Miki Hara-Yokoyama

Presence of a Complex Containing Vesicle-associated Membrane Protein 2 in Rat Parotid Acinar Cells and Its Disassembly upon Activation of cAMP-dependent Protein Kinase

Amylase release from parotid acinar cells is mainly induced by the accumulation of intracellular cAMP, presumably through the phosphorylation of substrates by cAMP-dependent protein kinase (PKA). However, the molecular mechanisms of this process are not clear. In a previous study (Fujita-Yoshigaki, J., Dohke, Y., Hara-Yokoyama, M., Kamata, Y., Kozaki, S., Furuyama, S., and Sugiya, H. (1996) J. Biol. Chem. 271, 13130–13134), we reported that vesicle-associated membrane protein 2 (VAMP2) is localized at the secretory granule membrane and is involved in cAMP-induced amylase secretion. To study the formation of the solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex containing VAMP2 in parotid acinar cells, we prepared rabbit polyclonal antibody against the peptide corresponding to Arg47-Asp64 of VAMP2 (anti-SER4256). The recognition site of anti-SER4256 overlaps the domain involved in binding target membrane SNAREs (t-SNARES). Then we examined the condition of VAMP2 by immunoprecipitation with anti-SER4256. VAMP2 was not included in the immunoprecipitate from solubilized granule membrane fraction under the control conditions, but incubation with cytosolic fraction and cAMP caused immunoprecipitation of VAMP2. The effect of cytosolic fraction and cAMP was reduced by addition of PKA inhibitor H89. Addition of both the catalytic subunit of PKA and the cytosolic fraction allowed immunoprecipitation of VAMP2, whereas the PKA catalytic subunit alone did not. These results suggest that (1) the t-SNARE binding region of VAMP2 is masked by some protein Xand activation of PKA caused the dissociation of X from VAMP2; and (2) the effect of PKA is not direct phosphorylation ofX, but works through phosphorylation of some other cytosolic protein.

cGMP production is coupled to Ca2+-dependent nitric oxide generation in rabbit parotid acinar cells

We investigated the mechanism of guanosine 3,5-monophosphate (cGMP) production in rabbit parotid acinar cells. Methacholine, a muscarinic cholinergic agonist, stimulated cGMP production in a dose-dependent manner but not isoproterenol, a beta-adrenergic receptor stimulant. Methacholine-stimulated cGMP production has been suggested to be coupled to Ca2+ mobilization, because intracellular Ca2+ elevating reagents, such as thapsigargin and the Ca2+ ionophore A23187, mimicked the effect of methacholine. The cGMP production induced by Ca2+ mobilization has also been suggested to be coupled to nitric oxide (NO) generation because the effects of methacholine, thapsigargin and A23187 on cGMP production were blocked by NG-nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of nitric oxide synthase (NOS), and hemoglobin, a scavenger of nitric oxide (NO). Sodium nitroprusside (SNP), a NO donor, stimulated cGMP production. Furthermore, methacholine stimulated NO generation, and NOS activity in the cytosolic fraction in rabbit parotid acinar cells was exclusively dependent on Ca2+. These findings suggest that cGMP production induced by the activation of muscarinic cholinergic receptors is coupled to NO generation via Ca2+ mobilization.

Endogenous inhibitor of the ADP-ribosylation of (a) G-protein(s) as catalyzed by pertussis toxin is present in rat liver

The inhibitor activity of the ADP‐ribosylation of (a) G‐protein(s) as catalyzed by pertussis toxin was found in the membrane extract of rat liver. The inhibitor activity was found in the fractions of DEAE‐Sephacel column chromatography at 50–120 mM NaCl. The inhibitor activity is not due to the degradation of NAD nor to the reverse reaction of pertussis toxin (removal of incorporated ADP‐ribose). The present result suggests the presence of an endogenous inhibitor of the ADP‐ribosylation reaction of (a) G‐protein(s).

An endogenous inhibitor of the ADP-ribosylation of GTP-binding proteins by pertussis toxin is present in bovine brain.

The ADP-ribosylation of GTP-binding proteins (G-proteins) catalyzed by pertussis toxin was inhibited by endogenous inhibitor activity in the membrane extract of bovine brain. Most of the activity appeared in the fractions eluted from a DEAE-Sephacel column by 0.5 M NaCl. The activity was heat-stable and sensitive to pronase K. The results suggest the presence of an endogenous inhibitor of pertussis toxin in bovine brain.