NEW FRAGMENT OF BOVINE HEPARIN FBH001 DECREASES INTRACELLULAR CALCIUM IN HEPATOCYTES SUBMITTED TO CALCIUM OVERLOAD
Ênio R. Vasques*1, José Eduardo M. Cunha1, Helena Nader2, Ivarne S. Tersariol2, Marcelo A. Lima2, Luiz Augusto C. D'Albuquerque1, Eleazar Chaib1, Tiago Rodrigues3
1GASTROENTEROLOGY, FMUSP, São Paulo, São Paulo, Brazil; 2INFAR, FEDERAL UNIVERSITY OF SAO PAULO, SAO PAULO, Brazil; 3FEDERAL UNIVERSITY OF ABC, SANTO ANDRE, Brazil
Background: Synthetic and porcine heparin fragments have action on the sodium-calcium exchanger accelerating the release of intracellular calcium into the extracellular medium, providing liver cell protection in situations of intracellular and calcium overload determined by ischemia and tissue reperfusion. The predominance of bovine herds in Brazil promotes abundant raw material for bovine heparin fragments (BHF), but the production and applicability of BHF should be conditioned by the similarity of effects, both in the coagulation mechanisms (which have already been proven) or in the dynamics of calcium, not yet determined. A novel 8000 Kd BHF produced at our institution, herein designated as BHF001, acts on coagulation mechanisms. However, its action on calcium dynamics is still unknown.
Aim: To evaluate in hepatocyte culture the action dynamics of BHF001 in cells submitted to calcium overload. Material and Method: Rattus norvegicus BRL3A liver cells were cultured in modified Dulbecco's medium and supplemented for 24 hours and exposed to the Fura-2 / AM Calcium fluorescent marker (4 μM) for 30 minutes at 37 ° C in 5% CO2. They were washed twice with 1.0 ml of CaCl2, 130 mM NaCl, 5.6 mM KCl, 0.8 mM MgSO 4, 1 mM Na2 HPO4, 25 mM glucose, 2 mM HEPES, and 2.5 mM NaHCO 3 (pH 7.3). analyzed and acquired by open field microscopy and digitally photographed. Changes in cytosolic calcium levels were measured at 37 ° for 15 minutes. To promote an increase in cytosolic calcium, 200 μM Thapsigargine was used and 50 μM of BHF001 was added after 150 ms of the action of tapsgargine. Finally, submission to ionophore action (0.15 μM) for hydrophilic pore formation allowing intracellular calcium elevation from the extracellular medium. Results: The addition of BHF001 decreased intracellular calcium levels, with maximum effect around 60 ms. Conclusion: BHF001 decreases the intracellular calcium overload determined by the action of tapsigargine on hepatocyte cell culture.
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