Lecture 12: Intracellular Receptors Flashcards
10th February 2025
Give a general overview of the action of intracellular receptors
- An extracellular ligand diffuses across the plasma membrane and binds and activates its receptor.
- The activated receptor converts its substrate into product.
- The activity of downstream targets is altered.
When was Nitroglycerine (NG) discovered by Ascanio Sobrero in Turin?
In 1846
What did Ascanio Sobrero note about Nitroglycerine (NG)?
- He noted the ‘violent headache’ produced by minute quantities of NG on the tongue.
- His face was badly scarred because of an explosion in his lab.– ‘I am almost ashamed to admit to be its discoverer’.
What was Nitroglycerine (NG) used as in 1849?
NG was used as a headache cure – a homeopathic use following the doctrine of ‘like cures like’.
1867…
Lauder Brunton, the father of modern pharmacology, used amyl nitrate to relieve angina, noting pharmacological resistance to repeated doses
1876…
William Murrell tried NG for angina. William Martindale, a pharmaceutical chemist, prepared ‘a more stable and portable preparation’: 1/100th of a grain in chocolate.
Overview of nitric oxide…
Explosive, homeopathic ‘cure’, and angina relief
1851…
Alfred Nobel began manufacturing NG in Sweden as a commercially useful explosive.
1864…
Nobel’s younger brother and several factory workers were killed in an explosion at the Nobel factory.
1865…
Nobel founded Alfred Nobel & Company in Germany
and built an isolated factory near Hamburg. Explosions destroyed the buildings of the factory twice.
1896…
A few months before his death, Alfred Nobel was prescribed NG for angina
“Isn’t it the irony of fate that I have been prescribed
nitro-glycerin, to be taken internally! They call it Trinitrin,
so as not to scare the chemist and the public.
Your affectionate friend
A. Nobel”
~1900
The NG industry flourished, exposing workers to high levels of organic nitrites; recognition of ‘Monday disease’ (dizziness, tachycardia, and headache) and of nitrate withdrawal and overcompensation by ‘Sunday Heart Attacks
1977
Ferid Murad discovered the release of nitric oxide (NO*: the star denotes that it is a free radical, with an unpaired electron) from NG – and its mode of action. It relaxes vascular smooth muscle.
1980…
Robert Furchgott demonstrated that acetylcholine induces the release of EDRF (endothelium-derived relaxing factor
1987…
Louis Ignarro demonstrated that EDRF is NO*.
How does nitiric oxide signal?
- The gas nitric oxide (NO*) diffuses across the plasma membrane and binds and activates guanylate cyclase, which is its receptor.
- The activated receptor (GC) converts GTP into cGMP.
- cGMP is a second messenger that alters the activity of target proteins.
Today, what remains the treatment of choice for relieving angina?
Glycerol trinitrate (nitroglycerine) remains the treatment of choice for relieving angina.
Why does Glycerol trinitrate (nitroglycerine) remain as the treatment of choice for relieving angina?
Because it dilates the blood vessels (makes them larger).
Why is NG preferred as an angina treatment over other organic esters and inorganic nitrates ?
Because the rapid action of NG and its established efficacy make it the mainstay of angina pectoris relief.
How do blood vessels normally dilate?
- Blood vessels dilate in response to high blood pressure.
- Smooth muscle will relax and dilation will increase the volume of the vessel and lower the blood pressure.
What is NO* production in vivo stimulated by?
NO* production in vivo is stimulated by high blood pressure
How is NO* production in vivo stimulated by high blood pressure?
- Autonomous nerves in the vessel wall respond to high blood pressure (high shear force) and release acetylcholine (Ach).
- Acetylcholine binds its receptors (AchR) on the plasma membrane of endothelial cells.
- Stimulation by acetylcholine increases endothelial cell cytosolic [Ca2+].
-Ca2+ is a second messenger , so high [Ca2+] activates nitric oxide synthase (NOS).
- NOS catalyses the conversion of arginine to citrulline and nitric oxide.
