Effect of cholesterol liposomes on calcium mobilization in muscle cells from the rabbit sphincter of Oddi

Abstract

AIM: To analyze the influence of cholesterol liposome on the Ca²⁺ mobilization of cultured muscle cells in rabbit sphincter of Oddi’s.

METHODS: New Zealand rabbit was sacrificed and the sphincter of Oddi (SO) segement was obtained aseptically. The SO segment was cut into pieces and cultured in DMEM solution. Then the smooth muscle cells were subcultured, and the 4th-7th passage cells were used for further investigation. The intracellular Ca²⁺ increase was measured under confocal microscope after the addition of 20 mmol·L^-1 KCl, 10^-7 mol·L^-1 acetylcholine and 10^-7 mol·L^-1 cholecystokinin, and different antagonists were added to analyze the Ca²⁺ mobilization pathway. After the cells were incubated with 1 g·L^-1 cholesterol liposome (CL)(molar ratio was-2:1), the intracellular Ca²⁺ increase was measured again to determine the effect of CL on cellular Ca²⁺ mobilization.

RESULTS: The resting cellular calcium concentration of cultured SO cell was 108 nmol·L^-1± 21 nmol·L^-1. The intracellular Ca²⁺ increases induced by 20 mmol·L^-1 KCl, 10^-7 mol·L^-1 ACh and 10^-7 mol·L^-1 CCK were 183% ± 56%, 161% ± 52% and 130% ± 43%, respectively. When the extracellular Ca²⁺ was eliminated by 2 mmol·L^-1 EGTA and 5 μmol·L^-1 verapamil, the intracellular Ca²⁺ increases induced by KCl, ACh and CCK were 20% ± 14%, 82% ± 21% and 104% ± 23%, respectively. After the preincubation with heparin, the Ca²⁺ increases were 62% ± 23% and 23% ± 19% induced by ACh and CCK, as for preincubation with procaine they were 72% ± 28% and 85% ± 37% induced by ACh and CCK, respectively. Pretreatment with CL for 18 h, the resting cellular Ca²⁺ concentration elevated to 152 nmol·L^-1± 26 nmol·L^-1, however, the cellular Ca²⁺ increase percentages in response to these agonists were 67% ± 32%, 56% ± 33% and 34% ± 15%.

CONCLUSION: KCl elicite the SO cellular Ca²⁺ increase depends on influx of extracellular Ca²⁺, ACh evoked the SO celllular Ca²⁺ increase is through the mobilization of intracellular Ca²⁺ pool and influx of extracellular Ca²⁺ as well, CCK excites the SO cells mainly through mobilization of intracellular IP3-sensitive Ca²⁺ store. After the incorporation with cholesterol liposome, KCl,ACh and CCK induced cellular Ca²⁺ increase percentages decreased.

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