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Effect of adenosine on metabolic rate and HSP 70 expression in anoxic hepatocytes
S115
P26-1
P26-3
Effect of Adenosine on Metabolic Rate and HSP 70 Expression in Anoxic Hepatocytes. Buck, L.T., J. Balasub ramaniam & N.Salem, University of Toronto, Dept. of Zoology, 25 Harbord St. Toronto, Ontario. Canada, M5S 3G5. Adenosine is known to reduce neuronal excitability, heart rate, ion channel open probability, and possibly the expression of heat shock proteins. These effects are consistent with a key role for adensine in the transition to a hypometabolic state during anoxia. We investigated the effect of adensine and the specific A1 receptor agonist (CPA) and antagonist (DPCPX) on normoxic and anoxic metabolic rates and expression of HSP 72 in turtle hepatocytes. Adenosine resulted in a 10% decrease in hepatcyte 02 consumption. This response was inhibited by DPCPX and reproduced by CPA. From the rate of lactate accumulation during anoxia we determined that adenosine and A1 specific receptor modulators had a similar effect on anoxic metabolic rate. Using Northern blot analysis we detected HSP 72 mRNA in hepatocytes following heat shock, exposure to heavy metals or free radicals and 1,2,4 and 8 hr anoxia. The inclusion of DPCPX during anoxia blocked the appearance of HSP 72 mRNA, conversely CPA could not induce HSP 72 during normoxia. Our results suggest that adenosine plays a role in the expression of HSP 72 and arresting metabolism during the transition to anoxia in turtle hepatocytes Work supported by NSERC Canada.
Gap junctions are not involved in the reduction in whole-cell conductance in anoxic turtle neurons. Ghai, H. & Buck L.T. University of Toronto, Dept. of Zoology, 25 Harbord St. Toronto, Ontario. Canada, M5S 3G5. We tested the effect of anoxia, a mimic aCSF consisting of high [Ca”] and Frgz’], low pH, and adenosine perfusions on whole cell conductance (Gw) in turtle brain sheets using a whole-cell patch-clamp technique. G, was unchanging throughout a 130 min normoxic control recording period. G, decreased during anoxia or mimic aCSF?erfusions, and this was EGTA sensitive, pointing to a Ca + mediated second messenger cascade. The A1 receptor agonist CPA reduced G, in a dose dependent manner whereas the antagonist DPCPX blocked both the adenosine and anoxic mediated changes in Gap junctions provide a possible non-synaptic G,. mechanism by which anoxia, Ca2+, and adenosine could exert Whole cell capacitance (C,) their effects on G,. measurements were obtained from tissue exposed to anoxic, high [Ca2T, and adenosine perfusions. Control values for C, did not change significantly over a period of 13Omin. Anoxia, Ca2’, and adenosine perfusion did not result in any significant changes in C,. These data suggest a mechanism involving A1 receptor mediated changes in [C$‘]i resulting in acute changes in G, with anoxia. The response is independent of changes in gap junction permeability. Work supported by NSERC Canada.
P26-2 Hormonal modulation silver sea bream.
P26-4 of branchial
beat shock protein 70 ia
Deane EE, Kelly SP and Woo NYS Department of Biology, The Chinese University of Hong Kong, Shatin, NT., Hong Kong. Our previous study on hormonal modulation of silver sea bream (Spanrs samba) hepatic heat shock protein 70 (HSP70) expression revealed a down-regulatory effect of growth hormone and prolactin [Deane et al., J. Endocrinol., 161 (19!?9)]. In this study, HSP70 expression was assessed in branchial tissue of silver sea bream. Using a previously isolated HSP70 cDNA clone as a probe in HSP70 mRNA analysis, we detected a single heat-shock inducible transcript. For our studies on hormonal modulation of branchial HSP70, groups of fish were administered recombinant bream growth hormone (rbGH), ovine prolactin (oPRL) or cortisol, daily, over a seven-day period. Quantification of HSP70 transcript, using RNA dot blots revealed that rbGH and oPRL treatment increased HSP70 mRNA abundance by 1.7 and 1.6 fold respectively compared to controls. In order to provide data to link this rbGH- and oPRL-induced elevation in HSP70 mRNA abundance with protein levels we developed an ELISA using antibody to bovine HSP70. Data from ELISA showed that rbGH and oPRL elevated HSP70 by 1.8 and 2.5 fold respectively from controls. Cortisol treatment did not alter branchial HSP70 mFWA abundance or protein levels. The results from this study provide evidence for up-regulation of bronchial HSP70, as opposed to hepatic HSP70 down-regulation after GH and PRL administration to silver sea bream.
