Cell Biology and Molecular Biology - LE3_ CELLMOL PDF

CELL BIOLOGY AND MOLECULAR BIOLOGY DR. ESPERANZA CHUA CABRERA LE 2 Coverage: COVERAGE: ALL TOPICS GRADE CONSUL: Dec 6 EXAM TIME: 10 Le 2 start MAIN TOPIC SUBTOPIOC SUBTOPIC...

CELL BIOLOGY AND MOLECULAR BIOLOGY DR. ESPERANZA CHUA CABRERA LE 2 Coverage: COVERAGE: ALL TOPICS GRADE CONSUL: Dec 6 EXAM TIME: 10 Le 2 start MAIN TOPIC SUBTOPIOC SUBTOPIC Sha & Shem 1  Juxtacrine ○ A cell targets a cell connected by gap Signal Transduction: Chemical Messenger junctions RECORDING; 11/11/24 ○ The cell producing the chemical signal attatched to the s chemical signal but the chemical signal are transferred by your gap MAIN TOPIC junctions Paracrine SUBTOPIOC ○ A cell targets a nearby cell ○ The cells are not adjacent, ○ The signal cell (green) is the one producing SUBTOPIC the chemical signal but the target cell is very near (like a neighbor).They are not directly attatched to each other. SIGNAL TRANSDUCTION: CHEMICAL MESSENGERS ○ Example: Growth Factors Endocrine ○ A cell targets a distance cell through the blood stream ○ You have the signaling molecule producing the endocrine molecules (hornones) and they travel through the bloodstream. ENDOCRINE HORMONES ENDOCRINE HORMONES ○ They travel through longer distances and the bloodstream AMINO ACID DERIVATIVES: ○ Epinephrine Also called: adrenaline It is just from tyroxine ○ Norepinephrine From tyrosine (photo) simple form of chemical signaling ○ Thryoxine ○ Based on the distance covered by the From tyroxine chemical signal and the target size Peptieds:ADH (vassoprressin) Autocrine ○ Several amino acid ○ The cell produces the chemical signal and ○ Short peptides that signal affects itself (same cell) ○ Antidiuretic hormone ○ EXAMPLE: Macrophage Proteins: insulin, glucagon Macrophage: is a kind of monocyte ○ Made up of polypeptides in the tissues. They produce a lot of Steroids: androgens, estrogens, corticosteroid cytokines. They are actively ○ They are hydrophobic in nature involved in phagocytosis. ○ Sex hormones and corticosteroids (produced Additionally, they are the problem in by your adrenal cortexx) the SARS-COV2. Local mediators: In this case, you have cytokines ○ Amino acid derivatives: histamine from called interleukins. There are diff histidine types of interleukins (either pro or Histamine: as a molecule that will anti-inflammatory). activate parietal cells to have the This interleukin will be produced by proton pumps attatched be your macrophages, and secreted delivered across the membrane to the outside but then there are ○ Arachidonic Acid Derivatives: prostaglandi receptors of itself in the same cell. So this will now allow the macrophage to be activated.  ○ The molecule (ligand- anything that attaches) ○ There is a ligand sitting on the specific receptor There is a ligand-receptor specificity. It is not only a matter of physical attachment, you also have chemical acids in your receptors ○ With this, (your ligand on your receptor), causes a change in conformation sending changes to your downstream processes. ○ So the primary messenger as well as your secondary messenger can respond either from your cytosol or from your nucleus Epinephrine is just one molecule derivative of tyrosine From nucleus: cause change in ○ Norepinephrine is similar in structure your gene expression Antidiuretic hormone- a cyclic peptide with nine amino In the image, you see that your receptor is at the acid and one disulfide bond plasma membrane, but this is not always the case. ○ How many polypeptides? Just one Your receptor can be found inside the cell (in the Because it only has one N terminal cytosol). These receptors are for hydrophobic and one C terminal molecules so they do not need receptors on the And there are only 9 amino acids + plasma membrane. The hydrophobic ligand can pass disulfide bond through the lipid bilayer by simple diffusion. If you destroy the disulfide bond RECEPTOR AFFINITY then it will become nonfunctional In insulin, it is a longer polypeptide (2). The 2 HIGH AFFINITY polypeptide chains are bound by your disulfide bonds, ○ All receptors occupied at low concentrations And in your 1 polypeptide chain are also cysteine of ligands chain bound by your disulfide bond. ○ This means that the ligand binds easily to the receptor affinity= liking or attachment ○ High affinity of the receptor to a particular ligand, it would be easy for the ligands to attach to the receptor. Because of this, you do not need to have a high amount of ligands. Even if you have a small amount of ligands it will attach to the receptors. LOW AFFINITY ○ All receptors occupied at high concentrations of ligands ○ If the receptors have a low affinity for the ligand, it does not bind very well to the ligand Then in return you have to have a high concentration of the ligands. Because the ligands can hit or miss the receptors (bcs they dont bind well) ○ All of the receptors should be occupied by a What is a chemical signal transduction? ligand. (hence, high concentration of ligands ○ We have the plasma membrane, the at low affinity) receptor for the chemical messenger is Dissociation constant Kd found on the plasma membrane  ○ “Concentration of free ligand needed to attatches to that receptor it will produce a state in which half of the receptors prevent the natural messsenger are occupied” from attatching– this is called your ○ I competitive inihibition ○ The value that will tell us how easy or how hard it is for ligand to separate from the receptor If there is a high dissociation constant= there is high tendency of RECEPTOR DOWN- REGULATION the ligand to be removed from the receptor If there is a high Cells are made to sense CHANGES in ligand dissociation constant concentration therefore this receptor ○ The more that ligand is present it comes to doesn’t attach well to the the point wherein you need more ligands to ligand meaning= it has low activate the response affinity ○ You are increasing the