T. Ozawa et al., CHARACTERIZATION OF 2 DIFFERENT CA2-SENSITIVE CA2+ RELEASE MECHANISMSIN MICROSOMAL CA2+ POOLS OF RAT PANCREATIC ACINAR-CELLS( UPTAKE AND IP3), The Journal of membrane biology, 144(2), 1995, pp. 111-120
We have examined the effect of the Ca2+ (Mg2+)-ATPase inhibitors thaps
igargin (TG) and vanadate on ATP-dependent Ca-45(2+) uptake into IP3-s
ensitive Ca2+ pools in isolated microsomes from rat pancreatic acinar
cells. The inhibitory effect of TG was biphasic. About 40-50% of total
Ca2+ uptake was inhibited by TG up to 10 nM (apparent K-i approximate
to 4.2 nM, Ca2+ pool I). An additional increase of inhibition up to 8
5-90% of total Ca2+ uptake could be achieved at 15 to 20 nM of TG (app
arent K-i approximate to 12.1 nM, Ca2+ pool II). The rest was due to T
G-insensitive contaminating plasma membranes and could be inhibited by
vanadate (apparent K-i approximate to 10 mu M). In the absence of TG,
increasing concentrations of vanadate also showed two phases of inhib
ition of microsomal Ca2+ uptake. About 30-40% of total Ca2+ uptake was
inhibited by 100 mu M of vanadate (apparent K-i approximate to 18 mu
M, Ca2+ pool II). The remaining 60-70% could be inhibited either by va
nadate at concentrations up to 1 mM (apparent K-i approximate to 300 m
u M) or by TG up to 10 nM (Ca2+ pool I). The amount of IP3-induced Ca2
+ release was constant at approximate to 25% over a wide range of Ca2 filling. About 10-20% remained unreleasable by IP3. Reduction of IP3-
releasable Ca2+ in the presence of inhibitors showed similar dose-res
ponse curves as Ca2+ uptake (apparent K-i approximate to 3.0 nM for IP
3-induced Ca2+ release as compared to approximate to 4.2 nM for Ca2+ u
ptake at TG up to 10 nM) indicating that the highly TG-sensitive Ca2pump fills the IP3-sensitive Ca2+ pool I. At TG concentrations >10 nM
which blocked Ca2+ pool II the apparent K-i values were approximate to
11.3 and approximate to 12.1 nM, respectively. For inhibition by vana
date up to 100 mu M the apparent K-i values were approximate to 18 mu
M for Ca2+ uptake and approximate to 7 mu M for Ca2+ release (Ca2+ poo
l II). At vanadate concentrations up to 1 mM the apparent K-i values w
ere approximate to 300 and approximate to 200 mu M, respectively (Ca2 pool I). Both Ca2+ pools I and II also showed different sensitivities
to IP3. Dose-response curves for IP3 in the absence of inhibitors (co
ntrol) showed an apparent K-m value for IP3, at 0.6 mu M. In the prese
nce of TG (inhibition of Ca2+ pool I) the curve was shifted to the lef
t with an apparent K-m for IP3 at 0.08 mu M. In the presence of vanada
te (inhibition of Ca2+ pool II), the apparent K-m for IF, was 2.1 mu M
. These data allow the conclusion that there are at least three differ
ent Ca2+ uptake mechanisms present in pancreatic acinar cells: TG- and
IP3-insensitive but highly vanadate-sensitive Ca2+ uptake occurs into
membrane vesicles derived from plasma membranes. Two Ca2+ pools with
different TG-, vanadate- and IP3-sensitivities are most likely located
in the endoplasmic reticulum at different cell sites, which could hav
e functional implications for hormonal stimulation of pancreatic acina
r cells.