Sesh 3- Drug Action - Cellular Aspects

Question 1

Role of calcium ?

Answer 1

Critical role in signal transduction 
Essential enzyme co-factor
Neurotransmitter 
Essential in muscle contraction 
Essential in fertilisation 
Bone formation and homeostasis

Question 2

What is the role of calcium in signal transduction ?

Answer 2

For example a non-steroid as a first messenger binds it’s receptor normally to a GPCR, then GPCR elicits change in G protein

This leads to opening of calcium channel

Calcium channel sits across plasma membrane and allows calcium to flow into the cell.

Once inside cell the calcium binds to protein called calmodulin . When it is not bound the calcium is inactive but when bound it is active.

Question 3

What is low calcium regulated by ?

Answer 3

Regulated calcium entry
Controlled calcium Efflux
Exchange between Cytosol and stores

Question 4

Voltage gated calcium channels ? is one type of calcium entry

Answer 4

Allows calcium entry in response to membrane depolarisation

Specific to calcium - do not transport potassium and sodium

Five types-
L- long lasting 
T- transient 
N-neither long lasting nor transient 
P/Q- Purkinje 
R-resistant

Question 5

L type calcium channel?

Answer 5

L-type calcium channels are responsible for the excitation-contraction coupling of skeletal, smooth, cardiac muscle, and for aldosterone secretion in endocrine cells of the adrenal cortex.
They are also found in neurons, and with the help of L-type calcium channels in endocrine cells, they regulate neurohormones and neurotransmitters

Question 6

N type calcium channel

Answer 6

N-type calcium channels are important in neurotransmitter release like glutamate, GABA, acetylcholine, dopamine, and norepinephrine.

Question 7

P/Q calcium channels

Answer 7

P/Q-type calcium channels contribute to vesicle release at synaptic terminals.

malfunctioning of P/Q channels can result in for example Alzheimer’s disease.

Question 8

T type calcium channel ?

Answer 8

T-type calcium channels function to control the pace-making activity of the SA Node within the heart and relay rapid action potentials within the thalamus. These channels allow for continuous rhythmic bursts that control the SA Node of the heart.

Question 9

Examples of calcium channel blocks

Answer 9

Examples of calcium channel blockers include:

Amlodipine (Norvasc)
Diltiazem (Cardizem, Tiazac, others)
Felodipine
Isradipine
Nicardipine
Nifedipine (Procardia)
Nisoldipine (Sular)
Verapamil

Question 10

When are calcium channel blockers used ?

Answer 10

When calcium channel blockers are used

high blood pressure,

a doctor also may prescribe calcium channel blockers to prevent, treat or improve symptoms of conditions, such as:

Coronary artery disease
Chest pain (angina)
Irregular heartbeats (arrhythmia)
Blood vessel conditions, such as Raynaud’s disease

Question 11

What is ziconotide?

Answer 11

Ziconotide is an N-type calcium channel antagonist used to manage patients with severe chronic pain who cannot tolerate, systemic analgesics.

Question 12

MOA for ziconotide?

Answer 12

Ziconotide acts as a selective N-type voltage-gated calcium channel blocker. This action inhibits the release of pro-nociceptive neurochemicals like glutamate, calcitonin gene-related peptide (CGRP), and substance P in the brain and spinal cord, resulting in pain relief.

Question 13

What is the amlodipine ?

Answer 13

Amlodipine is a calcium channel blocker medication used to treat high blood pressure and coronary artery disease.

Question 14

Amlodipine MOA?

Answer 14

Amlodipine is considered a peripheral arterial vasodilator that exerts its action directly on vascular smooth muscle to lead to a reduction in peripheral vascular resistance, causing a decrease in blood pressure.

Amlodipine is a dihydropyridine calcium antagonist (calcium ion antagonist or slow-channel blocker) that inhibits the influx of calcium ions into both vascular smooth muscle and cardiac muscle.

In the heart -

Amlodipine has a dilating effect on peripheral arterioles, reducing the total peripheral resistance (afterload) against which the cardiac muscle functions. Since the heart rate remains stable during amlodipine administration, the reduced work of the heart reduces both myocardial energy use and oxygen requirements.

The dilatation causes an increase in myocardial oxygen delivery in patients experiencing coronary artery spasm (Prinzmetal’s or variant angina) and reduces coronary vasoconstriction caused by smoking

Question 15

What drugs block P type calcium channel ?