response level Since there is a low affinity because you are now causing the response of the receptors therefore needed to occur at a higher concentration if you need a lot of ligands to you are constantly exposed to that ligand. ensure tha receptors If your cell is often exposed to would be occupied. ligand x, then you need more ligand x next time to effect a response. Prolonged contact of ligand with receptor leads to loss APPLICATION OF RECEPTOR AFFINITY IN DRUG DESIGN of response due to Receptor down-regulation Receptor down-regulation DRUGS THAT ARE: ○ Receptor-mediated endocytosis of ○ AGONISTS: drugs that bind tightly and receptor selectively to receptors leading to their For example when you have a new activation perfume/scent that you are using at For example, you have the ligand first you can smell the perfume on (epinephrine), the body cannot you very well. But as you continue produce enough epinephrine. Then to use it sometimes you can’t smell you’ll have an agonist ( a drug ) that the perfume on you causing you to would mimick the activity of the use more. ligand This is the same for the ligands, this When that drug is attached is due to the receptor to the receptor, It mimics down-regulation the epinephrine ligand. If the cell is often or prolonged This will now lead to the contact with the ligand then the activation of the response. receptor may be taken into the cell, ○ ANTAGONISTS: drugs that bind to receptors and if you don't have enough without triggering the changes that occur receptors in the cell then the when the normal ligand binds response would not be optimum. They bind to the receptor BUT they Then the receptor may be prevent the response taken into the cell by your Inhibits the receptor by preventing receptor-mediated the naturally occurring messenger endocytosis. from binding and activating the You will have receptors at your cells receptor. “Competitive inhibition” and form a vesicle called vessel The drug have structures that are lined with clathrin. This would be similar to the natural messenger. taken onto the cell and it can merge This drug will compete with these and will be degraded. messenger sot hat when that drug ○ Desensitization of receptors  Example: phosphorylation lower endocytosis and it binds to the lysosome for affinity for ligand, or can’t initiate degradation. changes in the cell Receptors are desensitized. How TERMINATION does this happen? Phosphorylation by a kinase. When the receptor is Extracellular messenger eliminated phosphorylated then may result in a ○ If there is no more ligand, then you cannot lower affinity of the ligand, making it activate the receptors= terminated not that sensitive. Activated receptors internalized ○ The role of phosphorylation, is taken into the cell. ○ Receptors degraded with ligand ○ Receptor recycled Activated receptors inactivated: phosphorylation of receptor, followed by binding of arrestin, endocytosis, lysosomal degradation Intracellular signaling proteins or molecules inactivated DIFF TYPES OF LIGANDS You are going to take up a kind of receptor called GPCR (G-protein coupled receptor) , this receptor functions together with a G protein. If this receptor is bound to an Active G protein that receptor will not function. Here the main objective is to show how this receptor can be activated and removed from the plasma membrane due to phosphorylation In this case, you have a chemical messenger (ligand) that binds to the GPCR (the receptor) which will trigger a number of reactions. ○ Now let us say, the response is done and No need to memorize you want to down-regulate or end the Amplification of the response, it is very important how response. Because the constant exposure to economical it is the ligand will cause the down-regulation of ○ amplification= increase the receptor. ligand= epinephrine (adrenaline) There is a protein here called the GRK or GPCRK (G RECEPTION: If you have one molecule of protein-coupled receptor kinase) which is a kinase. epinephrine binding to one receptor (G protein-linked This kinase catalyzes the addition of the phosphate to receptor) it will trigger the succeeding reaction. the receptor. Triggered reaction/response: TRANSDUCTION There is a molecule called beta-arrestin, this Transduction (connect connect the reaction so molecule will recognize the phosphorylated receptor. what is the activated molecule before hand will The phosphorylation is for the arrestin to recognize it activate the succeeding protein in the reaction) and to bind to it for endocytosis. ○ Inactive G protein to active g protein Now that the receptor is phosphorylated (GPCR) then Activates 10^2 molecules it will activate the receptor to be taken into the cell via One molecule molecule of epinephrine binding to 1 G  protein receptors can G protein- couple receptor (GPCR) activate 100 G proteins ○ You have a receptor and the binding of the ○ Inactive adenylyl cyclase to active adenylyl ligand allow this receptor to undergo a cyclase conformational change. This conformational So the activated g protein from the change will cause the activation of a G previous response will now activate protein by binding of the GTP. adenylyl cyclase Receptor Kinase Activates 10^2 molecules ○ Example: receptor tyrosine kinase 100 G protein will ○ Once it is activated by the ligand, (by itself is activated 100 adenyly kinase), this will allow its own cyclase phosphorylation. ○ Atp to cyclic AMP Nuclear receptor Activates 10^4 molecules ○ Intracellular receptor ○ Inactive protein kinase A to Active Protein ○ You have molecules/ligands/messengers Kinase A that are hydrophobic, they can pass through Activates 10^4 molecules the lipid bi layer by simple diffusion. The ○ Inactive Phosphorylase Kinase to Active receptor is found inside the cell and not the phosphorylase kinase plasma membrane. Activates 10^5 molecules ○ Inactive glycogen phosphorylase to active GPCR glycogen phosphorylase Activates 10^6 molecules RESPONSE: