reperfusion events and septic injury altered intracellular The present study evaluated the effect of the benzothi-Ca 2/ homeostasis. 2 Whereas disturbance of cellular Ca 2/ regazepine Ca 2/ channel blocker diltiazem (DZ) on altered ulation is a well-studied mechanism of cellular dysfunction hepatocellular Ca 2/ regulation and oxidant injury durin excitable cells, there is growing evidence that these ing hemorrhagic shock/resuscitation. In anesthetized, changes also occur in parenchymal and endothelial cells. 3 male Sprague-Dawley rats, hemorrhagic shock was in-Previous studies showed significantly increased intracellular duced by rapid blood withdrawal and maintaining the Ca 2/ concentrations in hepatocytes isolated from rats undermean arterial blood pressure at 40 mm Hg over 60 mingoing endotoxic shock, 4 intraabdominal gram-negative seputes. Rats were then resuscitated with 60% of shed blood sis 5 or ischemia because of hemorrhage. 6 Moreover, these and and threefold the shed blood volume of Ringer's lactate. other studies also reported a protective effect by the benzothi-At the end of ischemia, and 60 or 300 minutes after resusazepine Ca 2/ channel blocker diltiazem (DZ) in preventing citation, hepatocytes were isolated by liver collagenase shock-induced organ failure 7 and restoring altered immune perfusion. Hepatocellular Ca 2/ exchange (Ca 2/ ex ), rate of functions. 8,9 cellular Ca 2/ influx (Ca 2/ in ), and Ca 2/ membrane flux Hemorrhagic shock and resuscitation induced a broad spec-(Ca 2/ flux ) were determined using 45 Ca incubation techtrum of cellular alterations, including adenosine triphosniques. Hepatocyte oxidant injury was evaluated by fluphate breakdown, pH changes, release of Ca 2/ agonistic hororometrically measuring thiobarbituric acid reactive mones (e.g., catecholamines, vasopressin, and cytokines) and substances and oxidized/reduced glutathione. Both hemfree radical-induced membrane injury. Altered plasma memorrhage and hemorrhage/resuscitation increased hepabrane integrity because of oxygen-free radical attack has tocellular Ca 2/ in , Ca 2/ ex , and Ca 2/ flux . In contrast to conbeen shown to enhance cellular Ca 2/ in . 10 Ca 2/ channel blocktrol and sham-operated rats, in vitro stimulation by the ers significantly protected against membrane lipid peroxida-Ca 2/ agonist epinephrine (100 nmol/L) of hepatocytes tion and permeability changes. 5,11 Cellular Ca 2/ in and altered from either hemorrhaged or resuscitated rats did not Ca 2/ regulation could lead to cellular Ca 2/ overload, which further increase Ca 2/ in . Administration of DZ (.8 mg/kg) in a variety of in vitro studies induced cell membrane destabiwith resuscitation significantly decreased cellular Ca 2/ ex lization, DNA damage and proteolytic processes leading to and Ca 2/
flux , but did not restore impaired epinephrinecell death. 12 Thus, disturbance of cellular Ca 2/ regulation induced Ca 2/ in . DZ prevented hepatocyte lipid peroxidacould be an essential pathomechanism of organ dysfunction tion and glutathione oxidation. These findings suggest following hemorrhagic shock. hepatocellular Ca 2/ overload and impaired Ca 2/ signal-Aim of the present study was to evaluate alterations of ing during hemorrhage/resuscitation. Increased Ca 2/ cellular Ca 2/ exchange (Ca 2/ ex ) and membrane Ca 2/ flux uptake could be because of a receptor-gated Ca 2/ influx (Ca 2/ flux ) following hemorrhage and hemorrhage/resuscitaand/or oxygen-free radical induced membrane Ca 2/ tion. We further assessed hepatocyte oxidant injury and inleaks. (HEPATOLOGY 1997;25:379-384.) vestigated whether the Ca 2/ channel blocker DZ could modulate altered cellular Ca 2/ regulation and oxidant injury Intracellular Ca 2/ regulation represents a pivotal signaling during hemorrhagic shock. system to control and modulate important physiological processes, e.g., cell proliferation and differentiation, glucose me-
Methods
tabolism, protein synthesis, enzymatic reactions, and secretory functions. Depending on the cell type, receptor-or Hemorrhagic Shock Model. In compliance with the institution's voltage-gated cellular Ca 2/ influx (Ca 2/ in ), Ca 2/ mobilization guidelines and after approval by the local review board for experi- mental animals, male Sprague-Dawley rats (range, 210-250 g body from intracellular stores such as endoplasmic reticulum and weight [bw], n ยข 6/group) were intraperitoneally anesthetized with Ca 2/ release from Ca 2/ binding proteins are necessary prosodium pentobarbital (50 mg/kg). In all rats, but not controls, an cesses to initiate these Ca 2/ -mediated responses. 1 Ischemia/ intratracheal polyethylene tube was inserted to guarantee free airways and spontaneous breathing. A polyethylene tube (PE-50), inserted in the right carotid artery, was connected to a Statham pressure transducer system to measure mean arterial blood pressure Abbreviations: Ca 2/ in , Ca 2/ influx; DZ, diltiazem; Ca 2/ ex , Ca 2/ exchange; Ca 2/ flux , Ca 2/ (MABP). Right jugular vein and right femoral artery were exposed membrane flux; bw, body weight; MABP, mean arterial blood pressure; ww, wet weight;