Lecture 11 - 15 Flashcards
What are the main types of cartilage?
- hyaline
- fibro
- elastic
What is the function of Haline?
- forms articular joints = precursor for bone
What is the only cell that is found in the matrix of cartilage?
chondrocyte
What are the two key components found in articular cartilage and what is their function?
- collagen type II
- aggrecan
important for strength and support
What is the role of PG in cartilage?
PG are highly charged = attract water and form a hydrated gel
- provides resistance to compression
- results in swelling pressure (turgor)
- provides strength and support
Why are PG highly charged?
due to GAG content
What are the characteristics of chondrocytes?
- only cells in the matrix of cartilage
- large and mature
- in groups of 2-8
- rich in RER and golgi
- secrete high amounts of type II collagen and aggrecan
What are the characteristics of the ECM where chondrocytes are found?
- avascular (hypoxic)
- alymphatic
- aneuronal
What are the key transcriptional factors that MSC have to differentiate into chondrocytes?
- expression of Sox-9 = leads to Col2A expression = makes cartilage
- TGF-b
- fibroblasts growth factor (FGF)
- insulin-like growth factor (IGF-1)
- parathyroid hormone-related protein (PTHrP)
What is the role of Sox-9?
induce Sox-9 to form a chondroycte and start making cartilage
How does spatial patterning of chondrocytes in bone formation occur?
orchestrated by morphogens via inductive signaling
What does inductive signalling require?
- requires morphogens passing between cells through the developing ECM
- involves members of the Hedgehog family of proteins (SHH), (DHH), (IHH), and (PTHrP).
How do IHH and PTHrP interact?
via positive feedback loops that maintain spatial chondrocyte proliferation
What makes cartilage more rigid?
hydroxyapatite with calcium makes the cartilage more rigid = rigidity expands = bone formation
What is the role of chondrocytes that keep receiving the PTHrP signal?
move to the top of the bone and keep receiving their signalling and forming cartilage only at the top of the bone
What are the key ECM breakdown events in osteoarthritis?
- Aggrecan = ADAMTS5 involved in cleaving aggrecan can be knocked out to see if arthritis develops
- Collagen = can be knocked out to see if arthritis develops
What is MMP-13?
a collagenase
What are the genetics of OA?
*GDF5 growth factor member of TGF-b family important in ECM homeostasis
*RUNX2 (6p 21.1) master transcription factor responsible for driving endochondrial ossification including MMP-13 expression
*PTHLH (12p 11.22) encodes PTHrP chondrocyte growth factor driven by IHH secretion
*SMAD3 intracellular signaling protein involved in TGF-b production
What is Rhemuatoid arthritis?
Characterised by inflammation of the synovium
progressive loss of ECM and chondrogenic phenotype in articular cartilage due to immune cell-mediated damage (genetic linkage to MHC; HLA-DR4 in particular)
What receptors activate signal transduction pathways resulting indirectly in the opening of ion channels?
- GABA B
- Muscarinic receptors
What receptors does acetylcholine bind to?
- muscarinic receptor
- nicotinic receptor
What molecules are part of G-protein coupled receptor signalling?
- adrenaline
- acetylcholine
- muscarine
- GABA B
What is the order of proteins involved in the signal transduction of a G-protein coupled receptor?
- Adrenaline
- G-protein
- Adenylyl cyclase
- cAMP
- PKA
What are the 3 key stages of signal transduction?
- an extracellular signal molecule activates a membrane receptor
- alters intracellular molecules to be transduced via a certain pathway
- activate a cellular response
What is the difference between the first messenger and the second messenger in signal transduction?
first messengers = extracellular signal molecules
secondary messengers = intracellular signal molecules
How do signal transductions occur?
membrane proteins act as transducers converting the message of extracellular signals into intracellular messenger molecules that trigger a response.
What is the difference between extracellular receptors and intracellular receptors?
Extracellular = FAST RESPONSE, hydrophilic signaling molecules activate intracellular signal transduction
Intracellular = SLOW RESPONSE, hydrophobic signaling molecules act as transcription factor in nucleus to regulate gene transcription
What are the 4 main classes of receptors?
- ligand-gated ion channel
- G-protein coupled receptors
- enzyme-linked receptors
- nuclear receptors
What are examples of the cell surface receptors and what are the time scales?
- ligand-gated = nicotinic & ACh receptor = milliseconds
- G-protein = Muscarininc & ACh receptors = seconds
- Kinase-linked = cytokine receptors = hours
What is the difference between Ionotropic and metabotropic receptors?
