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J J Lemasters

Publications and source records attributed to J J Lemasters.

At least 19 recordsLinked to original sources
PubMed2006

Endothelial nitric oxide synthase protects transplanted mouse livers against storage/reperfusion injury: Role of vasodilatory and innate immunity pathways.

Endothelial nitric oxide synthase (eNOS) plays a role in microcirculatory and immunomodulatory responses after warm ischemia/reperfusion. We hypothesized that eNOS is essential to maintain microcirculation, attenuate macrophage infiltration and decrease graft injury after liver transplantation. Liver transplantation was performed after 18 hours of cold storage in University of Wisconsin (UW) solution from wildtype and eNOS-deficient (B6.129P2-Nos3(tm/Unc)/J) donor mice into wildtype mice. Serum ALT, necrosis by histology, apoptosis by TUNEL, and macrophage infiltration by immunostaining against F4/80 antigen were determined 2 to 8 hours after implantation. Hepatic microcirculation was investigated after 4 hours by intravital confocal microscopy following injection of fluorescein-labeled erythrocytes. After sham operation, livers of wildtype and eNOS-deficient mice were not different in ALT, necrosis, apoptosis, macrophage infiltration, and microcirculation. After transplantation, ALT increased >3 times more after transplantation of eNOS-deficient livers than wildtype livers. Necrosis was >4 times greater, and TUNEL and F4/80 immunostaining in nonnecrotic areas were 2 and 1.5 times greater in eNOS-deficient donor livers, respectively. Compared with wildtype and eNOS sham-operated mice, sinusoidal blood flow velocity increased 1.6-fold after wildtype transplantation, but sinusoidal diameter was not changed. After transplantation of eNOS-deficient livers, blood flow velocity and sinusoidal diameter decreased compared with transplanted wildtype livers. These results indicate that donor eNOS attenuates storage/reperfusion injury after mouse liver transplantation. Protection is associated with improved microcirculation and decreased macrophage infiltration. Thus, eNOS-dependent graft protection may involve both vasodilatory and innate immunity pathways.

Animals↗
PubMed2004

Primary cirrhotic hepatocytes resist TGFbeta-induced apoptosis through a ROS-dependent mechanism.

BACKGROUND/AIMS: The cirrhotic liver manifests dysregulated hepatocyte growth by poor regenerative capacity, formation of regenerative nodules, and malignant transformation to hepatocellular carcinoma. The purpose of this study was to determine if dysregulated hepatocyte growth occurs through deficient apoptosis. METHODS: Hepatocytes were isolated from normal and CCl(4)-treated mice and treated with TGFbeta, TNFalpha, and UV-C, known apoptotic agents. RESULTS: Cirrhotic hepatocytes were less sensitive to TGFbeta- (45+/-5 vs. 15+/-3%; P<0.003), TNFalpha- (59+/-21 vs. 21+/-8%; P=0.02), and UV-C-induced (31+/-4 vs. 17+/-4%; P<0.03) apoptosis compared to normal hepatocytes. In normal hepatocytes, TGFbeta-induced apoptosis occurred through a ROS-, MPT-, and caspase-dependent pathway. Cirrhotic hepatocytes lacked caspase activation, had decreased procaspase-8 expression, failed to undergo the MPT, and had increased basal ROS activity compared to normal hepatocytes. After treatment with trolox, an antioxidant that reduced basal ROS activity, cirrhotic hepatocytes underwent apoptosis in response to TGFbeta treatment. CONCLUSIONS: These findings suggest that increased ROS activity in cirrhotic hepatocytes plays a critical role in mediating cirrhotic hepatocyte resistance to apoptosis. Cirrhotic hepatocyte resistance to TGFbeta-induced apoptosis is ROS-dependent and is a mechanism of dysregulated growth in the chronically inflamed liver.

Animals↗
PubMed2003

Role of the mitochondrial permeability transition in apoptotic and necrotic death after ischemia/reperfusion injury to hepatocytes.

Reperfusion of ATP-depleted tissues after warm or cold ischemia causes pH-dependent necrotic and apoptotic cell death. In hepatocytes and other cell types as well, the mechanism underlying this reperfusion-induced cell death involves onset of the mitochondrial permeability transition (MPT). Opening of permeability transition (PT) pores in the mitochondrial inner membrane initiates the MPT, an event blocked by cyclosporin A (CsA) and pH less than 7.4. Thus, both acidotic pH and CsA prevent MPT-dependent reperfusion injury. Glycine also blocks reperfusion-induced necrosis but acts downstream of PT pore opening by stabilizing the plasma membrane. After the MPT, ATP availability from glycolysis or other source determines whether cell injury after reperfusion progresses to ATP depletion-dependent necrosis or ATP-requiring apoptosis. Thus, apoptosis and necrosis after reperfusion share a common pathway, the MPT. Cell injury progressing to either necrosis or apoptosis by shared pathways can be more aptly termed necrapoptosis.

