What level of ATP depletion is needed for critical damage to cell?
<5-10% of normal levels
Name the mechanisms of cell injury:
1) ATP depletion
2) Mitochondrial damage
3) Influx of intracellular Ca and loss of Ca homeostasis
4) Accumulation of oxygen-derived free radicals
5) Defects in membrane permeability
Name the effects of ATP depletion:
1) Failure of E dependent NaK pump - accumulation of Na, influx of water, swelling of ER
2) Failure of Ca pump - influx of Ca - mitochondrial damage
3) Shift to anaerobic glycolysis - deplete glycogen stores, produce lactic acid, lower pH, decreased activity of enzymes and clumping of chromatin
5) Decreased protein synthesis d/t detachment of ribosomes, causes damage to mito and lysosome membranes and leads to cell necrosis
6) Decreased O2, glucose, stress result in abnormal protein folding --- unfolded protein response
What causes mitochondrial damage?
1) Increased Ca
2) Oxidative stress
3) Lipid peroxidation
What are the consequences of mitochondrial damage?
1) Formation of high conductive channel - mito permeability transition, cannot maintain protein gradient
2) Leakage of cytochrome c --- induces apoptosis
What are the consequences of increased cytosolic Ca?
1) Activation of: ATPases, proteases, phospholipases and endonucleases
2) Increased mitochondrial permeability - mito permeability transition - release of cyto c - apoptosis
Name 5 ways free radicals are produced.
1) Radiant energy
2) Metabolic byproducts of exogenous toxins/drugs.
3) Normal respiration - molecular water reduced by addition of 4 e to water and toxic intermediates produced (Superoxide anion, hydrogen peroxide, hydroxyl ions)
4) Transition metals donate or accept free e during intracellular rxns and create free radicals - Fenton rxn
5) Nitric oxide - produced by endothelial cells, macs, neurons - can act as free radical
During normal respiration molecular water is reduced by the addition of 4 e to water - name the 3 toxic intermediates produced.
1) Superoxide anion (O2-)
2) Hydrogen peroxide (H2O2)
3) Hydroxyl ion (OH-)
What three free radical reactions are relevant to cell injury?
1) Lipid peroxidation
2) Oxidative modifiation of proteins
3) DNA lesions
What are the protective mechanisms of the cell?
1) Antioxidants: vit E, vit A, ascorbic acid, glutathione
2) Binding of transition metals to transport proteinds
List 5 mechanisms that contribute to membrane damage.
1) Mito dysfunction - d/t decreased phospholipid synthesis, ATP depletion (increased uptake of Ca into mito activates phospholipases)
2) Loss of membrane phospholipids - increased degredation d/t activation of phospholipases d/t incr cytosolic Ca and decreased ATP
3) Cytoskeleton abnormalities - incr. Ca activates proteases - damage to fillaments which anchor membrane to cytoskeleton - when cell swells - stretches and ruptures
4) Reactive oxygen species - lipid peroxidation
5) Lipid breakdown products - detergent effect
What two phenomenoa characterize irreversibility of cell injury?
1) Inability to reverse mito dysfunction.
2) Profound membrane disturbances -- massive leak of intracellular materials and massive influx of Ca
Name the ultrastructural changes in reversible cell injury:
5) Fat necrosis - pancreatic lipases liquefy fat, split TG, FA combine with Ca - fat saponification -
Name two ways chemicals can damage cells.
1) Directly combine with molecular component of cell (ex. cyanide blocks oxidative phosphorylation)
2) Chemical converted to reactive metabolite - reactive free radicals that can result in lipid peroxidation (eg. CCl4 and acetominophen) - ex. with tylenol - detox in liver via sulfation and glucuronidation - small amounts converted to toxic metabolite by p450 - this toxic metabolite can be reduced by GSH (glutathione) but if high dose GSH depleted
and with CCl4 --- CCl3--- lipid radicals ---- leads to membrane damage to ER (rib detach, decr apoprot syn, fatty liver) AND plasma membrane (incr perm to Na, H2O and Ca, cell swelling, influx of Ca, inactivation of mito enzymes, denaturation of proteins).
