title: Role of guanine nucleotide regulatory proteins and inositol phosphates in the hormone induced mobilization of hepatocyte calcium.
abstract: Treatment of isolated hepatocytes with F- produced a concentration-dependent activation of phosphorylase, efflux of Ca2+, rise in [Ca2+]i, increase in Ins 1,4,5-P3 levels, decrease in PI-4,5-P2 levels, and increase in DAG levels. The levels of intracellular cAMP were decreased by NaF. The effects of NaF were potentiated by AlCl3. This potentiation was abolished by the Al3+ chelator deferoxamine. These results illustrate that AlF4- can mimic the effects of Ca2+-mobilizing hormones in hepatocytes and suggest that the coupling of the receptors for these hormones to the hydrolysis of PI-4,5-P2 is through a guanine nucleotide-binding regulatory protein. This is because AlF4- is known to modulate the activity of other guanine nucleotide regulatory proteins (Gi, Gs, and transducin). Calcium-sensitive inositide release in a purified rat liver plasma membrane preparation was increased by calcium-mobilizing hormones in the presence of guanine nucleotides. Vasopressin-stimulated inositide release was evident in the presence of GTP or GTP gamma S. The guanine nucleotide and hormonal stimulation was evident on both inositide production and PI 4,5-P2 degradation. Treatment of plasma membranes with cholera toxin or islet activating protein or prior injection of animals with islet activating protein did not affect stimulation of inositide release by GTP gamma S or GTP gamma S plus vasopressin. The results suggest that calcium-mobilizing hormones stimulate polyphosphoinositide breakdown in rat liver plasma membranes through a novel guanine nucleotide binding protein. The GTPase activity of rat liver plasma membranes was stimulated 20% by 10(-8) M vasopressin. The vasopressin-stimulated GTPase activity was not inhibited in plasma membranes that had been ADP-ribosylated with either cholera toxin or pertussis toxin. When membranes that had been solubilized after preincubation with [3H]vasopressin were subjected to sucrose gradient centrifugation, most of the protein-bound [3H]vasopressin migrated as a single band, also, there was a GTPase activity that migrated with the bound [3H]vasopressin. This peak of bound [3H]vasopressin was decreased 90% when the sucrose gradient centrifugation was run in the presence of 10 M GTP gamma S. Direct evidence that a GTP-binding protein was present in the [3H]vasopressin peak was obtained by the immuno-detection of a 35 kDa beta subunit of a GTP-binding protein and a 40 kDa alpha subunit. These results support the conclusion that liver plasma membranes contain a GTP-binding protein that can complex with the vasopressin receptor.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
authors.forename: P F
authors.initials: PF
authors.lastname: Blackmore
authors.forename: C J
authors.initials: CJ
authors.lastname: Lynch
authors.forename: R J
authors.initials: RJ
authors.lastname: Uhing
authors.forename: T
authors.initials: T
authors.lastname: Fitzgerald
authors.forename: S B
authors.initials: SB
authors.lastname: Bocckino
authors.forename: J H
authors.initials: JH
authors.lastname: Exton
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology*; D007295:Inositol Phosphates / Q000502:physiology*; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D012969:Sodium Fluoride / Q000494:pharmacology; D013403:Sugar Phosphates / Q000502:physiology*; D014667:Vasopressins / Q000494:pharmacology*
chemical_list: D007295:Inositol Phosphates; D013403:Sugar Phosphates; D014667:Vasopressins; D012969:Sodium Fluoride; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D002118:Calcium