Quantification of p38/synaptophysin in highly purified adrenal medullary chromaffin vesicles

p38/synaptophysin is a membrane protein present in clear (synaptic) vesicles of neurons and endocrine ceHs [1-4]. From the amino acid sequence deduced from cDNAs encoding p38/synaptophysin, a model with several membrane spanning polypeptide segments and a carboxy-terminal protein domain exposed to the cytoplasmic surface has been constructed [5-7]. The function of p38/synaptophysin is not known. It has been suggested to form a transmembrane channel for ions, or to interact with cytoplasmic factors via its cytoplasmic domain [7]. Since synaptophysin binds Ca2 +, it may also play a role in the release of neurotransmitters stored in clear (synaptic) vesicles [3]. Recently it has been reported [8] that p38/synaptophysin also occurs in hormone containing large dense core vesicles. This would imply that p38/synaptophysin could fulfill similar functions as described above in chromaffin and other secretory ceHs containing large dense core vesicles. In dear (synaptic) vesicles p38/synaptophysin constitutes 7.51Jfo of the vesicle membrane proteins [I]. The amount of p38/synaptophysin in large dense core vesides is not known. Here we report on the quantification of p38/synaptophysin in highly purified chromaffin secretory veside

Uptake of Ca2+ by isolated secretory vesicles from adrenal medulla

Intact secretory vesicles isolated from bovine adrenal medulla contain 94 nmol Na+ per mg of protein, and Ca2+ influx into the vesicles is inhibited by increasing concentrations of extravesicular Na+ (but not of K+, Li+ or choline+) or by addition of the Na+ ionophore monensin. Thus Ca2+ influx is determined by the Na+ gradient across the vesicular membrane. Half maximal inhibition of Ca2+ influx occurs with 34 mM Na+ extravesicularly. The fact that Ca2+ can also be released from the vesicles by inversion of the Na+ gradient provides direct evidence that an Na+-Ca2+ exchange may operate. According to an analysis of the inhibition of Ca2+ uptake by Na+ in a Hill plot 2 Na+ would be exchanged for 1 Ca2+. Ca2+ influx into the vesicles increases with temperature (energy of activation: 16 kcal/mol), can be observed already with 10−7 M free Ca2+ and increases up to 10−4 M Ca2+. Ca2+ influx is not affected by Mg2+ but Sr2+ is inhibitory. Since the process is only slightly influenced by the pH of the incubation medium and is insensitive to Mg2+-ATP or inhibitors of the proton translocating Mg2+-ATPase the electrochemical proton gradient across the vesicular membrane does not affect directly the Ca2+ influx into the secretory vesicles. Ca2+ uptake is insensitive to ruthenium red and oligomycin

Influx of Ca2+ into isolated secretory vesicles from adrenal medulla Influence of external K+ and Na+

Secretory vesic1es from adrenal medulla contain catecholamines, nuc1eotides and proteins, all of which are released into the extracellular fluid during exocytosis. Adrenal medullary secretory vesic1es also contain high concentrations of Ca'+ [1]. The mechanism of the aecumulation of Ca 2+ into the vesicles is largely unknown and the experimental data eoncerning the uptake of Ca'+ into isolated secretory vesicles are contradictory. It has been reported that secretory vesicle membranes are impermeable to Ca'+ [2], that secretory vesicles take up Ca 2+ independently of ATP [3] and that they possess an ATP-stimulated uptake system [4,5]. In earlier work relatively impure and unstable seeretory vesicle fractions were used for the determination of Ca 2+ -uptake. We have developed a method to isolate highly purified and stable secretory vesicles from bovine medulla [6]. With these vesic1es we repeated earlier Ca'+ -uptake experiments and found that: (i) The vesic1es take up <sCa2+ in K+-containingmedia; (ü) 4SCa2+ uptake is abolished in the presence ofNa+; (üi) nie Ca 2+ content of isolated secretory vesic1es is increased when incubated with Ca 2+ in media containing K+, but not in media containing Na +