Answer 15

Eliprodil is a NMDA receptor antagonists and act to block P-type channels.

Question 16

What is NMDA receptor ?

Answer 16

The NMDA receptor (NMDAR) is an ion-channel receptor found at most excitatory synapses, where it responds to the neurotransmitter glutamate, and therefore belongs to the family of glutamate receptors.

Question 17

What does NMDA do in the brain ?

Answer 17

NMDA receptor can regulate neurological functions, including breathing, learning and memory formation

Question 18

Overactivation of NMDA receptors ?

Answer 18

Overactivation of NMDA receptors, causing excessive influx of Ca2+ can lead to excitotoxicity

Question 19

X

Answer 19

Under normal physiological conditions, the neurotransmitter opens glutamate, NMDA and AMPA receptor channels, and voltage dependent Ca2+ channels (VDCC) with high glutamate release, which is taken up again by EAAT1 and EAAT2. This results in a small rise in intracellular calcium that can be buffered in the cell. In ALS, a disorder in the glutamate receptor channels leads to high calcium conductivity, resulting in high Ca2+ loads and increased risk for mitochondrial damage. This triggers the mitochondrial production of reactive oxygen species (ROS), which then inhibit glial EAAT2 function.

Question 20

How is NMDA receptor used in medicine

Answer 20

NMDA receptors inhibit the action of, the N-Methyl-D-aspartate receptor (NMDAR).

Used as anesthetics for animals and humans

The NMDA receptor is thought to be very important for controlling synaptic plasticity and mediating learning and memory functions.

The NMDA receptor is ionotropic,

Question 21

What drugs act on smooth muscle ?

Answer 21

Drugs such as adrenoceptor agonists, muscarinic agonists, nitrates, and calcium channel blockers all affect smooth muscle.

Question 22

How does alpha adrenoceptors act on smooth muscle ?

Answer 22

The alpha-1 receptor is of the Gq type, resulting in activation of phospholipase C, increasing IP3 and DAG, and ultimately increasing the intracellular calcium concentrations leading to smooth muscle contraction

There are also α2-adrenoceptors found on the smooth muscle.
These receptors are linked to Gi-proteins, binding can decrease intracellular cAMP, which causes smooth muscle contraction.

.

Question 23

MOA of muscarinic agonist on smooth muscle ?

Answer 23

Muscarinic agonists are agents that activate the muscarinic acetylcholine receptor.
their mechanism of action is different depending on which receptor is activated.

The M1, M3, and M5 are transmembrane receptors that couple to a Gq protein. The Gq protein upregulates phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into 1,2- diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). DAG activates protein kinase C, which activates downstream protein and causes calcium influx. IP3 causes the sarcoplasmic reticulum to release stored calcium. Increased intracellular calcium causes smooth muscle contraction and exocrine glandular secretions.

Question 24

What does the M2 and M4 receptors do ?

Answer 24

The M2 and M4 receptors are Gi receptor, which inhibits adenylyl cyclase. Inhibition of adenylyl cyclase decreases cAMP production from ATP. This decreases the activation of protein kinase A.

Question 25

What is another way calcium can enter cells? (One way was voltage gated)

Answer 25

• Ligand gated.
• They have poor specificity to calcium
• P2x receptor activated by ATP only true Ligand gated receptor in smooth muscle
• GPCRs indirectly affect calcium entry
• glutamate receptor NMDA has high calcium permeability

Question 26

Where are NMDA receptors located ?

Answer 26

within the hippocampus and the cerebral cortex

Question 27

What is the third type of calcium entry called ? (Vg, lg and..?)

Answer 27

Store operated calcium system ( SOCS)

Question 28

Further info about SOCS?

Answer 28

It allows entry into cell when calcium stores are depleted (used up)

Then a protein called STIM accumulates at the junctions between endoplasmic reticulum and the plasma membrane

This protein traps and activates a receptor called ORAI 1.

When ORAI1 is activated it allows extracellular calcium to flow to replenish the stores.

The STIM-ORAI system is sensitive to the depletion if calcium stores. And this is an active research target for drug treatments

Question 29

Calcium release/ extrusion ?

Answer 29

Calcium extrusion - this describes extrusion from the cell/cytosol and this can be inwards towards the sarcoplasmic or endoplasmic reticulum and this relies on the action of ATPase enzymes

Release-
Calcium can be released from ER/SR by the Ip3 receptor.
This is linked to G protein coupled receptor action.