KIND OF RECEPTOR: G PROTEIN LINKED RECEPTOR ○ Glycogen to glycogen-1- phosphate (GPCR) Activated by the 10^6 active Natural ligands: glycogen phosphorylase ○ Hormones This activates 10^8 molecules of ○ Neurotransmitters the glucose-1 phosphate ○ Odorants NOTE: 1 molecule epinephrine is capable of The reason why you can smell triggering the production of hundreds of millions of Because there are ligands or glucose-1- phosphate molecule molecules in the perfume or particles in the air that can attach to EXAMPLES OF BASIC TYPES OF SIGNALING PATHWAYS the receptors ○ Tastants ○ Photons ○ Chemoattractants (for phagocytes) ○ Opium derivatives - morphine, codeine, oxycodone, and heroin etc. Ligand-gated ion channel ○ Channels, that are open and closed with the presence or absence of ligands  Then the long light purple in the cytosol part, that part is where the GPCR Kinase will attach to be able to stop the reaction ○ ORR if the receptor has to be taken into the cell as a result of down regulation because of constant exposure to the ligand. The green part: G PROTEIN ○ G protein: are molecules that will be in the active stage if it is attached to the GDP Take note: once the GTP is replaced by the GDP GTP: nucleotide, Guanosine triphosphate Guanine: base Guanosine: nucleoside+ sugar = ribose, For it to be nucleotide: nucleoside + sugar, and (a) molecule of GPCR phosphate We see that the receptors are integral or intrinsic FIRST G PROTEIN: Trimer, Hetero Trimeric G Protein proteins. With one polypeptide (one C terminal, One ○ 3 subunits, which is different from each N terminal). With multi-pass polypeptide, in alpha other. helix form OTHER TYPE: Monomer, Monomeric G Protein ○ Usually, integral proteins are in alpha helix ○ 1 unit form What happens if you have a ligand? LIGAND- from outside the cell ○ First you have a receptor The receptor has a specific part where the messenger ○ Then you have a specific messenger binds AKA, the messenger-binding site. attaching to it (ligand binds to receptor) ○ There will be chemical interactions between What triggers to the activation to the ligand and receptor the response, is the attachment of ○ There is specificity here the ligand to the receptor. Dark purple- G PROTEIN If the ligand attaches to the specific The one with red red stars= segment in which the receptor it will undergo a GPCR kinase will add phosphate conformational change. ○ When the receptor undergoes a conformational change it will cause an attachment of the G protein. To be specific the beta-gamma subunit are the ones to be attached to the GPCR ○ The other subunit is the alpha subunit. Take note that in this kind of receptor, the GTP attaches to the alpha subunit NOT to the beta-gamma ○ Once activated, the GDP that is attached to inactive G protein is replaced by a GTP. ○ Once your G protein has a GTP attached to your alpha subunit then, the G protein now is ACTIVATED Once the G protein is activated, this will activate the succeeding events. The top left photo: GPCR that we have shown a while The specific ligand binds to a specific receptor. The ago receptor will undergo a conformational change. It ○ So the light purple that is coiled= GPCR causes the g protein attatctch to the beta-gamma sub  unit. Once the beta-gamma subunit is attached to the receptor then the alpha subunit now has the GDP replaced by the GTP.Once the G protein is activated (bcs it now has a GTP), the other units will detach itself. When the G protein is now active, you will now have the separation of the alpha subunit with the G protein (which is active). The beta-gamma is also active, even without GTP. ○ This can also affect a response The next step is that the activated beta-gamma subunit can bind to the next molecule involved in the response, same with your alpha subunit (will attach to the next molecule) ○ The two may not be activated at the same time it depends on the cell G protein subunits activate or inhibit target proteins For termination, the alpha subunit has GTPase Table is made to guide you activity. For termination to occur, the alpha subunit Target to discuss right now is the G protein can catalyze the hydrolysis of the GTP removing 1 ○ You have so many kinds of G protein phosphate so the GTP is converted to GDP (which is What is similar among the G proteins in the table are: inactive) G protein are active when you have GTP bound to the ○ Now that it is inactive, it will bind to the alpha subunit beta-gamma again. Then this beta-gamma ○ They will have different functions because will bind to the alpha subunit which will result different G proteins are couple with different for the whole unit to be inactive types of receptors There are molecules in the cytosol which you call If you add more ligands, you need to add more regulators of G protein signaling receptors ○ RGS proteins: regulator of G protein ○ Ligands are the same all are epinephrine ○ GTPase activating proteins (GAP) binds to ○ For the receptor it varies with beta and alpha the G alpha-subunit receptor (in the example) which has different ○ How is it regulated? G proteins and will have an different effector Example of this regulator is the each time molecule: GTPase activating For example: G protein: Gs protein (GAP). ○ If this G protein is activated (Gs), by the GAP will bind to the alpha subunit. attachment of the GTP to the alpha subunit And when it binds to the alpha it is stimulatory, it will stimulate the subunit it will activate and increase responses (s= stimulatory) the GTPase activity of the alpha ○ Epinephrine binds with the beta-adrenergic subunit receptor with the G protein Gs) which has an The alpha subunit has effector of adenylyl cyclase and has a 2nd GTPase activity that will messenger of cAMP activate the hydrolysis of G protein: Gi the GTP to the GDP ○ “i”= inhibitory To enhance the GTPase activity, ○ This G protein is activated by the addition of there is a protein called GAP that is the GTP to the alpha subunit (similar to the on the cytosol. Your alpha subunit Gs). BUT the result will inhibit the response. has an optimum GTPase activity ○ Epinephrine binds with the alpha 2- that will lead to a faster hydrolysis adrenergic receptor with Gi as the G protein, of the GTP to GDP. Which will then the effector inhibits the adenylyl cyclase ( no to a faster inactivation of the alpha effector = no 2nd messenger,cAMP) subunit which will cause the G protein: Gp or Gq (same lang just diff tawag inactivation of the beta-gamma per textbook) subunit.  ○ This G protein works together with an ○ The ester bond is low energy (between the enzyme called phospholipase. acid and the alcohol) ○ The epinephrine binds with the alpha How is cyclic AMP formed? 1-adrenergic receptor if it binds witht he ○ You have the enzyme adenylyl cyclase or phospholipase C(beta), the second adenylate cyclase (active form) , which is messenger would be DAG and IP3. bound to the plasma membrane. ○ BUT if the G protein is not Gp but Tranducin What activates the adenylyl then it will bind to the cGMP cyclase? It is activated once the Gs phosphodiesterase effector and the 2nd (G protein) is activated by binding messenger would be the linear GMP with a GTP. And the reason why There is specificity from the Gprotein and their this G protein is activated because receptors the ligand was bound to its ○ For example, in this particular case if you are receptor. talking about the ligand epinephrine but ○ What does the activated adenylyl cyclase there are different cells there will be different do? It actiavates the hydrolysis of your ATP. receptors. Then these different receptors at Where is the bound cleaved? THebound at different cells are coupled with different G the 2nd acid anhydride is cleaved (not the proteins. one atatched to your adenine and the ester ○ Epinephrine: a hormone that is released by bond). the adrenal gland (also called adrenaline). The result will be only one The epinephrine is synthesized by the phosphate, now you have medulla of the adrenal gland. adenosine monophosphate with the Basically, the ligand and receptor could change release of 2 inorganic phosphate (depends)but if there is g protein, then the effector (PPi). The PPi will be released to would def be adenylyl cyclase and catalyze cAMP. the cytosol and will be used again by the cell. CYCLIC AMP ○ Now with your adenosine monophosphate, you have a bond formed at your carbon 3 and the phosphate. Now you have a cyclic amp due to your bond at carbon 5 (with the phosphate by your ester bond) and the new formed bond at you carbon 3 with the phosphate. This will now serve as the second messenger. When cyclic AMP is activated it will activate a molecule called protein kinase A, This second messenger cAMP is released to the cytosol. ○ Let us say that you already have the response from your protein kinase A, now you need to terminate the response Here the enzyme cAMP phosphodiesterase. The cyclic AMP You have here the nitrogenous adenine bound to the has 2 esterbond (diesterbond) at sugar (ribose, bcs there is an OH in the carbon 2)= so carbon 5 and carbon 3. now when you combine the 2 it will be a nucleoside Now the phosphodiesterase and will be called as adenosine triphosphate. destroys only 1 ester bond with the ○ Adenosine triphosphate- high energy addition of water (since ester bonds molecule bcs it is made up acid anhydride are formed by the removal of 1 bond, formed between acid phosphate with water). The bond destroyed is at the removal of 1 water reason why it has a your carbon 3 (the one that was kinky line.  added when the adenylyl cyclase was activated) But now this is not cyclic anymore, it is now just a linear AMP (which is not active). Therefore, it will not activate your protein kinase A REVIEW : SUMMARY FOR cAMP (APPLICATION) The blue one now is your adenyly cyclase which catalyze the production of your cAMP Once the adenyly cyclcase is active, it has different shape and cause the hydrolysis of your ATP to produce cyclic amp What is the substrate of your adenyly cyclase? ○ Means what molecule is being acted on by your adenyly cyclase. ○ Therefore your substrate is ATP and the You have the plasma membrane, receptor and the product of it is cAMP. specific ligand attached to it, which causes a Is it (cAMP) bound to your conformational change to your receptor and cause membrane? NO, it is at your your G protein to bind to itself via the beta-gamma cytosol. subunit. With that, your cAMP will bind to your protein kinase The G protein after binding to the receptor at your A. beta-gamma subunit, will now be activated because it Then to terminate it, one way would be end the allows you alpha subunit to bind to a GTP. reaction by detaching the ligand from the receptor. Specific example: The receptor will now go back to its original form and ○ You have your epinephrine (ligand) which will then your alpha subunit GTPase activity is activated bind to your beta-adrenergic receptor which now it hydroylases your GTP to GDP. Now that GDP will cause your receptor to a conformational will detach from your cAMP. Will now attach to your change. And your G protein is activated by beta-gamma = inactive the binding of the ligand and the receptor. G Another way is the cAMP produced degraded by your protein attached to the receptor by the phosphodiesterase. You will still have your cAMP but beta-gamma subunit. This attachment will not be activated but linear. causes the alpha subunit to bind with your What is targeted by your adenyly cyclase/cAMP? GTP (exchanged to your GDP). Your Protein Kinase A ○ In this particular example the beta-gamma is not involved only the alpha subunit. The blue circle in the photo is your adenylyl cyclase which will catalyze the production of the cAMP. Adenyly cyclsae is membrane bound.  