ISSN:
1432-1424
Keywords:
Thapsigargin
;
Vanadate
;
Ca2+ pump
;
Ca2+ ATPase
;
SERCA
;
Endoplasmic reticulum
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Abstract We have examined the effect of the Ca2+ (Mg2+)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent 45Ca2+ uptake into IP3-sensitive 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 Ki≈4.2 nm, Ca2+ pool I). An additional increase of inhibition up to 85–90% of total Ca2+ uptake could be achieved at 15 to 20 nm of TG (apparent Ki≈12.1 nm, Ca2+ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent Ki≈10 μm). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca2+ uptake. About 30–40% of total Ca2+ uptake was inhibited by 100 μm of vanadate (apparent Ki≈18 μm, Ca2+ pool II). The remaining 60–70% could be inhibited either by vanadate at concentrations up to 1 mm (apparent Ki≈300 μm) or by TG up to 10 nm (Ca2+ pool I). The amount of IP3-induced Ca2+ release was constant at ≈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-response curves as Ca2+ uptake (apparent Ki≈ 3.0 nm for IP3-induced Ca2+ release as compared to ≈4.2 nm for Ca2+ uptake at TG up to 10 nm) indicating that the highly TG-sensitive Ca2+ pump fills the IP3-sensitive Ca2+ pool I. At TG concentrations 〉10 nm which blocked Ca2+ pool II the apparent Ki values were ≈11.3 and ≈12.1 nm, respectively. For inhibition by vanadate up to 100 μm the apparent Ki values were ≈18 μm for Ca2+ uptake and ≈7 μm for Ca2+ release (Ca2+ pool II). At vanadate concentrations up to 1 mm the apparent Ki values were ≈300 and ≈200 μm, 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 (control) showed an apparent Km value for IP3 at 0.6 μm. In the presence of TG (inhibition of Ca2+ pool I) the curve was shifted to the left with an apparent Km for IP3 at 0.08 μm. In the presence of vanadate (inhibition of Ca2+ pool II), the apparent Km for IP3 was 2.1 μm. These data allow the conclusion that there are at least three different 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 have functional implications for hormonal stimulation of pancreatic acinar cells.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00232797
