Smooth Muscle Pharmacology and Drug Interactions

Smooth Muscle Relaxation

Increasing cAMP or cGMP → PKA or PKG → phosphorylation of MLCK or phosphorylation of VOCCs. This phosphorylation prevents MLCK from phosphorylating MLC (reducing the amount of phosphorylated MLC) → ↓ [Ca²⁺] → This is a powerful “anti-contraction” mechanism → relaxation.

Smooth Muscle Contraction

Release of Ca²⁺ from intracellular stores → calcium-calmodulin complex. This activates myosin light chain kinase (MLCK), leading to phosphorylation of MLC (myosin light chain) → cross-bridge cycling then promotes contraction.

Vasodilation

1. Nitric oxide (NO): NO activates guanylyl cyclase, ↑ cGMP → relaxation of smooth muscle.
2. β₂-adrenoceptor (G_αs): Activates adenylate cyclase, ↑ cAMP, which opposes contraction by activating PKA which phosphorylates MLCK. This phosphorylation prevents MLCK from phosphorylating MLC (reducing amount of phosphorylated MLC). This is a powerful “anti-contraction” mechanism → smooth muscle relaxation.
3. PKA also causes phosphorylation of K⁺ channels causing greater opening of K⁺ channels – hyperpolarization of smooth muscle cells → less likely to contract.

Vasoconstriction

α₁-adrenoceptor (G_αq/11): PLC cleaves PIP₂ → DAG + IP₃. IP₃ acts on ligand-gated ion channel (IP₃ receptor) → α₁-adrenoceptor activation results in release of calcium from intracellular stores which, in turn, causes calcium to bind to calmodulin. The calmodulin binds to myosin light chain kinase (MLCK) which phosphorylates myosin light chain (MLC) leading to cross-bridging cycling → promotes contraction.

Bronchodilation – Relaxation

1. Agonist acting on β₂-adrenergic receptor (G_αs) → adenylate cyclase activation → ↑ cAMP → PKA → PKA phosphorylates and inhibits VOCCs + phosphorylates MLCK → ↓ in calcium influx + reducing MLCK’s ability to phosphorylate MLC → inhibits smooth muscle contractility = relaxation.
2. NO (Nitric Oxide): Bronchial epithelium can release NO, which stimulates guanylyl cyclase, ↑ cGMP → promoting relaxation.

Bronchoconstriction

Muscarinic ACh receptors (PNS) (G_αq/11) → PLC activation → DAG & IP₃ → ↑ Ca²⁺ → contraction.

Prostatic Smooth Muscle (BPH) Relaxation

α₁ adrenergic antagonist → preventing NA from increasing intracellular Ca²⁺ → inhibits MLCK from phosphorylating MLC → smooth muscle relaxation.

Prostatic Smooth Muscle (BPH) Contraction

α₁ adrenergic receptors (G_αq/11) → PLC activation → DAG & IP₃ → ↑ Ca²⁺ → contraction = unable to pee (BPH)

GI Smooth Muscle Relaxation

1. NA released from sympathetic nerve terminal in the gut acts on pre-synaptic α₂ adrenoceptors (G_αi) on cholinergic nerves (PNS) → inhibits adenylyl cyclase → ↓ cAMP → reduction in ACh release → decreasing muscle contractions = relaxation of GI smooth muscle → decrease in motility and secretion.
2. β₂-adrenergic receptors (G_αs) → adenylate cyclase activation → ↑ cAMP → PKA → PKA phosphorylates and inhibits VOCCs + phosphorylates MLCK → ↓ in calcium influx + reducing MLCK’s ability to phosphorylate MLC → smooth muscle relaxation.

GI Smooth Muscle Contraction

Muscarinic ACh receptors (PNS) (G_αq/11) → PLC activation → DAG & IP₃ → ↑ Ca²⁺ → contraction.

Cardiac Muscle Contraction

NA acting on β₁-adrenergic receptors (G_αs) on cardiomyocytes → adenylate cyclase activation → ↑ cAMP → PKA activation → phosphorylates VOCCs → ↑ activity/opening of VOCCs → ↑ Ca²⁺ → promoting contractility of heart muscle → increasing rate and force of contraction.