The photo shows us how the cyclic AMP can activate the protein kinase A The protein kinase A includes: 4 subunits ○ 2 subunits for catalytic ○ 2 subunits for regulatory Not involved in the catalysis but are allosteric sites If you have your regulatory subunit bound to your catalytic subunit the entire protein kinase A is inactive But once you have your cyclic AMP, which will bind to your regulatory subunit and the binding will cause the regulatory subunit to change conformation, then the affinity for the catalytic subunit is decrease ○ Affinity decreased your regulatory subunit will now separate with your catalytic subunit Once separated, your catalytic subunit will become active and cause to activate the next molecule= protein kinase A is active —-------------------------- END OF NOV 11, 2024 —-------------------  Continuation of online meeting F2F; 11/14 G PROTEINS ASSOCIATED WITH GPCR We are done with this slide, this table is so important ○ Different G proteins ○ Partner are found here G protein: molecule that is activated when GTP is attached to it ○ And when GTP is hydrolysed to GDP, this hydrolysis switches the G protein back to its inactive state What partner of GS? = adenylyl/adenylate cyclase ○ And when this is activated, the 2nd If the reaction has to be terminated, you need to messenger is cAMP (cyclic AMP) destroy cAMP cAMP will activate protein kinase A ○ To destroy it, you need enzyme : cyclicAMP Protein kinase A has different phosphodiesterase activities depending on cell ○ This activates the hydrolysis of 1 Example we will be taking up is epinephrine. phosphoester bond ! *Ms just read the table* And that's the phosphoester bond that links the carbon 3 OH and phosphate DIFFERENT WAYS IN ENDING REACTION/TERMINATING ○ Ester bond that holds the sugar to the phosphate is not cleaved, so you still have adenosine monophosphate (but it is now linear no longer cyclic anymore) ○ Only one bond is cleaved but the name of enzyme is “diesterase” U might think 2 ester bonds are catalyzed/hydrolyzed, just one It is name such because the substrate has 2 phosphoester bonds And to end the reaction there are different ways; ○ Can remove di bond / ligand (??) sa receptor  ○ Cyclic AMP is converted into linear AMP ADRENAL MEDULLA PRODUCTS ACTIVATION OF PROTEIN KINASE A Epinephrine; ○ Catecholamines: epinephrine, We also discussed that when theres cAMP, what norepinephrine, dopamine… molecule will be activated? GPCR, and we’re talking ○ Where are these produced? Adrenal medulla about the GS, when GS is activated = adenylate ○ For norepinephrine there are 2 locations of cyclase activated, and when adenylate cyclase production activated = cAMP will be produced, and when there's Adrenal medulla cAMP na what molecule will be activated? Protein Postganglionic neurons kinase A How will the presence of cAMP activate protein kinase A? CENTRAL NERVOUS SYSTEM; sympathetic & ○ We have 4 subunits in protein kinase A parasympathetic ○ 2 subunits are catalytic and 2 are regulatory ○ cAMP will bind to regulatory sites causing change in confirmation of the subunit. This now causes lower affinity of the regulatory subunits to the enzymatic/catalytic subunits. What will comprise the CNS (central nervous system)?  ○ Central nervous system ○ There are neurotransmitters ○ Peripheral nervous system ○ If parasympathetic it will release Motor and sensory neurons acetylcholine to target organ Sending from muscles ○ If its sympathetic system it will release different organs back to epinephrine or norepinephrine to target nervous system organ What we’re concerned here is motor neurons; it has Adrenal medulla releases both epinephrine and opposite direction norepinephrine ○ From the central nervous system to the Won't discuss dopamine different organs/muscles ○ We have 2 divisions Somatic (which we are not Parasympathetic and sympathetic concerned with) connections and effects Autonomic nervous system (involuntary responses) (concerned with this!) Sympathetic ○ Fight or flight and parasympathetic ○ Rest or digest We have here the autonomic nervous system, one column is parasympathetic the other is sympathetic Neurons transferring from different organs back to the CNS: afferent [Enters CNS] From CNS going down: efferent [Exits CNS] AUTONOMIC NERVOUS SYSTEM Then i think image with like sympathetic vs parasympathetic for example airflow in lungs if constrict or widen~ Went through this^ HORMONE (EPINEPHRINE), TARGET CELLS, & EFFECT Preganglionic neuron: found in brain stem and spinal cord (CNS) Preganglionic neuron will be releasing acetylcholine into the synapse between itself and the postganglionic neuron Postganglionic neuron: Neurons found in ganglion (group of nerve cell bodies outside of the central nervous system)  Molecule epinephrine here and different target cells; Phosphorylase kinase; will add phosphate group to adrenergic receptors (beta 1, beta 2, alpha 1, alpha 2 substrate etc.) ○ If your phosphorylase is activated, your ○ Depending on the cell type glycogen now is broken down into the ○ Results in heart cells? subunits Epinephrine increases heart rate ○ What are the subunits of glycogen; glucose ○ Results in liver cells? ○ Aside from that it adds phosphate to glucose Wil activate glycogenolysis in the at carbon 1 liver cell to produce more glucose ○ So you have glucose-1-phosphate that will —------------------—------------------—------------------—------------------ Technically, you have the same ligand, epinephrine, but when it targets different receptors/cells its responses are different. LIVER CELL AND GLUCOSE FOCUSED What happens in the liver when you have epinephrine bound to the receptor You have your alpha adrenergic receptor Role of liver cell ○ Is it a GPCR? Yes ○ If low glucose levels in blood, then it supplied ○ We are discussing G proteins, GPCRs are glucose receptor associated with G proteins ○ So what happens is that GS is a Heterotrimer (trimer: 3 subunits) (hetero glucose-1-phosphate is formed, and different components) because of an isomerase, it becomes ○ G protein binds to alpha subunit