Second messengers and their systems

Question 1

First messengers are the extracellular hydrophilic ligands that bind onto membrane receptors. What are second messengers?

Answer 1

A second messenger is a small non-protein water soluble molecule or ion that participates in pathways initiated by GPCRs and RTKs.
In many cases, the second messenger acts by stimulating or inhibiting protein phosphorylation/dephosphorylation cascades.

Question 2

State the features of second messengers.

Answer 2

1. They can be synthesized/released and broken down again in specific reactions by enzymes or ion channels.
   e.g. Adenylyl cyclase catalyses synthesis of cAMP, while cyclic nucleotide phosphodiesterase (PDE) inactivates cAMP.
2. An appropriate temporal relationship must exist among the hormone, mediator and hormonal effect.
   Some (like Ca2+) can be stored in special organelles and quickly released when needed.
3. The hormone must induce elevated levels of the mediator. Their production and destruction can be localized, enabling the cell to limit space and time of signal activity.
4. The mediator or its analog must mimic the action of the hormone, which cannot enter the cell.
5. If drugs are available to modulate the endogenous level of the mediator, they should also mimic or inhibit, as appropriate the effects of the hormone.

Question 3

Describe the adenylyl cyclase system. cAMP here is the 2nd messenger.

Answer 3

1. Binding of a ligand (e.g., epinephrine, vasopressin) to its receptor activates the specific heterotrimeric G protein–coupled receptor (GPCR).
2. The activated receptor interacts with a heterotrimeric G protein (a GTP-binding protein made up of α, β, and γ subunits. Both the α and the γ subunits have lipid modifications that anchor these proteins to plasma membrane), leading to exchange of GDP with GTP on the α subunit (activated G protein). The α subunit is bound to GDP.
3. The activated G protein dissociates from the receptor into a GTP-bound α subunit (‘Gs’, means stimulatory α subunit) and a separate βγ subunit complex, both of which can regulate downstream effectors.
4. Here, the activated α subunit is a stimulator subunit (as opposed to an inhibitory subunit) which stimulates adenylyl cyclase enzyme that catalyzes the conversion of ATP into cAMP.
5. cAMP, in turn, binds to the regulatory subunit of its downstream effector, protein kinase A (PKA), leading to dissociation of PKA into catalytic and regulatory subunits.
6. The catalytic subunit of PKA is then free to catalyze the phosphorylation of a range of down-stream effector proteins, including glycogen phosphorylase kinase, glycogen synthase, phosphodiesterases, phosphoprotein phosphatases, ion channels, and certain nuclear transcription factors (e.g., cAMP-responsive element-binding protein [CREB]), thereby controlling downstream cellular process associated with the phosphorylated proteins.
7. cAMP also regulates some effector proteins directly, such as ion-gated channels.
8. In contrast, when a ligand binds to a receptor that interacts with a G protein composed of an α subunit of the αi class, adenylyl cyclase is inhibited, thereby reducing cAMP levels and consequently reducing PKA levels.

Question 4

hormones that use cAMP

Answer 4

⚚ Epinephrine and norepinephrine
⚚ Glucagon
⚚ Luteinizing hormone
⚚ follicle stimulating hormone,
⚚ thyroid-stimulating hormone
⚚ Calcitonin
⚚ parathyroid hormone
⚚ antidiuretic hormone

Question 5

Describe the role of cAMP as a transcription factor.

Answer 5

In addition to its importance in activating PKA, which phosphorylates specific serine and threonine residues on proteins, cAMP stimulates the transcription of many genes, including those that code for hormones, including somatostatin, glucagon, and vasoactive intestinal polypeptide. Many genes activated by cAMP have a cAMP response element (CRE) in their DNA. Increases in cAMP stimulate PKA, which translocates to the nucleus, where it phosphorylates cyclic AMP-responsive element-binding protein (CREB) and thereby increases its affinity for CREB-binding protein (CBP). The CREB-CBP complex activates transcription. The response is terminated when PKA phosphorylates a phosphatase that dephosphorylates CREB.

