HEPATOLOGY Vol, 22, No. 4, Pt. 2, 1995 AASLD A B S T R A C T S 307A

8 0 1 MODULATION OF INTRACELLULAR CALCIUM BY DEPOLA- RIZATION AND G-COUPLED AGONISTS IN ISOLATED CULTU- RED RAT LIVER ENDOTHELIAL CELLS. E. de Broin l, P.-M. Huet 1 and P. Haddad 2, Andr6-Viallet Clinical Research Center l, H6pital St-Luc and Membrane Transport Research Group 2, U. de Montr6al, Montr6al, Qu6bec.

Liver endothelial cells (LECs) are particularly sensitive to cold preservation injury and their dysfunction may underlie, in part, primary liver graft non- function that still occurs in a significant proportion of transplantations. One hypothesis postulates that intracellular calcium ([Ca2÷]s) homeostasis may be perturbed in LEC but, to date, little direct information is available. Endo- thelial celis were thus isolated from rat livers by standard centdfugatioa/elu- triation techniques and seeded onto collagen-coated glass coverslips. LEC purity was 95-99% as determined by monoclonal anti-LEC antibodies. Cells were cultured for 24~72 hrs, loaded with the calcium dye FURA 2-AM for 45 min at 37°C, and then observed with an inverted microscope. Fluorescence ratios were obtained from several cells with a video imaging system (MCID M2 v2. ltx by Imaging Research). LECs perifused with Krebs-Henseleit buffer at 37°C exhibited mean [Ca2+|t values of 70 to 85 nM. We increased buffer K ÷ from 5 to 50 mM (isotonic replacement for Na ÷) to depolarize the ceils or used 1 pM ATP as a classical endothelial ceU Ca2÷-mohilizing agonist. A 1- min high K ÷ peffusion caused a rapid bell-shaped increase in cytosolic Ca 2. in 80% of the cells. Generally, moderate elevations averaged 103 +17 nM (n=21) whereas large increases were more rarely seen (1" by 1210 + 142 nM, n=3). Ninety percent of cultured LECs also responded to 1 ~M ATP with an instantaneous rise in [Ca2÷]i averaging 90 ± 13 nM (n=lg) in moderate responders and 1206 ± 603 nM (n=4) in rare high responders. When ATP administration was repeated, we found no desensitization of the [CaZ+]~ response after a 2-3 rain washout period (1" 100 ± 22 ruM for the F t versus 1" 86 ± 21 nM for the 2 ~d, N.S.). The results suggest that cultured LECs possess voltage-operated calcium channels and G-coupled purinergic receptors as do endothelial cells in other tissues. Most importantly, the data clearly demonstrate the usefulness of this model to study sinusoidal LEC physiology and pathophysiology in the context of ischemia-reperfusion injury, Supported by Canadian MRC.

802 THE ROLE OF PLASMA IN THE DISTURBANCE OF BASAL CYTOSOLIC CALCIUM IN PLATELETS FROM PATIENTS WITH CIRRHOSIS. EH Forrest, JF Dillon, IAD Bouchier, PC Haves. Liver Research Laboratories, Department of Medicine, Royal Infirmary, Laufiston Place, Edinburgh. Scott and

Platelets from patients with cirrhosis have abnormal basal cytosolic calcium concentrations ([Ca2+]i). As well as impairing platelet aggregation, these changes may reflect those in vascular smooth muscle leading to vasodilatation. We assessed the role of plasma in the abnormal [Ca2+]i of platelets.

Platelets were obtained, from 26 control subjects and 26 patients with cirrhosis. After washing platelets were incubated in both patient and control fresh platelet poor plasma. Platelets from controls were also incubated in plasma which had been previously stored at -20 °C. The incubation was for 45 mins in the presence of FURA-2AM to allow calculation of [Ca2+]i by single wavelength spectrofluorimetery.

Results: Platelet Cytosolic Calcium Control Platelets Patient Platelets

Fresh Control Plasma 128 (7.8) 165.4 (I9.8)# Fresh Patient Plasma 93.0 (9.3)* 95.7 (10.0)** Stored Patient Plasma 117.6 (2=1) Mean +/- SEM. *p=0.005, **p=0.02 compared with Control Platelets in Fresh Control Plasma, #p=0.01 compared with Patient Platelets in Fresh Patient Plasma

Conclusions: Fresh plasma from patients with cirrhosis contains a factor which causes a fall in platelet [Ca2+]i. However when previously frozen plasma is used this effect upon [Ca2÷]i is absent. Isolation from patient plasma reverses the abnormal patient platelet basal [Ca2÷]i.