Hayfever Mechanism

1. Allergen enters nasal mucosa and binds to IgE (antibody) on mast cells.
2. Causing the release of histamine (degranulation) from mast cells. Degranulation is dependent on elevation of intracellular Ca²⁺ concentration in the mast cell.
3. Histamine sensitizes localized pain fibers, making them itchy.
4. Histamine acts on H₁ receptors (G_αq) on endothelial cells of blood vessels → phospholipase C (PLC) → PIP₂ hydrolysis = DAG + IP₃ → IP₃ binds to IP₃ receptors on the endoplasmic reticulum, causing the release of Ca²⁺ into the cytoplasm → Ca²⁺ binds to calmodulin → Ca²⁺-Calmodulin complex activates endothelial nitric oxide synthase (NOS) → catalyzes production of nitric oxide (NO) → NO diffuses out of endothelial cells and into underlying vascular smooth muscle cells, where it activates soluble guanylate cyclase (GC), promoting the formation of cGMP and activation of protein kinase G (PKG) → promotes dephosphorylation of MLC which leads to vascular smooth muscle relaxation – vasodilation → can lead to hypotension.

Drug-Drug Interactions

Beta Blockers and Asthma Therapy

• Effect: Non-selective beta blockers (e.g., propranolol β₁&β₂) can cause bronchoconstriction by acting at β₂-adrenoceptors, worsening asthma symptoms.
• Recommendation: Use selective beta blockers (e.g., metoprolol (β₁)) and monitor asthma control closely. Could also use a corticosteroid to reduce inflammation.

Phosphodiesterase Inhibitors and Blood Pressure Meds

• Effect: PDE inhibitors (e.g., sildenafil) enhance vasodilation, potentially causing severe hypotension when combined with blood pressure medications (β-blockers), which also lower blood pressure.
• Consideration: Avoid co-administration with nitrates (nitro-vasodilators), which also cause vasodilation; be cautious about timing and dosage adjustments for blood pressure medication to avoid dangerously low blood pressure.

Agonists: Affinity/Potency + Efficacy

1. Angiotensin II: AT₁ receptor agonist – raises blood pressure. Binds to AT₁ receptors on blood vessels, causing vasoconstriction and increasing peripheral resistance, which raises blood pressure.
2. Phenylephrine: Alpha-1 adrenergic receptor agonist – vasoconstriction.
3. GABA: Agonist at GABA_A (ionotropic) and GABA_B (metabotropic) receptors.
4. Nicotine: Nicotinic acetylcholine receptor agonist.
5. Noradrenaline: Adrenergic agonist (both alpha and beta receptors).
6. Acetylcholine: Muscarinic and nicotinic receptor agonist.

Antagonists

Antagonists do not activate the receptor, blocking the effect of agonists or endogenous ligands. Affinity but no efficacy.

1. Beta-blockers (e.g., Propranolol, Atenolol): Block beta-adrenergic receptors – reduce rate and force of contraction, reducing cardiac output.
2. Alpha-1 antagonists (e.g., Prazosin, tamsulosin): Block alpha-1 adrenergic receptors; prazosin is used to treat BPH and hypertension, tamsulosin is used to treat BPH.
3. Cocaine, SSRIs: Uptake inhibitors.
4. RTK inhibitors: Block receptor tyrosine kinases, preventing downstream signaling.

Autonomic Nervous System Effects

Asthma

Agonists (like β₂-agonists such as salbutamol) promote bronchodilation. Antagonists: Muscarinic antagonists (like ipratropium) inhibit bronchoconstriction.

Hayfever

Antihistamines (like loratadine) block histamine H₁ receptors, reducing allergic responses – competitive reversible – opposing vasodilation.

Blood Pressure

Agonists: Alpha-adrenergic agonists (like phenylephrine) increase blood pressure by vasoconstriction. Antagonists: Beta-blockers (like propranolol (β₁&β₂)) reduce blood pressure by decreasing heart rate and contractility. (metoprolol, atenolol are selective for β₁)

BPH

Alpha-1 antagonists (like tamsulosin) relax the smooth muscle in the prostate and bladder neck, improving urine flow.

Angina

G_αs: β₁-adrenergic receptors → adenylate cyclase → increasing cAMP and enhancing cardiac contractility and heart rate. Beta-blockers target this pathway to reduce oxygen demand and relieve angina.

Tumor Cell Growth

RTK inhibitors: Block pathways like the RTK/Ras signaling pathway, inhibiting cell proliferation.

Depression

SSRIs (like fluoxetine) inhibit the reuptake of serotonin, increasing its availability in the synaptic cleft → enhances neurotransmission and receptor activation, which is thought to contribute to mood elevation and the alleviation of anxiety symptoms.