-Nitric oxide diffuses from endothelium into smooth muscle.
- NO* activates soluble guanylate cyclase by binding to its haem group, causing a conformational change.
- GC converts GTP to cGMP
cGMP is a second messenger
cGMP is indirectly activated by nitrous oxide. cGMP is a second messenger. What does cGMP do?
cGMP activates protein kinase G in smooth muscle
PKG is a cGMP-dependent protein kinase.
It phosphorylates myosin light chain.
Muscle cells with phosphorylated myosin light chain relax.
Smooth muscle relaxation causes dilation of the blood vessel.
Dilation increases the volume of the vessel and lowers blood pressure.
Is it true that blood pressure homeostasis requires multiple co-ordinated signalling pathways?
Yes
How is NO* used in many signalling pathways downstream of nerves?
Control of blood vessel dilation
Control of peristaltic movement through the gut
Regulation of glomerular capillary pressure
Regulation of blood flow in the adrenal glands
Neurons of the corpus cavernosum (the erectile tissue in a penis): regulation of muscle contraction and blood flow
What are the 4 types of nitric oxide synthase?
NOS1, nNOS, neuronal isoform;
NOS2, iNOs, inducible isoform;
NOS3, eNOS, endothelial isoform;
(bNOS, bacterial NOS;
NOS1, nNOS, neuronal isoform…
development of the nervous system, protection against cardiac arrhythmia induced by myocardial infarction (‘heart attack’), peristalsis and sexual arousal
NOS2, iNOs, inducible isoform…
- produces large amounts of NO* as a defence mechanism used by macrophages (chemical warfare)
- It is also the cause of septic shock (see Immunology lectures).
NOS3, eNOS, endothelial isoform…
- controls vascular tone, insulin secretion, and regulates angiogenesis (growth of new blood vessels).
- plays a critical role in embryonic heart development and morphogenesis of coronary arteries and cardiac valves.
bNOS, bacterial NOS…
NO produced by bacterial NOS protects against oxidative damage, immune attack.
What are cyclic nucleotides?
Cyclic nucleotides are important secondary messengers that control many physiological processes, including smooth muscle contractility.
What are Phosphodiesterases (PDEs)?
a superfamily of metallophosphydrolases that specifically cleave the 3’,5’-cyclic phosphate moiety of cAMP (PDE4, PDE 7, PDE 8), cGMP (PDE5, PDE6, PDE9) or both cAMP and cGMP (PDE1, PDE2, PDE3, PDE10, PDE11) to produce the corresponding 5’ nucleotide.
What does PDE5 cleave?
PDE5 specifically cleaves cGMP.
Describe the tissue distribution of PDE5
The tissue distribution of PDE5 is relatively restricted
Is PDE5 the target for sucessful drug, sildenafil citrate?
Yes
What did Phase I clinical trials of Sildenafil show about angina?
Phase I clinical trials suggested that the drug had little effect on angina.
How is Sildenafil citrate a cGMP mimic?
- It’s a potent inhibitor of cGMP phosphodiesterases. particularly phosphodiesterase-5 (PDE-5), which is the principal phosphodiesterase in the corpus cavernosum, the erectile tissue of the penis.
- PDE-5 normally causes blood vessels to narrow.
- By inhibiting PDE-5, Sildenafil allows blood vessels to relax, improving blood flow.
What trade name is sildenafil sold under?
Viagra
What are the side effects of viagra?
Since PDE-5 is also found in vascular smooth tissue, Viagra can lower blood pressure and cause headaches.
It also inhibits PDE-3, which affects heart contraction, potentially leading to heart palpitations and increased heart rate.
It inhibits PDE-6, which regulates blue-green color vision, causing blue-tinted vision as a side effect.
What are the 2 types of intracellular receptors?
- Intracellular receptors with enzyme activity.
- Intracellular receptors that are transcription factors.
What are the primary female sex hormones?
Oestrogens
What are the 4 types of oestrogens and what are their functions?
