Unit III Flashcards
5 properties of a malignant cancer cell
- unresponsive to signals for proliferation control
- de-differentiated
- Invasive
- Metastatic
- Clonal in origin
Describe the multi-step process of carcinogenesis
Cancer cells are the accumulation of mutations in the DNA over an individuals lifetime.
E.g. - somatic mutations early in life (such as from UV light damage) that mutate genes involved in the DNA repair pathway may lead to cells with accumulated DNA damage over time (genetic instability).
- Cancer cells are “selected for” in that, of the cells with damaged DNA, the cells that have mutations related to carcinogenesis survive and proliferate much more effectively than surrounding tissue.
- Cancer is not often considered to be inherited in a Mendelian fashion, but rather the susceptibility to cancer is inherited
Types of genes that are often mutated in tumor initation
Oncogene: drive cellular proliferation
Anti-oncogenes (tumor suppressors): inhibit cellular proliferation
Cytogenetic abnormalities associated with malignancy
- translocation/deletions that activate oncogenes or inactivate tumor suppresors (ex. CML)
- Loss of heterozygosity —> inactivation of tumor suppressors
Describe the inheritance pattern of retinoblastoma
While the disease is considered to have an autosomal dominant inheritance, the mechanism of expression of the disease is actually autosomal recessive.
- One mutated copy of the gene may through the rare event of mitotic recombination become expressed twice in the same cell (LOH) - this cell proliferates - is selected for - and leads to tumorogensis
- Sporadic cases are extremely rare! these events require two independent mutation events.
Rb gene (location in genome)
13q14
Mechanisms for loss of heterozygosity
Rare events!
- Mitotic recombination
- errant recombination during DNA repair (homologous recombination pathway)
- chromosome loss and subsequent duplication
- point mutation on wild-type allele
Biochemical properties of Rb protein
- Rb is hypophosphorylated in non-proliferating cells (G0 or G1 phase)
- Rb is hyperphosphorylated in rapidly proliferating cells (S or G2 phase)
- hypophosphorylated form inhibits entry of cells to S phase, phosphorylation by CDK2/cycE “inhibits the inhibitor” allowing cell to continue into S phase
Rb protein and cell cycle
Rb protein inhibits entry of cell into S phase - and is controlled by phosphorylation by CDKs. Mutation or loss of Rb protein lead to uninhibited growth (in absence of growth-factors, DNA checkpoint inhibition etc.)
Why does the Rb mutation affect the eye over other systems?
For cells in the eye, the Rb pathway is the only inhibition pathway for its cell cycle, whereas many other cells have backup mechanisms with genes in the Rb family (p107, p130, p53) - Fun fact - in mice, Rb deletion leads to 100% penetrant pituitary tumors!
What is the associated cancer of the APC gene?
Familial Adenomatous Polyposis
Describe the APC tumor suppression mechamism
Inhibitory protein of the Wnt signaling pathway - APC protein binds and signals for the degradation of the beta-catenin protein - a transcription factor that is activated in the Wnt pathway
- when beta-catenin is present in the nucleus it upregulates the oncogene c-myc
Therefore loss of APC protein –> higher levels of beta-catenin in the cell –> transcription of the oncogene c-myc —> unchecked proliferation of cells
-mutations of the APC gene tend to be clustered at the 5’ and 3’ ends of the gene.
BRCA1 and BRCA2 - tumor suppression mechanism
BRCA1 and BRCA2 are involved in the DNA repair pathway and in regulate checkpoint during DNA replication (damaged cells cannot proliferate until repaired) - mutations lead to accumulative damage that increases risk of developing cancer - particularly in breast cancers
-most BRCA1 mutations are frame shift resulting in a truncated BRCA1 protein. Mutational events near teh 5’ end are asociated with breast and less likely, ovarian cancer.
