Abstract
The plasma membranes of isolated guinea pig hepatocytes were made permeable with saponin. The cells were then suspended in a medium resembling cytosol in which the level of ATP was kept constant with an ATP-regenerating system. Intracellular ATP-dependent 45Ca and 40Ca sequestration was then followed at various concentrations of Ca2+ in the medium. It was found that ATP-dependent Ca uptake could be divided into two mechanisms: a low affinity high capacity uptake sensitive to 2,4-dinitrophenol (DNP) and oligomycin, thought to be mitochondrial, and a low capacity high affinity uptake, which was insensitive to DNP and oligomycin, thought to be mainly endoplasmic reticulum (ER). The threshold for ATP-dependent Ca uptake by the latter pool was about 20 nM Ca2+. The process had an EC50 value of 0.3 microM (for 45Ca) and a capacity of 2.7 nmol/45Ca/mg of protein. The "ER" mechanism also had a high affinity for ATP (EC50, about 43 microM). There was no significant accumulation of Ca by the postulated mitochondrial pool until the [Ca2+] of the medium was greater than 1 microM. The concentration of Ca2+ in the cytosol of normal unstimulated hepatocytes was estimated from measurements of phosphorylase a activity to be about 0.18 microM. At this [Ca2+], the ER pool of the saponin-treated hepatocytes accumulated Ca but there was no evidence of any Ca uptake into the "mitochondrial" pool. This suggests that most of the exchangeable Ca in a normal cell may be in DNP and oligomycin-insensitive pools (presumably the ER or possibly the plasma membrane) and suggests that these pools are likely to be involved in the increase in cytosolic [Ca2+] which occurs after stimulation by Ca-mobilizing hormones.
Highlights
The plasma membranes of isolated guinea pig hepa- channels and a net release of Ca, mediated by tocytes were made permeable withsaponin
We have tried to measure the intracellular distribution mechanism alsohad a high affinity for ATP (ECSO, of exchangeable Ca inguinea pig hepatocytes as this obviously about 4 3 PM)
Phosphoryluse Measurements-The purpose of this investigation was to study the localization of Ca inguineapig hepatocytes by using saponin-treated cells as a model
Summary
The plasma membranes of isolated guinea pig hepa- channels (most species) and a net release of Ca, mediated by tocytes were made permeable withsaponin. Intracellular ATP-depend- cytes and because the hormone responsiveness of cells incuent 45Caand 40Casequestration was followed at bated in Ca-freemedium is only partially impaired As shown in other tissues (Glauert et centration of Ca2+in the cytosol of normal unstimu- al., 1962; Bangham and Horne, 1962; Blaustein et al, 1978a, lated hepatocyteswas estimated from measuremenotfs 1978b; Wakasugi et al, 1982) saponin interacts with cholesphosphorylase a activity to be about 0.18 PM At this terol in the cholesterol-rich plasma membrane to form pores [Ca2+],the ER pool of the saponin-treatedhepatocytes which make the membrane freely permeable to small moleaccumulated Ca but there was no evidence of any Ca cules and ions.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have