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Effect of Hg2+ on cytosolic Ca2+ (Cai2+) in hepatocytes isolated from the little sakte Raja erinacea
Al132
AASLD ABSTRACTS
• CHARACTERIZATION AND FUNCTION OF ATP RECEPTORS ON HEPATOCYTES FROM THE LITTLE SKATE RAJA ERINACEA. Michael H. Nathanson and Kavita Mariwaila. Mt. Desert Island Biological Laboratory, Salsbury Cove, ME 04672 and Liver Study Unit, Yale'Univ. School of Med., New Haven, CT 06520. It is well established in higher life forms that ATP receptors can regulate hepatocyte function, and that this is mediated via increases in cytosolic Ca2+ (Cai2+). Three types of G protein-linked P2 purinergic receptors have been described, which display preferential sensiUvity to UDP, ADP, and 2-methylthio ATP (2MeSATP), respectively. Hormonal regulation of elasmobranch liver has not been investigated, so we examined Cai2+ signaling in hepatocytes from the little skate, rata erinacea. Hepatocytes were isolated by collagenase perfusion, then CaiZ+ was measured both in populations of hepatocytes and in individual cells. For measurements of CaiZ+ in populations of hepatocytes, cells were loaded with the Ca2+-sensitive dye indo-1 and examined by ratio spectrofluorometry. For measurements of Cai2+ in single hepatocytes, cells were loaded with the Ca2+-sensitive dye rhod-2 and examined by confocal line scanning microscopy. In hepatocyte populations, ATP (100 ~tM) induced a rapid increase in Caj z+, followed by a decrease to a level that remained elevated above baseline, similar to the pattern seen in mammalian hepatocytes. The initial phase of this Cm2+ increase occurred even in Ca2+-free medium, while the late, sustained(plateau) phase of the increase did not. Similar dose-response curves were seen upon stimulation with ATP, ADP, UTP, and 2MeSATP. ATP-yS was likewise effective at increasing Cai2+, but AMP, adenosine, 13-y-methylATP, CTP and GTP each induced much less or no Cai2+ increase. In single hepatocytes, ATP, ADP, UTP, and -2MeSATP each predominantly induced a sustained increase in Cai2+ at high concentrations (10-100 p2Vl),a single transient Cai2+ increase at a concentration of 1 llM, and repetitive transient Cai2+ increases (i.e., Cai2+ oscillations) at the lowest concentrations that elicited a response (10100 nM). ATP (100 I.tM) also induced a marked, transient increase in bile flow in the isolated perfused skate liver, from a baseline rate of 3.8+1.7 I.tl/gm liver/hr to a peak flow rate of 11.1+2.5 ~tl/gm liver/hr (mean+SD, p<0.005). In contrast, 100 liM adenosine had no such effect on bile flow. These findings demonstrate that skate hepatocytes possess P2 purinergic receptors which transduce signals that result in intracellular plus extracellular Ca2+ mobilization. One effect of these signals is to stimulate bile secretion. The broad specificity of the response to ATP and related compounds" suggests that multiple types of P2 receptors may be expressed on skate hepatocytes. Alternatively, these cells may possess a single, primitive purinergic receptor from which other P2 subtypes subsequently evolved.
GASTROENTEROLOGY, VoI. I O 8 , No. 4
• EFFECT OF Hg 2+ ON CYTOSOLIC Ca 2+ (Cai2+) IN HEPATOCYTES ISOLATED FROM THE LITTLE SKATE RAJA ERINACEA. Michael. H. Nathanson Kavita Mariwalla, Nazzareno Ballatori and James L. Boyer. Mt. Desert Island Biol. Laboratory, Salsbta'y Cove, ME 04672, Dept. of Environ. Med., Univ. of Rochester School of Med., Rochester, NY 14642, and Liver Center, Yale Univ. School of Med., New Haven, CT 06520. Hg2÷ is an environmental pollutant that adversely affects a number of secretion-related functions in skate hepatocytes, including cell volume regulation, Na+-alanine co-transport, and plasma membrane ion permeability. Cai2~ regulates bile secretion in rodents, and our preliminary work suggests that Cai2+ signals regulate bile secretion in skates as well. The purpose of this study was to examine whether Hg2+ affects Ca~2+ in isolated skate hepatocytes, and if so, to define the responsible mechanisms. Isolated hepatocytes were loaded with the Ca2+-sensitive dye indo-1 and examined by ratio spectrofluorometry. At lower concentrations (100 nM-1 pM), Hg 2+ induced no detectable change in Cai2+. At higher concentrations (5 ~M-1 mM), H g2+ induced a dose-dependent, progressive increase in Caiz+. This Caiz+ increase began within seconds after addition of Hg2+ and occurred even in Ca2+-free medium. Pre-treatment of hepatocytes with the membrane-impermeantHg2+ chelator glutathione (GSH, 5 