typical pattern of cancer inheritance mechanism - dominant vs recessive
- Dominant conditions (heterozygotes develop cancer) are typically gain-of function mutations in oncogenes
- Recessive conditions (homozygotes develop cancer) are typically loss-of-function mutations in tumor suppressor genes
Why p53 was originally incorrectly thought to be an oncogene
heterozygotes had the cancer phenotype! this pattern is seen because p53 forms a tetramer of 4 homologous subunits, and if 1 of these subunits is mutated then the whole protein will cease to function - in fact - mutant form of p53 is often more stable, exacerbating the effect
Oncogenic viruses - examples
Adenovirus, HPV
Ongogenic viruses, mechanism
These viruses have oncogenes that inactivate p53, and can also inactivate Rb protein. This is done in order to drive the cell to proliferate and produce viral proteins - and bypass of cells normal inhibitions is necessary for this process for some viruses
Oncogene discovery
Oncogenes were discovered looking at oncogenic retroviruses in animals
- virus inserts cDNA into host genome via reverse transcriptase in proximity to a proto-oncogene (example c-src)
- even if not necessary for viral reproduction, c-src may be transcribed and added to the viral RNA genome (now v-src)
- if mutation in this process occurs, v-src will become oncogenic
Many of these genes were tested in the laboratory by using the property of cancer cells called anchorage independence - aka cells with oncogenic properties can grow regardless of the medium of the culture, and in much higher density
Describe the mechanisms/effects of viral oncogenes
Viral oncogenes interfere with the pathways of cell growth in response to environmental stimulation via growth factors. The viral oncogenes can interfere in any part of this pathway. e.g.
- increase receptors that respond to GF
- increase effect of cytoplasmic signal transduction in response to GF
- create new receptors that bind a different ligand or bind ligand differently
Can either cause quantitative changes (increased number of proteins) or qualitative changes (altered function)
What does the v-src gene do?
v-src gene codes for a protein kinase that phosphorylates tyrosine –> which affects gene expression
What does the v-erb-B gene do?
v-erb-B gene codes for protein that mimics cell surface receptors for epidermal growth factor (EGFR)
Specifics: V-ABL
v-abl codes for protein kinase, similar in function to ABL gene seen in BCR-ABL CML.
Oncogenes as molecular markers for prognosis
Gene amplification of certain c-onc genes can be detected in some cancers, with increased amount of gene expression indicating a poorer prognosis. Underlines a quantitative mechanism of carcinogenesis. Cytogenetic analysis indicating translocation of c-onc genes to regions that increase expression also indicate poor prognosis (aka BCR-ABL translocation)
oncogenetic molecular markers example: N-myc
FISH analysis of the N-myc gene shows amplification in cases of neuroblastoma. Those with less than 10 copies of N-myc have a much better prognosis.
oncogenetic molecular markers example: HER2/neu/erbB2
HER2/neu/erbB2 shows amplification in 20% of breast cancer
Targeted therapy example: HER2/neu/erbB2
Monoclonal antibodies (Herceptin) specific to a protein product of the HER2/neu/erbB2 gene enhances the immune response against those specific tumors that overexpress a specific protein on the cell surface. Used in concert with radiation increases effectiveness
Targeted therapy example: CML
Gleevac can inhibit the tyrosine kinase activity of BCR-ABL protein by acting as an ATP analogue and binding in the ATP site. However, the protein can develop resistance - Gleevac should be used in concert with other methods
- BCR-ABL is otherwise known as the philadelphia chromosome results in Chronic Myeloid Leukemia (CML).
Oncogene addiction and target therapy
It is thought that cancer cells are overly reliant on certain pathways of proliferation, where normal cells have many processes that work in concert. Targeted therapy has less toxicity because cancer cells have oncogene addiction, and affected greatly by inhibiting that specific pathway - normal cells will not be as greatly affected
Li-Fraumeni Syndrome (LFS) is most often associated with a mutation in what gene?
Associated with mutations of CHEK2 and TP53. They code for checkpoint kinase 2 and p53 protein respectively. Over 250 mutations have been identified
- clinical heterogeneity of syndrome may be associated with this spectrum of mutations.
- p53 is nicknamed “The guardian of the genome”.
Diagnostic Criteria for LFS
- LFS = Li Fraumeni Syndrome
- Proband with sarcoma diagnosed before age 45 AND
- 1st degree relative with any cancer under age 45 AND
- 1st or 2nd degree relative with cancer under age 45. or a sarcoma at any age
Diagnostic Criteria for Li-Fraumeni Like Syndrome (LFL)
- Proband with any childhood cancer or sarcoma, brain tumor, or adrenal cortical tumor before age 45 AND
- 1st or 2nd degree relative with a typical LFS cancer (sarcome, breast cancer, brain tumor, adrenal cortical tumor, leukemia) AND
- 1st or 2nd degree relative with any cancer under age 60
Molecular testing of LFS
Often involves direct sequencing of entire p53 gene (will move onto other genes if no mutation identified)
- provides diagnostic certainty of a complicated clinical diagnosis
- can avoid delay of diagnosis in another tumors, and informs clinician to avoid radiation.
