1. 1 HOW DRUGS ACT: CELLULAR ASPECTS OF DRUG ACTION

2. 2 OVERVIEW The link between a drug interacting with a molecular target and its effect at the pathophysiological level, such as a change in blood glucose concentration or the shrinkage of a tumour, involves events at that cellular level. Whatever their specialized physiological function, cells generally deploy much the repertoire of signalling mechanisms.

3. 3 Learning Objective At the end of this session the student should be able to describe the cellular mechanisms, particularly excitation, contraction and secretion.

4. 4 The cellular mechanisms account for many physiological responses and operate mainly over a short timescale (milliseconds to hours). The short term regulation of cell function depends mainly on the following components and mechanisms, which regulate, or are regulated by, the free concentration of Ca 2+ in the cytosol [ Ca 2+ ] i

5. 5 Components and mechanisms, which regulate, or are regulated by, the free concentration of Ca 2+ in the cytosol [Ca 2+ ] i Ion channels and transporters in the plasma membrane The storage and release of Ca 2+ by intracellular organelles Ca 2+ -dependent regulation of a variety of functional proteins, including enzymes, contractile proteins and vesicle proteins.

6. 6 Because [ Ca 2+ ] i plays such a key role in cell function, a wide variety of drug effects results from interference with one or more of these mechanisms. If love makes the human world go round, then [ Ca 2+ ] i does the same to cells i.e. makes the cell world go around

7. 7 REGULATION OF INTRACELLULAR CALCIUM LEVELS

8. 8 Many drugs and physiological mechanisms operate, directly or indirectly, by influencing the free intracellular concentrations of [ Ca 2+ ] i Most of the Ca 2+ in a resting cell is sequestered in organelles, particularly the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) and the mitochondria, and the free [ Ca 2+ ] i is kept to a low level about 10 -7 mol/l

9. 9 The Ca 2+ concentration in tissue fluid [ Ca 2+ ] o, is about 2.4 mmol/l so there is a large concentration gradient favouring Ca 2+ entry. Free intracellular concentrations of Ca 2+ [ Ca 2+ ] i is kept low by; a)The operation of active transport mechanisms which eject Ca 2+ through the plasma membrane, and pump it into the ER, and b)The normally low Ca 2+ permeability of the plasma and ER membranes.

10. 10 Regulation of intracellular calcium levels [ Ca 2+ ] i Involves three main mechanisms –Control of calcium entry mechanisms –Control of calcium extrusion mechanisms –Exchange of calcium between the cytosol and the intracellular stores

11. 11 Calcium Entry Mechanisms There are four main routes/channels by which Ca 2+ enters cells across the plasma membrane: –Voltage-gated calcium channels –Ligand-gated calcium channels –Sodium-calcium (Na + -Ca 2+ ) exchange (can operate in either direction –Store-operated calcium channels (SOCs)

12. 12 Voltage-gated calcium channels Are highly selective for Ca 2+ Allow substantial amounts of Ca 2+ to enter a cell Do not conduct Na+ or K+ Allow substantial amounts of Ca 2+ ions to enter the cell when the membrane is depolarized by a conducting action potential

13. 13 Voltage gated calcium channels… A combination of electrophysiological & pharmacological criteria suggests that there are 5 distinct sub types of Voltage-gated Ca2+ channels named as : L,T,N, P/Q & R channels (see next slide) L-long lasting T-transient N-neither long nor transient P, Q & R-carry on alphabetically from N, with O (omitted)

14. 14 Voltage gated Ca+ channels---- Gated by main channel Type Characteristics Voltage L High activation threshold; Slow inactivation N low activation threshold; Slow inactivation T Low threshold; fast inactivation P/Q Low activation threshold; Slow inactivation R Low threshold fast inactivation

15. 15 Voltage gated Ca+ channels----- channel type Location/function drug effects L Plasma membrane; main Ca2+ source for muscle contraction blocked by Verapamil & diltiazem; activated by ByK 8644 N Main Ca2+ source for transmitter release by nerve terminals Blocked by w-conotoxin (component of Conus snail venom)

16. 16 Voltage gated Ca+ channels--- T channel -This is widely distributed; is of particular function in cardiac pace maker & atria (role in dysrythmias) Drug effect: The receptor is blocked by mibefradil P/Q –channel- Is present in nerve terminals; is involved in neuro-transmitter release. It is blocked by w- agatoxin from web spider venom R channel Location = ? Drug effects ? ( Not known)

17. 17 Ligand gated Ca 2+ channels Most are activated by excitatory neurotransmitters Are relatively non selective Conduct Ca 2+ as well as other cations E.g. The glutamate receptor of the N- methyl-D-aspartate (NMDA) type, which has a high permeability to Ca 2+

18. 18 Ligand gated Ca+ channels---- It is a major contributor to Ca 2+ uptake by postsynaptic neurons and also glial cells Allows so much Ca 2+ entry inside the cell The excess Ca 2+ inside the cell may induce cell death by triggering apoptosis and/or via activation of Ca 2+ dependent proteases

