PHAR3306 Notes - Week 2 - Pharmacodynamics II - PDF

Wk 2: Pharmacodynamics II Cell Signalling Three stages of Cell Signalling 1. Reception when the agonist binds to the receptor 2. Transduction when the message is relayed 3. Activation of cellular responses...

Wk 2: Pharmacodynamics II Cell Signalling Three stages of Cell Signalling 1. Reception when the agonist binds to the receptor 2. Transduction when the message is relayed 3. Activation of cellular responses Stages - A signal is detected when the molecular signal (also known as a ligand) binds to a Reception receptor protein on the surface of the cell or inside the cell. - Receptor will transmit information from extracellular to intracellular environment by changing its shape which helps to propagate the signal into the cell - When an agonist induces a conformational change in the Transduction receptor it initiates the process of transduction. - Each molecule in the pathway acts upon the next molecule in the pathway. - These molecules are often enzymes, which can catalyse multiple reactions each. - This process leads to amplification of the signal. Response - Kinases play a central role in signal transduction. - Kinase cascades are triggered by GPCRs, guanylyl cyclase-linked (GC) receptors, or by catalytic (kinase-linked) receptors. - Kinase cascades regulate various target proteins, leading to numerous cellular events. Summary Smooth Muscle Found in - Throughout the body arteries and veins, bronchi, bladder, Iris, Urethra and Prostate, in Skin, Uterus and the Gastrointestinal tract - A muscle fibre contracts when the actin fibres are pulled and slide past the myosin Contraction fibres. This process requires ATP and calcium. How is Smooth Muscle Contraction & Relaxation Regulated? Heterotrimeric - G proteins bind to the intracellular G proteins surface of these G protein coupled receptors, which is the beginning of the intracellular signalling cascade. - G proteins are hetero trimeric proteins consisting of an alpha subunit, a beta subunit and a gamma subunit. - Both the alpha subunit and the beta gamma hetero dimer independently induce their own signalling outcomes. - There are multiple different subtypes of the alpha G protein that each have unique effects on second messenger molecules. Gαq signalling 1. GPCR activates Gαq – smooth 2. IP3 produced, and opens IP3R on SR muscle 3. Ca2+ released from SR contraction 4. Ca2+ binds and activates calmodulin (CaCM) 5. CaCM binds and activates the myosin light chain kinase (MLCK) 6. The activated MLCK catalyses the transfer of phosphate to myosin heads, activating myosin head ATPases 7. Phosphorylated myosin heads form cross bridges with actin, shortening occurs 8. Muscle contraction Steps 1-3 Gαq signalling Agonists bind to G protein coupled receptors, leading to the conformational change. Then GDP dissociates while GTP binds to Gαq, which causes the band γ and β to dissociate. The Gαq binds to the enzyme called PLCβ, which helps PIP2 change into DAG and IP3. DAG will activate enzyme call PKC, while the second messenger IP3 combined to the ligand channel in SR, leading to the gate open, which increase the intracellular calcium Steps 4-8 The intracellular calcium combines with calmodulin to form complexes. These complexes activate myosin light chain kinase (MLCK) to bring about phosphorylation of the myosin light chains, which generates ATPase activity to cause sliding of myosin over the actin fibrils and contracting the muscle. - Activating Gαq coupled receptors leads to increased cytoplasmic calcium. The calcium is released by IP3 ligand gated ion channels on the SR membrane. - Image 2: Adding the agonist for the M3 muscarinic receptor, causes calcium release from the sarcoplasmic reticulum. The release of calcium causes contraction of the  smooth muscle Gαq signalling Parasympathetic innervation of the sphincter muscle: - - Acetylcholine (ligand) activation of M3 muscarinic receptors on the iris sphincter smooth Pharmacology muscle resulting in smooth muscle contraction and consequent pupillary constriction. in the iris Sympathetic innervation of the radial muscle: - Noradrenaline (ligand) activation of α1 -adrenoceptors on the iris radial smooth muscle resulting in smooth muscle contraction and consequent pupillary dilation. Clinically used drugs: Pilocarpine (M3 receptor agonist), phenylephrine (α1-adrenoceptor agonist), tropicamide (M3 receptor antagonist). Gαs signalling 2 methods of relaxation – smooth 1. Gαs muscle 2. Nitric oxide relaxation 1.Gαs Signalling Agonists bind to Gαs coupled receptors, leading to the conformational change. Then GDP dissociates while GTP binds to Gαs, which causes the band γ and β to dissociate. Then Gαs bind to the enzyme called Adenylate cyclase which leads to the production of a second messenger cAMP from ATP. cAMP activates protein kinase A (PKA). - (This pathway is inhibited by Gαi-coupled GPCRs, which activate Gi to inhibit adenylyl  cyclase, thereby reducing the amount of cAMP in the cell) 2 ways for PKA lead to muscle relaxation - PKA catalyses the transfer of phosphate to the phosphatase enzyme. This activates the dephosphorylation of myosin light chain. - PKA catalyses the transfer of phosphate to myosin light chain kinase. This prevents the phosphorylation of the myosin light chain. - TOGETHER, THE BALANCE TIPS TOWARDS RELAXATION Gαq