- can assist with prenatal counseling and diagnosis
Knudson Two Hit Model
Idea that inheritance of a mutation in a tumor suppressor gene leads to susceptibility of cancer, but does not cause phenotype itself. Must have 2 hits, aka 2 mutated alleles (recessive-type expression) in order to develop cancer, and inheritance of mutated gene counts as already having 1 hit.
LFS and 2 hit model
Differs from Knudson model in that second hit can be from:
- a duplication of the same defect
- a different mutation that cause a different loss-of-function in the same associated gene
- a mutation in a totally separate gene (aka hit 2A occurring in amplification of HER2 gene increasing the risk of cancer)
- epigenetic silencing of a tumor suppressor gene (aka CDKN2A)
- insertion of an oncogene by a retrovirus
more complex than Knudson/Rb model!
p53 signaling pathway
DNA damage: ATM/ATR - CHK1/CHK2 —> p53 also activates MDM2 (which is inhibited by oncogenes)
-p53 binds directly to DNA. p53 decides whether to allow DNA repair or apoptosis to take place.
Note:
-MDM2 gene encodes for an E3 ubiquitin ligase the can promote tumor formation by targeting tumor suppresor proteins such as p53.
What are the mechanistic downstream effects of p53?
Primarily acts as a transcription factor
- Cell cycle arrest
- apoptosis
- inhibition of angiogenesis and metastasis
- DNA repair and damage prevention
- Inhibition of IGF-1/mTOR pathway
- Exosome mediated secretion
- cellular senescence
Goal is genetic stability! “Guardian of the Genome”
Von Hippel-Lindau (VHL) inheritance pattern and expression
Autosomal dominant high penetrance (>95% by 65) high variability in expression and age of onset
VHL clinical manifestations
Formation of cystic and highly vascularized tumors in multiple organs. VHL associated lesions include:
- Cerebellar and spinal cord hemangioblastomas (60-80%)
- Retinal hemangioblastomas (50%)
- Bilateral kidney cysts, clear cell renal cell carcinomas (75%)
- Pheochromocytomas (adrenal gland) (25%)
- Pancreatic cysts (75%) and pancreatic neuroendocrine tumors (11-17%)
- Endolymphatic sac tumors (inner ear) (10-15%)
- Cystadenomas of genitourinary tract (epididymal, broad ligament)
Major cause of death for majority of VHL patients
clear cell renal cell carcinomas and CNS hemangioblastomas
VHL Clinical criteria for diagnosis
1 VHL associated lesion
AND
a positive family history of VHL associated lesion OR 2 VHL-associated lesions
Types of VHL?
Type I: Hemangioblastoma + ccRCC (due to total or partial loss of VHL)
Type II: Pheochromocytoma +/- Hemangioblastoma+/- ccRCC (due to VHL gene missense mutation)
Type IIA: HB + PHEO
Type IIB: HB + PHEO + ccRCC
Type IIC: PHEO only
VHL gene location
3p25-26
Actions of VHL protein
VHL is a tumor suppressor gene.
- Regulates hypoxia inducible transcription factor (HIF)
- Suppression of aneuploidy
- microtubule stabilization/primary cilia maintenece
Note: VHL gene loss or inactivation leads to HIF accumulation. Under hypoxic conditions HIF is not degraded and activates transcription factors for angiogenesis and other process that promote cancer growth and survival under low O2 conditions.
VHL protein mechanism
VHL complexes with HIF-alpha transcription factor when it is hydroxylated (by proline and asparagine hydroxylase) under conditions in which oxygen is present. This complex then ubiquinates HIF-alpha which tags it for destruction.
In hypoxic conditions - HIF-alpha is not hydroxylated, and not targeted by VHL. HIF then activates transcription of genes involved in angiogenesis, metabolism, apoptosis etc. (genes include VEGF, TGF, PDGF)
Describe the VHL disease mechanism
A mutated or absent VHL protein will have cells that behave as if they are under hypoxic conditions
Clear cell renal cell carcinoma characteristics
majority of ccRCC cases are sporadic (96%) but VHL mutations are responsible for 2/3 of these cases. Requires 2 hits to VHL gene (tumor suppressor!)
