title: Role of Ca2+ feedback on single cell inositol 1,4,5-trisphosphate oscillations mediated by G-protein-coupled receptors.
abstract: The dynamics of inositol 1,4,5-trisphosphate (Ins (1,4,5)P3) production during periods of G-protein-coupled receptor-mediated Ca2+ oscillations have been investigated using the pleckstrin homology (PH) domain of phospholipase C (PLC) delta1 tagged with enhanced green fluorescent protein (eGFP-PHPLCdelta1). Activation of noradrenergic alpha1B and muscarinic M3 receptors recombinantly expressed in the same Chinese hamster ovary cell indicates that Ca2+ responses to these G-protein-coupled receptors are stimulus strength-dependent. Thus, activation of alpha1B receptors produced transient base-line Ca2+ oscillations, sinusoidal Ca2+ oscillations, and then a steady-state plateau level of Ca2+ as the level of agonist stimulation increased. Activation of M3 receptors, which have a higher coupling efficiency than alpha1B receptors, produced a sustained increase in intracellular Ca2+ even at low levels of agonist stimulation. Confocal imaging of eGFP-PHPLCdelta1 visualized periodic increases in Ins(1,4,5)P3 production underlying the base-line Ca2+ oscillations. Ins(1,4,5)P3 oscillations were blocked by thapsigargin but not by protein kinase C down-regulation. The net effect of increasing intracellular Ca2+ was stimulatory to Ins(1,4,5)P3 production, and dual imaging experiments indicated that receptor-mediated Ins(1,4,5)P3 production was sensitive to changes in intracellular Ca2+ between basal and approximately 200 nM. Together, these data suggest that alpha1B receptor-mediated Ins(1,4,5)P3 oscillations result from a positive feedback effect of Ca2+ onto phospholipase C. The mechanisms underlying alpha1B receptor-mediated Ca2+ responses are therefore different from those for the metabotropic glutamate receptor 5a, where Ins(1,4,5)P3 oscillations are the primary driving force for oscillatory Ca2+ responses (Nash, M. S., Young, K. W., Challiss, R. A. J., and Nahorski, S. R. (2001) Nature 413, 381-382). For alpha1B receptors the Ca2+-dependent Ins(1,4,5)P3 production may serve to augment the existing regenerative Ca2+-induced Ca2+-release process; however, the sensitivity to Ca2+ feedback is such that only transient base-line Ca2+ spikes may be capable of causing Ins(1,4,5)P3 oscillations.
authors.affiliation: Department of Cell Physiology and Pharmacology, Medical Sciences Building, University of Leicester, University Road, Leicester LE1 9HN, United Kingdom. KWY1@LE.AC.UK
authors.forename: Kenneth W
authors.initials: KW
authors.lastname: Young
authors.forename: Mark S
authors.initials: MS
authors.lastname: Nash
authors.forename: R A John
authors.initials: RA
authors.lastname: Challiss
authors.forename: Stefan R
authors.initials: SR
authors.lastname: Nahorski
mesh_terms: D000818:Animals; D016466:CHO Cells; D020013:Calcium Signaling / Q000502:physiology*; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology; D025461:Feedback, Physiological / Q000502:physiology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis / Q000378:metabolism*; D009638:Norepinephrine / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D064529:Receptor, Metabotropic Glutamate 5; D043587:Receptor, Muscarinic M3; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism; D018094:Receptors, Metabotropic Glutamate / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D064529:Receptor, Metabotropic Glutamate 5; D043587:Receptor, Muscarinic M3; D018340:Receptors, Adrenergic, alpha-1; D018094:Receptors, Metabotropic Glutamate; D011976:Receptors, Muscarinic; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D011493:Protein Kinase C; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D019311:Staurosporine; D009638:Norepinephrine