Molecular Cell Biology Lecture 22: G Protein-Coupled Receptors III

Molecular Cell Biology: G Protein-Coupled Receptors III

Lecture Information

  • Instructor: Mitra Esfandiarei, Ph.D.

  • Email: mesfan@midwestern.edu

  • Date: October 15, 2025

Learning Objectives

  • Explain the production of nitric oxide (NO).

  • Understand the different types of nitric oxide synthase (NOS), their localizations, and functions.

  • Name primary receptors (and their ligands) responsible for endothelial nitric oxide synthase (eNOS) activation in endothelial cells.

  • Recognize the function of receptor tyrosine kinase in eNOS activation.

  • Explain the mechanism through which acetylcholine induces eNOS activation in endothelial cells.

  • Understand the GPCR (G Protein-Coupled Receptor) and G alpha components involved in acetylcholine-induced eNOS activation.

  • Understand how eNOS is activated following calcium release from the endoplasmic reticulum (ER).

  • Understand how NO activates soluble guanylate cyclase in smooth muscle cells.

  • Explain how guanylate cyclase activation leads to protein kinase G (PKG) activation.

  • Understand mechanisms by which active PKG induces relaxation in smooth muscle cells, focusing on calcium entry and uptake.

  • Understanding how PKG controls the function of myosin light chain phosphatase (MLCP).

  • Trace the acetylcholine-induced signaling pathway from endothelial cells to smooth muscle cells in vascular walls.

  • Understand the role of nitric oxide in erectile function.

  • Know the general function of nitroglycerin and Sildenafil (Viagra).

  • Understand the concept of eNOS uncoupling caused by oxidative stress.

  • Explain how nitric oxide regulates inflammation and thrombosis.

  • Understand the general mechanisms of activation for ion channel-coupled receptors.

  • Explain how acetylcholine induces skeletal muscle contraction.

  • Illustrate the pathway downstream of acetylcholine binding to nicotinic receptors on skeletal muscles and the resulting action potential generation down the sarcolemma.

  • Discuss how acetylcholine binding to nicotinic receptors on skeletal muscle contributes to increased cytoplasmic calcium concentration and muscle contraction.

  • Explain the role of acetylcholine esterase in terminating muscle contraction.

  • Discuss consequences of deficient or excessive acetylcholine esterase activity at the neuromuscular junction leading to flaccid or spastic paralysis.

Nitric Oxide (NO) as A Signaling Molecule

  • An inorganic gas synthesized by nitric oxide synthase (NOS).

  • There are three isoforms of NOS:

    • Endothelial NOS (eNOS)

    • Neuronal NOS (nNOS)

    • Inducible NOS (iNOS)

  • Activation of eNOS and nNOS is calcium-dependent; iNOS is calcium-independent.

  • eNOS & nNOS produce low levels of NO for a short duration (beneficial), while iNOS produces a significant amount of NO for a prolonged period in response to stressors such as tissue injury and infection.

    • Chemical Reaction:

    • ext{L-Arginine + O}2 + ext{NADPH} + ext{BH}4
      ightarrow ext{Citrulline + NO}

  • Half-life of NO: 5-10 seconds.

Nitric Oxide Synthase (NOS) Family Characteristics

  • eNOS: Constitutively expressed; Ca2+-dependent; primarily located in endothelial cells; involved in vasorelaxation.

  • nNOS: Constitutively expressed; Ca2+-dependent; significant for memory and learning, as well as cardiac function.

  • iNOS: No basal expression; Ca2+-independent; produces NO in response to immune challenges.

Primary Receptors for eNOS Activation

  • G Protein-Coupled Receptors (GPCRs):

    • Muscarinic (M3) acetylcholine receptors on endothelial cells.

    • Bradykinin receptors (B2 receptors) on endothelial cells.

    • Serotonin receptors (5-HT2 receptors) on endothelial cells.

    • Prostacyclin receptors (IP receptors) on endothelial cells.

  • Tyrosine Kinase Receptors (RTKs):

    • Vascular Endothelial Growth Factor (VEGF) receptors and insulin receptors on endothelial cells (through PI3K/Akt pathway).

    • Estrogen receptors (ERs) on endothelial cells (to be discussed later).

    • Mechanosensitive receptors on endothelial cells sensitive to changes in blood flow and vessel wall stretching.

Activation of eNOS by Acetylcholine

  • Ligand: Acetylcholine (ACh)

  • Secreted by: Nerve cells

  • Target Cells: Endothelial cells

  • Receptor: M3 muscarinic receptor (a GPCR)

  • End Effects: Smooth muscle relaxation

  • Acetylcholine is released by parasympathetic nerve endings and binds to M3 muscarinic acetylcholine receptors on endothelial cell membranes, activating the Gqα protein.

