Azzarone, A

Borle, AB

Caraceni, P

Fagiuoli, S

Farghali, H

Gasbarrini, A

Starzl, TE

Van Thiel, DH

English

PubMed

Name not available

Anoxia/reoxygenation injury in hepatocytes is not prevented by calcium channel blockers

D-Scholarship@Pitt

AnoxiaiReoxygenation Injury in Hepatocytes Is Not Prevented By calcium Channel Blockers A. Gasbarrini, AB. Borle, P. Caraceni, H. Farghali, A. Azzarone, S. Fagiuoli, T.E. Starzl, and D.H. Van Thiel SEVERAL investigators have reported that calcium channel blockers may protect the liver from anoxic injury or from other hepatotoxic agents. I•Z Because the putative protective effects of calcium channel blockers have been obtained in perfused whole liver, it is impossible to determine which cells within the liver were actually the target of the calcium antagonists: vascular smooth muscle cells, endothelial cells, Kupffer cells, or parenchymal cells. In addition. most of the protective effects observed have occurred after reoxygenation and not during anoxia, suggesting that calcium antagonists act to prevent reperfu-sion injury and not anoxic injury. The objective of the present experiments was to determine whether calcium channels are involved in the damage induced by anoxia in perfused isolated rat hepatocytes. Specifically the studies described were designed to determine whether the massive rise in cytosolic-free calcium (Ca/+) and the increase in loss of cytosolic lactate dehydrogenase (LDH) evoked by anoxia can be blocked by specific Ca2+ channel blockers. 11tree classes of Ca2+ antagonists (nifedipine, verapamil, and diltiazem) were studied. Each was studied at his ~ (10- 7) and at concentrations 10 and 100 times above K j • MATERIALS AND METHODS Hepatocytes Frcsbly isolated hepatocytes were prepared from adult male Sprague-Dawley rats weighing between 200 and 250 g. The cells were isolated from fed animals using co11ageuase (Sigma type IV, Sigma Chemicals. St Louis. Mo) by the perfusion method de-scribed previously.J Cell viability assessed by trypan blue exclu-sion averqed 90%. The cells were embedded in agarose threads and were perfused at a rate of 0.6 mUmin with standard Krebs-Henseieit bicarbonate buffer (KHB) at 37"C. In all experiments the hepatocytes were first perfused for I hour in control KHB saturated with 9.5% Or.5% carbon dioxide (C02), During the 2 boars of experimental anoxia. the cells were perfused with KHB salW'lIted with 9.5% Nr .5% CO2, After 2 hours of anoxia. the cells were reperfused with oxygenated KHB for I hour. In the exper-imental studies, the cells were exposed. during the 2 hours of anoxia, to KHB containing 10-7, 10-6, 10-5 mollL verapamil, ~K or diltiazem. CytosoIic Ionized Calcium ~w+ was measured with the Ca2+ -sensitive photoprotein ae-quorin4 incorporated into the hepatocytes by gravity-loading. The aequorin-loaded ceils were imbedded in agarose gel threads. placed in the cuvette of an aequorin luminescence photometer. and wntinuously perfused with KHB. Lactate Dehydrogenase Cell injury was monitored by measuring LDH release from the cells into the perfusate emuent before. during and after the anoxic period. Because the concentration of perfused cells imbedded in the agamse threads varied slightly between individual experi-ments, LDH release was expressed as the percent increase above the control value measured during the I-hour control period at the beginning of each experiment. RESULTS Under control conditions, the Ca/+ was 126 ± 10 nmoUL. In the control group anoxia increased Cllj2+ in two distinct phases, reaching a maximum value of 1,215 ± 78 nmollL after 1 hour. Lactate dehydrogenase release increased sixfold during the second hour of anoxia. During reoxy-genation, Cllj2+ and LDH returned to control levels within 45 minutes. To determine whether the source of Ca2+ responsible for the increase in Cllj 2+ during anoxia was intra- or extracellular these experiments were repeated in the absence of extracellular Ca2+ (Ca., 2+) during the 2 hours of anoxia. The initial smaller peak in Cllj2+ seen previously in the first 10 minutes of anoxia was present even in the absence of Cao2 +. This suggests that the source of Ca2+ responsible for the initial Cllj 2+ peak is probably intracellular. The second major Cllj2+ peak, previously observed 1 hour after the initiation of anoxia, was com-pletely abolished in the absence of extracellular Ca2+. indicating that this second increase in Cllj 2+ was due to an influx of Ca2+ from the perfusate to the cytosol. Vera-pamil, nifedipine, and diltiazem (10-7 to 10-5 moUL) added to the perfusate during the anoxic period did not inhibit the biphasic rise in Cllj2+. Furthermore, the calcium antagonists, at all three concentrations used, did not protect hepatocytes from anoxic injury measured by the release of LDH in the perfusate etlluent. DISCUSSION These experiments demonstrate that anoxia causes a bi-phasic increase in ~ 2+. The second surge in Cllj 2+ is totally abolished when Ca2+ is removed from the perfus-From the Departments of Surgery (A.G., A.B.B .• P.C., H.F., AA. S.F., T.E.S., D.H.vn and Physiology (A.B.B.), University of Pltts-burgh, Pittsburgh, Pennsylvania; and Patologia Medica I (P .C.), Univenllty of Bologna, Bologna, Italy. Address reprint requests to A. Gasbarrini, MD, Patologia Medica I, Pollcllnlco Sant'0rs01a, Via Massarenti, 40100 Bologna, Italy. © 1992 by Appleton & Lange 0041-13451921$3.001+0 2812 Transplantation PlTJCtHldings, Vol 24, No 6 (December), 1992: pp 2812--2813 --:,-----.--I ·.ir " ,.. ;-' --ate, indicating that the source of Ca2+ responsible for the second increase in CIlj 2 + is the extracellular fluid. The lack of an effect on CIlj 2+ observed with the three Ca2+ channel blockers verapamil, diltiazem. and nifedipine in concen-trations up to 10-5 mollL strongly suggests that pathways other than L-type voltage-operated Ca2+ channels are involved in the influx of Ca2+ responsible for the increase in C1lj2+ evoked by anoxia. Furthermore. concentrations of verapamil, diltiazem, and nifedipine ranging from 10-7 to 10-5 moUL did not block the LDH release induced by anoxia. Thus, Ca2+ antagonists do not protect liver paren-chymal cells against anoxic injury. The reported cytopro-._ kdL=X ;uu -----= - 52 . .. 2813 tective effects of Ca2+ channel blockers seen in ischemic perfused whole liver must occur as a result of an effect on cells other than hepatocytes (ie, endothelial cells or smooth muscle cells of the liver vasculature). REFERENCES 1. Landon EJ. Naukam RJ. Rarna Sastri BV: Biochem Phar-rnaco135:697. 1986 2. Takei Y. Mani I. Kauffman FC. et al: Transplantation 50: 14. 1990 3. Studer RK. Boric AB: J Bioi Chern 257:7987. 1982 4. Borle AB. Snowdowne KW: Methods EnzymoI124:90. 1986 

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Anoxia/reoxygenation injury in hepatocytes is not prevented by calcium channel blockers

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