Question 6

Explain how generated cAMP is removed from the cells.

Answer 6

Cyclic AMP is formed from ATP by the action of the enzyme adenylyl cyclase and converted to physiologically inactive 5’AMP by the action of the enzyme phosphodiesterase. Some of the phosphodiesterase isoforms that break down cAMP are inhibited by methylxanthines such as caffeine and theophylline and drugs such as Sildenafil. Consequently, these compounds can augment hormonal and transmitter effects mediated via cAMP.

Question 7

What are some of the roles of protein kinase A?

Answer 7

Protein kinase A (a serine threonine kinase) is an important enzyme in cell metabolism by phosphorylating specific enzymes in metabolic pathway, also regulates specific gene expression, cellular secretion and membrane permeability.

Question 8

cGMP serves as the second messenger for?

Answer 8

1. Atrial natriuretic peptide (ANP).
2. Nitric oxide (NO).
3. Response of the rods of the retina to light.

Question 9

Guanylyl cyclases are a family of enzymes that catalyze the formation of cGMP. They exist in two forms. Describe these forms. Which form receives which molecule between ANP and NO?

Answer 9

1st form; has an extracellular amino terminal domain that is a receptor, a single transmembrane domain, and a cytoplasmic portion with guanylyl cyclase catalytic activity. ANP binds to the extracellular domain of the plasma membrane receptor guanylyl cyclase and induces a conformational change in the receptor that causes receptor dimerization and activation of guanylyl cyclase, which metabolizes GTP to cGMP. cGMP activates cGMP-dependent protein kinase (PKG), which phosphorylates proteins on specific serine and threonine residues. In the kidney, ANP inhibits sodium and water reabsorption by the collecting duct.

2nd form; soluble, contains heme, and is not bound to the membrane. NO activates a soluble receptor guanylyl cyclase that converts GTP to cGMP, which relaxes smooth muscle. Because nitroglycerin increases NO production, which increases cGMP and thereby relaxes smooth muscle in coronary arteries, it has long been used to treat angina pectoris (i.e., chest pain caused by inadequate blood flow to heart muscle).

Question 10

Just like cAMP, cGMP is inactivated by phosphodiesterase enzyme. What is the significance of inhibitors of this enzyme?

Answer 10

Drugs that inhibit cGMP-specific phosphodiesterase type 5, such as sildenafil (Viagra), Cialis (tadalafil), and Levitra (vardenafil), prolong the vasodilatory effects of NO and are used to treat patients with erectile dysfunction and pulmonary arterial hypertension.

Question 11

The calcium ion channels in photoreceptors are cGMP-gated i.e. cGMP binds to them and keeps them open. In this case when cGMP is bound onto the calcium, explain what happens when it is inactivated by PDE.

Answer 11

Degradation of cGMP causes calcium channels to close, which leads to the hyperpolarization of the photoreceptor’s plasma membrane and ultimately to visual information being sent to the brain.

Question 12

What is the difference between PKA and PKG during activation of these enzymes?

Answer 12

Unlike with the activation of PKA where the catalytic and regulatory units disassociate, the PKG is activated but the catalytic and regulatory units do not disassociate.

Question 13

The best-characterized role of cGMP is in the vertebrate eye, where it serves as the second messenger responsible for converting the visual signals received as light to nerve impulses. Describe the mechanism of action.

Answer 13

🩸 The photoreceptor in rod cells of the retina is a G protein-coupled receptor called rhodopsin.
🩸 Rhodopsin is activated as a result of the absorption of light by retinal, which then isomerizes inducing a conformational change in the rhodopsin protein.
🩸 Rhodopsin then activates the G protein transducin, and the α subunit of transducin stimulates the activity of cGMP phosphodiesterase, leading to a decrease in the intracellular level of cGMP.
🩸 This change in cGMP level in retinal rod cells is translated to a nerve impulse by a direct effect of cGMP on Ca++ ion channels in the plasma membrane.

