Bipn140 Lect 10 Figs Oct10

Bipn140 Lect 10 Figs Oct10 - BIPN 140: Cellular...

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Unformatted text preview: BIPN 140: Cellular Neurobiology LECTURE #10: Intracellular Signaling [Website:] INSTRUCTORS Nicholas C. Spitzer ([email protected]) Darwin K. Berg ([email protected]) ANNOUNCEMENTS Prob Set #4 was posted on the Web 10/25/10. Prob Set #5 will be posted 11/1/10. Fig 7.1 Chemical signaling mechanisms Fig 7.2 Amplification in signal transduction pathways Fig 7.3 Three classes of cell signaling molecules Fig 7.4 Categories of cellular receptors Fig 7.5 Types of GTP-binding protein Fig 7.6 Effector pathways associated with Gprotein–coupled receptors Fig 7.7b Neuronal second messengers Fig 7.7c Neuronal second messengers Fig 7.7d Neuronal second messengers Fig 7.8 Regulation of cellular proteins by phosphorylation Fig 7.9 Mechanism of protein kinase activation Box 7Bb,c Dendritic Spines Box 7Bd/e Dendritic Spines Fig 7.10 Steps involved in transcription of DNA into RNA Fig 7.11 Transcriptional regulation by CREB •\ Fig 7.12 Mechanism of action of NGF Fig 7.13 Signaling at cerebellar parallel fiber synapses Fig 7.14 Regulation of TH by protein phosphorylation AN ENDOGENOUS MORPHINE-LIKE FACTOR IN MAMMALIAN BRAIN by Gavril W. Pasternak, Robert Goodman and Solomon H . Snyder Life Sciences 16:1765-1769 (1975) BACKGROUND The nervous system was known to react to opiates (e.g. morphine) and to contain receptors that bind opiates. These so-called opiate receptors which were later shown to be GPCRs and potentially of hugh biomedical relevance had no known endogenous ligands. EXPERIMENTS Take advantage of the fact that opiate receptors can be quantified by their ability to bind radioactive opiate analogs (e.g. 3H-naloxone). The labeled receptorligand complex can be separated from free ligand and measured for bound radioactivity. The strategy: Test whether unidentified compounds present in brain extracts could specifically block the binding of the 3H-naloxone to the receptor. The hypothesis: Brain contains endogenous natural ligands for opiate receptors, and these ligands should be effective competitors against radiolabeled analogs in binding assays when receptor is limiting in amount. Fig 1: A naturally occuring morphine-like factor (MLF) competes with 3H-naloxone for binding to the opiate receptor Shown is the amount of inhibition (%) of total 3H-naloxone (35,000cpm/ml) binding caused by calf MLA by itself (“None”; 60%) or in the presence of additives (Mn, Na, K, & EDTA). Sodium, unlike other cations or EDTA, is known to inhibit opiate binding. (Note: % inhibition was always determined as the difference between MLA alone and MLA + additive, thereby correcting for sodium effects on both MLA and 3H-naloxone.) Table 2 MLF Inhibition of 3H-Naloxone Binding in Membranes Treated with Enzymes and Reagents Rat brain membranes were prepared and treated with trypsin (0.5 µg/ml), chymotrypsin (50 µg/ml), N-ethylmaleimide (10 µM), iodoacetamide (5 mM), and p-chloramercuribenzoate (10 µM), for 20 min at 25°C and the reagents or enzymes washed out by centrifugation. Aliquots of the tissue were then assayed with 3H-naloxone and 1 mM MgCl2 in the presence and absence of calf MLF (100 µl), RESULTS Mammalian brain extracts have a protease-sensitive component that competes with radiolabeled opiates for binding the opiate receptors. Subsequent purification and characterization has identified the component(s) as the peptide enkephalin which comes in two variants: metenkepalin and leu-enkephalin. TAKEHOME: Enkephalins are neuropeptides that represent the normal ligands of opiate receptors. They activate the receptors to drive downstream signaling pathways. ...
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This note was uploaded on 10/30/2010 for the course BIPN BIPN 140 taught by Professor Spitzer during the Fall '07 term at UCSD.

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