Animals↗
PubMed2001

Electrical properties and conduction in reperfused papillary muscle.

The reversibility of ischemia-induced changes of extracellular K(+) concentration ([K(+)](o)), resting membrane potential (E(M)), and passive cable-like properties, ie, extracellular resistance and cell-to-cell electrical coupling, and their relationship to recovery of conduction and contraction is described in 25 reperfused rabbit papillary muscles. No-flow ischemia caused extracellular K(+) accumulation, depolarization of E(M), an increase in whole-tissue (r(t)), external (r(o)), and internal (r(i)) longitudinal resistances, and failure of conduction and contraction. Muscles were reperfused 10 minutes after the onset of ischemia related cell-to-cell electrical uncoupling, ie, 26+/-1 minutes after arrest of perfusion. In 11 muscles, incomplete reflow occurred with only partial recovery of [K(+)](o) and r(t). In the remaining 14 muscles, reperfusion caused a rapid and parallel decrease in [K(+)](o), r(t), and r(o). When complete tissue reperfusion occurred, cell-to-cell electrical uncoupling was largely reversible. Thus, cell-to-cell electrical uncoupling did not indicate irreversible injury. Reperfusion induced a depolarizing current widening the difference between the K(+) equilibrium potential and the E(M). This difference decreased after longer periods of reperfusion. Conduction was restored and conduction velocity approached preischemic values as cell-to-cell electrical interaction was reestablished and E(M) recovered. The recovery of r(o) preceded r(i), decreasing the ratio of the extracellular to intracellular resistance early in reperfusion, an effect predicted to influence the amplitude of the extracellular voltage field and electrocardiographic ST segments during reperfusion.

Animals↗
PubMed2001

The mitochondrial permeability transition initiates autophagy in rat hepatocytes.

Cells degrade excess and effete organelles by the process of autophagy. Autophagic stimulation of rat hepatocytes by serum deprivation and glucagon (1 M) caused a fivefold increase of spontaneously depolarizing mitochondria to about 1.5% of total mitochondria after 90 min. Cyclosporin A (CsA, 5 M), an immunosuppressant that blocks the mitochondrial permeability transition (MPT), prevented this depolarization. Depolarized mitochondria moved into acidic vacuoles labeled by LysoTracker Red. These autophagosomes also increased several-fold after autophagic stimulation. CsA blocked autophagosomal proliferation, whereas tacrolimus, an immunosuppressant that does not block the MPT, did not. In conclusion, the MPT initiates mitochondrial depolarization after autophagic stimulation and the subsequent sequestration of mitochondria into autophagosomes.

Animals↗
PubMed2001

Screening assays for the mitochondrial permeability transition using a fluorescence multiwell plate reader.

Opening of permeability transition (PT) pores in the mitochondrial inner membrane causes the mitochondrial permeability transition (MPT) and leads to mitochondrial swelling, membrane depolarization, and release of intramitochondrial solutes. Here, our aim was to develop high-throughput assays using a fluorescence plate reader to screen potential inducers and blockers of the MPT. Isolated rat liver mitochondria (0.5 mg/ml) were incubated in multiwell plates with tetramethylrhodamine methyl ester (TMRM, 1 microM), a potential-indicating fluorophore, and Fluo-5N (1 microM), a low-affinity Ca(2+) indicator. Incubation led to mitochondrial polarization, as indicated by uncoupler-sensitive quenching of the red TMRM fluorescence. CaCl(2) (100 microM) addition led to ruthenium red-sensitive mitochondrial Ca(2+) uptake, as indicated by green Fluo-5N fluorescence. After Ca(2+) accumulation, mitochondria depolarized, released Ca(2+) into the medium, and began to swell. This swelling was monitored as a decrease in light absorbance at 620 nm. Swelling, depolarization, and Ca(2+) release were prevented by cyclosporin A (1 microM), confirming that these events represented the MPT. Measurements of Ca(2+), mitochondrial membrane potential, and swelling could be made independently from the same wells without cross interference, and all three signals could be read from every well of a 48-well plate in about 1 min. In other experiments, mitochondria were ester-loaded with carboxydichlorofluorescein (carboxy-DCF) during the isolation procedure. Release of carboxy-DCF after PT pore opening led to an unquenching of green carboxy-DCF fluorescence occurring simultaneously with swelling. By combining measurements of carboxy-DCF release, Ca(2+) uptake, membrane potential, and swelling, MPT inducers and blockers can be distinguished from uncouplers, respiratory inhibitors, and blockers of Ca(2+) uptake. This high-throughput multiwell assay is amenable for screening panels of compounds for their ability to promote or block the MPT.