8 0 3 GLYCINE PREVENTS ELEVATION IN INTRACELLULAR Ca 2+ IN KUPFFER CELLS BY ENDOTOXIN (LPS) VIA ACTIONS ON A CHLORIDE CHANNEL. K Ikejima and RG Thurman. Laboratory of Hepatobiology and Toxicology, Dept. of Pharmacology, Univ. of North Carolina, Chapel Hill, NC 27599.

Glycine has been reported to be protective against hypoxia in renal tubules and hepatocytes, and it improves survival after rat liver transplantation. We reported that a diet containing glycine improves survival and reduces liver injury and serum TNF-¢¢ levels after injection of a lethal dose of LPS (Ikejima and Thurman. FASEB J. 9; A885, 1995). Since intracellular Ca 2+ ([CaZ+]i) is thought to be essential for the production of chemical mediators such as TNF-a and eicosanoids in Kupffer cells, we investigated the effect of glycine on [CaZ+]i in cultured Kupffer cells stimulated with LPS. Kupffer cells were isolated by collagenase digestion and differential centrifugation with Pereoll gradients. Calls were suspended in D'MEM with 10% FBS, cultured for 24 hr prior to experiments and [CaZ+] i was measured fluor0metrically using fura-2. Addition of LPS (10 p~g/ml) increased [Ca2+]i innnediately with peak values reaching 204 + 29 nM (n=5). In contrast, addition of glycine (1 mM) 3 rain prior to LPS blunted the increase significantly (peak [CaZ+]i; 56 + 9 nM, n=5, P<0.05). Strychnine (1 ~M), an antagonist of the glycine receptor, reversed the inhibitory effect of glycine completely (peak [Ca2+]i; 245 -+ 73 nM, n=5, P<0.05). Moreover, the effect of glycine on the increase in [CaZ+]i due to LPS was prevented totally when cells were incubated in chloride-free buffer. We postulate that glycine affects Kupffer cells through activation of a chloride channel, leading to hyperpolarization of the plasma membrane thereby restricting the opening of voltage-dependent Ca 2+ channels. Prevention of an increase in [Ca2+] i due to LPS by glycine may reduce synthesis and release of chemical mediators by Kupffer cells, leading to improved survival and decreased liver injury in endotoxin shock (AA-03624).

804 GLYCINE BLOCKS THE INCREASE IN INTRACELLULAR FREE Ca 2÷ DUE TO PROSTAGLANDIN E 2 AND PHENYLEPHRINE IN HEPATIC PARENCHYMAL CELLS. We..j Qu and Ronald G. Thurmau. Lab. of Hepatobiology & Toxicology, Dept. of Pharmacology, Univ. of North Carolina, Chapel Hill, N.C.

Glyciue protects renal tubular cells, isolated hepatocytes and perfused livers against hypoxic injury, Recently, glycine has been shown to prevent liver injury after endotoxin treatment in vivo (K.Ikejima and R.G. Thurman. FASEB J. 9; A885,1995). The aim of this study was to determine if glycine prevents the increase in intraeellular free Ca 2+ ([Ca2+]i) by agonists released during stress such as prostaglandin E 2 (PGE2) and adrenergic hormones. Parenchymal ceils isolated from female Sprague-Dawley rats were cultured for 4 hours in DMEM/F12 medium and [Ca2+]i in individual cells was assessed using the fluorescent calcium indicator, fura-2. PGE 2 caused a dose-dependent increase in [Ca2+]i from basal values of 130 + 10 to maximal levels of 434 + 55 nM. TMB-8, an agent that prevents the release of Ca 2+ from intracellular stores, blocked the increase in [Ca2+]i due to PGE 2. This increase was also blunted in the absence of extracelhdar Ca 2+, suggesting that both intracellular and extracellular Ca 2+ pools are involved. Interestingly, glycine, which activates anion channels, blocked the increase in [Ca2+]i due to PGE 2 in a dose-dependent manner. Strychnine, a glycine antagonist in the central nervous system, reversed the inhibition by glycine partially. When extracellular CI" was omitted, glycine was much less effective. Phenylephrine, an alpha 1-type adrenergic receptor agonist, increased [Ca2+]i as expected from 159 -+ 20 to 432 + 43 nM. Glycine also blocked the increase due to phenylephrine, and the effect was also reversed by strychnine. Taken together, these data indicate that glycine blocks the increase in [Ca2+]i due to agonists released during stress, most likely by actions on a glycine-sensitive anion channel (AA-09156 and AA-03624).