glucose-6-phosphate. —------------------—------------------—------------------—------------------ ○ Now more glucose in the liver than in the blood so it goes out through facilitated diffusion. Phosphorylase kinase There are other methods through which glucose concentration is increased ○ glycogenolysis (is the one discussed above) ○ Gluconeogenesis Protein kinase can now phosphorylase a transcription factor (these are molecules that regulate transcription) Part of the gene to which transcription factor sits : response element What is the name of the transcription factor that will be phosphorylated by your protein Kinase A: B = binding. CREB (cAMP response element-binding  protein) is the molecule that would bind to CRE Fatty acids can now undergo beta oxidation (cAMP response element) You have your acetyl CoA enzyme produced and this ○ When it binds to the response element it will can enter Krebs cycle activate the transcription of enzymes that are PRoduce more NADPH and NADH2 involved in gluconeogenesis. More ATP under stressful conditions In gene, there is a portion called transcription element called CRE. —------------------—------------------—------------------—------------------ ○ CRE made up of DNA (part of the gene) ○ What do you call transcription factor that will HORMONES AND GLUCAGON bind to CRE = CREB ○ What will activate CREB? By addition of phosphate Enzyme that adds phosphate: kinase Protein kinase A in particular —------------------—------------------—------------------—------------------ ○ What is the problem if glucose level inside the liver cell increases? There will be a feedback mechanism ○ Need to inhibit glycogenesis AKA synthesis of glycogen Activated by addition of phosphate If you want glycogen synthesis activated then remove phosphate. ○ You can also destroy the 2nd messenger as Do you know how important it is during a fight or flight mentioned in previous discussion situation to have a better blood circulation, increase of supply in oxygen blood, and remove waste —------------------—------------------—------------------—------------------ Look at the effect of increase cyclic AMP on smooth muscle cells. If you have smooth muscle cells and the Very cluttered table (the slide image), different receptor is a beta adrenergic then the result is your examples; smooth muscle will contract. ○ What's in SER and liver cell that is not in ○ Baliktad sa heart muscle muscle cell: Antidiuretic hormone Glucose 6 phosphatase ○ Target is kidney: involved in urine production If its not epinephrine then you don't call it adrenergic ○ antidiuretic : want to lessen the urine output Epinephrine again is fight or flight so you want more It acts on the nephron so water glucose in the blood during challenging situations absorption is increase G protein involved?: GS (because we are talking Parathyroid hormone about Cyclin A and B) ○ Very important in regulation of calcium levels All involved protein kinase A from examples, only in the blood differ in receptors ○ Target tissue is bone, so it increases bone resorption —------------------—------------------—------------------—------------------ Let's say blood calcium level is low, so the homeostasis should be Adipose cell store glucose in the form of fat maintained Triglyceride backbone: glycerol One main way is bone resorption ○ 3 carbons You have cells called the ○ How many fatty acids: 3 osteoclasts that can break ○ Is it a phospholipid? No way no no down the bone materials We see that epinephrine can cause the hydrolysis to so that the calcium now is produce the fatty acids from your triglyceride  released into the will dilate / relaxation bloodstream from the bone ○ Opposite effect compared to in heart So important to have Sometimes epinephrine will sit on a different sufficient levels of calcium adrenergic receptor, not beta, alpha. Glucagon ○ If im smooth muscle cell and my adrenergic ○ In contrast to insulin receptor is alpha not beta, my results will be Insulin: enhances the absorption of that smooth muscle will contract glucose from the blood by insulin If different receptor, then different G proteins, then sensitive cells different result ○ Glucagon opposite ○ When the blood level drops. Glucagon —------------------—------------------—------------------—------------------ hormone will now pack to increase glucose levels in the blood ○ 2nd messenger involved in the activity of ASTHMA SITUATION EXAMPLE FOR ENHANCED glucagon is also cyclic AMP AIRFLOW Protein is GS But receptor is not adrenergic receptor, it is glucagon receptor In asthma attacks, doctors will give epinephrine so Its the different situations that will that the cAMP will decrease, causing the smooth call for the different responses; muscles lining the bronchioles to increase in glucagon and epinephrine diameter/widen → better airflow Prtotein kinase A results in high cAMP Partner of beta-adrenergic is GS protein ○ Adenylate Cyclase will be activated ○ And what will be produced? cAMP (cyclic AMP) ○ What will be activated? Protein kinase A Protein kinase can phosphorylate myosin → to form cross links allowing muscles to contract. —------------------—------------------—------------------—------------------ —------------------—------------------—------------------—------------------ SUMMARY OF DISCUSSION Summarise what we have discussed When we have epinpehirne again on beta adrenergic receptor in the heart muscle ○ Faster tibok ○ Higher cAMP we have higher contractions, and faster If certain smooth muscle cells, has beta adrenergic receptor ○ Elevated levels of cAMP = smooth muscle  ○ It can cause glycogen degradation, gluconeogenesis, inhibit glycogen synthesis Signal Transduction: Chemical Messenger ○ Bounded by beta-adrenergic receptor RECORDING; 11/25 ○ Epinephrine acts when there is a fight or flight situation ○ Glucagon, even if there is no figh or flight EPINEPHRINE situation, the glucagon will take over. MEDICATION Epinephrine on beta-1 (heart) and beta-2 (smooth