Expression
of the a,-acid glycoprotein gene in fetal rat liver during acute phase response
Mihailovic MI, Petrovic Ml, Milicevic 22 and Bogojevic D’ ‘Institute for Biological Research, 2The Institute of Nuclear Sciences “Vinca”, 11000 Belgrade, Yugoslavia
al-acid glycoprotein (AGP) is one of the most prominent mammalian acute phase proteins with pleotropic biological functions consisting of regulation of immune response and transportation of cationic compounds. The AGP gene expression mainly occurs in the liver. Although AGP is classified as a gene dominantly expressed after birth, we have examined the activation of this gene using the fetal livers of untreated and 12h-turpentine treated dams on day 19 of gestation. The 3-fold increase of AGP plasma concentration in fetuses during acute phase response was the consequence of the elevated level of corresponding mRNA and transcriptional activity, respectively. pointed at the activation of the examined gene in prenatal liver also. Knowing that transcriptional regulation of the AGP gene in adult rat hepatocytes depends on constitutive trans-acting proteins, South-Western assay showed participation of constitutive nucleoproteins with molecular masses of 68, 53, 35 and 29 kD, as well as new ones of 33, 20 and 18 kD having increase binding affinity to distal regulatory element of the examined gene (-5300/-5150) in acute phase fetal livers. Immune-Western analysis with antibodies to transcriptional factor ~53 as well as high mobility protein 1 (HMGI) identified presence of homologues of these proteins in modulation of AGP transcription.
P26-1
P26-3
Effect of Adenosine on Metabolic Rate and HSP 70 Expression in Anoxic Hepatocytes. Buck, L.T., J. Balasub ramaniam & N.Salem, University of Toronto, Dept. of Zoology, 25 Harbord St. Toronto, Ontario. Canada, M5S 3G5. Adenosine is known to reduce neuronal excitability, heart rate, ion channel open probability, and possibly the expression of heat shock proteins. These effects are consistent with a key role for adensine in the transition to a hypometabolic state during anoxia. We investigated the effect of adensine and the specific A1 receptor agonist (CPA) and antagonist (DPCPX) on normoxic and anoxic metabolic rates and expression of HSP 72 in turtle hepatocytes. Adenosine resulted in a 10% decrease in hepatcyte 02 consumption. This response was inhibited by DPCPX and reproduced by CPA. From the rate of lactate accumulation during anoxia we determined that adenosine and A1 specific receptor modulators had a similar effect on anoxic metabolic rate. Using Northern blot analysis we detected HSP 72 mRNA in hepatocytes following heat shock, exposure to heavy metals or free radicals and 1,2,4 and 8 hr anoxia. The inclusion of DPCPX during anoxia blocked the appearance of HSP 72 mRNA, conversely CPA could not induce HSP 72 during normoxia. Our results suggest that adenosine plays a role in the expression of HSP 72 and arresting metabolism during the transition to anoxia in turtle hepatocytes Work supported by NSERC Canada.
