Abstract
Saponin-treated liver cells and a microsomal fraction were used to characterize the mechanism of the Ca2+ release induced by different bile acids. The saponin-treated cells accumulated 0.8-1 nmol/mg of protein of the medium Ca2+ in a nonmitochondrial, high affinity, and inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3)-sensitive Ca2+ pool. Three of five bile acids tested, lithocholate and the conjugates taurolithocholate and taurolithocholate sulfate, released 85% of the Ca2+ pool within 45-60 s and with ED50 from 16 to 28 microM. Ins(1,4,5)P3 released 80% from the same Ca2+ pool with an ED50 of 0.3 microM. The Ca2+-Mg2+-ATPase inhibitor vanadate (1 mM) had no effect on the Ca2+ released by the bile acids and Ins(1,4,5)P3. The Ins(1,4,5)P3-binding antibiotic neomycin (1 mM) and the receptor competitor heparin (16 micrograms/ml) abolished the releasing effect of Ins(1,4,5)P3 but had no effect on the bile acid-mediated Ca2+ release. The 45Ca2+ accumulated by the microsomal fraction (8 nmol of 45Ca2+/mg of protein) was released by the bile acids within 45-90 s and with an ED50 of 17 microM. In contrast, the bile acids had no effect on the Ca2+ permeability of other natural and artificial membranes. The resting 45Ca2+ influx of intact cells (0.45 nmol/mg of protein/min), the 45Ca2+ accumulated by mitochondria (2-13 nmol of 45Ca2+/mg of protein), and the 45Ca2+ trapped in sonicated phosphatidylcholine vesicles (5 mM 45Ca2+) were not altered by the different bile acids. These results suggest that the Ca2+ release initiated by lithocholate and its conjugates results from a direct action on the Ca2+ permeability of the Ins(1,4,5)P3-sensitive pool. It is not mediated by Ins(1,4,5)P3 or via activation of the Ins(1,4,5)P3 receptor, and it is specific for the membrane of the internal pool.
Highlights
Kinetic Properties of the Different Bile Acids on Suponintreated Cell Cu2+-Experiments were performed to characterize further the kinetic properties of several bile acids in rat liver cells treated with 50 pg of saponin/ml and incubated in the internal medium containing K+, ATP, and themitochondrial uncoupler CCCP (5 p ~ (1)1, 20)
The first part of the results reported here have characterized the kinetic properties of several natural bile acids on the Ca2+sequestered in saponin-treated hepatocytes
Consistentwith a high affinity Ca2+pump, the medium Ca2+was rapidly reduced to about 150-200 nM and accumulated in theinternal pool(0.8-1 nmol/mg of cell protein) which accounts for 20-30%of the total cell Ca2+.Both data are reasonably close to the resting [Ca2+]; and tothe quantity of the Ca2+ mobilizedby Ins(l,4,5)P3-dependenthormones in intact hepatocytes (1-4)
Summary
Kinetic Properties of the Different Bile Acids on Suponintreated Cell Cu2+-Experiments were performed to characterize further the kinetic properties of several bile acids in rat liver cells treated with 50 pg of saponin/ml and incubated in the internal medium containing K+, ATP, and themitochondrial uncoupler CCCP (5 p ~ (1)[1, 20]). Fig. 1Bshows that theCa2+uptake could bereversed instantaneously by adding the Caz+ionophore ionomycin (5 p ~ )su, ggesting that a large Ca2+concentration gradient had been attained within the intracellular compartment. Perimental Procedures” amounted to 0.87 & 0.10 nmol/mg of cell protein ( n = 12). Consistent with our recent results (20), TLC at 100 p~ promoted a rapid and massive release of Ca2+ (Fig. lA). The sulfated derivative TLC-S (100 p ~ an)d the nonconjugated lithocholate (100 p ~ )a,lso induced a fast loss from the internal pool (Fig. lA).The Ca2+released by TLC amounted to 0.79 f 0.09 nmol/mg of cell protein ( n = 12) which approaches 85-90% of the ionomycin-sensitive Ca2+pool. Our results revealed no inhibitory effect of cell Ca2+on Caz+release initiated by the bileacids.
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