and Gαs Parasympathetic innervation of bronchial smooth muscle: signalling - Acetylcholine activation of the M3 muscarinic receptor causes smooth muscle pharmacology contraction and narrowing of the airways. in the lung Sympathetic innervation of bronchial smooth muscle: - Adrenaline activation of the β2 -adrenoceptor causes smooth muscle relaxation and widening of the airways. Clinically used drugs: - Salbutamol (short-acting β2 -adrenoceptor agonist), salmeterol (long-acting β2 -adrenoceptor agonist), ipratropium and tiotropium (M3 muscarinic receptor antagonists) Nitric oxide - Activation of the Gαq coupled receptor on the endothelium cell results in the production (NO) – smooth of nitric oxide (NO). NO travels to the smooth muscle cell where it binds to soluble muscle guanylyl cyclase (GC), this induces a conformational change resulting in the activation relaxation of the enzyme and conversion of GTP to cGMP. cGMP activates cGMP dependent kinase (PKG). Nitric oxide - NO activates soluble guanylyl cyclase (GC), converting GTP to (NO) causes cGMP. relaxation of - cGMP activates cGMP dependent kinase (PKG). smooth - PKG phosphorylates myosin light chain phosphatase (MLCP), muscle cells which activates this enzyme. via PKG - MLCP catalyses the removal of phosphate from myosin light chain, resulting in smooth muscle relaxation. - PKG also leads to MLCK inactivation. Summary Terminating Signalling GPCR - Reduction in smooth muscle contraction when the drug was washed out and the muscle Desensitisatio given time to recover stimulation with the same drug. n/Tachyphylax - The same concentration caused an is equivalent level of smooth muscle contraction as the first drug administration, so this process here, where our contractile response reduces upon re repeated administration of the same drug is known as desensitisation/tachyphylaxis. Causes Desensitisatio - Receptor modifications (phosphorylation/arrestin binding) n/Tachyphylax - Down-regulation of receptors (internalisation/reduced cell-surface expression) is - Depletion of mediators - Increased metabolic breakdown GPCR Desensitisatio n: turning off the signal Gao and Gai interact with its target enzyme to produce a second messenger. After a while, GRK activates GPCR and phosphorylates them, which means GPCR are less likely to bind G protein, leading to stop in signal. Also, β-arrest would bind to the complex and stop the binding of G protein. Which also stops signalling. Practice Question  Lecture 2: Pharmacological modulation of the ANS I Autonomic Nervous System - The autonomic nervous system controls the involuntary functions and influences the activity of internal organs Sympathetic and Parasympathetic Homeostasis Sympathetic vs When adrenaline acts on Sympathetic Nervous System, we get far vision, inhibition of Parasympathetic waste excretion and stimulates ‘Fight or Flight’ response whereas acting on the Parasympathetic nervous system, it stimulates near vision, reduced heart rate, increased food digestion and waste removal mechanisms as part of the ‘Rest and Digest’ functions of the body Site of drug action Each stage of Neurotransmission is a Potential Site of drug action - Places where a drug can be targeted Cholinergic Neurotransmission and drug targets Exocytosis of - SNARE proteins, SNAP-25, syntaxin, synaptobrevin (VAMP), acetylcholine (Ach) on the vesicle and the inner surface of the nerve terminal containing vesicles membrane mediate the fusing of the vesicles containing acetylcholine (Ach) to the membrane. - Release of Ach from the vesicles requires influx of calcium and is mediated by an action potential. - Calcium interacts with the SNARE protein complex on the vesicle membrane and triggers fusion of the vesicle membrane and release of the Ach into the synaptic cleft Inhibition of - A neurotoxin produced by Clostridium exocytosis of ACh botulinum, a bacterium that causes containing vesicles: food poisoning (botulism, lethal) Botulinum toxin - Botulinum toxin, taken up into (BOTOX) vesicles, cleaves the SNARE proteins, preventing assembly of the fusion complex and preventing the release of acetylcholine Botulinum toxin Mechanism of Action - Paralyses the muscles by blocking the release of acetylcholine at the neuromuscular junction Indications - Upper-limb spasticity in stroke patients, focal spasticity of the upper and lower limbs due to cerebral palsy, cervical dystonia in Parkinson's patients, strabismus, blepharospasm, overactive bladder (due to spinal cord injury or multiple sclerosis), prophylaxis of headaches in adults with chronic migraine, primary hyperhidrosis and cosmetic indications (temporary improvement in the appearance crow's feet and forehead lines) Contraindications - Myasthenia gravis or Eaton Lambert myasthenic syndrome (conditions in which the immune system attacks the neuromuscular junctions). Side Effects - weakness of adjacent muscles (this is expected for any injection procedure), urinary retention and urinary infections when used for overactive bladder and eyelid ptosis, dry eye & photophobia when used to treat strabismus and blepharospasm. Route(s) of administration Injection Cholinesterase inhibitors Effects after AChE inhibited: - Enhance the actions of ACh and producing actions similar to ACh agonists - Autonomic actions: enhancement of ACh activity at parasympathetic synapses (bradycardia, increased saliva secretion, increased smooth muscle contractility) - Neuromuscular junction: repeated firing of the muscle fibre leading to twitching and increased muscle contraction - If cross blood brain barrier (e.g. physostigmine), they can cause profound CNS effects AChE inhibitors - - Clinically used AChE inhibitors are medium duration, and their inhibitory actions clinical uses are reversible by adding muscarinic receptor antagonists. Myasthenia Gravis (MG) Neostigmine Cholinergic Receptors Acetylcholine - Nicotinic receptors are pentameric (5) Nicotinic Receptors- proteins. Ligand Gated Ion - Made up of combinations of α,β,γ,δ and ε Channels subunits - Muscle-types (NM) are formed with α,β,γ,δ,ε - Neuronal-types (NN) are formed with α or α &β subunits. Skeletal Muscle Neuromuscular Junction Neuromuscular Depolarising blocking agents blocking agents - Agonists at nicotinic receptors (Effects mostly due - Causes muscle twitching before paralysis to motor paralysis) - Maintains muscle depolarization. Non-depolarising agents Competitive antagonists at nicotinic receptors - Can block pre- and postsynaptic nicotinic receptors. Leading to tetanic fade. - The majority of clinically used neuromuscular blocking agents are non-depolarizing Non-depolarising blocking agents  - Sites of action of drugs that modulate the function of noradrenaline Uptake - Inhibiting NET-1 leads to an accumulation of NE in the synaptic cleft and 1/Norepinephrine increased activation of the postsynaptic adrenoceptors Transporter (NET) Inhibitors Indirectly Acting - Taken up into the nerve terminal via NET-1 and Sympathomimetic enter the synaptic vesicles via VMAT. Both these Amines transporters are usually unidirectional but the indirectly acting sympathomimetic amines alter their function converting them into bidirectional / exchange transporters leading to the removal of noradrenaline from the vesicle and then the synapse and its accumulation in the synaptic cleft activating post synaptic receptors. Uptake 1/Norepinephrine Transporter (NET) Inhibitors Monoamine Oxidase Inhibitors: Moclobemide Feedback control of noradrenaline release α2-Adrenergic Agonists: Clonidine Practice question  Lecture 3: Pharmacological modulation of the ANS II Muscarinic Receptors - GCPRS + Effects on tissues (Parasympathetic) Adrenergic Receptor Subtypes - GPCRs + Effects on tissues (Sympathetic) Signalling: Side effects Undesired effects that occur when a drug is (Adverse Effects) administered. - Unintended secondary effects that a drug will predictably cause - Side effects often stop people taking medication as the side effects are unpleasant. - Known to occur in a percentage of patients Side effects can be due to actions at the intended target (on-target) or an unintended target (off-target) Selectivity A drug’s ability to preferentially produce a particular effect and is related to the structural specificity of the drug binding to receptors. Selective drugs have greater affinity for one receptor over another. - At low concentrations, an agonist selective for receptor A will only activate receptor A and not receptor B. - At high concentrations, an agonist selective for receptor A will activate both receptor A and receptor B. - Agonist or antagonist drugs that are ‘selective’ for the intended receptor can still produce significant effects at other related receptors if a high enough dose is given. - Selectivity is useful in clinical practice only when the ratio of the affinity of a drug at the target receptor versus other related receptors is 100 x or more. When selectivity is lower, it is difficult to predict drug doses that will exploit the difference in subtype activity. Selectivity reduces off-target side effects. Adrenaline - Given during an asthma attack (Sympathetic) ASTHMA - To eliminate some of the side effects we can rely on beta receptor  Using a b2 agonist leads to relaxation → less side effects Turning off the Parasympathetic Nervous System  Less side effects: Autonomic Receptors - Eye Muscarinic Receptor - Mainly used in the treatment of glaucoma and to induce miosis Agonists Muscarinic receptor agonists stimulate muscarinic M3 receptors on ciliary muscle and GLAUCOMA sphincter pupillae to cause these smooth muscle contraction - In open angle glaucoma - increase trabecular outflow by contracting the longitudinal part of ciliary muscle - In angle closure - contraction of the sphincter pupillae causes miosis (pupil constriction), pulling iris away from trabecular meshwork and opens the angle  Reducing effects Sympathetic Diurnal variation: day 2.5 μl/min during day and 1.5 ul/min during the night Aqueous regulation of humour formation during rest (parasympathetic) is at basal levels and increases with aqueous humour activity (sympathetic) due to activation of β-adrenergic receptors (increased cAMP) and production inhibition occurs via α2-adrenergic receptors activation (decreased cAMP). Active ion transport mechanisms in ciliary epithelial cells result in fluid movement. This is dependent on HCO3−-dependent ion transport mechanisms, along with Na+/K+/Cl− cotransport and Cl− channels, which drive net Na+ and Cl− flux into the posterior chamber and the movement of fluid. cAMP production, has been shown to activate the Na+/K+/Cl− symporter and the Cl− channel. - Reducing IOP Angina Pectoris Practice question