Ionotropic:
- form an ion channel pore
- examples = Nicotinic ACh receptors & Gaba A
Metabotropic:
- indirectly linked with ion channels on the plasma membrane through signal transduction pathways
- examples = Muscarinic & GABA B
What are the characteristics of G-protein coupled receptors?
- G-protein receptors have 7 transmembrane domains
- consist of 3 polypeptide chains (alpha,beta and gamma)
- alpha subunit contains a guanine nucleotide binding site that binds GTP or GDP
- play role in regulation of cell function
What is the G-protein cycle?
- Adrenaline unbinds from receptor and alpha subunit can hydrolyze GTP into GDP
- Alpha subunit gains high affinity for beta and gamma and binds to them to restart a new cycle
- RGS proteins activate GTPase activity of alpha subunit = RGS proteins can make a G-protein more inactive by stimulation of the hydrolysis of GTP to GDP
What are the different types of G-proteins and what do each of them stimulate?
alpha s = stimulates Adenyl Cyclase
Alpha i = inhibits adenyl cyclase
B- adrenoreceptors, vasopressin receptors, A2A/B adenosine receptors couple to Gs
A2 adrenoreceptors, m and d opioid receptors, A1/3 adenosine receptors couple to Gi
What does the pertussis toxin act on?
Alpha i subunit of the G-protein = prevents inhibitory control over adenyl cyclase/PKA complex
What is protein kinase C (PKC) directly activated by?
DAG secondary messenger
What two receptors are coupled with a Gi protein?
Alpha2 adrenergic receptor and M2 muscarinic receptor
What receptors are associated with JAK2 kinases?
Tyrosine kinase associated receptor
What receptors involve dephosphorylation of target proteins rather than phosphorylation?
Receptor tyrosine phosphatase
How does the cholera toxin affect G-proteins?
- Active alpha subunit has GTP bound
- Hydrolysis of GTP leads to inactivation of alpha subunit
- Cholera toxin acts on alpha subunit and causes ADP-ribosylation
- Prevents hydrolysis of GTP = persistent activation of alpha subunit
- cAMP can activate chloride channels present in apical membrane
What is the role of cAMP in cholera toxin?
cAMP activate chloride channel and cause chloride ions and Na+ ions to secrete into lumen = attracts water = patient looses water
What is the difference between IP3 and DAG?
IP3:
- hydrophilic
- moves into cytosole = stimulates the release of calcium in the ER
DAG:
- hydrophobic
- remains in the membrane = activate protein kinase C
What is the role of a CAM?
Bind 4 ion calcium molecules and activates PDE = enzymes that degrades cAMP
What is the role of Ca2+-CaM complex?
Activates CaM kinases = involved in smooth-muscle contraction
What is the role of alpha-1 adrenoreceptor?
Gq couples protein receptor which mediates vascular smooth muscle contraction by increasing intracellular free Ca2+
What are the effects of DAG in relation to calcium ions?
DAG increases the activity of Ca2+ dependent kinase & evokes cellular response by phosphorylation other proteins.
What are the roles of alpha1 and beta2 adrenoreceptors in relation to blood pressure control?
Alpha1:
- cause vascular smooth muscle tone contraction (vasoconstriction)
- via Gq-PLC-IP3-CaMK
- makes blood pressure increase
Beta2:
- causes relaxation of vascular mouth muscle (vasodilation)
- via Gs-cAMP-PKA
- makes blood pressure decrease
What type of G-proteins are muscarinic receptors?
GQ and Gi couples receptors
1-3-5 Gq coupled stimulatory = work by activating phospholipids enzyme in using IP3 and DAG in increasing the level of calcium
2-4 Gi coupled inhibitory = linked to a Gi proteins and work to block adenyl cyclase
What are the mechanisms of signaling for the receptor tyrosine kinases (RTKs)?
- Binding of 2 molecules of insulin cause the receptor to dimerise
- Receptor use their cytoplasmic tyrosine kinase activity to phosphorylate each-other at multiple tyrosine residues creating “phosphotryosine motifs”
- These motifs recruit intracellular signaling molecules leading to the response
- Example of response: insulin mediated glucose uptake and storage in liver and muscles
How do RTKs lead to MAP kinase signaling pathway?
- Activated RTK cause activation of adaptor protein which activates Ras-GEF = causes exchange of GDP to GTP in a target protein
- Ras-GEF causes exchange of GDP to GTP so that RAS can bind GTP
- RAS_GTP activates MACC kinase kinase = phosphorylate MAC kinase = phosphorylate target protein
What are the Tyrosine kinase-associated mechanisms of signaling?
