Bile Acids Do Not Contribute to the Altered Calcium Homeostasis of Platelets from Rats with Biliary Cirrhosis

Abstract

Previously, we have found that intracellular calcium homeostasis is altered in platelets from an experimental model of liver cirrhosis, the bile-duct ligated (BDL) rat; these alterations are compatible with the existence of a hypercoagulable state and related to an enhanced intracellular calcium release evoked by thrombin and an increased amount of calcium stored in the intracellular organelles. In the present study we have investigated the role of bile acids in those alterations of the BDL cirrhotic model. Cholic acid (CA) or deoxycholic acid (DCA) did not change P-selectin expression or platelet aggregation in any group but elevated baseline platelet calcium levels. Incubation with both bile acids reduced calcium release after stimulation with thrombin in the absence of extracellular calcium. Pretreatment with CA but not with DCA reduced significantly thrombin-induced calcium entry in all three experimental groups. The capacitative calcium entry was also significantly lower in platelets pretreated with both bile acids. The simultaneous addition of thapsigargin and ionomycin to estimate the total amount of calcium in platelet internal stores was decreased by pretreatment with both CA and DCA, although these changes were significantly different in the control rats only with CA and in the BDL platelets with DCA. These results indicate that CA and DCA reduce calcium movements in platelets of control and BDL animals, thus suggesting that bile acids do not participate in the alterations observed in the BDL cirrotic model.

Keywords: bile-duct ligation; calcium signaling; capacitative calcium entry; cholestasis; fura-2; liver cirrhosis; thapsigargin; thrombin.

Figure 1

(A) Changes in [Ca²⁺]_i in platelets of control (left column), BDL (middle column) and BDL with ascites rats (right column) in response to thrombin (0.3 U/ml, added at time 0). Top, in the absence of extracellular Ca²⁺. Bottom, in the presence of extracellular Ca²⁺. Data were obtained in untreated platelets and after incubation with CA or DCA (100 μM each). (B). Area under the curve of the calcium changes after thrombin administration (0.3 U/ml, added at time 0) in the absence (top panels) and in the presence (bottom panels) of extracellular Ca²⁺. Data were obtained in untreated platelets and after incubation with CA or DCA (100 μM each). Results are means ± S.E.M. ^*p < 0.05 vs. Untreated.

Figure 2

(A) Changes in [Ca²⁺]_i after thapsigargin administration in the absence of extracellular Ca²⁺ (time 0 seconds) and after addition of calcium to allow for capacitative Ca²⁺ entry (time 180 s). Data were obtained in untreated platelets and after incubation with CA or DCA (100 μM). (B) Area under the curve of the calcium changes after thapsigargin administration in the absence of extracellular Ca²⁺ (top panels) and after addition of calcium to allow for capacitative Ca²⁺ entry (bottom panels). Data were obtained in untreated platelets and after incubation with CA or DCA (100 μM). Results are means ± S.E.M. ^*p < 0.05 vs. Untreated.

Figure 3

(A) Changes in [Ca²⁺]_i after thapsigargin and ionomycin administration (added at time 0) to allow for estimation of total calcium in intracellular stores. Data were obtained in untreated platelets and after incubation with CA or DCA (100 μM). (B) Area under the curve of the calcium changes after thapsigargin and ionomycin to estimate total calcium in intracellular stores. Data were obtained in untreated platelets and after incubation with CA or DCA (100 μM). Data are means ± S.E.M. ^*p < 0.05 vs. Untreated.