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Cellular Physiology - Dr. Rylett.docx

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Physiology 3140A
Donglin Bai

Cellular Physiology: Dr. Jane Rylett Cyclic Nucleotides: Cyclic AMP & Protein Kinase A Introduction • Cyclic AMP (cAMP) o Synthesized from ATP by plasma membrane-bound adenylate cyclase o Degraded to 5’-AMP by cyclic AMP-specific phosphodiesterase o Function as intracellular messengers – [ ] (normally <10 M) must be able to change rapidly in response to extracellular signals (ex. hormone can change [cAMP] by 5-fold within few seconds) -7  Need at least 10 [cAMP] to activate pathways o Primary physiological function is to serve as intracellular second messenger to bind & activate protein kinase A (PK-A) o Another role acts as ligand for a specific class of odorant cation channels in olfactory neurons Adenylate Cyclase • Membrane bound protein, found at plasma membrane • Consists of 2 alternating hydrophobic & hydrophilic domains o Hydrophobic domains each containing 6 membrane-spanning domains o Hydrophilic regions having 2 catalytic domains • Multiple isoforms • In general – stimulating bs G and inhibited iy G • At least 1 isoform complexes with calcium bound calmodulin leading to modulation of enzymes activity • Converts ATP to camp =  [cAMP] Cyclic AMP-Dependent Protein Kinase A (PK-A) Phosphorylation of Cellular Proteins • Inactive PK-A is a hetero-tetrameric complex: o 2 catalytic subunits o 2 regulatory subunits • Heterotetramer – inactive form – high binding affinity to catalytic subunits  catalytic domains are hidden within regulatory subunit • 2 cAMP binds to each regulatory subunits  conformational change but still are attached  release active catalytic subunits (no affinity) • Separated catalytic subunits = amplification (12) • Constitutively active (no other activating mechanism – no phosphorylation) • Activating of cAMP-dependent PKA o Allosteric mechanism enhances response of PK-A to changes in cAMP concentration nd  First molecule of cAMP – alters the ability of 2 molecule of cAMP to bind  Increases affinity  If you didn’t have allosteric binding – 2 molecule wouldn’t bind because phosphodiesterase would degrade and reduce [ ] o Heterotetramers are tethered/anchored to cytoplasm constituents (aKAP – A Kinase Anchor Protein) o When catalytic subunits are freed & active  Can stay in cytoplasm or migrate into nucleus  Serine/threonine kinase  phosphorylate transcription factors  Regulatory subunits remain in the cytoplasm • Inactivation of cAMP-dependent PKA o Dissociation of cAMP from regulatory subunits associated with decrease [cAMP] in the cell  Leads to binding of regulatory & catalytic subunits to make inactive heterotetramer o Catalytic subunits can be inhibited by endogenous cellular proteins: protein kinase inhibitors  Bind irreversibly to catalytic subunits thereby inhibiting their catalytic functions  Precise physiological role is not entirely known but appear to compete with regulatory subunits for binding to and inactivating of catalytic subunits  Need more transcription/translation to get more signaling Activation of Gene Transcription • DNA-binding proteins whose transcriptional functions are activated by phosphorylation by PK-A • Proteins bind to specific DNA sequences located in the promoters of many genes • DNA binding sequences - CRE (cAMP response element) • Protein transcription factor - CREB (cAMP response element protein) • CREB - membrane of a large superfamily of DNA-binding proteins known as bZIP proteins o Proteins get their name because their DNA-binding domain consists of  Basic region (b) involved in recognition & binding to DNA  Leucine zipper (ZIP) consisting of long repeats of leucines responsible for dimerization – transcription factors always work as dimers • Transcriptional activity of CREB is regulated by cAMP-dependent signaling pathway & represents final communication link in regulation of gene expression in response to activation of this signaling system Representation Cellular Actions of cAMP-Dependent Protein Kinase A Glycogen Phosphorylase & Glucose Metabolism • Circulating adrenaline induces muscles cells to break down glycogen to glucose-1-phosphate & stop synthesizing glycogen • Glucose is then oxidized by glycolysis to provide ATP for sustained muscle contraction • Mechanisms o Adrenaline causes a β-adrenergic (on cell surface)-induced increase in cellular cAMP causing PKA to phosphorylate 2 other enzymes  Phosphorylase kinase  phosphorylates glycogen phosphorylase  activated to release glucose residues from glycogen molecules  Glycogen synthase (performs final step in glycogen synthesis from glucose) is inhibited by phosphorylation shutting off glycogen synthesis Phosphatases Rapidly Reverse Effects of Protein Kinase A • Dephosphorylation of phosphorylated serine & threonine residues is catalyzed by a group of 4 serine/threonine phosphatases • Protein Phosphatase-1 (PP-1) dephosphorylates proteins phosphorylated by PKA • Activity of PP-1 in adrenaline-stimulated skeletal muscle cells is suppressed by phosphatase inhibitor protein (PIP) which is itself a substrate for PKA • When PIP is phosphorylated by PKA it binds to & inactivates PP-1 • By simultaneously activating phosphorylase kinase & inhibiting the opposing action of PP- 1 – PKA causes a much larger change in glycogen metabolism than could be obtained by one mechanism alone PKA – constitutively active Phosphorylase kinase – not Active not phosphorylation constitutively active constitutively activee – not Cyclic GMP & Nitric Oxide in Cell-Cell Signaling Introduction • cGMP is formed in many cell types from GTP by a catalytic reaction with guanylate cyclase • Amount of cGMP in a cell is determined by the balance between its synthesis and degradation by a cGMP- specific phosphodiesterase Guanylate Cyclase • 2 forms of guanylate cyclase in cells: cytosolic form & membrane-associated form • Unlike a number of other enzymes (ex. PKC) – cytosolic & membrane-bound forms do not inter-convert by translocation of enzyme protein between compartments • 2 forms have different gene products & distinct forms of protein both of which convert GTP to cGMP Cytosolic Guanylate Cyclase: Regulation of cGMP in Vascular Endothelium Experimental Design & Results • Blood vessels have a smooth muscle wall which is separated from blood by a endothelial layer • Several hormones transiently increase cGMP levels in vascular smooth muscle & cause relaxation of muscle cells (vasodilation) • 2 anomalous pieces of information made it difficult to determine the mechanism of action of hormones o Relaxation required participation of vascular endothelial cells, since muscle in which endothelium was removed were no longer sensitive to hormones (remained sensitive to cGMP analogues) o Hormone effects on cGMP could not be demonstrated in cell homogenates (grind it up/lysates) even when endothelial cells were present in the homogenate • Indicated that hormones acted on endothelial cells through release on some secondary transmitter or substance that in turn activated cGMP synthesis in mu
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