mM) blocked the Cai2+ increase induced by 50 I.tM Hg2+, while addition of GSH 2 min after exposure to Hg 2+ slowed but did not prevent further increases in Cai2+. As with GSH, pre-treatment with the membrane-permeantHg 2+ chelator dithiothreitol (DTT, 500 liM) blocked Hg2+-induced increases in C~ 2+. Unlike GSH, however, addition of DTT 2 rain after 50 liM Hg2+ significantly decreased Cai2+, returning it to near-baseline levels. The Ca2+-ATPase inhibitor thapsigargin (2 I.tM) caused a sustained increase in Ca~2+ and addition of Hg 2+ resulted in a further, progressive Cai2+ increase." Acute effects of Hg2+ on toxicity were examined in two ways. First, Hg2+ (100 llM) induced no increase in propidium iodide uptake over 4 min, relative to untreated (control) hepatocytes. Second, no morphological changes were detected by light microscopy in Hg2+-treated hepatocytes over this same r these findings suggest that: (1) Hg2+ increases Cai2+ time period, T ogethe, in skate hepatocytes, (2) Hg 2+ must enter the hepatocytes for this Ca~2+ increase to occur, and (3) this increase is mediated by release of Ca2+t~rom endogenous stores which are distinct from the thapsigar~in-sensitiveCa2+ stores. Furthermore, this acute effect of Hg2+ on CaiZ+ does not cause or result from acute toxicity to hepatocytes. Additional work will be needed to define the intracellular source from which Hg2+ releases Ca2+ into the cytosol, and to determine if previously described effects of Hg2+ on hepatocyte metabolism are mediated by these Hg2+-induced Cai2+ signals.
OHIGH PREVALENCE AND PROGNOSTIC SIGNIFICANCE OF HEPATOCELLULAR CARCINOMA (HCC) IN CIRRHOTIC PATIENTS WITH SPONTANEOUS BACTERIAL PERITONITIS (SBP). M Navasa, E Moitinho, JM Llovet, R Bataller, MP Rodriguez-lglesias, A Castells, R Planas, J Bruix, MA Gasull, J Rod6s. Liver Unit, Hospital Clinic and Gastroenterology Unit, Hospital Germans Trias i Pujol.. Barcelona. Spain.
ORmL UFLuxALIN VS IN[RAVEIX~DUS CEFO]AXIME IN [HE IREAfMEN[ OF PE]N-C~PLICA~ED SPONTANEOUS BACTERIAL_ P E R / f O N I T I S tSBP) iN C I R ~ S i S . HESULIS OF A MULFICENTER, R~DC#IIZED ~RIAL. M Navasa, R__P~l_anas, G Clemente, V Vargas, C Guarner, A Follo, dl't Llovet, JM 'fiver, A Obrador, L Rodrigo, A Rimola, I'$A Gassuii, V Arroyo, J Redes and members of the C o l l a b o r a t i v e broup /or tl-e Study ot SBP. SPAIN.
SBP carries a poor prognosis and therefore this complication is considered an indication for liver transplant in cirrhotic patients with ascites. However, its grim prognosis may be influenced not only by the impaired liver and renal function, but also by the presence of an undedying HCC, a neoplasm affecting mainly cirrhotics. The present study was aimed at investigating if SBP is associated with HCC existence and to assess if HCC determines the short and long term prognosis in a sedes of 169 consecutive cirrhotic patients diagnosed with SBP. The diagnosis of SBP was based on an ascitic polymorphonuclear count > 250 cell/mm 3 together with compatible clinical signs and its treatment was based on 3rd generation cephalospodns, followed by quinolones as secondary prophylaxis. HCC was screened by ultrasound and confirmed by biopsy and/or increased alpha-fetoprotein levels. Thirty-four patients (20%) were diagnosed with HCC at the time of SBP and 3 additional patients were diagnosed eady during follow-up. The clinical pattern of HCC patients was not different from non-HCC patients. Furthermore, the SBP resolution was also similar between both groups (89% vs 94%, respectively). However, there were significant differences in survival both during hospitalization (51% vs 78%, p<0.01) and during follow-up, the 1-yr survival rate (Kaplan-Meier) being 12% and 48%, respectively (p<0.001). These results show that the prognosis of SBP is markedly heterogeneous and that the clinical outcome of the patients is influenced both by the degree of liver and renal function impairment and by the existence of HCC. Thereby, the 1-yr survival of the 50 patients with bilirubin <6mg/dL, serum creatinine <1.2 rag/alL and without HCC was significantly better than that of the remaining patients (66% vs 29%, p<0.0001). These results show that HCC comp|icates liver cirrhosis in 20% of the patients with SBP and that its presence significantly affects eady and long term survival. Accordingly, the design and analysis of therapeutic trials in SBP patients should consider this variable. Furthermore, the use of SBP as a marker for liver transplant indication should be carefully temPered.
