Microvesicles Isolated from Bovine Posterior Pituitary Accumulate Norepinephrine

• Published in: The Journal of biological chemistry Vol. 270; no. 19; pp. 11424 - 11429
• Main Authors: Moriyama, Yoshinori, Yamamoto, Akitsugu, Yamada, Hiroshi, Tashiro, Yutaka, Tomochika, Ken-Ichi, Takahashi, Masami, Maeda, Masatomo, Futai, Masamitsu
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 12.05.1995; American Society for Biochemistry and Molecular Biology
• Subjects: Acetylcholine - metabolism; Adenosine Triphosphatases - analysis; Adenosine Triphosphatases - metabolism; Animals; Anti-Bacterial Agents - pharmacology; Cattle; Cell Fractionation; Centrifugation, Density Gradient; Electrochemistry; gamma-Aminobutyric Acid - metabolism; Glutamic Acid - metabolism; Glycine - metabolism; Macrolides; Microscopy, Electron; Neurotransmitter Agents - metabolism; Norepinephrine - metabolism; Organelles - metabolism; Organelles - ultrastructure; Pituitary Gland, Posterior - metabolism; Pituitary Gland, Posterior - ultrastructure; Proton-Translocating ATPases - antagonists & inhibitors; Synaptophysin - analysis; Vanadates - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.19.11424

Histochemical study indicated that the posterior pituitary possesses numerous microvesicles (MVs) containing synaptophysin, a marker protein specific for brain synaptic vesicles (Navone, F., Di Gioia, G., Jahn, R., Browning, M., Greengard, P., and De Camilli, P. (1989) J. Cell Biol. 109, 3425–2433). By monitoring cross-reactivity with anti-synaptophysin antibody, the MVs were highly purified from bovine posterior pituitaries by a combination of differential and sucrose density gradient centrifugations. The purified MVs had an average diameter of about 60 nm and were associated with synaptophysin as revealed by immunoelectron microscopy. The vesicles contained ATPase activity partially sensitive to bafilomycin A1 and to vanadate. The membrane fraction immunoisolated with anti-synaptophysin antibody also exhibited similar ATPase activity. The two ATPases could be purified separately; the vanadate-sensitive enzyme was identified as a 115-kDa polypeptide immunochemically similar to chromaffin granule P-ATPase (forming phosphoenzyme intermediate), and the bafilomycin A1-sensitive ATPase showed essentially the same properties as those of vacuolar type H+-ATPases. Upon addition of ATP, the MVs formed an electrochemical gradient of protons and took up norepinephrine in a reserpine-sensitive manner, indicating the presence of secondary monoamine transporter coupled with vacuolar type H+-ATPase. No uptake of L-glutamate, γ-aminobutyrate, glycine, or acetylcholine was observed. The identification of MVs as organelles responsible for storage of monoamines is important for understanding the physiological function of the posterior pituitary.