Clinical presentation of ccRCC in VHL cases vs sporadic cases
Familial ccRCC: multifocal, bilateral, early onset (up to 600 tumors per kidney!)
Sporadic ccRCC: solitary, unilateral, late age of onset
Therapeutic approaches to ccRCC
Management often involves surgical resection (partial or radical nephrectomy).
For localized cases - adjuvant therapy is not the SOC, but ongoing surveillance is critical to catch possible relapse for metastatic cases
- systemic therapies are used in addition to surgery including, radiotherapy, cytokines
- many more therapies in the works including targetting of VEGF-R, mTOR, and immunotherapy
Molecular components and overview of membranes
lipids, carbohydrates, proteins! cell membranes are lipid bilayers, with proteins that span the membrane with many different functions. water-soluble molecules are usually not able to freely diffuse through cell membranes. Carbohydrates on proteins/lipids important for development, immunological response, and proper protein folding.
Describe Membrane fluidity and the typical lipid molecule.
depends on the composition of the membrane (amount of cholesterol) and temperature
The typical lipid molecule–> very fluid membrane; exchanges place with neighboors 10^7 times/second; diffuses several mm/sec; phospholipids do not spontaneously flip-flop
three classes of membrane lipids
Phospholipids, sphingolipids, cholesterol
all are amphipathic!
Phospholipid structure and common examples
made up of a hydrophilic polar head group, and hydrophobic fatty acyl tail. primarily derived from glycerol (glycerol froms backbone for polar head group)
exs. phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS)
Sphingolipid structure
similar to phospholipids (hydrophilic head, hydrophobic tail) but the backbone is derived from sphingomyelin rather than glycerol.
ceramide is a sphingosine + fatty acid via amide linkage
glycosphingolipid is ceramide + a sugar (ex gangliosides)
Cholesterol structure
polar hydroxyl group, and rigid steroid ring with a hydrocarbon tail. interalated among membrane phospholipids
- interaction with hydrophobic tails immobilize lipid and decrease fluidity
- cholesterol also affects the thickness of the membrane (interactions force straightening of membrane lipids)
Distribution of different lipids in membranes
In plasma membrane:
internal surface: PS, PE, PI (movement of PS to outer membrane signals destruction by macrophages!)
external surface: PC, sphingomyelin, glycolipids cholesterol distributed equally enzyme (Flippase) needed to flip lipids to exoplasmic/cytoplasmic face of membrane
Levels of cholesterol in different membranes
Plasma membrane (thickest and least fluid) > Golgi membrane > ER membrane (thinnest and most fluid)
Means of obtaining cholesterol
- Diet (depends on LDLR receptor)
- synthesis in liver (from acetate - requires 30 enzymes!) HMGCoA reductase is the rate-limiting enzyme in this pathway - inhibited by statins
tightly regulated process to keep cholesterol levels stable
Regulation of cholesterol uptake and synthesis
regulated by sterol regulatory element binding protein (SREBP)
- contains transcription factor regulating low-density lipoprotein receptor (LDLR) for uptake, as well as all 30 enzymes need for synthesis
- SREBP is in ER membrane where cholesterol levels are lowest (proportional changes are biggest here)
- if cholesterol low - transcription factor released..How? : Insig-1 dissociates from SCAP–> SCAP escorts SREBP to the Golgi by veisuclar transport–> at the golgi, bHLH transcription factor is released from SREBP by two step proteolysis “RIP” (Regulate dintramembrane proteolysis” (S1P is luminal , S2P is within the membrne: cleavage by both is required for activation).
SREBP regulation specifics
- The bHLH transcription factor is held as on part of the SREBP in the membrane of the ER.