intravenous cefotaxime is the gold standard for the [rearmost of SliP. lhe introduction of the new qulnolones, which have high bioavailability after oral administration and eflioacy against the bacteria usually responsible for SBP, have opened n e w perspectives for the oral treatment of this infection. Allv__j: To c o m p a r e the efficacy of oral oflaxacin vs intravenous c e f o E a x i m e in the treatment o~ SBP. PAl IENIS ~ D I~L]HODS: One hundred anO thirty-two patients w i t ~ non-complicated SBP (compatible clinical data and F~IN count ;. 250/mm3 in the absence of severe complications: stock , i leus, gastrointestinal bleeding and serum creatinine > 3 mg/dl) have been included in a controlled trial in 8 hospitals. Patient w e r e randomized to receive oral ofloxacin 400 mg b.i.d. (69 patients, Group I) or intravenous c e f o t a x i m e 2g/6h (63 patients, Group I I). RESULIS: At inclusion there w e r e n o significant d i f f e r e n c e s between the two groups in age, sex, and data related ta the irtfection and underlying liver disease. After inclusion, secondary bacterial peritonitis was diagnosed in 8 cases and there was a voluntary drop-out. [hese 9 patients (Group I:% Group If:41 w e r e not included in the analysis of efficacy. /here were no significant differences in infection resolution between the two groups: 54/64 (84.4%) Group I, =0/59 (84.7%) Group II; (95% C o n f i d e n c e interval for the difference: -13.1 to 12.4%). There w e r e also no significant differences in treatment duration (median: 9 days in G r o u p 1 vs 8 days in Group II ) and survival at the end of hospital ization (55/69; 79.7% vs 5i/63; 80.9%; respectively ). C(~xCLUSION: [hese results indlcate that oral ofloxacin is as effective as intravenous cefotaxime in the treat/nent of non-complicated SBP.
AASLD ABSTRACTS
• CHARACTERIZATION AND FUNCTION OF ATP RECEPTORS ON HEPATOCYTES FROM THE LITTLE SKATE RAJA ERINACEA. Michael H. Nathanson and Kavita Mariwaila. Mt. Desert Island Biological Laboratory, Salsbury Cove, ME 04672 and Liver Study Unit, Yale'Univ. School of Med., New Haven, CT 06520. It is well established in higher life forms that ATP receptors can regulate hepatocyte function, and that this is mediated via increases in cytosolic Ca2+ (Cai2+). Three types of G protein-linked P2 purinergic receptors have been described, which display preferential sensiUvity to UDP, ADP, and 2-methylthio ATP (2MeSATP), respectively. Hormonal regulation of elasmobranch liver has not been investigated, so we examined Cai2+ signaling in hepatocytes from the little skate, rata erinacea. Hepatocytes were isolated by collagenase perfusion, then CaiZ+ was measured both in populations of hepatocytes and in individual cells. For measurements of CaiZ+ in populations of hepatocytes, cells were loaded with the Ca2+-sensitive dye indo-1 and examined by ratio spectrofluorometry. For measurements of Cai2+ in single hepatocytes, cells were loaded with the Ca2+-sensitive dye rhod-2 and examined by confocal line scanning microscopy. In hepatocyte populations, ATP (100 ~tM) induced a rapid increase in Caj z+, followed by a decrease to a level that remained elevated above baseline, similar to the pattern seen in mammalian hepatocytes. The initial phase of this Cm2+ increase occurred even in Ca2+-free medium, while the late, sustained(plateau) phase of the increase did not. Similar dose-response curves were seen upon stimulation with ATP, ADP, UTP, and 2MeSATP. ATP-yS was likewise effective at increasing Cai2+, but AMP, adenosine, 13-y-methylATP, CTP and GTP each induced much less or no Cai2+ increase. In single hepatocytes, ATP, ADP, UTP, and -2MeSATP each predominantly induced a sustained increase in Cai2+ at high concentrations (10-100 p2Vl),a single transient Cai2+ increase at a concentration of 1 llM, and repetitive transient Cai2+ increases (i.e., Cai2+ oscillations) at the lowest concentrations that elicited a response (10100 nM). ATP (100 I.tM) also induced a marked, transient increase in bile flow in the isolated perfused skate liver, from a baseline rate of 3.8+1.7 I.tl/gm liver/hr to a peak flow rate of 11.1+2.5 ~tl/gm liver/hr (mean+SD, p<0.005). In contrast, 100 liM adenosine had no such effect on bile flow. These findings demonstrate that skate hepatocytes possess P2 purinergic receptors which transduce signals that result in intracellular plus extracellular Ca2+ mobilization. One effect of these signals is to stimulate bile secretion. The broad specificity of the response to ATP and related compounds" suggests that multiple types of P2 receptors may be expressed on skate hepatocytes. Alternatively, these cells may possess a single, primitive purinergic receptor from which other P2 subtypes subsequently evolved.