E1: oestrone: predominant in the menopause
E2: oestradiol: predominant during the reproductive years
E3: oestril: predominant in pregnancy
E4: oestetrol: also produced during pregnancy.
What are natural oestrogens?
Natural oestrogens are steroid hormones, synthesized from androgens (‘male’ sex hormones) such as testosterone by the enzyme aromatase.
What does ER stand for?
Estrogen receptor
What is the Estrogen receptor?
The ER (oestrogen receptor) is a cytosolic protein
Describe the estrogen receptor
The ER has an N-terminal transactivation domain, a DNA-binding domain, and a C-terminal hormone-binding domain that can bind oestrogens.
It is stored in the cytosol in complex with a dimeric chaperone protein called Hsp90.
Hsp90 binds near the ligand-binding site and maintains the ER in a soluble state.
The Hsp90: ER complex is too large to enter the nucleus.
What is meant by the fact that the estrogen receptor is a cytosolic protein?
- When oestrogen binds, DNA binding domain becomes available to bind DNA.
- The transcription binding domain also becomes available.
How does Oestrogen bind to the Oestrogen receptor?
① Oestrogen diffuses across the PM and binds ER.
② ER is released from Hsp90.
③ ER-oestrogen complex enters the nucleus and binds oestrogen response elements (EREs) as a dimer.
Why is oestrogen signalling unusual?
There are no amplification steps via protein cascades or via second messengers.
This means that ONE protein is both receptor and effector.
This is because there’s 2 isoforms of the ER ( alpha and beta).
What are the physiological roles of oestrogen signalling?
Reproduction: In females: induction of the progesterone receptor; cell differentiation; ovulation; implantation; pregnancy maintenance; childbirth.
In males: normal testicular function; fertility; sperm morphology
Cardiovascular system: Lipid profiles, fat distribution, the tone of vascular and smooth muscle cells, endocrine factors produced by the vascular wall (endothelins, nitric oxide), fibrinogen levels, blood platelets, inflammatory factors and coagulation.
Immune system: ERs are expressed in primary lymphoid organs and in peripheral immune cells; thymus development; enhancement of the humoral immune response.
Central nervous system: Oestrogens influence cognitive function; male/female - specific differentiation of the hypothalamus; growth factors that influence brain development, cell survival, and neural plasticity.
Skeletal system: Bone modelling in adolescence, initiating pubertal bone growth and later limiting longitudinal bone growth in females; maintenance of bone mass.
ONE protein is both receptor and effector.
How can one receptor/effector regulate so many different processes?
How can one receptor/effector respond to four different oestrogens?
- There are two different forms of the ER (α and β) each encoded by a separate gene (ESR1 and ESR2, respectively).
- They can form ERα (αα) or ERβ (ββ) homodimers or ERαβ (αβ) heterodimers.
- Different DNA-binding properties
- Different trans-activation properties
- Different oestrogens bound
- Plus: splicing variants – at least 3α and 5β isoforms are known. At least 15 different combinations
Describe how the GPCR GPER binds to the ER
Oestrogen binds GPER and multiple pathways are stimulated:
① ligand-independent activation of ER.
② release of EGF via Ca2+ as a second messenger and
③ stimulation of the MAPK signalling pathway via interaction with Grb2
What drug is used for the treatment of both early and advanced ER+
(estrogen receptor positive) breast cancer females and it is the most common
hormone treatment for male breast cancer?
Tamoxifen
Oestrogens and breast cancer
Oestrogens are agonists that activate ER by stimulating specific shape changes (estradiol is not visible here, as it binds under the green loops).
Tamoxifen is a non-steroidal ER antagonist that is larger than estradiol and so the loops cannot take on their active conformation. Oestrogen signalling via ERs is inhibited. Tamoxifen also causes cells to remain in the G0 and G1 phases
of the cell cycle.
Why are some breast cancers resistant to tamoxifen?
- GPER can stimulate oestrogen-responsive growth independently of ER, and tamoxifen does not inhibit GPER.