- When cholesterol if high, Insig protein binds SCAP (SREBP cleavage activating protein) and blocks signaling
- when cholesterol drops, Insig releases SCAP which has a signal domain recognized by COPII - which packages SCAP/SREBP complex to Golgi
- at the Golgi, SREBP is cleaved to release the transcription factor that then moves to nuclease (cleavage by regulated intramembrane Proteolysis - RIP)
Note - SCAP is the protein that detects level of cholesterol itself
How membrane proteins associate with membrane
- extend single alpha helix across membrane
- multiple alpha helices that span membrane
- rolled up beta sheet (beta barrel) that spans membrane
- anchorage to cytosolic surface via amphipathic alpha helix
- covalently attached lipid chain in sytosolic monlayer
- oligosaccharide anchor to PI on outer surface
- attached to other transmembrane proteins
Total volume of fluid in the body
proportions of water, ions, and other
42 Liters = Total body fluid volume
28 Liters= ICF
14 Liters= ECF
99% water, .8 % Na, K, CL, and .2% everything else
Volume of intracellular fluid (ICF)
28 liters - 2/3 of total
Volume of extracellular fluid (ECF)
14 liters - 1/3 of total
Volume of fluid in “3rd space”
Special fluid in GI, kidney, sweat etc accounts for a total of 5 liters
Volume of plasma (subset of ECF)
3 liters
ICF and ECF concentrations (mM) and membrane permeability: Na
ICF - 14mM
ECF - 140 mM
functionally impermeable (pumped out of the cell to maintain concentration gradient)
ICF and ECF concentrations (mM) and membrane permeability: K
ICF - 150mM
ECF - 5mM
permeable to the membrane
ICF and ECF concentrations (mM) and membrane permeability: Cl and HCO3-
ICF - 5mM
ECF - 145mM
permeable to membrane
ICF and ECF concentrations (mM) and membrane permeability: Other anions (HP04, SO4)
ICF - 126mM
ECF - 0mM
impermeable to membrane
ICF and ECF concentrations (mM) and membrane permeability: Water
ICF - 55,000mM
ECF - 55,000mM
permeable to membrane
2 Important membrane properties conferred by lipids
- hydrophobic lipids make membrane impermeable to charged particles (including molecules with a dipole)
- electrically strong: can keep opposing electrical charges separated without collapsing (100,000V/cm)
Important membrane properties conferred by channels
Proteins can selective transport charged or polar molecules across the membrane.
Channels act as passive pores
- selective for particular ions
- contain molecular gates that can be regulated by temperature, mechanical stimulation, chemical ligand binding, or electric field (membrane potential)
Important membrane properties conferred by transporters
- Needed to transport large molecules selectively (ex glucose)
- Needed to pump molecules against their energy gradient (chemical, electrical)
Much slower than channels
Primary active transport vs secondary active transport
primary = direct metabolism (direct hydrolysis of ATP) secondary = other method (capture energy released from Na ions moving down gradient)
Definition of osmosis
movement of water across a semi-permeable membrane. rate of movement is faster than that predicted by diffusion alone water will always move according to its concentration gradient. i.e. more solute in the cell (less water) and water will move into the cell - causing it to swell
What can change the volume of a cell?
Only the movement of water!
3 mechanisms that have evolved to keep cells from swelling and bursting
- water impermeable membrane - not very feasibile as cells need to increase volume while growing, but some cells are observed with very low permeability to water
- cell wall that counters osmotic force with hydrostatic force, as seen in plants. Energetically expensive, and greatly limit cell shape
- balance the osmotic force by having solute molecules in the ECF in roughly equal concentration of the solute in the ICF, as seen in animal cells.
Van’t Hoff equation
Pressure needed to balance force of osmotic suction is directly proportional to the difference in solute concentration (ΔC)
π = RTΔC, where π = osmotic pressure
two factors important to osmotic balance
- chemistry doesn’t matter i.e. solutes inside can be different than outside (just need to be same concentration)
- ECF solute must be nonpermeating (same as ICF)
osmolarity
concentration of solution in cell burst problems, calculated at beginning of experiment
tonicity
The way that a solution cause a cell to respond in terms of change in volume (depends on permeability of solute to the membrane)
hypertonic - solution that makes a cell shrink
hypotonic- solution that makes a cell swell
REMEMBER! What effect do permeating solutes have on cell volume?
none!
rate of volume change in solutions with permeating solute
Water will initially leave the cell if concentration of a permeating solute is higher in the ECF, but as solute diffuses into the cell, water will eventually follow. The rate of volume change is dependent on the permeability of the membrane to the solute.