The CICR causes ryanodine receptor to open to allow stores to replenish with calcium

Question 30

What is bradykinin ?

Answer 30

a compound released in the blood causes contraction of smooth muscle and dilation of blood vessels.

Question 31

Check graph

Answer 31

X

Question 32

Calcium homeostasis ;

Answer 32

X

Question 33

Action potential .

Answer 33

• The inside potential of the cell is negative ranging from -30 - -80mV.
• The membrane is relatively impermeable to sodium ions
• the sodium ions are actively removed from the cell in exchange by the sodium / potassium pump

Net result is there’s higher intracellular concentration of potassium ions compared to sodium.

Chloride are actively transported .
Potassium chloride are often open so so membrane is permeable to potassium ions , resting potential settles at -60mv to -80mv near equilibrium potential for potassium ions

Question 34

Action potential.

Answer 34

When permeability of sodium ions increase there is a rapid influx of sodium ions and a rapid efflux of potassium ions .

The influx of sodium ions = depolarising current

The efflux of potassium ions = repolarising current

Question 35

Action potential graph . What happens ?

Answer 35

When stimulus activated.
The Membrane depolarises and sodium channels open allow influx of sodium ions

The channels quickly inactivate so sodium conductance is transient (lasting short time), and so membrane quickly returns to its resting rate .

Potassium channels are involved in depolarisation. These channels open after sodium channels are closed, it is voltage dependent.

Potassium channels contribute to the termination of the action potential and then repolarisation and hyperpolarisation.

Question 36

General rule of action potential?

Answer 36

Action potentials are initiated at the membrane and the action potential is governed by the excitability of the cell.
This in turn is influenced by an INCREASE in the number of sodium or calcium channels at the membrane. Increases EXCITABILITY

Opposing side - anything that increase membrane conductance to potassium channels REDUCES the excitability.

Question 37

What is batrachotoxin ?

Answer 37

Batrachotoxin binds to, and irreversibly opens, the sodium channels of nerve cells and prevents them from closing, resulting in paralysis and death.

Question 38

What is veratridine ?

Answer 38

is widely used as a sodium channel opener to increase sodium ion (Na+) accumulation inside the cells,

Question 39

What is tetrodotoxin?

Answer 39

Tetrodotoxin (TTX) is a potent toxin that binds to voltage gated sodium channels. it blocks the flow of sodium ions through the channel,
causes an increasing paralysis of the muscles of the body

Question 40

What is ivabradine ?

Answer 40

Ivabradine is used to treat adults who have chronic heart failure to reduce their risk of hospitalization for worsening heart failure.

ivabradine reduces the diastolic depolarization rate of sinoatrial node pacemaker cells through the inhibition of Hyperpolarization‐activated cyclic‐nucleotide gated (HCN)‐channel mediated “funny” current, If, which results in bradycardia

Question 41

Use dependant drug ?

Answer 41

Drugs that bind the inactive state and increase refractory periods is jnow as use dependant drugs as they only function when channel are used .

Question 42

What is refractory period ?

Answer 42

The refractory period is the time after an action potential is generated, during which the excitable cell cannot produce another action potential.

Question 43

Calcium plays a critical role in exocytosis. What is exocytosis?

Answer 43

Exocytosis is the key mechanism for neurotransmitter release, which occurs in response to elevated intracellular calcium. It is also an important component of the mechanism of release of a number of hormones and enzymes.

Question 44

Cell cycle

Answer 44

Dividing cells such as those in the gastrointestinal tract epithelium or in bone marrow, progress through the cell cycle.

Question 45

What are the stages of cell cycle ?

Answer 45

The cell cycle refers to a series of phases that occur in sequence.

G0- cells not actively dividing.

G1 - During this phase the cell prepares to synthesise DNA and replicate it’s chromosomes.

The second phase is called S-phase – synthesis. During this phase, the cell synthesises the DNA and replicates it’s chromosomes.

The third phase is called G2 – During this period the cell prepares to physically divide.

The final phase is called M-phase, or mitosis, which is the active phase of cell division.

Question 46

Stages of mitosis?

Answer 46

Prophase – this is where the duplicated chromosomes condense and the nuclear envelope degrades

Metaphase – this is where the chromosomes align at the equator of the cell

Anaphase – this is where the mitotic spindle – which is a component of the cytoskeleton, separates the chromatids

Telophase – this is where the nuclear envelope reforms around the two sets of separated chromosomes.

Question 47

What is the final step of the cell cycle?

