Clinical Science (2007) 112, 167-174 - N.M. Atucha and others - Platelet calcium in cholestatic and cirrhotic rats

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Clinical Science (2007) 112, (167–174) (Printed in Great Britain)

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Altered calcium signalling in platelets from bile-duct-ligated rats

Noemí M. ATUCHA*, David IYÚ*, Antonia ALCARAZ*, Vladimir ROSA*, Concepción MARTÍNEZ-PRIETO*, M. Clara ORTIZ*, Juan Antonio ROSADO† and Joaquín GARCÍA-ESTAÑ*

*Department of Physiology, Faculty of Medicine, University of Murcia, 30100 Murcia, Spain, and †Department of Physiology, University of Extremadura, Cáceres 10071, Spain

Key words: calcium signalling, bile-duct ligation, capacitative Ca²⁺ entry, cholestasis, liver cirrhosis, sarcoplasmic/endoplasmic-reticulum Ca²⁺-ATPase (SERCA), thrombin.

Abbreviations: 2-APB, 2-aminoethoxydiphenyl borate; AUC, area under the curve; BDL, bile-duct ligated; BDL group, rats at 4 weeks after bile-duct ligation; CCE, capacitative Ca²⁺ entry; cholestasis group, rats at 11–13 days after bile-duct ligation; fura-2/AM, fura 2 acetoxymethyl ester; IP₃, inositol 1,4,5-trisphosphate; PMCA, plasma membrane Ca²⁺-ATPase; SERCA, sarcoplasmic/endoplasmic-reticulum Ca²⁺-ATPase.

In the present study, we have analysed the mechanisms of Ca²⁺ entry and release in platelets obtained from BDL (bile-duct-ligated) rats, 11–13 days and 4 weeks after surgery. Platelets were washed and loaded with fura-2, and [Ca²⁺]_i (cytosolic Ca²⁺ concentration) was determined in cell suspensions by means of fluorescence spectroscopy. Basal [Ca²⁺]_i was similar in platelets from BDL rats compared with those from their respective controls, both in the absence and presence of extracellular Ca²⁺. Platelet stimulation with thrombin in the absence and presence of extracellular Ca²⁺ induced a rapid rise in [Ca²⁺]_i that was of greater magnitude in platelets from BDL rats than in controls. Ca²⁺ storage was significantly elevated in platelets from BDL rats, as well as the activity of SERCA (sarcoplasmic/endoplasmic-reticulum Ca²⁺-ATPase). Capacitative Ca²⁺ entry, as evaluated by inhibition of SERCA with thapsigargin, was also altered in platelets from BDL rats, having lower rates of Ca²⁺ entry. In conclusion, chronic BDL alters intracellular Ca²⁺ homoeostasis in platelets, such that an enhanced Ca²⁺ release is evoked by thrombin, which may be due to an increased amount of Ca²⁺ stored in the intracellular organelles and secondary to an enhanced activity of SERCA. These alterations are already evident before cirrhosis has completely developed and occurs during the cholestasis phase.