Animals↗
PubMed2001

Glycine blocks opening of a death channel in cultured hepatic sinusoidal endothelial cells during chemical hypoxia.

Using confocal microscopy, we investigated mechanisms underlying loss of plasma membrane integrity during necrotic death of cultured hepatic sinusoidal endothelial cells exposed to 2.5 mM potassium cyanide (chemical hypoxia). After 2-3 h, the anionic fluorophore calcein abruptly began to enter the cytosol, and nuclei labeled with cationic propidium after another 2-5 min. As calcein permeated, growth of blebs on the plasma membrane accelerated. Lucifer yellow, another anionic fluorophore, entered identically to calcein, whereas high molecular weight dextrans (40-2000 kDa) entered like propidium. Glycine slowed, but did not prevent calcein entry, whereas permeation of propidium and high molecular weight dextrans was blocked completely by glycine. These findings suggest that opening of a glycine-sensitive organic anion channel, or death channel, precipitates a metastable state characterized by rapid cell swelling and bleb growth. This metastable state culminates in non-specific breakdown of the plasma membrane permeability barrier and irreversible cell death.

Cell Death↗
PubMed2001

NF-kappaB stimulates inducible nitric oxide synthase to protect mouse hepatocytes from TNF-alpha- and Fas-mediated apoptosis.

BACKGROUND AND AIMS: Hepatocyte apoptosis is induced by tumor necrosis factor alpha (TNF-alpha) and Fas ligand. Although nuclear factor-kappaB (NF-kappaB) activation protects hepatocytes from TNF-alpha-mediated apoptosis, the NF-kappaB responsive genes that protect hepatocytes are unknown. Our aim was to study the role of NF-kappaB activation and inducible nitric oxide synthases (iNOSs) in TNF-alpha- and Fas-mediated apoptosis in hepatocytes. METHODS: Primary cultures of hepatocytes from wild-type and iNOS knockout mice were treated with TNF-alpha, the Fas agonistic antibody Jo2, a nitric oxide (NO) donor (S-nitroso-N-acetylpenicillamine), an NO inhibitor (N(G)-methyl-L-arginine acetate), and/or adenovirus-expressing NF-kappaB inhibitors. RESULTS: The IkappaB superrepressor and a dominant-negative form of IkappaB kinase beta (IKKbeta) inhibited NF-kappaB binding activity by TNF-alpha or Jo2 and sensitized hepatocytes to TNF-alpha- and Jo2-mediated apoptosis. TNF-alpha and Jo2 induced iNOS messenger RNA and protein levels through the induction of NF-kappaB. S-nitroso-N-acetylpenicillamine inhibited Bid cleavage, the mitochondrial permeability transition, cytochrome c release, and caspase-8 and -3 activity, and reduced TNF-alpha- and Fas-mediated death in hepatocytes expressing IkappaB superrepressor. N(G)-methyl-L-arginine acetate partially sensitized hepatocytes to TNF-alpha- and Fas-mediated cell killing. TNF-alpha alone or Jo2 alone induced moderate cell death in hepatocytes from iNOS(-)/(-) mice. CONCLUSIONS: NO protects hepatocytes from TNF-alpha- and Fas-mediated apoptosis. Endogenous iNOS, which is activated by NF-kappaB via IKKbeta, provides partial protection from apoptosis.

Animals↗
PubMed2001

Ischemic preconditioning of rat livers against cold storage-reperfusion injury: role of nonparenchymal cells and the phenomenon of heterologous preconditioning.