GPCR Steps

1. Agonist binds to GPCR.
2. Conformational change in the GPCR.
3. Transmembrane 5 and 6 move out of the way, and the C-terminal tail gets pushed aside, making room for G proteins.
4. G protein (heterotrimer) binds – three G proteins – alpha, beta, gamma.
5. Binding of the heterotrimeric structure to the GPCR triggers the activation of the alpha subunit, which undergoes a conformational change, leading to the replacement of GDP with GTP bound to the alpha subunit.
6. GTP binding catalyzes the break-up of the α-subunit-GTP complex from the βγ-subunit complex.
7. The attachment of the α-subunit-GTP complex to an effector molecule modulates the effector protein’s activity and increases the GTPase activity of the complex, which then converts GTP to GDP (+ phosphate), thus inactivating the α subunit.

• N-terminus inside, C-terminus outside.

G_αs & G_αq Coupled Receptor Activation

The activation of either G protein is the same. Their signal transduction pathways are different, but the activation process is the same.

Striated (Skeletal) Muscle Contraction

1. ACh is released due to the influx of extracellular Ca²⁺ in the pre-synaptic motor neuron. Membrane depolarization occurs due to the binding of ACh to muscle fiber nicotinic receptors, causing ligand-gated Na⁺ channels to open and Na⁺ influx – propagation of action potentials.
2. Action potential propagates down T-tubule.
3. Inside the T-tubule is a voltage-operated calcium channel (VOCC).
4. VOCC opens in response to the localized depolarization, letting Ca²⁺ into the cell.
5. Calcium activates the ryanodine receptor (RyR) on the sarcoplasmic reticulum membrane, triggering calcium release from intracellular stores.
6. Released calcium binds to troponin, creating the troponin-Ca²⁺ complex, causing contraction. Muscle relaxes when ACh is broken down by acetylcholinesterase in the synaptic cleft, stopping the signal.

Cell Signaling

Paracrine – cell signaling to a neighboring cell.
Autocrine – a cell signaling itself.
Endocrine – cells signaling to tissues far from themselves – secreted and transported to the site of action.
Neurocrine – neurotransmitter and hormone release.

Equilibrium Constant and Drug Properties

K_E = equilibrium constant = a measure of the affinity of an agonist for a receptor = agonist concentration at 50% of maximal response (EC₅₀)
Response = [agonist] / ([agonist] + K_E)
Partial Agonist: Binds to a receptor and produces a weaker response than a full agonist. It can act as an antagonist in the presence of a full agonist due to its lower efficacy.
Affinity: The strength with which a drug binds to its receptor. High affinity often correlates with greater potency.
Indirect Agonist: Enhances the release or action of a neurotransmitter or hormone without directly activating receptors – e.g., accessing uptake transporter to get into the pre-synaptic nerve and cause the release of an endogenous ligand.
Inverse Agonist: Binds to a receptor with constitutive activity and reduces its baseline activity, producing the opposite effect of an agonist.
Measure of agonist efficacy α = max response partial agonist / max response full agonist
Lower EC₅₀ = more potent; Higher EC₅₀ = less potent
Relative Potency = EC₅₀ Drug B / EC₅₀ Drug A

Ventolin and Bricanyl

Since the two devices are equi-effective and the two chemicals have similar molecular weights and PK/PD, it is likely that potency (affinity/efficacy) is the reason for the need to administer different doses to achieve the desired effect. Both are short-acting β₂-adrenoceptor full agonists acting at β₂ adrenoceptors located on airway smooth muscle cells and mediate relaxation to dilate the airway. If the EC₅₀ for salbutamol on an isolated airway preparation is 1 nM, the EC₅₀ for terbutaline is likely to be 5 nM. Five times more terbutaline is required than salbutamol to elicit the same response because salbutamol is five times more potent than terbutaline. Both are likely to be full agonists since, during bronchospasm, maximal airway dilation and, therefore, maximal O₂ intake is more desirable than subtle changes.