Question 14

Describe the role of inositol triphosphate and diacylglycerol as second messengers.

Answer 14

✪ When ligands like vasopressin (ADH), thyroid-stimulating hormone (TSH), angiotensin, and neurotransmitters like GABA bind onto GPCRs, it activates the αq subunit of the G protein which in turn activates phospholipase C (PLC) on the inner surface of the membrane.
✪ Note however that PLC has at least eight isoforms; PLCβ is activated by heterotrimeric G proteins, while PLCγ forms are activated through tyrosine kinase receptors.
✪ Activated PLCs catalyze the hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to form inositol triphosphate (IP3) and diacylglycerol (DAG).
✪ The IP3 diffuses to the endoplasmic reticulum, where it triggers the release of Ca2+ into the cytoplasm by binding the IP3 receptor, a ligand-gated Ca2+ channel.
✪ DAG is also a second messenger; it stays in the cell membrane, where it activates one of several isoforms of protein kinase C (PKC).
✪ Phosphorylation by PKC is important inregulating a variety of cellular events such as cell proliferation and the regulation of gene expression.

Question 15

Which hormones use Ca2+/IP3 system?

Answer 15

✪ epinephrine and norepinephrine
✪ angiotensin II
✪ antidiuretic hormone aka. vasopressin
✪ gonadotropin-releasing hormone
✪ thyroid-releasing hormone.

Question 16

Criteria for Ca++ to be considered a second messenger.

Answer 16

1. Intracellular Signal Amplification:
   Amplifying External Signals: Ca²⁺ is considered a second messenger when it amplifies the signal initiated by the binding of an extracellular ligand (first messenger) to a cell surface receptor. This leads to a significant intracellular response from a small extracellular signal.
2. Downstream Effector Activation:
   Triggering Cellular Responses: As a second messenger, Ca²⁺ directly or indirectly activates various intracellular proteins, enzymes, or signaling cascades that bring about a specific cellular response. For example, Ca²⁺ can activate protein kinases, phosphatases, and other effector molecules that mediate processes like muscle contraction, secretion, or gene expression.
3. Concentration Fluctuation:
   Transient Changes: Ca²⁺ functions as a second messenger due to its ability to transiently increase in concentration within specific cellular compartments. These fluctuations can be localized or propagate as waves or oscillations, enabling precise control over cellular processes.
4. Source of Release:
   Release from Stores or Entry from Extracellular Space: Ca²⁺ acts as a second messenger when it is released from intracellular stores (like the endoplasmic or sarcoplasmic reticulum) or enters the cell through plasma membrane channels in response to a signal from a first messenger.

Question 17

Describe the roles of calcium as a second messenger.