List physiologic causes of apoptosis.
1) Programmed cell death during embryogenesis
2) Hormone dependent involution
3) Cell deletion in proliferating cells (intestine)
4) Cells that have served purpose - ex. neuts in acute inflammation
4) Cell death in tumor regression and active growth
Name the morphologic features of apoptosis.
1) Cell shrinkage
2) Chromatin condensation (most characteristic)
3) Cytoplasmic blebs and apoptotic bodies
4) Phagocytosis of apoptotic bodies by macs
Describe the three biochemical steps/features of apoptosis.
1) Protein cleavage - via caspases (proenzymes that need to be activated to induce apoptosis), when activated caspases cleave lamins breaking up nuclear scaffold and cytoskeleton AND activate DNAses
2) DNA breakdown
3) Phagocytic recognition - express phosphatidylserine on outer layers - binds Annexin V --- leads to early recognition by macs without release of pro-inflam components
What are the two phases of apoptosis?
1) Initiation (activation of caspases) - extrinsic (death receptor) and intrinsic (mito) pathways
2) Execution (caspases cause cell death)
Describe the sequence of events of the extrinsic pathway.
1) Death receptors on cell surface (TNF receptor family - ex. TNFR1 and Fas)
2) FasL binds 3 or more Fas molecules - come together and cytoplasmic domains form binding site for FADD - Fas assoc death domain)
3) FADD bound to death domain activates pro-caspase 8
4) Caspase 8 cleaves and activates other pro-caspases
NOTE: caspase 8 can be inactivated by FLIP - protects against Fas-mediated apoptosis
Describe the sequence of events in the intrinsic pathway.
1) GF and survival signals stim production of anti-apopotic mol - Bcl-2, Bcl-x
2) When cells stressed or deprived of signals - Bcl-2 and Bcl-x lost from mito memb and replaced by pro-apoptotic molecules (Bax, Bak, Bim)
3) Decr Bcl-2 and Bcl-x cause incr. mito permeability and release of pro-apoptotic proteins: cyto c, AIF
4) Cyto c binds to apaf-1 and activates caspase 9
5) AIF binds and neutrolizes (IAP - inhib of apop) which block caspases activation
NOTE: Bcl-2 and Bcl-x inhibit apaf-1 so when they decrease get more activation of caspase 9
Describe the sequence of events of the execution phase of apoptosis.
1) Caspase 3 and 6 are activated
2) Cleave cytoskeletal and nuclear matrix proteins - breakdown cytoskeleton and nucleus
3) Nucleus - they cleave proteins needed for transcription, DNA replication and repair
4) Caspase 3 activates DNAse by cleaving inhibitor of this enzyme - cleaves DNA
How are apoptotic cells removed?
1) Early stages cells release soluble factors which recruit macs
2) Apoptotic cells have surface markers
3) Viable cells produce CD31 (PECAM-1) which prevents phagocytosis
4) Macs can produce substances that bind and opsonize apop cells and not viable cells
Name pro-apoptotic molecules:
Bax
Bak
Bim
Bid
Cyto c
AIF
apaf-1
p53
Name anti-apoptotic molecules
FLIP
Bcl-2
Bcl-x
IAP
Name 3 signals that induce apoptosis
1) Lack of GFs - triggers intrinsic path - d/t excess pro-apoptotic vs anti-apoptotic molecules
ex. hormone sens cells, lymphs not stim by cytokines/Ag, neurons deprived of nerve GF
2) Engagement of death receptors
a. FasL prod by cells of immune system - elim self-reactive lymphs
b. similar to Fas mediated, TNF binds to TNFR1, TRADD binds FADD and leads to caspase activation BUT.... TNF also is anti-apoptotic since it activates NF-kb ---- survival
c. CTLs - when recognize foreign Ag presented on cells - release perforin - perforate cell and allow Granzyme B to enter cell - this cleaves proteins and activates caspases - CTLs bypass the initiating phase and go directly to effector phase of apop AND CTLs can express FasL and induce apop via Fas
3) Injurious agents
DNA damage (ex. radiation, chemo) -- results in accum of p53 --- arrests cell at G1 to repair, but if no repair then p53 increases transcription of Bax, Bak and Apaf-1 (if p53 mutated can be tumor causing)
What is lipofuscin?