Reflection coefficient and modified Van’t Hoff
π = σ RT ΔC where σ ranges from 0 (same as water) to 1 (non-permeating) osmotic pressure will be diminished for permeating solutes, depending on how permeable the membrane is to the solute relative to water
greater σ means greater osmotic force!
Describe why membrane permeability and the blood brain barrier is important with injection of insulin in Type I diabetics and cerebral edema
diabetics have hyperosmotic body fluids due to high blood concentrations of glucose that cant be taken up by the cells. when insulin is injected, cells uptake glucose and ECF concentration falls, but at a slower rate in the brain due to the blood brain barrier. higher glucose levels in ECF of brain relative to blood will osmotically suck water in, cause pressure to rise (bad!). Insulin must be injected slowly to avoid this rapid change
Is membrane fusion energetically favorable or unfavorable?
Unfavorable though it will occur spontaneously (very slowly) hydrophilic polar head groups of lipids are hydrated in solution, so displacing these water molecules from each membrane is the largest energy barrier for fusion
Name 3 SNARE proteins that are targeted specifically by the Clostridium Botulinum toxin, and function primarily in skeletal muscle
VAMP (on vesicle membrane), SNAP25, syntaxin (on target membrane)
Describe the function of SNARE proteins and their structure that allows for this
- SNARE proteins functions in membrane fusion
- SNARE proteins bring the vesicle and target membrane in close contact to each other, displacing water molecules and allowing membranes to fuse spontaneously. This is done by forming a very stable amphipathic alpha helix structure (a coiled coil) in which 4 alpha helices come together to form a larger helix - aka “zippering”
How is the process of membrane fusion mediated by the NSF protein
- NSF is a hexameric ATPase that regulates membrane fusion and allows for the unwinding of the coiled-coil formed by the SNARE protein complex. NSF requires 6 ATP (1 for each subunit) to unwind the SNARE complex.
- The SNARE complex is very stable, and needs to be actively disassembled before it can function again.
How is the process of membrane fusion mediated by n-sec1 protein
- n-sec1 is needed to properly refold the SNARE proteins so that they are functional for the next fusion event.
- This is achieved by the n-sec1 protein which stays fused to syntaxin (complex also inactive) until a specific vesicle/VAMP comes along, then the n-sec1 protein will dissociate from syntaxin (syntaxin switches to active conformation) allowing for fusion to occur. Another level of regulation (?)
- Note: Syntaxin has 3 associated alpha helices (Ha, Hb, Hc) that can form a stable form of a coiled coil within itself which would not allow for any other interactions with the SNARE complex.
Describe viral fusion
enveloped viruses need to fuse with the host membrane in order to infect it. Proteins on the envelope of a virus recognize a specific target cell and resemble the SNARE proteins (though they are evolutionarily unrelated).
The principle of fusion is the same (bring membranes into close contact with each other so they can fuse spontaneously).
ex. HIV-1 gp41 has two alpha helix domains that form an anti-parallel coiled-coil.
Name steps of viral fusion
Pre-fusion
Extended Intermediate
Collapse of intermediate (forming coiled coil with self)
Hemifusion Fusion
Viral fusion regulation
different for different viruses. HIV by recognition of CD4 cells, influenza by changes in pH (low pH mediates conformational change for fusion)
Which is stronger, osmotic force or electric force?
Electric force (about 10^18 times stronger) strongest force in biology!
What two forces dictate movement of an ion across a membrane
the concentration gradient and the electrical gradient both dictate the movement of ions
Nernst Equation
E = (62/z) x log(Co/Ci)
[RT= 62 at body temperature]
E = equilibrium potential (inside cell with respect to outside)
z = valence
Co= concentration outside
Ci = concentration inside
Equilibrium potential
The electric potential difference across the membrane that must exist for the ion to be in equilibrium at a given concentration
Membrane potential (Vm)
the real voltage difference across a membrane
Vm = E if the cell is in equilibrium
if Vm != E then the membrane is either impermeable to a given ion, or that ion is being pumped across the membrane to maintain a state away from equilibrium (ions will always try to diffuse down their electrochemical gradient)
EXAMPLE TIME: Vm= -40mV and cell is permeable to Cl [Cl]o = 130mM and [Cl]i = 13mM. Is there a Cl pump, if so which direction does it pump?
