thus, these enzymes are sometimes called
hydroxylases.
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The general reaction equation for monooxygenases
R-H NADPH H O-O R-OH
NADP H2O
Monooxygenases require two substrates to serve as
reductants of the two oxygen atoms of dioxygen.
The main substrate accepts one of the oxygen
atoms, and a cosubstrate furnishes hydrogen atoms
to reduce the other oxygen atom. Monooxygenases
are sometimes called mixed-function oxidases or
mixed-function oxygenases to indicate that they
oxidize two different substrates simultaneously.
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Steroidogenesis in the adrenal cortex
The synthesis of all steroid hormones begins with
cholesterol. Plasma lipoproteins (especially LDL)
accounts for the majority of the cholesterol
delivered to the adrenal glands. A small pool of
free cholesterol within the adrenal is available
for rapid synthesis of steroids when the adrenal
is stimulated.
Stimulation of the adrenal results in hydrolysis
of stored cholesterol esters to free cholesterol,
and increased uptake of cholesterol from plasma
lipoproteins. Synthesis of cholesterol within
the adrenal cells is also increased.
Steroidogenesis begins in the mitochondrial
matrix.
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Cholesterol (from LDL) is esterified to
polyunsaturated fatty acids (acetyl-CoA
cholesterol acyltransferase, ACAT) in the
endoplasmic reticulum, and stored in lipid
droplets within the cell.
Fatty acid esters of cholesterol are hydrolyzed
by cholesterol esterase (CE).
Lipid droplet
cholesterol esters
ACAT
CE
cholesterol
cholesterol
Tissue-specific hormones (eg. ACTH, LH, and FSH)
stimulate the esterase and inhibit the
acyltransferase resulting in an increased
accumulation of free cholesterol.
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Sterol-carrier protein-2 (SCP-2) transports free
cholesterol within the aqueous cytoplasm for
incorporation into the outer mitochondrial
membrane.
The outer mitochondrial membrane is
cholesterol-rich. The inner mitochondrial
membrane is cholesterol-poor. Transfer of
cholesterol between these membranes is dependent
on the steroidogenic acute regulatory protein
(StAR).
StAR is phosphorylated by protein kinase A
(activation and/or increased stability), and is
also transcriptionally regulated in response to
an increase in cAMP levels.
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cholesterol-sterol carrier protein complex
Outer mitochondrial membrane
Inner mitochondrial membrane
cholesterol
steroidogenic acute regulatory protein (StAR)
mediated transport of cholesterol to inner
mitochondrial membrane
cholesterol
Mitochondrial matrix
Steroid biosynthesis
Mutations in the StAR gene are associated with
congenital lipoid adrenal hyperplasia, a disorder
with severe cortisol and aldosterone deficiency
at birth.
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Steroid Biosynthesis
Cytochrome P-450 side chain cleavage enzyme
(P-450scc)
Cholesterol
red
ox
red
2 NADPH
2 H
2 O2
Adrenodoxin reductase (flavoprotein)
adrenodoxin (Fe-S)
cyt P-450
2 H2O
ox
red
2 NADP
ox
Pregnenolone
20,22-Dihydroxycholesterol
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endoplasmic reticulum
Intermediates in steroid biosynthesis are
shuttled back and forth between the mitochondria
and the endoplasmic reticulum
Cholesterol
Mitochondrial matrix
P-450scc
Pregnenolone
Rate limiting step in steroidogenesis
It is not the activity of this enzyme that is
critical, but the supply of cholesterol crossing
from the outer mitochondrial membrane
(StAR-mediated transport).
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SER smooth endoplasmic reticulum
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Cortisol (glucocorticoid) synthesis in the zona
fasciculata and the zona reticularis
Cholesterol
P-450c17
(17a-hydroxylase)
P-450scc
17a-OH Pregnenolone
Pregnenolone
3bHSD
3bHSD
P-450c17
(17a-hydroxylase)
Progesterone
17a-OH Progesterone
P-450c21 (21-hydroxylase)
P-450c21 (21-hydroxylase)
11-Deoxycorticosterone (DOC)
11-Deoxycortisol
P-450c11 (11b-hydroxylase)
P-450c11 (11b-hydroxylase)
Corticosterone
Cortisol
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Cholesterol
Aldosterone (mineralocorticoid) synthesis in the
zona glomerulosa
P-450scc
Pregnenolone
3bHSD
The glomerulosa lacks the 17a-hydroxylase
activity and cannot produce cortisol or the
androgens.