Undigested material derived from cellular lipid peroxidation
Name 5 intracellular accumulations.
1) Lipids
2) Proteins
3) Hyaline change
4) Glycogen
5) Pigments
Describe the events of normal lipid metabolism
1) FFA from adipose or food transported to hep
2) Esterified to TG OR converted to chol OR phospholipid OR oxidized to KB
3) Associate with apolipoproteins
4) Exported from liver
DEFECTS IN ANY OF THESE STEPS LEAD TO ACCUMULATION IN HEPATOCYTES --- FATTY LIVER
Describe 3 situations resulting in cholesterol accumulation in cells.
1) Xanthomas (birds, cats on high fat diets) - chol in macs in subepith CT of skin and tendons
2) Inflammation and necrosis - macs full of lipid d/t phagocytosis of chol from cell membranes
3) Niemann-pick dz - type C - lysosomal storage dz, lack enzyme needed for chol traficking (cats)
Describe 3 situations resulting in accumulation of proteins in cells.
1) Prox renal tubular cells - small amounts resorbed with prot loss by pinocytosis, but if lots, pinocytotic vesicles fuse with lysosomes --- see pink hyaline droplets in tubular cells
2) Russel bodies - excess Ig production
3) Defects in prot folding - chaperones (protect unfolded proteins from degredation, guide them to organells for folding and if needed bring them to ubiquitin for degredation) - some chaperones are normally expressed others induced by heat stress (hsp70/90), abherent proteins can accum in ER ---- get unfolded protein response --- can result in slowing down of protein translation and increase in chaperones OR get activation of caspase 12 and apoptosis
1) Lipofuscin - insoluble byproduct of lipid peroxidation, sign of free radical injury and lipid perox
2) Melanin
3) Hemosiderin
4) Bilirubin
Describe the pathogenesis of dystrophic calcification
1) Initiation - mito accumulate Ca and interact with vesicles containing phospholipids of dying/dead cells, Ca binds to PL in vesicle membrane, phosphatases in membrane generate Phos from the PL and Phos binds to Ca forming Ca-Phos containing hydroxyapatite
2) Propagation - continued formation depending on the concentration of Ca and Phos and presence of inhibitors and other proteins
What is metastatic calcification?
Occurs in normal tissues d/t hypercalcemia - hyperPTH, PTHrp, osteolysis, excess Vit D, renal fail
Can occur anywhere but mostly in tissues that lose acid (GI, kidney, lungs, arteries, pulm v)
Name biochemical changes associated with cellular aging.
1) Decreased oxidative phos, prot and NA synthesis
2) Decreased nutrient uptake
3) Decreased chromosomal repair
Name morphologic features of aging cells.
1) Abnormally lobed nuclei
2) Vacuolated mito
3) Decreased ER
4) Distorted golgi
5) Accum of lipofuscin, misfolded proteins
What is telomere shortening?
Telomeres: short repreated DNA sequences at ends of chromosomes, when DNA is replicated a short section of telomere is not duplicated and so the telomere shortens, eventually leads to cell cycle arrest
Some cells, like germ cells have telomerase which is able to use its own RNA as a template to add nucleotides to the end of the chromosome
NOTE; immortal cancer cell lines have reactivated telomerase --- NB in tumor formation
T or F - SOD and catalase and DNA repair enzymes increase lifespan