- Binding of first messenger to the receptor = induce a conformational change that causes dimerization of the receptor
- Dimerization causes activation of the associated tyr kinases
- Kinases phosphorylate tyrosine residues = phosphotyrosine motifs
- Motifs recruit intracellular signaling molecules leading to the response
What is the difference between G-protein coupled receptors and Receptor tyrosine Kinases?
GPCR:
- a monomer
- 7 transmembrane domains
- cause production of secondary messenger that can be cAMP or IP3 and DAG
RTK:
- a dimer
- has 2 subunits and each subunit has 1 transmembrane domain
- intrinsic enzymatic activity in their tail
- kinase pathways
What type of G-proteins are the secondary messengers associated with?
cAMP = Gs protein
IP3 & DAG = Gq protein
What is glucose homeostasis?
- glucose absorbed from GI tract
- enters circulation
- used to fuel metabolism in many tissue
- body stores glucose as glycogen in muscles and liver
What happens to glucose after a meal?
- increased glucose absorption = increased glucose in circulation
- stimulates metabolism
- increase O2 demand
What happens to glucose in-between meals?
- glucose absorption is minimal
- lower glucose in circulation
- limits metabolism
- reduced O2 demand
What happens when glucose is low?
- glucose is released from glycogen
- glycogen in the liver is converted into glucose to recirculate in the body
What does insulin do?
Promotes glucose storage as glycogen = reduce the level of glucose in circulation
What does glucagon do?
Promotes glucose release from stored glycogen = increases the level of glucose in circulation
What are the 3 main cell type in each islet and what are their roles?
- Alpha cells = produce glucagon
- Beta cells = produce insulin and amylin
- Gamma cells = produce somatostatin
What releases insulin and glucagon?
Released by the pancreas in the Islets of Langerhans
When is insulin released and by what?
Insulin is released after meals by the beta cells of the pancreas
What transport protein is a target of PKB activity?
GLUT-4
What does PKB mediate?
PKB mediates the effects of insulin on the liver
Which of the following is NOT an effect of insulin?
A. Promoting glucose uptake in liver
B. Promoting storage of glycogen liver
C. Promoting storage of fats
D. Promoting use of fatty acids instead of glucose as metabolic substrate
E. Promoting protein synthesis
D
Promoting use of fatty acids instead of glucose as metabolic substrate
What is the glucagon receptor?
A Gs protein coupled receptor
Which of the following tissues/organs do NOT have a glucagon receptor?
A. Liver
B. Skeletal muscle
C. Adipose tissue
D. Pancreas
E. Kidney
B. Skeletal muscle
Which of the following situations can lead to release of both insulin and glucagon at the same time?
A. High level of glucose
B. Low level of glucose
C. High level of amino acids
D. Low levels of amino acids
E. All of them
C. High levels of amino acids
What synthesizes insulin?
Synthesized within pancreatic islet beta cells as a polypeptide processed within the golgi
What is the structure of insulin?
- polypeptide hormone
- 2 polypeptide chains held together by disulfide bridges
- alpha chain = 30 amino acids
- beta chain = 21 amino acids
What do beta cells express?
Express a type 2 glucose transport system (GLUT2)
- this system is hormone-insensitive = always active
Express ATP-sensitive K+ channels
Express voltage gated Ca2+ channels
What happens to glucose in beta cells?
Glucose is phosphorylate to glucose 6-P by glucose Ashe and metabolized by glycolysis and mitochondrial oxidation to generate ATP/ADP
What happens to beta cells exposed to low glucose?
- Normal glucose
- Normal internal ATP
- K+ channels open
- Vm is hyperpolarised
- Ca2+ channels are closed
- beta cell does not secrete insulin
What happens to beta cells exposed to high glucose?
- High glucose
- High internal ATP
- Vm is depolarized
- Ca2+ channels open
- beta cell secrete insulin
How does insulin receptor signaling occur?
- cellular effects of insulin are due. To activation of PI3K
- IRS-1 also activates MAPK cascade = stimulating cell growth and survival
How is insulin uptake promoted?
- Insulin induces activation of PI3K
- PI3K activates PKB
- PKB induced translocation of GLUT4 to plasma membrane
- GLUT4 allows glucose entry to the liver
What happens if glycogen reserves are full?
Glucose is metabolism to fatty acids