muscle) adrenergic receptors (couple to Gs) Cardiac muscle- higher cAMP, greater, faster cAMP phosphodiesterase inhibitors for asthma contraction patients ○ ○ Methylxanthines (theophyline, theobromine, Smooth muscle- higher cAMP, relaxation, vasodilation caffeine) ○ Smooth muscles in arterioles supplying the Beta-antagonists for asthma patients heart, the skeletal muscle- vasodilation ○ Salbutamol (or albuterol, ventolin), ○ Smooth muscles lining the bronchioles terbutaline (bricanyl, pulmoxcel) ○ Smooth muscles lining the GI- less motility Beta-1 blockers for heart patints: with tachycardia, hypertension, arrhythmia etc. Example: ○ You are an asthmatic patient, since you have a severe case of asthma, the doctor will give you epinephrine so that the cAMP in the cells lining the bronchioles will relax the smooth muscle cell, because you’ll need more air when you are having an asthmatic attack ○ But wont it also affect the heart? The heart now, since there is a high level of cAMP, you heart will now contract faster What if the patient has a heart problem? If you are a doctor or pulmonologist ylu have to know the entire well-being of your patient. We said that it is not only epinephrine that is Because when you treat the considered as a ligand. asthma, which can cause a side This table shows that it is not only epinephrine, that effect to your heart, it will affect the will attatch to an adregenic receptor tas is associated patient. with the GS. ○ There are different ligands This table is very useful Shows other receptor such as ADH receptor and parathyroid receptor (parathyroid as ligand) All of these are associated with the Gs ○ Because here the second messenger is cAMP Depends on the cell (target tissue) even if the same ligand, the response would be different. ○ Why? Even if all of them activates pKA, there are different types of things the pKA will act on (the downstream) For example, glucagon as ligand, glucagon has the same effect with epinephrine in the liver.  activation of the PRotein kinase A. In the muscle cell, what the protein kinase A phosphorylates is the myosin light chain kinase (MLCK) The protein kinase A occupies part of the MLCK causing it to be phosphorylated which affects its affinity. ○ The MLCK affinity to the calcium calmodulin decreases which inhibits the contraction, that’s what cause it to relax. Whereas when we go back to the heart, the protein kinase A does not phosphorylate the MLCK. Because the MLCK in the heart is different from the one in the smooth muscle. ○ The protein kinase A phosphorylates the actual chain and not the Kinase. ○ Smooth muscle: myosin light chain KINASE ○ Heart: Myosin light CHAIN In the heart, when there is a high cAMP it will contract, while in the smooth muscle, it will relax. Because we are talking the same the epinephrine and EPINEPHRINE: INHIBITORY beta-adrenergic receptors , which is associated with Gs Adenylate cyclase will be activated and produce the protein kinase A TO CLARIFY: in the heart, the pkA phosphorylates is the myosin light CHAIn (which is part of the myosin molecule, which is a link). This will enhance the contraction ○ The pka in the heart is not phosphorylating the myosin light chain KINASE. Go back Here we have our ligand: epinephrine You have the same ligand, that is attaching to different receptors even if they are occupying the beta-adrenergic receptor In the smooth muscle, if there is high cAMP the effect ○ Alpha 2 / alpha-1 would be relaxation. Note, alpha-2 the partner is Gi, which is inhibitory. ○ Why relaxation? You have Gs (we are talking ○ Alpha-2= inhibits adenylate cyclase= lower of the beta-adrenergci receptor) cause the cAMP= will cause it to contract, especially increase in the cAMP. The cAMP cause the the vascular smooth muscle  ○ Because the cAMP is low, the calcium concentration in the cell will be low as well Calcium is important in the exocystosis, such as the release in the neurotransmitter. If the calcium is low, the release of cellular products goes down. If neuron is involved, there is also a decrease or inhibition in the production of neurotransmitter. EPINEPHRINE: GP/GQ In the first one, the cell has beta-adrenergic receptors, the partner G protein would be the Gs. In the second cell, there is alpha-adrenergic receptor. (alpha-1) ○ Partner would be Gp/Gq ○ Gp/Gq= has the similar structure with your Gs, they are hetero trimer (3 molecules with different sturcutures). Activation of the response? The binding of the correct ligand to the correct receptor. The binding of you ligand (epinephrine) to your adrenergic receptor (alpha-1) will cause conformational change to your receptor. ○ This allow the attachment of the beta gamma subunit to the receptor which cause the GDP 3rd G protein- this time, Gp/Gq to be replaced by GTP which causes the G ○ p= phospholipase protein to be active. Receptor: alpha-1 adrenergic receptor When your G protein is active, the alpha subunit with If the G protein is Gp/Gq the receptor that is activated the attached GTP will dissociate from the is the Phospholipase C-beta beta-gamma. Second messenger produced: DAG, IP3 The alpha subunit of the Gp/Gq will not activate adenylyl cyclase instead it will activate the enzyme called phospholipase C-beta. ○ This will catalyze the formation of DAG and IP3 after this you will have downstream processes ○ IP3 is associated with the increase in calcium that calcium can come from your SER and will work with another molecule called calmodulin. ○ Protein Kinase C will be activated by the DAG  ○ This receptor is also found in the smooth muscle of your ureter , urethral sphincter, vas defrens and arrector pili muscle. You have your 2 kidneys each one would have its own ureter that will bring the urine to your urinary bladder. The urinary bladder would have your urtehtral sphincter All of this will contract during a fight or flight case. ○ smooth muscle in urinary bladder (but greater relaxing effect of B3 adrenergic