Progesterone
P-450c21 (21-hydroxylase)
11-Deoxycorticosterone
P-450aldo
P-450aldo aka 18-hydroxylase and 18-hydroxy
steroid dehydrogenase
Corticosterone
P-450aldo
18-Hydroxycorticosterone
P-450aldo
Aldosterone
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C11 hydroxyl group
C18 aldehyde group
C21 hydroxyl group
Aldosterone
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C11 hydroxyl group
C17 hydroxyl group
C21 hydroxyl group
Cortisol
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Androgen synthesis in the zona fasciculata and
the zona reticularis
P-450c17
(17a-hydroxylase)
DHEA
(Dehydroepiandrosterone)
17a-OH Pregnenolone
3bHSD
3bHSD
P-450c17
(17a-hydroxylase)
Androstenedione
17a-OH Progesterone
The adrenal androgens DHEA, DHEA-sulfate, and
androstenedione have minimal androgenic activity.
They are however converted in peripheral tissues
(gonads) to the more potent testosterone and
dihydroxytestosterone.
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Central Nervous System
Regulation
ACTH is the major regulator of the cortisol and
androgen production by the adrenal cortex.
Hypothalamus
CRH
Anterior Pituitary
ACTH
Adrenal Cortex
Cortisol, Androgens
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Central Nervous System
Hypothalamus
Feedback inhibition by ACTH on CRH secretion
(short feedback)
Feedback inhibition by cortisol on CRH and ACTH
secretion (long feedback)
CRH
Anterior Pituitary
ACTH
Adrenal Gland
Cortisol
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ACTH
adenylyl cyclase
plasma membrane
ACTH receptor (adrenal gland)
ATP
cholesterol esterase
cAMP
cholesterol ester synthase
activates cAMP-dependent protein kinases (PKA)
lipoprotein uptake
Phosphorylates (activates) StAR
Steroidogenesis
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Fluctuations in plasma levels of ACTH and
glucocorticoids
ACTH
Glucocorticoids
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Blood transport proteins
Cortisol binds to corticosteroid-binding globulin
(CBG, transcortin), and to a lesser extent
albumin.
Bound steroids are biologically inactive.
The plasma proteins may provide a pool of
circulating cortisol by delaying metabolic
clearance. This prevents marked fluctuations in
free cortisol levels during episodic secretions.
Androgens circulate weakly bound to albumin.
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Signaling through a steroid-receptor complex
Hormone response element (HRE)
Heat-shock protein (HSP)
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Metabolic effects of glucocorticoids (cortisol)
Effects are minimal in the fed state
During fasting
Glucocorticoids contribute to the maintenance of
blood glucose levels (this is how they got their
name).
Hepatic glucose production is enhanced.
The effects on muscle are catabolic.
Lipolysis is stimulated in adipose tissue.
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Metabolic effects of glucocorticoids (cortisol)
Liver
gluconeogenesis
activates phosphoenolpyruvate carboxykinase
activates glucose-6-phosphatase
responsiveness to glucagon
Muscle
lactate release
Adipose Tissue
free fatty acid and glycerol release by lipolysis
amino acid uptake
protein synthesis
glucose uptake
glucose uptake
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Metabolic effects of glucocorticoids (cortisol)
intestinal calcium absorption
urinary calcium excretion
lowers serum calcium levels
secondary PTH secretion
calcium resorption from bone
Glucocorticoids in excess result in a negative
calcium balance. (blood levels are maintained
however)
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Metabolic effects of aldosterone
The principle effects of the mineralocorticoids
(aldosterone) are on the maintenance of normal
Na and K concentrations and extracellular fluid
volume.
Aldosterone acts at the transcriptional level
(mRNA synthesis) after binding to intracellular
receptors.
Aldosterone induced proteins include factors that
regulate the luminal Na channel, facilitating
movement of Na into cells and components of the
Na K ATPase pump.
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Angiotensin II acts directly on the adrenal
cortex to stimulate aldosterone secretion (and in
most cases is the most important regulator of
aldosterone secretion).