Calculate E First E = 60 log (130/13) = 60 log10 = (+/-)60mV
- is 60 negative or positive? since the concentration of Cl ions is much greater outside the cell than inside, for the electric gradient to balance that force and put the cell in equilibrium, the electric force must work to keep Cl in the cell. Since Cl is a negative ion, the E will be -60mV (attracting negative anions)
- if the membrane potential is -40mv, that means we want the cell to be more positive that what Cl would do under its own will. Vm != E and we know Cl is permeating, therefore we know there is a pump!
Since the cell wants to be more positive than the E for Cl, how could a Cl pump do this? Cl is negative, so we need to pump it OUT of the cell to make the inside more POSITIVE
The Principle of Electrical Neutrality
bulk solutions inside and outside of the cell have to be electrically neutral
Even for a cell with Vm = -80mV, for every 100,000 cations there will be 100,001 anions - so the approximation is still a good one in these cases
Donnan Rule
If a cell is permeable to both K and Cl ions, the cell will be at electrochemical equilibrium when Ek=ECl [K]o{Cl]o = {K]i[Cl]i Explains an unequal distribution of charged ions when there is a semipermeable membrane
Na / K pump: how many ions per cycle?
3 Na for 2 K requires an ATP for each cycle
Describe a cell at equilibrium vs a cell in a steady state
Because the cell is permeable to Na that must constantly be pumped out, to maintain the correct concentrations of ions there is work being done and the cell is not in equilibrium. Rather, the cell is in a steady state - ion concentrations are not changing over time but a constant input of energy is required.
Describe how relative permeability of ions affects the resting membrane potential Vm
Na and K are both trying to reach equilibrium (their value for E) which are around +60 and -90 respectively. The true value for Vm depends on the relative permeability of the membrane to each of these ions. A cell more relatively permeable to sodium will have a more positive Vm, and cell more relatively permeable to K will have a more negative Vm
Ohms law and relation to Vm and relative permeabilities
Ohms Law: V = IR V is the “driving force” (it is the difference between Vm and E for that ion), I (current) is the movmement of the given ion, and R is membrane resistance to the ion. Conductance (G) = 1/R and is proportional to the number of channels for a given ion (higher the permeability, higher the conductance) When relating membrane potential and substituting using Ohms law, the conclusion is made that relative permeabilities (G) are directly proportional to Vm
Vm= Ek + Gr(ENa) / Gr + 1 [where Gr = GNa/GK)
Why do extracellular changes in Na have relatively little effect compared to extra cellular changes in K?
If [Na] is reduced in the ECF, value for ENa will decrease, and so Vm will also decrease a proportional amount (because the bulk solution is always electrically neutral, so only concentrations of Na are affected) There is little effect, partly because the cell is relatively impermeable to Na (Vm lies closer to the natural value of EK, and is proportionally effected less by changes in Na)
If [K] in the ECF is increased, EK will increase (think of Nernst equation concentration changes) and so Vm will also increase. However, because the cell is much more permeable to K, and the small proportional change in K has greatly changed the value of EK, the value of Vm will also change significantly! A increase in as little as 10mM K can cause depolarization of cells in the heart due to changes in the Vm that are more sensitive to voltage changes.
Treatment of hyperkalemia
C BIG K
C = Calcium: relieves cardiac arrhythmias
B = Bicarbonate: alkalinizing the blood encourages cells to take up K
I = Insulin: increase glucose uptake, increase ATP, increase activity of NA/K pump
G= Glucose: works with insulin to increase NA/K pump activity
K = Kayexalate: ion exchanger that selectively binds K
Law of Mass Action
- Keq = kf/kr= [C][D] / [A][B]
- the rxn above is the same as Keq=[Products]/[Reactants]
- At equilibrium, the rate of the forward and reverse reactions are equal, and the rate is proportional to the concentration of products or reactants.
define pH
Water is in equilibrium with [H] and [OH]
Keq for water is 1.8X10^-16 and water is 55 M so [H][OH]=1X10^-14
pH = -log[H+] which is equal to 7 in a neutral solution (when pH=pOH because log[H] + log[OH]= -14)
Concentration of [H] and pH relationships
[H] increases logrithmically with pH
pKa
measurement for the strength of an acid or bases ability to donate or accept protons
Ka = [H+][A-] / [HA]
pKa = -logKa
low pKa = strong acid!
high pKa = strong base!