Answer 17

✪ Activation of Signaling Pathways: Ca²⁺ acts as a key signal transducer in various cellular pathways, translating extracellular signals into intracellular actions.
✪ Triggering of Downstream Responses: The influx or release of Ca²⁺ initiates a cascade of downstream signaling events within the cell.
✪ Skeletal and Cardiac Muscle: Ca²⁺ binding to troponin C facilitates the interaction between actin and myosin, leading to muscle contraction.
✪ Smooth Muscle: Ca²⁺ interacts with calmodulin to activate myosin light chain kinase, which phosphorylates myosin and induces contraction.
✪ Synaptic Transmission: Ca²⁺ entry into presynaptic neurons through voltage-gated channels triggers vesicle fusion and the release of neurotransmitters into the synaptic cleft.
✪Regulation of Transcription Factors: Ca²⁺ influences the activity of various transcription factors (e.g., NFAT) by promoting their translocation to the nucleus and modulating gene expression.
✪ Cell Cycle Regulation: Ca²⁺ modulates the activity of enzymes and proteins that control the cell cycle, influencing cell division.
✪ Induction of Apoptosis: Elevated intracellular Ca²⁺ levels can activate apoptotic pathways, leading to programmed cell death.
✪ Kinases and Phosphatases: Ca²⁺ activates various enzymes, including protein kinases (e.g., CaMK, PKC) and phosphatases, which play roles in diverse cellular functions.
✪ Exocytosis and Endocytosis: Ca²⁺ is crucial for the regulation of vesicle trafficking processes like exocytosis and endocytosis, impacting membrane dynamics and cellular communication.
✪ Cytoskeletal Reorganization: Ca²⁺ affects the dynamics of the cytoskeleton, influencing cell motility and migration by modulating actin and microtubule structures.
✪ Mitochondrial Function: Ca²⁺ plays a role in regulating mitochondrial metabolism and ATP production by influencing mitochondrial enzymes.
✪ Role in Embryogenesis: Ca²⁺ signaling is essential for the regulation of developmental processes and cellular differentiation during embryogenesis.
✪ Vision: In photoreceptor cells, Ca²⁺ is involved in the phototransduction cascade, affecting visual processing.
✪ Taste and Smell: Ca²⁺ signaling is crucial for the transduction of chemical signals in taste and olfactory cells.
✪ T Cell Activation: Ca²⁺ is vital for the activation of T cells and the regulation of immune responses, including cytokine production.
✪ Gap Junction Regulation: Ca²⁺ influences the permeability of gap junctions, affecting intercellular communication and the propagation of calcium waves.
✪ Endocrine and Exocrine Functions: Ca²⁺ plays a pivotal role in the secretion of hormones and other substances by endocrine and exocrine glands.

Question 18

Describe mechanisms that operate to terminate calcium action.

Answer 18

The IP3 generated by the activation of PLC can be dephosphorylated by cellular phosphatases leading to inactivation of this second messenger. Because the movement of Ca2+ outside of the cytosol (ie, across the plasma membrane or the membrane of the internal store) requires that it move up its electrochemical gradient, it requires energy. Ca2+ movement out of the cell is facilitated by the plasma membrane Ca2+ ATPase. Alternatively, it can be transported by an antiport (Na+ - Ca2+ antiport) that exchanges three Na+ for each Ca2+ driven by the energy stored in the Na+ electrochemical gradient, by Ca-H ATPase and by Ca-Mg ATPases. Ca2+ movement into the internal stores is through the action of the sarcoplasmic or endoplasmic reticulum Ca2+ ATPase (SERCA pump).

Question 19

Describe the most common calcium-binding proteins.

Answer 19

Ca2+-binding proteins include: troponin, calmodulin, and calbindin. Troponin is the Ca2+- binding protein involved in contraction of skeletal muscle. Calmodulin contains 148 amino acid residues and has four Ca2+-binding domains. It is unique in that amino acid residue 115 is trimethylated, and it is extensively conserved, being found in plants as well as animals. When calmodulin binds Ca2+, it is capable of activating five different calmodulin-dependent kinases, among other proteins. One of the kinases is myosin light-chain kinase, which phosphorylates myosin. This brings about contraction in smooth muscle. Ca2+/calmodulin kinase I (CaMKI) and Ca2+/calmodulin kinase II (CaMKII) are concerned with synaptic function, and Ca2+/calmodulin kinase III (CaMKIII) is concerned with protein synthesis. Another calmodulin-activated protein is calcineurin, a phosphatase that inactivates Ca2+ channels by dephosphorylating them. Calcineurin also plays a prominent role in activating T cells.

Question 20

Describe NO as a second messenger in vascular smooth muscle relaxation.