19. 19 ‘Store operated’ Ca 2+ channels (SOCs) Occur in the plasma membrane Open to allow Ca 2+ entry inside the cell when Endoplasmic reticulum (ER) stores are depleted Indirectly coupled to ER/SR Ca 2+ stores i.e. Activated indirectly by agents that deplete intracellular stores

20. 20 Calcium extrusion mechanisms Active transport of calcium outwards across the plasma membrane and inwards across the membranes of the ER or SR depends on activity of a calcium- dependent ATPase similar to the Na + /K + ATPase that pumps Na + out of the cell in exchange for K +. Ca 2+ is also extruded from cells in exchange for Na + (Na + - Ca 2+ exchange ) Three sodium ions are exchanged for one calcium ion producing a net hyperpolarising current when calcium is extruded SR=Sarcoplasmic reticulum

21. 21 Calcium extrusion mechanisms--- The energy for Ca 2+ extrusion comes from the electrochemical gradient for Na +, not directly from ATP hydrolysis. i.e A reduction in the Na + concentration gradient resulting from Na + entry will reduce Ca 2+ extrusion by the exchanger, causing a secondary rise in [Ca 2+ ] i, a mechanism that is particularly important in cardiac muscle.

22. 22 Calcium extrusion mechanisms--- The exchanger can function in reverse if [Na + ] i rises excessively, resulting in increased Ca 2+ entry into the cell The effect of digoxin on cardiac muscle involves this mechanism

23. 23 Calcium Release Mechanisms Calcium channels in the ER and SR membrane play an important part in controlling the release of calcium from intracellular stores

24. 24 Two main types of Ca+ channels exist in ER/SR these are: 1.Ligand-gated ion channel through the inostol triphosphate receptor (IP 3 R).  This is activated by inositol triphosphate( IP 3), a second messenger produced by the action of many ligands on G-protein-coupled receptors.  IP 3 R) is a ligand-gated ion channel, though its molecular structure differs from that of ligand- gated channels in the plasma membrane.  This is the main mechanism by which activation of G-protein-coupled receptors cause an increase in Ca 2+

25. 25 Two main types of Ca+ channels exist in ER/SR these are: 2. Calcium-induced-calcium release (CICR) through the ryanodine receptor (RyR), so called because it was first indentified through the specific blocking action of plant alkaloid, ryanodine

26. 26 Calmodulin It is Ca 2+ binding protein Calcium exerts its control over cell functions by virtue of its ability to regulate activity of many different proteins including enzymes (especially kinases and phosphatases), channels, transporters, transcription factors, synaptic vesicle proteins etc In most cases, a calcium-binding protein serves as an intermediate between calcium and the regulated cell functional proteins

27. 27 The ubiquitous calmodulin is the best known such binding protein, which modulates about 40 different functional proteins Calmodulin is a dimeric protein with 4 calcium- binding sites When all sites are occupied, calmodulin undergoes conformational change, exposing a hydrophobic domain that attracts many proteins into association thereby affecting their functional properties

28. 28 Excitation Excitability describes the ability of a cell to show regenerative all-or-nothing electrical response to depolarization of its membrane. This membrane response is known as an action potential (an electrical response following cell membrane depolarization-excitation). It is a characteristic of most neurons & muscle cells ( striated, cardiac & smooth muscles) Revise your physiology on ions involved in the genesis and propagation of an action potential

29. 29 Release of chemical mediators Fall into 2 main groups: - Preformed and packaged in storage vesicles from which they are released by exocytosis Mediators that are produced on demand -are released by diffusion or by membrane carriers Ca+ plays a key role in both cases

30. 30 Exocytosis Occurs in response to an increase in intracellular Calcium [ Ca 2+ ] i It is the principal mechanism of transmitter release in both peripheral and CNS Involves fusion between the membrane and synaptic vesicles which are preloaded with the stored transmitter and the inner surface of the plasma membrane

31. 31 Muscle contraction Mechanisms involved in muscle contraction –Skeletal muscle –Cardiac muscle –Smooth muscle Involve a link between events and an increase in intracellular Ca+ and mechanisms which Ca+ regulates contraction Muscle contraction occurs in response to a rise in intracellular Ca+ In skelatal muscle depolarization causes rapid Ca+ release from SR Please revise your physiology on role of Ca+ in muscle contraction

32. 32 Epithelial ion transport Many epithelia (e.g. from renal tubules, exocrine glands, airways etc) are specialized to transport specific ions This type of transport depends on a class of Na+ channels called ENaCs (epithelial sodium channels) ENaCs allows Na+ entry into cells on one surface coupled to active extrusion of Na+ or exchange for another ion, from the opposite surface

33. 33 Epithelial ion transport---- epithelial sodium channels (ENaC) expression is increased by aldosterone ENaCs are blocked by amiloride The activity of channels, pumps and exchange transporters is regulated by various second messengers and nuclear receptors, which control the transport of ions in specific ways.

34. 34 Further reading