MICROVESICLES ISOLATED FROM BOVINE POSTERIOR PITUITARY ACCUMULATE NOREPINEPHRINE

Histochemical study indicated that the posterior pituitary possesses n umerous 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 Carnilli, P. (1989) J. Cell B iol. 109, 3425-2433). By monitoring cross-reactivity with anti synapto physin antibody, the MVs were highly purified from bovine posterior pi tuitaries by a combination of differential and sucrose density gradien t centrifugations. The purified MVs had an average diameter of about 6 0 nm and were associated with synaptophysin as revealed by immunoelect ron microscopy. The vesicles contained ATPase activity partially sensi tive to bafilomycin A(1) and to vanadate. The membrane fraction immuno isolated with anti-synaptophysin antibody also exhibited similar ATPas e activity. The two ATPases could be purified separately; the vanadate -sensitive enzyme was identified as a 115-kDa polypeptide immunochemic ally similar to chromaffin granule P-ATPase (forming phosphoenzyme int ermediate), and the bafilomycin A(1) sensitive ATPase showed essential ly the same properties as those of vacuolar type H+-ATPases. Upon addi tion of ATP, the MVs formed an electrochemical gradient of protons and took up norepinephrine in a reserpine-sensitive manner, indicating th e presence of secondary monoamine transporter coupled with vacuolar ty pe H+-ATPase. No uptake of L-glutamate, gamma-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.