Gap junctions are not involved in the reduction in whole-cell conductance in anoxic turtle neurons. Ghai, H. & Buck L.T. University of Toronto, Dept. of Zoology, 25 Harbord St. Toronto, Ontario. Canada, M5S 3G5. We tested the effect of anoxia, a mimic aCSF consisting of high [Ca”] and Frgz’], low pH, and adenosine perfusions on whole cell conductance (Gw) in turtle brain sheets using a whole-cell patch-clamp technique. G, was unchanging throughout a 130 min normoxic control recording period. G, decreased during anoxia or mimic aCSF?erfusions, and this was EGTA sensitive, pointing to a Ca + mediated second messenger cascade. The A1 receptor agonist CPA reduced G, in a dose dependent manner whereas the antagonist DPCPX blocked both the adenosine and anoxic mediated changes in Gap junctions provide a possible non-synaptic G,. mechanism by which anoxia, Ca2+, and adenosine could exert Whole cell capacitance (C,) their effects on G,. measurements were obtained from tissue exposed to anoxic, high [Ca2T, and adenosine perfusions. Control values for C, did not change significantly over a period of 13Omin. Anoxia, Ca2’, and adenosine perfusion did not result in any significant changes in C,. These data suggest a mechanism involving A1 receptor mediated changes in [C$‘]i resulting in acute changes in G, with anoxia. The response is independent of changes in gap junction permeability. Work supported by NSERC Canada.
P26-2 Hormonal modulation silver sea bream.
P26-4 of branchial
beat shock protein 70 ia
Deane EE, Kelly SP and Woo NYS Department of Biology, The Chinese University of Hong Kong, Shatin, NT., Hong Kong. Our previous study on hormonal modulation of silver sea bream (Spanrs samba) hepatic heat shock protein 70 (HSP70) expression revealed a down-regulatory effect of growth hormone and prolactin [Deane et al., J. Endocrinol., 161 (19!?9)]. In this study, HSP70 expression was assessed in branchial tissue of silver sea bream. Using a previously isolated HSP70 cDNA clone as a probe in HSP70 mRNA analysis, we detected a single heat-shock inducible transcript. For our studies on hormonal modulation of branchial HSP70, groups of fish were administered recombinant bream growth hormone (rbGH), ovine prolactin (oPRL) or cortisol, daily, over a seven-day period. Quantification of HSP70 transcript, using RNA dot blots revealed that rbGH and oPRL treatment increased HSP70 mRNA abundance by 1.7 and 1.6 fold respectively compared to controls. In order to provide data to link this rbGH- and oPRL-induced elevation in HSP70 mRNA abundance with protein levels we developed an ELISA using antibody to bovine HSP70. Data from ELISA showed that rbGH and oPRL elevated HSP70 by 1.8 and 2.5 fold respectively from controls. Cortisol treatment did not alter branchial HSP70 mFWA abundance or protein levels. The results from this study provide evidence for up-regulation of bronchial HSP70, as opposed to hepatic HSP70 down-regulation after GH and PRL administration to silver sea bream.
Expression
of the a,-acid glycoprotein gene in fetal rat liver during acute phase response
Mihailovic MI, Petrovic Ml, Milicevic 22 and Bogojevic D’ ‘Institute for Biological Research, 2The Institute of Nuclear Sciences “Vinca”, 11000 Belgrade, Yugoslavia
al-acid glycoprotein (AGP) is one of the most prominent mammalian acute phase proteins with pleotropic biological functions consisting of regulation of immune response and transportation of cationic compounds. The AGP gene expression mainly occurs in the liver. Although AGP is classified as a gene dominantly expressed after birth, we have examined the activation of this gene using the fetal livers of untreated and 12h-turpentine treated dams on day 19 of gestation. The 3-fold increase of AGP plasma concentration in fetuses during acute phase response was the consequence of the elevated level of corresponding mRNA and transcriptional activity, respectively. pointed at the activation of the examined gene in prenatal liver also. Knowing that transcriptional regulation of the AGP gene in adult rat hepatocytes depends on constitutive trans-acting proteins, South-Western assay showed participation of constitutive nucleoproteins with molecular masses of 68, 53, 35 and 29 kD, as well as new ones of 33, 20 and 18 kD having increase binding affinity to distal regulatory element of the examined gene (-5300/-5150) in acute phase fetal livers. Immune-Western analysis with antibodies to transcriptional factor ~53 as well as high mobility protein 1 (HMGI) identified presence of homologues of these proteins in modulation of AGP transcription.