receptors) SO BASICALLY, in emergency situations the smooth muscle, lining the bronchioles (they need to expand, relax), blood vessels feeding the heart and skeletal muscle (they also need to relax, so they have more supply), they have to be activated during fight or flight ○ But during fight or flight, the organs that are not needed will be constricted with blood Overview flow (so it will be redirected to parts that The Ligand binds to your alpha-1 adrenergic receptor need it) The associated g protein would be your Gp/Gq The phospholipase C will be activated and cause the production of your IP3 and DAG When IP3 is produced calcium is increased ○ When calcium is increase= smooth muscle cell contracts EPINEPHRINE ON a-1 adrenergic receptor couple to Gp - higher Cat+, greater contraction, vasoconstriction ○ If there is an increase in IP3, there will be an increase in calcium Because it will activate the release of calcium from your SER ○ When there is a high concentration of calcium then your muscle will contract ○ If your muscle is in your blood vessel, will Alpha1 (Gq): will increase calcium release from constrict. calcium storage structure alpha 1 adrenergic receptors on ○ Smooth muscle will constrict in blood ○ Alpha-1 adrenergic receptors are found in vessels, in intestine etc (vasocontsrict) your: smooth muscle in your blood vessels ○ Pyloric sphincter contraction (smooth which supplies your intestines, skin, kidney muscle): stomach. and other visceral organs that do not need to The sphincter on top, from the response to an emergency situation esophagus: cardiac sphincter Therefore there will be less blood If you have problem inyour flow to this organ so that it will be cardiac sphincter it can redirected to those require more cause gerd (since the food blood such as your heart or skeletal goes back to your muscle esophagus) The pyloric sphincter leads to the small intestines  ○ Pupillary dilation: iris dilatory muscle will This includes phosphatidylinositol and with 2 contract causing the pupil to increase in size phosphate (added to the backbone) This is a fight or flight response, ○ Called the: phosphatidylinositol-4,5- making you more alert biphosphate (PIP2) ○ Urinary sphincter contraction: The enzyme involved: phospholipase C (for your Gq/Gp protein) ○ Complete name: phospholipase C beta PHOSPHOGLYCERIDES Activates and catalyze the cleavage of your PIP2 into 2 molecules. 2 molculecues, one will be IP3 (will go to the cytosol). The other one that will be left behind, DAG, which is attatchted to the plasma membrane (doesnt get dettatched), will remain in the plasma membrane WHAT HAPPENS? Review : 2 backbone of the phospholipid ○ Glycerol and sphingosyl The orange molecule: ○ Glycerol Green molecule: ○ Fatty acid Inositol: alcohol sugar ○ One encircled below: phosphotadyl-inositol ○ Has only 1 phosphate (ulit ulit) Epinephrine/norepinephrine binds to the alpha-1 adrenergic receptor. This will be couple with the G protein: Gp/Gq Gp/Gq activates the phospholipase C beta ○ It will catalyze the cleavage of your PIP2, to form 2 molecules and this are the IP3 and DAG  Cellular metabilism includes stimulation of cell growth and differentiation. Regualtion of ion channels, changes in cytoskeleton, secretion of susbtances AND increases calcium entry into your smooth muscle cell. IP3:Inositol triphosphate Your IP3 will go to your cytosol, and DAG will remain in the plasma membrane When there is inositol triphosphate (InsP3/IP3) your calcium level in the cytosol will increase. ○ Even if your calcium level increases in your cytosol, the calcium level on the other side of the cytosol (such as SER and extracellular) Remember that the concentration of calcium in your will remain higher. cytosol is always lower compared to the other side What does Diacylglycerol do (DAG)? ○ Even if there is an increase in the calcium ○ It activates the protein kinase C level, such as when the SER releases calcium, it will still be lower in the cytosol. ○ It i also important that when the calcium level increases in response to the stimulus it should always go back to its old level. What type of transport is involved in the transport of calcium from your SER to cytosol? ○ High to low ○ Passive transport : facilitated diffusion Because you are transporting calcium which is an ion. The calcium channel opens up when there is a ligand attatched to it ○ Ligand: IP3, binds to the receptor (IP3 receptor) ○ Result: calcium will increase in your cytosol In your SER you also have calcium channel whcih is the ryanodine calcium channel. ○ This channel is sensitive to the molecule ryanodine. ○ When this channel is open , it will release calcium ○ This channel releases calcium when there is What does protein kinase C do? muscle contraction : cardiac, skeletal, ○ PKC phosphorylates traget proteins which smooth muscles, neuron etc. results cellular metabolism  There is a phenomenon called calcium induced calcium release ○ If calcium is being release to your cytosol it will cause more increase in the calcium level ○ Example: if you have IP3 that causes calcium release from the SER we now have a higher calccium in your cytosol which will also activate your ryanodine channel to release calcium which will activate MORE IP3 channel to release calcium. You have to stop the response (calcium release) to lower the calcium level ○ You have the Calcium ATPase pump. What type of transport? Active transport Low to high ○ What is the mode of transport from your cytosol to the outside of the cell? Calcium is often paired with calmodulin in response Low to high Active transport ○ How does calcium go in thru the plasma membrane via the calcium channel? High to low Outside cell high than cytosol Facilliated diffusion ○ How about in your sodium calcium pump? From cytosol to outside Active transport Low to high Since this is not an ATPase, where does your metabolic energy come from? From your sodium gradient Because you have a