Brief periods of ischemia followed by reperfusion render tissues resistant against subsequent prolonged ischemia, a phenomenon called ischemic preconditioning. The effect of ischemic preconditioning on liver transplantation was investigated in relation to sinusoidal endothelial cell injury and Kupffer-cell activation, which are prominent features of storage and reperfusion injury leading to liver graft failure. Rat livers were preconditioned by 5 or 10 minutes of ischemia and 5 minutes of reperfusion and stored in University of Wisconsin (UW) solution for 30 hours. Livers were then reperfused for 15 minutes with physiological buffer containing trypan blue. Under these conditions, injury occurs predominantly to sinusoidal endothelial cells, reflected by trypan blue staining of nonparenchymal cells in histological sections. Ischemic preconditioning decreased nonparenchymal cell killing by more than 50%. When half the liver was preconditioned, sinusoidal endothelial cells were also protected in the contralateral half. Other stored livers were reperfused with nitroblue tetrazolium, which is converted to insoluble formazan by superoxide radicals. Ischemic preconditioning decreased the intensity of formazan deposition over Kupffer cells. Finally, stored livers were transplanted into nontreated rats. Ischemic preconditioning improved recipient long-term survival after 30 hours of cold ischemic storage in UW solution from 30% to 80% and decreased serum tumor necrosis factor-alpha levels in posthepatic blood 4 hours postoperatively from 98 to 54 pg/mL. In conclusion, ischemic preconditioning protects sinusoidal endothelial cells and suppresses Kupffer-cell activation after storage and reperfusion. As a result, graft survival improves after liver transplantation. Moreover, ischemia to half the liver confers protection to the other half. Such heterologous preconditioning provides a new means to protect liver tissue against ischemia-reperfusion injury without imposing ischemia on the target tissue.

Animals↗
PubMed2001

Alteration in Kupffer cell function after mild hemorrhagic shock.

Functional changes in Kupffer cells occur after profound hemorrhagic shock. This study was performed to demonstrate if Kupffer cell changes also occur after mild hemorrhagic shock. Sprague-Dawley rats were bled to a systolic blood pressure of 60 to 70 mmHg and resuscitated with Lactated Ringers solution (twice the shed blood volume) after 30 min. Resuscitation produced immediate recovery of blood pressure and allowed long-term recovery of the animals. Sham animals received anesthesia and monitoring only. Thirty minutes after resuscitation, Kupffer cells were isolated by centrifugal elutriation and cultured for 48 h. In Kupffer cells isolated from shocked animals, phorbol ester-stimulated superoxide production increased 7-fold and lipopolysaccharide- (LPS) stimulated prostaglandin E2 (PGE2) production increased 4-fold. Tumor necrosis factor-alpha (TNFalpha) production, on the other hand, was decreased by 50%. A non-significant trend toward increased phagocytosis was also observed, whereas LPS-stimulated nitric oxide production was unchanged. In conclusion, mild hemorrhagic shock produced increases in superoxide and PGE2 production, and decreases in TNFalpha production by Kupffer cells, changes that may be appropriate to defend against the infectious challenges that often follows trauma and hemorrhage.

Animals↗
PubMed2001

Activated Kupffer cells cause a hypermetabolic state after gentle in situ manipulation of liver in rats.

Harvesting trauma to the graft dramatically decreases survival after liver transplantation. Since activated Kupffer cells play a role in primary nonfunction, the purpose of this study was to test the hypothesis that organ manipulation activates Kupffer cells. To mimic what occurs with donor hepatectomy, livers from Sprague-Dawley rats underwent dissection with or without gentle organ manipulation in a standardized manner in situ. Perfused livers exhibited normal values for O(2) uptake (105 +/- 5 micromol. g(-1). h(-1)) measured polarigraphically; however, 2 h after organ manipulation, values increased significantly to 160 +/- 8 micromol. g(-1). h(-1) and binding of pimonidazole, a hypoxia marker, increased about threefold (P < 0.05). Moreover, Kupffer cells from manipulated livers produced three- to fourfold more tumor necrosis factor-alpha and PGE(2), whereas intracellular calcium concentration increased twofold after lipopolysaccharide compared with unmanipulated controls (P < 0.05). Gadolinium chloride and glycine prevented both activation of Kupffer cells and effects of organ manipulation. Furthermore, indomethacin given 1 h before manipulation prevented the hypermetabolic state, hypoxia, depletion of glycogen, and release of PGE(2) from Kupffer cells. These data indicate that gentle organ manipulation during surgery activates Kupffer cells, leading to metabolic changes dependent on PGE(2) from Kupffer cells, which most likely impairs liver function. Thus modulation of Kupffer cell function before organ harvest could be beneficial in human liver transplantation and surgery.

Animals↗
PubMed2000

Kupffer cells mediate increased anoxic hepatocellular killing from hyperosmolarity by an oxygen- and prostaglandin-independent mechanism.