GASTROENTEROLOGY, VoI. I O 8 , No. 4
• EFFECT OF Hg 2+ ON CYTOSOLIC Ca 2+ (Cai2+) IN HEPATOCYTES ISOLATED FROM THE LITTLE SKATE RAJA ERINACEA. Michael. H. Nathanson Kavita Mariwalla, Nazzareno Ballatori and James L. Boyer. Mt. Desert Island Biol. Laboratory, Salsbta'y Cove, ME 04672, Dept. of Environ. Med., Univ. of Rochester School of Med., Rochester, NY 14642, and Liver Center, Yale Univ. School of Med., New Haven, CT 06520. Hg2÷ is an environmental pollutant that adversely affects a number of secretion-related functions in skate hepatocytes, including cell volume regulation, Na+-alanine co-transport, and plasma membrane ion permeability. Cai2~ regulates bile secretion in rodents, and our preliminary work suggests that Cai2+ signals regulate bile secretion in skates as well. The purpose of this study was to examine whether Hg2+ affects Ca~2+ in isolated skate hepatocytes, and if so, to define the responsible mechanisms. Isolated hepatocytes were loaded with the Ca2+-sensitive dye indo-1 and examined by ratio spectrofluorometry. At lower concentrations (100 nM-1 pM), Hg 2+ induced no detectable change in Cai2+. At higher concentrations (5 ~M-1 mM), H g2+ induced a dose-dependent, progressive increase in Caiz+. This Caiz+ increase began within seconds after addition of Hg2+ and occurred even in Ca2+-free medium. Pre-treatment of hepatocytes with the membrane-impermeantHg2+ chelator glutathione (GSH, 5 mM) blocked the Cai2+ increase induced by 50 I.tM Hg2+, while addition of GSH 2 min after exposure to Hg 2+ slowed but did not prevent further increases in Cai2+. As with GSH, pre-treatment with the membrane-permeantHg 2+ chelator dithiothreitol (DTT, 500 liM) blocked Hg2+-induced increases in C~ 2+. Unlike GSH, however, addition of DTT 2 rain after 50 liM Hg2+ significantly decreased Cai2+, returning it to near-baseline levels. The Ca2+-ATPase inhibitor thapsigargin (2 I.tM) caused a sustained increase in Ca~2+ and addition of Hg 2+ resulted in a further, progressive Cai2+ increase." Acute effects of Hg2+ on toxicity were examined in two ways. First, Hg2+ (100 llM) induced no increase in propidium iodide uptake over 4 min, relative to untreated (control) hepatocytes. Second, no morphological changes were detected by light microscopy in Hg2+-treated hepatocytes over this same r these findings suggest that: (1) Hg2+ increases Cai2+ time period, T ogethe, in skate hepatocytes, (2) Hg 2+ must enter the hepatocytes for this Ca~2+ increase to occur, and (3) this increase is mediated by release of Ca2+t~rom endogenous stores which are distinct from the thapsigar~in-sensitiveCa2+ stores. Furthermore, this acute effect of Hg2+ on CaiZ+ does not cause or result from acute toxicity to hepatocytes. Additional work will be needed to define the intracellular source from which Hg2+ releases Ca2+ into the cytosol, and to determine if previously described effects of Hg2+ on hepatocyte metabolism are mediated by these Hg2+-induced Cai2+ signals.
OHIGH PREVALENCE AND PROGNOSTIC SIGNIFICANCE OF HEPATOCELLULAR CARCINOMA (HCC) IN CIRRHOTIC PATIENTS WITH SPONTANEOUS BACTERIAL PERITONITIS (SBP). M Navasa, E Moitinho, JM Llovet, R Bataller, MP Rodriguez-lglesias, A Castells, R Planas, J Bruix, MA Gasull, J Rod6s. Liver Unit, Hospital Clinic and Gastroenterology Unit, Hospital Germans Trias i Pujol.. Barcelona. Spain.