G_αi-GPCR Modulation of Neurotransmitter Release

Neurotransmitter binds to pre-synaptic G_αi GPCR and activates it → Conformational change → facilitates replacement of GDP with GTP → Leads to the dissociation of the G_αi subunit from the βγ-subunit. → G_αi inhibits adenylyl cyclase → decreases intracellular cAMP → reduced activation of PKA → decrease in phosphorylation of VOCCs → decrease in Ca²⁺ influx into the pre-synaptic terminal when the neuron is depolarized → Elevated intracellular Ca²⁺ is essential for neurotransmitter release into the synaptic cleft; however, this decrease leads to a decrease in neurotransmitter release into the synaptic cleft → This modulates synaptic transmission and signal strength – negative feedback loop.

Cardiac Muscle Contraction

1. Sodium channel propagating a signal to L-type VOCC.
2. Calcium floods in through the open VOCC.
3. Ca²⁺ activates ryanodine receptor (RyR) – calcium-sensitive receptor (ligand-gated ion channel).
4. RyR receptor promotes the release of calcium from intracellular stores.
5. Released Ca²⁺ binds to troponin.
6. Ca²⁺-troponin complex promotes contraction.

Physiological Antagonism (Anaphylactic Shock)

Allergen exposure → massive histamine release → Vasodilation = hypotension & Bronchoconstriction: histamine activates G_αq-coupled H₁ receptors in bronchial smooth muscle → PLC → Ca²⁺ release. Ca²⁺ activates MLCK → MLC phosphorylation → bronchoconstriction, narrowing airways = respiratory depression.
Treatment with Adrenaline: Stimulates α₁-adrenoceptors → vasoconstriction, stimulates β₂-adrenoceptors → bronchodilation, stimulates β₁-adrenoceptors → increased heart rate and force.

pA₂ Value

The pA₂ value is a measure of a competitive reversible antagonist’s potency. It is the negative log of the molar concentration of an antagonist required to produce a 2-fold shift in an agonist concentration-response curve. The pA₂ value is the negative logarithm of the concentration of antagonist required to produce a log [CR-1] = 0.
pA₂ = -log [antagonist conc M].
A higher pA₂ (right side of graph) = more potent antagonist as a lower concentration of antagonist is needed to achieve a given effect. A lower pA₂ value (left side of graph) = less potent antagonist.
Allosteric Modulator: Modulates efficacy or affinity by altering receptor structure.
To determine the nature of antagonism: Repeat the experiment with increasing concentrations of the “antagonist”.

Toxic Contexts

1. On-target: Toxicity due to the interaction of the drug with the same target that produces the desired pharmacological response (e.g., statins = inhibition of HMG-CoA reductase in muscle = myopathy).
2. Hypersensitivity & immune response: Allergic reaction to penicillins.
3. Off-target toxicity: Toxicity caused by drug binding to an alternative target (e.g., terfenadine blocking hERG channel causing arrhythmias).
4. Bioactivation: Drugs being converted into reactive metabolites or ultimate toxicants (e.g., paracetamol producing toxicant NAPQI, which is toxic to cells).
5. Idiosyncratic or individual reactions: Responses are rare and theorized to depend upon multiple factors, including genetic polymorphisms and/or hapten development (promoting antibody production) (e.g., halothane).

Dopamine Toxicity

1. Off-target effects: L-dopa → Dopamine → Noradrenaline → Adrenaline = Dopamine and NA can then have many off-target effects in the periphery. Treatment: Peripheral L-dopa effects can be minimized by the concomitant use of carbidopa (a peripheral dopa decarboxylase inhibitor), which penetrates weakly into the CNS.
2. Toxic (ROS production): Dopamine is oxidized and converted to a quinone and/or semiquinone (P450), which has the potential for redox cycling. Molecular oxygen is then used to produce superoxide radical anion from the semiquinone radical anion, which then gets catalyzed by SOD (superoxide dismutase) to make hydrogen peroxide, which catalase and glutathione peroxidase can turn into H₂O. However, in the presence of transition metals, this reactivity can change and generate the reactive species, a hydroxyl radical – super reactive – which can cause lipid peroxidation/DNA damage = cellular damage and death.

Glutathione = principle detoxification mechanism: Detoxifies H₂O₂ into H₂O and conjugates with quinones, making them far less reactive and easily excretable.

Paracetamol Toxicity

NAPQI (reactive intermediate) is produced from paracetamol breakdown by CYP450 enzymes → liver toxicity. With sufficient amounts of glutathione, NAPQI can be neutralized and then excreted in urine; however, with insufficient levels of glutathione, NAPQI accumulates and can bind to anything with a cysteine (sulfur-containing) amino acid → ultimately leading to necrosis.