Answer 47

The final step of the cell cycle is cytokinesis, which is the physical splitting of the cell into two daughter cells.

Question 48

Purpose of checkpoints in cell cycle and where are they located? Failure of checkpoints?

Answer 48

Precise timing of each of the cell cycle phases is critical and this is mediated by CHECK POINTS. The first check point sits between G1 and S-phase and the second is at the start if mitosis. At these checkpoints, any damage to the DNA can be sensed and cause cell cycle arrest. Failure of these checkpoints leads to genomic instability.

Question 49

what does Quiescent cells mean ?

Answer 49

Quiescent means that they are sitting outside the cell cycle.

Quiescent cells can exit G0 and enter G1 in response to damage – for example in the case of the need to heal a wound. Often, this re-entry into the active cell cycle is driven by the action of growth factors.

Question 50

What 2 proteins allow progression through the cell cycle?

Answer 50

Progression through the cell cycle is regulated by two families of proteins – the CYCLINS and the CYCLIN DEPENDENT KINASES (CDKs).

Question 51

what does CDKs do in cell cycle ?

Answer 51

CDKs phosphorylate a range of enzymes to co-ordinate the progression of the cell cycle.

Question 52

When are CDK’s active?
What are the groups of cyclines in terms of cell cycle?
What happens when a specific CDK is activated?

Answer 52

CDKs are only active when bound to cyclins.

There are 8 groups of cyclins, although the best characterised in terms of the cell cycle are given the names CYCLIN A, CYCLIN B, CYCLIN D and CYCLIN E.

After a specific CDK has become activated and phosphorylated its target, it is degraded –degradation important component in ensuring that the cell cycle proceeds in only one direction.

Question 53

Cell cycle process ?

Answer 53

Quiescent cells can exit G0 and enter G1 in response to damage – for example in the case of the need to heal a wound. Often, this re-entry into the active cell cycle is driven by the action of growth factors.

• stimulating re-entry into the cell cycle is an active field of research in regenerative medicine
• Progression through the cell cycle is regulated by two families of proteins – the CYCLINS and the CYCLIN DEPENDENT KINASES (CDKs).
• CDKs are only active when bound to cyclins and CDKs have specific cyclin binding partners., best characterised in terms of the cell cycle are given the names CYCLIN A, CYCLIN B, CYCLIN D and CYCLIN E
• After a specific CDK has become activated and phosphorylated its target, it is degraded. Degradation is an important component in ensuring that the cell cycle has directionality and proceeds in only one direction.
• In response to a growth factor signal, a cell in G0 will begin to increase the levels of cyclin D, which leads to an increase in levels of cyclin D/CDK complexes which phosphorylate key proteins necessary for DNA synthesis.
• Cyclin E and it’s CDK2 binding partners rise. This is necessary to cross the first cell cycle check point and enter into S-phase.

In the S-phase, the cell is committed to DNA replication. Cyclin E/CDK2 and Cyclin A CDK 1&2 regulate progression through S-phase

By G2, the cell has duplicated its DNA and Cyclin a/CDK permits the crossing of check point 2 into mitosis. Cyclin B/CDK2 is necessary for entry into mitosis.

• The Rb protein is a key negative regulator of the cell cycle. It prevents progression from G0 to G1. When phosphorylated in response to growth factor-induced G protein action it allows progression into G1.
• The CDKs can be inhibited if DNA damage is detected. For example, the protein p21, which is under the control of the tumour suppressor p53 can inhibit a number of CDKs.

Question 54

What is apoptosis?

Answer 54

Apoptosis is regulated controlled means of cell loss and it is vital for normal development and homeostasis.

However, defective apoptosis has been implicated in a range of diseases including autoimmune diseases, neurodegenerative disorders, myocardial infarction, stroke and evasion of the immune response by tumour cells.

Question 55

What is the name of the proteins called that regulate apoptosis?

Answer 55

Caspases.

Question 56

What do caspases initiate?

Answer 56

These proteins initiate a cascade.
whereas some actually do the work of apoptosis – so called effector proteins. A key effector protein is caspase 3, which cleaves cell components including protein kinase C and components of the cytoskeleton. In apoptosis, genomic DNA is cleaved and apoptosis concludes.

As these intracellular processes occur, the cell rounds up, the chromatin condenses, the cytoplasm shrinks and the membrane blebs. The cell is then transformed into small fragments which are phagocytosed

Question 57

what are the 2 ways apoptosis can be triggered?