(produced in kidneys)
(produced in liver)
ACE angiotensin converting enzyme (greatest
concentration in lung)
Half-life of angiotensin II in plasma less than
1 minute
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Low Na levels lead to reduction in extracellular
fluid volume
stimulates
renin-angiotensin system
Angiotensin II
Adrenal cortex
Aldosterone secretion
vasoconstriction of peripheral arterioles
Na retention
(prevent further volume loss)
maintain blood pressure
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Adrenal medulla
The adrenal medulla consists of masses of neurons
that are a highly specialized part of the
sympathetic nervous system. These neurons
function under stress or marked deviation from
homeostasis to secrete catecholamines
(epinephrine and norepinephrine) into the blood.
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Tyrosine hydroxylase is the rate limiting step in
catecholamine biosynthesis. It is
transcriptionally activated by acetylcholine via
cAMP/protein kinase A.
Tyrosine hydroxylase
(Dihydroxyphenylalanine)
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Aromatic L-amino acid decarboxylase (dopa
decarboxylase) is found in all tissues with the
highest concentrations in liver, kidney, brain,
and vas deferens.
Dopa decarboxylase
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This is a mixed-function oxidase (monooxygenase)
requiring molecular oxygen and external electron
donor. The membranes of the storage vesicles that
synthesize and store catecholamines contain
dopamine b-hydroxylase, cytochrome P-561 and
cytochrome P-561NADH reductase.
Dopamine b-hydroxylase
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PNMT uses S-adenosylmethionine (SAM) as
methyl donor.
Phenylethanolamine N-methyltransferase (PNMT)
Synthesis of catecholamines is coupled to
secretion so that constant stores are available
in the neurons.
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Catecholamines are released by stressful stimuli
Exercise, angina pectoris, myocardial infarction,
hemorrhage, hypoglycemia, asphyxia
When released into the circulation, the amines
are bound to albumin or a closely related protein
with low affinity and high capacity.
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The actions of catecholamines are terminated
rapidly. The free hormone is removed by several
mechanisms Reuptake by the nerve
endings Metabolism by catechol-O-methyltransferas
e (COMT) and monoamine oxidase Conjugation with
sulfate ion (liver, gut, red blood
cells) Direct excretion by the kidney
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COMT requires S-adenosylmethionine as methyl
donor. About 70 of epinephrine is methoxylated
in liver and kidney.
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Mechanism of action of catecholamines
The adrenergic receptors are G protein coupled.
Most cells in the body have adrenergic receptors.
a1-adrenergic receptors mediate vascular and
other smooth muscle contraction
a2-adrenergic receptors found in platelets,
adipose tissue and smooth muscle
phosphatidyl inositol system (Gq, phospholipase C)
cAMP/adenylyl cyclase system using Gi to inhibit
adenylyl cyclase and decrease cAMP
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Epinephrine
a1-adrenergic receptor
Gq
GDP
Phospholipase C
Gq
Phosphatidylinositol-4,5-bisphosphate
GTP
Cytosol
Diacylglycerol
IP3
Ligand-gated
Ca2 channel
Ca2
Endoplasmic reticulum
Protein kinase C
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b-adrenergic receptors
cAMP/adenylyl cyclase system using Gs to
stimulate adenylyl cyclase and increase cAMP
b1 receptor mediates direct cardiac effects
b2 receptor mediates vascular, bronchial, and
uterine smooth muscle contraction
(phosphorylation of myosin light chain kinase)
b3 receptors regulate energy expenditure and
lipolysis
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b-Adrengeric receptor
Epinephrine bound to receptor
Adenylyl cyclase (AC)
Plasma membrane
G protein (a, b, and g subunits)
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Protein kinase A (PKA)
Cyclic AMP response element binding protein (CREB)
Cyclic AMP regulatory element (CRE)
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Imagine being trapped in Jurassic Park when the
power went off.

• Increased rate and force of contraction of heart
  muscle (epinephrine acting through b1-receptors)
• Constriction of blood vessels (norepinephrine,
  in particular, causes widespread
  vasoconstriction, and hence an increase in blood
  pressure)
• Dilation of bronchioles (more air)
• Stimulation of lipolysis in fat cells (mobilize
  fatty acids for energy)
• Increased metabolic rate (oxygen consumption
  increases, glycogen breakdown in skeletal muscle)
• Dilation of the pupils (so you can see T. rex in
  dim light)
• Inhibition of non-essential processes
  (gastrointestinal secretions)

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