Henderson-Hasselbalch Equation
pH = pKa + log([A-] / [HA])
Note: if 50% dissociated, then: pH =pKa
Relationships between pH and pKa
when [A-}=[HA] then log([A-]/[HA] = 0 (i.e. “Log(1) = 0”)
pH = pKa when species is 50/50 protinated/deprotonated
if pH > pKa; species is mostly deprotonated
if pH < pKA; species is mostly protonated
define buffer solution
- mixture of a weak acid and its conjugate base
- resist small changes in pH
- most effective within one pH unit of pKa
Reaction for bicarbonate buffer system
H + HCO3 <===> H2C03 <===> CO2(d) <===> C02 (g)
Henderson-Hasselbalch for bicarbonate buffer system
pH = 6.1 + log[HCO3-]mM / .03(PCO2 mmHG)
ex. normal conditions are 24mM HC03- and 40mmHG for pCO2
pH = 6.1 + log(24/.03*40) = 6.1 + log20 = 7.4
Normal blood pH = 7.4!
regulation of blood pH and buffer system - contributions of lung and kidney
Kidney excretes H+ and HC03 - Lung excretes C02 (g)
Allows bicarbonate buffer to be effective 1.3 units above its pKa of 6.1
What two conditions include idiopathic ‘Inflammatory Bowel Disease’?
Crohn’s Disease and Ulcerative Colitis
Incidence (US) of IBD and age of onset
1.4 million Americans with peak onset by 15-30 yrs
Tobacco and IBD
Smokers have increased risk for Crohn’s and disease is more severe
former smokers and nonsmokers at greater risk for ulcerative colitis
Crohn’s Disease general symptoms
Hematochezia: Rarely
Location: Ileum
Pattern: Discontinuous lesions
Upper GI Tract: Yes
Extra GI manifestations: Common
Fistulas: Common
Inflammation: Transmural
Ulcerative colitis general symptoms
Hematochezia: Common
Location: Rectum
Pattern: Continuous lesions
Upper GI Tract: No
Extra GI manifestations: Common
Fistulas: Rare
Inflammation: Mucosal
Extra-intestinal manifestations of IBD
Pleuritis, myocarditis, pancreatitis, sacroileitis, arthritis, *erythema nodosum and pyoderma gangrenosum*, tendinitis
IBD etiology
inappropriate inflammatory response to intestinal microbes
genetic factors: NOD2, interleukin-23-type 17 helper T-cell pathway, autophagy genes
Rising prevalence due to changes in diet, antibiotic use, altered intestinal colonization
Characteristic presentation of Diabetic Ketoacidosis (DKA)
Ill appearance
rapid, deep breathing with tachycardia
nausea, vomitting, belly pain
dehydration (combined with polyuria and polydipsia)
fruity odor to breath (ketones)
Metabolic disturbances in DKA: hyperglycemia
Type I diabetes results from an immune reaction against beta cells of the pancreas. These beta-cells can no longer produce insulin to signal glucose uptake in cells throughout the body.
DKA- plasma glucose > 200 mg/dl since glucose is not entering cells. Other methods of obtaining energy are used which include lipolysis and fatty acid oxidation in the liver, this leads to elevated levels of ketoacids in the blood.
Insulin - pathway to production
- glucose enters beta cell through GLUT2 transporter
- stimulates glucose metabolism, and leads to an increase in ATP produced
- increased ATP inhibits an ATP-sensitive K channel
- inhibition of K into cell leads to depolarization
- depolarization activates voltage-gated Ca channel in plasma membrane
- increase in Ca leads to exocytosis of preformed insulin secretory granules
Insulin actions on the Liver
Insuling effects on the Liver:
Results in increase of glucose uptake, glycogen synthesis. Also decrease of gluconeogenesis, decrease of ketogenesis, and increase of lipogenesis
Insulin actions: muscle
on muscle cells, insulin has the effect of increasing glucose uptake, glycogen synthesis and increasing protein synthesis
Insulin actions: adipose tissue
Adipose
increase glucose uptake, increase triglyceride uptake, increase lipid synthesis
Metabolic disturbances in DKA: acidosis
result of beta oxidation of fatty acids in the liver. process generates H+ ions and ketone bodies (measurable). result is low blood pH (7.28 - 2.32) and low bicarbonate (18-26mEq/L) and high levels of ketones measured in the urine