Answer 57

Apoptosis can be triggered by the extrinsic pathway or through the action of mitochondria.

Question 58

Explain the two pathways of how apoptosis can be triggered?

Answer 58

An example of the extrinsic pathway can be seen by the action of tumour necrosis factor, which acts on the TNFR (also called the Fas ligand receptor.) Upon binding a ligand, these receptors trimerise allowing intracellular death domains to bind adaptor proteins which activate the caspase cascade and trigger apoptosis.

By contrast, the mitochondrial pathway can be triggered by DNA damage. If the cell senses DNA damage, and ‘choses’ apoptosis, p53 activates pro-apoptotic members of the BCL2 family of proteins, including a protein called Bax. These proteins form pores in the mitochondrial membrane, allowing the release of cytochrome C. This then complexes with the cytosolic protein apoptotic protease-activating factor 1 which in turn triggers the caspase cascade resulting in apoptosis

Question 59

Exocytosis?

Answer 59

Exocytosis is the key mechanism for neurotransmitter release, which occurs in response to elevated intracellular calcium. It is also an important component of the mechanism of release of a number of hormones and enzymes.

Question 60

When does exocytosis occur?

Answer 60

Exocyctosis occurs when the membrane storage vesicle – the granule – fuses with the plasma membrane, allowing the release of the vesicle contents.
Release of the vesicle contents is highly dependent on calcium.

Question 61

What happens to vesicles in fast acting synapses (exocytosis)?

Answer 61

For example, in fast acting synapses, vesicles are docked at specific regions of the pre-synaptic plate, and release their contents in response to calcium influx from the extracellular compartment, through N and P/Q calcium channels.

In slower nerves, calcium may increase in response to intracellular store release.

Answer 62

For example, the fast neurotransmitter glutamate is released in response to influx of extracellular calcium, causing release of docked vesicles, and then a slow neuropeptide is release later from vesicles further away from the active site.

Let’s talk about the actual release.

The common feature of vesicle release is elevated intracellular calcium. A group of proteins called SNARES mediate the vesicle release, by binding the calcium. Synaptotagmin binds calcium.

This facilitates binding to the SNARE proteins synaptobrevin and synaptotaxin on the inner membrane of the vesicle. This association of proteins leads to vesicle/plasma membrane fusion and vesicle release its contents in response to increased calcium. Interference with this mechanisms can inhibit exocytosis.

Not all transmitters are released through vesicles. For example, lipid-based signals such as prostaglandins are able to cross the plasma membrane freely. In such cases, it is the synthesis of the messenger that is regulated, rather than the messengers release, and the regulation of the synthetic enzymes is often mediated by calcium.

Question 63

Excitable cells

Answer 63

Excitable cells lead to muscle contraction

There are important differences in the control of calcium-regulated muscle contraction in the different types of muscle

Question 64

Smooth muscle contraction involving calcium?

Answer 64

-Spontaneous calcium oscillations in smooth muscle produces rhythmic contractions.

Action potential is generated by L-type Calcium channels allowing calcium to enter the cells.

Many smooth muscle cells express the P2X receptor, which allows calcium entry when they bind ATP,
Agonists can also act on the P2x receptors aswell as GPCR.

Intracellular calcium levels in smooth muscle can also be elevated in response to G-protein coupled receptors activating IP3, leading to sarcoplasmic reticulum release of stored calcium through Ip3 receptor

Smooth muscle contraction can occur when receptors are activated

The elevation of intracellular calcium leads to binding to the protein calmodulin. Calmodulin bound to calcium can bind the protein myosin light chain kinase which phosphorylates myosin light chain, thus leading to contraction.

Relaxation occurs when the phosphorylation of MLCK is reversed by myosin phosphatase.

Question 65

Muscle contraction in skeletal muscles

Answer 65

In skeletal muscles:
Transverse T-tubules extend into the cell, they contain DIHYDROPYRIDINE receptors, which are voltage gated calcium channels that respond to an action potential.

The DHPRs are located next to the ryanodine receptors on the sarcoplasmic reticulum.

The sarcoplasmic reticulum can release calcium ions into the cytosol in response to ryanodine receptor activation from the DHPR

Because the DHPR and ryanodine receptors are coupled, rapid membrane depolarisation following an action potential causes a very fast burst of calcium release from the sarcoplasmic reticulum.

calcium binds the protein TROPONIN, releasing troponin inhibition of actin/myosin, allowing contraction to occur.