ORmL UFLuxALIN VS IN[RAVEIX~DUS CEFO]AXIME IN [HE IREAfMEN[ OF PE]N-C~PLICA~ED SPONTANEOUS BACTERIAL_ P E R / f O N I T I S tSBP) iN C I R ~ S i S . HESULIS OF A MULFICENTER, R~DC#IIZED ~RIAL. M Navasa, R__P~l_anas, G Clemente, V Vargas, C Guarner, A Follo, dl't Llovet, JM 'fiver, A Obrador, L Rodrigo, A Rimola, I'$A Gassuii, V Arroyo, J Redes and members of the C o l l a b o r a t i v e broup /or tl-e Study ot SBP. SPAIN.
SBP carries a poor prognosis and therefore this complication is considered an indication for liver transplant in cirrhotic patients with ascites. However, its grim prognosis may be influenced not only by the impaired liver and renal function, but also by the presence of an undedying HCC, a neoplasm affecting mainly cirrhotics. The present study was aimed at investigating if SBP is associated with HCC existence and to assess if HCC determines the short and long term prognosis in a sedes of 169 consecutive cirrhotic patients diagnosed with SBP. The diagnosis of SBP was based on an ascitic polymorphonuclear count > 250 cell/mm 3 together with compatible clinical signs and its treatment was based on 3rd generation cephalospodns, followed by quinolones as secondary prophylaxis. HCC was screened by ultrasound and confirmed by biopsy and/or increased alpha-fetoprotein levels. Thirty-four patients (20%) were diagnosed with HCC at the time of SBP and 3 additional patients were diagnosed eady during follow-up. The clinical pattern of HCC patients was not different from non-HCC patients. Furthermore, the SBP resolution was also similar between both groups (89% vs 94%, respectively). However, there were significant differences in survival both during hospitalization (51% vs 78%, p<0.01) and during follow-up, the 1-yr survival rate (Kaplan-Meier) being 12% and 48%, respectively (p<0.001). These results show that the prognosis of SBP is markedly heterogeneous and that the clinical outcome of the patients is influenced both by the degree of liver and renal function impairment and by the existence of HCC. Thereby, the 1-yr survival of the 50 patients with bilirubin <6mg/dL, serum creatinine <1.2 rag/alL and without HCC was significantly better than that of the remaining patients (66% vs 29%, p<0.0001). These results show that HCC comp|icates liver cirrhosis in 20% of the patients with SBP and that its presence significantly affects eady and long term survival. Accordingly, the design and analysis of therapeutic trials in SBP patients should consider this variable. Furthermore, the use of SBP as a marker for liver transplant indication should be carefully temPered.
intravenous cefotaxime is the gold standard for the [rearmost of SliP. lhe introduction of the new qulnolones, which have high bioavailability after oral administration and eflioacy against the bacteria usually responsible for SBP, have opened n e w perspectives for the oral treatment of this infection. Allv__j: To c o m p a r e the efficacy of oral oflaxacin vs intravenous c e f o E a x i m e in the treatment o~ SBP. PAl IENIS ~ D I~L]HODS: One hundred anO thirty-two patients w i t ~ non-complicated SBP (compatible clinical data and F~IN count ;. 250/mm3 in the absence of severe complications: stock , i leus, gastrointestinal bleeding and serum creatinine > 3 mg/dl) have been included in a controlled trial in 8 hospitals. Patient w e r e randomized to receive oral ofloxacin 400 mg b.i.d. (69 patients, Group I) or intravenous c e f o t a x i m e 2g/6h (63 patients, Group I I). RESULIS: At inclusion there w e r e n o significant d i f f e r e n c e s between the two groups in age, sex, and data related ta the irtfection and underlying liver disease. After inclusion, secondary bacterial peritonitis was diagnosed in 8 cases and there was a voluntary drop-out. [hese 9 patients (Group I:% Group If:41 w e r e not included in the analysis of efficacy. /here were no significant differences in infection resolution between the two groups: 54/64 (84.4%) Group I, =0/59 (84.7%) Group II; (95% C o n f i d e n c e interval for the difference: -13.1 to 12.4%). There w e r e also no significant differences in treatment duration (median: 9 days in G r o u p 1 vs 8 days in Group II ) and survival at the end of hospital ization (55/69; 79.7% vs 5i/63; 80.9%; respectively ). C(~xCLUSION: [hese results indlcate that oral ofloxacin is as effective as intravenous cefotaxime in the treat/nent of non-complicated SBP.