The purpose of this study was to evaluate the roles of Kupffer cells, prostaglandin biosynthesis, and glycolytic metabolism in accelerated anoxic cell killing by hyperosmolar stress. Isolated rat livers were perfused with anoxic normosmolar Krebs-Heinseleit bicarbonate buffer (KHB) or anoxic hyperosmolar KHB (+40 mM NaCl). Hyperosmolar KHB accelerated LDH release during anoxia in livers from both fed and fasted rats by as much as 3.7-fold. GdCl(3) pretreatment to inactivate Kupffer cells substantially delayed anoxic LDH release during normosmolar perfusions and blocked entirely the hyperosmolarity-induced acceleration of LDH release. Cyclooxygenase inhibition with indomethacin failed to alter LDH release during anoxia in hyperosmolar KHB. Neither GdCl(3) nor hyperosmolarity changed glycolytic flux during hypoxia, and hyperosmolarity did not change basal oxygen uptake. We conclude that accelerated cell killing in hyperosmolar buffer is a Kupffer cell-dependent event that is independent of oxygen-requiring prostaglandin synthesis, changes of glycolytic flux, and activation of cellular ATP demand. Another as yet unidentified Kupffer cell product appears to mediate the effect of hyperosmolarity of anoxic hepatocellular injury.

Animals↗
PubMed2000

Molecular cloning and characterization of a human mitochondrial ceramidase.

We have recently purified a rat brain membrane-bound nonlysosomal ceramidase (El Bawab, S., Bielawska, A., and Y. A. Hannun (1999) J. Biol. Chem. 274, 27948-27955). Using peptide sequences obtained from the purified rat brain enzyme, we report here the cloning of the human isoform. The deduced amino acid sequence of the protein did not show any similarity with proteins of known function but was homologous to three putative proteins from Arabidospis thaliana, Mycobacterium tuberculosis, and Dictyostelium discoideum. Several blocks of amino acids were highly conserved in all of these proteins. Analysis of the protein sequence revealed the presence at the N terminus of a signal peptide followed by a putative myristoylation site and a putative mitochondrial targeting sequence. The predicted molecular mass was 84 kDa, and the isoelectric point was 6.69, in agreement with rat brain purified enzyme. Northern blot analysis of multiple human tissues showed the presence of a major band corresponding to a size of 3.5 kilobase. Analysis of this major band on the blot indicated that the enzyme is ubiquitously expressed with higher levels in kidney, skeletal muscle, and heart. The enzyme was then overexpressed in HEK 293 and MCF7 cells using the pcDNA3. 1/His-ceramidase construct, and ceramidase activity (at pH 9.5) increased by 50- and 12-fold, respectively. Next, the enzyme was characterized using lysate of overexpressing cells. The results confirmed that the enzyme catalyzes the hydrolysis of ceramide in the neutral alkaline range and is independent of cations. Finally, a green fluorescent protein-ceramidase fusion protein was constructed to investigate the localization of this enzyme. The results showed that the green fluorescent protein-ceramidase fusion protein presented a mitochondrial localization pattern and colocalized with mitochondrial specific probes. These results demonstrate that this novel ceramidase is a mitochondrial enzyme, and they suggest the existence of a topologically restricted pathways of sphingolipid metabolism.

Amidohydrolases↗
PubMed2000

The mitochondrial permeability transition augments Fas-induced apoptosis in mouse hepatocytes.