Answer 20

☯️ Endothelial cells lining blood vessels are stimulated by factors such as acetylcholine (ACh), bradykinin, or shear stress from blood flow.
☯️ These stimuli lead to the activation of endothelial nitric oxide synthase (eNOS). Increased intracellular Ca²⁺ levels in endothelial cells activate eNOS. eNOS requires cofactors such as tetrahydrobiopterin (BH4) and the presence of L-arginine.
☯️ eNOS catalyzes the conversion of L-arginine to L-citrulline and releases nitric oxide (NO) as a by-product.
☯️ NO rapidly diffuses across endothelial cells and the adjacent smooth muscle cell membranes due to its gaseous nature. NO diffuses into the underlying vascular smooth muscle cells.
☯️ NO binds to the heme moiety of soluble guanylate cyclase (sGC) within smooth muscle cells. This activates sGC, increasing its catalytic activity.
☯️ Activated sGC converts guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cGMP).
☯️ The intracellular concentration of cGMP increases within smooth muscle cells.
☯️ cGMP activates protein kinase G (PKG) by binding to it.
☯️ PKG phosphorylates and inhibits voltage-gated Ca²⁺ channels, reducing Ca²⁺ influx.
☯️ PKG enhances the activity of the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA), increasing Ca²⁺ sequestration into the sarcoplasmic reticulum.
☯️ PKG phosphorylates myosin light chain kinase (MLCK), decreasing its activity and reducing phosphorylation of myosin light chains, leading to muscle relaxation. [note that phosphorylation of MCLK by calcium-calmodulin complex and by PKA produces different effects; the former results in activation and the latter results in inhibition]
☯️ The overall decrease in intracellular Ca²⁺ levels and the reduced phosphorylation of myosin light chains result in smooth muscle relaxation.
☯️ Relaxation of smooth muscle cells leads to the dilation of blood vessels (vasodilation), increasing blood flow and reducing blood pressure.

Question 21

Describe intracellular receptors and how they function in the cells.

Answer 21

Intracellular receptors bind ligands that can diffuse readily across the cell membrane; that is, the ligands are lipophilic. This class includes steroid hormones (e.g., estrogen, progesterone, testosterone, aldosterone, corticosteroid), vitamin D, retinoic acid (vitamin A), and thyroid hormone. When these ligands diffuse into a cell and bind to their intracellular receptors, the complex becomes an activated transcription factor that enhances or represses the expression of various target genes by binding to specific DNA sequences called hormone responsive elements. These DNA sequences may be located in the immediate vicinity of the gene’s promoters or at a considerable distance from them. The various nuclear receptors display specific cell and tissue distributions so that considerable diversity may exist in the response of one cell and that of another in regard to what gene is enhanced or repressed. Typical cellular responses include inflammatory responses, proliferation, and differentiation.

Question 22

Explain how nuclear receptor-linked signal transduction pathways are regulated.

Answer 22

The family of nuclear receptors includes more than 30 genes and has been divided into two subfamilies based on structure and mechanism of action: (1) steroid hormone receptors and (2) receptors that bind retinoic acid, thyroid hormones (iodothyronines), and vitamin D. When ligands bind to these receptors, the ligand-receptor complex activates transcription factors that bind to DNA and regulate the expression of genes. The location of nuclear receptors varies. Glucocorticoid and mineralocorticoid receptors are located in the cytoplasm, where they interact with chaperones (i.e., heat shock proteins). Binding of hormone to these receptors results in a conformational change that causes chaperones to dissociate from the receptor, thereby uncovering a nuclear localization motif that facilitates translocation of the hormone-bound receptor complex to the nucleus. Estrogen and progesterone receptors are located primarily in the nucleus, and thyroid hormone and retinoic acid receptors are located in the nucleus bound to DNA. When activated by hormone binding, nuclear receptors bind to specific DNA sequences in the regulatory regions of responsive genes called hormone response elements. Ligand-receptor binding to DNA causes a conformational change in DNA that initiates transcription. Nuclear receptors also regulate gene expression by acting as transcriptional repressors. For example, glucocorticoids suppress the transcription activator protein-1 (AP-1) and nuclear factor κB (NF- κB), which stimulate the expression of genes that cause inflammation. By this mechanism glucocorticoids reduce inflammation.

Question 23

Illustrate, with a well-labelled diagram, the mechanism of action of steroid hormones.

Answer 23

[Diagram]