Mechanism of Acetylcholine-Induced eNOS Activation in Endothelial Cells

  • The binding of acetylcholine to M3 muscarinic receptor leads to a cascade:

    • Gqα activates phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol (DAG).

    • Role of IP3:

    • It binds to IP3 receptors on the endoplasmic reticulum (ER) membrane, leading to calcium ion release (Ca²⁺) into the cytoplasm.

  • The increase of intracellular Ca²⁺ activates eNOS via a calcium/calmodulin complex, enabling the conversion of L-arginine to L-citrulline and the production of NO in the endothelial cells.

NO's Role in Smooth Muscle Cell Relaxation

  • Once produced in endothelial cells, NO diffuses into adjacent smooth muscle cells:

    • Activation of Soluble Guanylate Cyclase (sGC):

    • NO binds to and activates sGC, which catalyzes the conversion of GTP to cyclic GMP (cGMP).

    • cGMP's Role:

    • Acts as a second messenger to activate protein kinase G (PKG).

    • PKG Effects:

    • Decreased intracellular calcium levels:

      • PKG activates calcium pumps (e.g., SERCA, plasma membrane Ca²⁺-ATPase) to sequester calcium into the sarcoplasmic reticulum or pump it out, leading to vasodilation.

    • Increased MLCP activity leading to myosin light chain dephosphorylation, resulting in muscle relaxation.

Clinical Relevance of Nitric Oxide: Angina Pectoris

  • Definition: A condition characterized by chest pain due to reduced blood flow to the heart muscle, typically from coronary artery disease.

    • Types of Angina:

    • Stable Angina: Occurs with exertion or stress and managed by rest or nitroglycerine.

    • Unstable Angina: Occurs unpredictably, more severe and long lasting, a precursor to myocardial infarction (heart attack).

Angina Treatment Using Nitroglycerin

  • Mechanism: Nitroglycerin converts to nitric oxide in smooth muscle cells, enhancing vasodilation and blood flow.

  • Administration Routes: Sublingual, intravenous, transdermal.

Clinical Applications: Erectile Dysfunction

  • Erectile Dysfunction (ED): The inability to maintain an erection; prevalent among older men and individuals with cardiovascular/metabolic disorders.

    • Role of NO in Erection:

    • Sexual stimulation leads to acetylcholine release, activating NO signaling in the penile corpus cavernosum, facilitating increased blood flow and erection.

Clinical Applications: Sildenafil (Viagra)

  • Mechanism: Inhibits phosphodiesterase-5 (PDE-5), preventing cGMP breakdown, enhancing NO-mediated smooth muscle relaxation and prolonging erection.

  • Note: Requires initial sexual stimulation for effectiveness; does not independently induce erection.

Nitric Oxide Functions in the Body

  1. Smooth Muscle Cell Contraction Regulation (anti-hypertensive effects).

  2. Regulation of intercellular adhesion in endothelial cells (anti-inflammatory effects).

  3. Inhibition of platelet aggregation (anti-thrombotic effects).

eNOS Uncoupling and Endothelial Dysfunction

  • Definition: eNOS becomes dysfunctional and produces superoxide (O₂⁻) instead of NO, leading to vascular health deterioration associated with cardiovascular diseases.

  • Required for NO production:

    • L-arginine as substrate and tetrahydrobiopterin (BH₄) as cofactor.

  • Mechanisms of eNOS uncoupling:

    • BH₄ depletion and increased oxidative stress.

    • Result: Less NO availability, increased oxidative stress, leading to a vicious cycle of endothelial dysfunction.

Ion Channel-Coupled Receptors

  • Also known as ligand-gated ion channels; they modify membrane permeability to ions upon ligand binding.

  • Function: Trigger physiological responses (e.g., depolarization/hyperpolarization).

  • Examples of ligands include acetylcholine, GABA, glutamate, and serotonin.

Nicotinic Acetylcholine Receptor Activation in Skeletal Muscle

  • AChR found in CNS and neuromuscular junctions:

    • Binding results in membrane depolarization and muscle contraction.

Excitation-Contraction Coupling in Skeletal Muscle

  • Action potentials trigger ACh release, causing depolarization, calcium release from the sarcoplasmic reticulum, and ultimately muscle contraction through interactions with actin and myosin filaments.

Termination of Muscle Contraction

  • Acetylcholinesterase breaks down ACh, ceasing the signal. Calcium is re-sequestered into the SR, allowing muscle relaxation.

  • Nerve Agents: Such as Sarin, inhibit ACh-esterase, leading to spastic paralysis. Excessive ACh-esterase results in flaccid paralysis due to insufficient ACh activity.