Tumor necrosis factor-alpha receptor 1 and Fas recruit overlapping signaling pathways. To clarify the differences between tumor necrosis factor alpha (TNFalpha) and Fas pathways in hepatocyte apoptosis, primary mouse hepatocytes were treated with TNFalpha or an agonist anti-Fas antibody after infection with an adenovirus expressing an IkappaB superrepressor (Ad5IkappaB). Treatment with TNFalpha induced apoptosis in Ad5IkappaB-infected mouse hepatocytes, as we previously reported for rat hepatocytes. Ad5IkappaB plus anti-Fas antibody or actinomycin D plus anti-Fas antibody rapidly induced apoptosis, whereas anti-Fas antibody alone produced little cytotoxicity. The proteasome inhibitor (MG-132) and a dominant-negative mutant of nuclear factor-kappaB-inducing kinase also promoted TNFalpha- and Fas-mediated apoptosis. Expression of either crmA or a dominant-negative mutant of the Fas-associated death domain protein prevented TNFalpha- and Fas-mediated apoptosis. In addition, the caspase inhibitors, DEVD-cho and IETD-fmk, inhibited TNFalpha- and Fas-mediated apoptosis. In Ad5IkappaB-infected hepatocytes, caspases-3 and -8 were activated within 2 h after treatment with anti-Fas antibody or within 6 h after TNFalpha treatment. Confocal microscopy demonstrated onset of the mitochondrial permeability transition (MPT) and mitochondrial depolarization by 2-3 h after anti-Fas antibody treatment and 8-10 h after TNFalpha treatment, followed by cytochrome c release. The combination of the MPT inhibitors, cyclosporin A, and trifluoperazine, protected Ad5IkappaB-infected hepatocytes from TNFalpha-mediated apoptosis. After anti-Fas antibody, cyclosporin A and trifluoperazine decreased cytochrome c release but did not prevent caspase-3 activation and cell-death. In conclusion, nuclear factor-kappaB activation protects mouse hepatocytes against both TNFalpha- and Fas-mediated apoptosis. TNFalpha and Fas recruit similar but nonidentical, pathways signaling apoptosis. The MPT is obligatory for TNFalpha-induced apoptosis. In Fas-mediated apoptosis, the MPT accelerates the apoptogenic events but is not obligatory for them.

Animals↗
PubMed2000

Mitochondrial calcium transients in adult rabbit cardiac myocytes: inhibition by ruthenium red and artifacts caused by lysosomal loading of Ca(2+)-indicating fluorophores.

A cold/warm loading protocol was used to ester-load Rhod 2 into mitochondria and other organelles and Fluo 3 into the cytosol of adult rabbit cardiac myocytes for confocal fluorescence imaging. Transient increases in both cytosolic Fluo 3 and mitochondrial Rhod 2 fluorescence occurred after electrical stimulation. Ruthenium red, a blocker of the mitochondrial Ca(2+) uniporter, inhibited mitochondrial Rhod 2 fluorescence transients but not cytosolic Fluo 3 transients. Thus the ruthenium red-sensitive mitochondrial Ca(2+) uniporter catalyzes Ca(2+) uptake during beat-to-beat transients of mitochondrial free Ca(2+), which in turn may help match mitochondrial ATP production to myocardial ATP demand. After ester loading, substantial amounts of Ca(2+)-indicating fluorophores localized into an acidic lysosomal/endosomal compartment. This lysosomal fluorescence did not respond to electrical stimulation. Because fluorescence arose predominantly from lysosomes after the cold loading/warm incubation procedure, total cellular fluorescence failed to track beat-to-beat changes of mitochondrial fluorescence. Only three-dimensionally resolved confocal imaging distinguished the relatively weak mitochondrial signal from the bright lysosomal fluorescence.

Adenosine Triphosphate↗
PubMed2000

Contribution of adenosine A(2) receptors and cyclic adenosine monophosphate to protective ischemic preconditioning of sinusoidal endothelial cells against Storage/Reperfusion injury in rat livers.

A brief period of liver ischemia decreases sinusoidal endothelial cell killing after cold liver storage and improves graft survival after liver transplantation, a phenomenon called ischemic preconditioning. In this study, we investigated the mechanism of sinusoidal endothelial cell protection after ischemic preconditioning. Livers were preconditioned by 5 minutes of ischemia and 5 minutes of reperfusion. Subsequently, livers were stored for 30 hours in cold University of Wisconsin (UW) solution and reperfused briefly with physiological buffer containing Trypan blue. Ischemic preconditioning decreased sinusoidal endothelial cell killing after storage/reperfusion, as assessed by Trypan blue staining of nonparenchymal cells. Adenosine A(2) receptor blockade prevented the protective effect of ischemic preconditioning. By contrast, adenosine A(1) receptor blockade did not prevent protective ischemic preconditioning. Other rat livers were treated with adenosine A(1) and A(2) receptor agonists or dibutyryl-cyclic adenosine monophosphate (DB-cAMP) before storage. The adenosine A(2) receptor agonist, CGS-21680, and DB-cAMP decreased sinusoidal endothelial cell killing to the same extent as ischemic preconditioning, but the adenosine A(1) receptor agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA), had no effect. The adenosine A(2) agonist and prostaglandin E(2), another agent that preconditions sinusoidal endothelial cells against storage/reperfusion injury, but not the adenosine A(1) agonist, increased cAMP levels in cultured sinusoidal endothelial cells. In conclusion, an adenosine A(2) receptor pathway coupled to increased cAMP mediates sinusoidal endothelial cell protection by ischemic preconditioning.

Adenosine↗